Behavioral Neurogenetics

Current Topics in Behavioral Neurosciences
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Current Topics in Behavioral Neurosciences provides critical and comprehensive

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Behavioral Neurogenetics
123
Editors
John F. Cryan Prof. Dr. Andreas Reif
Department of Anatomy and Neuroscience Department of Psychiatry
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Preface
The field of behavioural neurogenetics has developed significantly over the past
two decades. This has been largely driven by technical advances in the field of
molecular genetics both in model systems and in clinical analysis. Indeed, it is
hoped that the elucidation and ongoing functionalisation of the human genome
may provide new insights into the aetiology, course and, ultimately, treatment of
psychiatric illnesses.
This book covers a wide array of topics relevant to behavioural genetics from
both a preclinical and clinical standpoint. Indeed in juxtaposing both areas of
research the reader will appreciate the true translational nature of the field. Topics
covered range from technical advances in genetic analysis in humans and animals
to specific descriptions of advances in schizophrenia, attention disorders, depres-
sion and anxiety disorders, autism, aggression, neurodegeneration and neurode-
velopmental disorders. The importance of gene–environment interactions is
emphasised and the role of neuroimaging in unravelling the functional conse-
quences of genetic variability described.
Part I of this book focuses on advances in the basic sciences of behavioural
neurogenetics with a strong emphasis on animal models of psychiatric illness. It
opens with a chapter by Robert Gerlai highlighting the use of model organisms and
specifically zebrafish (Danio rerio) in modelling complex human psychiatric
disease and its applicability for behavioural genetic studies. Lisa Tarantino follows
by giving the state of the art on forward genetic approaches to elucidate the
contribution of genetic variation to complex behavioural phenotypes. Carola and
Gross illuminate the relative contribution of environment and genetics to psy-
chopathology and how this is informing translational studies in animals and
humans. Miczek and colleagues discuss the neurobiological mechanisms under-
lying aggression and how it can be modified in genetically modified mice. We-
gener, Mathe and Neumann describe the utility of various selectively bred rodent
strains to ask key questions regarding the underlying pathophysiology of depres-
sion and anxiety. Next up is a chapter on how genetic manipulations in rodent
models have allowed for analysis of the impact of specific roles of glutamate
receptors and transporters in cognitive and emotional behaviours shown to be
v
vi Preface
altered by stress. The final two chapters are relevant to schizophrenia. First,
O’Tuathaigh and colleagues describe how phenotypic characterisation of genetic
models of candidate risk genes and/or putative pathophysiological processes
implicated in schizophrenia, as well as examination of epidemiologically relevant
gene 9 environment interactions in these models, can illuminate molecular and
pathobiological mechanisms involved in schizophrenia. Powell, on the other hand,
reviews the literature on genetic models of sensorimotor gating as they apply to
schizophrenia and other neuropsychiatric disorders and discusses the utility of
prepulse inhibition as a tool in phenotyping mutant mouse models.
Part II of this volume focuses on advances in clinical genetic analysis as applied
to various neuropsychiatric disorders. Bayes and colleagues describe how second
generation sequencing technologies are generating unprecedented amounts of
sequence data very rapidly and at relatively limited costs. They also describe the
challenges associated with such data generation in terms of data interpretation,
analysis and management in addition to highlighting where such technologies are
moving to in the future. Hakonarson and colleagues follow on with a description of
the role of copy number variations in a number of neurodevelopmental disorders
including autism, attention-deficit/hyperactivity disorder and schizophrenia. They
also discuss relevant methodological considerations for such analysis. The appli-
cation of neuroimaging has transformed modern neuroscience research, thus Hariri
and colleagues describe how such approaches can be used to understand the
interplay between genes and behaviour in shaping individual variability in brain
function. Christine Freitag, Philip Asherson and Joahnnes Hebebrand discuss in
detail the behavioural neurogenetics of childhood disorders including autism
spectrum disorders, attention deficit/hyperactivity disorder, nocturnal enuresis and
obesity. A chapter follows this on the use of new technologies to identify genes
relevant to Schizophrenia by Dan Rujescu. As highlighted in the preclinical sec-
tion there is growing appreciation of gene–environment as an emerging area in
psychiatry research. Katja Karg and Srijan Sen give a comprehensive introduction
to the field from a clinical context emphasising theoretical and practical problems
that are worth considering. The behavioural neurogenetics of affective and anxiety
disorders is expertly reviewed by Katharina Domschke and Andreas Reif in the
subsequent chapter. This is followed up by a discussion by Quinn and colleagues
of the contribution of variable number tandem repeat polymorphisms to a range of
psychiatric disorders. Individual variability in response to stimulant drugs has long
been known and Amy Hart, Harriet De Wit and Abraham Palmer examine the
evidence for the contribution of genetic polymorphisms to this response. The
penultimate chapter in the book focuses on the cognitive genetics of psychiatric
disorders and reviews evidence for the heritability of the main cognitive pheno-
types and early progress in the field using cytogenetic, linkage and candidate gene-
based research methodologies. The book closes with a chapter from Daniela
Galimberti and Elio Scarpini on the behavioural genetics of neurodegenerative
disorders with a special focus on susceptibility genes for Alzheimer’s Disease and
Frontotemporal Lobar Degeneration.
Preface vii
These chapters, either individually or as a whole, provide a broad overview of

the current status of a rapidly evolving and exciting field. Both the basic scientist
and clinician alike will value this volume. It will also be of use to the novice to the
field, to whom it will serve as an in-depth introduction to this area of research.
Finally, it is worth noting that the advances made in behavioural neurogenetics to
date have been very promising and we are particularly optimistic that the parallel
advances in both basic and clinical neurogenetics fields will lead to a better
understanding and eventual treatment strategies for complex neuropsychiatric
disorders where there remains a large unmet medical need.
Cork, Ireland John F. Cryan

Würzburg, Germany Andreas Reif
Contents
Part I Preclinical Behavioral Genetics
Using Zebrafish to Unravel the Genetics of Complex

Brain Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Robert Gerlai
Forward Genetic Approaches to Understanding

Complex Behaviors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Lisa M. Tarantino and Amy F. Eisener-Dorman
Mouse Models of the 5-HTTLPR 3 Stress Risk Factor

for Depression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Valeria Carola and Cornelius Gross
Behavioral and Pharmacogenetics of Aggressive Behavior . . . . . . . . . 73

Aki Takahashi, Isabel M. Quadros, Rosa M. M. de Almeida
and Klaus A. Miczek
Selectively Bred Rodents as Models of Depression and Anxiety . . . . . . 139

Gregers Wegener, Aleksander A. Mathe and Inga D. Neumann
Stress-Induced Deficits in Cognition and Emotionality:

A Role for Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Carolyn Graybeal, Carly Kiselycznyk and Andrew Holmes
ix
x Contents
Susceptibility Genes for Schizophrenia: Mutant Models,

Endophenotypes and Psychobiology . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Colm M. P. O’Tuathaigh, Lieve Desbonnet, Paula M. Moran
and John L. Waddington
Genetic Models of Sensorimotor Gating: Relevance

to Neuropsychiatric Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Susan B. Powell, Martin Weber and Mark A. Geyer
Part II Clinical Behavioral Genetics
Applications of Second Generation Sequencing Technologies

in Complex Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Mònica Bayés, Simon Heath and Ivo Glynne Gut
Rare Genomic Deletions and Duplications

and their Role in Neurodevelopmental Disorders . . . . . . . . . . . . . . . . 345
Joseph T. Glessner, John J. M. Connolly and Hakon Hakonarson
Toward a Mechanistic Understanding of How Variability

in Neurobiology Shapes Individual Differences in Behavior. . . . . . . . . 361
Ryan Bogdan, Justin M. Carré and Ahmad R. Hariri
Behavioural Genetics of Childhood Disorders. . . . . . . . . . . . . . . . . . . 395

Christine M. Freitag, Philip Asherson and Johannes Hebebrand
Schizophrenia Genes: On the Matter of Their Convergence . . . . . . . . 429

Dan Rujescu
Gene 3 Environment Interaction Models

in Psychiatric Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Katja Karg and Srijan Sen
Behavioral Genetics of Affective and Anxiety Disorders . . . . . . . . . . . 463

Katharina Domschke and Andreas Reif
Behavioural Genetics of the Serotonin Transporter . . . . . . . . . . . . . . 503

K. Haddley, V. J. Bubb, G. Breen, U. M. Parades-Esquivel and J. P. Quinn
Contents xi
Genetic Factors Modulating the Response to Stimulant

Drugs in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Amy B. Hart, Harriet de Wit and Abraham A. Palmer
The Cognitive Genetics of Neuropsychiatric Disorders . . . . . . . . . . . . 579

A. Corvin, G. Donohoe, A. Hargreaves, L. Gallagher and M. Gill
Behavioral Genetics of Neurodegenerative Disorders . . . . . . . . . . . . . 615

Daniela Galimberti and Elio Scarpini
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
Part I
Preclinical Behavioral Genetics
Using Zebrafish to Unravel the Genetics
of Complex Brain Disorders
Robert Gerlai
Abstract The zebrafish has been prominently utilized in developmental biology

for the past three decades and numerous genetic tools have been developed for it.
Due to the accumulated genetic knowledge the zebrafish has now been considered
an excellent research tool in other disciplines of biology too, including behavioral
neuroscience and behavior genetics. Given the complexity of the vertebrate brain in
general and the large number of human brain disorders whose mechanisms remain
mainly unmapped in particular, there is a substantial need for appropriate laboratory
research organisms that may be utilized to model such diseases and facilitate the
analysis of their mechanisms. The zebrafish may have a bright future in this research
field. It offers a compromise between system complexity (it is a vertebrate similar in
many ways to our own species) and practical simplicity (it is small, easy to keep,
and it is prolific). These features have made zebrafish an excellent choice, for
example, for large scale mutation and drug screening. Such approaches may have a
chance to tackle the potentially large number of molecular targets and mechanisms
involved in complex brain disorders. However, although promising, the zebrafish is
admittedly a novel research tool and only few empirical examples exist to support
this claim. In this chapter, first I briefly review some of the rapidly evolving genetic
methods available for zebrafish. Second, I discuss some promising examples for
how zebrafish have been used to model and analyze molecular mechanisms of
complex brain disorders. Last, I present some recently developed zebrafish
behavioral paradigms that may have relevance for a spectrum of complex human
brain disorders including those associated with abnormalities of learning and
memory, fear and anxiety, and social behavior. Although at this point co-application
R. Gerlai (&)
Department of Psychology, University of Toronto Mississauga,
3359 Mississauga Road North, Rm 3035,
Mississauga, ON L5L 1C6, Canada
e-mail: robert_gerlai@yahoo.com
Curr Topics Behav Neurosci (2012) 12: 3–24 3

DOI: 10.1007/7854_2011_180
Published Online: 17 January 2012
4 R. Gerlai
of the genetics and behavioral approaches is rare with zebrafish, I argue that the
rapid accumulation of knowledge in both of these disciplines will make zebrafish a
prominent research tool for the genetic analysis of complex brain disorders.

Keywords Zebrafish High throughput behavioral screening Fetal alcohol

syndrome Alcoholism Learning and memory Fear and anxiety
Contents
1 Zebrafish, a Novel Research Tool with Some Advantages............................................. 4

2 Zebrafish, the Favorite of Geneticists .............................................................................. 5
3 Reverse Genetic Tools ...................................................................................................... 6
4 Forward Genetic Tools...................................................................................................... 8
5 Parkinson’s Disease........................................................................................................... 10
6 Tauopathy and Alzheimer’s Disease ................................................................................ 10
7 The Anxiety ‘‘Cluster’’...................................................................................................... 11
8 Autism Spectrum Disorders .............................................................................................. 12
9 Schizophrenia..................................................................................................................... 12
10 Expanding Our Horizon: The Need for Sophisticated Behavioral
Test Paradigms in Zebrafish Research ............................................................................. 13
11 Learning and Memory....................................................................................................... 14
12 Fear and Anxiety ............................................................................................................... 17
13 Social Behavior ................................................................................................................. 19
14 Concluding Remarks ......................................................................................................... 21
References.................................................................................................................................. 21
1 Zebrafish, a Novel Research Tool with Some Advantages
Numerous laboratory organisms are available for one to employ in the analysis of
how genes influence behavior or how certain biochemical processes affect the
functioning of the brain. For example, rats have been traditionally utilized to screen
libraries of compounds to identify drugs that may be beneficial in a range of human
brain and behavioral disorders both in academic and biopharmaceutical preclinical
research. The house mouse has been employed in a range of behavior genetic
applications including those that probe the effects of specific genes (reverse
genetics) and their roles in behavior and brain function [e.g. Gerlai et al. (1995),
Pekhletski et al. (1996)]. But other, simpler, laboratory organisms including the flat
worm (Giles and Rankin 2009), the sea slug (Bailey and Kandel 2008), or the fruit
fly (Sokolowski 2001) have also been successfully utilized to study the biological
bases of brain function and behavior. Compared to these laboratory model organ-
isms the zebrafish is quite novel. I use the term ‘‘model’’ here in a very loose sense
and only mean that the reason for the use of animal species in the laboratory is to
isolate and mimic some aspects of complex biological phenomena, a reductionist
approach that may yield results faster than in human due to the simpler features of
Using Zebrafish to Unravel the Genetics 5
the studied organism and to the precisely controlled laboratory conditions. In this
sense, zebrafish may be an ideal model organism.
The zebrafish strikes an optimal compromise between system complexity and
practical simplicity. It is a vertebrate species with a physiology (Alsop and Vijayan
2008), brain anatomy (Tropepe and Sive 2003), and neurochemistry (Chatterjee and
Gerlai 2009) characteristic of the prototypical vertebrate, and thus translationally
relevant to our own species. Most importantly, the nucleotide sequence of zebrafish
genes is often found highly similar (70–80% homology) to the mammalian (and
human) counterparts, and the amino acid sequence of functionally relevant domains
of its proteins has been found to be even more evolutionarily conserved, i.e. similar
to mammalian sequences (Reimers et al. 2004; Renier et al. 2007). It is thus quite
probable that a gene identified in zebrafish as involved in particular functions/dys-
functions of its brain, will have a human homolog serving similar functions and vica
versa. Briefly, the translational relevance of zebrafish research is expected to be high.
The number of animals one can screen is a crucial factor in forward genetics
where one does not know which and how many genes may influence the pheno-
typical function in question. In case of brain disorders or behavioral function, the
number of such genes may be quite large and thus one may have to analyze
thousands of mutants to tackle this complexity and identify appropriate mutations
and thus the genes involved. One can, of course, generate the same number of
zebrafish and mice for screening purposes but there are several reasons why
zebrafish may be preferred. First, a single female zebrafish can produce 200
offspring at every spawning and can spawn multiple times a week. Second,
zebrafish is small (4 cm long) and is highly social and thus a large number of
subjects may be housed cheaply in a small animal holding room. For example, a
standard zebrafish stand-alone high density rack system (e.g. Aquatic Ecosystems
Inc, FL, or Aquaneering Inc. CA) with six shelves and about 23 liter tanks per shelf,
can house about 2,000 zebrafish, and a 40 m2 standard vivarium room may be fitted
up with up to 10 such racks. Briefly, the same room that may house a couple of
hundred mice can have about 20,000 zebrafish in it. Therefore, when it comes to
large scale screening, the zebrafish has a definite cost advantage. Given the relative
simplicity of this vertebrate species and the fact that it is a phylogenetically older
‘‘design’’ compared to mammals, one may also argue that it may allow the analysis
of fundamental core mechanisms of the chosen brain function. Last, adding zebrafish
to the list of already well studied vertebrates (e.g. the mouse and the rat) should
facilitate cross species comparison and finding common characteristics and mech-
anisms, which should also enhance our ability to translate the findings to human.
2 Zebrafish, the Favorite of Geneticists
Genetics is one of the strengths of zebrafish and excellent reviews have been
published on numerous genetic techniques developed for this species (Amsterdam
and Hopkins 2006; Chen and Ekker 2004; Lekven et al. 2000; Patton and Zon 2001).
6 R. Gerlai
Here I will discuss these techniques only briefly. In addition to sophisticated gene
expression analyses including quantitative reverse transcriptase polymerase
chain reaction (q-RT-PCR) and DNA microarrays (gene chip), both reverse genetic
and forward genetic methods are available with zebrafish, although the former, the
forward genetic approaches, have been more prevalent in zebrafish research. Reverse
genetic analysis allows one to study the phenotypical effects of targeted manipulation
of known genes. The main goal of forward genetic studies, on the other hand, is to
discover novel genes by the introduction of random mutations.
3 Reverse Genetic Tools
Among the reverse genetic tools, TILLING has been employed successfully in
zebrafish (Moens et al. 2008). Targeting induced local lesions in genomes
(TILLING) allows the identification of mutations in specific genes of interest in
chemically mutagenized zebrafish populations. The method was first described for
mutation detection in Arabidopsis about a decade ago but since then it has
successfully been adopted for zebrafish too (Moens et al. 2008). The essence of the
TILLING method is the screening of chemically mutagenized populations of
zebrafish using the polymerase chain reaction (PCR) for mutations in the gene
of interest. Unlike in gene targeting with the use of homologous recombination in
embryonic stem cells employed in the mouse (Capecchi 1989), and most recently
in the rat (Tong et al. 2010), the actual mutagenesis conducted in TILLING is
random, i.e. not targeted. The ‘‘trick’’ of TILLING is then the identification of the
mutation(s) in the target gene. The identification of the mutation may be achieved
using two different approaches. One is the resequencing of every single mutage-
nized genome (the gene of interest and its sequence is known and thus alterations
in the sequence can be detected), a brute force approach that is becoming
increasingly feasible with the ever improving speed of sequencing methods. The
other method is based upon the use of cell, a plant-specific extracellular glyco-
protein that cleaves heteroduplex DNA at single nucleotide mismatches (resulting
from the introduced point mutation). Using fluorescently labeled primers to
detect cell cleavage products on a LiCor acrylamide slab gel, cell can identify a
heterozygous single nucleotide mismatch [for further details of the TILLING
methodology and its use, see Moens et al. (2008)].
Another approach that is principally a reverse genetic method, i.e. it is also
aimed at the characterization of the function of known genes, is a knock down
method using morpholinos (Bill et al. 2009). Morpholinos are antisense oligonu-
cleotides (usually 25 bases long) composed of a phosphorodiamidate backbone
with a morpholine ring and the same bases as DNA (Bill et al. 2009). Morpholinos
are injected into the target cells and act by steric hindrance to block ribosome entry
and hence prevent protein production. The antisense technology has been long
employed in mammalian species but the efficiency and specificity of the oligo-
nucleotide approach has been controversial. In zebrafish, however, the morpholino
approach has been successfully employed. For example, due to the altered
backbone of the morpholinos they are not affected by nucleases and are, therefore,
highly resistant to breakdown in vivo. Furthermore, because of their small size and
unusual chemistry, morpholinos remain undetected for the immune system.
Morpholinos are traditionally introduced into the yolk of 1–8 cell-stage embryos in
which the cytoplasmic bridges connecting the embryonic cells allow rapid diffu-
sion of the hydrophilic morpholinos leading to ubiquitous delivery. The analysis of
the result of the gene expression knock down is usually limited to the embryonic
stage of zebrafish. However, examples already exist suggesting that modified
morpholino chemistry (the VIVO-morpholino, which allows penetration of the
oligonucleotide into adult zebrafish cells) may be successfully employed in adult
zebrafish as well [e.g. Kim et al. (2010)]. This is a crucial novel development
considering that most complex brain disorders and higher behavioral functions can
be best modeled, observed, and analyzed in the adult zebrafish.
RNA-interference, or RNAi, is yet another intriguing possibility for targeted
modification of gene expression in zebrafish. RNAi is a transcriptional gene
silencing mechanism-induced by short (21–23 bases long) double stranded
RNA whose main mechanisms are believed to be gene expression regulation via
miRNA’s (endogeneous microRNA’s) and defense against viral genetic material
mediated by dsRNAs (double stranded RNAs), terms that represent structurally
indistinguishable mRNA species. Irrespective of the physiological function, a few
years ago it was realized that the cell’s RNAi mechanism could be utilized for the
induction of targeted knock down of gene expression by delivering double
stranded short RNA sequences specific to the chosen target gene. Although
zebrafish cells have been shown to possess the RNAi machinery, the functional
consequences, especially the specificity of the RNAi-based manipulation, have
been questioned [for review see Skromne and Prince (2008)] and thus whether this
technology will lead to success in zebrafish remains to be seen. Another tech-
nology, transgenic methods, however, may offer a currently existing true and tried
alternative.
In the mammalian neurobehavioral genetics field, transgenic methods have
been perhaps the most fruitful reverse genetic approaches. Transgenic technologies
have also been successfully employed with zebrafish [for a recent review see
Skromne and Prince (2008)]. These techniques make use of a variety of methods
(e.g. enzymatic approaches, transposons, and retroviruses) to enable the delivery
and increase the efficiency of incorporation of foreign DNA into the genome of
zebrafish thereby generating stable transgenic fish lines in which the foreign DNA
is expressed. Irrespective of the mode of delivery, transgenic zebrafish may be
divided into two main classes: transgenic overexpressors and dominant negative
transgenics. In the former, overexpression of the transgene is achieved, for
example, by delivering multiple copies of the transgene or using a strong promoter,
and is used to test the functional consequences of the excess amount of translated
gene product. In the latter, the expressed transgene product interferes with or
blocks the function of the endogenous gene and thus allows one to test the effect of
loss-of-function at the phenotypical level.
8 R. Gerlai
In addition to constitutive transgene expression, inducible expression systems

are also available in zebrafish. Given that zebrafish tolerate a broad range of
temperatures (in its natural geographical range temperatures may vary between
10 and 35°C), heatshock promoters have been successfully employed to induce
transgene expression in a temporally controlled manner, and the use of focal
heating has also allowed the induction of transgene expression in a spatially
restricted manner, at least in superficial structures. Furthermore, the Gal4-UAS
system (Scott et al. 2007), well developed for the fruit fly, the tetracycline trans-
activator system (Huang et al. 2005), as well as the Cre/loxP system (Langenau
et al. 2005) used, for example, in the temporal and spatial control of knock out of
genes in the mouse, have all been utilized in the control of transgene expression in
zebrafish.
Although the classical knock out technology based upon homologous recom-
bination in embryonic stem cells as employed in the mouse (Capecchi 1989) and
most recently in the rat (Tong et al. 2010) is not yet available in the zebrafish,
research in this direction is also progressing. For example, germline transmitting
embryonic stem cells have been isolated from zebrafish (Fan and Collodi 2006)
and methods alternative to classical gene targeting are also being explored. For
example, nuclear transfer of genetically modified cultured embryonic fibroblast
cells has been achieved in zebrafish, which implies that targeted genetic modifi-
cation for in vivo analysis of gene function may be possible via this route (Lee
et al. 2002). Furthermore, zinc finger nuclease-based knockout technology is also
being developed for zebrafish (Ekker 2008). Zinc finger nucleases are genetically
engineered restriction enzymes that cut the DNA sequence of interest according to
their specific design. Zebrafish embryos injected with the specific custom designed
zinc finger nuclease-encoding mRNA are reared to adulthood and crossed with
wild type fish. As much as 25% of the resulting offspring has been shown to
transmit the induced mutation, usually a frameshift allele in the germline (Ekker
2008). Last, a gene-breaking transposon-based method to generate mutations is
also being developed for zebrafish (Sivasubbu et al. 2006) to mention but a few
reverse genetic technologies.
The range of reverse genetic approaches discussed above is somewhat mis-
leading, however. Although they do demonstrate how fast zebrafish genetics is
evolving, many of these methodologies are not mature enough for reliable use
even for embryonic or developmental biology phenotypes, the focus of most of
these investigations. For complex neurobehavioral traits, virtually none of these
methods have been employed.
4 Forward Genetic Tools
Unlike reverse genetic approaches, forward genetics has provided decades of

consistent success with zebrafish. The first two large scale comprehensive forward
genetic screens the Tubingen (Haffter et al. 1996) and Boston (Driever et al. 1996)
screens were conducted about 15 years ago and set the stage for subsequent
screening studies. Since then, most forward genetic studies have utilized a
chemical mutagen, ethyl-nitroso-urea (ENU), which is expected to induce single
nucleotide point mutations, and when dosed appropriately, one mutation per
genome on average. The advantage of ENU mutagenesis is that ENU is efficient
and with it one can achieve a good coverage of the entire genome, i.e. can expect
to hit a large proportion of genes as long as a large enough number of animals
(thousands) are mutagenized and analyzed. The Achilles heel of ENU mutagen-
esis, however, is the subsequent linkage analysis, which requires several genera-
tions of crosses and cumbersome mapping. Nevertheless, due to the availability of
high resolution markers developed for zebrafish, the genes carrying the induced
mutations can be successfully identified using linkage analysis-based positional
cloning (Knapik 2000; Patton and Zon 2001). Another concern with ENU-based
forward genetic analysis is that the point mutation induced by ENU is often
recessive and thus may not be observable unless bred into a homozygous form,
which requires three generations of breeding to create an F3 or a backcross seg-
regating population (Patton and Zon 2001). Although not without technical
complications, an alternative that can speed up the generation of recessive
homozygous mutants does exist, it is gynogenesis. For gynogenesis one may use
heat shock or pressure to manipulate the cell division cycle at the earliest
embryonic stage leading to haploid to diploid genome conversion and allowing the
generation of homozygous fish in essentially one step without the cumbersome
breeding [for review of experimental examples using gynogenesis, see Patton and
Zon (2001)].
ENU has been the most frequently employed mutagen for zebrafish, but other
mutagenesis methods have also been successfully utilized. Perhaps the most
promising among them is retrovirus mediated insertional mutagenesis (Amsterdam
and Hopkins 2006). This method has the advantage over ENU because insertion of
the viral genetic material into the zebrafish genome not only induces a mutation
but by leaving the unique viral sequences in the genome allows fast and efficient
localization and cloning of the mutated gene (Amsterdam and Hopkins 2006).
A potential concern with forward genetic approaches in zebrafish is that this
species has undergone a partial genome duplication event in its evolutionary past.
The concern is that the genes whose locus is on the duplicated region of the
genome (approximately 20% of the genome) may be able to compensate for the
induced mutation and could mask its effects if the sister gene remained active and
if its function has not changed much since the gene duplication event. However,
some argue that the partial ‘‘tetraploidy’’ may be viewed as an advantage in
zebrafish for forward genetics especially when it comes to the analysis of the
genetics of complex traits: the induced mutations are not expected to be lethal and
may only have small quantitative effects on the phenotype, thus making functional
phenotypical identification and characterization feasible. Whether these arguments
turn out to be correct will have to be seen. But the few already existing examples
suggest that we have some reason for optimism. Below I review such examples
organized according to their disease relevance.
10 R. Gerlai
5 Parkinson’s Disease
Parkinson’s disease is the second most prevalent human neurodegenerative dis-

ease, which primarily affects the nigro-striatal dopaminergic system. Numerous
genes have been identified associated with Parkinson’s disease in humans three of
which are believed to underlie early onset of Parkinson’s Disease: PARK2
encoding Parkin, PINK1 encoding PTEN-induced putative kinase 1, and PARK7
encoding DJ-1 [for review see Bandmann and Burton (2010)]. The zebrafish
orthologs of the human genes have been identified and, for example, the PARK2
gene product is known to be a 458 amino acid long protein which is 75% similar to
the human protein with functionally important regions having 93% similarity.
The similarities between the other two human genes with their zebrafish orthologs
are also substantial. Morpholino-induced knock down of expression of these genes
have led to significant dopaminergic neuron loss and/or to increased sensitivity of
these neurons to toxins [although the selectivity of the effects have been ques-
tioned in the case of PARK6, reviewed by Bandmann and Burton (2010)].
Behavioral impairments resulting from the knock down have not been shown in
zebrafish with one exception: Morpholino antisense knock down induced reduction
of PINK1 expression led to loss of tyrosine hydroxylase staining in distinct groups
of dopaminergic neurons in the zebrafish brain and when challenged with normally
sub-effective concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrapyradine (MPTP)
the affected larval zebrafish showed reduced locomotion. Detailed swim path
analysis of these larvae was not conducted, however.
6 Tauopathy and Alzheimer’s Disease
Another class of neurodegenerative diseases is the tauopathies. These diseases are

all defined by their underlying molecular pathology: the presence of hyperphos-
phorylated insoluble forms of the microtubule associated protein Tau, which is
deposited in neurons in the form of observable neurofibrillary tangles. Most of these
diseases are sporadic [e.g. progressive supranuclear palsy (PSP), corticobasal
degeneration (CBD), and Pick’s disease (PiD)], but neurofibrillary tangles have also
been associated with familial early onset of Alzheimer’s disease. Both transient as
well as stable transgenic zebrafish lines overexpressing Tau have been generated and
in both structures resembling neurofibrillary tangles have been reported [for review
see Bandmann and Burton (2010)]. Behavioral analysis of the effects of these
changes has not been conducted except for a rudimentary, observation-based,
judgment as to whether tactile stimulation induced a normal escape response in the
48 h post-fertilization embryo expressing a mutant form of Tau (Paquet et al. 2009).
Alzheimer’s disease is often mentioned under tauopathies but there are other
prominent known genetic factors associated with this disease too. Alzheimer’s
disease is the most common form of dementia affecting over 25 million people
worldwide (Querfurth and LaFerla 2010). The histological hallmarks of the disease
are neurofibrillary tangles (mentioned above) and, perhaps even more character-
istically, amyloid plaques. Although the familial (heritable) form of the disease
only makes up 4–5% of all cases, clearly demonstrating the importance of
environmental factors, the genetic analysis of Alzheimer’s disease has led to major
discoveries and clarified some of the biological mechanisms core to the disease.
The genes encoding amyloid-b precursor protein (APP), and presenilins PS1 and
PS2 have been found to underlie familial Alzheimer’s disease, and the e4 allele of
the gene encoding apolipoprotein E (ApoE) has been found to be a risk factor in
sporadic (non-familial) Alzheimer’s cases. The APP, PS1, PS2, and ApoE genes
have all been identified in zebrafish and have been found to be highly homologous
to their mammalian, and human counterparts, with certain functionally relevant
regions approaching 100% identity with human (e.g. the transmembrane region of
APP). Furthermore, some of the components of the c-secretase complex mediating
the processing and cleavage of APP, which may lead to the generation of the toxic
Ab (40, 42) peptide have started to be examined in zebrafish (reviewed in Xia
2010). To characterize the involvement of APP in zebrafish embryonic develop-
ment, the expression of the APP gene was reduced using morpholinos, which
led to significant shortening of the body length of the embryos (Joshi et al. 2009).
The involvement of overexpression of APP (as in Down’s patients, for example,
who also develop Alzheimer’s disease) or of the expression of mutant forms of
APP (identified in familial Alzheimer’s cases), or the role of presinilins have not
been investigated in zebrafish, nor has been any studies conducted for the potential
behavioral consequences of transgenic manipulations of these genes. The function
of ApoE and its potential role in Alzheimer’s disease related abnormalities also
has not been investigated with zebrafish.
7 The Anxiety ‘‘Cluster’’
Perhaps the largest cluster of human brain disorders in terms of prevalence are
neuropsychiatric conditions including anxiety and stress disorders, depression,
obsessive compulsive disorders, and several forms of phobias. Although both
academic and pharmaceutical and biotechnology companies have been studying
the potential mechanisms of these disorders and several treatment options have
been developed, these diseases still represent an enormous unmet medical need.
This is because the causative factors (both genetic and environmental) behind
these diseases are difficult to trace and the mechanisms of the diseases are also
complex. A recent review paints an optimistic picture as to the potential use of
zebrafish in the analysis and modeling of neuropsychiatric conditions (Mathur and
Guo 2010) and I share this optimism. For example, zebrafish possess a gluco-
corticoid and a mineralocorticoid receptor that have been cloned and sequenced,
and has a corticoid signaling pathway highly similar to that of mammals
(Amsterdam and Hopkins 2006; Denver 2009). Also, the major components
12 R. Gerlai
necessary for success, including the genetic tools discussed above and the novel
behavioral paradigms, some of which are presented below, already exist for
zebrafish. Nevertheless, one must also acknowledge that these studies with zeb-
rafish are only starting now and thus have not produced major breakthroughs.
8 Autism Spectrum Disorders
The zebrafish has also been suggested for the analysis of the mechanisms of autism
spectrum disorders (Tropepe and Sive 2003). The number of genetic factors
underlying autism spectrum disorders is believed to be much less than in such
neuropsychiatric conditions as anxiety or schizophrenia, and thus animal genetic
models have been generated with much hope [for a review, see e.g. Gerlai and
Gerlai (2003)]. Importantly, several of the genes implicated in the human disease
have been shown to have homologs in zebrafish [for a most recent review, see
Mathur and Guo (2010)]. It may therefore be possible to recapitulate some aspects
of autism spectrum disorders by selectively targeting these genes and testing the
effect of the genetic or pharmacological manipulations on developmental as well
as behavioral characteristics in zebrafish. It is also notable that the zebrafish is a
highly social species, and the novel behavioral paradigms that are being developed
to induce and quantify social behavioral responses in zebrafish may also contribute
to this research, for example, by allowing large scale mutaganesis screening-based
identification of molecular mechanisms involved in vertebrate social behavior.
9 Schizophrenia
Schizophrenia is a neurodevelopmental disorder that often manifests first during

adolescence. Unlike in the case of autism spectrum disorders the number of genes
involved in schizophrenia may be extremely large (hundreds) and many of these
genes may only have a minor ‘‘predisposing’’ effect, which have hindered the
unraveling of the mechanisms of the disease. Nevertheless, some of the genes
implicated in schizophrenia have been identified in zebrafish. For example, DISC1,
a schizophrenia susceptibility gene, has been shown to play roles in cell migration
and differentiation in the zebrafish neural crest (Drerup et al. 2009) as well as in
the development of oligodendrocytes and neuronal lineages developing from olig2
expressing precursor cells. In addition to delineating the cellular and molecular
roles of some schizophrenia associated genes, there is already one example for a
genetic manipulation to affect zebrafish behavior. SHANK3 is a synaptic scaf-
folding protein whose gene was recently identified to carry mutations in some
patients suffering from schizophrenia and was also found in autistic patients.
Morpholino-induced knock down of the expression of the corresponding gene
resulted in robust morphological abnormalities as well as impaired swimming in
response to tactile stimulation in the zebrafish larva (Gauthier et al. 2010). It may
be noted, however, that the specificity of the morpholino-induced changes may be
questionable given that the attempt to rescue the phenotype by injection of wild
type or mutant SHANK3 mRNA sequences led only to partial success at best.
Last, psychopharmacological experiments have already started to be utilized with
zebrafish in the analysis and modeling of schizophrenia. One behavioral endo-
phenotype often argued to be an important aspect of schizophrenia is reduced
prepulse inhibition, or PPI. Prepulse inhibition is believed to be a measure of
sensory gating. It is induced by employing a weak stimulus (the prepulse), which is
expected to inhibit the reaction to a subsequent stronger startling stimulus (the
pulse). Larval zebrafish exhibit PPI of the acoustic startle response similarly to
what has been demonstrated in rodents [reviewed in Mathur and Guo (2010)]. PPI
can be disrupted by dopamine agonists in the zebrafish larvae, an alteration that is
reversed by antipsychotic drugs similarly to the mammalian situation (Braff et al.
2001). In addition to these promising psychopharmacology results, a forward
genetic screen has already isolated a mutant ‘‘Ophelia’’, which exhibited reduced
PPI (Burgess and Granato 2007). In summary, the first examples showing how
zebrafish may be utilized in the investigation of the genetic mechanisms of
complex brain disorders already exist. However, in most of these studies the
behavioral consequences of the employed experimental manipulations were not
analyzed or were studied in a rudimentary manner. This is, in general, the current
weakness of the zebrafish as an experimental tool: its genetics and neurobiology
have been traditionally powerful but its behavioral characteristics are largely
unmapped because only a few behavioral test paradigms are available (Sison et al.
2006).
10 Expanding Our Horizon: The Need for Sophisticated

Behavioral Test Paradigms in Zebrafish Research
Sophisticated behavioral paradigms may be crucial for two main reasons: first, the
construct and face validity of the genetic (reverse genetics) or pharmacological
models may only be fully established using behavioral tests; and two, behavioral
paradigms may represent unbiased screening tools for forward genetic applications
(Gerlai 2002). Arguably, behavioral analysis can efficiently probe a broad spec-
trum of brain functions in a large number of subjects. It is not limited to particular
brain regions or neurobiological mechanisms, and it is simple and cheap to
conduct (Gerlai and Clayton 1999). Arguably, high throughput behavioral screens
may be able to systematically reveal mutation or drug-induced functional changes
in the brain. Fortunately, for the past few years a clear upsurge of zebrafish
behavioral studies is evident, indicating that behavioral neuroscience and behavior
genetics has started to acknowledge the utility of this species. Below I discuss
some of these recent studies focusing on the question of how behavioral analysis
may be utilized for the discovery of novel genes and compounds affecting brain
14 R. Gerlai
dysfunction associated with complex human brain disorders. Three main behav-
ioral focus areas will be represented below: learning and memory, which is rele-
vant for a number of neurodegenerative diseases including Alzheimer’s disease;
fear and anxiety, which are important behavioral responses and behavioral states
relevant for a spectrum of neuropsychiatric conditions; and social behavior, whose
abnormalities may be important in the analysis of autism spectrum disorders and
schizophrenia, to mention but the two most important diseases in this domain.
11 Learning and Memory
Learning and memory has been extensively studied by scholars of several scien-
tific fields. Numerous mechanistic questions related to how learning occurs, and
what memory is, have been successfully tackled. For example, by now a large
number of genes and biochemical mechanisms underlying learning and memory
have been identified (Sweatt 2010). Can zebrafish add anything to this wealth of
knowledge? Although hundreds of genes involved in learning and memory have
been identified, the mechanisms of these complex processes are far from being
understood. It is likely that the number of undiscovered genes that play roles in
learning and memory is large. For example, according to conservative estimates,
most vertebrate genomes contain about 30,000 genes. Recent microarray studies
suggest that at least 50% of all the genes of the genome are expressed in the
vertebrate brain (e.g. in zebrafish), i.e. about 15,000 genes (Pan et al. 2010). Given
that plasticity is perhaps the most complex aspect of brain function, it is likely that
a large proportion of these genes, i.e. potentially thousands of them, are involved
in some mechanisms subserving plasticity, i.e. learning and memory.
A number of laboratories have realized that the cheap and easy to breed zeb-
rafish may offer a solution for high throughput screening which would be costly
with traditional laboratory rodents. Investigators have started to characterize the
cognitive capabilities of zebrafish and have already developed several test methods
that can measure learning and memory efficiently and fast [for examples see Sison
et al. (2006), Sison and Gerlai (2010)]. The key in these paradigms concerns
automatability. Even if one needs to employ several repeated training trials, as is
the case in most learning paradigms, if these trials are administered in an auto-
mated manner, and if the behavioral responses that reflect learning and memory
performance are easy to measure and do not require the constant presence and
attention of an experimenter, the paradigm may be run in multiple test apparati in
parallel and thus become high throughput.
A successful high throughput learning task design utilizes moving (animated)
images of conspecifics, which are shown on a computer screen placed by each side
of the experimental tank (Pather and Gerlai 2009). Previously, access to view a
shoal (group) of zebrafish has been shown to represent a reward for experimental
zebrafish and that this visual stimulus (the sight of a group of zebrafish) can support
good learning performance (Al-Imari and Gerlai 2008). Subsequently, it has been
demonstrated that computer animated images of zebrafish can serve as a positive

reinforcement (Gerlai et al. 2009a). The simple manner with which the reward
could be administered allowed the development of an automated learning task
(Pather and Gerlai 2009). The task is also simple. For a short period of time [20 s in
Pather and Gerlai (2009)] the image of the moving shoal is shown and then it is
turned off for 90 s. After this 90 s no-image period the image of the moving shoal is
shown on the opposite side of the test tank for again 20 s, and the sequence repeats
itself. As a result of the alternating image presentation sides, zebrafish have to make
a choice during the no-image period as to whether they stay close to the side where
the image was just shown, or move to the opposite side, where the image will
appear. The natural tendency of zebrafish is to stay close to its conspecifics and thus
initially zebrafish spend the highest amount of time near the side where the image
was shown last. However, as the training proceeds, zebrafish spend increasingly
longer amount of time near the side that will show the image. There are several
important points to make about these results. First, the motivation to stay close to
conspecifics does not habituate over time and thus the experimental subjects remain
motivated to perform in this task, a major advantage compared to the use of food,
which satiates zebrafish quickly. Second, the stimulus is a visual cue administered
precisely using consumer grade (i.e. cheap) video-equipment. Third, the behavioral
response (distance from stimulus screen) is easy to quantify using video-tracking
systems and/or motion detectors (e.g. photocell detector arrays). Fourth, multiple
trials [in Pather and Gerlai (2009), thirty trials] can be administered without
the intervention by the experimenter. The fish stays in the test tank and is given the
stimuli and their responses are measured repeatedly across the continuous sequence
of trials. As a result of all these above features, the paradigm is fully automated and
thus multiple set-ups can be run in parallel. Although the 30 trials required 3,300 s
(55 min) per experimental fish (Giles and Rankin 2009), one can easily set up
several such test apparati. Briefly, the throughput of the task can be dramatically
increased by scaling up. In our facility a 20 m2 test room could be easily fitted with
50 such test apparati, i.e. in an eight work hour day, one can test 400 zebrafish in a
single room using this learning task, a sufficiently high throughput even for large
scale mutagenesis screens (Haffter and Nüsslein-Volhard 1996).
The above paradigm is new and thus there are numerous unexplored questions
one may need to address. For example, we do not know whether zebrafish can
forecast the future, i.e. whether their performance improvement was due to better
timing of their responses (knowing when and where the shoaling image will appear
in the near future). It is possible that the performance improvement of the fish
was simply due to acquisition of CS-US association: disappearance of the stimulus
on one side serving as the conditioned stimulus predicted the reappearance
of the unconditioned stimulus, the shoal image, on the opposite side. There are
many questions that concern possible optimization of the task as well: is the
20/90 s stimulus/no-stimulus interval ratio the best? Could longer tanks
(the original experiment was conducted in a 50 cm long tank, a distance that can
be easily traversed by the fast zebrafish) be more appropriate allowing more
sensitive detection of performance improvements/deficits? Also, how would other
16 R. Gerlai
stimulus/no-stimulus schedules (random versus fixed ratio, increasing stringency

versus constant) affect the behavior of the fish? Last, mechanistic questions as to
what neuroanatomical structures subserve the task, what drugs may influence
performance in it, and how sensitive it may be to detect mutation-induced changes
all will have to be explored. Clearly, there are many questions when one intro-
duces a new paradigm. Nevertheless, the above example demonstrates how one
can utilize species-specific perceptual, motor and motivational characteristics to
design relatively simple and high throughput behavioral test methods that may
allow addressing many of the above questions in the future.
The above learning task have an important temporal component, the delay between
the stimulus presented on one versus the other side of the tank and as such may allow
the analysis of a complex forms of learning in zebrafish known to be associated with
the hippocampus in mammals, trace conditioning (McEchron and Disterhoft 1999)
and/or acquisition of relational memory (Cohen et al. 1997). Although fish do not have
a structure whose circuitry resembles that of the mammalian hippocampus, they do
possess a brain region, the lateral pallium that is believed to be a structure homologous
to the mammalian hippocampus (Vargas et al. 2009). Furthermore, fish without the
classical mammalian hippocampal circuitry have also been found to be able to learn
spatial learning paradigms, a class of tasks that is associated with hippocampal
function in mammals (Salas et al. 1996). Spatial learning has also been demonstrated
in zebrafish (Sison and Gerlai 2010), however, the spatial task employed (learning to
find a particular location in a plus maze) was extremely time consuming. It required
many repeated trials which could be administered only manually. Could one design a
high throughput spatial task for zebrafish?
This question was answered in a recent study (Gómez-Laplaza and Gerlai 2010)
that demonstrated good learning performance of zebrafish in a latent-learning
paradigm. The paradigm consisted of two phases, a long training phase and a brief
probe trial. During the training phase zebrafish were allowed to explore a complex
maze which consisted of a starting chamber that was connected to a goal chamber
by a left and right tunnel. Zebrafish were allowed to explore the maze in groups of
ten (a shoal) for 16 consecutive days, each day once for 50 min. Allowing zeb-
rafish to swim around the maze in ten-member shoals facilitated active exploration
and reduced passive fear responses. During the exploration of the maze certain
shoals were allowed to go through only one of the tunnels, i.e. there was a set of
fish for which only the left tunnel was open and the right tunnel was blocked and
another for which the left tunnel was blocked and only the right tunnel was open,
and yet another group for which both tunnels were open, a spatial exploration task.
The second part of the paradigm was a short (10 min long) probe trial, during
which both tunnels of the maze were open, a shoal of stimulus fish was placed
inside a transparent container and into the goal chamber of the maze, and the
experimental fish were tested singly in the maze. Given the social nature of
zebrafish, the experimental subject was highly motivated to get as close to the
stimulus fish in the goal chamber as possible. Which route, the right versus the left
tunnel, the experimental fish took was video-recorded and analyzed. The results
showed that those fish that experienced the right tunnel open during the maze
exploration phase of the paradigm also used the right tunnel during the probe trial,
those fish that experienced the left tunnel open used the left tunnel during the
probe trial and those fish that experienced both the left and right tunnel open chose
randomly. Why is this paradigm high throughput? Although the exploration phase
of the paradigm took 16 days, because the fish were not monitored and their
behavior was not analyzed, one could set up a large number of mazes and train a
large number of fish every day. The probe trial was conducted for every fish
separately, but it lasted only for 10 min per fish and the swim path of the fish could
be quantified using automated video-tracking techniques (Blaser and Gerlai 2006).
Thus this phase of the paradigm could also be made high throughput. Furthermore,
given the spatial nature of the task, this paradigm is likely to be capable of tapping
into complex forms of learning and memory.
There are again many questions about this novel paradigm. What motivates the
fish to learn the maze? In other words, why fish remember the tunnel they explored
before? This form of learning is termed latent learning because apparently there is
no external experimenter controlled motivator (positive or negative reinforcement)
presented. However, it has been argued (Gómez-Laplaza and Gerlai 2010), based
on prior supporting evidence, that exploration of novelty itself is rewarding in this
task and the novel aspect of the maze is what kept the fish motivated to explore and
learn. The results of this study also suggested that learning in this paradigm was
likely based upon acquiring and remembering external visual cues, i.e. spatial
learning, a hypothesis that will need to be proven in the future. But again, despite
the novel aspect of the task and the fact that there may be numerous questions one
could explore with it, the paradigm does appear to be appropriate for high
throughput screening of learning and mnemonic characteristics of zebrafish and
mutation-induced changes in these characteristics.
There are numerous human disorders associated with memory loss and/or
impairment of cognitive function, perhaps the most devastating and prevalent is
Alzheimer’s disease discussed above. But milder forms of memory problems, mild
cognitive impairment (MCI) and age-related memory decline also affect a large
percentage of the aging human population in the twenty-first century. Given
the large unmet medical need associated with these diseases and the potential
complexity of the genetic mechanisms underlying them (Haffter et al. 1996), the
importance of appropriate screening tools with which mutation-induced changes in
learning and memory processes may be identified is unquestionable.
12 Fear and Anxiety
Fear (induced by particular negative stimuli) and anxiety (a more diffuse and
prolonged behavioral state not associated with particular induction stimuli) affects
a large percentage of the human population (Weisberg 2009) and despite concerted
efforts by pharmaceutical research companies and academic laboratories and
despite the existence of several drugs, proper treatment is still not available for a
18 R. Gerlai
proportion of patients. It has been argued by several researchers that zebrafish may
be successfully utilized to study and model some of the mechanisms of vertebrate
fear and/or anxiety (Gerlai 2010). For example, fear responses have been reliably
induced in zebrafish using a chemical cue, the alarm substance (Speedie and Gerlai
2008). Alarm substances have been shown to elicit fear and panic reactions in a
broad range of fish species (Speedie and Gerlai 2008). These substances, which are
produced by epidermal club cells in the skin of many fish species, are released
when the skin is cut or damaged. In nature, the alarm substance is believed to
signal danger, perhaps the presence of an actively hunting predator (piscivore fish
species or a bird of prey). In the laboratory, the alarm substance has been suc-
cessfully utilized to experimentally induce fear responses. Zebrafish have also
been shown to reliably respond to this chemical cue with alarm reactions that
include erratic movements (zig-zagging), jumping (or leaping) and freezing
(complete immobility) (Speedie and Gerlai 2008). From the perspective of muta-
genesis screening, however reliable these responses may seem, the alarm substance
approach suffers from a major disadvantage. This substance has to be extracted
from the skin of conspecifics which entails cutting or homogenizing the skin of
freshly sacrificed fish and washing, diluting the extract. Because of the variability
inherent in this extraction process, the exact dose and potency of the substance can
not be ascertained across multiple experiments (multiple extractions). Recently,
however, zebrafish has been shown to respond to a synthetic alarm substance that
shares a key chemical structural element with that of natural alarm substances from
several fish species (Parra et al. 2009). Hypoxantin-3-N-Oxide, H3NO, has been
found to induce alarm reactions in zebrafish similar to those elicited by the natural
alarm substance (Parra et al. 2009). Thus, it is now possible to precisely control the
dose of the alarm substance and reduce unwanted experimental error variation, a
crucial requirement for high throughput mutagenesis screens.
Although the synthetic alarm substance, H3NO, now allows precise and rep-
licable fear induction, this method suffers from a drawback. Olfactory cues are
notoriously difficult to work with. The onset (delivery) of the cue and now also its
dose can be precisely controlled, however, its offset (washout) is difficult to
achieve. In most behavioral paradigms, experimenters want to introduce the
subject to its test chamber (tank) and let the subject habituate, establish a stable
baseline behavior before administering the cue (the alarm substance in this case).
This allows pre- and post-cue delivery periods to be compared and thus is a more
powerful experimental design. Ideally, after the delivery of the cue and recording
the effects of this delivery, one would like to turn it off and again compare periods
during and after cue delivery. But this is quite cumbersome with olfactory cues.
Furthermore, even if the experimental paradigm does not require turning off the
cue during the behavioral session, the cue may be difficult to remove from the tank
for the next subject. Residual amounts of the alarm substance may remain in the
test tank even after emptying and refilling the test tank. As even trace amounts of
the alarm substance may influence the behavior of the fish, this olfactory cue is
difficult to work with especially when one wants to run a large number of fish as
required for mutagenesis screening.
To circumvent the above issues, cues of other modalities have been tried for the
induction of fear responses. Zebrafish, being a diurnal vertebrate, has excellent
vision and respond well to visual cues. Zebrafish have been demonstrated to
respond differentially to the sight of live fish according to whether the fish species
shown were predatory or harmless and whether they were sympatric (coinhabiting
the geographical region) or allopatric with zebrafish (Bass and Gerlai 2008). The
latter study also demonstrated that zebrafish uniquely responded to a sympatric
predator, the Indian leaf fish (Nandus nandus) and that the sight (solely visual
stimuli) of the predator was sufficient to induce a maximal fear response (erratic
movements and jumps). Utilizing this finding, subsequently zebrafish have been
found to exhibit significant antipredatory responses not only to the sight of live
Indian leaf fish but also to animated (moving) computer images of this species
(Gerlai et al. 2009b). In this latter paradigm, both the presentation of stimuli and
the recording and analysis of the fear responses were conducted in an automated
computerized manner, i.e. the test paradigm was scalable and thus potentially
appropriate for high throughput screening. Although numerous parameters of this
automated fear paradigm will have to be optimized (e.g. size of the test tank, size
and speed of movement of the predator image, presence or absence of hiding
places, level of illumination, etc.), the results demonstrate the feasibility of high
throughput screening for agents (mutations or pharmaceutical compounds) that
may have fear altering properties.
13 Social Behavior
The last behavioral focus area I discuss in this paper is social behavior. Social
behavior is a common term for a range of complex behavioral phenomena from
agonistic (aggressive) encounters to reproductive (courtship) behaviors. Here I
focus on a behavior within this broad range termed affiliative behavior, social
cohesion or group forming. Affiliative or group forming behaviors are character-
istic of our own species. Humans tend to form groups, which in modern history led
to the development of the complex society where a set of rules govern. We are
particularly sensitive to social signals and tend to spontaneously follow a large
number of complex social rules. Briefly, being social is an inherent human trait.
There are numerous human disorders that are associated with abnormalities in
social behavior, one prominent example is the autism spectrum disorders (ASD).
Treatment for ASD and other forms of abnormal social behaviors is lacking for
two main reasons. One, the mechanisms underlying these diseases are unclear.
Two, the mechanisms underlying social behaviors in general are not understood.
Laboratory model organisms have been proposed to speed up the discovery of such
mechanisms [for review see Gerlai and Gerlai (2003)]. The question as to whether
autism may be modeled using animals is not trivial, however. Some may be skep-
tical and say that in order to model autism in animals one would need to understand
its mechanism first in humans, so what is the use of animal research? Nevertheless, it
20 R. Gerlai
is becoming clearer that even such complex phenomena as social behavior has not
only face but also construct validity in animal models, i.e. not only looks similar in
animals but may also be mechanistically similar to that of our own species. Briefly,
it may make sense to study social behavior in vertebrates other than humans,
discover the underlying mechanisms in the laboratory organism, and look for
translational aspects of the work, i.e. human homologs. Zebrafish is perhaps the most
social vertebrate model organism currently under study in the laboratory. Zebrafish
are found swimming in groups in nature, a behavior that they maintain under the
artificial confines of the laboratory (Engeszer et al. 2007). It is this swimming
together response, or shoaling, that may be an excellent behavioral phenomenon
to study from a translational perspective. Answering such questions as to what
neurobiological mechanisms (circuits, synaptic processes, biochemical interactions)
underlie group forming or social cohesion in zebrafish may help us understand
human social behavior and ultimately perhaps the mechanisms of the abnormalities
of human social behavior. The first step in this research could be the characterization
of social behavior in zebrafish followed by the development of behavioral test
paradigms that could detect mutation or drug-induced changes in brain function at
the level of social behavior. Below I present some examples of recent discoveries
with zebrafish that may be useful to make the first steps in this direction.
Zebrafish forms groups and swims in group formation but due to unavailability
of appropriate behavioral quantification methods, the complexities of this behavior
were not properly described in the past. By now, however, methods have been
designed that allow the quantification of numerous parameters of shoaling
behavior, including moment to moment changes of the distances among every
possible pairs of fish within the shoal (Miller and Gerlai 2007, 2008). A periodic
(cyclical) fluctuation of shoal cohesion has been discovered in zebrafish (Miller
and Gerlai 2008). Analysis of shoaling is now further developed to allow high
throughput automated tracking of multiple fish and thus the precise description of
how the entire shoal behaves. This method may enable one to screen for mutations
but would require the use of a group of fish that carry the same mutation, which
would necessitate breeding an extra generation (i.e. testing not the individual
mutant fish but its offspring). Perhaps a faster behavioral screening method may be
to test single fish and its response to social stimuli. The disadvantage of the latter
approach is that complex group dynamics may not be detected but the advantage is
that the test would save the extra generation of breeding.
Testing responses of individual fish to social stimuli has been achieved with an
experimental set up similar to the predator visual stimulus paradigm (Gerlai et al.
2009a). Here the computer monitor placed on the side of the experimental tank
shows animated (moving) images of zebrafish (five fish in this case). Each fish on
the monitor moves independently and in different randomized directions and with
a speed that changes from second to second while remaining within the range of
the speed of normally swimming zebrafish. This artificial ‘‘shoal’’ elicits a robust
behavioral response. The single experimental fish placed in the test tank usually
does not exhibit a preference for any sides of the tank, explores the entire tank,
and thus its position when averaged over a period of time (e.g. for one minute
intervals) ends up to be in the middle of the tank, which is 25 cm away from the
computer screen in case of a 50 cm long tank. However, as soon as the computer
screen shows the artificial shoal, the experimental zebrafish moves closer to the
computer screen and on average stays about 10 cm away from it, a distance that is
similar to what has been obtained with freely moving zebrafish in a real shoal
(Miller and Gerlai 2008). Given that the visual stimulus that elicits the response
is computer controlled and the subject’s distance from the stimulus screen is
recorded using computerized video-tracking, the entire test paradigm is automated,
i.e. does not require the presence of the experimenter during the behavioral
recording session. The paradigm therefore is high throughput and has utility in
screening for mutation or drug-induced changes in social behavior. Indeed, this
paradigm has been already used to detect strain (genetic) differences between
populations of zebrafish, and alcohol and dopamine receptor antagonist-induced
changes in social behavior [Gerlai et al. (2009a) and unpublished results].
14 Concluding Remarks
The excellent genetic tools developed for zebrafish have already provided promising
results in the analysis of complex brain disorders. Importantly, several genes impli-
cated in a number of human brain disorders have been shown to have zebrafish
homologs. The function of these genes in embryonic development, and in a few cases
in behavioral responses has started to be investigated. The genetic tools are constantly
refined. Increasing number of genetic markers is becoming available for linkage
analysis-based gene localization in random mutagenesis. Reverse genetic tools are
also rapidly developing. In addition, but also very importantly, numerous novel
behavioral paradigms are being developed and our understanding of the behavioral
responses and capabilities of zebrafish has been exponentially increasing over the past
few years suggesting that efficient and high throughput phenotypical screening
applications (Gerlai 2002) are becoming reality for zebrafish. Given the fact that
behavior is the output of the brain and that vertebrates share numerous biological
features, including nucleotide sequence homologies, it is likely that zebrafish
neurobehavioral genetics will facilitate the identification of numerous genes and
compounds leading to the understanding and better treatment of human brain disorders.
Acknowledgments Supported by NIH/NIAAA (USA) and NSERC (Canada).
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Forward Genetic Approaches
to Understanding Complex Behaviors
Lisa M. Tarantino and Amy F. Eisener-Dorman
Abstract Assigning function to genes has long been a focus of biomedical research.
Even with complete knowledge of the genomic sequences of humans, mice and other
experimental organisms, there is still much to be learned about gene function and
control. Ablation or overexpression of single genes using knockout or transgenic
technologies has provided functional annotation for many genes, but these tech-
nologies do not capture the extensive genetic variation present in existing experi-
mental mouse populations. Researchers have only recently begun to truly appreciate
naturally occurring genetic variation resulting from single nucleotide substitutions,
insertions, deletions, copy number variation, epigenetic changes (DNA methylation,
histone modifications, etc.) and gene expression differences and how this variation
contributes to complex phenotypes. In this chapter, we will discuss the benefits and
limitations of different forward genetic approaches that capture the genetic variation
present in inbred mouse strains and present the utility of these approaches for
mapping QTL that influence complex behavioral phenotypes.
Keywords Mouse Genetics Behavior Mapping QTL Mutagenesis
Contents
1 The Importance of Phenotype............................................................................................. 27

2 Forward Genetics and Inbred Strains: The QTL Approach .............................................. 27
3 QTL Mapping: The Basics ................................................................................................. 28
4 Initial QTL Identification: Standard Mapping Populations ............................................... 29
L. M. Tarantino (&) A. F. Eisener-Dorman

Department of Psychiatry, University of North Carolina,
Chapel Hill, NC 27516, USA
e-mail: lisat@med.unc.edu

DOI: 10.1007/7854_2011_189
Published Online: 2 February 2012
26 L. M. Tarantino and A. F. Eisener-Dorman
4.1 Recombinant Inbred Lines ......................................................................................... 29

4.2 Intercross and Backcross Mapping Populations ........................................................ 32
4.3 Consomic Lines or Chromosome Substitution Strains.............................................. 34
5 QTL Fine Mapping ............................................................................................................. 35
5.1 Congenic Strains ......................................................................................................... 35
5.2 Advanced Intercross Lines ......................................................................................... 36
5.3 Heterogeneous Stocks................................................................................................. 37
5.4 Outbred Mice .............................................................................................................. 39
5.5 The Collaborative Cross ............................................................................................. 40
6 Haplotype Association Mapping......................................................................................... 41
7 Mutagenesis ......................................................................................................................... 43
8 Beyond QTL Analysis: Finding the Quantitative Trait Gene ........................................... 47
9 The Future of Forward Genetics......................................................................................... 47
References.................................................................................................................................. 48
Animal models, and rodents in particular, have been used extensively to study
behaviors that model human psychiatric diseases. While rats have been particu-
larly well-suited to this research, the mouse has recently emerged as the primary
model for studying genetic and genomic aspects of human disease (Cryan and
Holmes 2005). This is due, in large part, to the availability of a vast array of
genetic and genomic resources for such endeavors in the mouse.
The ability to introduce genes into the mouse (transgenics) and retain tissue-
specific expression patterns as well as the ability to disrupt gene function
(knockouts) represented a significant advance in a field that has now become
known as ‘‘functional genomics.’’ Reverse genetic approaches such as these have
been useful for identification and analysis of genes believed to increase risk for
schizophrenia (Desbonnet et al. 2009), anxiety (Holmes et al. 2003; Lesch et al.
2003), depression (Savitz et al. 2009; Cryan and Slattery 2010) and autism (Sudhof
2008) but are not without limitations. The types of genetic lesions introduced are
often severe, completely knocking out gene function over the course of the life-
span, and may not accurately reflect the genetic basis of many complex human
diseases. The ability to make conditional knockouts that limit loss-of-function to
specific tissues or developmental timepoints has overcome some of these limita-
tions. However, the reverse genetic approach is generally hypothesis-driven,
depending on some prior knowledge regarding a gene’s role in a disease or
pathway. Understandably, the genes interrogated are often those in pathways
that are already targeted by current drug treatments, making the approach inher-
ently biased. More comprehensive phenotyping might result in identification of
gene function not yet attributed to a specific gene. Recently, large-scale reverse
genetic projects have been launched in the United States (knockout mouse
project (KOMP), Texas A&M Institute for Genomic Medicine (TIGM)), Europe
(European Conditional Mouse Mutagenesis (EUCOMM)) and Canada (North
American Conditional Mouse Mutagenesis (NorCOMM)) to systematically
interrogate the role of every gene by producing targeted knockouts and analyzing
the resulting mutants in a variety of phenotypic assays (Collins et al. 2007;
Gailus-Durner et al. 2009). These efforts to catalog gene function in a non-
hypothesis-driven, unbiased manner will surely provide new insights into gene
Forward Genetic Approaches to Understanding Complex Behaviors 27
function. However, studying induced mutants exclusively fails to account for

naturally occurring variation, genetic modifiers and gene-gene interactions that
may be of primary importance in complex human diseases. In addition, recent
research in human populations indicates that loss-of-function variants can occur
far more frequently than expected with no discernable effect on health (Macarthur
and Tyler-Smith 2010), suggesting that null mutations may not be the best
approach for modeling many human diseases.
1 The Importance of Phenotype
In the earliest days of mouse genetics, the phenotype was the primary starting
point for genetic analysis. Observations of spontaneous, chemical- or radiation-
induced mutations progressed to identification of linkage groups and, eventually,
to chromosomal assignment (for excellent reviews, see (Lyon 2002; Paigen
2003)). As molecular tools advanced, starting with the identification of genetic
markers and progressing to complete sequencing of the mouse genome, the means
for exact gene localization were realized. Knowledge regarding the organization of
the genome increased along with an appreciation for the complexities therein,
resulting in a renewed interest in the study of complex traits from a systems
biology perspective. Recent years have witnessed a resurgence of forward genetic
approaches that consider naturally occurring genetic and phenotypic variation and
place more emphasis on the genetic complexities that are likely to explain a large
portion of human phenotypic variation.
As opposed to reverse genetics, forward genetic studies start with the mea-
surement or observation of a phenotype followed by mapping of the causative loci
or genes. A major advantage of this approach is that the role of the gene or genes
in a specific biological process is/are proven a priori. In addition, forward genetic
approaches are, by definition, unbiased in terms of gene identification since the
phenotype is the primary level of measurement. The term ‘‘forward genetics’’
applies to any phenotype-driven mapping approach, including the use of standard
inbred strain crosses and haplotype association analysis—both of which result in
the identification of quantitative trait loci (QTL).
In this chapter, we will describe forward genetic approaches for gene identi-
fication and functional analysis, detailing the advantages, disadvantages, successes
and failures of several commonly used forward genetic models.
2 Forward Genetics and Inbred Strains: The QTL Approach
Early mouse geneticists, including pioneers of the field William Castle and
Clarence Little, utilized visible characteristics such as coat color and tumor sus-
ceptibility to study Mendelian inheritance. These studies eventually led to the
development of inbred mouse strains in the early 1900s (reviewed in (Crow

2002)). These strains are the starting point for most forward genetic approaches.
Inbred strains are produced by brother-sister mating for 20 or more consecutive
generations to fix all loci in a homozygous state. There are now hundreds of inbred
strains available, and while mice within a single inbred strain are genetically
identical, substantial genetic and phenotypic variation exists across strains. This
phenotypic and genetic variation has been used for decades to further our
understanding of the genetic basis of human disease. Inbred lines and the crosses
derived from them have been instrumental in the search for genes and genomic
loci that contribute to the phenotypic variability for complex behavioral traits.
These loci, termed quantitative trait loci, or QTL, are regions of the genome that
are associated with the phenotypic expression of complex traits, and the genomic
locations of these regions can be identified using a process known as QTL
mapping.
3 QTL Mapping: The Basics
In its simplest form, QTL mapping can be described as a statistical association

analysis between phenotype and genotype at specific locations across the genome.
In order to achieve this, two things are necessary—phenotypic and genetic vari-
ation. Phenotypic variation is easily observed among inbred strains (Hamilton and
Frankel 2001), and experimental crosses between inbred strains provide both
phenotypic and genetic variation. Genetic variation across the genome is measured
using a set of markers that can distinguish between the two or more strains used in
an experimental cross. The source of these markers has changed over the years
with advances in technology and knowledge of genomic structure. Genetic
markers initially took the form of linked visible characteristics such as coat color
and progressed to restriction fragment length polymorphisms (RFLPs). With the
advent of the polymerase chain reaction (PCR), scientists had access to a trove of
genetic markers called simple sequence length polymorphisms (SSLPs) that were
more numerous and provided dense genetic maps. Single nucleotide polymor-
phisms (SNPs) are single nucleotide changes that are also detectable by PCR, and
millions of them exist between inbred strains, providing even greater resolution for
genetic mapping. SNPs are now the primary source of genetic variation measured
in mapping studies. Genetic markers need not be causative alleles and simply serve
as a means of ascertaining allelic state at a genomic location.
Once genotypes and phenotypes are collected, association of genotype with
phenotype can be performed. This aim can be accomplished by single-marker
analysis using basic statistical techniques (ANOVA, marker regression) and
comparing marker status at each genomic location with phenotype. In the late
1980s a technique for analyzing the interval between two genotyped markers was
developed by Lander and Botstein (1989), called interval mapping (IM). This
technique involves calculating the likelihood of a particular genotype based on the
recombination frequency and genetic distance between two markers. A LOD score
is calculated to indicate the strength of the association between the phenotype and
genotype. This technique has been refined over the years—Jansen (1993) and Zeng
(1993) developed an algorithm for composite interval mapping (CIM) that allows
for interval mapping in combination with multiple-regression analysis to account
for background QTL. The specifics of QTL mapping algorithms are beyond the
scope of this chapter, but several books are available that provide a thorough
discussion (Broman and Sen 2009; Wu et al. 2010). Linkage and mapping pro-
grams are freely available, including mapmaker QTL (Lander et al. 1987), QTL
Cartographer (Basten et al. 1994, 2002), map manager QTX (Manly et al. 2001)
and R/qtl (Broman et al. 2003) or its graphical user interface, J/qtl (Smith et al.
2009). Most mapping programs were designed to analyze the basic mapping
populations described below, but special attention must be given to populations
frequently used for fine mapping QTL.
4 Initial QTL Identification: Standard Mapping Populations
4.1 Recombinant Inbred Lines
Donald Bailey and Benjamin Taylor developed RI lines at the Jackson Laboratory
with the goal of creating a recombinant population for linkage mapping. RI lines
are created by intercrossing the F1 hybrids of two inbred progenitor strains to
generate F2 progeny, which are then sib-mated for at least 20 generations,
resulting in homozygosity at each locus and a mixture of the parental alleles
providing genetic variation necessary for QTL mapping. Each RI line consists of
animals that are genetically identical but distinct from other RI lines in the set
(Fig. 1c) (Bailey 1971).
The first murine RI panel, the CXB line, was constructed by Bailey from a
BALB/cBy 9 C57BL/6By cross. Bailey used the seven lines resulting from the
initial crosses to identify loci associated with histocompatibility factors and coat
color (Taylor 1978). Twelve CXB lines now exist and have been used to map
QTL that influence exploratory activity (Blizard and Bailey 1979; Crabbe et al.
1982; Neiderhiser et al. 1992), circadian rhythms of locomotion (Schwartz and
Zimmerman 1990), avoidance (Neiderhiser et al. 1992) and sleep behaviors (Tafti
et al. 1997). Taylor developed more RI panels, such as the BXH (C57BL/6J 9
C3H/HeJ) and AKXL (AKR/J 9 C57L/J) RI sets, which were used in the study of
murine leukemia virus (Taylor et al. 1971), drug-induced seizures (Taylor 1976)
and lipopolysaccharide response (Watson et al. 1977). There are now RI panels for
crosses between A/J and C57BL/6J (AXB and BXA), B6(Cg)-Tyrc-2 J/J and C3H/
HeJ (BXH) and C57BL/6J and DBA/2J (BXD), as well as RI lines derived from
mice selectively bred for righting response to ethanol—the Long Sleep and Short
Sleep mice, ILS/IbgTejJ and ISS/IbgTejJ (ILSXISS) (http://jaxmice.jax.org).
The BXD RI panel is by far the most frequently used, beginning with an
initial set of 25 lines (Taylor et al. 1977) but now consisting of 79 strains (Peirce
et al. 2004) (http://www.genenetwork.org). QTL for alcohol (Cunningham
1995; Phillips et al. 1994, 1995; Rodriguez et al. 1994, 1995) and drug-related
(Alexander et al. 1996; Grisel et al. 1997; Jones et al. 1999; Mogil et al. 1997;
Phillips et al. 1998) behaviors were among the first to be dissected using BXD
lines. More recently, concerted efforts have been made to characterize the full
BXD panel for neurobehavioral phenotypes, particularly those relating to pain,
b Fig. 1 a–h Breeding schemes of various genetic mapping populations. Backcross mice (a) are
generated by crossing two inbred strains to recover F1 mice and then backcrossing the F1 mice to
one of the parental strains, resulting in one copy of a chromosome that has undergone meiotic
recombination and another copy from the inbred strain to which the F1 was backcrossed.
Intercross mice (b) result from crossing F1 mice, resulting in two recombined chromosomes.
Recombinant inbred strains (c) are produced by continuous brother-sister mating among F2s,
resulting in homozygosity at each locus but with a reassortment of alleles from each parental
strain. Advanced intercross lines (d) are also produced by crossing F2s, but inbreeding is avoided
by restricting breeding partners to exclude common parents or grandparents and a large breeding
population is maintained to avoid allelic drift. Congenic strains (e) have a small portion of one
chromosome from a donor strain introgressed onto a recipient background strain, while
chromosome substitution strains (f) have an entire donor chromosome introgressed onto a
recipient background strain (examples of a congenic interval on Chr 9 (e) and a Chr 9 CSS (f) are
shown). Heterogeneous stocks (g) are similar to advanced intercross lines but are derived from
eight different inbred strains to increase genetic diversity. The collaborative cross (h) also has
eight parental strains, but the breeding scheme is similar to recombinant inbred lines to maximize
recombination while breeding each CC line to homozygosity
drug addiction, anxiety, stress and locomotor activity (Philip et al. 2010). One
innovative approach uses brain-specific gene expression data from a microarray
analysis of the BXD panel to map neurobehavioral QTL (Bao et al. 2007).
RI lines offer several advantages for QTL mapping. RI lines are genetic
reference populations—genetically stable over time and infinitely reproducible.
Therefore, each line must be genotyped only once, and that data becomes
available to anyone using the line in the future (Taylor 1978). Comparisons of
phenotypic data can be made across laboratories enabling genetic correlations
and investigation of similarities among phenotypes. Phenotypic data can be
examined retrospectively as marker density increases and more phenotypes are
collected. This process has been automated by Williams and colleagues, who
established the GeneNetwork database that provides tools for RI-QTL mapping
and correlations among more than 1,200 phenotype datasets, including gene
expression (Wang et al. 2003). RI lines are also particularly well-suited for
mapping QTL for phenotypes for which genetic variance is small compared
to environmental variance. This feature of RI lines is derived from the ability to
conduct repeated sampling from individuals with the same genetic background,
resulting in strain means that reduce environmental noise and more accurately
reflect the strain phenotype.
The ability to map QTL for complex traits is dependent upon the availability of
a sufficiently large RI panel that provides enough power to detect QTL of small
effect size (Gora-Maslak et al. 1991). However, the RI strains were developed to
investigate Mendelian traits, and the small number of strains per line has limited
their usefulness for identifying QTL for complex phenotypes. Prior to the devel-
opment of dense sets of genetic markers, the utility of RIs was also limited by the
availability of loci for mapping. The expansion of the BXD line, along with the
development of a more dense set of polymorphic markers, now allows for mapping
of QTL responsible for 10% of the phenotypic variance to approximately 1–2
megabases (Mb) (Peirce et al. 2004).
However, even the largest RI sets have several characteristics that mitigate
their usefulness in complex trait analysis. Existing RI panels were produced by
crossing only two inbred strains and from only a limited number of inbred
strains, resulting in limited genetic variability both within and across panels.
Both the advantages and disadvantages of RI lines were considered when
planning the collaborative cross (CC) (Churchill et al. 2004), an experimental
population akin to RI strains but with added genetic variability. The CC is
discussed in more detail below.
4.2 Intercross and Backcross Mapping Populations
The limited power for mapping provided by existing RI lines led to the use of other
types of mapping populations—either for replication of RI-identified QTL or as
alternative crosses for initial QTL identification. The most commonly used QTL
mapping populations are intercross (F2) or backcross (BC) lines produced by
breeding two inbred strains known to differ for the complex trait(s) of interest. The
resulting F1 hybrid progeny are either intercrossed to produce an F2 generation
(Fig. 1b) or backcrossed to one of the parental strains to produce a BC generation
(Fig. 1a). At each locus, an F2 animal will have one of three genotypes: homo-
zygous for either of the parental alleles or heterozygous, possessing one allele
from each of the parental strains. A BC animal has only two genotype possibilities:
heterozygous or homozygous for the allele of the parental strain to which the F1
progeny were backcrossed.
Unlike RI animals, intercross and backcross progeny are genetically hetero-
geneous; each F2 or BC individual within the population is characterized by a
unique pattern of genome-wide recombination. Therefore, each set of animals
must be genotyped and the informativeness of these genotypes is limited to the set
of phenotypes collected for any particular set of animals. When genotyping costs
were prohibitive, selective genotyping of animals from the top and bottom 15% of
the phenotypic distribution was frequently performed using DNA pools (Darvasi
and Soller 1994; Wang and Paterson 1994). The genetic (DNA) contribution of
each individual to the pool was often weighted toward individuals at the pheno-
typic extremes (Taylor et al. 1999, 2001). Individuals in each pool were genotyped
only at loci for which a significant or suggestive QTL were identified, and the
entire mapping population was only genotyped for those loci that proved signifi-
cant. As genotyping costs have decreased, the use of pooling has waned.
The number of markers required to genotype either an F2 or BC is relatively
low based on the limited number of recombinations in these populations.
A polymorphic marker every 40 Mb provides adequate coverage, as each marker
will sweep 20 Mb in each direction. The choice of which type of cross to use is
dependent on the genetic architecture of the phenotype of interest. Using an F2
mapping population is recommended to obtain an overview of the number, loca-
tion and estimated effect sizes of both additive and dominant QTL segregating in
the population, as well as QTL interactions (Darvasi 1998). Approximately 30%

fewer F2 animals are required to detect additive QTL in comparison with a BC
population due to the increased number of recombinations in the F2. However,
BC mapping populations are generally considered more efficient for QTL identi-
fication, specifically for QTL with dominant effects. The decreased genetic
variance in a BC—a population with a 1:1 genotypic ratio and only two pro-
spective genotypes at any given locus—results in fewer genetic interactions than in
an F2. The LOD threshold for significance is therefore lower for a BC, and gene
effects are more prominent because the decreased genetic variance results in fewer
genetic interactions in the BC (Darvasi 1998; Silver 1995).
Regardless of the cross type, QTL mapping has been successful in identifying
thousands of loci. Conventional F2 populations derived from various inbred
parental strains have been used to identify QTL for behaviors such as contextual
fear conditioning (Wehner et al. 1997), spatial learning (Steinberger et al. 2003),
alcohol preference (Fernandez et al. 2000) and consumption (Boyle and Gill
2008), anxiety (Bailey et al. 2008; Eisener-Dorman et al. 2010; Henderson et al.
2004; Turri et al. 2001a; Turri et al. 2004), home cage activity (Turri et al. 2001b;
Umemori et al. 2009), ethanol-induced (Hitzemann et al. 1998) and cocaine-
induced (Boyle and Gill 2009) locomotor activation and locomotor activity in the
open field (Bailey et al. 2008; Eisener-Dorman et al. 2010; Gershenfeld et al. 1997;
Kelly et al. 2003; Koyner et al. 2000). QTL for locomotor activity in the open field
(Eisener-Dorman et al. 2010), alcohol preference (Melo et al. 1996) and body
weight regulation (Zhang and Gershenfeld 2003) have been identified using BC
populations.
The deluge of QTL for various phenotypes caused concerns regarding the
possibility of numerous false positives (Type I Error). A paper published in 1995
proposed thresholds for reporting significant or suggestive QTL loci (Lander and
Kruglyak 1995). Some researchers worried that the thresholds were too restrictive
and that it would be cost prohibitive to phenotype and genotype populations of the
size necessary to result in the levels of significance proposed (Yoon 1996). Others
recommended the use of multiple strategies, including provisional mapping and
replication of suggestive and significant QTL in independent populations and
pooling results (Atkins 2001). At the time, however, the significance levels pro-
posed by Lander and Kruglyak were almost universally adopted when reporting
QTL. Advances in computational power now make it more common to generate
significance thresholds based on individual experimental populations by running
permutation tests (Churchill and Doerge 1994), and this function is present in most
QTL mapping software packages.
Large F2 or BC populations have substantial power for detecting QTL, but they
also have limitations. Neither F2 nor BC mapping panels are capable of providing
high-resolution QTL localization due to the relatively low number of recombi-
nations present in these populations. This results in coarsely mapped QTL with
broad peaks that can encompass half of a chromosome and contain hundreds of
candidate genes. Because the QTL regions identified are so large, the individual
effects of closely-linked QTL are difficult to dissect. QTL comprised of multiple
closely-linked, small-effect QTL may fail to be detected if the loci have opposing
phenotypic effects. Consequently, the fine mapping necessary to narrow QTL
intervals and, ultimately, identify quantitative trait genes (QTG) must be carried
out in specialized populations.
4.3 Consomic Lines or Chromosome Substitution Strains
Consomic lines, or chromosome substitution strains (CSS), are produced by int-

rogressing a single chromosome from a donor strain onto a recipient strain
background by repeated backcrossing for at least 10 generations (Fig. 1f). A full
CSS panel consists of 21 lines—one for each of the 19 autosomes and 2 sex
chromosomes. Established CSS lines are genetic reference populations that allow
for relatively fast localization of a QTL or group of QTL to a single chromosome
and have several advantages over standard mapping populations. Because statis-
tical comparisons are made across 21 CSS lines and the background strain rather
than across hundreds of markers in hundreds of F2 or BC animals, the P-value
required for significance (corrected for multiple comparisons) in a CSS experiment
will be lower. The total number of mice necessary for mapping QTL to a single
chromosome in a CSS panel is only slightly lower than in an F2 cross based on
sample size estimates required to detect a QTL that accounts for a similar amount
of the variance in either population (Belknap 2003). However, no genotyping is
required in the initial scan, making the CSS more economical. In addition, the
effect size of a QTL will be amplified in the CSS due to the presence of only
homozygotes.
These advantages make the CSS an attractive option for initial identification of
QTL. However, the end result is an entire chromosome, or multiple chromosomes,
on which reside QTL that must be localized and fine-mapped. CSS provide a
starting population that requires fewer generations of breeding to produce interval-
specific congenic strains or mice for recombinant progeny testing. Since QTL were
identified and present on a homogeneous genetic background in the CSS, their
effect size should be sufficient for recovery in congenic lines. However, if a
significant result on a single chromosome represents a cluster of two or more QTL,
the effect might be lost upon construction of congenic or subcongenic lines, as
described below.
Constructing a CSS panel is costly and takes years of breeding. Realistically,
therefore, most researchers are limited to existing CSS panels and the genetic and
phenotypic variability that exists therein. Three CSS panels have been reported in
the literature, beginning with the B6. A panel (A/J introgressed on to C57BL/6J)
(Singer et al. 2004). This panel has been the most extensively used for mapping
QTL for behavioral traits such as prepulse inhibition (Leussis et al. 2009;
Petryshen et al. 2005), anxiety (Singer et al. 2005) and sleep-related epilepsy
(Strohl et al. 2007) and other complex phenotypes, such as onset of puberty
(Nathan et al. 2006) and resistance to diet-induced obesity (Buchner et al. 2008).
Takada et al. produced a CSS panel with MSM/Ms introgressed onto C57BL/6J
(Takada et al. 2008) that has been used to identify QTL for emotionality
(Takahashi et al. 2008), social interactions (Takahashi et al. 2010) and home cage
activity (Nishi et al. 2010). Finally, Gregorova et al. (Gregorova et al. 2008) have
introgressed PWD/Ph onto C57BL/6J and have characterized the lines for blood
chemistry-related phenotypes.
5 QTL Fine Mapping
The identification of QTL is fairly straightforward, as evidenced by the thousands

that now exist in online databases and in the literature (Flint et al. 2005). However,
identifying the gene, or genes, underlying a QTL peak has been a persistent
limitation of commonly used QTL methods. The limited resolution of standard
QTL crosses (RIs, F2s, BCs) results in identification of large genomic regions,
usually [ 20 Mb, containing hundreds of genes. To identify the quantitative trait
gene (QTG), the QTL region must be narrowed considerably before a manageable
list of candidate genes can be interrogated. Fine mapping of QTL has been the
focus of a great deal of research in recent years (Flint et al. 2005). Initially,
congenic and subcongenic strains were pursued and have resulted in some success.
However, partitioning QTL in this way is not always possible, and efforts toward
the use of primary crosses have resulted in the development of advanced intercross
lines (AILs), heterogeneous stocks (HS), outbred mice and the CC.
5.1 Congenic Strains
Congenic strains are derived from repeated backcrossing of a donor strain to a

recipient strain until only a small region of the QTL-containing donor strain
chromosome is introgressed onto the recipient strain background (Fig. 1e). This
process, aided by genotyping the QTL region of interest, takes 13 generations
(12 backcrosses followed by an intercross to obtain founders), but the process has
been accelerated by marker-assisted selection of both the introgressed region and
the remaining recipient background in a strategy called ‘‘speed congenics’’
(Markel et al. 1997; Wakeland et al. 1997). Consequently, it is now possible to
produce congenics in less than half the number of generations. QTL intervals in
congenic strains can be further narrowed by backcrossing the congenic founders to
break up the introgressed region, thereby producing subcongenics.
There have been some successes in using congenic and subcongenic mice to
narrow QTL regions. Berrettini and colleagues identified a morphine preference
QTL (Mop2) in a C57BL/6J (B6) 9 DBA/2J (D2) F2 (Berrettini et al. 1994). The
20-Mb QTL was confirmed using a marker-assisted breeding strategy to generate a
pair of reciprocal congenic mouse strains—the B6 QTL interval was introgressed
onto the D2 recipient background (D2.B6-Mop2), and the D2 QTL interval was
introgressed onto the B6 recipient background (B6.D2-Mop2) (Ferraro et al. 2005).
Heterozygous D2.B6-Mop2 congenic mice were backcrossed to D2 mice, thereby
fragmenting the 28.8-Mb QTL interval of the congenic into smaller, discrete
intervals of 9.5 and 17.2 Mb in the resulting subcongenic strains (Doyle et al.
2008). Mice from one of these two subcongenic strains exhibited morphine
preferences similar to both the D2.B6-Mop2 congenic and the B6 parental strain
mice, thereby narrowing the Chr 10 QTL interval to a 9.5-Mb region containing 39
genes (Doyle et al. 2008). In another study, the obesity-related QTL Fob3 on Chr
15 was identified in an F2 cross originating from two outbred mouse lines selected
for either high or low body fat (fat and lean lines) (Horvat et al. 2000). One mouse,
selected because it was recombinant within the Fob3 interval, was backcrossed for
10 generations to the Fat line to create two subcongenic strains with the Lean line
Fob3 QTL interval introgressed onto the Fat line recipient background (Stylianou
et al. 2004). Further backcrossing of the Fob3 subcongenic strains to the Fat
line yielded additional recombinants, allowing the isolation of two smaller QTL
(Fob3a and Fob3b) within the original Fob3 QTL interval (Stylianou et al. 2004).
Continued backcrossing of select recombinants within the 22.39-Mb Fob3b interval
generated six additional subcongenic strains with overlapping donor intervals. Four
subcongenic strains were intercrossed with the Fat line to generate four F2 mapping
populations, which identified two closely-linked QTL, Fob3b1 (4.98 Mb) and
Fob3b2 (7.68 Mb), within the Fob3b QTL interval (Prevorsek et al. 2010).
However, QTL effects often disappear during the construction of congenics or
subcongenics. Reasons for this loss of QTL effect could be the removal of the QTL
from a heterogeneous background, thereby eliminating epistatic interactions that
increase QTL effect size. It is also likely that QTL that disappear during congenic
production might actually be groups of two or more smaller effect size QTL that
appear as a single peak in an F2 or BC but do not have sufficient power alone to
result in a statistically significant change in phenotype (Legare et al. 2000; Legare
and Frankel 2000).
5.2 Advanced Intercross Lines
Although the ability to restrict genetic heterogeneity to only two strains has
advantages (Cheng et al. 2010), the limited number of recombinations present in
F2 and BC mice also limits the resolution of these mapping resources. AILs are
produced by intercrossing two parental strains to obtain an F2 and then inter-
crossing each successive generation (Fig. 1d). AILs have the advantage of
expanding the genetic map by increasing the number of breakpoints, thereby
increasing the recombination fraction between markers. The increased number of
recombinations in an F10 can reduce the size of a QTL interval by five-fold in
comparison with a similarly-sized population of F2 animals (Darvasi and Soller
1995). However, the increase in resolution comes at the cost of power to detect
QTL. This loss in power is due to the increased number of markers necessary to
account for the reduction in linkage disequilibrium between markers and genetic
drift that results in altered allele frequencies. The latter can be controlled some-
what by choosing an appropriate breeding strategy (Rockman and Kruglyak 2008).
In general, maintaining large population sizes and controlling for inbreeding can
mitigate concerns about power in an AIL.
One of the largest AILs is the LG/J 9 SM/J (LG, SM) AIL produced
by Cheverud and colleagues at Washington University in St. Louis, Missouri
(Norgard et al. 2008). The F9-F10 LG, SM AILs have been used successfully to
identify dozens of QTL for bone-length (Kenney-Hunt et al. 2008; Norgard et al.
2008, 2009). More recently, F34 LG, SM AILs were used to replicate and refine
QTL from previous studies. Norgard et al. were able to replicate almost 80% of
QTL identified in both F2-F3 and F9-F10 populations. However, QTL interval
reduction in the F34 was less than expected compared to previous studies. QTL
intervals ranged from 0.6 to 14 Mb with half of the QTL having confidence
intervals from 2 to 5 Mb. The authors postulated that family structure bias inflated
the QTL peaks and decreased the confidence interval sizes reported in earlier
studies. An alternative explanation is the presence of multiple linked loci at the
QTL peaks—a problem that is encountered with any fine mapping population.
Cheng et al. (2010) took advantage of the power of an F2 and the mapping
resolution of the LG, SM AIL to identify QTL for methamphetamine sensitivity.
QTL were identified for both methamphetamine- and saline-induced locomotor
behavior in a large LG 9 SM F2 and the F34 LG, SM AIL. Both populations were
also combined and analyzed as one large intercross. Cheng et al. found that the
population structure present in AILs must be considered when mapping to reduce
Type I errors. By using a mixed model that considered relatedness of individuals in
the AIL for both mapping and significance threshold determination, the authors were
able to identify several QTL intervals at sub-centimorgan resolution—one of which
contains only a single gene that is now being assessed for its role in methamphet-
amine sensitivity. Samocha et al. (2010) used a similar approach to map QTL for
acoustic startle response, habituation and prepulse inhibition of the startle response.
A large set of F2 identified QTL for multiple behaviors. The population size of the
AIL used in this study did not provide enough power to detect significant QTL on its
own, but combined analysis of both the F2 and AIL populations narrowed QTL
intervals identified initially in the F2. Taken together, these studies indicate that the
approach of using both an F2 and AILs, along with taking into account population
structure, is an effective method for identification and fine-mapping of QTL.
5.3 Heterogeneous Stocks
Heterogeneous Stocks (HS) lines are produced by pseudo-random breeding over

multiple generations with the aim of increasing the number of meiotic crossovers
and, thus, the genetic resolution (Fig. 1g). The term ‘‘heterogeneous stock’’ is
commonly used to describe two independently established eight-way inbred mouse

crosses. The Boulder HS was established in 1970 from an eight-way cross between
C57BL/6, BALB/c, RIII, AKR, DBA/2, I, A/J and C3H strains and has been
breeding for more than 60 generations (McClearn et al. 1970). Initially, the
Boulder HS mice were conceived as a normative population of mice with increased
genetic variation in comparison to individual inbred strains (McClearn et al. 1970).
The Northport HS, derived from A/J, AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/
J, DBA/2J and LP/J strains, was established in 1994 as a stock for selective breeding
(Hitzemann et al. 1991) and has been breeding for more than 50 generations
(Demarest et al. 2001; Hitzemann et al. 1994). More recently, two additional HS lines
have been reported. A collaborative cross HS (CC-HS), derived from the same eight
founder strains that were used to establish the CC, was recently reported by the
Hitzemann laboratory and is currently at the 12th generation of outbreeding (Iancu
et al. 2010). In addition, a cross between four inbred strains, C57BL/6J, DBA/2J,
BALB/cJ and LP/J, called the HS4 has also been produced and maintained in the
Hitzemann laboratory for 19 generations (Malmanger et al. 2006).
It is estimated that HS mice at 60 generations of random breeding could
increase mapping resolution by 30-fold over an F2 or BC. Talbot et al. (1999) used
the Boulder HS to fine map a QTL for open field behavior identified on Chr 1 in
previous studies (Caldarone et al. 1997; Flint et al. 1995; Gershenfeld et al. 1997;
Wehner et al. 1997) and were able to refine the chromosomal location to a 1.6-Mb
region. Using single-marker association analysis, they also replicated a QTL on
Chr 12 but failed to replicate three additional QTL on Chrs 1, 10 and 15. The
inability to replicate QTL in the HS was attributed to the inability to distinguish
between identical alleles contributed by different progenitor strains. This short-
coming was overcome by the development of a multipoint mapping model that
takes into account information from flanking markers and progenitor haplotypes.
This mapping algorithm, called HAPPY, is available for download or use as a
web-based program (http://www.well.ox.ac.uk/*rmott/happy.html). Using the
multipoint mapping method, all three previously undetected loci were mapped.
The Northport HS has been used to fine map a QTL on Chr 2 for ethanol-
induced locomotor activity. The QTL was initially identified in the BXD RIs and
replicated in a B6XD2 F2. Using a G32-35 HS, Demarest et al. (2001) were able to
replicate the Chr 2 QTL and resolve the region into three separate peaks.
Mott and Flint (2002) also developed a technique, called the inbred-outbred
cross, for using HS mice to both detect and fine map QTL. The technique involves
generating an F2 cross using HS and a genetically-distinct line (inbred strain,
knockout, transgenic), detecting QTL using standard methods (low marker reso-
lution) and then dense genotyping in candidate QTL regions to take advantage of
the increased resolution of the HS-contributed genetic material. Through simula-
tions, Mott and Flint showed that a 5% effect size QTL could be detected and fine
mapped with 50% probability to within 6 Mb by genotyping 1,500 animals.
HS mice have been used primarily to fine map previously identified QTL
(Demarest et al. 2001; Malmanger et al. 2006; Talbot et al. 1999; Turri et al. 1999),
although their increased genetic variability and expanded genetic map also make
them suitable for genome-wide association studies. However, several procedural

and analytical hurdles had to be addressed before genome-wide QTL identification
in the HS could be realized. First, because of the expansion of the genetic map, 100
times more markers and 10 times more animals are required to have the power to
detect QTL with 5% effect or less (Flint et al. 2005; Valdar et al. 2006b), making
genome-wide mapping in the HS an expensive proposition. Also, unknown selective
pressures and random fluctuations in allele frequencies during production and/or
maintenance of the stock may affect the resolving power of the HS. These issues are
detailed in the first publication of genome-wide association in the Northport HS
(Valdar et al. 2006b). Costs for line production and genotyping were reduced by
collecting over 100 phenotypes in parallel for each mouse (Solberg et al. 2006), and
model-averaging techniques were developed to analyze multiple QTL models and
overcome genotype correlations (linkage disequilibrium) and family structure.
Hundreds of QTL for dozens of phenotypes were detected with confidence intervals
averaging 2.8 Mb—a substantial improvement over standard F2 crosses.
5.4 Outbred Mice
Outbred stocks are closed populations of genetically variable animals that are bred
to maintain maximum heterozygosity (Chia et al. 2005). Dozens of outbred stocks
exist, and like HS mice, they offer the advantage of increased mapping resolution
but suffer some of the same drawbacks—increased sample size and marker density
are required for QTL detection. In addition, outbred stocks (with the exception of
HS lines) do not have the advantage of progenitor allele information to derive the
origin of alleles in the offspring. This limitation introduces difficulties in the use of
these stocks for QTL mapping. Only one example of a behavioral QTL has been
published thus far using outbred stocks. The Rgs2 gene that influences anxiety in
mice was identified using outbred MF1 mice (Yalcin et al. 2004). Yalcin et al.
determined that the haplotype patterns in MF1 mice were very similar to those
found in standard inbred strains. Thus, they were able to use similar techniques for
mapping as those used with HS mice.
Aldinger et al. (2009) recently examined genetic variation and population
structure in CD-1 outbred stocks and determined that CD-1 mice are reasonably
outbred, polymorphic at a significant number of loci and resemble human popu-
lations in terms of complex genetic history. Although CD-1 mice have population
substructure that may affect mapping results, the authors believe this structure
could be exploited and that the CD-1 mice represent a valuable genetic mapping
resource. Williams et al. (2009) used CD-1 mice to confirm the effect of a
duplication of the Glo1 gene on anxiety-related behavior in mice. However, out-
bred mice have not yet been used for genome-wide association mapping of
behavioral traits. Perhaps as genomic and analytical resources are developed, these
untapped resources of genetic variability will become utilized more frequently in
the identification and fine mapping of QTL.
5.5 The Collaborative Cross
In the late 1990s and early 2000s, the discussion in the mouse genetics community
started to focus on the problem of QTG identification; it was determined that
current mouse resources were not suitable for tackling such a difficult problem.
Various standard mapping populations had the features necessary for identifying
complex trait loci, but not one mapping resource had all of the features that were
necessary if such an endeavor was to be successful down to the point of gene
identification. Mouse geneticists began to envision a reference population of mice,
the CC, that would capitalize on the advantages of existing resources by offering
genetic diversity, mapping power and high-resolution. The CC would also provide
a platform for systems genetic studies and modeling of complex networks,
including gene by gene and gene by environment interactions. The CC was to be
derived from eight divergent inbred strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/
LtJ, NZO/HlLt, CAST/EiJ, PWK/PhJ and WSB/EiJ) to maximize genetic diversity
and ensure that the resulting population would provide the phenotypic diversity
necessary to study any trait of interest. Like the AIL and HS lines, the CC would
be subjected to multiple generations of breeding to increase meiotic recombination
and provide the necessary mapping resolution for fine mapping QTL (Fig. 1h).
However, the CC would be inbred and, upon completion, would provide a fully-
genotyped reference population that could be provided to individual investigators.
Finally, the CC would be sufficiently large (*1,000 lines) to provide the power
necessary to map QTL of relatively small-effect size.
The initial and largest crosses to produce the CC were begun in 2005 at the Oak
Ridge National Laboratory (ORNL). Separate, smaller breeding populations were
located in Tel Aviv, Israel and Western Australia. In late 2008, the Department of
Energy announced that the ORNL mouse genetics program was being phased out,
and the ORNL CC population was transferred to the University of North Carolina
(UNC). The Tel Aviv population has recently joined the ORNL lines at UNC.
Several CC lines have already reached the inbred state due to an acceleration of the
process using marker-assisted inbreeding. At least 100 lines will be completed by
the end of 2012, and additional lines will follow over the next several years
(personal communication, Darla Miller).
It is likely that the final population of the CC will be closer to 500 rather than
1,000 lines, as first proposed. Simulation studies have shown that 500 lines provide
both adequate power and fine resolution for QTL mapping (Broman 2005; Valdar
et al. 2006a). A multi-stage strategy has also been proposed using an initial
mapping panel of 100 strains followed by a second set of 100 strains that have
informative allele combinations and recombination events based on QTL locali-
zation from the first stage (Churchill et al. 2004). This strategy will make the
CC more accessible for smaller laboratories. The completion of fully-genotyped
CC-RI lines would also enable the production of many more CC recombinant
inbred intercrosses (CC-RIX) produced by crossing different CC lines. Each
CC-RIX mouse will inherit a chromosome from each CC parent and, thus,
genotype information can be extrapolated. Heterozygosity will be recaptured in the

CC-RIX lines, making them a more realistic model for human populations and
allowing for more accurate modeling of complex human diseases.
6 Haplotype Association Mapping
Inbred strain surveys are an important initial step in understanding the genetic and
phenotypic architecture of complex traits. Until recently, however, strain surveys
had become less frequently used, meaning that researchers had a limited set of data
on which to base experiments. In 2000, the Mouse Phenome Project was started as
an effort to collect phenotypic data from a core set of inbred strains. These data
would be stored in a central location, be publicly available on the Mouse Phenome
Database (MPD; http://phenome.jax.org/ and Bogue et al. (2007)) and would offer
investigators a wide range of phenotypes with which to inform research decisions
for complex trait analysis (Bogue 2003; Paigen and Eppig 2000).
The effort to collect phenotype data for inbred strains coincided with efforts to
increase the number of single nucleotide polymorphisms (SNPs) identified in
inbred mouse lines and construct a SNP haplotype map of the mouse similar to the
HapMap being constructed in humans (2003) (http://hapmap.ncbi.nlm.nih.gov/).
This convergence of phenotype data and SNP genotype data presented an obvious
opportunity to take advantage of both the phenotypic and genotypic diversity
among inbred strains. In the context of genetic mapping, inbred strains offer
several advantages over existing mapping populations. Inbred strains are, of
course, a reference population of mice enabling collection and storage of pheno-
typic and genetic data that can be utilized repeatedly. In addition, inbred strains,
like RIs, allow for repeated sampling within a strain to reduce environmental
variance. Inbred strains also have increased genetic and phenotypic diversity in
comparison to RI or standard F2 or BC populations, as well as a dense genetic map
due to increased recombination.
In 2001, Grupe et al. used existing inbred strain phenotype data from the MPD
and over 3,000 SNPs identified in their laboratory and at the Whitehead Institute
(Lindblad-Toh et al. 2000) to conduct ‘‘in silico QTL mapping’’ in mouse. The
basic approach is straightforward—as described previously, inbred strains are
genetically identical within a strain but genetically diverse across strains. There-
fore, associating SNP genotypes across the genome with strain phenotypes, similar
to an RI-QTL study, can be expected to yield chromosomal regions that are
associated with the trait of interest. The in silico mapping technique was successful
in identifying QTL that overlapped with previously published reports using
standard crosses (Grupe et al. 2001). Nevertheless, subsequent commentary on the
method exposed several weaknesses, including lack of power, insufficient genetic
variability and failure to control for both Type I and II errors (Chesler et al. 2001;
Darvasi 2001). Darvasi (2001) estimated that between 40 and 150 strains would be
necessary to identify a QTL affecting a quantitative trait with a heritability of 50%,
and the number of strains used in the Grupe study ranged from 4 to 8. However,
Grupe et al. argued that this estimate was based on power calculations using
Lander and Schork significance levels based on an infinite density of genetic
markers and was not applicable to their study.
Regardless, it was generally agreed that in silico mapping, more recently
termed haplotype association mapping (HAM), might be a useful tool for identi-
fication of QTL. Three factors were necessary for its successful implementation:
dense SNP coverage across the genome for more than just a few strains, phenotype
data for multiple strains and appropriate analysis tools for genotype/phenotype
associations. Pletcher et al. (2004) addressed two of these problems by identifying
over 10,000 SNPs in 48 strains and developing a HAM algorithm called SNPster
(http://snpster.gnf.org) to perform genotype/phenotype associations across the
genome using a three-SNP sliding haplotype window. Using this method, the
authors were able to identify QTL peaks for several Mendelian traits, including
coat color and retinal degeneration. They were also able to replicate previously
identified QTL for more complex traits such as saccharin preference, high-density
lipoprotein (HDL) levels and gallstone formation. In addition, most map locations
spanned intervals of 1 Mb or less—a significant improvement over standard F2 or
BC mapping.
However, several hurdles still remained for HAM. As more dense SNP panels
were genotyped and more was known about the genetic structure of the inbred
strains, it became clear that an extensive family structure existed that could lead to
spurious associations. In addition, only a limited number of strains existed for which
there was both phenotype and genotype data, resulting in limited power to detect
QTL for complex phenotypes. McClurg et al. (2007) attempted to address family
structure by calculating a genetic similarity matrix along with a weighted bootstrap
method to decrease the significance of nonspecific associations. In addition, gene
expression data were used to optimize the power of the SNPster algorithm to
identify cis-acting expression QTL that are considered, by some, to be a highly-
enriched set of true positives with a low false-positive rate (Chesler et al. 2006).
Additional analysis programs have been developed to perform association mapping
in inbred strains, including Efficient Mixed-Model Association (EMMA; (Kang
et al. 2008)) and hmmSNP, which uses a hidden Markov model (HMM)-based
algorithm (Tsaih and Korstanje 2009). Each program uses a different algorithm and
has a slightly different way of dealing with population structure. However, most
programs give qualitatively similar results (unpublished data, Tim Wiltshire).
The full power of HAM has not yet been realized, as the resources for
performing such analyzes are still being compiled. The quantity of SNPs in mice
now number in the millions across more than 100 inbred strains. The number of
phenotypes in the mouse phenome database continue to grow, with over 2,000
phenotypes stored and an average of 19 mouse strains tested per phenotype.
However, the HAM technique has been used successfully to narrow down previ-
ously identified QTL intervals (Burgess-Herbert et al. 2009; Cervino et al. 2005;
Harrill et al. 2009; Park et al. 2003; Wang et al. 2005) and to identify new QTL
(Bopp et al. 2010; Liao et al. 2004; Liu et al. 2006; Pletcher et al. 2004).
Few HAM studies have been published for behavior. Webb et al. conducted a
whole genome association study for prepulse inhibition and identified QTL on
Chrs 1 and 13 (Webb et al. 2009) that overlap with genes that alter PPI when
knocked out or regions of the human genome that contain genes that have been
implicated in schizophrenia. Miller et al. (2010) measured tail suspension, an
animal model of behavioral despair or depression, in 33 inbred strains of mice and
identified four QTLs, including one that overlapped a human region that contains a
locus associated with major depressive disorder and bipolar disorder.
Initial reports of QTL mapping using HAM resulted in concern about the fate of
standard QTL studies (Chesler et al. 2001; Darvasi 2001). However, these con-
cerns have not been realized. In general, HAM is viewed as one step in QTL
identification that can be used for initial identification of QTL regions that must
then be replicated using standard approaches. More commonly, HAM can be used
to both replicate and narrow QTL that were identified using standard approaches
(Burgess-Herbert et al. 2008, 2009; DiPetrillo et al. 2005). This technique is
particularly effective when using strains that are genetically similar. Although
genetic similarity may result in phenotypic similarity, QTL can be detected even
when using parental strains that are phenotypically similar (Bailey et al. 2008;
Eisener-Dorman et al. 2010). This is due to transgressive segregation, the
reshuffling of alleles that occurs when two strains are crossed (Rieseberg et al. 1999).
QTL identified in these crosses may be fine mapped to relatively small genomic
regions using haplotype comparisons since regions of shared haplotype can be
eliminated from consideration (Bailey et al. 2008; Eisener-Dorman et al. 2010).
As more phenotype data are collected for larger numbers of inbred strains and
more information is gathered on the genomic structure of the laboratory mouse,
HAM analysis will become an even more vital tool with which to identify complex
disease genes.
7 Mutagenesis
In the late 1990s, with QTL accumulating in the literature and very few identified
genes, the scientific community was looking for alternative approaches to iden-
tification of complex disease genes. Mutagenesis, the induction of mutations by
chemicals or radiation, was particularly attractive. Mutagenesis in the mouse was
not new—radiation-induced mutagenesis experiments to determine the genetic
effects of radiation on mammals had been ongoing at the Department of Energy’s
Oak Ridge National Laboratory (ORNL) since just after World War II. The use of
N-ethyl-N-nitrosourea (ENU) as a chemical mutagen in mice began in 1978 when
it was shown to have a mutation rate 12 times higher in male spermatogonia than
that of X-rays (Justice 2004).
ENU is an alkylating agent that causes single base pair mutations—usually
A–T to T–A transversions or A–T to G–C transitions. ENU induces random
heritable mutations at a rate of approximately 1.4 9 10-6 per nucleotide site
(Takahasi et al. 2007), although this rate varies depending on the protocol and the
species or inbred strain background. Because of the random nature of mutations
resulting from ENU, it was proposed as an unbiased way to induce a single
mutation that would affect a complex phenotype. Thus, unlike transgenic and
knockout mouse technologies, which target a specific gene of interest, a strength of
the ENU mutagenesis approach is that it is not hypothesis-driven and is not limited
to the study of known genes previously associated with specific biological path-
ways. Downstream analysis for ENU studies included mapping of the causative
mutation followed by identification of the genetic lesion. The presence of only one
causative mutation was anticipated to render the gene easier to identify.
In 1997, several large-scale ENU mutagenesis centers were set up in the UK
and Germany to produce and screen ENU-generated mutants using batteries of
phenotypic tests, including dysmorphologies, behavior, neurological abnormali-
ties, immunology, clinical chemistry and hearing (Hrabe de Angelis et al. 2000;
Nolan et al. 2000). In 2000, the NIH awarded grant funding to three large-scale
mutagenesis centers in the United States at the University of Tennessee at
Memphis, Northwestern University and the Jackson Laboratory with a mandate on
production, identification and dissemination of ENU-induced mutants displaying
alterations in nervous system function and behavior (collectively called the
Neuromice.org Consortium). Additional ENU mutagenesis centers were formed
outside the auspices of NIH, both small and large-scale, in academia and industry
(for a complete list see (Cordes 2005)).
The mutagenesis centers quickly began producing hundreds of mutants for
dozens of disease-related domains. However, it quickly became obvious that
identification of the causative mutation would not be as straightforward as hoped.
There have been many successes in gene identification for ENU-induced mutants
in the areas of immunology (Hoebe and Beutler 2008; Sandberg et al. 2005; Tabeta
et al. 2006; Theodoratos et al. 2010), development (Herron et al. 2002; Garcia–
Garcia et al. 2005; Stottmann et al. 2009) and metabolism (Lloyd et al. 2005, 2006,
2010; Wilkes et al. 2009). ENU-disrupted genes responsible for neurological traits
that might be considered less susceptible to environmental fluctuations have also
been successfully identified in the areas of deafness (Grillet et al. 2009; Mackenzie
et al. 2009; Parker et al. 2010; Schwander et al. 2009a, b), ataxia (Sharkey et al.
2009; Swanson et al. 2010; Xie et al. 2010) and epilepsy (Frankel et al. 2009;
Tokuda et al. 2011). Mapping mutants for more complex behavioral phenotypes,
on the other hand, has proven problematic.
The basic steps in ENU mutagenesis (Fig. 2) include induction of mutations by
injection of the mutagen in male mice. These G0 mice are then bred with wildtype
females, and the offspring of that cross, the G1, can be screened for dominant
mutations. Further breeding of the G1 males to wildtype females produces G2
females that can be bred back to the G1 male. The resulting G3 animals will carry
recessive mutations at a rate of one per eight G3s. Once an outlier is identified, the
standard course of action is to prove heritability by crossing the animal back
to a wildtype, produce F1 mice and then intercross to recover the mutation in the
F2 progeny—one-quarter of which should exhibit a fully-penetrant, recessive
Dominant/recessive ENU mutagenesis screen
ENU
G0: . .
random point
mutations
G1:
dominant
. . .
mutations
detected
G2:
dominant
. .
mutations
detected
G3:
recessive . . . .
mutations
detected
Fig. 2 Standard ENU mutagenesis. The standard ENU mutagenesis scheme includes injecting
male mice (G0) with ENU. Doses vary depending upon strain, but generally, three weekly
injections of 85–100 mg/kg ENU works well in B6 mice. ENU-treated males become infertile
following treatment but many will regain fertility after 10–12 weeks. Loss of fertility is often
used as an indicator that the ENU was effective. After regaining fertility, ENU-treated males are
bred to wildtype females of the same strain or a different strain. G1 animals produced from this
cross can be tested for dominant mutations. G1 mice will inherit a mutagenized genome from the
father and the mutations will differ between G1s. To recover recessive mutations, the G1 females
are crossed back to the G1 father to generate G2s. Two copies of any mutations that the father and
daughter share will be recovered in the G3 at a rate of one mutation per eight G3s. For this reason,
G3 pedigrees are often screened for phenotypes in order to recover at least one or two affected
animals with which to propagate the new mutant colony
phenotype. If no F2 mice exhibit the expected phenotype, it is assumed that the

original outlier had multiple alleles contributing to the phenotype (ENU produces
one mutation approximately every 1–2 Mb; Kile and Hilton 2005) that were
dissociated by outcrossing OR that the original outlier displayed an abnormal
phenotype due to non-genetic reasons (environmental variability, etc.). If the ratio
of affected mice in the F2 is lower than expected, reduced penetrance may be an
issue that needs to be considered during the mapping process. If, however, the
heritability cross proves successful, the mutants enter into a mapping funnel that
starts with outcrossing a proven mutant to a different inbred mapping strain and F2
or BC animals are produced and phenotyped. Here we start to enter into familiar
‘‘QTL’’ territory—if the phenotype produced by the mutation is too weak to be
observed on a mixed background or interacts with QTL in the mapping cross, the
phenotype may disappear and mapping attempts will be unsuccessful.
One way to deal with a non-robust phenotype is to cross the mutant to a number
of inbred strains in order to find a heterogeneous background that allows for the
recovery of the mutant phenotype in the mapping cross, but this increases mapping
costs. Alternatively, sufficient SNP data are now available that allow mapping
to strains that are closely related to the background strain and might reduce
the number of interacting QTL (Bailey et al. 2008; Eisener-Dorman et al. 2010;
Xia et al. 2010). The mutant phenotype might also be preserved by limiting the
amount of the mapping strain background by backcrossing the F1 animals to a
mutant. This strategy also increases the number of animals in the mapping cross
that carry the causative mutation. Finally, a contemporaneous population of mice
not carrying the mutation can be produced, phenotyped and genotyped to provide a
control for both choosing outliers in the mutant cross and identifying background
QTL.
Even with these strategies, however, identification of genes in ENU mutants
that exhibit behavioral anomalies has been painfully slow, even when the phe-
notype appears robust. Many mutants with abnormal behavioral profiles have been
reported (Rastan et al. 2004; Reijmers et al. 2006; Hamre et al. 2007; Mathews
et al. 2009), yet gene identification for ENU behavioral mutants has been slow to
materialize.
Recent publications, however, indicate that ENU-induced behavioral mutants
are beginning to yield genes. In 2007, Keays et al. identified an ENU-induced
mutation in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin
(Tuba1). Tuba1 mice exhibited hyperactive behavior but also had a secondary
correlated phenotype—body weight. The presence of a secondary phenotype that
was more stable and reproducible than the hyperactivity phenotype aided in fine
mapping and identifying the gene. Speca et al. (2010) recently reported on the
identification of an ENU-induced nonsense mutation in the Unc-79 gene in a
mouse mutant (Lightweight) initially identified in a screen for animals with
enhanced locomotor activity. The screen was conducted on a sensitized genetic
background (mice heterozygous null for dopamine transporter), but the Light-
weight mutation acted independently of this background. As the name implies, this
mutant also had a correlated phenotype of low body weight. Peaks for both weight
and locomotor activity overlapped, and the use of both phenotypes contributed to
the identification of the causative ENU-induced mutation. Finally, an ENU-
generated missense mutation in the Grin1 gene was identified in a mouse mutant
that displayed increased spontaneous locomotor activity (Furuse et al. 2010). It
should be noted that all three of these mutants were identified in screens for
dominant ENU mutations, and all were initially generated on a mixed genetic
background (i.e. ENU mutagenized males were crossed to females of a different
inbred strain). These results suggest that dominant mutations are ultimately easier
to identify than recessive mutations. Furthermore, using a mixed genetic back-
ground in the primary ENU screen may increase the chance of recovering mutant
mice in the mapping cross, thereby hastening identification of the mutated gene.
The goals of the NIH-funded ENU centers included generation and distribution
of mutagenized mice with the idea that individual researchers could follow up on
phenotypes of interest and map the mutated genes. Although gene identification of
ENU behavioral mutants has been slow, improvements in next-generation
sequencing technologies and decreased costs may accelerate the process. Deep
sequencing of ENU-generated mutants, in combination with or independent of
mapping information, could yield the causative mutations. Several large libraries
of cryopreserved sperm and/or embryos from G1 mice are also available at RIKEN
(Yoshiki et al. 2009), Harwell (Glenister and Thornton 2000), the Australian
Phenomics Network and the German ENU mutagenesis center. As sequencing
prices decrease, these libraries could be screened to identify mutations genome-
wide. Cryo-recovery and phenotyping of mice with mutations in specific genes
would complement efforts such as the KOMP.
8 Beyond QTL Analysis: Finding the Quantitative Trait Gene
The ultimate goal of QTL mapping is identification of the underlying polymor-

phism that can provide insight into the biology of the phenotype/disease.
Regardless of how a QTL is identified, members of the complex trait community
have determined that several methods are appropriate and necessary for QTL
validation (Abiola et al. 2003). These criteria include relating the gene function
to the QTL phenotype, identifying allelic polymorphisms or assessing gene
homology to determine if the sequence is evolutionarily conserved across species.
Manipulation of the gene of interest is also advantageous for QTL validation and
includes the study of knockout, knockin, transgenic or otherwise genetically-
altered mouse models. Genetic or functional complementation of different inbred
strains or a genetically-deficient mouse model serves as an additional confirmation
of the QTL (Kono et al. 2003; Yalcin et al. 2004).
Identification of the QTG represents the beginning of a new phase of analysis
that explores the mechanisms underlying alterations in gene expression and bio-
logical pathways (Flint 2003). For example, Rgs2 was mapped as an anxiety QTL
and was later genetically dissected and confirmed using quantitative comple-
mentation (Yalcin et al. 2004). These findings have since been translated to human
anxiety research, with the human RGS2 ortholog correlating with introversion and
social anxiety, thereby becoming a potential target for the treatment of social
anxiety disorders (Smoller et al. 2008).
9 The Future of Forward Genetics
As knowledge of the genomic sequence and organization of both the human and
mouse genomes increases, an appreciation for the complexities that will likely
explain the genetic components of complex behaviors has become more apparent.
Single-gene, reverse genetic approaches like knockouts and transgenics still have
much to offer toward functional annotation of genes. However, it is now widely

accepted that naturally occurring genetic variation, along with epigenetic effects
and environmental variation, may act synergistically to increase risk for complex
neuropsychiatric diseases. Although the complex trait community has made con-
sistent advances in the search for QTL that influence behavior, the progress from
QTL to QTG has been painstakingly slow. However, genomic tools in the mouse
are being developed at a rapid pace and promise to transform complex trait
analysis. For example, dense SNP maps now available make it possible to narrow
QTL regions using haplotype comparisons—the substantial reduction of a QTL
interval that used to take years can now be accomplished in hours depending on
the parental strains utilized (Eisener-Dorman et al. 2010).
New sequencing technologies are driving down costs and allowing for more
accurate sequencing of entire genomes. As acquiring sequence becomes less cost
prohibitive, the ability to sequence individual mice in a specific genomic interval
or genome-wide will become a real option even for smaller laboratories. This will
be especially useful for gene identification in ENU mutagenized lines.
Using new sequencing technologies, the Sanger Institute is sequencing the entire
genome from 17 different inbred mouse strains, including the CC parental strains, as
part of its Mouse Genomes Project (http://www.sanger.ac.uk/resources/mouse/
genomes/). These data will provide an invaluable resource for mapping and hap-
lotype analysis in the CC as it comes online. These sequence data, along with the
increased genetic variation and mapping resolution offered by the CC, will provide
access to QTL that have been unrepresented in current mapping populations.
This is a particularly exciting time for mouse genetics as resources and
technology advance at a rapid pace. Behavioral scientists are poised to gain
new insights into the biology and genetic control of complex behaviors with the
ultimate goal of translational studies in humans.
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Mouse Models of the 5-HTTLPR 3 Stress
Risk Factor for Depression
Valeria Carola and Cornelius Gross
Abstract The incidence of mood disorders is known to be influenced by both

genetic as well as environmental factors. Increasingly, however it is becoming
clear that few genetic and environmental factors act alone, but that instead they
regularly act in concert to determine predisposition to psychiatric disorders. Quite
a few cases now have been reported in which stratification of subjects by exposure
to environmental pathogens has been shown to alter the association between
specific genetic variants and mental illness. The best studied of such measured
gene-by-environment risk factors for mental illness is the increased risk for
major depression reported among persons carrying the short variant (S allele) of a
functional polymorphism in the serotonin transporter (5-HTT, SLC6A4) gene
promoter and who have been exposed to stressful life events. Recently, a large
number of laboratories have tried to model the interaction between 5-HTTLPR
genotype and early/adult stress in mouse. Findings from their studies have helped
to define the rodent orthologs of the environmental stressors and behavioral traits
involved in risk for depression. Furthermore, several of these studies attempted to
identify changes in molecular substrates that might underlie the 5-HTT x stress
risk factor, pointing to the hippocampus and frontal cortex as critical brain
structures involved in the interaction between 5-HTT gene variation and early and
adult stress, respectively. These results will serve to help inform clinical research
V. Carola (&)
Santa Lucia Foundation, Via del Fosso di Fiorano 64/65,
00143 Rome, Italy
e-mail: valeria.carola@uniroma1.it
V. Carola
Department of Psychology, University of Rome,
‘‘La Sapienza’’, Via dei Marsi 78, Rome, Italy
C. Gross
Mouse Biology Unit, European Molecular Biology Laboratory (EMBL),
Via Ramarini 32, Monterotondo, Italy

DOI: 10.1007/7854_2011_190
60 V. Carola and C. Gross
into the origins of major depression and other mental illnesses with interacting
genetic and environmental risk factors.
Keywords Gene-by-environment Serotonin transporter Mouse models

Early/adult stress risk factor Anxiety-depression
Contents
1 Challenges of Designing Mouse Models of the 5-HTTLPR 9 Stress Risk Factor ......... 60
2 Mouse Models of the 5-HTTLPR 9 Early Stress Risk Factor for Depression................ 63
3 Mouse Models of the 5-HTTLPR 9 Adult Stress Risk Factor for Depression ............... 65
4 Physiological and Molecular Substrates of the 5-HTT 9 Stress Risk Factor in Mice .... 66
5 Promises of Mouse Models of Gene 9 Environment Risk Factors
for Clinical Research........................................................................................................... 68
References.................................................................................................................................. 70
1 Challenges of Designing Mouse Models

of the 5-HTTLPR 3 Stress Risk Factor
Risk for mood disorders is moderated by genetic as well as environmental factors.

Twin studies suggest that genetic factors contribute only moderately to the vari-
ance in incidence of these disorders, while environmental factors presumably
explain the majority of their incidence. Increasingly, however, it is becoming clear
that few genetic and environmental factors act alone, but that instead they function
in concert to determine predisposition to mental illness. Several cases now have
been documented in which stratification of subjects by exposure to measured
environmental pathogens, ranging from childhood maltreatment to cannabis
use, have been shown to alter the association between specific genetic variants and
mental illness (Caspi and Moffitt 2006).
Arguably the best studied of such measured gene-by-environment risk factors
for mental illness is the increased risk for major depression reported among
persons carrying the short variant (S allele) of a functional polymorphism in the
serotonin transporter (5-HTT, SLC6A4) gene promoter (called serotonin trans-
porter gene-linked polymorphic region, 5-HTTLPR) and who have been exposed
to stressful life events. Originally, the short variant was associated with a small,
but significant increase in anxiety-related personality traits, such as neuroticism
and disagreeableness (Lesch et al. 1996). However, larger effects of the short
variant were found following stratification of subjects by exposure to either
childhood maltreatment or adult stress. When environmental exposure was
taken into account a significant interaction between 5-HTTLPR genotype and
stress exposure was uncovered (Caspi et al. 2003). Subjects carrying one or two of
the short variants (S/S or S/L individuals) demonstrated increased risk for major
Mouse Models of the 5-HTTLPR 9 Stress Risk Factor 61
depression at age 26 when exposed to either childhood maltreatment or stressful

life events in the five preceding years. L/L individuals, on the other hand, showed
low incidence of depression regardless of environmental exposure. Intriguingly,
they observed significant interactions between the short variant and both early (age
0–11) and adult (age 21–26) stress, suggesting that genetic influences on serotonin
homeostasis fundamentally altered the physiological or emotional response of an
individual to stress throughout life. The increased risk for depression in stressed S
allele carriers was later replicated by several laboratories although there have also
been failures and controversy (Risch et al. 2009; Kaufman et al. 2004, 2010; for
review, see Uher et al. 2011). Studies in non-ill subjects have also shown evidence
of a similar 5-HTTLPR 9 stress effects on physiological and behavioral traits,
including heart rate reactivity and anxiety (Caspi et al. 2010; Williams et al. 2009).
Thus, the 5-HTTLPR S allele is likely to act by favoring basic stress coping
strategies that subsequently carry an increased risk for depression.
Using electroencephalogram and functional magnetic resonance imaging (fMRI),
several groups were able to identify brain regions that showed significantly altered
neural activity in response to emotional stimuli in 5-HTTLPR short variant carriers
(Hariri et al. 2002; Heinz et al. 2005; Pezawas et al. 2005; Canli et al. 2005, 2006).
The most common finding has been an apparent increase in amygdala fMRI signal
reactivity to emotional stimuli (Hariri et al. 2002; Canli et al. 2005). However,
several studies have suggested that this is a reflection of an increase in baseline neural
activity in S allele carriers in a much wider range of forebrain areas, an interpretation
supported by perfusion imaging techniques (Canli et al. 2006; Heinz et al. 2007).
Several fMRI studies have also shown altered functional connectivity between
frontal cortical regions and amygdala in S carriers (Heinz et al. 2005; Pezawas et al.
2005). In one of these studies (Canli et al. 2006) subjects were explicitly stratified by
exposure to life stress events. Neural activity in a wide range of forebrain regions
showed evidence of a significant 5-HTTLPR 9 life stress effect following visual
exposure to emotional words. In many brain regions, including amygdala and hip-
pocampus, neural responses in S allele carriers increased with life stress events while
they decreased in L/L allele carriers so that under high stress load the genotype effect
on neural activation was magnified. In other brain nuclei, including regions impli-
cated in imitative behavior such as superior temporal gyrus and superior parietal
lobule, L/L subjects experiencing high stress showed increased neural activity
responses compared to S allele carriers. Such regions might mediate the stress
resilience seen in L/L persons. Moreover, a similar 5-HTTLPR 9 life stress effect
was seen on absolute baseline brain activity as measured by perfusion imaging (Canli
et al. 2006), suggesting that 5-HTTLPR genotype moderates the effect of life stress
on stable levels of neural activity.
These studies have transformed the psychiatric genetics field and have compelled
researchers to incorporate information about environmental exposure into the study
of genetic risk factors. The new approach to human association studies has also had
a significant impact on the pre-clinical research (Caspi and Moffitt 2006). For the
very first time, researchers applying animal models to study mood disorders have a
genetic variant in their hands that under the right environmental conditions is
expected to show significant and reproducible associations with depression-relevant

traits. This is a significant advance because until then no reproducible measured
genetic risk factor for mood disorders or associated traits existed.
The mouse has become the premier mammalian model organism for genetic
studies due to the amenability of its genome to targeted genetic manipulation.
However, because the upstream promoter region harboring the 5-HTTLPR variant
is not present in rodents, designing an appropriate mouse model of the
5-HTTLPR 9 life stress risk factor for depression is not straightforward. This
deficit means that studying the polymorphism in mice requires ‘‘humanization’’ of
the mouse gene. However, although present gene targeting technology makes it
relatively straightforward to produce a constitutive knockout of a gene or to replace
a gene with a variant, the need for a selectable marker (typically a neomycin
resistance gene) during embryonic stem (ES) cell targeting means it is difficult to
seamlessly replace a specific sequence in the mouse genome without leaving behind
a mark of the engineering process. Consequently, precise ‘‘humanization’’ of the
mouse promoter region is tricky and has so far not been reported. As a compromise
researchers have used constitutive 5-HTT knockout mice (Bengel et al. 1998) as an
approximative model either in the gene’s homozygous, complete knockout, or its
heterozygous, partially reduced, form. Studies on homozygous 5-HTT knockout
mice have been extremely helpful in better understanding the physiological function
of the transporter, while heterozygous mice are arguably a better model of the
human promoter polymorphism because the 50% reduction of 5-HTT mRNA in
heterozygous knockouts mimics that are found in 5-HTTLPR S allele carriers when
compared to L allele carriers (Lesch et al. 1996; Bengel et al. 1998).
The use of heterozygous mice as a model of 5-HTTLPR, however, remains
problematic because it remains unclear how and in what way 5-HTTLPR affects
gene expression. Data documenting an effect of 5-HTTLPR on gene expression in
humans derive primarily from studies in cultured non-neuronal cell lines (Lesch et al.
1996) and only a few studies have examined 5-HTT gene expression in L and S allele
carriers in neuronal cell lines or postmortem brain tissue (Greenberg et al. 1999;
Mann et al. 2000; Eley et al. 2004; Sugden et al. 2009). Moreover, autoradiography
and positron emission tomography studies using 5-HTT selective ligands have failed
to find consistent differences in 5-HTT protein binding between the variants, sug-
gesting a potential differential effect on mRNA and protein expression. One possi-
bility is that 5-HTTLPR controls transcription in a cell-type specific or temporally
controlled manner and that these features have masked differences in gene expres-
sion. It may be, for example, that 5-HTTLPR controls transcription primarily during
brain development, when serotonin is known to have a critical role in brain wiring
(Trowbridge et al. 2010; Ferreira et al. 2010) but only weakly in adulthood. More
controversially, it is possible that 5-HTTLPR affects 5-HTT expression in only a
subset of cells expressing the gene. Such issues could be addressed using humanized
mice carrying the L and S alleles. A more faithful mouse model of 5-HTTLPR could
also serve as a test platform to understand the transcription factor binding and
chromatin remodeling mechanisms that are presumed to mediate the transcriptional
effects of this variant and about which little or nothing is known.
2 Mouse Models of the 5-HTTLPR 3 Early Stress

The first study to incorporate measured environmental stress in the study of

behavioral phenotypes in 5-HTT knockout mice involved daily exposure to foot
shock (Carroll et al. 2007). From postnatal day 7–13, mouse pups were exposed
daily to three mild footshocks over a period of 150 s and then tested in adulthood
in a battery of anxiety and depression-related behavioral tests (elevated plus-maze,
light/dark test, open field, forced swim test). Although 5-HTT knockout mice
showed increased avoidance in anxiety tests and increased behavioral despair in
the forced swim test, no interaction between genotype and environmental stressor
was detected.
Using a similar battery of behavioral tests a more recent study (Carola et al.
2008) showed that levels of maternal care as measured by maternal licking and
grooming of pups during the first 2 weeks of life could significantly modulate the
effect of heterozygous 5-HTT knockout mutations on anxiety and depression
behavior. Mice were raised by mothers that were genetically identical but nev-
ertheless provided high and low maternal care (C57BL/6 9 BALB/c vs. BALB/
c 9 C57BL/6, respectively). Heterozygous 5-HTT knockouts showed equivalent
level of avoidance and behavioral despair as wild-type littermates when exposed to
high levels of maternal care, but when exposed to low maternal care showed
significantly more avoidance and behavioral despair than controls. Furthermore,
heterozygous knockout mice exposed to low maternal care also showed enhanced
fear in response to partially conditioned cues, suggesting enhanced cognitive
processing of ambiguous threatening cues. This study suggested that novelty
avoidance, behavioral despair, and ambiguous fear conditioning in mice may be
useful surrogates of risk for depression in humans (Fig. 1). It also suggested that
environmental stressors that perturb critical social cues such as maternal care
might be better models of childhood maltreatment than physical stressors such as
foot shock. It is important to keep in mind, however, that low maternal care in
mice and childhood maltreatment in humans act on only partially overlapping
stages of brain development (Fig. 1). Because mice are born relatively prematurely
compared to primates the first week of life in rodents when maternal care is most
pronounced corresponds to the late gestational period in primates (Clancy et al.
2007). Thus, rodent studies using maternal care as a surrogate for childhood
experiences must be interpreted in light of the caveat that the neural substrates of
these manipulations are not necessarily equivalent across species.
Another study evaluated anxiety behavior in heterozygous and homozygous
5-HTT knockout mice following exposure to adverse olfactory stimuli across both
pre- and post-natal periods (Heiming et al. 2009). During pregnancy and lactation
mothers were exposed to soiled bedding from the cage of an unfamiliar adult male.
Because adult males tend to attack and kill pups that are not their own, the
olfactory cues were assumed to be aversive to both the mother and her offspring.
Consistent with previous studies, homozygous 5-HTT knockout mice showed
short allele
5-HTTLPR
long allele 5-HTT

•Negligent or demanding mother Problems with –
•Harsh discipline •Employment
childhood •Finances
maltreatment •Change in caregive life stress
•Physical abuse •Housing
•Sexual abuse
events •Health
•Relationships
birth 14 yrs 21 yrs 26 yrs
human Incidence of major
Caspi et al., 2003 depression
birth 3 wks 8 wks 11 wks Social avoidance, risk

assessment, behavioral
despair
poor •Low licking and daily social •Continuous visual and
maternal grooming of pups olfactory contact with
mouse defeat dominant male
care +/– knockout •10 min daily exposure to
dominant male
Carola et al., 2008 5-HTT

Bartolomucci et al., 2010
+/+ wild-type
Fig. 1 Mouse models of 5-HTTLPR 9 early and adult stress risk factors for depression.
In humans, the combination of either childhood maltreatment or adult stress and the 5-HTLPR S
allele results in increased risk for major depression (Caspi et al. 2003). In mice, the 5-HTTLPR S
allele was modeled by a heterozygous knockout (-/+) mutation in 5-HTT that shows a similar
twofold decrease in mRNA expression. Childhood maltreatment was modeled by low maternal
care (Carola et al. 2008) and adult stressors were modeled by daily social defeat (Bartolomucci
et al. 2010). In both cases the low-expressing heterozygous knockout mice were more susceptible
to the effects of stress on anxiety and depression-related behavior, mimicking the human findings.
These studies suggest that reduced 5-HTT function is associated with increased responsivity to
environmental stressors in mice and humans (dotted arrows indicate orthologous developmental
time periods)
increased anxiety in the open field, elevated plus maze, and dark-light tests.
Exposure to unfamiliar male odor was also associated with increased anxiety in all
mice. However, the environmental effect was significant in homozygous knockout
mice and not in wild-type mice, indicating greater susceptibility of the knockout to
threatening olfactory cues than the wild-type. This study confirmed a role for
serotonin homeostasis in regulating the long-term effects of early exposure to
environmental stressors. Furthermore, these findings are intriguing because they
highlight a susceptibility that spans embryonic and early postnatal development
and thus points to a relatively non-specific modulation by serotonin of environ-
mental responsivity during development. It will be important to determine whether
environmental pathogens in humans are similarly effective across development in
modulating the effect of 5-HTTLPR genotype.
3 Mouse Models of the 5-HTTLPR 3 Adult Stress

A much larger number of studies have tried to model the interaction between
5-HTTLPR genotype and adult stress. The first study used a chronic mild stress
procedure involving daily exposure to several stressors (forced swimming, wet
bedding, light cycle reversal, unfamiliar cage mate, tilted cage, restraint stress, no
bedding material, etc.) each day for 4 weeks (Joeyen-Waldorf et al. 2009). Chronic
mild stress was associated with increased anxiety in the novelty suppressed
feeding test and this effect was potentiated in both heterozygous and homozygous
5-HTT knockout female mice, while males showed no genotype-by-environment
interaction effect. A similar genotype by stress effect was observed following
exposure of heterozygous and homozygous 5-HTT knockout mice to a winner or
loser experience in a resident-intruder paradigm over a period of 3 days (Jansen
et al. 2010). Both winners and losers exhibited increased anxiety behavior in the
elevated plus maze, dark-light test, and open field demonstrating an anxiogenic
effect of adverse social exposure regardless of outcome. Homozygous, but not
heterozygous 5-HTT knockout mice showed exaggerated anxiety when exposed to
either repeated winning or losing. These data suggest that 5-HT homeostasis
modulates responses to a wider range of social experiences that are previously
thought and warrants closer investigation in future human association studies.
Although heterozygous 5-HTT knockout mice did not show interactions with
repeated winning or losing in the above study, they did show enhanced response to
a continuous psychosocial stress paradigm where mice were exposed to brief daily
defeat by an aggressive male mouse with whom they were housed in continuous
sensory contact (Bartolomucci et al. 2010), possibly due to the greater intensity of
the later stressor. Following 3 weeks of psychosocial stress mice were tested for
social interaction toward an unfamiliar male mouse in a novel environment.
Stressed mice showed strong avoidance of the unfamiliar mouse, but this effect
was significantly greater in heterozygous knockout mice than wild-type litter-
mates. An important feature of this study was the monitoring of behavioral and
physiological responses to stress in the home cage on a daily basis. Heterozygous
5-HTT knockout mice showed similar responses to stress as wild-type littermates,
including decreased home cage locomotion between encounters, increased body
weight, and increased temperature responses to daily social defeat. These data
suggest that 5-HTT genotype modulates the generalization of avoidance behavior
rather than the direct physiological and behavioral consequences of stress. Such an
interpretation is in line with a role of 5-HTT in determining not the physical
responses to stress, but in modifying stress coping strategies. Moreover, these
findings confirm that a 50% decrease in 5-HTT function is sufficient to modulate
the response to environmental stressors if those stressors are sufficiently prolonged
or salient. Continuous exposure to psychosocial stressors in particular was most
effective in interacting with serotonin homeostasis to increase susceptibility to
anxiety and depression behavior.
4 Physiological and Molecular Substrates

of the 5-HTT 3 Stress Risk Factor in Mice
As discussed above, fMRI studies have identified numerous brain regions whose
activity is modulated by 5-HTTLPR genotype and life stress events and which
are thus implicated in mediating the effect of this risk factor on behavioral traits.
In particular, neural activity in both amygdala and hippocampus was shown to be
significantly modulated by 5-HTTLPR 9 stress, with S allele subjects showing a
positive correlation between life stress events and L/L subjects showing a negative
correlation (Canli et al. 2006). Neural activity in the superior temporal gyrus and
superior parietal lobule, on the other hand, showed a positive correlation with life
stress events in L/L subjects, suggesting a possible role of this structure in the
stress resilience of this genotype. In the same study, the authors also presented
evidence that resting state brain activity in hippocampus and amygdala as mea-
sured while the subjects fixated on a spot was also significantly enhanced in
stressed S allele carriers when compared to all other groups. The modulation of
resting state brain activity is important as it suggests that serotonin signaling
moderates the capacity of stress to reset resting brain activity, and raises the
possibility that changes in neural activity homeostasis in selected brain regions
may underlie the 5-HTTLPR 9 stress risk factor. Unfortunately, this hypothesis
has not yet been examined in animal models.
Nevertheless, two mouse model studies did attempt to identify changes in
molecular substrates that might underlie the 5-HTT 9 stress risk factor. In the first
study, where heterozygous 5-HTT knockout mice were shown to be more sus-
ceptible to the anxiogenic and depressogenic effects of low maternal care (Carola
et al. 2008), molecular substrates were identified in the adult that correlated with
the effect of genotype (G), early environment (eE), and genotype 9 environment
(G9eE; Table 1). First, levels of GABA-A receptors (as measured by binding to
the benzodiazepine flunetrazepam) were reduced in the central nucleus of the
amygdala of mice exposed to low maternal care regardless of the genotype. This
finding replicated earlier data in rats exposed to low maternal care (Caldji et al.
2003) and suggested that decreased inhibition in this nucleus might underlie the
increased risk assessment behavior associated with low maternal care. Second,
serotonin turnover as measured by the ratio of the serotonin metabolite 5-hydroxy-
indole-acetic acid (5-HIAA) to serotonin (5-HT) in the hippocampus was
decreased in heterozygous 5-HTT knockout mice regardless of environmental
exposure. This finding was consistent with earlier studies showing decreased
turnover and serotonin neuron firing in this genotype (Mathews et al. 2004). The
absence of an effect of environment on serotonin turnover suggested that stress
acts downstream of serotonin homeostasis to affect neural circuit function. Finally,
levels of brain-derived neurotrophic factor (BDNF) mRNA in the CA1 region of
the hippocampus (but not in other parts of the hippocampus) showed an interacting
effect of genotype and environment, with heterozygous 5-HTT knockouts exposed
to low maternal care showing the highest BDNF levels. Intriguingly, elevated
Table 1 Physiological and Molecular Correlates of the 5-HTT 9 Stress Risk Factor in Mice.
Summary of the effects of gene (G), environment (E), and gene-by-environment (G9E) on
molecular substrates as reported by studies measuring anxiety and depression-related behavior in
5-HTT +/+ and ± mice exposed to low maternal care (Carola et al. 2008, 2011) and chronic
psychosocial stress (Bartolomucci et al. 2010). Note that molecular substrates following exposure
to low maternal care were studied at two time points, postnatal day 10 and adulthood (– no effect;
n.d. not determined)
Molecular substrate Low maternal care Chronic psychosocial stress
Adult Postnatal day 10
FCtx 5-HT turnover G GxE GxE
AMY GABA-A receptor E - n.d.
HIP 5-HT turnover G G G
BDNF mRNA GxE E -
AMPA receptor - GxE n.d.
BDNF levels in forebrain are associated with elevated anxiety behavior (Yee et al.
2007) and are a candidate molecular substrate for the elevated neural activity seen
in this structure in human 5-HTTLPR S allele carriers exposed to life stress events
(Canli et al. 2006). Moreover, genetic variation in BDNF itself moderated the
long-term effects of maternal care on anxiety behavior (Carola and Gross 2010).
In humans too a non-synonymous variant in BDNF (Val66Met) with reduced
functionality is able to reverse the neural activity correlates of associated with the
5-HTTLPR S allele (Pezawas et al. 2008). Consistent with a causal role for these
neural substrates in behavior, levels of GABA-A receptor binding, serotonin
turnover, and BDNF expression were significantly correlated with anxiety
behavior in the mice and estimated to each explain about 15–30% of the variance
of relevant behavioral measures (Carola et al. 2008).
A follow-up study by the same group (Carola et al. 2011) investigated the
origins of these changes by examining molecular substrates in young mice during
the period of maternal care (postnatal day 10). Unlike in adult mice, BDNF mRNA
in the hippocampus was elevated in all low maternal care offspring regardless of
genotype (Table 1). This finding suggests that the low functioning 5-HTT variant
may act to maintain elevated BDNF expression induced by low maternal care.
Thus, 5-HTT genotype may not modulate the immediate impact of environment on
neurophysiology, but rather to control its perseverance. As a result, 5-HTTLPR S
allele carriers might be more vulnerable to repeated stressors and, consequently,
recurrent illness, a feature supported by recent epidemiological evidence (Uher
et al. 2011). At the same time, levels of excitatory neurotransmitter receptors
(of the AMPA class) in the hippocampus at postnatal day 10 showed a G9eE
effect and were elevated in heterozygous 5-HTT knockout mice exposed to low
maternal care. These differences did not persist to adulthood (V. Carola, unpub-
lished observations) and thus enhanced excitatory neurotransmission in hippo-
campus may serve as an immediate substrate for early stress and 5-HTT variation
that subsequently triggers long-term G9eE effects in this or other brain circuits.
Neural substrates of the 5-HTT 9 adult stress risk factor were investigated in
one study (Bartolomucci et al. 2010). These authors observed that although
genotype alone determined serotonin turnover in most brain regions, in the frontal
cortex serotonin homeostasis showed a significant GxaE effect, with the lowest
serotonin turnover in heterozygous 5-HTT knockout mice experiencing chronic
social defeat (Table 1). Intriguingly, serotonin turnover in the frontal cortex was
best correlated with levels of 5-HTT protein in the amygdala, suggesting cross-talk
between these two connected structures. This finding contrasts with serotonin
turnover data from experiments with 5-HTT 9 early stress (Carola et al. 2008),
where no such interaction was observed. Why frontal cortical serotonin turnover is
more plastic in adulthood than in childhood following stress is not clear, but could
reflect differences in the predominant circuits engaged by stress at these ages or by
the longer time since childhood stress. This difference suggests that the 5-HTTLPR
9 early stress and adult stress risk factors are associated with different patterns of
neural activity and that these forms of depression may show differential thera-
peutic responses. It also suggests that, at least following adult stress, altered
serotonin homeostasis may directly explain the altered activity and/or connectivity
of frontal cortical circuits seen in 5-HTTLPR S carriers by fMRI.
5 Promises of Mouse Models of Gene 3 Environment

Risk Factors for Clinical Research
Animal models of gene by environment risk factors for mental illness hold several
promises for the pre-clinical research aimed at developing new diagnostic markers
and therapeutic approaches. One important outcome of a successful animal model
is its ability to point out homologous animal and human behavior. Until now, the
primary avenue to match animal and human behavioral traits associated with
mental illness was pharmacological validation, in which drugs that are used to treat
behavioral disorders in humans induce homologous behavioral changes in animals.
However, pharmacological validation has several problems. The benzodiazepine
class of anxiolytics, for example, is frequently used to validate behavioral tests of
anxiety in rodents, and these drugs cause increased exploration of novel envi-
ronments and reductions in contextual fear (Garner et al. 2009). However, because
these drugs act non-selectively to boost GABA-A receptor function throughout the
brain, they act on a wide range of behaviors beyond anxiety and just because a
particular rodent behavior is modulated by benzodiazepines does not mean it
reflects anxiety. In the field of schizophrenia the situation is even less clear since it
is not obvious how to measure psychotic symptoms in animals. Some behaviors
such as head twitches and scratches selectively induced in mice by hallucinogenics
have been reported (González-Maeso et al. 2007), but the relevance of these
behaviors to the positive symptoms of schizophrenia remain speculative.
For these reasons, a genetic validation approach using a mouse model incor-
porating environmental exposure could offer a parallel avenue for the identification
of human–animal behavioral homologs. An excellent example for the use of a

genetic validation approach is a mouse engineered to carry a deficiency of a
1.3 Mb region on mouse chromosome 16 whose orthologous region in humans
when deleted results in a 30% incidence of schizophrenia (Stark et al. 2008). These
mice have deficits in working memory; a negative symptom found in many
schizophrenics and electrophysiological recordings in these mice during a working
memory task revealed a deficit in connectivity between hippocampus and
medial prefrontal cortex, two regions known to contribute to working memory
(Sigurdsson et al. 2010). Poor connectivity between these structures is now a
candidate for an endophenotype in the wider population of schizophrenics. As this
example shows, genetic models offer a powerful approach to validate both
behavior and their associated physiological and molecular substrates. Moreover,
because genetic models (whether by modeling the gene alone or a gene 9 envi-
ronment risk factor) model the etiology of the disorder rather than its treatment,
they are likely to be more specific and informative.
Mouse genetic models have the advantage that current gene targeting tech-
nology allows for essentially unrestricted temporal and spatial regulation of
transgene expression. Thus, once a genetic model is constructed, follow-up studies
can use conditional gene targeting to map the circuitry and time periods involved
with a resolution not achievable with pharmacological manipulations. Such
approaches then allow for the mapping of G9E risk factors to specific brain
circuitry as a prelude to understanding the cellular and network mechanisms
involved. Moreover, genetic manipulation can be used to pinpoint the brain cir-
cuitry critical for the processing of stressful environmental stimuli with a speci-
ficity not possible with the direct manipulation of the stress itself. In this way,
mouse models of G9E risk factors can help unravel the molecular mechanisms of
susceptibility to stress, invariably a major risk factor across all mental illnesses.
In summary, mouse models of the 5-HTTLPR 9 stress risk factor for depres-
sion have helped to define the rodent orthologs of the environmental stressors and
behavioral traits involved in risk for depression. Mouse models have also been
able to pinpoint several specific physiological and molecular deficits involved that
can now serve to inform clinical research into the origins of major depression.
Nevertheless, much still needs to be done to expand on these findings. Additional
early stressors (e.g. social defeat, negligence) should be tested for their ability to
precipitate depression-related behaviors and determine whether differences
between the neural substrates of early and adult stressors are due to their timing or
quality. More careful time course analyses are also needed to pinpoint the critical
periods for different stressors. These studies will need to be accompanied by
physiological, molecular, and anatomical studies aimed at aligning mouse and
human development in the postnatal period so as to better understand the human
developmental transitions implicated. Work on aligning rodent and primate
development during the prenatal period should be used as a model for such
research (Clancy et al. 2007).
A more faithful genetic model of the human 5-HTTLPR is needed to help
answer outstanding questions about where and when this polymorphism affects
5-HTT expression and the mechanism by which it acts to alter transcription.

Conditional genetic and/or pharmacological (Ansorge et al. 2004) manipulations of
5-HTT will be needed to identify the cell types and developmental periods involved.
Pharmacological blockade of 5-HTT has been used to restrict the critical window
of 5-HTT activity on depression-related behavior in adulthood to the first 2 weeks of
mouse development (Ansorge et al. 2004). However, it remains to be determined
whether the 5-HTT 9 stress effects will map to a similar period, particularly as the
first weeks of mouse development corresponds to mid-gestation in humans, rather
than the postnatal period implicated in human association studies.
Finally, further work on identifying physiological and molecular substrates of
the 5-HTT 9 stress effects is needed. How is brain activity altered during exposure
to stressful or anxiogenic stimuli? Is there evidence for similar widespread effects
of the 5-HTT 9 stress risk factor on brain activity in mice and humans? What are
the serotonin receptors and circuit mechanisms involved in such effects? Are there
developmental consequences of the 5-HTT 9 stress risk factor on brain wiring that
might explain treatment resistance and/or relapse in susceptible individuals?
Can novel therapeutic targets be identified in the circuits specifically affected by
the 5-HTT 9 stress risk factor? And finally, it will be imperative to show that
mechanisms identified in rodent studies can be extrapolated back to the clinic so as
to develop improved depression medications.
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Behavioral and Pharmacogenetics
of Aggressive Behavior
Aki Takahashi, Isabel M. Quadros, Rosa M. M. de Almeida

and Klaus A. Miczek
Abstract Serotonin (5-HT) has long been considered as a key transmitter in

the neurocircuitry controlling aggression. Impaired regulation of each subtype
of 5-HT receptor, 5-HT transporter, synthetic and metabolic enzymes has been
linked particularly to impulsive aggression. The current summary focuses
mostly on recent findings from pharmacological and genetic studies. The
pharmacological treatments and genetic manipulations or polymorphisms of a
specific target (e.g., 5-HT1A receptor) can often result in inconsistent results
on aggression, due to ‘‘phasic’’ effects of pharmacological agents versus ‘‘trait’’-
like effects of genetic manipulations. Also, the local administration of a drug
using the intracranial microinjection technique has shown that activation of
specific subtypes of 5-HT receptors (5-HT1A and 5-HT1B) in mesocorticolimbic
A. Takahashi
Mouse Genomics Resource Laboratory,
National Institute of Genetics (NIG),
Mishima, Shizuoka, Japan
I. M. Quadros
Departamento de Psicobiologia, Universidade Federal de São Paulo (UNIFESP),
São Paulo-SP, Brazil
R. M. M. de Almeida
Instituto de Psicologia, Universidade Federal do Rio Grande do Sul (UFRGS),
Porto Alegre-RS, Brazil
K. A. Miczek (&)
Departments of Psychology, Psychiatry, Pharmacology and Neuroscience,
Tufts University, Bacon Hall, 530 Boston Ave,
Medford and Boston, MA 02155, USA
e-mail: klaus.miczek@tufts.edu

DOI: 10.1007/7854_2011_191
Springer-Verlag Berlin Heidelberg 2012
74 A. Takahashi et al.
areas can reduce species-typical and other aggressive behaviors, but the same
receptors in the medial prefrontal cortex or septal area promote escalated forms
of aggression. Thus, there are receptor populations in specific brain regions that
preferentially modulate specific types of aggression. Genetic studies have shown
important gene-environment interactions; it is likely that the polymorphisms in
the genes of 5-HT transporters or rate-limiting synthetic and metabolic enzymes
of 5-HT (e.g., MAOA) determine the vulnerability to adverse environmental
factors that escalate aggression. We also discuss the interaction between the
5-HT system and other systems. Modulation of 5-HT neurons in the dorsal
raphe nucleus by GABA, glutamate and CRF profoundly regulate aggressive
behaviors. Also, interactions of the 5-HT system with other neuropeptides
(arginine vasopressin, oxytocin, neuropeptide Y, opioid) have emerged as
important neurobiological determinants of aggression. Studies of aggression in
genetically modified mice identified several molecules that affect the 5-HT
system directly (e.g., Tph2, 5-HT1B, 5-HT transporter, Pet1, MAOA) or indi-
rectly [e.g., BDNF, neuronal nitric oxide (nNOS), aCaMKII, Neuropeptide Y].
The future agenda delineates specific receptor subpopulations for GABA, glu-
tamate and neuropeptides as they modulate the canonical aminergic neuro-
transmitters in brainstem, limbic and cortical regions with the ultimate outcome
of attenuating or escalating aggressive behavior.
Keywords Aggression Serotonin Trait State Neuropeptides GABA
Contents
1 Introduction.......................................................................................................................... 75
2 Definition and Measurement of Aggression ...................................................................... 76
3 Aggressive ‘‘Trait’’ Versus ‘‘States’’ .................................................................................. 81
4 5-HT Receptors ................................................................................................................... 84
4.1 5-HT1 Family ............................................................................................................. 84
4.2 5-HT2 Family ........................................................................................................... 100
5 Serotonin Transporter (5-HTT)......................................................................................... 102
5.1 Pharmacological Approach....................................................................................... 102
5.2 Genetic Approach ..................................................................................................... 103
6 Monoamine Oxidase A ..................................................................................................... 104
6.1 Pharmacological Approach....................................................................................... 104
6.2 Genetic Approach ..................................................................................................... 104
7 Modulation of Serotonergic Activity by Other Systems ................................................. 106
7.1 Excitatory and Inhibitory Amino acids ................................................................... 107
7.2 Neuropeptides ........................................................................................................... 109
7.3 Other Molecules that Directly or Indirectly Affect 5-HT Pathways
and Aggression ......................................................................................................... 112
8 Concluding Remarks ......................................................................................................... 114
References................................................................................................................................ 116
Behavioral and Pharmacogenetics 75
1 Introduction
One of the oldest roots of the neurogenetic analysis of aggressive behavior can be
traced to the domestication of feral animals, as illustrated experimentally in the
elevated brain levels of serotonin in tame silver foxes (e.g., Popova et al. 1991).
No other transmitter system has been more consistently implicated in the neuro-
biological mechanisms mediating impulsive aggressive behavior than serotonin
(Miczek et al. 2002, 2007; Takahashi et al. 2011). Serotonin emerged as the focus in
the initial phase of ‘‘top-down’’ genetics of aggressive behavior, when the brains of
individuals that were selected for high aggressive traits revealed lower levels of
serotonin, its major acidic metabolite and some of the receptor or transporter mol-
ecules upon which this amine acts (Brown et al. 1979; Garattini et al. 1967; Linnoila
et al. 1983). It continues to be the key transmitter system of interest in the more recent
phase of ‘‘bottom-up’’ genetics, when the gene for a specific receptor or transporter
was deleted and the behavioral phenotype indicated a higher than normal level of
aggressive behavior (Bouwknecht et al. 2001a; Saudou et al. 1994).
The current understanding of serotonergic mechanisms in aggressive behavior
has to accommodate two different but interacting sources of influence. Classically,
serotonin has been studied for its role in the predisposition to engage in impulsive
violent and antisocial behavior. The question of interest continues to be which
features of serotonergic transmission—its rate of synthesis, uptake, metabolism,
receptor and transporter expression—are characteristic of an aggressive heritable
trait that runs in families (Brunner et al. 1993a). It has become clear that the
seductively simple serotonin deficiency hypothesis that links low serotonin activity
to the propensity for aggressive behavior is being replaced by more sophisticated
and detailed schemes. Recent clinical and preclinical data point to important allelic
differences in the genes for specific serotonin receptor subtypes, transporter
molecules and metabolic enzymes in impulsively aggressive individuals (de Boer
and Koolhaas 2005; Lesch and Merschdorf 2000; Miczek et al. 2002, 2007; Nelson
and Chiavegatto 2001; Takahashi et al. 2011).
A second role for serotonin is to modulate aggressive behavior by its phasic
activity, particularly during the anticipation of aggressive and defensive acts and at
the termination of an aggressive bout. Superimposed on the serotonergic tone are
transient changes in release, impulse flow and receptor activation that are syn-
chronized with the initiation and termination of bouts of aggressive and defensive
acts (Ferrari et al. 2003). Phasic serotonergic activity appears to be linked to bouts
of several species-normative consummatory behaviors ranging from feeding,
drinking and sexual acts to fighting and to pathological excesses of these activities.
Neuroplastic changes in serotonergic pathways from the dorsal raphé neurons to
the neo- and paleocortical terminals result from repeated aggressive experiences as
reflected in serotonin receptor regulation, firing rate and release. To further our
understanding of the interaction between tonic and phasic serotonergic activities
ideally requires real-time measurements over the course of a circadian cycle and
arranging conditions that provide a challenge behaviorally and neurochemically.
During the last decade, epigenetic mechanisms for the expression of certain
genes that encode for serotonergic metabolic enzymes, transporter proteins and
receptors have been scrutinized for their role in impulsively violent individuals.
Stressful experiences such as abusive maltreatment during a critical developmental
period can lead young adults to engage in more impulsive and violent behavior,
particularly in those individuals who are characterized by the expression of a
certain allelic variety of Monoamine Oxidase A, an important metabolic enzyme
for serotonin (Caspi et al. 2002).
The last decade has seen also a concerted effort to translate more readily
between preclinical data and clinical observations, and vice versa. This is evident
from both an increasing focus on escalated, atypical forms of aggressive behavior
in animal models, and by efforts to define aggressive behaviors using operational
and functional definitions and assessments in clinical settings (DSM-V).
2 Definition and Measurement of Aggression
Most research on the neurogenetics of serotonin and aggression in both human and
veterinary medicine seeks to understand and control pathological aggression
(Volavka et al. 2005), while laboratory experiments using animal models typically
deal with adaptive, species-typical aggressive behavior. When studying aggression
in animals, it is important to consider the ethological significance of the behavior,
including its phylogenetic and ontogenetic development and the functions it serves
for individuals and the species. Aggressive signals, postures and acts are used to help
an animal obtain specific goals, or to defend against threats actual attacks (Miczek
et al. 2002). These behaviors occur when individuals compete for food, water and
other resources necessary for survival and reproduction (resident-intruder aggres-
sion), when they defend their territory or offspring (territorial and maternal
aggression), or in response to frustration or fear (Miczek et al. 2001). Engaging in
aggressive behavior is often beneficial to the individual and the species. For example,
dominance hierarchies are established and maintained through confrontations
between rival males (Fig. 1), and so-called isolation-induced aggression mimics
many elements of the behavior used by resident males to exclude other breeding
males from their home territory and their mates (Brain and Benton 1979; Miczek
et al. 2001, Table 1). However, it should be noted that isolated housing results in
social avoidance in a large proportion of a sample of mice, while aggressive behavior
is seen in varying numbers of isolated animals depending on species and strain.
Aggressive behavior can be classified as offensive or defensive based upon the
distal and proximal conditions that precipitated it, the topography of the behavior,
and its consequences (Blanchard and Blanchard 1977; Brain 1979). Defensive
behaviors occur in response to threatening or fear-inducing stimuli and often result in
escapes (Brain 1979). In male rodents, specific defensive behaviors include escape,
freezing, defensive postures and threats, which occur in response to attacks by either
predators or conspecifics (Blanchard et al. 2003; Pellis and Pellis 1988; Rasia-Filho
Fig. 1 Mouse agonistic

behavior. Behaviors of
resident and intruder mice
engaged in an aggressive
confrontation: a the resident
leaps and bites the intruder as
the intruder attempts to
escape; b the resident (right)
threatens as the intruder (left)
holds a defensive upright
posture; c the resident
investigates the intruder’s
anogenital region; d the
resident pursues the fleeing
intruder; e both resident and
intruder engage in a mutual
upright defensive posture.
Reprinted with permission
from Miczek and O’Donnell
(1978)
et al. 2008). Postpartum female rodents engage in maternal aggression, which

includes both defensive and offensive elements, to protect their newborn offspring
from male intruders (Lucion and de Almeida 1996; Parmigiani et al. 1998).
Table 1 Types of aggressive behavior in preclinical models
78
A. Species-typical aggressive behavior

Situational or experimental Agonistic behavioral References
variable measurements
Dominant resident, In a stable colony where dominance Frequency and duration of agonistic acts, postures Bennett et al. (2002)
mainly in primates hierarchy is to be established and displays including displacement, threat, Bernstein et al. (1974)
and rats and maintained pursuit and fight Fairbanks et al. (1999)
Higley et al. (1996)
Mehlman et al. (1994)
Steiniger (1950)
Vandenbergh (1967)
Territorial resident (resident- Requires an established territory. In the Frequency of attack bite, sideways Crawley et al. (1975)
intruder test), mainly in mice laboratory, home-cage of experimental threat, tail-rattle, pursuit, Eibl-Eibesfeldt (1950)
and hamsters male (resident) where it is pair-housed upright posture. Miczek and O’Donnell
with a female. A male stimulus animal (intruder) that Latency to the first bite (1978)
is group housed with other Van Oortmerssen and
males is introduced into resident’s cage Bakker (1981)
Maternal aggression, mainly in Home-cage of lactating females from postpartum Frequency of attack bite (especially Haney et al. (1989)
rats, mice and hamsters day 1–7. Either male or female of intruder is directed at the snout and the face), sideways Hurst (1987)
introduced into dam’s cage threat, tail-rattle, pursuit, upright posture. Lonstein and Gammie
Latency to the first bite (2002)
Noirot et al. (1975)
Sgoifo et al. (1992)
Female aggression, Dominance hierarchy among female monkeys. Harrassing attacks by dominant female. Frequency DeBold and Miczek (1981)
mainly in primates and In the laboratory settings, female rodent pair-housed with of attack bite, sideways threat, tail-rattle, pursuit, Palanza et al. (2005)
rodents a breeding male. Sexually mature female is introduced as upright posture. Smuts (1986)
an intruder Latency to the first bite Zitzman et al. (2005)
Isolation-induced aggression Male isolated for 24 h to 8 weeks prior to resident- Frequency of attack bite, sideways threat, tail- Cairns and Nakelski (1971)
(similar to territorial intruder encounter rattle, pursuit, upright posture. Malick (1979)
aggression) Latency to the first bite Valzelli and Bernasconi
(1979)
Yen et al. (1959)
(continued)
A. Takahashi et al.
Table 1 (continued)
B. Escalated aggressive behavior
Situational or experimental Agonistic behavioral References
variable measurements
Alcohol-heightened Ethanol (1.0 g/kg) is injected Frequency of attack bite, sideways Blanchard et al. (1987)
aggression, mainly intraperitoneally or orally or threat, tail-rattle, pursuit, upright posture. Miczek and de Almeida
in rats and mice self-administered before the Latency to the first bite (2001)
resident-intruder encounter Targets of attack bites (head, Miczek et al. (1992, 1998a)
dorsal areas, ventral areas, appendages) Peeke and Figler (1981)
Social provocation A resident male is exposed to another Fish et al. (1999)
(instigation), mainly male behind a protective screen in his Heiligenberg (1974)
in hamsters, mice, and rats home-cage prior to the resident- Potegal (1991)
intruder encounter Potegal and Tenbrink (1984)
Frustration-heightened A resident male is positively reinforced. Berkowitz (1993)
aggression The reinforcement is abruptly omitted De Almeida and Miczek
before the resident-intruder encounter (2002)
Aggression induced by low Resident rats are adrenalectomized and Haller et al. (2001)
glucocorticoids implanted with a low-dose
corticosterone pellet
Affective defense Electrical stimulation (0.2–0.8 mA, Hissing, arching of the back, Hess (1954)
(‘‘rage’’), mainly in cats 63 Hz, 1 ms per half cycle duration) retraction of the ears, piloerection, Leyhausen (1979)
delivered in medial hypothalamus or unsheathing of the claws, Siegel et al. (1999)
midbrain periaqueductal gray papillary dilatation and paw striking
79
‘‘Violence’’ is a controversial concept among ethologists. The term has been

used to describe escalated, pathological and abnormal forms of aggression char-
acterized by prolonged and frequent attack bites and aggressive behavior with brief
latencies (Miczek et al. 2002, 2003). The differences are quantitative rather than
qualitative, with violence marked by shorter attack latencies and higher frequencies
and longer durations of fighting than adaptive aggression. Violence has been
described to be qualitatively different from adaptive aggression. For instance,
attack bites aimed at vulnerable parts of the opponent’s body are considered
abnormally aggressive, when adaptive aggression consists of less-injurious bites
directed at the intruder’s back and flanks (Haller et al. 2005). Additional qualitative
distinctions that have been proposed include context-independent attacks, which
are directed at an opponent regardless of its sex, responsiveness (free-living/
anaesthetized/dead) or the test environment (home/neutral cage) (Koolhaas 1978),
and lack of ritualistic behaviors, quantified as attack/threat (A/T) ratios (Haller
et al. 2005). Therefore in principle ‘‘violence’’ could refer either to quantitatively
escalated or hyper-aggression, or to qualitatively abnormal forms of aggression,
or even rarely to aggression that is both escalated and abnormal [for review see
Natarajan and Caramaschi (2010)].
Human and non-human aggressive behaviors have some common features, but
most animal aggression is less complex. Social norms establish boundaries for
what is accepted as appropriate aggressive behavior, but the array of inappropriate
interpersonal behaviors classified as violence is a serious social and mental health
issue (Ferris et al. 2008). Numerous psychiatric disorders defined in the DSM-IV R
and the new DSM-V (scheduled for publication in May 2013), including schizo-
phrenia, brief reactive psychosis, anxiety disorder, adjustment disorder, impulse
control disorder, antisocial personality disorder, attention deficit disorder, mania/
depression, PTSD, autism and substance abuse, specify aggressive behavior
among their symptoms (Boles and Miotto 2003; Raine 2002; Rydén et al. 2009;
Volavka et al. 2005).
It can be useful to classify human aggressive behavior as either defensive, pre-
meditated or impulsive-hostile in nature (Stoff and Vitiello 1996; Vitiello and Stoff
1997). The premeditated (e.g., predatory and instrumental) and impulsive forms of
aggression are especially likely to be diagnosed as pathological and in need of
treatment. Impulsive, but not premeditated, aggression is linked to biological and
environmental causes, as well as pharmacological or psychological treatment
response factors, by a growing body of empirical data (Coccaro et al. 2010).
Aggression as a behavioral state in humans has usually been measured by
provoking subjects in competitive situations with fictitious opponents and then
giving them opportunities to engage in quantifiable responses that are defined as
aggressive [see Table 2, for review see Miczek et al. (2002)]. Human aggression
as a trait is assessed using psychometric measures including inventories, ques-
tionnaires and scales. The role of 5-HT in human aggression has been success-
fully investigated using such laboratory techniques (Table 2), but relating
laboratory indices of aggression to actual violence and aggression outside the
laboratory remains a critical challenge for such approaches; identifying subtypes
of human aggression with psychometric and laboratory methods is also difficult.

The psychometric instruments used to differentiate between individuals with
contrasting aggressive traits such as the impulsive–reactive–hostile–affective
subtype versus the controlled–proactive–instrumental–predatory subtype (Stoff
and Vitiello 1996) are summarized in Table 2.
3 Aggressive ‘‘Trait’’ Versus ‘‘States’’
A frequently reiterated theory, based on early clinical and preclinical studies, links
impulsive, hostile and violent behavior to a serotonin deficiency (Brown and
Goodwin 1986; Goldman et al. 1992; Lesch and Merschdorf 2000; Linnoila and
Virkkunen 1992; Mann 1999; Valzelli 1977). Individuals exhibiting such behavior
may benefit from pharmacological treatments aimed at inhibiting 5-HT trans-
porters (e.g., SSRIs such as fluoxetine or citalopram), activating 5-HT1A receptors
(e.g., buspirone) or blocking 5-HT2A receptors (e.g., risperidone). Acute treatment
with these drugs induces phasic changes in 5-HT function that are associated with
their transient anti-aggressive effects. In vivo microdialysis allows transient
changes in extracellular 5-HT levels to be monitored in anticipation of, during, and
after aggressive encounters in rats. One study reported reduced 5-HT levels in the
prefrontal cortex during and after the aggressive confrontation, while no changes
were detected in the nucleus accumbens, another terminal region (Van Erp and
Miczek 2000, Fig. 2). By contrast, chronic treatment with these anti-aggressive
compounds may promote as yet undefined neuroadaptive changes in 5-HT
function such as autoreceptor desensitization, which may in turn be associated
with the emergence of therapeutic effects.
On the other hand, aggression as a ‘‘trait’’ is the focus of genetic studies. While
these aggressive traits are clearly polygenic, it is remarkable that several studies
have found an interaction between genotypes such as TPH2, MAO-A and 5-HTT
polymorphisms and environmental triggers such as social stress underlying an
increased likelihood of violent outbursts (see below). For example, a SNP in TPH2
gene (A2051C) has been linked to aggressive behavior in rhesus monkeys.
Monkeys with the AA/AC genotype that were reared without their mother (peer-
reared) showed increased aggressive acts compared to those with a CC genotype,
but the difference disappeared when infants of both genotypes were reared by their
mothers (Chen et al. 2010). This review will focus on the interaction between
salient environmental events and genes and the subsequent effects on aggressive
behaviors.
Gene–gene interactions are also of interest and need to be explored. For
example, Passamonti et al. (2008) showed interactions between 5-HTT and
MAOA polymorphisms that affected the activity of the anterior cingulate cortex, a
brain area that has been implicated in impulsivity, including impulsive aggression.
Many other genes may have subtle effects on aggressive phenotypes, and stronger
effects may emerge as a result of complex epistatic interactions between those
82
Table 2 Experimental protocols for assessing 5-HT effects on human aggressive behavior
A. Experimental manipulations
Experimental manipulation Measurement Trait/state References
Aggressive responses toward a Activate buttons at 5–10 settings, State Buss (1961)
competitor are measured in the each corresponding to Godlaski and Giancola (2009)
form of electric shock settings a different intensity or duration
of electric shock
A fictitious instigator or competitor is the target of Setting of electric shock level on State Chermack and Giancola (1997)
aggressive responses that are measured in the a scale from 1–10 Taylor (1967)
form of electric shock deliveries
The subjects are provoked by having points Number of point subtractions from State Cherek and Heistad (1971)
subtracted in a competitive task. The point losses a fictitious competitor Cherek and Lane (1999)
are attributed to a fictitious opponent, but are Gowin et al. (2010)
actually random. Subjects
responded by retaliation of
point subtractions
(=aggressive responses)
Aggression was defined as Uses a modified version of the State Giancola et al. (2009)
delivery of electric shocks Buss aggression machine. Zeichner and Pihl (1979)
to a fictitious opponent Setting of shock level on a
scale from 1–5
B. Psychometric inventories
Psychometric assessment Instrument Trait/state References
Aggression, impulsivity Inventory Trait McKinley et al. (1948)
and hostility are measured Nagtegaal and Rassin (2004)
by Minnesota multiphasic
personality inventory (MMPI)
(continued)
A. Takahashi et al.
Table 2 (continued)
B. Psychometric inventories
Psychometric assessment Instrument Trait/state References
Buss–Durkee hostility inventory Inventory Trait Buss and Durkee (1957)
(BDHI), a self-rating scale of
anger and hostility. Sixty six items
with false/true answers; also contains
seven scales: assault, indirect aggression,
irritability, negativism, resentment,
suspicion and verbal aggression
Anger and anxiety are measured Inventory State/trait Kim et al. (2009b)
by state–trait anger expression Spielberg et al. (1973)
inventory (STAXI)
Aggression is measured by beck Inventory Trait Beck et al. (1961)
anxiety inventory and beck Lamar et al. (2009)
depression inventory
83
genes (Miczek et al. 2001). In the past 15 years, most rodent studies on genetics
and aggressive behavior have used conventional knockout techniques, in which the
expression of a gene is completely deleted, affecting the whole body through all
developmental stages and inducing compensatory changes in other genes (trait-like
change; see Table 3). Newer, more subtle methods, including conditional knock-
out, viral vector microinfusion, and drug-inducible knockout techniques, can
produce transient and local changes in gene expression, allowing the study of more
‘‘phasic’’ changes in gene expression and how they influence aggression. Such
studies may explain some of the discrepancies in the results from previous genetic
and pharmacological studies of 5-HT function and aggression.
4 5-HT Receptors
Considerable evidence suggests that serotonin receptors regulate aggressive

behaviors in various animal species. Among the multiple 5-HT receptor subtypes,
it is mainly the first two families of serotonin receptors (5-HT1 and 5-HT2) which
have been studied in regard to their role on aggression (Miczek et al. 2002; Olivier
2004). There is some, but less, information on the involvement of 5-HT3 receptors
on aggressive behaviors (McKenzie-Quirk et al. 2005; Ricci et al. 2004; Rudissaar
et al. 1999). Further development of adequate pharmacological tools that can
activate or inhibit a certain subtype of 5-HT receptor specifically is required to
study the role of other receptor subtypes (e.g., 5-HT5, 6, 7, 1e, 1f) on aggression.
4.1 5-HT1 Family
4.1.1 Pharmacological Approach
The 5-HT1A receptor partial agonist buspirone has been used clinically to reduce
general anxiety. It was shown that buspirone also reduces aggressive behavior in
mentally retarded patients (Kavoussi et al. 1997; Ratey et al. 1991), and this
compound has been used for the management of aggressive outbursts associated
with neuropsychiatric disorders in adults and children (Connor and Steingard
1996; Pabis and Stanislav 1996). Similar findings have been reported in preclinical
studies; systemic administration of 5-HT1A receptor agonists (e.g., 8-OH-DPAT,
repinotan, alnespirone) dose-dependently decrease aggressive behaviors in a wide
range of animal species, including fish, amphibians, birds, rodents, guinea pigs and
non-human primates (Bell and Hobson 1994; Blanchard et al. 1988; Clotfelter
et al. 2007; de Boer et al. 1999; de Boer and Koolhaas 2005; Dompert et al. 1985;
Haug et al. 1990; Joppa et al. 1997; Lindgren and Kantak 1987; McMillen et al.
1988; Miczek et al. 1998b; Muehlenkamp et al. 1995; Nikulina et al. 1992; Olivier
et al. 1992; Sanchez et al. 1993; Sperry et al. 2003; Ten Eyck 2008; Tompkins
Fig. 2 Dopamine and serotonin during aggression. Measurements of extracellular dopamine and
serotonin via in vivo microdialysis in resident male rats before, during and after a confrontation with
an intruder. a In the nucleus accumbens (top panel), dopamine levels (gray circles) rise and remain
elevated after the confrontation, while serotonin levels (black diamonds) do not significantly
change. b In the prefrontal cortex (bottom panel), dopamine levels rise after the confrontation, while
serotonin decline and remain lower after the confrontation. Samples were collected every 10 min
and levels are expressed as mean percent of baseline ±SEM. Baseline was measured for 50 min
before the fight. The vertical light gray bar indicates the occurrence of the 10-min fight. Asterisks
and double asterisks represent significant differences from baseline (dashed line) at the p \ 0.05 and
0.01 levels, respectively. Reprinted with permission from Van Erp and Miczek (2000)
et al. 1980). One exception are fruit flies (Drosophila melanogaster), as they
showed increased aggressive behavior after 8-OH-DPAT treatment (Johnson et al.
2009). Selective antagonists of 5-HT1A receptors such as WAY-100635 success-
fully reversed the effect of 5-HT1A agonists, while the administration of the
antagonist by itself did not show any effect on aggression (de Boer and Koolhaas
2005; Mendoza et al. 1999; Miczek et al. 1998b). Notably, in laboratory studies the
anti-aggressive effects of most of 5-HT1A agonists are accompanied by nonspecific
Table 3 Genes and aggressive behavior in mice
86
Gene Abb Chr Background strain Type of Effects Serotonin function References
(generations of aggression
backcross)
Plasmacytoma expressed transcript 1 Pet1 1 Mix C57BL/6 Isolation- Increased 90% reduction Hendricks et al.
(Fev) and 129 Sv induced of brain 5-HT (2003)
resident
aggression
Arginine vasopressin receptor 1B V1bR 1 Mix C57BL/6 Isloation- Suppressed Wersinger et al.
and 129/SvJ induced (2002)
aggression
Neutral cage Suppressed
aggression
Aadenosine A1 receptor A1AR 1 Mix C57BL Isolation- Increased Gimenez-Llort
and 129/OlaHsd induced et al. (2002)
resident
aggression
Regulators of G protein signaling 2 Rgs2 1 C57BL/6J (N5) Social Suppressed Oliveira-dos-
dominance Santos et al.
test (2000)
v-abl Abelson murine leukemia viral Arg 1 Mix C57BL/6 Resident Suppressed Koleske et al.
oncogene homolog 2 (Abelson- and 129/SvJ aggression (1998)
related gene)
Glutamic acid decarboxylase 2 GAD65 2 Mix C57BL/6 Isolation- Suppressed Stork et al.
and CBA2 induced (2000)
resident
aggression
Calcium channel, voltage-dependent, Cav2.2 2 Mix C57BL/6J Isolation- Increased Increased hypothalamus Kim et al.
N type, a1B subunit and 129S4/SvJae induced 5-HT (2009a)
resident
aggression
A. Takahashi et al.
(continued)
Table 3 (continued)
backcross)
Dopamine b-hydroxylase Dbh 2 Mix C57BL/6J Isolation- Suppressed Marino et al.
and 129/SvEv induced (2005)
resident
aggression
Brain-derived neurotrophic factor [±] BDNF 2 C57BL/6J ([N10) Isolation- Increased Decreased brain 5-HT Lyons et al.
induced (1999)
resident
aggression
b2-microglobulin b2 m 2 129S2/SvPas Resident Suppressed Loconto et al.
(Mix C57BL/6J?) aggression (2003)
Oxytocin Oxt 2 Mix C57BL/6J Isolation- Increased Winslow et al.
and 129SvEv induced (2000)
resident
aggression
Resident Increased
aggression Suppressed DeVries et al.
(1997)
Membrane metallo endopeptidase NEP 3 Mix C57BL/6J Resident Increased Fischer et al.
(enkephalinase) and 129 Sv aggression (2000)
Cannabinoid receptor 1 CB1 4 CD1 (N15) Isolation- Increaseda Martin et al.
induced (2002)
resident
aggression
Preproenkephalin Enk 4 Mix CD1 Isolation- Increaseda Konig et al.
and 129 induced (1996)
resident
aggression
87
(continued)
Table 3 (continued)
88
backcross)
Endothelial nitric oxide synthase eNOS 5 Mix C57BL/6 Resident Suppressed Increased 5-HT turnover Demas et al.
and 129 Sv aggression (1999)
aggression
Interleukin-6 IL-6 5 Mix C57BL/6, Isolation- Increasedb No difference in brain Alleva et al.
129/SvEv induced 5-HT concentration (1998)
resident
aggression
Adrenergic receptor, a2c Naa2C 5 C57BL/6J (N7) Isolation- Increased Sallinen et al.
induced (1998)
resident
aggression
Neuronal nitric oxide synthase nNOS 5 Mix C57BL/6J, Resident Increased Reduced brain 5-HT Nelson et al.
129 Sv, DBA2 aggression turnover (1995)
Maternal Suppressed Gammie and
aggression Nelson
(1999)
Acetylcholinesterase AChE 5 129S6/SvEvTac Home-cage Suppressed Duysen et al.
hierarchy (2002)
Adenylate cyclase activating PAC1 6 Mix C57BL/6J Resident Suppressed Nicot et al.
polypeptide 1 receptor 1 and 129 Sv aggression (2004)
Tachykinin receptor 1 NK-1r 6 Mix C57BL/6 Isolation- Suppressed De Felipe et al.
and 129 Sv induced (1998)
resident
aggression
A cluster of vomeronasal receptor V1Ra/b 6 129/SvEv Maternal Suppressed Del Punta et al.
genes aggression (2002)
A. Takahashi et al.
(continued)
Table 3 (continued)
backcross)
Oxytocin receptor Oxtr 6 Mix C57BL/6 Isloation- Increased Takayanagi
and 129 Sv induced et al. (2005)
aggression
Cage-mate injury Increased
Histamine receptor H1 H1 6 Mix C57BL/6J Isolation- Suppressedb Increased 5-HT turnover Yanai et al.
and 129 induced (1998)
resident
aggression
Amyloid b(A4) precursor protein- X11L 7 C57BL/6 Competitive Subordinate Increased Hypothalamus Sano et al.
binding, family A, member 2 feeding 5-HT (2009)
Transient receptor potential cation TRP2 7 Mix C57BL/6J Resident Suppressed Stowers et al.
channel, subfamily C, member 2 and 129Sv aggression (2002)
Neuropeptide Y receptor Y1 Y1 8 Mix C57BL/6 Resident Increased Reduced TPH mRNA Karl et al.
and 129SvJ aggression expression in the (2004)
raphe nuclei
Prostaglandin E receptor 1 EP1 8 C57BL/6 (N5) Neutral cage Increased No difference in 5-HT Matsuoka et al.
(subtype EP1) aggression turnover (2005)
Norepinephrine transporter NET 8 Mix C57BL/6J Isolation- Increaseda Haller et al.
and 129SvJ induced (2002)
resident
aggression
Neural cell adhesion molecule 1 NCAM1 9 C57BL/6J ([N5) Isolation- Increased Stork et al.
induced (1997)
resident
aggression
(continued)
89
Table 3 (continued)
90
backcross)
Aromatase (Cyp19a1) Ar 9 Mix C57BL/6 Resident Suppressed Matsumoto
and 129S/SvEv aggression et al. (2003)
Aggression Increased
toward
female
5-hydroxytryptamine 5-HT1B 9 129S2/SvPas Resident Increased Deleted 5-HT1B receptor Saudou et al.
(serotonin) receptor 1B aggression expression (1994)
Maternal Increased Brunner and
aggression Hen (1997)
Estrogen receptor-a ERa 10 Mix C57BL/6J Resident Suppressed Ogawa et al.
and 129 aggression (1998b)
Female Increased Ogawa et al.
aggression (1998a)
Fyn tryrosine kinase Fyn 10 Mix C57BL/6 and Isolation- Suppressed Miyakawa
CBA induced et al. (2001)
resident
aggression
(continued)
A. Takahashi et al.
Table 3 (continued)
backcross)
Nuclear receptor subfamily 2, Nr2e1 10 C57BL/6J ([ N6) Resident Increased Young et al.
group E, member 1 aggression (2002)
Adenosine A2a receptor A2AR 10 CD1 (N4) Isolation- Increased Ledent et al.
induced (1997)
resident
aggression
Tryptophan hydroxylase 2 Tph2 10 FVB/N (N7) Home-cage Increased Reduced brain 5-HT Alenina et al.
injury (2009)
Glutamate receptor, ionotropic, GluR-A 11 C57BL/6J (N5) Isolation- Suppressed No difference in brain 5- Vekovischeva
AMPA1 (a1) induced HT level et al. (2004)
aggression
aggression
5-hydroxytryptamine (serotonin) SERT 11 C57BL/6J (N8) Isolation- Suppressed Increased extracelluler Holmes et al.
transporter induced 5-HT (2002)
resident
aggression
Estrogen Receptor-b ERb 12 Mix C57BL/6J Resident Increaseda Ogawa et al.
and 129 aggression (1999)
Dopamine transporter DAT 13 Mix C57BL/6J Dyadic Increased Rodriguiz et al.
and 129SvJ encounter (2004)
aggression
5-hydroxytryptamine (serotonin) 5-HT1A 13 129S1/Sv Resident Suppressed Deleted 5-HT1A receptor Zhuang et al.
receptor 1A aggression expression (1999)
Catechol-O-methyltransferase 1 [±] COMT 16 Mix C57BL/6J Neutral cage Increased No difference in brain Gogos et al.
and 129SvJ aggression 5-HT level (1998)
(continued)
91
Table 3 (continued)
92
backcross)
Calcium/calmodulin-dependent protein aCaMKII 18 Mix C57BL/6, Defensive Increased Reduced 5-HT release in Chen et al.
kinase II alpha [±] 129/OU, aggression the dorsal raphe (1994)
BALB/c nucleus
Melanocortin-5 receptor MC5R 18 C57BL/6 ([N7) Neutral cage Suppressed Morgan et al.
aggression (2004)
Monoamine oxidase A MAOA X C3H/HeJ Resident Increased Increased brain 5-HT up Cases et al.
aggression to ninefold (1995)
Cage-mate injury Increased
Cyclic nucleotide–gated channel a2 Cnga2 X Mix C57BL/6J Resident Suppressed Mandiyan et al.
and 129SvEv aggression (2005)
Guanosine diphosphate (GDP) Gdi1 X C57BL/6J (N5) Isolation- Suppressed D’Adamo et al.
dissociation inhibitor 1 induced (2002)
resident
aggression
Androgen receptorc AR X C57BL/6 and Isolation- Suppressedb Raskin et al.
129SvEv induced (2009)
resident
aggression
b
[±] Data obtained from heterozygote of knockout mouse, a behavioral change only at the first encounter, behavior change after repeated exposure,
c
Nestine-Cre-LoxP conditional knockout mice that selectively lack AR expression in the nervous system
A. Takahashi et al.
Fig. 3 a Effects of social instigation on aggressive behavior by a resident mouse toward a male
intruder. Bars represent the mean frequency ±SEM (vertical lines) of attack bites under control
(light gray) and instigated (dark gray) conditions. Asterisks denote statistical significance from
control (double asterisks P \ 0.01). b Preferential reduction of instigated aggressive behavior by
the 5-HT1B agonist anpirtoline (left panel, filled circles) and CP-94,253 (right panel, filled
squares). Symbols represent the mean frequency of attack bites, expressed as a percentage of
vehicle (V) baseline, ±SEM. Light gray symbols represent non-instigated fighting and dark gray
symbols represent instigated levels of fighting. Asterisks denote significance from vehicle
baseline (P \ 0.05). Adapted from Fish et al. (1999) and de Almeida and Miczek (2002)
behavioral side effects including sedation, motor inactivity, stereotypic behavior or

reduced social interest (de Boer and Koolhaas 2005; Miczek et al. 1998b; Olivier
et al. 1995). However, some 5-HT1A agonists (i.e., alnespirone and S-15535)
reduced aggressive behavior specifically without changing the other non-aggres-
sive behaviors, at least in a wild-derived rat strain (de Boer et al. 1999, 2000;
de Boer and Koolhaas 2005). These compounds are suggested to have different
effects on 5-HT receptors on presynaptic and postsynaptic terminals (de Boer and
Koolhaas 2005), and thus it is possible that a subpopulation of 5-HT1A receptors
on which those compounds acts is involved in anti-aggressive effect specifically.
The 5-HT1B receptor agonists seem more specific in their anti-aggressive effect
than 5-HT1A agonists. In mice and rats, the systemic administration of 5-HT1B
agonists (e.g., CP-94253, CP-93129, CGS-12066B) reduces aggressive behavior
without sedation, or motor or sensory impairment (de Almeida et al. 2001a;
de Almeida and Miczek 2002; de Boer and Koolhaas 2005; Fish et al. 1999;
Miczek et al. 2002, 2004; Mos et al. 1993; Olivier et al. 1990; Olivier 2004, Fig. 3).
These effects were antagonized by 5-HT1B/1D antagonist GR-127935, further con-

firming that the anti-aggressive effects of these compounds were mediated by 5-HT1B
receptors (de Boer and Koolhaas 2005). However, there are no clinically approved
drugs that target 5-HT1B receptors. There is an amino acid difference in the binding
domain of 5-HT1B receptors of humans and rodents, and therefore the pharmaco-
logical sensitivity and specificity of 5-HT1B agonists in rodents may not compare
with those in humans (Olivier 2004).
The precise functions and sites of action for the anti-aggressive effects of
5-HT1A and 5-HT1B receptors still remain to be resolved. One site of inhibitory
action for 5-HT is the 5-HT1 autoreceptors that are localized at either the somata or
the synaptic terminal of serotonin neurons. Stimulation of 5-HT1 autoreceptors
inhibits serotonergic neural activities and therefore reduces 5-HT impulse flow and
release. On the other hand, 5-HT1 receptors are also localized at the postsynaptic
terminal as heteroreceptors at the projection sites of serotonin neurons. By con-
trast, the effect of agonists that act on 5-HT1 heteroreceptors can be interpreted as
increasing 5-HT neurotransmission.
Microdialysis studies have shown that both systemic and intra-raphé adminis-
tration of 5-HT1A and 5-HT1B agonists decreased extracellular levels of 5-HT in
the forebrain areas including striatum, hippocampus and frontal cortex (Adell et al.
2001; Bonvento et al. 1992; De Groote et al. 2003; Dekeyne et al. 2000; Gobert
et al. 1998; Hjorth and Sharp 1991; Johnson et al. 2001; Knobelman et al. 2000;
Kreiss and Lucki 1994; Sprouse and Aghajanian 1987). These data suggest that the
inhibition of 5-HT release by 5-HT1A and 5-HT1B agonists is mediated by the
5-HT1 autoreceptors. Note that these findings suggest that compounds which
decrease 5-HT neurotransmission concurrently reduce aggressive behavior, which
challenges the serotonin deficiency hypothesis of aggression.
On the other hand, the importance of the 5-HT1A and 5-HT1B heretoreceptors on
projection sites has been emphasized in studies using lesion or depletion of raphé
5-HT neurons either by systemic administration of the tryptophan hydroxylase
inhibitor PCPA or by intracerebral injection of the 5-HT neurotoxin, 5,7
-dihydroxytryptamine (5,7-DHT). Since these neurotoxic treatments destroy the
function of serotonergic neurons, one can observe the effect of 5-HT1 agonists on
postsynaptic receptors exclusively. If only somatodendritic and presynaptic auto-
receptors are the site of action of 5-HT1A and 5-HT1B agonists, PCPA or 5,7-DHT
treatment should eliminate the anti-aggressive effect of 5-HT1A and 5-HT1B agonists.
However, lesions or depletion of 5-HT neurons did not affect the anti-aggressive
effects of 5-HT1A and 5-HT1B agonists (de Almeida et al. 2001b; Miczek et al. 1998b)
or even had pro-aggressive effects (Sanchez and Hyttel 1994; Sijbesma et al. 1991).
While these data may suggest postsynaptic 5-HT1 receptors as critical sites of action,
it is important to note that these pharmacological depletions spared a subpopulation
of receptors.
The intracranial microinjection technique has been used to determine the crit-
ical brain regions underlying the anti-aggressive effects of 5-HT1A and 5-HT1B
receptor agonists (Table 4). 5-HT in the mammalian central nervous system
derives mainly from the dorsal and median raphé nuclei (DRN and MRN,
Table 4 Modulation of aggressive behaviors after local infusion of drugs targeting 5-HT receptors in selected brain regions
Brain region Type of aggression, species Target; drugs and doses Pharmacological effects References
DRN Resident aggression, male rats 5-HT1A: 8-OH-DPAT, ; aggressive behavior, with inactivity and decreased Mos et al.
1–10 lg social interaction (1993)
5-HT1A/5-HT1B:
Eltopronazine, 1–30 lg
(agonists)
Resident aggression, male rats 5-HT1A: Alnespirone, 25 lg ; aggression; no side effects Van der Vegt
(agonist) et al.
(2003)
Alcohol-escalated aggression, 5-HT1A: 8-OH-DPAT, 8-OH-DPAT and CP-94253: ; baseline aggression, Faccidomo
male mice 1.0 lg with reduced motor activity; no effects on alcohol- et al.
5-HT1B: CP-94253, 1.0 lg related aggression (2008)
(agonists)
Schedule-heightened aggression, 5-HT1B: CP-93129, ; escalated aggression; reduced walking behavior Bannai et al.
male mice 0.1–1.0 lg (agonist) (2007)
Alcohol-escalated aggression, 5-HT1B: CP-93129, ; baseline and alcohol-related aggression (0.5–1.0 lg); Faccidomo
male mice 0.1–1.0 lg (agonist) concomitant reduction in motor activity et al.
2012
Maternal aggression, rats 5-HT1A: 8-OH-DPAT, : maternal aggression (0.56 lg); no motor effects Veiga et al.
0.56 lg (agonist) DPAT-escalated aggression prevented by infusion (2011)
of CP-93129 (1.0 lg) into the orbitofrontal cortex
MRN Maternal aggression, rats 5-HT1A: 8-OH-DPAT, ; maternal aggression; no side effects De Almeida
0.2–2.0 lg (agonist) and
Lucion
(1997)
PAG Maternal aggression, rats Dorsal PAG ; maternal aggression (0.2–2.0 lg); no side effects de Almeida
5-HT1A: 8-OH-DPAT, and
0.2–2.0 lg (agonist) Lucion
(1997)
(continued)
95
Table 4 (continued)
96
Maternal aggression, rats Dorsal PAG a-methyl-5-HT maleate: ; maternal aggression; de Almeida
5-HT2A/2C: a-methyl-5- no motor effects et al.
HT maleate, 0.2–1.0 lg (2005)
(agonist)
5-HT2A/2C: ketanserin, Ketanserin: no effects on aggression,
1.0 lg (antagonist) decreased motor activity
Hypothalamic-stimulated PAG 8-OH-DPAT: ; defensive hissing (0.66–1.0 lg), effect Shaikh et al.
defensive aggression, cats 5-HT1A: 8-OH-DPATa, prevented by antagonist p-MPPI. No motor effect (1997)
0.016 ng–1.0 lg DOI: facilitation of defensive hissing (0.54 lg)
5-HT2C: DOIa, 3.57 ng–
0.54 lg (agonists)
Septal nuclei Maternal aggression, rats Medial septal nucleus : maternal aggression (0.2–0.5 lg); reduced activity de Almeida
5-HT1A: 8-OH-DPAT, only with highest dose (2.0 lg) and
0.2–2.0 lg (agonist) Lucion
(1997)
Maternal aggression, rats Medial septal nucleus No effects on aggressive or non-aggressive behaviors de Almeida
5-HT2A/2C: alpha-methyl- (agonist) et al.
5-HT maleate, Ketanserin: no effects on aggression, but decreased (2005)
0.2–1.0 lg (agonist) motor activity
5-HT2A/2C: ketanserin,
1.0 lg (antagonist)
Resident aggression, male mice Lateral septal nucleus 8-OH-DPAT: no behavioral effects Cologer-
(castrated males with hormonal 5-HT1A: 8-OH-DPATa, CGS: ; aggression with CGS, only in androgen- Clifford
replacement) 0.33 ng replacement condition; no side effects et al.
5-HT1B: CGS12066a, (1997)
18.0 ng
(agonists)
Alcohol-escalated aggression, Lateral septal nucleus No effects on alcohol-related or baseline aggression Faccidomo
male mice 5-HT1B: CP-94253, et al.
1.0 lg (agonist) (2008)
A. Takahashi et al.
(continued)
Table 4 (continued)
Hypothalamus Resident aggression, male mice Medial pre-optic area 8-OH-DPAT: ; aggression in androgen- and Cologer-
(castrated males with hormonal 5-HT1A: 8-OH-DPATa, estrogen-replacement conditions; no motor effects Clifford
replacement) 0.16 ng CGS: ; aggression in androgen- and estrogen- et al.
5-HT1B: CGS12066a, replacement conditions; no motor effects (1997)
9.0 ng (agonists)
Vasopressin-escalated aggression, Anterior hypothalamus 8-OH-DPAT: ; escalated aggression (0.33, 3.28 ng); Ferris et al.
male hamsters 5-HT1A: 8-OH-DPATa, no apparent motor effects (1999)
0.03–3.28 ng
5-HT1B: CGS12066a, 5-HT1B: no effects on aggression (trends toward
4.5–45.0 ng inhibition)
(agonists)
PAG-stimulated defensive Medial hypothalamus 8-OH-DPAT: ; defensive hissing (0.33–1.0 lg); Hassanain
aggression, cats 5-HT1A: 8-OH-DPATa, effect prevented by antagonist p-MPPI et al.
0.03–1.0 lg (2003)
5-HT2C: DOIa, DOI: facilitates defensive hissing (0.36 lg); effect
0.036–0.36 lg (agonists) prevented by antagonist LY-53,857
Amygdala Maternal aggression, rats Corticomedial amygdaloid ; maternal aggression (0.5–2.0 lg); no side effects De Almeida
nucleus and
5-HT1A: 8-OH-DPAT, Lucion
0.2–2.0 lg (agonist) (1997)
Prefrontal Resident aggression, male mice 5-HT1B: CP-94253, 1.0 lg No effects on aggressive or non-aggressive behaviors de Almeida
cortex (agonist) et al.
(2006)
Alcohol-escalated aggression, 5-HT1B: CP-94253, : alcohol-related aggression (1.0 lg); mild increases Faccidomo
male mice 0.5–1.0 lg (agonist) in activity; no effects on baseline aggression et al.
(2008)
(continued)
97
Table 4 (continued)
98
Alcohol-escalated aggression, 5-HT1B: CP-93129, ; baseline and alcohol-related aggression (0.1–1.0 lg); Faccidomo
male mice 0.1–1.0 lg (agonist) no motor effects et al.
2012
Orbitofrontal Resident aggression, male mice 5-HT1B: CP-94253, ; aggression (0.56–1.0 lg); no side effects. De Almeida
cortex 0.1–1.0 lg (agonist) CP-94253 effects prevented by 1B antagonist, et al.
GR-127935. (2006)
Maternal aggression, rats 5-HT1B: CP-93129, 1.0 lg CP-93129: ; maternal aggression (1.0 ug); no side Veiga et al.
5-HT1B: CP-94253, effects (2007)
0.56–1.0 lg (agonists) CP-94253: no behavioral effect
Alcohol-escalated aggression, 5-HT1B: CP-94253, No effects on alcohol-related or baseline levels of Faccidomo
male mice 0.5–1.0 lg (agonist) aggression et al.
(2008)
Social instigation-escalated 5-HT1A: 8-OH-DPAT, 8-OH-DPAT: ; instigated aggression (0.56–1.0 lg); no Centenaro
aggression, male mice 0.1–1.0 lg side effects et al.
5-HT1B: CP-93129, CP-93129: ; instigated aggression (only 0.1 lg); no (2008)
0.1–1.0 lg (agonists) side effects
DRN dorsal raphé nucleus, MRN median raphé nucleus, PAG periaqueductal gray area
a
doses calculated based on the following molecular weights (MW) of the compounds (as available at Sigma–Aldrich): 8-OH-DPAT hydrobromide,
MW = 328.29; CGS-12066 maleate salt, MW = 450.41; DOI hydrochloride, MW = 357.62
A. Takahashi et al.
respectively). Activation of 5-HT1A and 5-HT1B receptors in the DRN with

microinfusion of selective receptor agonists consistently reduced aggressive
behavior in rats and mice, but with concomitant reduction of motor activity and
social interactions (Bannai et al. 2007; Faccidomo et al. 2008; Mos et al. 1993;
Van Der Vegt et al. 2003). Infusion of a 5-HT1A agonist into the MRN also
reduced aggressive behavior of lactating female rats (de Almeida and Lucion
1997). In projection sites of 5-HT neurons, 5-HT1B receptors likely modulate 5-HT
release from synaptic terminals as autoreceptors, whereas both 5-HT1A and
5-HT1B receptors modulate postsynaptic neurons (Olivier et al. 1992). In most
studies, local activation of 5-HT1A and 5-HT1B in projection regions (e.g., medial
preoptic area, lateral septum, orbitofrontal cortex, anterior hypothalamus, medial
hypothalamus, periacqueductal gray) reduced aggressive behavior in various
procedures and species (see Table 4). Interestingly, under conditions that may
escalate aggression, such as consumption of moderate doses of alcohol or maternal
aggression, a 5-HT1A or 5-HT1B agonist further increased levels of aggressive
behavior when infused into the medial prefrontal cortex (Faccidomo et al. 2008) or
the medial septal area (de Almeida and Lucion 1997), respectively. Further studies
are required to delineate the mechanisms for such pro-aggressive effects.
4.1.2 Genetic Approach
The 5-HT1B receptor was the first molecule to be linked to aggression by using the
gene knockout technique. Male mice with disrupted 5-HT1B receptor expression
(Htr1b-/-) increased isolation-induced aggressive behavior compared to the wild-
type mice (Bouwknecht et al. 2001b; Saudou et al. 1994). However, the aggressive
behavior of the background strain of mice (129/Sv-ter) was very low, close to zero,
and thus the frequency of attack bites in Htr1b-/- was low and the latency to
initiate fighting was very long compared to other strains of mice. These mice
displayed behavioral disinhibition in other behavioral tests including hyperloco-
motor activity (Brunner et al. 1999; Ramboz et al. 1995), drug intake (Crabbe et al.
1996; Rocha et al. 1998), measures of anxiety-like behavior (Brunner et al. 1999;
Malleret et al. 1999) and autonomic hyperreactivity to novelty (Bouwknecht et al.
2001a). Females of Htr1b-/- increased their aggressive behavior during the
postpartum period (Brunner and Hen 1997). These results suggest a role for
5-HT1B receptors in the inhibition of some aggressive and impulsive behaviors.
The level of tissue 5-HT concentration in Htr1b-/- mice was lower than wild-type
in nucleus accumbens and spinal cord (Ase et al. 2000).
There are several polymorphisms in the human 5-HT1B receptor (HTR1B) gene.
The most frequently studied HTR1B polymorphism in relation to aggressive
phenotype is a single nucleotide polymorphism (SNP) G861C. This SNP had
significant linkage with aggressive behavior in antisocial alcoholism (Lappalainen
et al. 1998). Specifically, 861C, the SNP with lower 5-HT1B receptor expression
(Huang et al. 1999), was related to antisocial behavior. This SNP is actually a
synonymous polymorphism, in which no amino acid change occurred, and thus

this SNP seems not to have any function by itself. Thus, G861C polymorphism
may have a linkage with other functional polymorphisms that causes the expres-
sion change of 5-HT1B receptors. Jensen et al. (2009) showed that a SNP
(A1997G) in a non-coding regulatory region of the HTR1B gene is actually related
to the 5-HT1B expression pattern. This SNP is in the binding site for the microRNA
miR-96 that inhibits the translation or degrades the HTR1B mRNA. College
students homozygous for the A-allele, who have reduced 5-HT1B receptor
expression, reported a greater history of aggressive behaviors than G-allele indi-
viduals. Also, another SNP in the 50 UTR region of the HTR1B gene, A161T, was
found to correlate significantly with a history of aggression in subjects who
completed violent suicides (Zouk et al. 2007). Individuals with the T161 locus had
more lifetime aggressive behaviors, and again this polymorphism was linked to
reduced transcriptional activity of 5-HT1B receptors (Sun et al. 2002). However,
these associations between the HTR1B polymorphisms (G861C, G261T, or
C129T) and aggression and antisocial behavior are not seen in other studies
(Huang et al. 1999; Kranzler et al. 2002; New et al. 2001; Sinha et al. 2003; Van
den Berg et al. 2008). Based on the findings from pharmacological studies and
gene knockout and polymorphism studies, it is likely that the 5-HT1B receptor has
an important role in the inhibition of certain types of aggression.
In contrast to the strong pharmacological evidence implicating the 5-HT1A
receptor in aggression, no linkage has yet been reported between the 5-HT1A gene
polymorphism and aggression. Also, deletion of 5-HT1A gene (Htr1a-/-) reduced
aggressive behavior in mice (Zhuang et al. 1999), which is the complete opposite
of the findings with 5-HT1A agonists. However, there is evidence for a correlation
between 5-HT1A receptor expression and aggression. A human PET study found a
higher 5-HT1A receptor distribution in prefrontal cortex of subjects with higher
aggression scores based on a self-report questionnaire (Witte et al. 2009). Also,
mice selected for short latency to attack showed higher 5-HT1A receptor expres-
sion and receptor sensitivity (Korte et al. 1996; Van Der Vegt et al. 2001; van Riel
et al. 2002; Veenema et al. 2005), while rats selected for higher defensive reactions
showed reduced 5-HT1A receptor expression in several brain areas (Popova et al.
1998). It is possible that the polymorphisms which directly or indirectly affect
5-HT1A receptor transcription may be associated with either aggressive or
defensive responses.
4.2 5-HT2 Family
4.2.1 Pharmacological Approach
Atypical antipsychotic agents (e.g., risperidone) with significant antagonist action

at 5-HT2A receptors have been successfully used to reduce aggressive outbursts in
patients diagnosed with various neuropsychiatric disorders (Buckley et al. 1997;
Buitelaar et al. 2001; Czobor et al. 1995; De Deyn et al. 1999; Fava 1997; Keck
et al. 2000; Zarcone et al. 2001). However, some reports cast doubt on the routine
use of antipychotics (Swanson et al. 2008; Tyrer et al. 2008). In one study, the
placebo control group showed the greatest reduction in aggressively challenging
behavior compared to antipsychotic drug treatments in people with intellectual
disability (Tyrer et al. 2008). In animal models, selective 5-HT2A antagonists
(e.g., ketanserin, ritanserin and MDL 100907) reduce aggressive behaviors in a
behaviorally non-specific manner (Rodriguez-Arias et al. 1998; Sakaue et al. 2002;
Shih et al. 1999; White et al. 1991).
Activation of 5-HT2A and 5-HT2C receptors by DOI and other substituted
phenylisopropylamines also reduces aggressive behavior in several species
including flies, amphibians, mice and rats (Bonson et al. 1994; de Almeida and
Lucion 1994; Johnson et al. 2009; Muehlenkamp et al. 1995; Olivier et al. 1995;
Sanchez et al. 1993; Ten Eyck 2008). However, the anti-aggressive effects of
5-HT2 ligands are accompanied by sedative effects in the same dose range. Local
infusion of a 5-HT2A/2C agonist into the PAG reduces maternal aggression in rats
(de Almeida et al. 2005), whereas microinjections into the medial hypothalamus
and into the PAG increased defensive aggression in cats (Hassanain et al. 2003;
Shaikh et al. 1997, see Table 4). This latter effect is likely linked to the role of
5-HT2A/2C receptors in anxiety-like behavior (Lucki and Wieland 1990; Nogueira
and Graeff 1995). The development of a more selectively acting pharmacological
tools will allow a more adequate differentiation of 5-HT2 receptor subtypes, and
promises to dissociate the anti-aggressive and sedative effects.
4.2.2 Genetic Approach
Platelet 5-HT2A receptor binding is increased in patients with personality disorders

and in a psychiatric population with greater lifetime aggression scores (Coccaro et al.
1997; McBride et al. 1994). Positive correlation between impulsive physical
aggression and 5-HT2A receptor expressions in orbitofrontal cortex has been reported
using PET (Rosell et al. 2010) and a similar finding was reported in a postmortem
study of suicide victims (Mann et al. 1986; Oquendo et al. 2006). However, another
PET study reported opposite changes in 5-HT2A receptor expression (Meyer et al.
2008). It is possible that polymorphisms that affect the level of expression of 5-HT2A
receptors can be associated with self-directed aggression. In some samples, a sig-
nificant linkage was found between polymorphisms in the 5-HT2A receptor (HTR2A)
gene, T102C, A1438G and His452Tyr, and aggressive-impulsive trait or adolescent-
onset antisocial behavior in humans (Assal et al. 2004; Bjork et al. 2002; Burt
and Mikolaiewski 2008; Nomura et al. 2006), but others have reported no such
link between aggression and HTR2A polymorphisms (Khait et al. 2005; Van den
Berg tet al. 2008). Again, the successful pharmacotherapeutic management of
aggressive patients using compounds with affinity for 5-HT2A receptors would
suggest that violence-prone individuals may be characterized by distinctive HTR2A
polymorphisms.
Linkage of a minor polymorphism in the 5-HT2B receptor (HTR2B) gene with

antisocial behavior was reported recently. The HTR2B Q20* allele, which is found
exclusively in the Finnish population, contains a stop codon in HTR2B and
blocked the expression of the 5-HT2B receptor. Males with the Q20* allele are
prone to show more impulsive violence or impulsive suicidal behavior interacting
strongly with alcohol than control (Bevilacqua et al. 2010). Male mice with dis-
rupted 5-HT2B receptors (Ht2b-/-) also showed increased impulsive behavior.
Interestingly, these mice had three times higher testosterone level in the cere-
brospinal fluid, which is also consisted with the Q20* individuals among humans.
5 Serotonin Transporter (5-HTT)
5.1 Pharmacological Approach
Aggressive behavior in humans and animals can be reduced and prevented by

blocking serotonin transporter molecules, which in turn presumably increases 5-HT
levels in the brain. Clinically, chronic treatment (i.e., [3 weeks) with selective
serotonin reuptake inhibitors (SSRIs) has been shown to reduce aggressive outbursts
and violent behavior in psychiatric patients (Barkan et al. 2006; Blader 2006; Bond
2005; Coccaro and Kavoussi 1997; New et al. 2004; Reist et al. 2003; Walsh and
Dinan 2001). However, SSRIs have occasionally been reported to increase the
incidence of aggressive and suicidal behavior, and the causes of these paradoxical
effects remain unknown (Spigset 1999; Troisi et al. 1995).
Both acute and chronic SSRI treatment can produce a dose-dependent reduction
in aggressive behavior in animal models (Carrillo et al. 2009; Delville et al. 1996;
Olivier et al. 1989; Pinna et al. 2003). When given acutely to rodents and non-
human primates, several SSRIs including fluoxetine, fluvoxamine and sertraline
reduced aggression in various contexts (Abbadie et al. 1994; Carrillo et al. 2009;
Cutler et al. 1997; Delville et al. 1996; Fairbanks et al. 2001; Ferris et al. 1997;
Fuller 1996; Ho et al. 2001; Sanchez and Meier 1997). Mice treated with the SSRI
citalopram daily for three weeks no longer showed increased levels of aggression
after consuming a moderate dose of alcohol, and baseline levels of aggression also
showed modest reductions (Caldwell and Miczek 2008). On the other hand, the
very low levels of agonistic behavior typically seen in laboratory rats may be
restored to species-typical levels by chronic SSRI treatment (Mitchell et al. 1991;
Mitchell 2005; Mitchell and Redfern 1992, 1997). Thus, SSRIs are more likely to
exhibit anti-aggressive effects in conditions of escalated agonistic behavior, such
as fighting enhanced by alcohol (Caldwell and Miczek 2008).
Mechanistically, extracellular levels of 5-HT in the prefrontal cortex of rats are
elevated by both acute and chronic administration of citalopram or the more potent
isomer escitalopram, implying that SSRIs’ effects on aggression and other mood
disorders is a result of increased cortical 5-HT (Ceglia et al. 2004). However, the
anti-aggressive effects of fluoxetine, another SSRI, may be mediated primarily by

acting on neurosteroids and GABA transmission, and only secondarily by
elevating 5-HT (Pinna et al. 2003, 2006). In addition, long-term SSRI treatment
probably recruits both pre- and post-synaptic mechanisms and neuroplastic events
that may also have therapeutic actions (Benmansour et al. 1999; Blier and de
Montigny 1998; Ceglia et al. 2004; Pineyro et al. 1994).
5.2 Genetic Approach
The serotonin-transporter-gene-linked polymorphic region (5-HTTLPR) is a varia-

tion in the length of the 50 -flanking transcriptional control region (promoter) of the
5-HTT gene that affects the gene’s transcriptional activity. The variation has been
found in humans (Heils et al. 1996) and also in great apes and rhesus monkeys (Lesch
et al. 1997). The short-length (s) allele reduces 5-HTT expression in vitro compared
to the long length homozygote (l/l) and lowers the prolactin response to clomipra-
mine in humans, indicating reduced 5-HT function (Heils et al. 1995; Lesch et al.
1996; Whale et al. 2000). Studies in humans have found that males and females with
one or two copies of the s allele (s/s, s/l) exhibit more hostility, aggression, anxiety
and depression and lower agreeableness than l/l homozygotes (Lesch and Mersch-
dorf 2000). ‘‘Type 2’’ alcoholics who displayed high impulsivity and antisocial
behaviors showed a higher frequency of the s allele than either ‘‘Type 1’’ alcoholics
without antisocial behavior or healthy controls (Hallikainen et al. 1999). As in
humans, rhesus monkeys with the s allele were more aggressive than l/l individuals
(Jarrell et al. 2008; Lesch and Merschdorf 2000).
5-HTT polymorphism also exhibits a genotype-environment interaction. Rhesus
monkeys with the s allele that were peer-reared without their mothers had lower
CSF levels of 5-hydroxyindoleacetic acid (5HIAA) than l/l individuals, but this
difference was not present in maternally reared monkeys (Bennett et al. 2002).
Peer-reared male monkeys also showed both altered CSF 5HIAA levels and an
increase in aggression-related behavior (Higley et al. 1991; Kraemer et al. 1989).
Humans carrying the s allele exhibited more suicidal ideations or attempts in
response to stressful life events than l/l homozygotes, but did not differ in less
stressful situations (Caspi et al. 2003). Therefore, it is possible that animals with
the s allele are more vulnerable to stressful challenges, and subsequently escalate
their aggressive behaviors toward others and themselves. However, the presence of
the s allele did not affect human aggression consistently across sexes (Cadoret
et al. 2003) or cultures (Baca-Garcia et al. 2004).
Deletion of the 5-HTT gene in mice (Slc6a4) produced seemingly contrary
results. Wild-type C57BL/6J mice attacked more and showed shorter latencies to
start fighting in the resident-intruder test than homozygote and heterozygote 5-HTT
knockouts (Holmes et al. 2002, Table 3). 5-HTT knockout mice have higher
extracellular 5-HT concentrations and lower 5-HT uptake in the forebrain compared
to wild-type (Mathews 2004). Knockout mice with reduced or absent 5-HTT
function also exhibited more than 50 phenotypic changes, including alterations of

behavioral, physiological, morphological and sensory functions (Murphy and Lesch
2008), and those pleiotropic changes in various other phenotypes may contribute to
the reduction of aggression in these mice. Comparable findings on 5-HTT and
aggression have also been reported in rats; 5-HTT knockout rats on a Wistar/Crl
background showed reduced offensive behaviors and longer attack latencies
compared to wild-types (Homberg 2007). Thus genetic ablation of 5-HTT has
consistently been shown to reduce aggressive behaviors in rodents.
6 Monoamine Oxidase A
6.1 Pharmacological Approach
Inhibition of MAOA leads to a reduction in the oxidative metabolism of mono-

amines, presumably making 5-HT and other monoamines more available in the
brain. Although the importance of MAO inhibitors as antidepressants was soon
recognized, only a few studies have evaluated the effects of MAO inhibitors on
aggression in preclinical models (Miczek 1987). Non-selective inhibitors of both
MAOA and MAOB (e.g., phenelzine, isocarboxazid, tranylcypromine) produce
acute anti-aggressive effects only at doses that also produce sedation and alter
other non-aggressive behaviors (DaVanzo et al. 1966; Sofia 1969; Valzelli et al.
1967; Welch and Welch 1968). Non-selective MAO inhibitors or selective MAOB
inhibitors can be clinically useful for treating patients with personality disorders
who exhibit suicidal tendencies and impulsive aggression, but the drugs also have
a profile of undesirable side effects (Hollander 1999; Raj 2004).
6.2 Genetic Approach
The gene for monoamine oxidase A (MAOA) was the first to be identified as a
possible determinant for pathological aggression in humans, and it has remained
the focus of most genetic and epigenetic studies. A Dutch family with a syndrome
of borderline mental retardation and dysregulated impulsive aggression was
identified by Brunner et al. (1993b). Aggressive outbursts were seen in all affected
males in the kindred, and some exhibited aberrant sexual behavior, attempted
murder and arson. Linkage and sequence analyses identified one missense muta-
tion in the MAOA gene on the X chromosome, so that MAOA function was
completely disturbed; the affected males had more serotonin and lower levels of
norepinephrine, dopamine and 5-HT metabolites in their urine (Brunner et al.
1993a). MAOA is also an important factor in animal aggression. Aggressive
behaviors (skin wounds among cage mates and briefer attack latency in the resi-
dent-intruder test) escalated in male mice with a disrupted MAOA gene on either
Fig. 4 Means on the composite index of antisocial behavior as a function of MAOA activity and
a childhood history of maltreatment. MAOA activity is the gene expression level associated with
allelic variants of the functional promoter polymorphism, grouped into low and high activity;
childhood maltreatment is grouped into three categories of increasing severity. The antisocial
behavior composite is standardized (z score) to a M = 0 and SD = 1; group differences are
interpretable in SD unit differences (d). Reprinted with permission from Caspi et al. (2002)
C3H/He or 129 Sv background compared to wild-type mice (Cases et al. 1995;

Scott et al. 2008). Mice with an MAOA deficiency also showed a large increase in
5-HT and norepinephrine and a slight elevation in dopamine in the brain and liver
(Cases et al. 1995; Kim et al. 1997, Table 3); the behavioral changes in the
MAOA-deficient mice are probably caused by this change in 5-HT function.
Escalated aggression in MAOA mutant mice was blocked by 5-HT2A receptor
antagonists such as ketanserin and MDL100907 (Shih et al. 1999). Some of the
behavioral and brain structural abnormalities in the MAOA-deficient mice were
ameliorated when 5-HT was depleted by PCPA early in the developmental process
(Cases et al. 1995, 1996).
MAOA expression can also be affected by variable-number tandem repeat
(VNTR) polymorphism on the upstream region of the MAOA gene. The number of
tandem repeats determines MAOA levels: alleles with 3.5 or 4 repeats have 2–10
times higher transcription than 3–5 repeat alleles in vitro (Denney et al. 1999;
Sabol et al. 1998). A powerful interaction between MAOA genotype and envi-
ronment on aggressive behavior has been reported (Caspi et al. 2002, Fig. 4).
Individuals with low MAOA expression (MAOA-L) polymorphisms who suffered
from abuse, neglect or traumatic life events in the first 15 years of their lives
were more likely to have a history of adolescent conduct disorder, violence and
criminal arrests, and also scored higher on aggressive disposition in a self-report
questionnaire compared to MAOA-L individuals without abuse or trauma, or
individuals with higher MAOA expression (MAOA-H) (Caspi et al. 2002; Foley
et al. 2004; Frazzetto et al. 2007; Kim-Cohen et al. 2006; Weder et al. 2009;
Widom and Brzustowicz 2006). The effect of MAOA genotype disappeared if all
subjects were lumped together regardless of rearing environment (Fresan et al.
2007), and MAOA-H individuals sometimes reported higher aggression in inter-
views and on questionnaires (Manuck et al. 2000, 2002). The finding that indi-
viduals with the MAOA-L allele are vulnerable to environmental factors, showing
a high propensity to engage in aggressive behaviors when in a stressful environ-
ment, is consistent in males but not females (Sjoberg et al. 2007). Rhesus monkeys
have a similar repeat length variation polymorphism in the MAOA gene
(rhMAOA-LPR) which is also linked to aggression. Monkeys with a low-activity
allele who were reared by their mother showed more aggressive behavior and
attained higher dominance rank than monkeys with the low-activity allele who were
peer-reared separately from their parents (Newman et al. 2005). Higley and Suomi
(1986) attributed this inhibition of aggression to increased fear and anxiety in peer-
reared monkeys.
Substantial differences in both volume and activity of limbic system and
neocortical areas between MAOA-L and MAOA-H individuals have been found in
neuroimaging studies [see Buckholtz and Meyer-Lindenberg (2008) for a review].
In healthy male human volunteers with the MAOA-L variant, fMRI showed
smaller limbic and orbitofrontal volumes and higher activity in amygdala and
hippocampus during aversive recall (Meyer-Lindenberg et al. 2006), which may be
related to violent behavior. Lower MAOA activity in cortical and subcortical brain
areas is associated with elevated aggression as measured by a self-report ques-
tionnaire, with no effect of MAOA polymorphism (Alia-Klein et al. 2008). These
data show that the MAOA activity is one determinant of the propensity to
aggression. The interaction between MAOA polymorphism and stressful social
experiences is especially critical, since it can escalate aggression and can also
change relevant brain structures.
7 Modulation of Serotonergic Activity by Other Systems
The 5-HT neurons in the raphé nuclei are modulated by other amines, acids,
peptides and steroids (Adell et al. 2002). Several efforts have been undertaken to
uncover the nature of the neural systems that modulate 5-HT neurons to promote
escalated aggressive behaviors. Here we will focus briefly on inhibitory and
excitatory neurotransmitters and some neuropeptides in terms of their interaction
with 5-HT system. Especially, dorsal raphé nucleus (DRN), the largest 5-HT
nucleus in the brain, will be highlighted because of its important role for
aggression (Bannai et al. 2007; Faccidomo et al. 2008; Jacobs and Cohen 1976;
Koprowska and Romaniuk 1997; Mos et al. 1993; Sijbesma et al. 1991; Van Der
Vegt et al. 2003; Vergnes et al. 1986).
7.1 Excitatory and Inhibitory Amino acids
7.1.1 c-Aminobutyric Acid
c-Aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the

brain. Large numbers of GABA interneurons and distal GABAergic afferents are
found in the DRN (Belin et al. 1983; Gervasoni et al. 2000; Nanopoulos et al.
1982; Wang et al. 1992), and both GABAA and GABAB receptors are expressed in
the DRN (Bowery et al. 1987). In vivo electrophysiology studies have shown that
the activation of either GABAA or GABAB receptors can inhibit the cell firing of
serotonergic neurons (Colmers and Williams 1988; Gallager and Aghajanian 1976;
Innis and Aghajanian 1987; Judge et al. 2004). On the other hand, in vivo
microdialysis studies have shown that the 5-HT release may be differentially
modulated by GABAA receptors and GABAB receptors depending on the
projection sites (Tao et al. 1996). In addition, microinjection of a GABAB receptor
agonist into the DRN can induce either increases or decreases of 5-HT neuronal
activity (Abellan et al. 2000; Takahashi et al. 2010b; Tao et al. 1996).
Systemic administrations of positive modulators of GABAA receptors such as
benzodiazepines, barbiturates and neurosteroids exert dose-dependent biphasic
effects on aggressive behaviors, from escalation to inhibition (Miczek et al. 2003).
Low to moderate doses of GABAA positive modulators escalate aggression in
mice, rats, pigs and monkeys (Arnone and Dantzer 1980; Cole and Wolf 1970;
Ferrari et al. 1997; Fish et al. 2001; Gourley et al. 2005; Miczek 1974; Miczek and
O’Donnell 1980; Mos and Olivier 1989; Rodgers and Waters 1985; Weerts and
Miczek 1996). On the other hand, pharmacological activation of GABAA receptors
in the DRN inhibits aggressive behaviors in rats (Van Der Vegt et al. 2003) but has
no effect in mice (Takahashi et al. 2010a). However, we found an interaction
between alcohol and GABAA receptor in the DRN on aggressive behavior. Only
under the influence of alcohol, local administration of GABAA receptor agonist
muscimol in the DRN also heightened aggressive behaviors (Takahashi et al.
2010a). Therefore, GABAA modulation of serotonergic neurons may connect to
drug-induced escalated aggression (e.g., alcohol, benzodiazepines) but not to
species-typical aggression.
GABAB receptors in the DRN are also involved in the escalation of aggression
in mice. Pharmacological activation of GABAB receptors in the DRN escalates
intermale aggression (Takahashi et al. 2010b). The pro-aggressive effect of
GABAB agonist baclofen was blocked by selective agonists of GABAB receptors
(phaclofen, CGP54626). Additionally, the systemic administration of baclofen also
escalates aggression in male mice. Therefore, both GABAA and GABAB receptors
are involved in escalated aggression via different mechanism to modulate different
types of aggression. In vivo microdialysis showed that GABAB activation in the
DRN increased extracellular 5-HT level in the medial prefrontal cortex (Takahashi
et al. 2010b, Fig. 5). This result suggests that the phasic activation of 5-HT system
may be able to promote certain types of escalated aggressive behaviors in mice.
(a) 5-HT in mPFC

200 *
% Baseline
150
100
Baseline Saline Baclofen

50
-110 -90 -70 -50 -30 -10 0 10 30 50 70 0 10 30 50 70 90 110 130 min
(b) (c)
60
Saline
Bregma * Baclofen
+2.10mm
Bregma
4.36mm
*
Attack bites
40
+1.94mm
4.60mm
20
+1.70mm 4.84mm
0
PFC DRN 10 40 100 min
Fig. 5 Extracellular 5-HT concentration in the medial prefrontal cortex (mPFC) of mice after
GABAB receptor activation in the dorsal raphe nucleus (DRN). a Baclofen microinjected into the
DRN increased the 5-HT level in the mPFC whereas saline injection did not change the 5-HT
level. Twenty-minute samples were collected: five samples for baseline, three samples after saline
injection and six samples after baclofen (0.06 nmol) injection. Data are means ± SEM expressed
as percentage of baseline (n = 7); aisterisks = p \ 0.05 compared to baseline. b Histological
representation of probe placement in the mPFC for the microdialysis (vertical bars: 2 mm probe
membrane) and drug injection site in the DRN (circles). c The effect of 0.06 nmol baclofen
(black bars) or saline (gray bars) on attack bites at different post-injection intervals (10, 40 and
100 min, corresponding to fractions 9, 11 and 14 in the microdialysis, respectively). Escalated
attack bites were observed both 10 and 40 min after the intra-DRN baclofen injection. Values are
means ±SEM; asterisks = p \ 0.05compared to corresponding vehicle control. From Takahashi
et al. (2010b)
7.1.2 Glutamate
The DRN receives glutamate input by the descending projections from the lateral
habenula, periaqueductal gray, lateral hypothalamus, interpeduncular nucleus
and medial prefrontal cortex (Aghajanian and Wang 1977; Behzadi et al. 1990;
Kalen et al. 1986; Maciewicz et al. 1981). Both the N-metyl-D-aspartate (NMDA)
and a-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate/kainate (AMPA/kainate),
ionotropic receptors for glutamate, are localized on serotonergic neurons and

increase the 5-HT release in the DRN and its projection areas (Celada et al. 2001;
Pallotta et al. 1998; Tao et al. 1996; Tao and Auerbach 2000; Vandermaelen et al.
1986). Metabotropic receptors of glutamate (mGluRs) are also found in the DRN,
but their function on serotonergic neurotransmission and aggressive behavior
needs to be characterized by further investigations. Systemic administrations of
classic antagonists of NMDA receptors, including phencyclidine (PCP) and
dizocilpine (MK-801), can increase aggressive behavior (Burkhalter and Balster
1979; Krsiak 1974; McAllister 1990; Musty and Consroe 1982; Rewerski et al.
1971; Wilmot et al. 1987), while other studies find that these compounds are
suppressive and sedative due to their marked side effects (Belozertseva and Bes-
palov 1999; Lang et al. 1995; Miczek and Haney 1994; Tyler and Miczek 1982).
Memantine and neramaxane block the channel of NMDA receptors with different
characteristics and promote alcohol-heightened aggression in resident mice con-
fronting an intruder. Anatomically discrete analysis is required to identify the sites
of action for NMDA receptors that produce escalated aggressive behavior. The
5-HT system is one of the candidates, especially the descending glutamatergic
projection from the medial prefrontal cortex (mPFC) to the DRN will be inter-
esting to investigate. The prefrontal cortex (PFC) is implicated in the emotion
regulation including aggression (Davidson et al. 2000; Miczek et al. 2007). This
mPFC-DRN glutamatergic projection is involved in the controllability or emotion
regulation (Amat et al. 2005) and it is possible that this pathway is also involved in
the regulation of aggression.
7.2 Neuropeptides
7.2.1 Corticotropin-Releasing Factor
The DRN is innervated by corticotropin-releasing factor (CRF) immunoreactive

fibers, and is the site for both subtypes of CRF receptors, CRF1 and CRF2
(Chalmers et al. 1995; Potter et al. 1994; Swanson et al. 1983). CRF, CRF
receptors and other peptides of the CRF family (i.e., urocortins), play key mod-
ulatory roles on DRN 5-HT neurons (Valentino and Commons 2005). Electro-
physiological and microdialysis studies consistently report that i.c.v. or intra-DRN
microinjections of CRF, or drugs targeting CRF receptors, exert potent modulatory
control over 5-HT neural firing (Kirby et al. 2000; Lowry et al. 2000), and 5-HT
output to limbic, striatal and prefrontal cortical regions (Amat et al. 2004, 2005;
Forster et al. 2008; Lukkes et al. 2008; Meloni et al. 2008; Price et al. 1998; Price
and Lucki 2001).
CRF, CRF1 and CRF2 receptors are implicated in maternal and inter-male
aggression in mice and hamsters (D’Anna et al. 2005; Farrokhi et al. 2004;
Gammie et al. 2004, 2005, 2007; Gammie and Stevenson 2006). In rats, i.c.v. or
intra-amygdala infusions of low doses of CRF itself facilitated or induced
pro-aggressive effects (Elkabir et al. 1990; Tazi et al. 1987). Under conditions
of escalated aggression promoted by moderate doses of alcohol in male
mice, CRF1 receptors are a promising target for pharmacological intervention.
Systemic administration of the antagonists of CRF1 receptors reduce alcohol-
heightened aggression, but also reduce baseline levels of aggressive behavior
(Quadros et al. 2009b). When locally administered into the DRN, CRF1 antagonists
(e.g., CP-154526 or MTIP) prevent the escalated levels of aggression observed
after consumption of alcohol, with no detectable side effects on other behaviors.
Remarkably, such anti-aggressive effects of CRF1 antagonists can be abolished with
the infusion of 8-OH-DPAT into the DRN, which transiently slows 5-HT impulse
flow. On the other hand, microinfusion of a CRF2 antagonist (e.g., Astressin-2B) into
the DRN escalates aggressive behavior (Quadros et al. 2009a).
The modulation of aggressive behaviors by CRF systems depends on the spe-
cies such as mice or rats, and type of aggression (species-typical, maternal or
escalated aggression). Initial evidence suggests the 5-HT cells in the DRN as one
of the critical sites for such modulation in escalated aggression that is promoted by
alcohol, with presumably opposing roles for CRF1 and CRF2 receptors.
7.2.2 Neuropeptide Y
Neuropeptide Y (NPY) controls primarily food intake, energy balance and meta-
bolic regulation (Herzog 2003). In addition to the critical role for energy
homeostasis, NPY is also implicated in aggressive behavior (Emeson and Mor-
abito 2005). Male mice with deleted expression of Y1 receptor (Y1-/-) showed
obesity and reduced energy homeostasis (Kushi et al. 1998). These animals also
exhibited increased aggressive behaviors in the resident-intruder test (Karl et al.
2004). However, escalated aggression was observed only in the home-cage but not
in the novel environment in Y1-/- mice. Both NPY and its receptors can be found
in the DRN (Martel et al. 1990; O’Donohue et al. 1985; Saria et al. 1984), and
NPY inhibits both hyperpolarizing and depolarizing slow synaptic potentials in the
DRN (Kombian and Colmers 1992). Y1-/- mice showed reduced expression of
tryptophan hydroxylase mRNA in the raphé nuclei, and also 5-HT1A agonists
could suppress the heightened aggression of Y1-/- mice (Karl et al. 2004). These
results suggest that the disruption of Y1 receptor expression specifically increases
territorial aggression by altering 5-HT function.
7.2.3 Arginine Vasopressin
Arginine vasopressin (AVP) is a neuropeptide that modulates a variety of social

behaviors including pair-bonding, social recognition, maternal behavior, and
aggression (Albers and Bamshad 1998; Coccaro et al. 1998; Ferris et al. 1992;
Goodson 2008; Koolhaas et al. 1990; Neumann et al. 2010; Winslow and Insel
1993). Selective antagonists of vasopressin V1a receptors (SRX251, [d(CH2)
5Tyr(Me)AVP]) inhibited inter-male aggression (Ferris et al. 2006; Ferris and
Potegal 1988), suggesting the involvement of V1a receptors in aggressive
behaviors. Evidence points to a critical role of the interaction between AVP and
5-HT in certain types of aggressive behaviors. In humans, a positive correlation has
been observed between AVP concentrations in the CSF and the life history of
aggression, a composite measure of trait aggression. Also, there was a positive
correlation between AVP concentrations and prolactin responses to a challenge
with d-fenfluramine (Coccaro et al. 1998). This result indicates that individuals that
have higher aggression ratings tend to have a high AVP concentration in the CSF and
a hyporesponsive 5-HT system. Neuronal interactions between AVP containing
neurons and 5-HT neurons are found in the anterior hypothalamus (Ferris et al. 1997,
1999), and this AVP-5-HT link is implicated in aggression. Microinjection of AVP
into the anterior or lateral hypothalamus increased aggressive behavior in hamsters,
and systemic fluoxetine, a 5-HTT inhibitor, blocked the pro-aggressive effect of
AVP (Delville et al. 1996; Ferris et al. 1997) Therefore, 5-HT may have an inhibitory
effect on the AVP-heightened aggression. By contrast, mice with disrupted Ca2+
channel expression (Cav2-/-) showed escalated level of aggressive behavior and
also higher AVP concentration in the CSF. However, these animals showed over-
activation of the dorsal 5-HT neurons and increased 5-HT concentration in the
hypothalamus (Kim et al. 2009a). Further investigation will uncover how AVP and 5-
HT interact and whether there are specific types of aggression that require the activity
of either 5-HT or AVP systems independently.
7.2.4 Oxytocin
Oxytocin, which has a structure similar to vasopressin, differing in only two

amino acid positions, also plays an important role in the control of social
behaviors like vasopressin. Administration of the agonists of oxytocin receptor
increased aggressive behavior in lactating females (Bosch et al. 2005; Ferris
et al. 1992) but reduced territorial aggression in males (Harmon et al. 2002).
Mice in which the gene for oxytocin and its receptor were knocked out, showed
enhanced aggressive behaviors in both males and lactating females (Ragnauth
et al. 2005; Takayanagi et al. 2005; Winslow et al. 2000) but see (DeVries
et al. 1997). In humans, the level of oxytocin in the CSF is inversely correlated
with the life history of aggression (Lee et al. 2009). Oxytocin and its receptor
are expressed in the DRN, and the effect of local infusion of oxytocin can
facilitate passive avoidance in rats (Kovács et al. 1979). However, the exact
interactions between the oxytocin and serotonergic systems in their modulatory
effects on aggressive behavior remain to be discovered.
7.3 Other Molecules that Directly or Indirectly Affect 5-HT

Pathways and Aggression
7.3.1 Tryptophan Hydroxylase
Variations in Tryptophan hydroxylase (TPH) activity can directly affect 5-HT

neurotransmission. Polymorphisms in the TPH1 or TPH2 gene change the enzyme
activity and 5-HT level (Jonsson et al. 1997) for TPH1 gene; (Cichon et al. 2008;
Lin et al. 2007; Zhang et al. 2004, 2005) for TPH2 gene. Two SNPs in tight
linkage disequilibrium, A218C and A779C, located in the intron of TPH1 gene
[which are often referred to as U (upper) and L (lower) allele], have been shown to
be associated with state and trait anger as assessed by interview and self-report
questionnaires (Manuck et al. 1999; Rujescu et al. 2002). Lower CSF 5-HIAA
levels have been observed in healthy men, but not women, with the U allele
(Jonsson et al. 1997), whereas the lowest CSF 5-HIAA was observed in LL carriers
diagnosed with antisocial alcoholism (Nielsen et al. 1994). Apparently, both U and
L alleles may relate to different types of aggression. Hennig et al. (2005) per-
formed a factor analysis on the Buss-Durkee Hostility Inventory (BDHI) and found
two factors, named as ‘‘neurotic hostility’’ and ‘‘aggressive hostility’’. Neurotic
hostility is characterized by irritability, resentment, verbal hostility and guilt,
whereas aggressive hostility represents a more ‘‘cold’’ aggression including assault
and negativism but low guilt. Individuals with the UU allele showed higher
aggressive hostility but slightly lower neurotic hostility (Hennig et al. 2005),
whereas LL homozygote showed higher level of impulsivity in the measurements
which focused more on neurotic hostility (New et al. 1998). This may explain
controversial results about the association between the polymorphisms and
aggression; depending on which dimensions of aggressive behavior are measured,
the association pattern may change.
In mouse models, the focus has been on the Tph2 gene, because Tph2 is
preferentially expressed in the brain, whereas Tph1 is more widely distributed
throughout the body (Walther et al. 2003). Mice with an R439H point mutation
in the Tph2 gene have strongly reduced 5-HTP, 5-HT and its acidic metabolite
levels in (Beaulieu et al. 2008). This mutant mouse showed increased aggres-
sive behaviors in the social interaction test in a neutral arena where typically
very low levels of aggression are seen (Beaulieu et al. 2008). In contrast,
another polymorphism in Tph2, C1473G, which inhibits Tph2 activity in the
midbrain, reduced the intensity of aggression in mice (Osipova et al. 2009).
The mouse data on the R439H mutation in the Tph2 gene may be relevant to a
large association study in children with attention deficit/hyperactivity disorder
(ADHD), which identified a TPH2 gene polymorphism associated with
impulsivity (Oades et al. 2008).
7.3.2 Pet-1
Pet-1 (also known as Fev), one of the transcription factors, is specifically

expressed in the serotonergic raphé neurons, and has a critical role in 5-HT neural
development. Deletion of Pet-1 expression reduced 5-HT levels in the forebrain,
and also depleted expression of TPH, 5-HTT, and the vesicular monoamine
transporter 2 (Vmat2). In the resident-intruder test, Pet-1 knockout mice (Pet-1-/-)
engaged in a higher frequency and intensity of attacks toward a conspecific male
(Hendricks et al. 2003). These increases in aggressive behavior in Pet-1-/- mice are
embedded in broad behavioral disruptions that also extended to maternal and
anxiety-like behaviors (Hendricks et al. 2003; Lerch-Haner et al. 2008), and it is
possible that those other behavioral changes promote indirectly aggressive
behaviors. Since Pet-1 expresses specifically in the 5-HT neurons, the promoter or
enhancer regions of this gene are useful for gene manipulation of 5-HT neurons
(Scott et al. 2005).
7.3.3 Brain-Derived Neurotrophic Factor
Brain-derived neurotrophic factor (BDNF) has several important roles in neuronal

survival, development, differentiation and plasticity. Mice with decreased BDNF
expression including knockout (BDNF+/-) and conditional knockout (BDNF2L/
1LNes–Cre
and BDNF2L/2LCk-Cre) all showed increased inter-male aggression (Chan
et al. 2006; Lyons et al. 1999). Higher extracellular levels of 5-HT in the hippo-
campus was observed in BDNF+/- mice compared to wild-type (Deltheil et al.
2008). However, fluoxetine could reduce heightened aggressive behavior in
BDNF+/- mice (Lyons et al. 1999). All mutants changed 5-HT2A receptor
expression, however BDNF+/- showed increased 5-HT2A expression in the lateral
frontal cortex and hypothalamus (Lyons et al. 1999) whereas BDNF2L/1LNes-Cre
and BDNF2L/2LCk-Cre exhibit reduced 5-HT2A receptor expression in the prefrontal
cortex (Chan et al. 2006; Rios et al. 2006). A SNP in the BDNF gene, Val66Met,
has attracted considerable interest because of its association with mood disorders
and hippocampal function in humans (Egan et al. 2003; Neves-Pereira et al. 2002).
Knockin mice with this human Met allele also showed increased aggressive
behavior and changed the response to SSRI treatment (Chen et al. 2006). Con-
ditional BDNF knockout mice using kainate receptor promoter lack BDNA
expression especially in hippocampal CA3 area. These mice also showed
heightened aggression compared to wild-type, suggesting an important role of
hippocampal BDNF in aggression (Ito et al. 2011). In contrast to the consistent
results on aggressive behavior among BDNF mutant mice, studies on polymor-
phisms of the BDNF gene in aggressive behavior in humans remain to be resolved.
No association was observed between Val66Met polymorphism and proneness to
violence in a Chinese male sample (Tsai et al. 2005). Other SNPs in the BDNF
gene may be associated with high impulsivity in children with ADHD (Oades
et al. 2008). A further role of BDNF is evident in neuroadaptions in the
VTA-accumbens pathway of mice and rats that are repeatedly attacked and show
evidence for defeat, submission and disrupted social interactions (Berton et al.
2006; Miczek et al. 2011).
7.3.4 Neuronal Nitric Oxide
Nitric oxide, a free radical gas which diffuses across membranes, is involved in
several cellular functions [for review, see Calabrese et al. (2007)]. Mice lacking
neuronal nitric oxide synthase (nNOS-/-) show various deficits in their physio-
logical development and also behavior (Huang et al. 1993). nNOS-/- males, but
not females, showed higher duration of aggressive behavior and also displayed
much fewer submissive postures compared to wild-types (Nelson et al. 1995).
Serotonergic dysfunctions were observed in the nNOS-/- mice, specifically
reduced 5-HT turnover in the brain and deficient 5-HT1A and 5-HT1B receptor
function (Chiavegatto et al. 2001). Escalated aggression in the nNOS-/- was
rescued by 5-HTP treatment which increased 5-HT level and turnover. These
findings point to an important role of nitric oxide for the normal 5-HT function,
and thus increased aggression nNOS-/- may be induced by changing 5-HT
activity.
7.3.5 a-Calcium-Calmodulin Kinase II
a-Calcium-calmodulin kinase II (a-CaMKII) is a neural specific enzyme and has

been shown to be involved in long-term potentiation (LTP) (Silva et al. 1992).
Heterozygotes of a-CaMKII knockout mice showed escalated defensive aggres-
sion but not offensive aggression (Chen et al. 1994). In the resident-intruder test,
resident a-CaMKII heterozygotes showed similar aggressive behavior as wild-type
mice. In contrast, when the mutant was tested as an intruder, they exhibited highly
defensive reactions toward the resident. Reduced 5-HT release was observed in the
dorsal raphé of a-CaMKII mutant in vitro, and thus the changed 5-HT function
may be associated with defensive aggression in this mouse.
8 Concluding Remarks
• Activation of 5-HT1A, 5-HT1B and 5-HT2A/2C receptors in mesocorticolimbic

areas reduce species-typical and other aggressive behaviors. In contrast, agonists
at 5-HT1A and 5-HT1B receptors in the medial prefrontal cortex or septal area
can increase aggressive behavior under specific conditions. 5-HT exerts a
complex role in aggression that depends on (1) the subtypes of receptors and the
brain in which they are expressed (2) the types of aggressive behaviors, and
(3) the trait characteristics of the human subject or the mouse (i.e., Mus mus-
culus is a pugnacious species and many inbred strains are quite placid).
• Based on genetic studies, it is likely that the 5-HT1B receptor has an important
role in the inhibition of aggression. For other receptor subtypes such as 5-HT1A
or 5-HT2A receptors, the association between aggressive behavior and poly-
morphisms in receptor genes all failed to have a reliable relationship compared
to relatively consistent pharmacological data. It may be important to determine
where in the brain of aggressive individuals the gene expression is changed by
the polymorphisms.
• An important role of MAOA in aggressive behaviors is supported by genetic
studies of deleterious mutation of MAOA gene in humans and knockout mice.
In contrast to the serotonin deficiency hypothesis, these results suggest that
chronically increased 5-HT levels due to reduced MAOA function—trait-like
change—may promote or intensify escalated aggressive displays.
• Consistent anti-aggressive effects of SSRI treatment in several species strongly
point to the serotonin transporter as a promising therapeutic target for man-
agement of impulsive, escalated aggression. More studies are required to
determine the precise neurobiological mechanisms recruited for the anti-
aggressive effects of SSRIs to emerge, especially as a result of chronic treatment
that induces pre- and post-synaptic neuroadaptive processes.
• The genetic studies in human and nonhuman primates also suggest 5-HTT
polymorphisms such as the short allele as a risk factor for violent traits, which
seems to be particularly relevant in combination with environmental stress.
Results from transgenic mice lacking 5-HTT are more difficult to interpret due
to the wide variety of behavioral functions that are affected by the genetic
manipulation.
• Gene polymorphism studies of MAOA, 5-HTT and Tph2 revealed critical gene-
environment interactions as a risk factor for violent traits. Individuals with a
certain allele are particularly prone to engage in violent behavior when they
have a history of early life maltreatment, but the effect disappeared when they
are reared in an environment with low stress.
• Modulatory mechanisms of serotonergic neurons that induce escalated aggres-
sion have gradually been identified. So far, evidence points to the modulations
of activity of dorsal raphé nucleus by GABA, glutamate, CRF and its auto-
receptors as being particularly relevant to the display of escalated aggression.
Genetic analyses of aggressive individuals have identified several molecules that
affect the 5-HT system directly (e.g., Tph2, 5-HT1B, 5-HT transporter, Pet1,
MAOA) or indirectly (e.g., Neuropeptide Y, aCaMKII, NOS, BDNF).
In the current phase of advanced molecular genetic studies, it is necessary to
remind ourselves to match them with more sophisticated behavioral methodologies
of aggression. It is evident that the neurogenetic mechanisms differentiate types
and patterns of aggressive behavior requiring higher resolution. The current focus
on brain serotonin takes advantage of the most intensively studied neural system
that has been implicated in the neurogenetics of aggression. It remains astounding
how such trace amounts of this indole amine can engender such profound changes
in aggressive traits and states.
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Selectively Bred Rodents as Models
of Depression and Anxiety
Gregers Wegener, Aleksander A. Mathe and Inga D. Neumann
Abstract Stress related diseases such as depression and anxiety have a high degree
of co morbidity, and represent one of the greatest therapeutic challenges for
the twenty-first century. The present chapter will summarize existing rodent models
for research in psychiatry, mimicking depression- and anxiety-related diseases.
In particular we will highlight the use of selective breeding of rodents for extremes in
stress-related behavior. We will summarize major behavioral, neuroendocrine and
neuronal parameters, and pharmacological interventions, assessed in great detail
in two rat model systems: The Flinders Sensitive and Flinders Resistant Line rats
(FSL/FRL model), and rats selectively bred for high (HAB) or low (LAB) anxiety
related behavior (HAB/LAB model). Selectively bred rodents also provide an
excellent tool in order to study gene and environment interactions. Although it is
generally accepted that genes and environmental factors determine the etiology
of mental disorders, precise information is limited: How rigid is the genetic
disposition? How do genetic, prenatal and postnatal influences interact to shape adult
disease? Does the genetic predisposition determine the vulnerability to prenatal and
G. Wegener (&)
Centre for Psychiatric Research, Aarhus University Hospital,
8240 Risskov, Denmark
e-mail: wegener@dadlnet.dk
G. Wegener
Unit for Drug Research and Development, School of Pharmacy (Pharmacology),
North-West University, Potchefstroom, South Africa
A. A. Mathe
Department of Clinical Neuroscience, Karolinska Institutet,
Stockholm, Sweden
I. D. Neumann
Department of Behavioural and Molecular Neuroscience,
University of Regensburg, Regensburg, Germany

DOI: 10.1007/7854_2011_192
140 G. Wegener et al.
postnatal or adult stressors? In combination with modern neurobiological methods,

these models are important to elucidate the etiology and pathophysiology of anxiety
and affective disorders, and to assist in the development of new treatment paradigms.
Keywords Animal models Selective breeding Depression Anxiety
Contents
1 Introduction........................................................................................................................ 140
2 Selectively Bred Models on Depression .......................................................................... 143
2.1 The Flinders Sensitive and Resistant Line Rat ....................................................... 143
2.2 Learned Helplessness Rats (cLH/cNLH) ................................................................. 155
2.3 Fawn Hooded Rats ................................................................................................... 155
2.4 Wistar-Kyoto Rats .................................................................................................... 156
2.5 Swim Low-Active/Swim High-Active Rats ............................................................ 156
2.6 High/Low Stress Reactivity Mice ............................................................................ 157
3 Selectively Bred Models on Anxiety................................................................................ 157
3.1 Rats Selectively HAB and LAB Anxiety-Related Behavior .................................. 157
3.2 HAB LAB Mice ....................................................................................................... 169
3.3 Floripa H and L Rats................................................................................................ 170
3.4 Maudsley Reactive and Nonreactive Rats ............................................................... 170
3.5 High/Low Avoidance Rats: RHA/RLA; SHA/Bru and SLA/Bru .......................... 171
4 Conclusions........................................................................................................................ 171
References................................................................................................................................ 172
1 Introduction
It has been estimated that 127 million Europeans out of a population of 466 million
currently live with a brain disorder, with total annual costs (of brain disorders in
Europe) of €386 billion in 2004 (Andlin-Sobocki et al. 2005). Of these, mental
disorders constitute about 60% of the total costs reflecting the large socioeconomic
burden of these diseases. These numbers greatly emphasize the importance of
developing new strategies in treating mental disorders.
Stress-related diseases such as depression and anxiety, having a high degree of
co-morbidity, represent one of the greatest therapeutic challenges for the twenty-
first century. Although it is generally accepted that genes and environmental
factors determine depression, precise information is limited: How rigid is the
genetic disposition? How do genetic, pre- and post-natal influences interact to
shape adult depression? Does the genetic predisposition determine the vulnera-
bility to pre- and post-natal or adult stressors?
When depression, and in some degree anxiety, precipitates, the dominating
etiological hypotheses have focused at a dysregulation in the serotonergic and
noradrenergic system. This is emphasized by the mechanism of action of the
Selectively Bred Rodents as Models of Depression and Anxiety 141
currently marketed antidepressants, which almost exclusively act by direct mod-

ulation of these two systems. Unfortunately, the efficacy of the presently clinically
used antidepressant drugs are low, only approximately 30–35% after subtracting
the placebo effects. Thus, there exists a major unmet medical need, the resolution
of which is contingent on elucidating the disease etiology and pathogenesis.
Therefore, good model systems are needed to answer fundamental neurobiolo-
gical questions and to predict responses to novel therapeutic agents. Animal models
can be assessed on the basis of five major criteria (Willner 1984; Geyer and Markou
1995): face validity (how well the model resembles the disease/condition),
construct validity (how well the model is consistent with theoretical rationale),
etiological validity (how identical are the etiologies of the disease (phenomenon) in
the animal model and in humans), convergent/discriminant validity (the degree to
which a test correlates with other tests that attempt to measure the same construct/
the degree to which a test measures aspects of a phenomenon that are different from
other aspects of the phenomenon that other tests assess (Campbell and Fiske 1959),
and predictive validity (how well the model responds favorably to clinically
established drugs). An optimal model fulfills all the criteria. However, a model can
even be useful, even if not all conditions are met (Geyer and Markou 1995).
Genetic selection for behavioral and other phenotypic characteristics is a core
feature in evolution, and crucial for survival of any species. Selective breeding is
the process of breeding plants or animals for particular genetic traits, which
is desired by the researcher. Selective breeding has proven to be a valuable tool in
the advancement of science. In fact, one of the most commonly used animals in
laboratory research, the Wistar rat, may be considered––in the strict terms of
selective breeding––an example of selective breeding. This strain was developed
at the Wistar Institute in 1906 for use in biological and medical research, and it
was the first rat strain developed to serve as a model organism at a time when
biological laboratories primarily used mice (Clause 1998; Lindsay and Baker
2006). Several of the laboratory rat strains used today originate from the original
Wistar colony established by Donaldson, Greenman, and King (Clause 1998;
Lindsay and Baker 2006).
With this development, it is therefore not surprising that several selectively
bred animal models also have been established in neuroscience and psychiatric
research.
Selectively bred models are essential for studying underlying mechanisms of
the disease and have been established for various psychopathological entities/
phenotypes, as it appears from Table 1.
The present chapter will highlight the use of selective breeding in order to
establish animal models for research in psychiatry, with special focus on depression
and anxiety disorders. We will highlight two rat models: the Flinders Sensitive and
Flinders Resistant Line (FSL/FRL) and rats selectively bred for high (HAB) or low
(LAB) anxiety-related behavior (HAB/LAB model). A very brief description of
other existing selectively bred rodents, modeling depression and anxiety is also
given. For the remaining models on e.g., schizophrenia and epilepsy, a detailed
description can be found in the references listed in Table 1.
Table 1 Overview of some selectively bred animal models
142
Disease/phenotype Model Species Reference

ADHD/impulsivity Naples high/low excitability Rats Sadile et al. (1988); Cerbone et al. (1993); Viggiano et al. (2003)
(NHE/NLE)
Aggression Novosibirsk Rats Naumenko et al. (1989)
Turku Mice Sandnabba (1996)
SAL/LAL Mice van Oortmerssen and Bakker (1981)
Alcoholism HAD/LAD Rats Li et al. (1993); Li and Lumeng (1977); Murphy et al. (2002)
Fawn hooded (FH/Wjd) Rats Rezvani et al. (1990), (1991), (2007)
Anxiety Floripa H and L Rats Ramos et al. (1998), (2002), (2003)
HAB/LAB Rats See Main Text
Liebsch et al. (1998b); Landgraf and Wigger (2002); Landgraf et al. (2007)
HAB/LAB Mice Kessler et al. (2011); Kromer et al. (2005)
Maudsley MRS/MNS Rats Broadhurst (1960); Blizard and Adams (2002)
High/low avoidance: Rats Brush et al. (1985), (1989); Brush (2003)
syracuse SHA/BRU and SLA/Bru
Roman HA/LA
Depression WKY Rats Pare and Redei (1993); Lahmame et al. (1997); Will et al. (2003)
FSL/FRL Rats See Main Text
Overstreet et al. (2005); Overstreet (1986)
SwLo/SwHi Rats West and Weiss (1998b)
LR/HR Mice Touma et al. (2008)
cLH/cNLH Rats Vollmayr and Henn (2001); Vollmayr et al. (2001)
Fawn hooded (FH/Wjd) Rats Rezvani et al.( 2002)
Epilepsy WAG/Rij Rats Van Luijtelaar and Coenen (1986), (1989); Coenen and Van Luijtelaar (2003)
Gaers Rats Vergnes et al. (1982); Marescaux et al. (1992); Marescaux et al. (1984)
Schizophrenia APO-SUS/APO-UNSUS Rats Ellenbroek and Cools (2000); Costall and Naylor (1973); Ellenbroek and
Cools (2002)
G. Wegener et al.
Table 2 Symptoms in depressed individuals which can be modeled in FSL rats

Symptom/activity Patients FSL rats
Suicidal ideas Frequent Cannot be modeled
Activity Psychomotor retardation Reduced bar pressing for rewards
Anhedonia Yes Yes (following stress)
Appetite Reduced Reduced
Weight Weight loss Lower body weight
Cognitive performance Reduced Reduced/normal (dependent on test)
REM Sleep Elevated Elevated
Anxiety Not a core feature No anxiety
HPA axis dysregulation Yes Yes
Treatment response (see text) Yes Yes
Killer T-cell activity Reduced Reduced
Cardiovascular morbidity Increased Increased
2 Selectively Bred Models on Depression
2.1 The Flinders Sensitive and Resistant Line Rat
The Flinders Line rats were established by selective breeding for differential
responses to the anticholinesterase agent, diisopropyl fluorophosphate (DFP), at
Flinders University in Adelaide, Australia. The original rationale was to breed a rat
strain that would be genetically resistant to irreversible anticholinesterase agents,
DFP. However, the selective breeding of Sprague–Dawley (SD) rats, resulted in a
line more sensitive to DFP, the Flinders Sensitive Line (FSL), whereas the Flinders
Resistant Line (FRL) rats were not more resistant than an outbred control
(Overstreet et al. 1979; Russell et al. 1982). Being less tolerant to DFP, the FSL rat
were also found to be more sensitive to drugs targeting the cholinergic system, in
particular effects of directly acting muscarinic receptor agonists (Russell and
Overstreet 1987; Overstreet and Russell 1982; Overstreet 1986) and to have more
muscarinic receptors in several brain regions (Overstreet and Russell 1984).
As it also was reported that depressed individuals were more sensitive to
cholinergic agonists than normal controls, defined by behavior, neuroendocrine
measures and sleep (Janowsky et al. 1980, 1994; Risch et al. 1981), it was sug-
gested that the FSL rat might be a model for depression.
Today there are now breeding colonies of the FSL rats in Australia, Canada,
Denmark, Greece, Israel, Mexico, South Africa, Sweden and United States.
2.1.1 Key Features of the FSL Rat Depression Model
As mentioned, the FSL line phenotypically resembles a number of depression

symptoms and has been a useful tool to elucidate the endophenotype of depression.
Indeed, extensive work has demonstrated that many of the core symptoms of
depression can be reproduced in the FSL strain; the more salient characteristics are
shown in Table 1.
Depression-Related Behavior
Several observations of the unmotivated and motivated behavior of the FSL rat
suggest that it exhibits psychomotor retardation, a key behavioral characteristic of
depressed individuals (Lecrubier 2006). In particular, the FSL rat is less active in a
novel open field (Overstreet and Russell 1982; Overstreet et al. 1986), bar-presses
at a low rate for water or food reward (Overstreet and Russell 1982; Bushnell et al.
1995), and does not complete food-motivated nonmatching- to-sample learning
trials in a timely manner (Bushnell et al. 1995).
Importantly, the FSL rats show increased immobility in the forced swim test
(FST), which is the prototypic screening tool for depression-like behavior in
rodents (Overstreet and Russell 1982; Schiller et al. 1992; El Khoury et al. 2006).
Especially of interest for the predictive validity of the model, these behaviors are
reversible by chronic but not acute treatment with antidepressants.
Anhedonia, the inability to experience pleasure, is often regarded as a core
symptom of depression. Interestingly, under basal conditions the FSL compared to
FRL rats did not show signs of anhedonia, and signs of anhedonia were only found
when FSL rats were exposed to chronic mild stress (Pucilowski et al. 1993;
Matthews et al. 1996), supporting this model as being a candidate for Gene 9
Environment studies. We have replicated these findings, and found that group
housed FSL rats display a higher level of anhedonia following chronic mild stress
exposure, when compared with the FRL rats (Mathé et al. unpublished results).
A reduction in appetite is a classical symptom seen in most depressed indi-
viduals, while an increase in appetite and weight gain is observed in fewer.
Appetite and food intake in the FSL and FRL rats has not been studied in detail,
but the FSL rat weighs less than the FRL rat and in a recent study the FSL were
found to consume less food than FRL (Abildgaard et al. 2010). It, therefore,
appears that the FSL rats have a decreased appetite thus resembling the reduced
appetite in depressed individuals.
Anxiety-Related Behavior
Anxiety is not considered a core feature of depression, but there exist a high degree
of co-morbidity of anxiety with depression. Therefore, the behavior of FSL was
examined in the classical test of anxiety-like behavior, the elevated plus maze
(EPM), which is an unconditioned test for anxiety in rodents, and works by cre-
ating a conflict between an animal’s exploratory drive and its fear of open and
brightly-lit areas. Under baseline conditions no differences were discovered
between the FSL and FRL lines (Overstreet et al. 1995). Treatment with a
benzodiazepine exerted a comparable anxiolytic effect in both FSL and FRL rats
and did not differentiate between the two strains (Schiller et al. 1991; Mathé et al.
unpublished data).
However, our own recent results demonstrated that FSL rats had a reduced level
of unconditioned anxiety on the EPM compared to the FRL rats (Abildgaard et al.
2010). Specifically, the FSL spent more time on the open arms and had a higher
level of full entries onto open arms. Similar findings have been described in young
FSL rats compared to SD rats (Braw et al. 2006). It is, however, of interest to note
that FSL rats did exhibit some anxiogenic behavior in the social interaction task
(Overstreet et al. 2004b), which may reflect enhanced social anxiety, or alterna-
tively, reduced social motivation and social withdrawal.
Taken together, as anxiety does not seem to be a prominent feature of the FSL
strain, the FSL rats seem to be a model for depression without comorbidity of
anxiety. However, as anxiety can be judged from multiple paradigms, further
studies are warranted.
Cognition
Cognitive disturbances in depressed individuals can involve both learning

difficulties and memory loss. Most learning and memory studies on FSL rats used
foot shock as the motivating stimulus, where the FSL rat show greater difficulty in
acquiring a shock-motivated, active avoidance task (Overstreet et al. 1990). On
the other hand, the FSL rat exhibited normal memory of a shock-motivated
passive avoidance task (Overstreet et al. 1992; Russell et al. 1982). Also in a food-
motivated task similar completion rates between the FSL and the FRL were
obtained, achieved by reducing the size of the food pellet in the FSL rats (Bushnell
et al. 1995). Although the FSL rats did not perform this task as rapidly as the FRL
rats, they chose the correct bar just as efficiently (Bushnell et al. 1995). Thus, there
is no definitive evidence for cognitive disturbances in the FSL rats under basal
conditions, which is further underlined by a recent study from Aarhus, where the
FSL show similar spatial memory abilities as the FRL in the Morris Water Maze
(Wegener et al. unpublished).
Pain
Affective disorders have been repeatedly linked with alterations in thermal and
visceral pain perception (Haug et al. 2004; Vedolin et al. 2009; Robinson et al. 2009).
However, only a limited number of studies have been carried out in the FSL model. In
a model with partial denervation of the sciatic nerve (PSL model), which produces a
chronic decrease in touch and heat withdrawal thresholds (allodynia) and an
increased response to noxious mechanical and heat stimuli (hyperalgesia, Fujioka
et al. 2001; Fumagalli et al. 2007), the FSL rats expressed significantly lower levels of
tactile allodynia and less heat hyperalgesia following PSL injury compared to SD rats
(Shir et al. 2001). These studies have been carried out using a denervation model,
and no results for basal pain parameters are available.
Sleep Patterns
Sleeping disorders are very closely associated with depressive disorders, with both
insomnia and hypersomnia being observed. Two pronounced changes associated
with depression are increases in rapid eye movement (REM) sleep and decreases in
slow wave sleep (Jindal et al. 2002; Thase et al. 1995; Benca 1996; Benca et al.
1992; Adrien 2002). Basal sleep recordings in the FSL have demonstrated that the
FSL rat exhibited a reduced latency to––and greater amount of REM sleep than the
FRL rats (Benca et al. 1996; Shiromani et al. 1991), with no differences in slow
wave sleep patterns. Thus, the FSL rat resembled depressed individuals with
regard to the elevated REM sleep, but not with regard to the reduced slow wave
sleep (Jindal et al. 2002; Benca et al. 1992).
Hypothalamic-Pituitary Adrenal Axis
Distinct changes in the Hypothalamic-Pituitary Adrenal (HPA) axis reactivity and

cortisol levels in severe depression (melancholia) are well documented (Keck and
Holsboer 2001). However, studies in the FSL/FRL model are conflicting: Whereas
no differences in corticosterone levels under basal conditions or in response to a
chronic mild stressor have been found (Ayensu et al. 1995), in another study, the
FSL rats had significantly lower plasma ACTH concentrations compared with the
FRL, but still with no differences in plasma corticosterone concentrations between
the two groups (Owens et al. 1991). In the brain, it was found that the density
of anterior pituitary CRF receptor binding sites was elevated in the FSL rats
compared with the FRL (Owens et al. 1991).
This finding is further substantiated by later studies, where FSL and FRL, were
subjected to 1 h acute restraint and the effects of the stress exposure, including
possible strain specific changes were studied (Zambello et al. 2008). Under basal
conditions, no significant differences between FSL and FRL rats in the CRH
mRNA expression were found. However, an upregulation of the CRH mRNA
hybridization signal was detected in the central amygdala of the stressed FRL,
compared to the non-stressed FRL rats (Zambello et al. 2008). Following these
findings, it was hypothesized that, since a hypoactive mechanism of response to
stressful stimuli in the FSL rats was present, lack of amygdala CRH activation
following stress could suggest a subtype of allostatic load, which may alter the
interpretation of environmental stimuli by FSL rats and consequently influence
their behavioral response to stressful situations (Zambello et al. 2008). However,
these suggestions require further examinations.
Monoamine Metabolism
The monoaminergic hypothesis of depression (Schildkraut 1965) suggests that

there are distinct abnormalities of the serotonergic system in depressed individu-
als, although several inconsistencies exist. For example, both serotonergic 5HT1A
receptor overactivity (Arango et al. 1995) as well as 5HT2 receptor underactivity
have been reported (Mikuni et al. 1991).
In the FSL, several differences in the serotonin synthesis, 5HT1A receptor
sensitivity and the density of the serotonin transporter were found compared to the
FRL strains (Overstreet et al. 1994; Kanemaru et al. 2009; Nishi et al. 2009;
Kovacevic et al. 2010). However, whether these abnormalities are comparable to
those seen in depressed individuals are not known, and there are some data sug-
gesting inverse pharmacological responses in FSL rats and depressed individuals.
For example, studies have shown that the FSL rats are more sensitive to the
hypothermic effects of 5-HT1A receptor agonists (Wallis et al. 1988; Overstreet
et al. 1994), but depressed individuals are usually less sensitive to these effects of
similar agents (Lesch 1991). Moreover, in depressed individuals, both increases
(Reddy et al. 1992), decreases (Asberg et al. 1984) or no changes (Roy et al. 1985)
in the serotonin metabolite 5-hydroxy-indoleacetic acid (5-HIAA) in cerebrospinal
fluid have been reported. How this may relate to the FSL model remains to be
established.
The psychomotor retardation and anhedonia-like features following chronic
mild stress (CMS) in the FSL rats may suggest the dopaminergic system to be
involved. This has been supported by a few studies on dopamine metabolism and
release from selected brain regions of FSL rats (Zangen et al. 2001; Yadid
et al. 2001), and in behavioral responses to dopaminergic agents (Crocker and
Overstreet 1991). However, it is not clear how these findings can be translated to
human pathology.
Nitric Oxide Signaling
The atypical neurotransmitter nitric oxide (NO) possesses both neuroprotective

and neurodestructive properties (Dawson and Dawson 1996; McCaslin and Oh
1995). Nitric oxide has been implicated in the psychopathology of depression, as
postmortem studies on brains from the Stanley Consortium (Bethesda, MD, USA)
have demonstrated that patients suffering from depression have an increase in NO
synthase-immunoreactivity in the CA1 hippocampal area (Oliveira et al. 2008). By
virtue of its unpaired electron, NO promotes the formation of free radicals and has
been linked to various neurodegenerative processes (Ischiropoulos and Beckman
2003). Drugs that affect the major NO pathways have also been shown to possess
antidepressant-like properties (Wegener and Volke 2010), and antidepressants
have been shown to affect the NO signaling (Wegener et al. 2003). Investigation of
NO signaling in the FSL rats did not reveal any baseline FSL–FRL differences in
hippocampal constitutive NO synthase (cNOS) activity and neuronal nitric oxide
Fig. 1 Hippocampal constitutive nitric oxide synthase (cNOS) activity data under basal
conditions [FSL (n = 8), FRL (n = 7)], and following escapable stress/inescapable stress
(ES–IS) [FSL (n = 8), FRL (n = 7)]. Following ES–IS, cNOS activity is significantly elevated in
FSL rats compared to unstressed FSL controls (** p \ 0.005) and versus pre- and post-stress FRL
animals (* p \ 0.05). Pre- and post-stress activity levels for FRL rats did not differ from one
another. Values shown are means ? S.E.M. Reprinted from (Wegener et al. 2010) with
permission. Ó Cambridge University Press
synthase (nNOS) protein levels. However, following exposure to stress in the

escapable stress/inescapable stress paradigm, the FSL strain showed a larger
activation of the cNOS system (Fig. 1), confirming the NMDA–NO cascade as an
important vulnerability factor in the depression-like phenotype of the FSL rat
(Wegener et al. 2010). Furthermore, several distinct agents affecting NO synthesis
have also been shown to be effective antidepressants in the FSL (Wegener et al.
unpublished observations, see Table 3).
Neuropeptides
Neuropeptide Y (NPY) is one of the most abundant peptides in the mammalian

brain, interacting with the noradrenaline, serotonin and dopamine systems with
effects on multiple brain functions. Several findings suggest that NPY plays an
important role in the pathophysiology of depression and anxiety (Mathe et al.
2007; Heilig 2004).
Studying NPY in FSL has revealed marked similarities between vicissitudes of
NPY in the rat depression models and human subjects. Thus, we have shown
decreased NPY levels in the hippocampus of the FSL rats (Jimenez-Vasquez et al.
2000), and NPY protein and mRNA were found reduced in the CA1-2 regions and
the dentate gyrus of FSL rats compared with FRL (Jimenez-Vasquez et al. 2000).
Table 3 Effect of some antidepressants (common and experimental), in the forced swim test (FST) in FSL (See also Overstreet et al. (2005); and Overstreet
(1993))
Class Drug Response in FST References
TCA Desipramine + Overstreet et al. (2004b); Overstreet and Griebel (2004); Overstreet
et al. (2010a); Overstreet et al. (2004a); Overstreet et al. (2008)
Imipramine + (Schiller et al. (1992); Liebenberg et al. (2010)
Nortryptyline + Petersen et al. (2009)
NaSSA Nefazodone + Dremencov et al. (2004)
SSRI Citalopram + Overstreet et al. (2004b)
Escitalopram + El Khoury et al. (2006); Wegener et al. (unpublished)
Paroxetine + Zangen et al. (2001); Zangen et al. (2002); Zangen et al. (1999), (1997)
Sertraline + Pucilowski and Overstreet (1993)
Fluoxetine + Overstreet and Griebel (2004); Overstreet et al. (2004a); Overstreet
et al. (2008)
NOS inhibitors Methylene blue + (Wegener et al. (unpublished)
L-NAME + (Wegener et al. (unpublished)
7-Nitroindazole + (Wegener et al. (unpublished)
NPY R5 antagonist Lu AA33810 + Walker et al. (2009)
CRF antagonist CP-154,526 – Overstreet et al. (2004b)
SSR125543 + Overstreet and Griebel (2004)
NK2 antagonists Saredutant + Overstreet et al. (2010b)
Melatonin antagonists S 20304 + Overstreet et al. (1998)
Selectively Bred Rodents as Models of Depression and Anxiety
PDE inhibitors Rolipram + Overstreet et al. (1989)

Sildenafil + (together with atropine) Liebenberg et al. (2010)
Miscellaneous ECS + Jimenez-Vasquez et al. (2007); Wegener et al. (unpublished)
Tianeptine + Wegener et al. (unpublished)
Ketamine + Wegener et al. (unpublished)
Exercise + Bjornebekk et al. (2005)
Nerve growth factor + Overstreet et al. 2010a)
Nemifitide + Overstreet et al. (2004a)
Amibegron + Overstreet et al. (2008)
Lu AA21004 + Mørk et al. (2012)
Inositol + Einat et al. (2002)
149
Fig. 2 Messenger RNA samples from hippocampus and frontal cortex of FSL (%; n = 9) and
FRL (&; n = 9) rats were used for quantification of the expression levels of BDNF using real-
time qPCR. Values for each individual were normalized with the geometric mean of the reference
genes Ywhaz and Hmbs in the hippocampus and Ywhaz and Actb in the frontal cortex. Plotted
data show mean group values ? S.E.M. of mRNA expression as % of FRL rats. * Indicates
significant between-group differences (p \ 0.05). Reprinted from (Elfving et al. 2010a) with
permission. Ó Cambridge University Press
In contrast, local NPY-Y1 receptor binding was increased, indicating functional

significance of the changes in NPY availability (Jimenez-Vasquez et al. 2000a,
2000b; Husum et al. 2001; Caberlotto et al. 1999; Mathe et al. 2007).
Consistent with these data are the findings that antidepressants, lithium and ECS,
all increase NPY expression in selected brain regions, and that the increases are
larger in the FSL compared with the FRL strain (Husum et al. 2003; Husum et al.
2001; Jimenez Vasquez et al. 2000; Jimenez-Vasquez et al. 2007). For instance,
reduced NPY levels in the CSF of depressed patients and altered NPY and NPY
receptors mRNA expression in post-mortem brains have been shown (Widdowson
et al. 1992; Olsson et al. 2004; Hou et al. 2006; Heilig 2004; Caberlotto et al. 1999;
Caberlotto and Hurd 2001). Conversely, increased NPY concentrations in CSF
following successful treatment of depressed in-patients with citalopram or ECT have
been reported (Nikisch and Mathe 2008; Nikisch et al. 2005).
Neurotrophic Factors
Several studies have found decreased serum or plasma brain-derived neurotrophic

factor (BDNF) levels in depressed patients, and a positive correlation between
BDNF reduction and the severity of the disease has also been observed (Shimizu
et al. 2003; Karege et al. 2002, 2005; Aydemir et al. 2006). Moreover, in
post-mortem hippocampal tissue, increased levels of BDNF immunoreactivity

have been reported in subjects treated with antidepressants compared to untreated
subjects (Chen et al. 2001). These findings constitute the rationale for studying
BDNF also in the FSL model of depression. In a recent study from Aarhus, BDNF
expression in the hippocampus was significantly decreased in the FSL compared
with FRL rats (Fig. 2), while no differences were found in the frontal cortex or
CSF (Elfving et al. 2010a). Contraintuitively, BDNF levels in serum and whole
blood of the FSL rats were significantly increased compared with FRL rats
(Elfving et al. 2010a). Whether this finding is relevant, or a peculiarity of the FSL,
remains to be fully established. However, recent studies underline that multiple
factors must be taken into consideration when correlating serum BDNF with
clinical state (Elzinga et al. 2011; Bus et al. 2011; Gass and Hellweg 2010;
Sartorius et al. 2009). Nevertheless, the regulation of the BDNF levels in hippo-
campus, serum, and whole blood in FSL and FRL rats adds to the hypothesis that
neurotrophic factors may be related to the pathophysiology of depression.
Similar to findings with BDNF, we have recently characterized vascular
endothelial growth factor (VEGF) in the FSL rats. VEGF protein, but not mRNA,
expression in the hippocampus and frontal cortex were found to be significantly
decreased in the FSL compared with FRL rats, while no differences were found in
the striatum, hypothalamus or serum (Elfving et al. 2010b).
Neurogenesis and Cell Proliferation
Hippocampal neurogenesis has been implicated in the etiology of depression and

has been suggested to constitute the final common mechanism underlying antide-
pressant treatments (Santarelli et al. 2003). In order to further explore the
hypothesis that reduction in hippocampal neurogenesis contributes to the etiology
of depression, which was essentially based on studies on healthy rats exposed to
repeated or chronic stressors, the FSL model was tested under a variety of cir-
cumstances (Petersen et al. 2008, 2009; Husum et al. 2006; Bjornebekk et al. 2007).
We found that adult FSL rats have significantly more BrdU-immunoreactive
(IR) cells in the dentate gyrus compared with FRL, and aging caused an exacer-
bated loss of these cell types in the FSL. FSL animals treated chronically with
nortriptyline, there was no apparent effect on the number of BrdU-IR cells,
although it significantly decreased the immobility time in the FST (Petersen et al.
2009). Taken together, these results clearly demonstrate a dissociation of the
effects of antidepressants on behavior in the FST and cell proliferation. Thus
SSRIs and tricyclics can decrease immobility in the FST without affecting the
cytogenesis and, conversely, increased cytogenesis is not necessarily reflected in
decreased depression-like behavior in FST. These data are of importance since
they indicate that changes in cell proliferation may be sufficient, but are not
necessary for antidepressant effects of currently used antidepressants in the
FSL/FRL model.
Fig. 3 Effect of Imipramine on the non-perforated spine synapses in FSL and FRL rats. The total
number of spine synapses in the CA1 stratum radiatum was significantly smaller in the FSL rats
compared to the FRL rats. Following 3 weeks of Imipramine (Imi, 15 mg/kg/day) there were a
significant increase in the spine synapses in both FSL and FRL, thereby normalizing the FSL
spine synapse numbers. (* p \ 0.05; *** p \ 0.001). Modified from (Chen et al. 2010) with
permission. Ó John Wiley & Sons
Also other aspects of brain remodeling have been proposed to be essential for
development of disease. Thus, both the hippocampal volume (Videbech and
Ravnkilde 2004) and synaptic morphology may play a role (Nestler et al. 2002).
Therefore, we have investigated changes in hippocampal volume, neuron and
synapse numbers in the FSL and FRL following chronic imipramine therapy, using
design-based stereological methods (Chen et al. 2010). We found that the volume
and the number of neurons and synapses were significantly smaller in the FSL
saline group compared with the FRL saline group, a feature which was reversed
following imipramine treatment (Fig. 3). Our experiments illustrate the impor-
tance of using a disease model to study cell proliferation and effects of treatments
that could potentially be translated to human condition.
Cardiovascular and Metabolic Function
Depression is a well-known risk factor for the development of ischemic heart

disease and is associated with increased cardiovascular morbidity and mortality
(Barefoot and Schroll 1996; Egede et al. 2005; Hemingway and Marmot 1999;
Rugulies 2002). Major depression doubles the risk of adverse cardiovascular
events within 12 months in patients with newly diagnosed coronary heart disease
(Carney et al. 1988) and increases the risk of mortality after acute myocardial
infarction (Frasure-Smith et al. 1993). The presence of diabetes has been found to
double the risk of co-morbid depression (Anderson et al. 2001), and a meta-
analysis has shown that depression increases the risk of developing type 2 diabetes
in adults by 37% (Knol et al. 2006). In a study comparing the myocardial

responsiveness to ischemia/reperfusion injury and the effects of ischemic pre-
conditioning in hearts from FSL rats using SD rats as controls, it was observed that
the myocardial infarct size was significantly larger in the FSL rats than in the SD
rats following ischemia/reperfusion injury, but have maintained cardioprotective
mechanism following ischemic preconditioning (Solskov et al. 2010). In the same
study, it was also demonstrated that FSL were hyperinsulinemic, with a strong
tendency in different levels of fasting glucose levels compared with SD rats
(Solskov et al. 2010).
However, in a recent study performed in Aarhus, we have not been able to
detect any difference in fasting glucose levels in FSL compared with FRL
(Abildgaard et al. 2010). Metabolic stress induced by a high fat diet increased
insulin levels during an oral glucose tolerance test in both FSL and FRL, with
fasting blood glucose levels significantly increased by high fat diet in the FSL rat
(Abildgaard et al. 2010). Interestingly, the metabolic changes were associated with
increased depression-like behavior in the FST and cognitive impairments in the
object recognition test in the FSL only. These findings confirm the FSL as a model
with a greater metabolic susceptibility, and further highlight the usefulness of the
model in translational interdisciplinary depression research.
2.1.2 Gene 3 Environment Interactions
Interactions with the environment, which can have negative or positive conse-
quences, have also been found to be a major determinant of disease. For example,
psychosocial stress in adulthood impairs the health condition of the individual
(Lupien et al. 2009; Bale et al. 2010; Reber et al. 2007), whereas social support or
exercise exert beneficial effects on somatic and mental health (Brene et al. 2007;
Dishman et al. 2006; Dunn and Dishman 1991; Young 1979; Neumann 2009).
The consequences of acute, subchronic or chronic stress are largely dependent
on the individual (and genetically determined) stress susceptibility, and there is
good evidence that FSL and FRL as well as HAB and LAB rats provide good
models to study gene 9 environment interactions. For example, as mentioned
above, a subchronic adult stress paradigm significantly upregulates the NO sig-
naling pathway in the FSL only (Wegener et al. 2010), and metabolic stress more
severely impacts the FSL compared with the FRL (Abildgaard et al. 2010).
In another series of experiments, we compared adult female FSL and SD rats in a
paradigm of 7 weeks of social isolation at the age of 29 weeks, and observed
increased number of BrdU-IR cells in the FSL, whereas it had no impact in the SD
strain (Bjornebekk et al. 2007). Other environmental stimuli may be experienced
positive. Thus, we have examined the effect of physical activity using running
wheels. We observed that voluntary wheel running had antidepressant effects
and selectively altered NPY and NPY Y1 receptor and opiate expression in the
FSL, but not FRL, rats further supporting a role of NPY in their phenotype
(Bjornebekk et al. 2006, 2010). These findings parallel and support the results from
human studies (Russo-Neustadt et al. 1999; Ransford 1982).
In addition to adult stress exposure, gene 9 environment interactions have been
described with respect to early-life stress, either prenatally or postnatally. Thus, a
large number of human and animal studies show a strong association between an
adverse fetal or immediate postnatal environment and behavioral and emotional
development later in life (Abe et al. 2007; Maccari et al. 2003; Nagano et al. 2008;
O’Connor et al. 2002; Tazumi et al. 2005; Van Den Bergh et al. 2005). Stressful
experiences during early life have been hypothesized to enhance susceptibility
(eventually triggered by adult stress) for mental illness (Cottrell and Seckl 2009;
Fumagalli et al. 2007; Maynard et al. 2001).
Prenatal stress studies have not yet been carried out in FSL/FRL. However, the
classical post-natal stress paradigm, maternal separation, in FSL and FRL have
been demonstrated to exacerbate the depression-like behavior of the FSL, but not
the FRL (El Khoury et al. 2006). Treatment with escitalopram selectively
decreased depression-like behavior in the FST in both maternally non-separated
and separated FSL, but not FRL rats (El Khoury et al. 2006). Maternal separation
in FSL has been also been found to reduce NPY in dorsal hippocampus of both
female and male FSL rats compared with FRL rats (Jimenez-Vasquez et al. 2001;
Wortwein et al. 2006).
In another study, we analyzed hippocampal synaptic transmission and plasticity
in vivo and ionotropic receptors for glutamate in FSL and FRL rats subjected to
maternal separation. A strong inhibition of long-term potentiation (LTP) and lower
synaptic expression of NR1 subunit of the NMDA receptor were found in FSL rats
(Ryan et al. 2009), and unexpectedly maternal separation induced a remodeling of
synaptic plasticity only in FSL rats, reducing inhibition of LTP accompanied by
marked increase of synaptic NR1 subunit and GluR2/3 subunits of AMPA
receptors (Ryan et al. 2009). This finding is in line with the demonstration that
maternal separation increased the hippocampal cell number, while consistently
with this increase, chronic escitalopram treatment reduced the cell number
(Petersen et al. 2008; Husum et al. 2008).
In a study of basal differences in synaptic signaling between FSL and FRL rats,
as well as on consequences of maternal separation in adulthood, it was found that
the FSL rats showed basal differences in the interaction/activation of distinct
synaptic mediators purified hippocampal synaptosomes (Musazzi et al. 2010).
In addition, following maternal separation, the FSL rats displayed a blunted
response of the mediators, suggesting a synaptic dysfunction in the FSL animals
(Musazzi et al. 2010). Escitalopram treatment restored some but not all alterations
observed in FSL rats after early-life stress, suggesting that early gene-environment
interaction may cause life-long synaptic changes affecting the course of depres-
sion-like behavior and response to drugs (Musazzi et al. 2010).
Finally, using an open-ended approach based on a proteomic analysis of serum,
maternal separation was found to induce changes in inflammation and transport
proteins in FSL rats (Carboni et al. 2010), changes that were partly reversed
following treatment with escitalopram or nortriptyline (Carboni et al. 2010).

No comparison between early-life stress in FSL and FRL was carried out.
These experiments underline that the consequences of environmental factors
are strongly determined by the genetic background, suggesting that a genetically
shaped phenotype can be further modulated by environmental factors.
2.1.3 Response to Treatment
A detailed review of the different studies, where the FSL rat has been used to test for
the antidepressant-like effects of drugs lies beyond the scope of this text, and only a
brief overview is given in the Table 3. A detailed review of the classical antide-
pressants and selective serotonin reuptake inhibitors (SSRIs) as well as a variety of
novel agents that presumably have different actions from the well-characterized
antidepressants, can be found elsewhere (Overstreet 2002, 2005).
2.2 Learned Helplessness Rats (cLH/cNLH)
The learned helplessness (LH) paradigm is a well characterized rat model of

depression, in which the animals are exposed to uncontrollable and unpredictable
aversive events, i.e., foot shock (Overmier and Seligman 1967). The model has
good face and predictive validity, including alterations in HPA axis activity and
REM sleep characteristic of depression (Maier 1991; Breier et al. 1987; Henn and
Vollmayr 2005). However, in outbred rats not the entire proportion of animals
become helpless. Therefore, breeding of helpless lines from Harlan SD outbred
rats was initiated in 1990 to achieve a higher yield of helpless animals following
inescapable shock-training (Vollmayr and Henn 2001; Henn and Vollmayr 2005).
This resulted in congenitally learned helpless (cLH) rats exhibiting a helpless
phenotype without exposure to uncontrollable shock, and a congenitally not
learned helpless (cNLH) strain being resistant to the effects of inescapable shock
(Vollmayr and Henn 2001).
2.3 Fawn Hooded Rats
A high degree of comorbidity between alcoholism and depression have been

reported (Merikangas and Gelernter 1990; Cloninger et al. 1979). The Fawn-
Hooded (FH/Wjd) rat is an inbred strain of rat, originally selected from a back-
ground of platelet serotonin storage abnormality (Tschopp and Zucker 1972). The
rat has been reported to exhibit both high immobility in the FST, elevated serum
corticosterone and high voluntary ethanol intake, measures that have been linked
with depression and alcoholism in humans (Rezvani et al. 2002, 2007).

For example, the FH/Wjd rat drinks up to 6 g/kg 10% ethanol per day, and
responds to drugs that are effective in humans with a reduction in alcohol intake
(Rezvani et al. 1999). Interestingly, the exaggerated immobility in the FST and the
hypercorticosterone levels can be also attenuated following chronic antidepressant
treatments (Aulakh et al. 1988, 1993; Rezvani et al. 1999). The FH/Wjd also
exhibits abnormalities in the central serotonergic function (Aulakh et al. 1994;
Bendotti and Samanin 1987; Arora et al. 1983; Dumbrille-Ross and Tang 1981),
but whether these serotonergic abnormalities contribute to both behaviors remains
to be determined. In addition, the first results showing decreased NPY in hippo-
campus in a model of depression were obtained in the FH, changes that were
reversed following ECS (Mathe et al. 1998). These findings were of great heuristic
value as they led to subsequent identification of reduced NPY expression in other,
both genetic and environmental models, including the FSL/FRL as described
above.
2.4 Wistar-Kyoto Rats
The Wistar-Kyoto (WKY) rat strain was developed as the normotensive control
strain for the spontaneously hypertensive rat, and bred from the Wistar strain
starting in 1963 (Okamoto and Aoki 1963). The WKY presents with hormonal,
behavioral, and physiological measures that mimic those found in depressed
patients, such as increased immobility in the FST (Lahmame et al. 1997;
Rittenhouse et al. 2002; Paré 1992, 1994) and dysregulation of the HPA and
hypothalamic–pituitary–thyroid axes (Solberg et al. 2001; Redei et al. 1994;
Gómez et al. 1996). However, the WKY responds with variable degree to anti-
depressants (López-Rubalcava and Lucki 2000; Lahmanie and Armario 1996;
Lahmame et al. 1997), and has therefore been proposed as a model of treatment-
resistant depression (Lahmame et al. 1997). Therefore, the model has been further
developed into ‘WKY most immobile’ (WMI) and ‘WKY least immobile’ (WLI)
rats (Will et al. 2003).
2.5 Swim Low-Active/Swim High-Active Rats
Since low motor activity and a condition of passive stress coping in a swim test
have been proposed to represent depression-like behavior in the rat, SD rats were
bred in accordance with the motor-activities starting in 1987 (Weiss et al. 1998).
Two rat lines have been obtained, Swim Low-Active (SwLo) and Swim High-
Active (SwHi) rats, which differ dramatically in FST behavior. The SwLo rats
show little struggling and much floating, while SwHi rats show the reverse (Weiss
et al. 1998). Importantly, when SwLo rats were given antidepressant, chronic but
not acute administration increased swim-test activity of SwLo rats (West and
Weiss 1998a). Information on neurotransmitter involvement was limited, but
studies suggest involvement of both glutamatergic (Tabb et al. 2007) and dopa-
minergic (West et al. 1999a, 1999b) mechanisms as well as alterations of the stress
axis (Gutman et al. 2008).
2.6 High/Low Stress Reactivity Mice
As mentioned before, dysfunctions (hyper- or hypo-activity) of the HPA axis

may play a prominent role in the development of major depressive disorders
(De Kloet et al. 1998; Holsboer 2000; Bale 2006). Therefore, attempts of gen-
erating animal models mimicking these neuroendocrine core symptoms have
been made in order to unravel parameters underlying increased or decreased
stress reactivity (Touma et al. 2008). Mice expressing a hyper- or a hypo-
reactivity of the HPA axis were selected for the ‘high reactivity’ (HR) and the
‘low reactivity’ (LR) breeding line. Compared with LR animals, the HR males
and females were ‘hyperactive’ in some behavioral paradigms (Touma et al.
2008), resembling symptoms of restlessness and agitation often seen in melan-
cholic depression. On the neuroendocrine level, the circadian rhythm of gluco-
corticoid secretion revealed a flattened diurnal rhythm (Touma et al. 2008),
mimicking findings from patients suffering from melancholic depression
(Deuschle et al. 1997; Keller et al. 2006).
3 Selectively Bred Models on Anxiety
3.1 Rats Selectively HAB and LAB Anxiety-Related Behavior
An adequate level of innate anxiety and fear is essential for survival of individuals
and species. Naturally, there exists a wide individual range in trait anxiety: from
extremely low to extremely high. Similarly, in humans, anxiety-related patholo-
gies including generalized anxiety, panic disorders or social phobia, reflect
extremes in trait anxiety with significant contributions of adverse life events
shaping the individual anxiety phenotype. In order to reveal neuroendocrine,
neurochemical and neurogenetic mechanisms of a complex behavioral phenotypes
such as anxiety, and in order to identify potential targets for psychotherapy,
we have established and extensively studied selectively bred HAB and LAB
rats (Landgraf and Wigger 2002; Landgraf et al. 2007; Neumann et al. 2010).
This approach is particularly promising to further our understanding of genetic
mechanisms underlying anxiety-related disorders. Other relevant rodent models for
anxiety-related behavior include exposure to early-life stress (Wigger and
Fig. 4 Anxiety-related behavior of male and female HAB, LAB and non-selected NAB rats on
the elevated plus-maze (EPM) which is consistent over the years between 2003 and 2010
Neumann 1999; Huot et al. 2001), chronic stress in adulthood (Barrot et al. 2005;
Reber et al. 2007; 2008), or transgenic modifications (Bale 2006; Mantella et al.
2003). Recently, the Landgraf group succeeded in establishing also mice lines
selectively bred for high (M-HAB) and low (M-LAB) anxiety-related behavior (see
Table 1; Landgraf et al. 2007).
Since 1993, we have selectively and bi-directionally bred outbred Wistar rats
for high (HAB) versus low (LAB) anxiety-related behavior based on their
behavioral performance on the EPM at the Max Planck Institute of Psychiatry
in Munich and, since 2002, at the University of Regensburg (Landgraf and
Wigger 2002; Landgraf et al. 2007; Neumann et al. 2010; see Fig. 4). Male and
female HAB and LAB rats are selected at the age of 9 weeks for further
breeding only, if the percentage of time spent on the open arms of the elevated
plus maze (EPM) is below 5% and above 40–45%, respectively. For experi-
mental purposes, HAB rats with an anxiety level of less than 10% and LAB
rats with more than 35% time on the open arms during testing at the age of
9 weeks are used.
3.1.1 Key Features of the HAB/LAB Model
Anxiety-Related Behavior
The behavioral profile of HAB and LAB rats with respect to the selection criteria
has been reliable and robust over the last 10–15 years (Liebsch et al. 1998b;
Neumann et al. 2010; see Fig. 4), is present over all seasons, independent of sex or
age and could be confirmed in different European laboratories. The extremes in
anxiety could be confirmed in a battery of relevant behavioral tests, including the
EPM, the open field, the light dark box and the holeboard (Slattery and Neumann
2010; Ohl et al. 2001; Henniger et al. 2000). In addition, HAB rats are unable to
properly extinguish inappropriate fear in tests for conditioned anxiety despite
similar acquisition of fear (Muigg et al. 2008). Thus, although bred for high innate
non-conditioned anxiety on the EPM, this finding reveals that HAB rats also
display exaggerated responses to conditioned fear, which makes them a potentially
useful model for posttraumatic stress disorder. Also, LAB rats were able to
extinguish this fear faster than non-selected Wistar rats (NAB), again showing
robust trait differences between these lines.
Effects of Anxiolytic Treatment

The predictive validity of the HAB/LAB model is substantial, and anxiolytic
agents reverse or attenuate the high anxiety phenotype seen in HAB rats.
Acute treatment with the reference anxiolytic drug, diazepam (1 mg/kg, i.p),
reduced anxiety in HAB rats on the EPM or in the light–dark box, whereas it was
without effect in LAB rats (Liebsch et al. 1998a; Jochum et al. 2007). In addition
to its effects on anxiety-related behavior in HAB rats, diazepam also corrected the
abnormal pain sensitivity seen in HAB rats (Jochum et al. 2007). Similar to
diazepam, we have repeatedly found a reliable and profound anxiolytic effect of
chlordiazepoxide (20 mg/kg, i.p.) with relatively low individual differences in
responsiveness (Slattery; Beiderbeck and Neumann, unpublished).
Also, manipulation of various relevant anxiogenic and anxiolytic neuropeptide
systems of the brain, such as arginine vasopressin, CRH, oxytocin and neuro-
peptide S, respectively, was found to be effective in reducing the high level of
anxiety seen in HAB rats.
Depression-Like Behavior
Importantly, mimicking the high degree of comorbidity of anxiety and depression

mentioned above, HAB rats are also characterized by depression-related behavior
in the FST, independent of sex (Keck et al. 2003b; Slattery and Neumann 2010;
Frank and Landgraf 2008).
Effects of Antidepressant Treatment

Chronic treatment with antidepressant drugs reduces the depression-like status in
HAB rats. Treatment of male HAB rats with the SSRI paroxetine during a period
of 8 weeks markedly increased active stress coping in the FST to a level similar to
that seen in LAB rats. The reversal of the depression-like phenotype was
accompanied by a reduction of the high level of hypothalamic vasopressin
expression and normalization of the Dexamethasone-suppression/CRH-challenge
test (DEX/CRH test) described below (Keck et al. 2003a). However, paroxetine
did not alter vasopressin expression or any anxiety-related behavior assessed in
LABs, underlining the impact of the genetic predisposition to trait anxiety and
comorbid depression-like behavior on drug effects.
Recently, landmark studies have shown that inactivation of Brodmann Area 25

(BA25) using deep brain stimulation alleviated depressive symptoms in severely
depressed patients (Mayberg et al. 2005). Interestingly, transient pharmacological
inactivation using muscimol of the infralimbic cortex, the rodent correlate of
BA25, decreased the high inborn depression-like behavior of the HAB rats sup-
porting their face and predictive validity for affective disorders (Slattery et al.
2010). It remains to be determined whether this region may also be involved in
their innate anxiety phenotype.
Several lines of evidence resulting from both preclinical and clinical studies
support the view that repetitive transcranial magnetic stimulation (rTMS) of left
frontal brain regions exerts antidepressant effects (Post and Keck 2001). Thus,
acute, subchronic or long-term rTMS sessions reduced the duration of immobility
in rodents in the Porsolt swim test, exert neuroprotective effects both in vitro and
in vivo and change the expression of BDNF and cholecystokinin similar to those
reported after antidepressant drug treatment (Post and Keck 2001). To test for
rTMS efficacy in a psychopathological rat model, HAB and LAB rats received
stimuli over two 3-day series (Keck et al. 2001a). The stimulation point was set at
the left frontal cortex in order to mimic clinical conditions. Repetitive transcranial
magnetic stimulation increased active stress coping in HAB rats, rendering these
animals indistinguishable from LAB rats. The rTMS-induced shift in HAB animals
towards active stress coping was markedly higher than has previously been
reported in ‘‘normal’’ Wistar rats (Zyss et al. 1997). Furthermore, rTMS treatment
resulted in a significant attenuation of the neuroendocrine hyper-response of the
HPA axis to and acute ethologically relevant stressors characteristic for HAB rats,
whereas their high anxiety level remained unchanged.
Cognition
Emotionality and cognition are closely inter-related, as the assessment of envi-

ronmental stimuli, in particular of potentially dangerous situations, is dependent on
the acquisition and storage of information. To further test this hypothesis, the
cognitive performance of HAB and LAB rats has been estimated on the modified
holeboard, where HABs showed a clearly improved declarative memory perfor-
mance indicating a better learning strategy despite (or because of) a more passive
performance during the test (Ohl et al. 2002). No differences in working memory
in a visual-spatial task were found (Ohl et al. 2002).
In confirmation, line-dependent differences were also seen with respect to social
memory and cognition abilities, as HAB, but not LAB, males could distinguish
between a known and an unknown juvenile after a 30-min inter-exposure interval
(Landgraf and Wigger 2002). The lack of social preference seen in LABs (Lukas
and Neumann, unpublished) may underlie their impaired social memory.
To which extent the high central vasopressinergic drive ( Wigger et al. 2004;
Bosch et al. 2006; Bosch and Neumann 2010) contributes to the improved learning
and memory performance in HAB rats, remains to be shown. Brain vasopressin is
an important neuromodulator promoting cognitive functions (Engelmann et al.

1996) and may thus facilitate the storage of adverse emotional situations and shape
future stress coping.
Social Behaviors
Other behavioral differences, which have been established over the years of
selective breeding for high versus low trait anxiety, include a variety of social
behaviors (Neumann et al. 2010), which is of interest as several psychopathologies
often are accompanied by various abnormalities in social interactions including
aggression, social phobia, or impaired social bonding.
Inter-Male Aggression
Male HAB and LAB rats differ in inter-male aggression with LAB males showing
an extreme high level of offensive behavior in the resident-intruder test (Veenema
et al. 2007), a finding which is also reflected by an abnormal aggression of LAB
males displayed towards females and anesthetized males compared with NAB rats
(Neumann et al. 2010). In contrast, HAB males rather display an intermediate level
of intermale aggression (Beiderbeck et al. 2007; Neumann et al. 2010; Veenema
et al. 2007). Thus, the selective breeding for low trait anxiety resulted in a socio-
behavioral phenotype characterized not only by low anxiety and fear responses,
reduced risk assessment and an active stress coping style, but also by abnormal
social behaviors in different social settings, thus providing an excellent model for
studying mechanisms of pathological aggression.
Social Phobia
In a relevant behavioral setup for social phobia/social preference, NAB and HAB
rats show social preference. In this test, rats are allowed to explore a small wired
cage placed into the cage of the experimental rat. Social preference is seen, when
the animal explores the small cage longer and more often, when it contains a
conspecific animal. Here, LABs, in general, do not search for social contact,
display reduced contact to cage mates (Ohl et al. 2001) and do not show social
reference, which can also be interpreted as social phobia (Lukas and Neumann,
unpublished).
Maternal Care
Line-differences in social behavior are also found in females with respect to
maternal care and maternal defense behavior (maternal aggression). Here, HAB
dams are more protective towards their pups, leave the nest less often, show more
arched back nursing and more maternal aggression (Bosch et al. 2005; Bosch and
Neumann 2010; Neumann et al. 2005a). A significant contribution of high activity
of the brain vasopressin system seen in HAB dams to their maternal behavior
profile could recently be identified (Bosch and Neumann 2008, 2010). Interest-
ingly, a correlation between high maternal trait anxiety and the intensity of
maternal care has also been found in mouse dams bred for high and low anxiety
and, again, line-dependent differences in brain vasopressin appear to underlie the
behavioral differences (Kessler et al. 2011).
Pain
Similar to depressed patients, HAB rats have been shown to exhibit decreased
sensitivity to thermal pain (Jochum et al. 2007). Acute administration of diazepam
partly reversed the abnormal pain response. Also, treatment of male HAB rats with
the SSRI citalopram, daily for 8 weeks, reduced anxiety-related behavior as
assessed on the EPM and reversed the abnormal thermal pain response in HAB rats
(Jochum et al. 2007). As, in contrast to the thermal pain sensitivity, the sensitivity
to visceral pain has been found to be elevated in patients suffering from affective
disorders (Haug et al. 2004), it would be of interest to test visceral pain threshold
in HAB versus LAB rats.
Sleep
In depressed patients, sleep disturbances are a common symptom, as mentioned

above. Analysis of sleep patterns in HAB and LAB rats revealed similar circadian
fluctuation in sleep-wake behavior, but differences in their spontaneous sleep–
wake behavior. HAB rats spend less time awake and more time in non-REM sleep
(Lancel et al. 2002). This difference is particularly pronounced during darkness
and this is in accord with the observed decreased locomotor activity of HAB rats
during the nighttime (Liebsch et al. 1998b). The larger amount of non-REM sleep
in the HAB group was not associated with an increased length of non-REM
episodes, but with a greater number of sleep episodes, suggesting a higher non-
REM sleep fragmentation. HAB rats also displayed less pre-REM sleep and REM
sleep than LAB rats during the light period. Thus, the sleep pattern of HAB rats
seems to be opposite to that found in depression, and further studies are needed.
Hypothalamic-Pituitary Adrenal Axis
The behavioral differences between HAB and LAB rats are accompanied by
distinct neuroendocrine underpinnings. Although basal levels of plasma ACTH or
corticosterone reflecting basal activity of the HPA axis do not differ, the respon-
siveness of the HPA axis to a mild emotional (and non-social) stressor is more
pronounced in HAB compared with LAB males (and with non-selected Wistar
rats; Landgraf et al. 1999; Neumann et al. 2010), thus resembling psychiatric
patients (Holsboer 2000). However, the neuroendocrine response to social stimuli
is aggravated in LABs, paralleling their abnormal social behavioral responses
described above (Neumann et al. 2010; Veenema et al. 2007). HAB rats also show
an aberrant hormonal secretion pattern during the DEX/CRH test (Keck et al.
2002; Neumann et al. 2010), a clinical test used for the neuroendocrine charac-
terization of depressed patients (Ising et al. 2005). Intravenous administration of
DEX revealed DEX-non-suppression and, thus, impairment of negative feedback
regulation in HAB rats. Subsequent administration of CRH resulted in pronounced
ACTH and corticosterone responses, which are absent in LAB and NAB rats.
This indicates a contribution of endogenous vasopressin, which––together with
exogenous CRH––triggered pituitary secretion of ACTH despite (impaired) DEX-
suppression. Consequently, and in support of this hypothesis, an acute intravenous
administration of a vasopressin V1a receptor antagonist abolished the ACTH and
corticosterone hyper-response and normalized the DEX/CRH test outcome in
HAB rats, pointing towards a significant contribution of the endogenous brain
vasopressin system in these neuroendocrine abnormalities (Keck et al. 2002).
Furthermore, chronic administration of paroxetine over 8 weeks prevented the
abnormal DEX/CRH response in HAB rats with a concomitant attenuation of the
vasopressin hyperdrive in the hypothalamic paraventricular nucleus (PVN, Keck
et al. 2003b).
Neuronal Activity
In addition to neuroendocrine responsiveness, neuronal responses within brain

regions belonging to the anxiety/fear circuitry to anxiogenic, social or pharma-
cological stimuli also differ between HAB and LAB rats (Muigg et al. 2007;
Salchner et al. 2006; Salome et al. 2004; Frank et al. 2006). For example, in
response to airjet stimulation, an escape-provoking stimulus, HAB rats show a
higher neuronal activity in various hypothalamic areas including the medial pre-
optic and anterior hypothalamic areas, and in the nucleus accumbens as estimated
by quantification of the expression of the early immediate gene fos (Salome et al.
2004). Similarly, in response to forced swimming, neuronal responses within
selected cortical, septal, and hypothalamic areas were more pronounced in HAB
males. These neuronal responses could be attenuated after chronic paroxetine
treatment (Muigg et al. 2007), which could underlie the reduction in depression-
like behavior seen after antidepressive treatment in HAB rats.
Neuropeptides
Vasopressin
Given the importance of brain vasopressin in the regulation of anxiety, depression,
and neuroendocrine stress coping (Landgraf and Neumann 2004; Frank and
Landgraf 2008), the vasopressin gene was considered a candidate gene in high trait
anxiety. Indeed, high vasopressin mRNA expression within the parvocellular part
of the PVN both under basal conditions and in response to stressor exposure is a
reproducible characteristics for male and female HAB rats (Keck et al. 2003b;
Wigger et al. 2004; Bosch et al. 2006; Bosch and Neumann 2010; Frank and
Landgraf 2008). Consequently, increased vasopressin immunoreactivity and local
neuropeptide release were both found in the HAB hypothalamus. In addition,
vasopressin V1a receptor binding is also elevated within the lateral septum of
HAB rats, a region relevant for the regulation of anxiety and social behaviors
(Keck et al. 2003b).
In line with our hypothesis of a substantial contribution of high endogenous
vasopressin activity to the high anxiety and depression-like phenotype of HAB rats,
blockade of vasopressin V1a receptors within the hypothalamic PVN reduced their
anxiety level and resulted in a more active coping style (Wigger et al. 2004). Further,
the V1a antagonist (i.v.) normalized the pathological outcome of the DEX/CRH test
in male HABs (Keck et al. 2002). These findings, reflecting both construct and
predictive validity, confirm the involvement of endogenous central vasopressin in
the behavioral and neuroendocrine phenomena of high trait anxiety and depression.
Interestingly, line-dependent differences in brain vasopressin also appear to
contribute to differences in social behavior, i.e. intermale aggression as well as
maternal behavior (Beiderbeck et al. 2007; Veenema et al. 2007; Bosch et al. 2010;
Bosch and Neumann 2008).
Given the robust increase in brain vasopressin activity in HAB rats in parallel to
their anxiogenic and hyperresponsive neuroendocrine phenotypes, underlying
genetic mechanisms are likely. Indeed, 10 single nucleotide polymorphisms
(SNPs) within the vasopressin promoter were found between the lines. In addition,
a single base pair substitution has been identified in the first intron of the
vasopressin gene of HAB rats itself (Murgatroyd et al. 2004). One of the SNPs
identified was found to be embedded in a potential transcription factor binding
site (CArG box), the locus of binding to the transcriptional repressor CBF-A.
Functional relevance of the SNP was identified by in vitro DNA binding assay and
revealed that CBF-A binding to the CArG box derived from the HAB allele was
indeed diminished, resulting in an attenuated transcriptional repression of the
vasopressin gene. Thus, this genetic mechanism may underlie vasopressin over-
expression in the PVN of HAB rats (Murgatroyd et al. 2004; Landgraf et al. 2007).
Interestingly, out of 100 outbred Wistar rats, the HAB allele was found in 3 rats
(heterozygeous) indicating a gene frequency of 1.5% in the general Wistar rat
population.
In addition to vasopressin, other brain neuropeptides such as CRH, oxytocin,
prolactin, NPY, or neuropeptide S are important neuromodulators of emotionality,
in particular of anxiety- and depression-related behaviors. Thus, an endophenotype
of high or low anxiety accompanied by differences in active or passive stress
coping style is likely to be accompanied by differences in the activity of several
endogenous neuropeptide and other neurotransmitter systems.
Corticotropin-Releasing Hormone
Corticotropin-Releasing Hormone (CRH) exerts anxiogenic and depression-like
effects, and CRH mRNA expression has recently been found to be up-regulated
within the PVN of HAB rats compared with LAB rats (Bosch et al. 2006). In contrast,
line-dependent differences in CRH receptor binding could not be identified. Thus,

differences in endogenous CRH system activity are likely to contribute to the
emotional phenotype of HAB rats, as suggested in depressed patients (Nemeroff
2004; Keck and Holsboer 2001). Indeed, acute peripheral administration of the non-
peptide CRH 1 receptor antagonist R121919 reduced anxiety levels on the EPM in
HAB, but not LAB, rats and reduced stress-induced corticotropin secretion in both rat
lines (Keck et al. 2001b).
A high level of anxiety could also be due to an attenuation of endogenous
anxiolytic neuropeptides such as oxytocin (Neumann et al. 2000; Waldherr and
Neumann 2007), prolactin (Torner et al. 2001; Donner et al. 2007) and/or neu-
ropeptide S (NPS; Xu et al. 2004).
Oxytocin
Besides its capacity to modulate complex social behaviors, oxytocin is an estab-
lished anxiolytic neuropeptide of the brain (Blume et al. 2008; Neumann 2008)
and has antidepressive properties (Slattery and Neumann 2010). However, dif-
ferences in central oxytocin expression or release were not found between HAB
and LAB rats, except in lactation (Bosch et al. 2007).
In order to study potential anxiolytic or antidepressive effects of oxytocin in a
psychopathological animal model, HAB and LAB rats were treated i.c.v. with
either oxytocin or an oxytocin receptor antagonist (Slattery and Neumann 2010),
which was only effective in female but not male rats: chronic oxytocin reduced the
high anxiety level of HAB females on the EPM, whereas chronic i.c.v. treatment
with the oxytocin antagonist increased anxiety only in female LAB rats without
any effect in HABs or males. In contrast, acute manipulation of the oxytocin
system did not alter anxiety-related behavior independent of sex and trait anxiety.
Also, passive/active stress coping in the FST was not altered by any manipulation
of the oxytocin system. Thus, chronic oxytocin seems to be a promising thera-
peutic strategy in particular for the treatment of anxiety disorders in women.
Prolactin
Prolactin has distinct anxiolytic properties, and the brain prolactin is involved
in the regulation of anxiety and stress coping (Torner et al. 2004; Bunck et al.
2009; Ditzen et al. 2010). Plasma prolactin levels were found elevated in HAB rats
in response to a mild emotional stressor (Neumann et al. 1998; Landgraf et al.
1999). The behavioral significance of this finding needs to be studied, as plasma
prolactin does not reflect intracerebral prolactin release patterns (Torner et al.
2004), but peripheral prolactin can cross the blood brain barrier. However,
differences in brain prolactin expression, release or receptor binding have not been
studied in HAB and LAB rats until now.
Neuropeptide S
Neuropeptide S (NPS) is another powerful anxiolytic neuropeptide (Xu et al. 2004;
Leonard et al. 2008; Vitale et al. 2008), and our preliminary results indicate substantial
genetic and activity differences of the endogenous brain NPS system between HAB
and LAB rats. For example, the latter express more NPS receptors in the hypothalamus
(Slattery, Wegener, Naik, Mathé, Neumann, unpublished). In order to confirm the
anxiolytic properties described in non-selected male mice (Xu et al. 2004) and rats
(Vitale et al. 2008) in a psychopathological animal model, we acutely treated HAB and
LAB rats with i.c.v. NPS 45 min prior to EPM testing can (Slattery et al. 2008). Indeed,
preliminary evidence suggest that NPS reversed the high trait anxiety of HABs and
exerted modest antidepressant effects (Slattery et al. 2008). Further, i.c.v. NPS
improves consolidation of extinguished learned fear (Sartori et al. 2009).
Other Neurochemical Differences
Serotonin Disturbances in serotonergic neurotransmission are likely to contribute to

the pathophysiology of anxiety and depression disorders and to underlie the hyper-
activity of the HPA axis (Holsboer 2000). In HAB rats, serotonin 1A receptor
expression was found to be reduced in the hippocampus, whereas the expression of the
serotonin transporter binding sites was increased. Further, the basal availability of
extracellular serotonin as estimated in microdialysates did not differ between the lines,
but serotonin release in response to emotional stress was abolished in HAB rats.
Chronic paroxetine markedly increased the stress-induced rise in hippocampal sero-
tonin release, but did not alter receptor expression (Keck et al. 2005). Thus, the reduced
raphe-hippocampal serotonergic transmission of HAB rats, which is evident both at the
presynaptic (release) and postsynaptic (receptor) level, are likely to contribute to their
high emotionality.
Hippocampal Neurogenesis
Consistent with the generally elevated stress responsiveness including HPA axis
hyperreactivity and impaired negative feedback functions, we found reduced
hippocampal cell survival and neurogenesis in 43-days old male HAB rats
(Lucassen et al. 2009). Specifically, the number of newly generated surviving
(BrdU-positive) cells in the subgranular cell layer/subgranular zone of the hip-
pocampal dentate gyrus was found to be lower in HAB versus LAB rats. Further,
the number of hippocampal doublecortin-positive cells reflecting neurogenesis is
lower in HAB rats (Lucassen et al. 2009).
These results show that the high level of anxiety and activity of the HPA axis
may affect cell survival in HAB rats, which may, in turn, also be partly responsible
for their behavioral phenotype.
3.1.2 Gene 3 Environment Interactions
Early life stress, such as prenatal and immediately postnatal stress is a well-
characterized risk factor for the development of affective disorders in adulthood,
Fig. 5 Gene 9 early environment interactions shape adult behavioral and neuroendocrine stress
responses as seen in rats with genetic determination of trait anxiety
and there are well documented interactions of genetic and environmental factors
(Caspi et al. 2003; Fig. 5, see also Sect. 2.1.2). Selectively bred rodents with clear
genetic determinants are valuable models for studying gene 9 environment
interactions. Both prenatal stress as well as postnatal maternal separation resulted
in line-dependent behavioral effects seen in adult HAB and LAB rats.
Gene 9 prenatal environment interactions: Surprisingly, after exposure to pre-
natal stress between pregnancy days 4 and 18 (exposure of the pregnant dam to
maternal defeat by an unknown lactating resident daily for 45 min between preg-
nancy days 4 and 10, and to restraint between pregnancy days 11 and 18 for 60 min
daily) adult male HAB rats became less anxious (see Fig. 6). This was confirmed in
two independent tests for anxiety-related behavior, i.e. the EPM and the modified
holeboard (Bosch et al. 2006). The opposite behavioral consequences of prenatal
stress were accompanied by opposing effects on central vasopressin and CRH mRNA
expression: whereas the genetically determined high level of hypothalamic vaso-
pressin mRNA expression of HAB rats was not altered by prenatal stress, it was
elevated in early life stressed LAB male offspring (Fig. 6). Similarly, the genetic
difference in CRH expression within the PVN with high levels in unstressed HAB
compared with LAB controls was also at least partly abolished by prenatal stress.
Further, after prenatal stress the high HPA axis response to a mild stressor found in
HAB rats was reduced and found to be indistinguishable from unstressed and pre-
natally stressed LABs (Bosch et al. 2006).
Opposing effects of prenatal stress were also found with respect to hippocampal
neurogenesis, which has been shown to be stress-sensitive and implicated in
depression (Pittenger and Duman 2008). As mentioned above, the survival of
Fig. 6 Opposite effects of prenatal stress on anxiety-related behavior on the EPM (% entries
open arms; left) and basal vasopressin mRNA expression in the hypothalamic PVN (right) in
male HAB and LAB adult offspring indicating gene 9 early environment interactions. # versus
LAB; * versus respective control. Adapted from (Bosch et al. 2006)
newly generated hippocampal cells is lower in young unstressed HAB compared

with LAB males (day 43 of life). Interestingly, while prenatal stress caused a
further reduction in the number of BrdU and doublecortin positive cells in the
subgranular zone of the hippocampal dentate gyrus in HAB rats, it did not affect
this parameter in the LAB rats (Lucassen et al. 2009). Although detailed mecha-
nisms underlying the opposing effects of prenatal stress in HAB and LAB rats
are unknown, line-dependent differences in the activity of the placental enzyme
11-beta hydroxysteroid dehydrogenase type 2, which catalyzes maternal cortico-
sterone to inert 11-dehydrocorticosterone are likely to contribute (Lucassen et al.
2009).
Gene 9 postnatal environment interactions: Opposite effects on emotionality
and neuroendocrine responsiveness and, consequently, approximation of the HAB
and LAB behavioral and neuroendocrine phenotypes were also found after
immediate postnatal stress, i.e. after maternal separation (Fig. 7). Daily 3-h
separation of HAB and LAB offspring from the mother between postnatal days 2
and 15 reduced anxiety in adult HABs as seen on the modified holeboard, but
rather increased (EPM) or had no effect (holeboard) in LABs (Neumann et al.
2005b). Further, the HPA axis hyper-responses seen in HAB control rats became
attenuated after postnatal stress, whereas maternal separation did not significantly
alter neuroendocrine responses in LAB rats (Neumann et al. 2005b; Fig. 7).
These experiments underline that the consequences of environmental factors
are strongly determined by the genetic background. Vice versa, even a robust
genetically determined individual behavioral phenotype can be shaped by envi-
ronmental factors; likely via epigenetic mechanisms (Murgatroyd et al. 2009).
Fig. 7 Opposite effects of postnatal stress (periodic maternal separation; PMD) on anxiety-
related behavior on the EPM (% time open arms; left) and plasma corticosterone response to
novel environment exposure (elevated platform, right) in HAB and LAB adult offspring
indicating gene 9 early environment interactions. # versus LAB; * versus respective control.
Adapted from (Neumann et al. 2005b)
The mitigating effect of early life experiences on behavioral and neuroendocrine

parameters in rats representing extremes in trait anxiety may also reflect an evo-
lutionary benefit, as the genetic variability among individuals of a species is
sustained, while maintaining adequate responses to potentially dangerous stimuli
in adulthood. Our results in HAB and LAB rats after pre- and postnatal stress
exposure further indicate that gene 9 environment interactions can be found at
behavioral, neuroendocrine, neuronal and gene levels indicating their complexity.
3.2 HAB LAB Mice
Although various selectively bred rat lines are useful tools for studying behavioral
and especially neuroendocrine parameters of depression and anxiety, as well as
environmental factors shaping innate stress coping style, genetic studies such as
the functional analysis of candidate genes underlying, for example, high trait
anxiety or depression-related behavior, are a priori limited in rats. Therefore,
Landgraf and co-workers also generated mice selectively bred for high (M-HAB)
versus low (M-LAB) anxiety-related behavior (Kromer et al. 2005). After 9
generations of continuous breeding (sibling mating), a robust behavioral diver-
gence had been achieved. High trait anxiety has been confirmed on the EPM and in
the light–dark box; further, M-HAB mice pups show more ultrasound vocalization,
which was reversed by diazepam (Kromer et al. 2005). In agreement to what has
been found in HAB and LAB rats, M-LAB mice are more active in several tests for
depression-like behavior (FST, tail suspension). Also, as seen in HAB and LAB
rats, differences in the hypothalamic expression of vasopressin are likely to

underlie their trait anxiety and also line-dependent social behavior (Kessler et al.
2011), but here M-LABs show signs of central diabetes insipidus, i.e. low vaso-
pressin expression and availability compared with M-HAB and non-selected CD1
mice (Kessler et al. 2007). A SNP in exon 1 of the vasopressin gene of LAB mice
causes an amino acid substitution in the signal peptide of the vasopressin pre-
cursor, and is likely to impair processing and trafficking of the precursor (Bunck
et al. 2009; Kessler et al. 2007). Besides vasopressin, differences in the expression
of the cytosolic enzyme glyoxalase-I, which is of potential interest in the context
of various psychopathologies, have been found in these mice (Kromer et al. 2005;
Hambsch et al. 2010). Thus, selectively bred mice have a high potential to reveal
novel candidate genes underlying high trait anxiety.
3.3 Floripa H and L Rats
The Floripa H and L rat lines, have been developed based on selection for high and
low locomotion in the central aversive area of an open field, an experimental
measure of fearfulness in rodents (Ramos and Mormede 1998). The Floripa H and
L lines differ from each other not only for the selected behavior, but also for other
experimental indices of anxiety, such as the approach towards the open arms of the
elevated plus maze and the white compartment of the black/white box (Ramos
et al. 2003). In addition, compared with Floripa L, the Floripa H rats show less
depression-like behavior in the FST (Hinojosa et al. 2006), suggesting the Floripa
rats to be a combined model of both anxiety and depression. The Floripa L female
rats consumed more ethanol than their Floripa H counterparts at concentrations of
6 and 10% in a two-bottle choice protocol (Izídio and Ramos 2007), however the
connection to the anxiety-like phenotype remains to be determined.
3.4 Maudsley Reactive and Nonreactive Rats
Based on reactivity in the open field, with defecation and urination being the
central variables, two lines of rats, later termed the Maudsley high reactive (high
defecation, MHR) and low, nonreactive (low defecation, MLR) rats, were bred
from Wistar in the 1950s aiming to model the human personality dimension of
emotionality (Broadhurst 1957, 1960, 1962, 1975). The overall conclusion of the
several studies published were that Maudsley reactive rats yielded higher scores in
several tests of anxiety-like/avoidance behavior than non-reactive animals (Blizard
and Adams 2002). Although this approach was useful to study individual differ-
ences in anxiety-like/avoidance behavior, it has not been pursued intensively in
recent years (Pawlak et al. 2008; Blizard and Adams 2002).
3.5 High/Low Avoidance Rats: RHA/RLA; SHA/Bru

and SLA/Bru
Several rat breeding lines have been selected for learning to actively avoid foot
shocks in a two-way shuttle-box. The Roman high avoidance (RHA) and
Roman low avoidance (RLA) Wistar rats (Bignami 1965), the Long–Evans rats
(Brush et al. 1979, 1985, 1989; Brush 2003), and Syracuse high and low
avoidance rats (SHA/Bru and SLA/Bru) are the most prominent examples.
Although these attempts differ in nature, some of the common characteristics
include differences in learning and memory, and a number of emotion-related
behavioral and neuroendocrine characteristics. A detailed review can be found
in (Brush 2003).
4 Conclusions
In conclusion, the existing data on selectively bred rodent models, in particular

of the FSL/FRL and HAB/LAB rats reviewed above, reveal the importance of
rodent breeding lines for studying neurobiological, neuroendocrine and genetic
mechanisms underlying anxiety- and depression-related diseases. Essentially,
the development of potentially novel therapeutic strategies targeting brain
neuropeptide systems such as NPY, vasopressin, CRH, oxytocin or neuropep-
tide S will be enabled and promoted using such relevant and complementary
animal models. Moreover, selectively bred rat and mouse lines provide an
important tool in order to provide further evidence for gene 9 environment
interactions demonstrating differential vulnerabilities, for example to prenatal or
immediate postnatal adverse life events. The combination of genetic models
with various stress paradigms is likely to mimic the human situation more
accurately.
Given their behavioral and neuroendocrine phenotype, the neurobiological
mechanisms underlying their anxiety- and depression-related behavior, as well as
successful pharmacological attempts to reverse the psychopathological phenotype,
the FSL/FRL and HAB/LAB rats fulfill the requirements of face, construct and
predictive validity of an animal model. Therefore, they should be further exploited
to discover potential novel therapeutic strategies.
Acknowledgments GW was supported by grants from The Danish Medical Research Council
(grant 271-08-0768) and the Research Foundation of County Midtjylland. AAM was supported
from the Swedish Medical Research Council (grant 10414) and the Karolinska Institutet. IDN
was supported by DFG, BMBF and Bayerische Forschungsstiftung.
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Stress-Induced Deficits in Cognition
and Emotionality: A Role for Glutamate
Carolyn Graybeal, Carly Kiselycznyk and Andrew Holmes
Abstract Stress is associated with a number of neuropsychiatric disorders, many

of which are characterized by altered cognition and emotionality. Rodent models
of stress have shown parallel behavioral changes such as impaired working
memory, cognitive flexibility and fear extinction. This coincides with morpho-
logical changes to pyramidal neurons in the prefrontal cortex, hippocampus and
amygdala, key cortical regions mediating these behaviors. Increasing evidence
suggests that alteration in the function of the glutamatergic system may contribute
to the pathology seen in neuropsychiatric disorders. Stress can alter glutamate
transmission in the prefrontal cortex, hippocampus and amygdala and altered
glutamate transmission has been linked to neuronal morphological changes. More
recently, genetic manipulations in rodent models have allowed for subunit-specific
analysis of the role of AMPA and NMDA receptors as well as glutamate trans-
porters in behaviors shown to be altered by stress. Together these data point to a
role for glutamate in mediating the cognitive and emotional changes observed in
neuropsychiatric disorders. Furthering our understanding of how stress affects
glutamate receptors and related signaling pathways will ultimately contribute to
the development of improved therapeutics for individuals suffering from neuro-
psychiatric disorders.
Keywords Glutamate Stress Emotionality Cognition
C. Graybeal C. Kiselycznyk A. Holmes (&)

National Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, 5625 Fishers Lane, Room 2N09,
Rockville, MD 20852-9411, USA
e-mail: holmesan@mail.nih.gov

DOI: 10.1007/7854_2011_193
190 C. Graybeal et al.
Contents
1 Introduction........................................................................................................................ 190
2 Stress-Induced Executive Dysfunction in Humans and Rodents .................................... 190
3 Structural Changes in Response to Stress ........................................................................ 193
4 Molecular Mechanisms of Structural Changes: Focus on Glutamate ............................. 195
5 Glutamate in Rodent Behavioral Correlates of Emotion and Cognition ........................ 196
6 Conclusions........................................................................................................................ 199
References................................................................................................................................ 200
1 Introduction
Stress is associated with a number of neuropsychiatric disorders either as a trigger,

as with post-traumatic stress disorder (PTSD), or a risk factor, as with drug
addiction or depression. Thus understanding the neurobiological consequences of
stress is critical to treating these diseases. The first part of this chapter focuses on
behavioral changes in animal models of stress and structural changes in key
cortical regions mediating these behaviors. The second part focuses on the role of
the glutamatergic system in mediating these changes and, through genetic
approaches, some of the advances made in our understanding of the molecular
underpinning of stress and mental disorders.
2 Stress-Induced Executive Dysfunction

in Humans and Rodents
Stress is a known risk factor of a number of neuropsychiatric disorders such as

depression, PTSD and addiction (Hammen 2005; Lupien et al. 2009; Schneider-
man et al. 2005; Sinha 2008). Stress can be defined as the expense or ‘‘allostatic
load’’ on the organism’s homeostatic regulating system (McEwen 2000a). When
faced with a stressor, the hypothalamic-pituitary-adrenal (HPA) axis is activated,
resulting in the release of corticosteroids (de Kloet et al. 2005; Joels et al. 2007;
McEwen 2000b). Though short bouts of stress can be tolerated or even be bene-
ficial, prolonged exposure to stress can be detrimental (McEwen 2000a). Early life
stress, such as an unstable childhood, is a strong predictor for the development of
depression or addictive behavior in adulthood (Lupien et al. 2009). A stressful life
event, a death in the family or chronic stress such as poverty is strongly linked with
the development of PTSD, depression and alcoholism (Hammen 2005; Schnei-
derman et al. 2005; Sinha 2008).
Characteristic of these stress-related neuropsychiatric disorders are deficits in
emotional regulation and cognition (Ferreri et al. 2011). Patients with panic dis-
order or PTSD are unable to normally regulate their emotions. These individuals
Stress-Induced Deficits in Cognition and Emotionality 191
express greater aversive feelings toward negative stimuli, find themselves preoc-
cupied with worry and are impaired in their ability to suppress feelings of fear
(Blechert et al. 2007; Michael et al. 2007). Individuals with attention deficit
hyperactivity disorder (ADHD), depression or schizophrenia have deficits in cer-
tain learning and memory functions such as spatial working memory and cognitive
flexibility (McLean et al. 2004; Murphy et al. 2003; Murray et al. 2008; Rogers
et al. 2004; Waltz and Gold 2007). Using various stress paradigms, animal models
have begun to show evidence for a direct link between stress and emotional or
cognitive dysfunction.
In rodents, exposure to stress can result in increased anxiety- and depression-
related behaviors (Sterner and Kalynchuk 2010). Adult rodents that experienced
maternal separation as pups, a model of early life stress, have heightened corti-
costerone secretion following stress and show greater anxiety in the elevated plus-
maze (e.g. Eiland and McEwen 2012; Holmes et al. 2005; Huot et al. 2001).
Rodents exposed to chronic unpredictable stress (CUS) or repeated restraint stress,
or simply injected with corticosterone, show elevated anxiety- like behaviors in the
light/dark exploration and the elevated plus-maze test, and a heightened startle
response relative to non-stressed controls (e.g. Mozhui et al. 2010; Pego et al.
2008). Cross-strain comparisons indicate the effect of stress is more pronounced in
mouse strains with greater trait anxiety-like behavior, suggesting genetic back-
ground influences stress susceptibility (Mizoguchi et al. 2000). In addition to
heightened anxiety, stress can evoke a behavioral profile reminiscent of depres-
sion. Rodents exposed to a stressor or those that received corticosterone orally or
systemically show reduced social interaction and increased behavioral despair (e.g.
Berton et al. 2006; Gourley et al. 2008; Shirayama et al. 2002; Wood et al. 2008).
History of maternal separation or oral corticosterone delivery can decrease sucrose
consumption or responding in an appetitive progressive ratio test, measures of
anhedonia (Gourley et al. 2009, 2008; Huot et al. 2001).
Also sensitive to stress are fear conditioning and extinction, measures of
emotional learning and regulation (Rodrigues et al. 2009). Stress prior to condi-
tioning has a potentiating effect on fear learning resulting in greater freezing
during fear conditioning and post-conditioning fear recall tests (Rau et al. 2005;
Sandi et al. 2001; Wood et al. 2008; Yamamoto et al. 2009). A series of experi-
ments examining the effect of footshock stress prior to fear conditioning found that
stressed rats showed enhanced fear recall when tested the following day (Rau et al.
2005). Using variants of this basic design, fear generalization and reinstatement
were ruled out as alternate explanations for the increase in fear. Rather it appears
that stress strengthens the fear memory, which may explain the impairing effects of
stress on fear extinction, the learned suppression and the fear response (Corcoran
and Quirk 2007; Quirk and Mueller 2008). Rats exposed to restraint stress or given
oral corticosterone fail to extinguish the fear response, retaining a greater level of
freezing after extinction training as compared to non-stressed subjects (Baran et al.
2009; Gourley et al. 2009; Miracle et al. 2006). In mice, a mere three days of
10 min forced swim stress was sufficient to impair fear extinction (Izquierdo et al.
2006). Stress does not have to immediately precede fear conditioning to affect
learning. Rat pups exposed to five days of footshock stress and then tested on fear
conditioning as adults fail to extinguish the fear response showing that stress can
have long-lasting repercussions on cognition (Judo et al. 2010).
The effects of stress are not limited to cognitive processes associated with
emotionality (Arnsten 2009; Holmes and Wellman 2009; Sterner and Kalynchuk
2010). Working memory, the ability to transiently store, recall and utilize recently
learned information is vulnerable to stress. Mice exposed to four weeks of cold
water submersion are impaired in a delayed alternation T-maze task (Mizoguchi
et al. 2000). In rats, four weeks or merely six days of CUS result in impaired
spatial working memory in the Morris water maze (MWM) (Cerqueira et al. 2005).
This deficit is replicated by four weeks of daily injections of dexamethasone, an
agonist of the glucocorticoid receptor, one of the two main corticosterone recep-
tors, suggesting that the effects of stress are mediated at least in part by gluco-
corticoid receptor activity (Cerqueira et al. 2005).
Spatial learning itself is disrupted by stress. Rats that experienced maternal
separation were impaired in an object placement task which examines the ability to
discriminate between objects placed in the same or a novel location (Eiland and
McEwen 2012). A single session of restraint stress or a corticosterone injection
resulted in mice having longer escape latencies in circular hole board maze
(Schwabe et al. 2010). In the hidden platform version of the MWM, tail shock
stressed rats had greater escape latencies than non-stressed rats (Kim et al. 2001).
Predator stress, where the rodent is exposed to either the odor of or an actual
predator, impairs spatial working memory in the radial arm version of the MWM
(RAWM) (Diamond et al. 1999; Park et al. 2008). In the RAWM, up to six swim
paths are available from a center start point, one of which leads to the escape
platform. An effect of stress was not present when just four arms were available.
However, increasing the difficulty of the task by presenting six arms reveals an
effect of stress, suggesting that observable effects of stress may be dependent on
task demands (Diamond et al. 1999). Pretreatment with the antidepressants ago-
melatine or tianeptine can rescue this effect (Campbell et al. 2008; Conboy et al.
2009).
Stress also interferes with cognitive flexibility, the ability to adjust previously
learned behavior in response to changing task demands. Rats subjected to either
four weeks of CUS or dexamethasone injections or six just days of CUS were
impaired on a spatial reversal variant of the MWM, where the escape platform is
switched to the quadrant opposite from where it was located during training
(Cerqueira et al. 2007; Cerqueira et al. 2005). Two weeks of CUS impaired
reversal and extra-dimension set shifting in a texture-odor attention set-shifting
task (Bondi et al. 2008). In this rodent adaptation of human cognitive flexibility
tasks, the rodent learns to discriminate between digging textures or odors to locate
a hidden reward. This stress-induced set-shifting impairment is robust, having
been replicated with a number of different stress protocols (Bondi et al. 2008;
Lapiz-Bluhm et al. 2009; Liston et al. 2006) and has been shown to be rescued
with the antidepressants desipramine and citalopram (Bondi et al. 2008;
Lapiz-Bluhm et al. 2009).
From the subset of work highlighted here, there is strong evidence to support
the link between stress exposure and altered emotionality and cognitive function.
Similar to what is seen in clinical populations, in rodent models stress increases
anxiety- and depression-like behaviors, and facilitates the acquisition of fear while
impairing control over fear expression. Stress also impairs certain cognitive
functions such as spatial learning, working memory and cognitive flexibility. How
stress may be affecting these changes via morphological and molecular alterations
will be discussed in the following sections.
3 Structural Changes in Response to Stress
Key substrates mediating emotionality and cognition include the prefrontal cortex,
hippocampus and amygdala. Changes in cortical volume have been recorded in
these regions in human neuropsychiatric patients suggesting possible loci for the
behavioral and cognitive pathologies (Sterner and Kalynchuk 2010; van Harmelen
et al. 2010). These regions are anatomically interconnected, often working in
concert to mediate emotional regulation and cognition (Thierry et al. 2000).
Preclinical studies in rodents have not only shown parallel changes but have
provided more precise information on morphological changes in these areas.
Loss of prefrontal cortex (PFC) function can impair working memory and
cognitive flexibility as well as other higher-order cognitive functions (Chudasama
and Robbins 2006; Dalley et al. 2004; Robbins 2007). In rodents, the prelimbic
and infralimbic cortices are subregions of the PFC thought to mediate fear
expression and fear extinction, respectively (Corcoran and Quirk 2007; Myers and
Davis 2007; Quirk and Mueller 2008). Four weeks of CUS result in neuronal
atrophy, specifically reduced volume, neuron number and apical dendritic length in
layers I–III in both these regions (Cerqueira et al. 2005; Dias-Ferreira et al. 2009).
This stress effect is replicated pharmacologically with four weeks of corticosterone
or dexamethasone, a corticosterone-receptor agonist, injections (Cerqueira et al.
2005). In addition to neuronal atrophy, decreases in spine density and spine surface
area have been observed in the medial PFC following exposure to three weeks of
restraint stress (Liston et al. 2006; Radley et al. 2004, 2006, 2008). Interestingly,
while restraint stress reduced apical dendritic length in the medial PFC, there was a
corresponding increase in the orbitofrontal cortex, suggesting that subregions of
the PFC may respond differently to stress (Liston et al. 2006). Shorter stress
exposures have highlighted the potential sensitivity of the PFC to stress. A reduced
restraint stress paradigm, ten days of two-hour restraint stress, reduced apical
dendritic length in rat infralimbic cortex (Shansky and Morrison 2009). Seven days
of only ten-minute daily restraint stress was effective in reducing apical branch
number and length in layer II/III of the cingulate cortex (Brown et al. 2005). Even
more limited, one bout of ten-minute forced swim stress was suffient to reduce
apical dendritic length in layer II/III of the mouse infralimbic cortex though no
change was seen in the prelimbic cortex (Izquierdo et al. 2006).
Also key in the fear learning circuitry are the hippocampus and amygdala (Maren
and Quirk 2004). The hippocampus has a well-established role in learning and
memory and is particularly important for spatial learning, as in the MWM or with
contextual fear learning (Bird and Burgess 2008; Ji and Maren 2007; Maren and Holt
2000). Stress has a similar effect on morphology in the hippocampus as it does in the
PFC (Joels et al. 2004; McEwen 2001). Five weeks of social conflict decreased cell
proliferation and survival in the rat dentate gyrus, an effect rescued by concomitant
fluoxetine treatment (Czeh et al. 2007). Rats exposed to either a brief (two days) or
chronic (21 days) social stressor induced morphology changes, though changes were
more robust following chronic stress (Kole et al. 2004). Four weeks of CUS or
pharmacological stress by corticosterone or dexamethasone injections resulted in
reduced CA3 and DG volume and decreased dendritic length of granule cells, CA3
and CA1 pyramidal cells (Cerqueira et al. 2007; Sousa et al. 2000). Shorter durations
of stress, 21 days of restraint stress or ten days of CUS, also results in hippocampal
dendritic retraction which was reversed when tianeptine was given in conjunction
with stress (Vyas et al. 2002; Watanabe et al. 1992b).
In contrast to the PFC and hippocampus, stress induces neuronal hypertrophy in
the amygdala, a region essential for the acquisition and consolidation of aversive
memories (Maren and Quirk 2004; Pare et al. 2004; Roozendaal et al. 2009). While
21 days of restraint stress decreased hippocampal dendritic length, this same stressor
increased dendritic length and branching in neurons of the basolateral amygdala
(BLA) as well as the bed nucleus of stria terminalis (Vyas et al. 2002, 2003). This
effect has been replicated with either CUS or corticosterone injections which lead to
increases in dendritic length and spine density in the BLA and bed nucleus of stria
terminalis (Pego et al. 2008). Stress-induced changes appear to be longer lasting in
the amygdala than in the hippocampus or PFC. Following 21 days of recovery from
stress, BLA pyramidal cells of stressed rats were still longer than controls, whereas
CA3 hippocampal neurons were no different and overextension was seen in proximal
dendritic arbors of IL neurons (Goldwater et al. 2009; Vyas et al. 2004).
As with behavior, stress results in different changes in neuronal structure in the
PFC, hippocampus and amygdala. Generally, stress-induced dendritic retraction in
the PFC and hippocampus but induced extension in the amygdala. This appears to
align with the differing effects of stress on behavior, with impairments in largely PFC
and hippocampus-associated behaviors and enhancements in amygdala-associated
behaviors. However, it is unlikely that structural changes alone are the cause of
stress-induced behavioral changes. More likely stress results in a cascade of changes
on a molecular level of which modification to structure is an observable consequence.
4 Molecular Mechanisms of Structural Changes: Focus

on Glutamate
It is clear that exposure to stress can alter neuronal morphology in regions asso-
ciated with the emotional and cognitive changes seen with psychiatric disorders.
Stress can alter multiple molecular signaling cascades that could explain the
observed structural changes (a subset of these are reviewed in Pittenger and

Duman (2008). Here, we focus on evidence for the role of the glutamatergic
system in mediating stress-induced changes because of its known role in mecha-
nisms of plasticity, cognition, and increasingly, emotional and psychiatric
disorders.
Glutamate is the main excitatory neurotransmitter in the CNS and binds to
multiple metabotropic receptors and three families of ionotropic receptors: alpha-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, kainate
receptors and N-methyl-D-aspartate (NMDA) receptors. Activity-dependent
alterations in glutamate transmission are a key mechanism of synaptic plasticity
that in turn influences multiple downstream signaling pathways, including those
activating neurotrophic factors involved in cell growth. Activation of synaptic
NMDA receptors appears to be crucial for activity-dependent changes in synapses
and even neuronal survival (Hardingham and Bading 2003). Changes in synaptic
plasticity have been shown to influence spine morphology, as LTP-inducing
protocols can lead to new dendritic spines (Engert and Bonhoeffer 1999; Matsu-
zaki et al. 2004). Conversely, elevated glutamate levels and excessive activation of
NMDA receptors can lead to cell damage and even death in many neuropatho-
logical conditions, (Dirnagl et al. 1999; Lipton and Rosenberg 1994; Olney et al.
1986; Sapolsky 2000a, 2000b). The opposing effects of glutamate transmission
most likely depend on the pattern of NMDA receptor activation and have been
reviewed in (Hardingham and Bading 2003). Variations in the pattern of glutamate
receptor activation could in part explain a shift in the activation of cell growth
versus cell death pathways that influence the observed morphological changes.
Stress is known to lead to changes in glutamate transmission in the same
regions showing stress-induced morphological changes. Various forms of stress as
well as acute injections of corticosterone lead to a rapid and transient increase in
extracellular glutamate in the hippocampus and PFC (Moghaddam 1993; Venero
and Borrell 1999). Interference with glutamate signaling using glutamatergic
receptor antagonists can attenuate the dendritic atrophy observed after chronic
restraint stress. The anti-epileptic drug phenytoin is known to reduce excitatory
amino acid release including glutamate and injections of phenytoin block stress-
induced atrophy of the CA3 hippocampal neurons (Watanabe et al. 1992a). More
specifically, blocking NMDA but not AMPA receptors attenuates CA3 dendritic
atrophy suggesting that the NMDA receptor has a particular role in mediating the
morphological changes seen with stress (Magarinos and McEwen 1995).
In the PFC, less is understood about how NMDA receptor antagonists alter
stress-induced atrophy. However, lesions resulting in cholinergic deafferentation
lead to dendritic remodeling and increased spine density in the PFC, an effect
shown to be dependent on NMDA receptors (Garrett et al. 2006). Interestingly,
systemic administration of the NMDAR antagonist CPP during chronic restraint
stress not only blocks dendritic atrophy in the mPFC, but actually induces den-
dritic hypertrophy in this region, an effect not observed in unstressed control rats
(Martin and Wellman 2011). Most recently, the NMDA receptor antagonist ket-
amine was found to block the loss of synaptic proteins in the PFC after stress
induced by the learned helplessness paradigm (Li et al. 2010). NMDA receptor
antagonists not only block stress-induced atrophy but have also been found to
increase spine density in medial PFC pyramidal cells 24 h after administration in
the absence of stress (Li et al. 2010). While additional data directly studying the
role of glutamate transmission in each region showing stress-induced structural
changes is sparse, much is known about the effects of glutamate transmission in
cognitive and emotional behavioral paradigms mediated by these regions.
5 Glutamate in Rodent Behavioral Correlates of Emotion

and Cognition
The NMDA receptors are heteromeric assemblies composed of an obligatory NR1

subunit and one or more NR2 (NR2A-NR2D) (Rosenmund et al. 1998) or NR3
subunits (Ciabarra et al. 1995). Postmortem studies of the brains of depressed
patients have found reduced NR1 mRNA in the hippocampus (Law and Deakin
2001) and decreased NR2A and NR2B protein levels in the PFC (Feyissa et al.
2009). In rodent models, a single exposure to forced swim stress leads to increases
in surface (but not total) NR1, NR2A, NR2B and AMPA receptor subunits in the
PFC (Yuen et al. 2009). As mentioned earlier, NMDA, but not AMPA receptor
antagonism, attenuates dendritic atrophy resulting from chronic stress or gluco-
corticoid injections (Magarinos and McEwen 1995). Similarly, NMDA receptor
antagonists block stress-induced alterations in long-term potentiation and long-
term depression in hippocampal CA1 neurons (Kim et al. 1996). Together, NMDA
receptors appear to be modulated by stress and necessary for some stress-induced
morphological changes. Further evidence has demonstrated that NMDA receptors
are also involved in the cognitive and emotion-related paradigms affected by
stress.
Genetic manipulations in rodent models have allowed for a subunit-specific
analysis of the NMDA receptor’s role in behaviors shown to be altered by stress.
While there are no NR2A subunit-specific antagonists, a mutant mouse line
lacking this subunit has been developed and displays decreased anxiety- and
depression-related behaviors (Boyce-Rustay and Holmes 2006). While wild-type
mice normally show restraint stress-induced changes in the light/dark exploration
task, this effect was blocked or even reversed in NR2A knockout mice, high-
lighting the importance of NR2A in behavioral changes consequential to stress
(Mozhui et al. 2010). Supporting the importance of NMDA receptors in mor-
phology, these knockout mice also demonstrated decreased spine density on
pyramidal neurons of the BLA (Mozhui et al. 2010). As mentioned earlier, stress-
induced increases in anxiety-like behavior are often paralleled by increases in
BLA spine density. The loss of spine density in the BLA NR2A KO mice could in
part explain their reduced anxiety-like behavior. Future studies could evaluate if
the increase of spines after stress is also blocked in these mice, and if local NR2A
knockdown in the BLA still blocks the stress-induced behavioral changes in the
light/dark exploration task.
Prenatal deletion of the NR1 or NR2B subunit is lethal. However, several
genetic models with reduced NR1 or NR2B levels have been successfully used.
With the development of a floxed NR1 mouse line, it has been possible to study the
effects of NR1 deletion in specific regions of the brain. One such model limited
NR1 deletion to hippocampal CA1 pyramidal cells and found a deficit in trace, but
not delay, fear conditioning and impaired spatial (MWM, T-maze), but not non-
spatial memory (Huerta et al. 2000; Tonegawa et al. 1996; Tsien et al. 1996a,
1996b). Mice with a deletion of NR1 restricted to hippocampal CA3 pyramidal
cells are impaired in retention of one-trial context discrimination at three h, but not
24 h after avoidance training (Cravens et al. 2006). Similar results were found in
mice with the NR2B subunit deleted in CA1 and cortical pyramidal cells (Brigman
et al. 2010). Mice lacking the NR2B subunit on pyramidal cells of the cortex and
CA1 also showed multiple learning and memory deficits, including in the MWM,
T-maze and trace, but not delay fear conditioning. These same mice additionally
showed decreased spine density in pyramidal cells of the CA1. While they dem-
onstrated normal depressive-like behavior in the six-minute forced swim test, these
mice developed less depressive-like behavior over the course of a novel 10 day
swim stress paradigm, indicating a phenotype specific to repeated stress
(Kiselycznyk et al. 2011).
Ketamine and other NMDA receptor antagonists have acute behavioral effects
in humans and rodent models that appear to resemble many of the symptoms of
schizophrenia. This has in part led to the NMDA receptor hypofunction hypoth-
esis, suggesting that NMDA receptor hypofunction on inhibitory neurons in the
PFC is responsible for excessive neuronal excitability in this region thus disrupting
executive functions. Mice that are viable with only 5–10% of the normal NR1
levels have been used to test the relationship between NR1 subunits and schizo-
phrenia. These mice have decreased prepulse inhibition, sociability and anxiety-
like behavior but increased locomotion, a constellation of behaviors similar to
those seen in patients (Halene et al. 2009). A recent study found that deletion of
NR1 on interneurons during early postnatal development resulted in schizophre-
nia-like phenotypes in mice. This included novelty-induced hyperlocomotion,
impaired nest building and mating, increased anxiety- and anhedonia-like
behaviors, behaviors that were exacerbated by social isolation stress (Belforte et al.
2010). The schizophrenia-like behavior was selective to mice with early postnatal
deletion (approximately equivalent to late gestation to age 2 in humans) and was
not observed in mice with post-adolescence NR1 deletion. This is consistent with
hypotheses proposing a developmental cause in schizophrenia, with stress exac-
erbating deficits in cortical development.
An additional post-synaptic receptor of glutamate, the AMPA receptor, is also
essential for synaptic plasticity. The AMPA receptor is a heteromeric receptor
composed of a combination of four subunits: GluR1-GluR4. Various anti-
depressants with diverse structures have been found to alter the phosphorylation
of GluR1 receptors, therefore altering glutamatergic synaptic transmission
(Svenningsson et al. 2007). In schizophrenia, studies have found decreased GluR1

expression in the hippocampus, striatum and PFC that is reversed by treatment
with neuroleptics (Sokolov 1998). Mice lacking the GluR1 subunit display nov-
elty-induced hyperlocomotion that is reversed by the antipsychotic haloperidol
(Wiedholz et al. 2008). These mice also demonstrated altered social behavior and
deficits in prepulse inhibition and impairments in a spatial reversal learning task.
In additional studies, GluR1 KO mice were found to demonstrate behaviors
indicative of mania-related phenotypes as they also showed stress-induced
hyperactivity, reduced immobility in the forced swim test, and alterations in
approach/avoid conflict tests. Treatment with lithium reversed the KO’s anxiety-
like phenotype and partially reversed their stress-induced hyperlocomotion
(Fitzgerald et al. 2010). However, deletion of GluR1 did not affect the stress-
induced changes in light/dark exploration, as was observed in NR2A knockout
mice (Mozhui et al. 2010). As mentioned earlier, AMPA receptor antagonists
alone do not block stress-induced changes in morphology as with the NMDA
antagonists (Magarinos and McEwen 1995). While this appears contradictory, it is
likely that activation of these two types of glutamatergic receptors activate a
different collection of downstream signaling pathways (such as calcium-activated
pathways), and there are some pathways specific to NMDA receptors that explain
stress-induced changes.
The amount of glutamate available to NMDA and AMPA receptors is partially
regulated by reuptake with glutamate transporters (EAAT1-5 in humans) located
both on neurons and neighboring glial cells (Anderson and Swanson 2000).
Postmortem studies of schizophrenic patients have found elevated expression of
mRNA but decreased protein expression of EAAT1 in the dorsolateral PFC and
anterior cingulate cortex (Bauer et al. 2008) and increased thalamic EAAT1 and
EAAT2 (Smith et al. 2001). Similarly, variants of the EAAT1 gene (SLC1A3)
have been linked with schizophrenia (Walsh et al. 2008). Conversely, microarrays
of postmortem samples of depressed patients found a decrease of EAAT1 and
EAAT2 mRNA in the frontal cortex (Choudary et al. 2005) and decreases in
EAAT3 and EAAT4 mRNA in the striatum (McCullumsmith and Meador-
Woodruff 2002). The antidepressant riluzole has been found to enhance glutamate
transporter activity (Fumagalli et al. 2008), and the mood stabilizer valproate has
been shown to increase EAAT1 but decrease EAAT2 levels in the hippocampus
(Hassel et al. 2001; Ueda and Willmore 2000).
Human EAAT1-4 corresponds to the rodent GLAST, GLT-1, EAAC1 and
EAAT4, respectively, (Arriza et al. 1994), with GLT-1 (human EAAT2) respon-
sible for the majority of extracellular glutamate regulation (Arriza et al. 1994;
Shigeri et al. 2004; Zarate et al. 2002). Complete GLT-1 knockout results in
hippocampal damage and spontaneous epileptic seizures and is often lethal. To
avoid these effects, antisense knockdown of GLAST, GLT-1 and EAAC1 have
been used and revealed that GLAST and GLT-1 knockdown cause an elevation in
extracellular glutamate, neurodegeneration and paralysis while EAAC1 knock-
down does not elevate extracellular glutamate and produces only mild neurotox-
icity (Rao et al. 2001a, 2001b; Rothstein et al. 1996). GLAST knockout mice are
viable and have a phenotype resembling the positive and negative symptoms of
schizophrenia, including increased novelty-induced hyperlocomotion that is
reversed by the antipsychotic haloperidol, abnormal sociability, reduced acoustic
startle response and impaired visual discrimination (Karlsson et al. 2008, 2009). It
is not known, however, how a deficit in glutamate transporters affects depression-
related behaviors or response to stress. Future studies could investigate the role of
glutamate transporters in buffering pyramidal cells to the increased glutamate
release during stress.
Because of their role in synaptic plasticity, these glutamatergic receptors have
been extensively studied in animal models of cognition. Animals lacking these
receptors often show deficits in cognitive-related tasks that overlap those seen in
models of neuropsychiatric disorders. Their role in emotional-related behaviors
and stress response, however, has not been systematically studied and still remains
untested in many of these mutant models. There have been examples of baseline
alterations in emotionality (as with the NR2A mice) and schizophrenia-related
behaviors, suggesting that dysfunction of the glutamatergic system alone can result
in pathology. A more nuanced approach, however, is seen in those studies ana-
lyzing changes in the normal stress response in animals with alterations in the
glutamatergic system. As mentioned earlier, stress can act as a predisposing factor
for many neuropsychiatric illnesses and it is important to see how stress can lead to
a disruption in cognition and emotional regulation. Here, we briefly discussed the
dysregulated stress response in NMDA, but not AMPA, receptor-related manip-
ulations. In the future, it will be interesting to see studies of stress response applied
to more models of glutamatergic function.
6 Conclusions
Stressful experiences are often reported in the life histories of neuropsychiatric

patients and it is well established that stress can be a risk factor for the devel-
opment of neuropsychiatric disorders. Typical to this clinical population are def-
icits in emotional regulation and cognitive function. Preclinical data showing
parallel behavioral and structural changes in the PFC, hippocampus and amygdala
following stress support a direct role for stress modifying these emotional and
cognitive functions. Multiple molecular mechanisms could explain these stress
evoked changes. Here, we have shown how the glutamate system can be linked
to the structural and ultimately behavioral changes seen in neuropsychiatric
disorders.
Glutamatergic transmission is integral to multiple levels of neural function. At
one level, alterations in glutamate transmission can influence downstream sig-
naling pathways involved in cell death or cell growth. Changes in glutamate
transmission during stress could lead to differential activation of these signaling
pathways, possibly explaining the morphological changes observed. One such
signaling pathway of interest involves brain-derived neurotrophic factor (BDNF).
In its mature form, BDNF promotes neuronal growth and survival and its release is
dependent on glutamatergic synaptic activity (Carvalho et al. 2008; Lu 2003).
BDNF itself also influences glutamatergic transmission, altering the release of
glutamate, glutamate receptor composition and synaptic plasticity (Carvalho et al.
2008; Kuczewski et al. 2009; Lu 2003). BDNF expression is reduced with stress,
an effect reversed by antidepressants, and has itself been shown to be reduced in
depression-like behaviors (Gourley et al. 2009; Nibuya et al. 1995; Shirayama
et al. 2002; Siuciak et al. 1997). Thus far few studies have collectively examined
the effects of stress, morphology and behavior in relation to BDNF. Preliminary
work has shown that genetically induced BDNF overexpression can prevent stress-
induced hippocampal dendritic retraction and reduced learned helplessness,
making BDNF a particularly relevant molecule in the study of stress and cognition
(Govindarajan et al. 2006). The contributions of BDNF and other downstream
signaling pathways will be important targets elucidating the mechanisms of stress-
induced morphological changes.
In addition, many studies have used genetic techniques to understand the
contribution of glutamate receptors in emotional and cognitive behavioral tasks.
These studies have shown that changes in activity and composition of glutamate
receptors can lead to changes in emotional regulation and cognitive deficits.
However, few studies have used these models to directly investigate the role of
receptors in the context of stress. These receptors are integrally involved in syn-
aptic plasticity mechanisms, thus examining the effects of stress on these receptors
could help explain why stress is a precursor to the development of neuropsychi-
atric disorders.
Furthering our understanding of how stress affects glutamate receptors and
alters downstream signaling pathways will ultimately contribute to the develop-
ment of improved therapeutics for individuals suffering from neuropsychiatric
disorders.
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Susceptibility Genes for Schizophrenia:
Mutant Models, Endophenotypes
and Psychobiology
Colm M. P. O’Tuathaigh, Lieve Desbonnet, Paula M. Moran

and John L. Waddington
Abstract Schizophrenia is characterised by a multifactorial aetiology that

involves genetic liability interacting with epigenetic and environmental factors to
increase risk for developing the disorder. A consensus view is that the genetic
component involves several common risk alleles of small effect and/or rare but
penetrant copy number variations. Furthermore, there is increasing evidence for
broader, overlapping genetic-phenotypic relationships in psychosis; for example,
the same susceptibility genes also confer risk for bipolar disorder. Phenotypic
characterisation of genetic models of candidate risk genes and/or putative patho-
physiological processes implicated in schizophrenia, as well as examination of
epidemiologically relevant gene 9 environment interactions in these models, can
illuminate molecular and pathobiological mechanisms involved in schizophrenia.
The present chapter outlines both the evidence from phenotypic studies in mutant
mouse models related to schizophrenia and recently described mutant models
addressing such gene 9 environment interactions. Emphasis is placed on evalu-
ating the extent to which mutant phenotypes recapitulate the totality of the disease
C. M. P. O’Tuathaigh L. Desbonnet J. L. Waddington

Molecular and Cellular Therapeutics,
Royal College of Surgeons in Ireland,
Dublin 2, Ireland
C. M. P. O’Tuathaigh (&)
School of Medicine, University College Cork,
Western Gateway Building, Cork, Ireland
e-mail: c.otuathaigh@ucc.ie
P. M. Moran
School of Psychology, University of Nottingham,
Nottingham, NG7 2RD, UK

DOI: 10.1007/7854_2011_194
210 C. M. P. O’Tuathaigh et al.
phenotype or model selective endophenotypes. We also discuss new developments

and trends in relation to the functional genomics of psychosis which might help to
inform on the construct validity of mutant models of schizophrenia and highlight
methodological challenges in phenotypic evaluation that relate to such models.

Keywords Schizophrenia Psychotic illness Susceptibility gene Mutant model

Phenotype Gene 9 environment interaction
Contents
1 Introduction........................................................................................................................ 211
1.1 ‘Missing Heritability’? Evaluating Genetic Evidence for Schizophrenia
Risk Genes ................................................................................................................ 211
1.2 The Influence of Technological Progress on Mutant Modelling of Psychosis ...... 212
1.3 Assessing Construct Validity of Mutant Models of Schizophrenia........................ 213
1.4 Gene 9 Environment Interactions in Schizophrenia: Implications
for Modelling ............................................................................................................ 214
2 Genetic Models Relating to Dopaminergic Dysfunction................................................. 215
2.1 Dopamine Receptors................................................................................................. 215
2.2 Dopamine Transporter .............................................................................................. 216
2.3 Catechol-O-Methyltransferase .................................................................................. 217
2.4 AKT1/GSK-3 ............................................................................................................ 218
3 Genetic Models Relating to Glutamatergic Dysfunction................................................. 219
3.1 NMDAR-Mediated Neurotransmission.................................................................... 219
3.2 Glycine ...................................................................................................................... 220
3.3 D-serine ..................................................................................................................... 221
3.4 NMDAR Signalling Complex .................................................................................. 222
3.5 Metabotropic Glutamate Receptors.......................................................................... 222
4 Genetic Models Relating to Candidate Risk Genes and Copy Number Variations ....... 223
4.1 Disrupted-in-schizophrenia-1.................................................................................... 223
4.2 Dysbindin .................................................................................................................. 225
4.3 Neuregulin................................................................................................................. 226
4.4 Copy Number Variations.......................................................................................... 228
5 Evaluation of G 9 E Hypotheses in Mutant Models of Schizophrenia ......................... 229
5.1 DISC1 9 Immune Challenge ................................................................................... 229
5.2 NRG1/COMT 9 Exposure to Drugs of Abuse ....................................................... 230
5.3 Genes Related to the Pathophysiology of Schizophrenia 9 Exposure
to Prenatal Stress ...................................................................................................... 231
5.4 Overview of G 9 E studies ..................................................................................... 232
6 Discussion .......................................................................................................................... 233
6.1 Divining gene-Phenotype Associations in Schizophrenia Risk Gene Models ....... 233
6.2 Integration of Novel Gene Manipulation Strategies ............................................... 234
6.3 Examination of Gene 9 Gene Interactions in Schizophrenia................................. 238
7 Conclusions........................................................................................................................ 239
References................................................................................................................................ 239
Susceptibility Genes for Schizophrenia 211
1 Introduction
1.1 ‘Missing Heritability’? Evaluating Genetic Evidence

for Schizophrenia Risk Genes
Schizophrenia is a highly heritable disorder [having a heritability estimate of

approximately 0.8] that is characterised by a multifaceted psychopathology of
psychotic symptoms, negative symptoms and cognitive deficits (Tandon et al.
2009; Waddington et al. 2011). The genetic basis of schizophrenia has proven
complex and difficult to capture, with the expected breakthrough from genome-
wide association studies (GWAS) failing to support the more prominent common
risk alleles identified to date (Gill et al. 2009; Owen et al. 2010). Lack of con-
sistent supportive data for specific genes or loci associated with schizophrenia has
lead investigators to speculate regarding the underlying causes of ‘missing heri-
tability’ (Manolio et al. 2007; International Schizophrenia Consortium 2009) in
schizophrenia.
Two main hypotheses of schizophrenia genetics have suggested that such
‘missing heritability’ reflects either or both of multiple common risk alleles, each
of small effect, or the impact of rare but highly penetrant alleles (Allen et al. 2008;
Gill et al. 2009; Bassett et al. 2010; Owen et al. 2010). The latter hypothesis is
supported by recent microarray findings which have highlighted the involvement
of copy number variations (CNVs) in susceptibility to schizophrenia (Xu et al.
2008, 2009; International Schizophrenia Consortium 2008; Walsh et al. 2008).
These are not mutually incompatible hypotheses and it is likely that both con-
tribute to risk. It has been noted that genetic studies of schizophrenia might be
handicapped by several factors, including genetic and phenotypic heterogeneity,
epistatic gene interactions and the role that environmental events play in the
development and expression of psychiatric illness (Burmeister 1999; Burmeister
et al. 2008). An additional factor is uncertainty as to the genetic-phenotypic
boundaries of schizophrenia, with both common risk alleles and rare CNVs
associated also with risk for bipolar disorder (Gill et al. 2009; International
Schizophrenia Consortium 2009; Grozeva et al. 2010; Steinberg et al. 2011), in
accordance with a dimensional, as opposed to a categorical, model of psychotic
illness (Waddington et al. 2011). Thus, henceforth, our use of the term ‘schizo-
phrenia’ should be interpreted as shorthand for a breadth of psychotic illness, the
boundaries of which remain to be defined.
Evidence to date indicates that while a number of genetic variants have been
associated with multiple diagnostic domains, it is clear that some contribute to
the expression of a limited set of endophenotypes (van Os et al. 2010). This
places considerable emphasis on dissection of the schizophrenia phenotype into
distinct and accessible endophenotypes that may relate more closely to under-
lying pathobiology. Endophenotypes are defined as measurable, intermediate
disease features that bridge the gap between the overt manifestations of
schizophrenia and underlying risk genes (Gottesman and Gould 2003; Braff et al.
2007). We have previously noted the utility of endophenotypes in relation to
mutant phenotypes, being relatively closer to the genetic basis of the disorder at
issue and, therefore, less complex to model; it is assumed that while a single
gene can affect multiple behavioural endpoints, an intermediate biological or
behavioural endophenotype is less susceptible to confounding influences and is
therefore more amenable to investigation (O’Tuathaigh et al. 2007a; Desbonnet
et al. 2009; Kirby et al. 2010). An endophenotype-based approach to schizo-
phrenia risk genes may explain how a gene with modest association support may
still have a function of potential relevance to the disorder, insofar as it may be
associated with one or more putative endophenotype in humans, or their
equivalents in mice. Among the most prominent candidate genes implicated in
risk for schizophrenia by association or CNV studies of risk for schizophrenia,
those for which the evidence is most strong and replicable are Disrupted-in-
Schizophrenia-1 (DISC1), dystrobrevin-binding protein 1 (DTNBP1; dysbindin),
and neuregulin 1 (NRG1) (Waddington et al. 2007; Allen et al. 2008; Bertram
2008; Gill et al. 2009; Owen et al. 2010).
1.2 The Influence of Technological Progress on Mutant

Modelling of Psychosis
Constitutive mutant models involving the construction of mice with gene dis-
ruption, either by deletion [i.e. knockout (KO)] or insertion/over-expression [i.e.
transgenic/knockin] have been used routinely to study the effect of developmental,
whole-body loss-of-target gene function. These techniques have not been without
drawbacks, with extrapolation of gene-phenotype relationships potentially con-
founded by: putative compensatory and redundancy mechanisms; the possibility of
embryonic lethality where the gene is functionally pleiotropic; potentially, whole-
body ablation or overexpression of a given gene may result in phenotypes that
involve both diverse brain regions and extracerebral mechanisms and thus com-
plicate interepretation of phenotypic data. However, each technique presents its
own profile of advantage and disadvantage.
Progress in mouse genetic technology over the last decade has facilitated the
development of conditional genetic models that are differentiated on the basis of
cell-type specificity, regional brain selectivity and temporal activation of the
mutation. Unsurprisingly, the evidence indicates that each of these factors can be
critical determinants of whether a specific gene disruption can give rise to a given
phenotypic outcome or otherwise; indeed, contradictory data have also been
reported for the same gene mutation depending on cell-, regional- and possibly
temporal specificity (e.g. Singer et al. 2011).
1.3 Assessing Construct Validity of Mutant Models

of Schizophrenia
Criteria employed to evaluate a mutant model of schizophrenia involve several

forms of validity: face validity [whether the model produces a behavioural
phenotype which resemble human disease features]; predictive validity [the degree
to which deficits or behavioural signs observed in the model are responsive to
agents which either ameliorate or worsen analogous symptoms in patients]; con-
struct validity [whether the experimental/genetic manipulation can be directly
related to the known aetiology or pathophysiological features of the illness].
Assessing predictive validity is problematic, due to the lack of efficacy of existing
antispsychotic drugs against negative symptoms and cognitive deficits in schizo-
phrenia, both of which have been most strongly associated with prognosis and
functional outcome.
The symptoms of schizophrenia, which emerge during adolescence, include
positive symptoms [i.e. hallucinations, delusions and thought disorder], negative
symptoms [e.g. avolition, anhedonia, blunted affect, poverty of speech and social
withdrawal], and cognitive dysfunction [e.g. impairment in working memory,
executive function and attention]. Phenotypic modelling of positive symptoms at
the level of behaviour is restricted to ‘proxy’ indices of positive symptoms, such as
hyperactivity in response to a novel stimulus and to psychotomimetics such as
amphetamine or phencyclidine (PCP); prepulse inhibition (PPI) and latent inhi-
bition (LI), involving sensory gating, selective attention to relevant over irrelevant
stimuli and attribution of salience, are at the interface of psychotic and cognitive
processes (Amann et al. 2010; Desbonnet et al. 2009; Arguello and Gogos 2010;
Kirby et al. 2010; O’Tuathaigh and Waddington 2010; van den Buuse 2010).
Modelling of negative symptoms has focused primarily on a restricted range of
social, emotional, and motivational behaviours that apply to, and are accessible in,
both humans and animals, i.e. asociality and, to a lesser extent, anhedonia
(Arguello and Gogos 2006; O’Tuathaigh et al. 2010a).
Although the issue of which cognitive domains are disrupted-in-schizophrenia
has proven controversial (Harvey et al. 2010), there is some agreement that
impairments in working memory, executive function and attention are core
features. Disruption to these domains of cognition [including PPI and LI, which
are at the interface of cognitive dysfunction and positive symptoms] is amenable to
investigation in animals. Particular emphasis has been placed on spatial working
memory and executive function, processes which can be accessed using the maze-
based and several operant reward-based tasks (Arguello and Gogos 2006, 2010;
Amann et al. 2010).
Evaluating the construct validity of a genetic model of psychosis places the
focus on (a) how accurately the genetic model mimics the human risk poly-
morphism, (b) the status of the clinical data linking the risk variant with the
entirety of the disorder and/or limited to disease-relevant endophenotypes, and
(c) the availability of a valid and reliable phenotypic armamentarium capable of
exploring hypothesised gene-phenotype relationships (Arguello and Gogos 2006;

Arguello and Gogos 2010; Desbonnet et al. 2009; Kirby et al. 2010; Low and
Hardy 2007).
1.4 Gene 3 Environment Interactions in Schizophrenia:

Implications for Modelling
With ongoing progress in the field of psychiatric genetics and the modifying
influence of environmental factors, current conceptualisations of schizophrenia
have addressed the complex interaction of genes (G) and environment (E) (van
Os et al. 2010). Focusing on studies examining the interaction of epidemiolog-
ically relevant risk factors and candidate gene mutant models, it has been noted
that such an approach has the potential to produce G 9 E models with increased
construct validity (Desbonnet et al. 2009; Kirby et al. 2010). Consistent with the
well-considered ‘stress-vulnerability’ aetiological model, which proposes an
interaction between inherited genetic vulnerability and subsequent exposure to
adverse environmental risk factors, there is a growing body of evidence linking
early pre-l and postnatal life trauma (e.g. maternal infection, childhood abuse),
societal factors (e.g. urbanicity, migration), and drug use (e.g. cannabis exposure)
with risk for schizophrenia (van Os et al. 2010). While clinical studies linking
specific genetic factors with the psychosis-inducing or precipitating effects of
environmental factors have been slow to emerge, there has been some supportive
evidence sensitive enough to detect the interaction of environmental adversity
with a putative risk gene within a young population followed prospectively
(Caspi et al. 2005).
Recent years have seen a movement towards the generation of animal models of
psychosis based on the interaction of genetic mutations and well-characterised
environmental factors (Ayhan et al. 2009; Gray and Hannan 2007). As outlined
elsewhere (Burrows et al. 2011), the utility of such G 9 E models depends upon
the degree to which the environmental models at a preclinical level possess con-
struct validity with respect to environmental factors which have been epidemio-
logically linked with increased risk for schizophrenia; thus, although issues of
validity have most frequently been explored in relation to the target gene mutation
employed in a given model, whether epidemiologically relevant environmental
factors have been successfully translated into current preclinical G 9 E models
constituting a particular challenge (Burrows et al. 2011).
The present chapter will discuss the evidence on (a) mutant models relating to
the putative pathophysiology of schizophrenia, primarily dopaminergic and
glutamatergic dysfunction, (b) mutant models involving candidate risk genes for
schizophrenia, with an emphasis on evaluating the extent to which mutant phe-
notypes recapitulate the totality of the disease phenotype or model selective
endophenotypes, and (c) mutant models addressing G 9 E interactions relevant to
schizophrenia.
2 Genetic Models Relating to Dopaminergic Dysfunction
2.1 Dopamine Receptors
Constitutive KO of dopamine (DA) receptor subtypes in mice has been an

important experimental tool in understanding their roles in behaviours considered
as endophenotypes for schizophrenia (O’Sullivan et al. 2010). Amphetamine has
been shown to disrupt sensory-motor gating, considered to be a behavioural proxy
for the impaired ability of schizophrenia patients to gate information flux appro-
priately. The indirect DA agonist d-amphetamine disrupts sensory gating in D1,
D3, and D4 KO mice, but not D2 KO mice, suggesting that the D2 receptor is
required for its disruption by amphetamine (Ralph et al. 1999; Ralph-Williams
et al. 2002). The KO approach has recently suggested dissociable roles for D1 and
D2 receptors in habituation and sensitisation to acoustic startle (Halberstadt and
Geyer 2009), which may be of relevance to subgroups of patients that display
habituation abnormalities. Antipsychotic drugs enhance low levels of LI. This
behavioural index of the ability to ignore irrelevant information is abnormal in
patients with schizophrenia and is enhanced in D2 KO mice (Bay-Richter et al.
2009), indicating the importance of the D2 receptor in the processes by which
antipsychotic drugs exert their behavioural effects. We have recently shown that
enhancement of LI by the antipsychotic drugs haloperidol and clozapine is pre-
vented in D2 KO mice, confirming directly that they act via D2 receptors to restore
the ability to ignore irrelevant information. However, we have also shown that
amphetamine impairment of LI and its restoration by haloperidol and clozapine is
intact in D2 KO mice, suggesting that the role of the D2 receptor differs depending
on how disruption to LI is produced. New therapeutic approaches are seeking to
circumvent direct effects at the D2 receptor, given their association with unde-
sirable motor and other side effects side effects, hence amphetamine disruption of
LI in D2 KO mice may prove to be a useful new application of the KO approach to
produce animal models for the identification and evaluation of new treatments
(Bay-Richter et al., unpublished data).
The D2 receptor has two isoforms, long (D2L) and short (D2S), derived from
alternative splicing. Studies in isoform-specific KOs have indicated the D2L iso-
form to be crucial for antipsychotic drug action (Xu et al. 2002) and may be of
greater importance in extrapyramidal side effects (Wang et al. 2000); however,
effects on complex cognitive behaviours more specifically related to schizophrenia
have yet to receive systematic evaluation.
Cognitive functions such as working memory and cognitive flexibility are
known to be impaired in schizophrenia and are underpinned by prefrontal [PFC]
cortical brain regions. There is a high density of D1 receptors in these regions,
hence it is not surprising that D1 KO mice show deficits in spatial working
memory and reversal learning (Holmes et al. 2004; El-Ghundi et al. 2007). The
D2 receptor may also be important in set shifting and reversal learning. D2 KO
mice show impairment in adjusting responding to previously reinforced stimuli
when unexpected outcomes are encountered and both D2 KO and antipsychotic

drugs have recently been shown to produce similar reversal learning deficits in a
set shifting paradigm (Glickstein et al. 2002; Kruzich and Grandy 2004; DeSteno
and Schmauss 2009). This raises the possibility that the D2 receptor is important
in producing drug-induced impairments in these functions. D3 KO mice also
show deficits in spatial working memory in some (Glickstein et al. 2002) but not
all studies (Xing et al. 2010). Mutants with selective overexpression of subcor-
tical D2 receptors show behavioural changes that include reduced incentive
motivation, as indexed by reduced lever pressing for food reward in operant
tasks, and abnormal timing behaviour, which may be relevant to perturbations in
timing seen in a number of psychiatric diseases, including schizophrenia (Drew
et al. 2009).
2.2 Dopamine Transporter
Extracellular DA concentration is regulated via a plasma membrane DA trans-

porter (DAT). Deletion of this entity dramatically alters both presynaptic
homeostasis and extracellular dynamics of DA. These models have been partic-
ularly useful in understanding the mechanism of action of psychostimulant drugs
(Gainetdinov 2008). While most studies have used DAT KO and heterozygous
mice (Giros et al. 1996), DAT knockdown (Zhuang et al. 2001) mice with mod-
erately increased DA levels have also been investigated (Gainetdinov 2008). As a
consequence of permanently enhanced DAergic tone, DAT KO mice show loco-
motor hyperactivity and stereotypy in a novel environment, as well as memory and
sensory gating deficits (Gainetdinov 2008). These mice also show social interac-
tion deficits related to behavioural inflexibility (Giros et al. 1996; Rodriguiz et al.
2004), a more positive bias towards a hedonically positive tastant (Costa et al.
2007) and enhanced resistance to extinction of food-reinforced operant behaviour
(Hironaka et al. 2004), indicating a role for DA in updating rewarding values, habit
learning and memory.
A variety of behavioural responses to psychostimulants and other drugs of
abuse have been reported in DAT KO mice and have been reviewed previously
(Gainetdinov 2008). Sex-dependent effects on ethanol preference and consumption
has been reported in DAT KO mice (Savelieva et al. 2002; Hall et al. 2003;
Mathews et al. 2006; Gainetdinov 2008) and have been interpreted as reflecting
altered hedonic mechanisms; KO mice showed greater motivation for the task than
WT (i.e. ‘wanting’), without affecting their responsivity for sucrose reward (i.e.
‘liking’). These data suggest that chronically elevated tonic DAergic activity
produces changes in incentive motivation; however, these are in the opposite
direction to those that might form part of the negative symptom profile of
schizophrenia (Cagniard et al. 2006). Rodriguiz et al. (2004) found several patterns
of abnormal social responsivity in DAT KO mice, including disruption of the
stability of social hierarchies. This suggests that chronic DAergic hyperfunction

may play an important role in abnormal social interactions.
2.3 Catechol-O-Methyltransferase
The enzyme catechol-O-methyltransferase (COMT) is involved in the catabolism of

catecholeamine neurotransmitters. COMT is expressed in the pyramidal neurons of
the prefrontal cortex and hippocampus and plays a specific role in the catabolism of
cortical DA but not noradrenaline (Papaleo et al. 2008). This specificity, together
with the COMT gene lying within a chromosomal region of interest for psychosis
[22q11] and showing polymorphism that alters activity of the enzyme, has made
COMT the subject of extensive study in schizophrenia (Craddock et al. 2006).
Several studies suggest that functional polymorphisms of the COMT gene are
associated with performance on frontal cortical tasks such as the Wisconsin card sort
task in patients with schizophrenia and controls and confer enhanced vulnerability to
environmental triggers for schizophrenia such as cannabis (Caspi et al. 2005).
Studies in COMT KO mice have investigated motor activity, anxiety, aggression,
and sensorimotor gating (Gogos et al. 1998, Huotari et al. 2002, Haasio et al. 2003,
Tammimäki et al. 2008). The findings include impaired emotional reactivity in
female but not male KO and increased aggression and ethanol consumption in male
but not female KO; these sex-specific effects support an emerging body of evidence
that the influence of COMT on a variety of phenotypes may be different in males and
females (Harrison and Tunbridge 2008). Babovic et al. (2007) have studied the
phenotypic ethogram of COMT mutants and found that heterozygous but not
homozygous, mice show abnormal exploration of and habituation to a novel envi-
ronment. It is likely that behavioural effects are influenced by DA state according to
the Yerkes-Dodson arousal curve, which indicates an inverted U-shaped relationship
between arousal and behavioural performance; this has been evoked previously to
explain paradoxical effects of prefrontal DAergic manipulations on behavioural
performance (Seamans and Yang 2004). Male COMT KO show improvement in
spatial working memory (Babovic et al. 2008; Papaleo et al. 2008), whereas a
transgenic COMT mutant involving overexpression of the human COMT-VAL
polymorphism shows deficits in attentional set shifting, recognition memory, and
working memory performance (Papaleo et al. 2008); low-dose amphetamine
restored recognition memory, confirming DAergic involvement and providing fur-
ther support for an inverted U-shaped relationship between cognitive function and
cortical DAergic activity (Seamans and Yang 2004; Tunbridge et al. 2006).
Recent studies across commonly used inbred strains have shown that a poly-
morphism in COMT, a B2 SINE insertion, is associated with behavioural differ-
ences related to exploration (Kember et al. 2010), altered expression of genes
implicated in synaptic function and intracellular signalling, as well as genes linked
with DAergic, glutamatergic, and GABAergic systems (Li et al. 2010).
2.4 AKT1/GSK-3
Striatal D2 receptors have been shown to exert their action in a cAMP-independent

manner by promoting the formation of a signalling complex composed of AKT,
protein phosphatase-2A (PP2A), and ß-arrestin 2 (Beaulieu et al. 2004, 2005).
Formation of this complex leads to the inactivation of AKT after the dephos-
phorylation of its regulatory threonine 308 (Thr-308) residue by PP2A (Beaulieu
et al. 2005). Inactivation of AKT in response to DA results in the activation of
glycogen synthase kinase 3 (GSK-3; see below). Antipsychotic drugs such as
haloperidol have been shown to activate AKT phosphorylation in the mouse brain
(Emamian et al. 2004). A number of studies have indicated a role for ß-arrestin,
AKT, and GSK-3 in DAergic regulation of behaviour. ß-arrestin KO mice show
reduced climbing and reduced sensitivity to the effects of amphetamine on loco-
motor activity, while AKT1 KO mice show enhanced sensitivity to the disruptive
effects of amphetamine on PPI (Beaulieu et al. 2009). Recently, deficits in PPI in
females only has been demonstrated in AKT1 KO mice (Chen and Lai 2011).
Several studies have identified SNPs in the DISC1 gene to be associated with
schizophrenia and DISC1 has been shown to regulate the phosphorylation of
certain GSK-3 substrates and neuronal progenitor proliferation via modulation of
GSK-3 signalling (Mao et al. 2009). Takashima (2009) have shown that GSK-3
KO mice show deficits in memory reconsolidation, but not acquisition deficits, in
water maze and contextual fear conditioning paradigms. While direct studies in
negative symptom models are awaited, the association between neuroplasticity,
neurodevelopment, and DA signalling makes this a very promising candidate for
understanding negative symptoms.
GSK-3 is also a central component of the developmentally important Wnt
signalling pathway, which has been suggested to be important for certain aspects
of neuronal functioning that may relate to negative symptoms, including syna-
ptogenesis (Hur and Zhou 2010). Mice lacking the homologue of the Drosophila
segment polarity gene Dvl-1, a Wnt signalling partner, are reported as having
deficiencies in PPI and social interactions (Lijam et al. 1997). The same group
replicated their study six years later and included some additional behavioural
tasks (Long et al. 2004). In common with the earlier study, DVL-1 mice were less
likely to huddle together and less likely to build nests, although no PPI deficits
were seen. Other behavioural tests showed that mice lacking Dvl-1 had other
impairments in behaviours related to group dynamics. It is not clear why, several
generations later, mice lost their deficit in PPI; an environmental effect was
considered most likely but an interaction with the background strain cannot be
excluded. It should be noted that there are very few research groups that have
performed follow-up studies of this type within colonies and it is possible that
many phenotypic differences reported for a variety of mutant mice could turn out
to show similar environmental sensitivity.
3 Genetic Models Relating to Glutamatergic Dysfunction
Hypoglutamatergic function may play an important pathobiological role in

schizophrenia (Coyle 2006; Balu and Coyle 2011). The supporting arguments are
based on: (a) the properties of NMDA receptor (NMDAR) antagonists to be
psychotomimetic in healthy humans (Adler et al. 1999; Kegeles et al. 2000; Coyle
2006) and to exacerbate or precipitate psychotic symptoms in patients with
schizophrenia (Malhotra et al. 1997a, 1997b); (b) reports of NMDAR abnormal-
ities in schizophrenia, both in postmortem brain (Stone et al. 2007) and in living
patients using SPECT imaging (Pilowsky et al. 2007); (c) limited evidence to
suggest that pharmacological enhancement of NMDAR activity may be thera-
peutically useful against the symptoms of schizophrenia (Javitt et al. 2004; Coyle
2006; Pinard et al. 2010). Clinical genetic evidence linking gene variants associ-
ated with NMDAR activity to schizophrenia has proven inconsistent (Allen et al.
2008).
3.1 NMDAR-Mediated Neurotransmission
Mice deficient in the NMDAR NR1 subunit exhibit deficiencies across several
schizophrenia-related phenotypes: decreased responsivity to the NMDAR antag-
onists PCP and MK-801, hyperactivity in a novel environment and deficits in PPI
that were reversible particularly by second-generation antipsychotics, and social
deficits that were less responsive to antipsychotics (Mohn et al. 1999; Fradley et al.
2005; Duncan et al. 2004, 2006). Mice containing a targeted mutation in NR1
which prevented NR1 phosphorylation at serine 897, shown to be dramatically
reduced in patients with schizophrenia (Emamian et al. 2004), induced a marked
disruption of NMDAR-mediated synaptic transmission; these mice displayed
deficits in both PPI and social interaction, as indexed by a modified sociability and
preference for social novelty paradigm, but no genotypic changes in locomotor
activity (Li et al. 2009). These mutants also displayed decreased AMPA receptor-
mediated transmission and decreased AMPAR GluR1 subunit expression in the
synapse.
A recent study further refined our understanding of the role NMDAR signalling
in modulation of schizophrenia endophenotypes by examining the effect of tar-
geted postnatal or post-adolescent ablation of the NR1 subunit in cortical and
hippocampal GABAergic neurons, based on evidence supporting corticolimbic
GABAergic interneurons as a site of NMDA receptor hypofunction (Belforte et al.
2010). Several schizophrenia-related phenotypes, including novelty-induced hy-
perlocomotion and nest-building behaviour, were found to be present in postna-
tally targeted NR1 mutants when assessed in adulthood but not before
adolescence. These NR1 mutants also displayed social recognition deficits and a
mild anhedonic phenotype, measured in the sucrose preference test, as well as
deficits in spatial working memory, assessed by spontaneous alternation behaviour.

Deficits in spontaneous alternation and PPI were reversed by risperidone; other
schizophrenia-related behaviours which were not reversed by risperidone included
nest building. Interestingly, the same schizophrenia-related phenotypic profile was
not observed in mice containing post-adolescent ablation of NR1, supporting the
neurodevelopmental hypothesis of schizophrenia and highlighting the importance
of developmental dysregulation of GABAergic neurons.
An alternative NMDAR mutant line, involving deletion of the NR1-associated
NR2A (GluR1) subunit, was associated with hyperactivity that was ameliorated by
haloperidol and risperidone, together with deficits in spatial and latent learning and
augmented DA metabolism in striatum and frontal cortex (Miyamoto and Nabe-
shima 2002). Additionally, mice with partial reduction of the gene SP4 (a member
of the SP1 family of transcription factors) evidence a reduction in the expression of
the NR1 subunit, accompanied by a specific learning deficit in the Barnes maze; no
genotypic differences were found in tests of social interaction, anxiety-related
behaviour or other measures of cognition (Zhou et al. 2010).
3.2 Glycine
NMDAR activation involves postsynaptic depolarisation and the binding of two

agonists, glutamate, and either glycine or D-serine at the glycine modulatory site
(Tsien 2000). Glycine acts at an accessory site necessary for NMDAR function to
facilitate NMDA-mediated transmission; thus, glycine agonists promote NMDA-
mediated transmission and, controversially, may evidence some efficacy against
the negative symptoms of schizophrenia (Tuominen et al. 2005; Patil et al. 2007;
Pinard et al. 2010). A mutant line carrying a point mutation in the NMDAR
glycine binding site, Grin1 (D481 N), has been shown to display hyperactivity in a
novel environment that was not reversed by antipsychotics, reduced sociability
that was reversed by D-serine and partially by clozapine, as well as abnormalities
in spatial learning and memory (Ballard et al. 2002; Labrie et al. 2008).
As the availability of glycine is partially modulated by glycine transporters
GlyT1 and GlyT2, with GlyT1 known to be co-expressed with NMDAR in
glutamatergic synapses in the forebrain, mutant studies have reported enhanced
NMDAR activity in mice with conditional GlyT1 deletion in forebrain neurons
(Yee et al. 2006). Forebrain GlyT1 deletion has been associated with enhancement
of associative learning (Yee et al. 2006), object recognition memory (Singer et al.
2007), selective attention, as measured by LI (Yee et al. 2006), and reversal
learning in a water maze task (Singer et al. 2009); no effect of the mutation was
observed on spatial working memory (Singer et al. 2009). Mice with constitutive
heterozygous GlyT1 KO (Tsai et al. 2004) or lacking GlyT1 in the hippocampus
and cerebellum (Singer et al. 2011) evidence unchanged PPI and non-responsivity
to the PPI-disruptive effects of the NMDAR antagonist MK-801. However, con-
ditional forebrain-specific GlyT1 mutants evidenced impaired PPI and intact
responsivity to the PPI-disruptive effects of MK-801 (Singer et al. 2011). Overall,

the diverse precognitive profile observed following enhancement of NMDA
transmission via GlyT1 inactivation supports a role for GlyT1 inhibitors in the
treatment of cognitive symptoms in schizophrenia.
3.3 D-serine
D-Serine is a highly selective endogenous activator of NMDAR signalling. A

mouse strain lacking activity of D-amino acid oxidase (DAO, an enzyme involved
in metabolism of D-serine), due to a spontaneous point mutation in the gene
encoding DAO, is accompanied by reduced behavioural sensitivity to MK-801
(Hashimoto et al. 2005) and PCP (Almond et al. 2006); these changes were
accompanied by an increase in NMDA receptor-mediated neurotransmission
(Maekawa et al. 2005). DAO mutants demonstrated no disruption to PPI but
reduced exploration in a novel environment, with attenuation of stereotype and an
increase in locomotor response to methamphetamine (Almond et al. 2006). More
recently, using a different procedure to that employed by Almond and colleagues,
DAO mutants showed enhanced PPI, increased sensitivity to the PPI-disruptive
effect of the NMDA receptor antagonist SDZ 220-581 and a subtle performance
deficit in the Morris water maze, but no change in anxiety-related behaviours
(Zhang et al. 2011).
Grin1 (D481 N) mutants were crossed with the same DAO mutant line in order
to examine whether genetic inactivation of DAO would increase availability of D-
serine and ameliorate schizophrenia-related phenotypes in a mouse model char-
acterised by deficient NMDA glycine site activation (Labrie and Roder 2010). In
agreement with their hypothesis, mice with both DAO inactivation and Grin
(D481 N) mutation showed normalisation of social affiliative behaviour, as mea-
sured in the sociability and preference for social novelty test, and of LI, with
partial reversal of spatial recognition and sensorimotor gating deficits. These data
would suggest that inhibition of DAO function may, in a domain-selective manner,
partially improve schizophrenia-related behavioural deficits caused by decreased
occupancy of glycine sites asociated with NMDA receptors.
Mutants unable to produce D-serine due to disruption of its synthetic enzyme
serine racemase display mild hyperactivity (Basu et al. 2009). While social
interaction and PCP- or amphetamine-induced hyperactivity and disruption of PPI
were found to be unaffected in serine racemase mutants, PCP-enhanced startle
reactivity was found to be disrupted in these mutants (DeVito et al. 2011; Ben-
neyworth et al. 2011). Serine racemase KO mice also demonstrate a subtle cog-
nitive phenotype characterised by spatial learning deficits (Basu et al. 2009) and
disrupted episodic memory performance in an object recognition paradigm, while
other aspects of task performance which measured novelty detection, recognition
memory, and relational memory were intact (DeVito et al. 2011); these deficits
were accompanied by dendritic morphological abnormalities of pyramidal neurons
in the medial prefrontal cortex. In overview, while the endpoint associated with
many of these lines is NMDA receptor hypofunction, the diverse modes for
achieving this outcome carry distinct phenotypic consequences compensatory
mechanisms may account for some of these differences. However, phenotypic
diversity may reflect (a) differential availability of D-serine and glycine at the
glycine modulatory site and (b) whether the mutation affects receptor function
directly or indirectly via limited availability in a subset of glycine modulatory site
ligands.
3.4 NMDAR Signalling Complex
Abnormalities in NMDAR-interacting protein function have been implicated in

schizophrenia; these include the glial glutamate and aspartate transporter
(GLAST), which has been shown to be expressed differentially in the dorsolateral
prefrontal cortex, anterior cingulate cortex and thalamus in postmortem brains
from patients with schizophrenia (Smith et al. 2001; Bauer et al. 2008). GLAST
KO exhibit antipsychotic-sensitive hyperactivity in a novel environment, increased
locomotor responsivity to the NMDAR antagonist MK-801 and impaired socia-
bility (Karlsson et al. 2008, 2009). Knockout of the NMDAR signalling molecule
SynGAP is associated with increased novelty-induced hyperlocomotion and
reduced behavioural sensitivity to MK-801 administration (Guo et al. 2009).
SynGAP mutants evidence intact sociability but impaired social novelty prefer-
ence, disrupted PPI, enhanced startle reactivity, and deficits in spatial working
memory (Guo et al. 2009).
3.5 Metabotropic Glutamate Receptors
Metabotropic glutamate receptor (mGluR) involvement in the pathophysiology of

schizophrenia, together with the diverse range of phenotypes associated with
mutation for members of this receptor family (mGluR1-8), are discussed in detail
elsewhere (Krivoy et al. 2008). Mutants with AMPA GluR1 KO display anti-
psychotic-sensitive hyperactivity in a novel environment but not in the home cage,
reduction of MK-801-induced hyperactivity and impaired PPI, in association with
reduced clearance of DA (Wiedholz et al. 2008). Examination of the behavioural
phenotype of mGluR4 KO mice revealed impaired PPI and enhanced sensitivity to
the locomotor stimulatory effects of the NMDAR antagonist MK-801; no differ-
ences, however, were observed in relation to social behaviour and spatial working
memory (Sagata et al. 2010). mGluR5 KO mutants show hyperactivity and
impaired PPI that are reversible by antipsychotics; hyperactivity induced by the
NMDA antagonist MK-801 was also increased following deletion of mGluR5
(Brody et al. 2004; Gray et al. 2009).
In summary, the evidence from metabotropic glutamate receptor mutants would

implicate this receptor family in PPI impairment. Other studies have documented
depression- and anxiety-related behavioural phenotypes across a variety of assays
(Feyder et al. 2007; Chourbaji et al. 2008). GluR1 KO also exhibited increased
novelty- or stress (injection, restraint or forced stress)-induced hyperactivity, as
well as reduced immobility in the forced swim test (Fitzgerald et al. 2010). These
deficits were accompanied by a reduced anxiety phenotype in the elevated plus
maze that was normalised following chronic (2 week) treatment with lithium, used
as a moods stabiliser in bipolar disorder. The authors suggested that the combi-
nation of schizophrenia-related phenotypes and affective/manic behavioural signs
in GluR1 KO mice, particularly under conditions of stress, may provide a novel
model of schizoaffective disorder.
4 Genetic Models Relating to Candidate Risk Genes

and Copy Number Variations
4.1 Disrupted-in-schizophrenia-1
Familial mutation in the disrupted-in-schizophrenia-1 (DISC1) gene, due to a

balanced chromosomal translocation at 1q42.1-1q42.3, has been associated
with schizophrenia and other psychiatric disorders across diverse populations
(Chubb et al. 2008; Johnstone et al. 2011); while there are initial reports of
associations between DISC1 and positive symptoms, these are inconsistent as to
whether the relationship is with delusions (DeRosse et al. 2007) or hallucinations
(Szeszko et al. 2008). Genetic lineage and association studies have further sug-
gested DISC1 as a general risk factor for schizophrenia, schizoaffective disorder,
bipolar disorder, major depression, autism, and Asperger syndrome (Chubb et al.
2008). The mechanism through which DISC1 dysfunction may contribute to a
wide spectrum of psychiatric disorders remains unknown (Hennah and Porteous
2009). During embryonic development, DISC1 appears to play an important role
in neurodevelopment and structural plasticity via interaction with several proteins,
including phosphodiesterase-4B, Fez1, NDEL1 and LIS1 (Duan et al. 2007; Chubb
et al. 2008; Muir et al. 2008).
While generation of a DISC1 KO model has not been achieved, largely due to the
complexity of exon usage of the DISC1 gene (Wang et al. 2008), several
mutant models of DISC1 gene function have been described in the literature: (1) A
naturally occurring DISC1 mutation (25 base pair deletion at exon 6) in the
commonly employed 129S6 Sv/Ev mouse strain, which additionally carries a
termination codon in exon 8 and a polyadenylation signal in the adjacent intron,
shows impairment in spatial working memory using a delayed-non-match-to-place
task, but not using the Morris water maze, tests of associative learning-contextual
conditioning or other alternative measures of memory-related processes
(Koike et al. 2006; Kvajo et al. 2008); (2) ENU mutagenesis in exon 2 of DISC1 is
characterised by hyperactivity, with antipsychotic-sensitive deficits in PPI and LI
and disrupted working memory in a T-maze task (Clapcote et al. 2007); (3) A
transgenic line with inducible expression of a DISC1 C-terminal fragment evi-
denced impaired spatial working memory using the delayed-non-matched-to-place
task, as well as reduced sociability (Li et al. 2007); (4) A transgenic line with
expression of a dominant-negative truncated form of DISC1 under the CaMKII
promoter, mutants showed hyperactivity but no changes in PPI, social interaction
or cognition (Hikida et al. 2007); (5) An inducible transgenic line with forebrain-
specific expression of mutant human DISC1 (hDISC1) under the CaMKII pro-
moter showed hyperactivity without material disruption to PPI, sex-specific
impairment of working memory using the Morris water maze, with a deficit in
dyadic social interactions but not sociability (Pletnikov et al. 2008); (6) A trans-
genic line with expression of truncated DISC1 exhibited normal exploratory
activity but disruption to LI (Shen et al. 2008).
Interestingly, lentiviral silencing of DISC1 expression in the adult dentate gyrus
was accompanied by an increase in novelty-induced hyperlocomotion; this
behavioural effect was reversed following treatment with the GSK-3b inhibitor
SB-216763, suggesting that increased GSK-3b activity secondary to DISC1 loss-
of-function might be associated with schizophrenia-like behaviours (Mao et al.
2009). Further support for this hypothesis has come from a recent report that
pharmacological or genetic inactivation of GSK-3 in the ENU-generated DISC1
mutant (L100P) reversed phenotypic deficits in PPI and LI, and normalised their
hyperactivity profile (Lipina et al. 2010). This further implicates impaired DISC1-
GSK-3 interplay in schizophrenia-relevant behaviours.
Employing the mutant hDISC1 transgenic model (Pletnikov et al. 2008), a
follow-up study assessed the effect of timing of the DISC1 mutation on the
expression of schizophrenia-related phenotypes (Ayhan et al. 2011). In this study,
the effect of mutant hDISC1 expression was compared during prenatal, postnatal,
or both periods. Regardless of timepoint of expression, hDISC1 mutants exhibited
fewer cortical parvalbumin-positive cells, as well as cortical hypodopaminergia
relative to controls. Combined pre- and postnatal mutant hDISC1 expression was
associated with the most prominent schizophrenia-related abnormalities; these
included decreased social interaction in a test of dyadic interaction, increased
sensitivity to psychostimulants and increased immobility in the tail suspension
test, with lateral ventricular enlargement and morphological abnormalities in
granule cells of the dentate gyrus. In contrast, neither pre- nor postnatal expression
alone fully recapitulated the phenotype associated with combined timepoint
expression.
Extending these findings and introducing regional specificity, Niwa and col-
leagues (2010), using in utero electroporation to produce selective knockdown of
DISC1 in pyramidal neurons in prefrontal cortex of the foetal mouse, reported
post-pubertal emergence of disrupted PPI, impaired object recognition memory
and delayed T-maze alternation; there was disturbed maturation of mesocortical
DAergic projections to and interneurons in the medial prefrontal cortex. Several of
these phenotypes, including impaired PPI and increased extracellular DA levels in

the prefrontal cortex, were normalised following administration of clozapine.
In summary, the diversity of DISC1 models available indicates that the variable
phenotypic effects of different DISC1 mutations appears to be highly dependent
upon time of expression, environmental factors (see 6.1) and, potentially, inter-
actions with other risk genes (Ross et al. 2006; Ayhan et al. 2011; see 6.3).
4.2 Dysbindin
Systematic review and meta-analysis has indicated dystrobrevin-binding protein 1

(DTNBP1; dysbindin) to be a replicable risk gene for schizophrenia (Allen et al.
2008; Gill et al. 2009). Initial data indicated that dysbindin mRNA expression was
decreased in schizophrenia in PFC and hippocampus (Weickert et al. 2004, 2008)
but subsequent studies have failed to replicate this finding in the dorsolateral
prefrontal cortex (Tang et al. 2009). In vitro work indicates that reduction in
dysbindin can lower glutamate release, while overexpression of dysbindin elevates
glutamate release, suggesting a modulatory role for dysbindin in glutamate
neurotransmission in cortical neuronal cultures (Numakawa et al. 2004).
The ‘sandy’(sdy) mouse, a spontaneous mutation identified in the DBA/2J
strain, carries a naturally occurring deletion that includes the DTNBP1 gene.
Behavioural phenotyping of sdy mutants has produced inconsistent findings, there
being reports of unaltered (Feng et al. 2008; Bhardwaj et al. 2009; Li et al. 2003),
decreased (Takao et al. 2007; Hattori et al. 2008) or increased (Cox et al. 2009; Ji
et al. 2009) spontaneous exploratory activity in the open field. Sensitisation to
amphetamine is enhanced, with both increased turnover (Murotani et al. 2007) and
decreased concentration (Hattori et al. 2008) of DA having been reported. While
the DBA/2J strain does not show robust PPI, precluding assessment therein,
backcrossing of the sdy mutation onto a C57BL6 line revealed either reduced PPI
(Talbot 2009) or enhanced PPI and startle reactivity (Papaleo et al. 2012).
Sdy mutants evidence reduction in social contacts and social contact time in a
dyadic paradigm (Feng et al. 2008; Hattori et al. 2008); genotypic disruption of
spatial learning and working memory using the Barnes maze, the Morris water
maze and the T-maze forced alternation task (Takao et al. 2007; Cox et al. 2009),
as well as deficits in recognition memory using the novel object recognition
paradigm, have also been observed (Feng et al. 2008; Hattori et al. 2008).
It has been suggested that reduction in DTNBP1 expression, associated
with decreased glutamatergic neurotransmission (Numakawa et al. 2004), may
contribute to aberrant prefrontal cortex function and consequent working memory
deficits in schizophrenia (Jentsch et al. 2009). These authors found modest dis-
ruption in working memory in Sdy mutants using the choice accuracy measure in a
delayed-non-match-to-position task; this was accompanied by disturbance of
excitatory neurotransmission in the PFC, as indexed by reduction in amplitude of
eEPSCs and frequency of miniature EPSCs, with abolition of paired-pulse
facilitation. However, abundant dysbindin expression in the hippocampus may

indicate an alternative mechanism for such dysfunction via synaptic plasticity and
associated memory functions (Takao et al. 2008; Cox et al. 2009). Mutation of
DTNBP1 also caused deficits in working memory, as measured by the delayed-
non-match-to-position task; this deficit was accompanied by a decrease in cortical
NR1 mRNA expression that was correlated with working memory performance
(Karlsgodt et al. 2011).
In a separate study, Sdy mice displayed enhanced learning in the acquisition
phase of a modified T-maze working memory task; with introduction of delay
intervals, DTNBP1 mutants then displayed worse performance (Papaleo et al.
2012). This genotype-specific and delay-dependent disruption was exacerbated by
exposure to a mild stressor during testing, where mice were housed in a new, clean
cage rather than the familiar home cage; these data are consistent with the
recognised ability of mild stressors to impair PFC mediated working memory
function. Cellular findings suggest that the interaction between CaMKII and
enhanced signalling at cortical D2 receptors may contribute, at least partially, to
observed working memory phenotypes (Ji et al. 2009; Papaleo et al. 2012).
4.3 Neuregulin
Neuregulin-1 (NRG1) has been associated functionally with numerous neurode-

velopmental processes, including neuronal migration, neurotransmitter receptor
expression/activation, myelination, and synaptic plasticity (Mei and Xiong 2008).
Multiple NRG1 isoforms have been described, the diversity of which has been
attributed to alternative splicing and the existence of multiple 50 flanking regula-
tory elements. NRG1 I–III share the EGF–like signalling domain; interaction of
these EGF-like domains with membrane-associated tyrosine kinases (ErbB
receptors) activates intracellular signalling pathways implicated in the pathobiol-
ogy of schizophrenia (Harrison and Law 2006; Mei and Xiong 2008; O’Tuathaigh
et al. 2009). Initially associated with increased risk for schizophrenia in an
Icelandic sample (Stefansson et al. 2002), NRG1 has been confirmed on meta-
analysis to be a replicable risk gene for schizophrenia (Bertram 2008). Further
evidence indicating disruption to NRG1-ErbB signalling in the pathobiology of
schizophrenia derives from studies in human postmortem brain tissue and cell lines
(Harrison and Law 2006; Mei and Xiong 2008).
A missense mutation in exon 11, which codes for the transmembrane region of
NRG1, has been associated with schizophrenia (Walss-Bass et al. 2006). The
phenotype of mutants with heterozygous deletion of the transmembrane
(TM)-domain of NRG1 [homozygous KO being lethal] is characterised by
hyperactivity, including sex-specific effects among individual topographies of the
murine repertoire, that is sensitive to amelioration by clozapine (O’Tuathaigh et al.
2006, 2007a; Karl et al. 2007); there is modest disruption to PPI (Stefansson et al.
2002; however, see Van den Buuse et al. 2009; O’Tuathaigh et al. 2008, for
conflicting data). Although initial studies indicated no effect of TM-domain NRG1

loss-of-function on various learning and memory measures such as Y maze
alternation and the Barnes maze (O’Tuathaigh et al. 2007b), a separate investi-
gation revealed mild domain-specific cognitive abnormalities in TM-domain
NRG1 mutants in novel object recognition memory and in contextual but not cue-
directed fear learning (Duffy et al. 2010). Consistent with earlier reports
(O’Tuathaigh et al. 2007b), no differences in spatial learning or working memory
were observed in this study.
Heterozygous KO of TM-domain NRG1 likely affects several NRG1 isoforms.
In terms of phenotypic comparisons with more isoform-specific NRG1 deletions,
type III NRG1 mutants exhibit greater deficits in PPI relative to TM-NRG1
mutants (Chen et al. 2007). However, neither type III NRG1 mutants nor those
with targeted disruption of type I/type II NRG1 display the hyperactivity that is
characteristic of TM-NRG1 mutants (Rimer et al. 2005; Chen et al. 2007). In
contrast, mutants with heterozygous deletion in the EGF-like domain [that might
be expected to impact on all NRG1 isoforms] exhibit an initial increase in loco-
motor activity and more rapid habituation of exploration in a novel environment;
in the same mutant line, showing baseline PPI to be unaltered, PPI was disrupted
by MK-801 but not by amphetamine (Duffy et al. 2008).
To assess the impact of over-expression of NRG1, mice carrying the transgene
of mouse type-1 NRG1 cDNA were examined for the presence of schizophrenia-
related abnormalities (Kato et al. 2010). Each of two separate transgenic lines
displayed: (a) increased novelty-induced hyperactivity, although habituation levels
did not differ across genotypes; (b) intact PPI and startle responsivity; (c)
impairment in contextual learning; (d) increased aggression and decreased social
behaviours in the resident-intruder paradigm. HPLC analysis in various brain
regions found decreased DA and DOPAC levels in the hippocampus of over-
expressor mutants. Paradoxically, both hypermorphic and hypomorphic expression
of NRG1 produce a common behavioural phenotypic profile. A different type-1
NRG1 over-expressor line, driven by a Thy-1 promoter in brain projection neu-
rons, displayed marked impairment of PPI; however, a tremorous phenotype likely
confounds behavioural assessment (Deakin et al. 2009). Phenotypic differences
between both NRG1 Type-1 over-expressor lines may be attributable to the use of
distinct gene promoters to regulate transgene expression (Kato et al. 2010).
Although it was initially shown that NMDAR expression is reduced in
TM-domain NRG1 mutants (Stefansson et al. 2002), recent evidence has ques-
tioned the role of NRG1 in NMDAR function, with brain-specific mutation of
ErbB2 and ErbB4 having no effect on the expression of NMDAR subtypes (Barros
et al. 2009; Gajendran et al. 2009). However, phosphorylation of NR2B receptor
subunits appears reduced in the TM-domain NRG1 mutant (Bjarnadottir et al.
2007). Interestingly, ablation of the ErbB4 receptor in parvalbumin-positive
interneurons in the prefrontal cortex prevented NRG1-induced stimulation of
GABA and produced a schizophrenia-related phenotype comparable to that
observed in heterozygous NRG1 or ErbB4 KO models; these included PPI deficits,
impaired working memory and hyperactivity (Wen et al. 2010). This conditional
line also showed impaired working memory in the 4- or 8-arm radial maze; a
similar profile has not been observed in NRG1 and ErbB4 mutants (O’Tuathaigh
et al. 2007a, b). These data would implicate parvalbumin-positive neurons as a
target for NRG1/ErbB4 modulation of behaviours relevant to schizophrenia.
Recent experiments demonstrate that ErbB4 also controls the formation and/or
maintenance of excitatory synapses on specific populations of GABAergic inter-
neurons and that postsynaptic ErbB4 function is probably required in this process
(Fazzari et al. 2010).
A potential role for NRG1 in G 9 E interactions related to schizophrenia (see
5.2) is highlighted by a recent study which examined HPA axis activity and stress
responsivity in a line of NRG1 mutant rats [generated using the sleeping beauty
transposon] with disruption of the NRG1 Type II isoform (Taylor et al. 2011).
NRG1 mutant rats displayed increased basal corticosterone levels and decreased
corticosterone levels after 30 min restraint stress relative to basal levels, with
increased expression of glucocorticoid receptor expression in the pituitary and
hippocampus and decreased expression in the paraventricular nucleus. These
mutants also displayed slower habituation in a novel environment, consistent with
that observed in NRG1 hypomorphic mice (O’Tuathaigh et al. 2006), which the
authors interpreted as increased reactivity to environmental stimuli.
4.4 Copy Number Variations
Studies now indicate the importance of rare structural mutations in schizophrenia,

although the size of their contribution or possible associations with symptom
subtypes remains unclear (Bassett et al. 2010; Owen et al. 2010). These mutations
usually consist of DNA sequences that vary between individuals due to either
duplication or deletion of chromosomal material. Initial support for CNVs came
from patients with velocardiofacial syndrome, caused by a microdeletion at
chromosome 22q11 and associated with substantive increase in risk for psychosis
(Karayiorgou and Gogos 2006). Mice containing heterozygous disruption in a
subset of 22q11.2 genes (Gnb1l, Dgcr8 and Tbx1) demonstrate significantly
reduced PPI (Long et al. 2006; Paylor et al. 2006; Stark et al. 2008). Other
schizophrenia-related phenotypes of mutants carrying a multigene deletion across
the 22q11 region include neuronal migratory defects and disruption of cortical
neurogenesis (Meechan et al. 2007a, 2007b; Stark et al. 2008).
There is now considerable evidence for an increased number of low-frequency
CNVs, across numerous loci, in individuals with schizophrenia (International
Schizophrenia Consortium 2008; Walsh et al. 2008; Kirov et al. 2009a; Levinson
et al. 2011). Several studies have identified common CNV loci which map to
chromosomes 1q21.1 and 15q13.2 (International Schizophrenia Consortium 2008;
Stefansson et al. 2002; Levinson et al. 2011). The 15q13.2 region contains the a7
nicotinic cholinergic receptor gene (CHRNA7); mice containing partial knockout
of the CHRNA7 gene evidence attentional deficits (Young et al. 2007), while
agonists at the a7 nicotinic cholinergic receptor may have some efficacy against
both the positive and negative symptoms of schizophrenia (Hajós and Rogers
2010).
In recent years, several studies have supported a link between increased sus-
ceptibility for schizophrenia and microdeletions affecting the gene neurexin 1
(NRXN1; Kirov et al. 2008, 2009b; Ikeda et al. 2010; Rujescu et al. 2009;
Levinson et al. 2011). Neurexins are presynaptic proteins that function as synaptic
recognition molecules and may contribute to various aspects of synaptic function
via binding to neuroligins (Ichtchenko et al. 1995). Mice containing deletion of
NRXN1a display behavioural and electrophysiological deficits relevant to
schizophrenia, including disruption to synaptic transmission and PPI (Etherton
et al. 2009). Interestingly, dysregulation of NRXN1 expression throughout
development has been observed in mice containing mutation of the DISC1 gene
(the ENU-generated L100P mutation), providing a measure of functional associ-
ation between two prominent risk genes.
5 Evaluation of G 3 E Hypotheses in Mutant Models

of Schizophrenia
According to the neurodevelopmental model of schizophrenia, genetic (G) and

environmental (E) influences combine to influence early CNS development and
function, producing pathophysiological deficits which precede the onset of
psychotic symptoms (Fatemi and Folsom 2009; Waddington et al. 2011). A variety
of early insults, ranging from viral infection during pregnancy, through psycho-
social adversities during childhood, to psychosocial stressors and substance abuse
over adolescence/young adulthood, have been associated with schizophrenia
(van Os et al. 2010) and shown to produce behavioural and neurobiological
changes reminiscent of schizophrenia in adult rodents and non-human primates
(O’Tuathaigh et al. 2007a, 2010a, O’Tuathaigh et al. 2010b). One important
consideration in the development of G 9 E models is the timing of the environ-
mental insult. The majority of preclinical G 9 E studies have sought to employ
environmental manipulations at specific periods of brain development, from early
stages of pregnancy through to adolescence, which have been identified as critical
to the pathogenesis of schizophrenia (Cannon et al. 2003; Waddington et al. 2011).
5.1 DISC1 3 Immune Challenge
A number of recent studies have examined the behavioural consequences of

manipulation of the pre- or post-natal environment in mice harbouring genetic
mutation. Epidemiological findings have found an association between elevated
cytokines in maternal serum and increased risk for schizophrenia in offspring
(Brown et al. 2004), leading some authors to suggest that G 9 E interaction in

schizophrenia may involve genetic vulnerability to abnormal immune responses
following environmental challenge (Ayhan et al. 2009). A recent study examined
the interaction of the schizophrenia risk gene DISC1 with neonatal exposure to the
immune system activating agent Poly I:C (Ibi et al. 2010). The authors proposed
that the neonatal period in mice corresponds to post-conception days 128–158 for
cortical events and 93–115 for limbic development in humans (Clancy et al. 2007;
Ibi et al. 2010). Transgenic mice expressing a dominant-negative form of DISC1
demonstrated a selective and more pronounced schizophrenia-related phenotype
across various cognitive measures (i.e. spontaneous alternation, object recognition
memory, contextual fear memory) following neonatal immune challenge; social
interaction and MK-801-induced hyperactivity were also selectively altered in
Poly I: C-treated DISC1 mutants. These behavioural deficits were accompanied by
a decrease in parvalbumin-positive interneurons in the medical prefrontal cortex.
Another model of G 9 E interaction related to schizophrenia has been
described recently in mice with inducible expression of mutant hDISC1 in fore-
brain neurons (Pletnikov et al. 2008; Abazyan et al. 2010). Rather than examining
neonatal immune challenge, this study employed the well-characterised prenatal
Poly I: C immune challenge paradigm (Meyer et al. 2009). These authors dem-
onstrated in male mice expressing mutant hDISC1 that treatment with Poly I:C at
gestational day (GD) 9, corresponding to the end of the first trimester of human
pregnancy, produced a behavioural profile that included elevated anxiety,
depression-like behaviours and disrupted social interaction; these behavioural
deficits were accompanied by altered 5-HT neurotransmission in the hippocampus,
decreased HPA axis reactivity and differential modulation of secretion of
inflammatory cytokines, as well as attenuation of genotypic enlargement of the
lateral ventricles. In contrast with the earlier study (Ibi et al. 2010), no changes in
cognition were observed. These data may suggest an environmental mechanism by
which the same DISC1 mutation can produce diverse neuropsychiatric phenotypes,
in a manner comparable to that observed with the original Scottish chromosomal
dislocation (Chubb et al. 2008; Hennah et al. 2009). With respect to potential
mechanisms underlying putative interactions between DISC1 dysfunction and
immune activation, it has been suggested that a key target may be disturbance of
neuronal maturation, which may be effected via proliferation, migration, early
dendritic development, and axonal outgrowth, as well as synapse formation and
maturation (Ayhan et al. 2009).
5.2 NRG1/COMT 3 Exposure to Drugs of Abuse
Epidemiological data have indicated an association between substance abuse and

risk for psychosis. Although clinical data have not indicated a relationship between
NRG1 genotype and behavioural responsivity to drugs of abuse, studies conducted
in mutant models of NRG1 function have indicated changes in behavioural and
neural sensitivity to cannabis and psychostimulant drugs in heterozygous NRG1

TM-domain mutants and COMT mutants (Boucher et al. 2007a, b; O’Tuathaigh
et al. 2010c).
NRG1 male mutants demonstrated increased sensitivity to the hypoactivity-
inducing and PPI-disruptive effects of an acute dose of D9-THC, the psychoactive
constituent of cannabis (Boucher et al. 2007a). In a separate follow-up study,
female NRG1 mutants were shown to be insensitive to D9-THC -induced social
withdrawal, although its hypoactivity-inducing effects were observed in both
genotypes (Long et al. 2010). Increased sensitivity to D9-THC-induced activation
of c-fos, a marker of neuronal activation, was also found in NRG1 male mutants
across several brain regions (Boucher et al. 2007b). Subchronic exposure to the
synthetic cannabinoid CP55, 940 for 15 days in NRG1 TM-domain mutants was
associated with more rapid tolerance to its hypoactivity-inducing and hypothermic
effects (Boucher et al. 2010); in contrast, NRG1 mutants were insensitive to its
anxiety-modulating effects. More recently, it was shown that the effects of
subchronic administration of NMDA receptor antagonists, a pharmacological
regimen known to produce schizophrenia-related deficits in rodents, were altered
in a paradoxical manner in NRG1 TM-domain mutants; specifically, the hyper-
activity and social interaction phenotypes reported in vehicle-treated mutants were
absent following NMDA receptor antagonist treatment (O’Tuathaigh et al. 2010b).
Cannabis consumption, particularly when used in adolescence, has been asso-
ciated with a doubling of risk for schizophrenia (Arseneault et al. 2004; Henquet
et al. 2005; Moore et al. 2007). A longitudinal birth cohort study has shown that
cannabis use was more likely to be followed by psychosis among those exposed
during adolescence and homozygous for the COMT Val108Met allele (Caspi et al.
2005). We have recently shown that in COMT KO, genotype exerted specific
modulation of responsivity to chronic D9-THC administration during adolescence
in terms of exploratory activity, spatial working memory and anxiety; this
illuminates the interaction between genes and adverse environmental exposures
over a particular stage of development in the expression of the psychosis pheno-
type (O’Tuathaigh et al. 2010c).
5.3 Genes Related to the Pathophysiology

of Schizophrenia 3 Exposure to Prenatal Stress
Mice containing either knockdown or a point mutation in the synaptosomal-

associated protein of 25 kDa (SNAP-25), a gene associated with synaptic trans-
mission and linked with schizophrenia in meta-analysis of genomic association
data (Lewis et al. 2003), were particularly vulnerable to the effects of prenatal
stressors on the emergence of schizophrenia-releveant behavioural phenotypes
(Oliver and Davies 2009). Specifically, during embryonic day 11.5–17.5, SNAP-25
mutants exposed to a combination of physical (restraint, swim), situational (open
field), and social stress, demonstrated significantly enhanced PPI deficits relative
to stressed WT; these deficits were reversed by haloperidol or clozapine. Inter-

estingly, social interaction deficits, in terms of both sociability and preference for
social novelty, were observed only in SNAP-25 point mutants that had been
exposed to prenatal stress; this indicates that combining genetic vulnerability with
exposure to an environmental stressor both modifies and produces novel pheno-
types relevant to schizophrenia. Prenatal stress increased anxiety-related behaviour
(elevated plus maze, open field) in all mice regardless of genotype.
Reelin is a protein that has been functionally linked with the control of synaptic
functions and a dysfunctional Reelin-mediated signalling pathway has been
suggested to play a role in the pathophysiology of schizophrenia (Knuesel 2010).
Several studies have examined the effect of early life adversity on the heterozy-
gous Reeler mouse phenotype. In Reeler mutants who had been prenatally exposed
over GD 14-16 to the neurotoxin chlopyrifos, development was altered in a par-
adoxical fashion, reversing genotypic abnormalities across a number of behav-
ioural domains: ultrasound vocalisation, increased amphetamine-induced
locomotion, and stereotype (Laviola et al. 2006). Interestingly, a similar effect was
observed in Reeler mutants who were exposed to early maternal separation
(PND2-6); maternally separated Reeler mutants failed to show abnormalities in
ultrasonic vocalisations and exploratory behaviour, as well as social interaction
(Ognibene et al. 2007). These data illustrate the emergence of unexpected and
novel phenotypes in risk gene mutants as a result of gene 9 environment
interactions.
5.4 Overview of G 3 E studies
Going forward, systematic examination of G 9 E risk factor manipulations in

genetic models will be necessary to identify common and distinct G 9 E risk
profiles at the level of endophenotypes and underlying neurobiology. With respect
to diagnostic categories which may be differentially susceptible to environmental
versus genetic influences, a recent review of clinical evidence for G 9 E inter-
actions in schizophrenia hypothesised that environmental factors associated with
schizophrenia may have a greater impact on affective and psychotic dimensions,
while developmental influences may have a greater impact on negative and
cognitive dimensions (van Os et al. 2010). One of the criticisms of the genetic
model data to date has focused on the translational success of modelling certain
environmental factors or stressors (e.g. psychosocial stressors) between human and
small animal species (van Os et al. 2010). Additionally, for logistical reasons, the
feasibility of studies combining exposure to several environmental factors with
any given genetic vulnerability factor(s) may be difficult to realise.
In teasing out the biological mechanisms underlying synergistic effects of
environmental and genetic factors on risk for disease, it is known from preclinical
studies that exposure to several environmental stressors implicated in schizo-
phrenia (prenatal immune challenge, early life adversity, psychosocial stressors,
adolescent cannabis/psychostimulant exposure) may be associated with disruption

to DAergic transmission. Given interactions between DAergic function and
GABAergic, glutamatergic and endocannabinoid signalling, future research on the
biological impact of these environmental insults should also examine neurode-
velopmental processes and functions implicated in schizophrenia, including
changes in myelination, synapse formation, and immune system function.
6 Discussion
6.1 Divining gene-Phenotype Associations in Schizophrenia

Risk Gene Models
6.1.1 Sex Differences
Despite considerable epidemiological evidence for sex differences in the inci-

dence, psychopathology and course of schizophrenia, the notable absence of
mutant studies which have systematically compared male and female murine
phenotypes has been noted (O’Tuathaigh et al. 2010a; Chen and Lai 2011).
Additionally, it has been noted that aside from the potential explanatory role of
sexually dimorphic biology in relation to the epidemiological findings, sex dif-
ferences in the nature of environmental interactions may also help to explain sex
differences in the expression of psychiatric symptoms (Accortt et al. 2008;
Burrows et al. 2011). The presence of sexually dimorphic effects of susceptibility
gene mutation on the expression of schizophrenia-related endophenotypes, which
have been noted throughout our review of the literature, might suggest the
involvement of sex hormonal regulation of AKT1, DISC1, NRG1 and other risk
gene function in a manner which may contribute to the pathogenesis and/or
expression of schizophrenia.
6.1.2 Improved Characterisation of Behavioural Models of Schizophrenia
As others have pointed out, progress in molecular and genomic techniques, and the
implications for genetic modelling in schizophrenia, has not been paralleled by
comparable advances in the generation of murine behavioural paradigms capable
of measuring specific forms of cognition and social and motivational behaviours
which are disrupted-in-schizophrenia; furthermore, these relate to clinical features
that are largely insensitive to existing antipsychotic treatments (Brigman et al.
2010). While the field has made incremental progress in generating novel murine
paradigms to assess these domains, including translating across species from rat to
mouse, significant progress have been made in some areas such as the develop-
ment of tasks employing touchscreen technology to measure executive function
processes (Brigman et al. 2010).
Despite cognitive deficits being considered a core feature of schizophrenia,

several aspects of cognition have not been addressed in existing behavioural
models. Recent studies have described novel assessment methods for problem-
solving behaviour, a form of executive functioning (Ben Abdallah et al. 2011).
In this paradigm, which is dependent upon a set of ethologically appropriate
escape behaviours within a limited time period, the greatest executive function
deficits were observed in mice deficient in the GluA1 subunit of AMPA receptors
or mice which had received hippocampal lesions. Mice with lesions of the medial
prefrontal cortex showed more subtle and specific performance deficits under one
of the task conditions, while similarly subtle difficulties in problem-solving
behaviour were found in mice containing overexpression of the D2 receptor or in
mice which were chronically treated with an NMDA receptor antagonist (Ben
Abdallah et al. 2011). Although variable performance in control groups across
each of these experimental conditions indicates that further work is required in
relation to standardisation of the procedure, the relatively simple box design and
experimental procedure indicates a promising new tool for assessment of cognitive
processes, including executive functions, relating to schizophrenia.
Stress responsivity has been proposed as a modulating trait which might
increase risk for psychotic disorders (Goodwin et al. 2004; Myin-Germeys et al.
2009). Individual differences in affective response to stressful stimuli, alongside
the associated physiological response, may play a role in induction of psychosis. It
has been suggested that inherited liability to psychotic disorder may be expressed
as altered responsivity to everyday stressful situations (Myin-Germeys et al. 2005).
6.1.3 Strain Differences
Even where a single gene mutation has been introduced, evidence indicates that a
mutant model bred onto different background genetic strains can show very dif-
ferent phenotypes, suggesting gene 9 gene interactions and/or genetic modifier
effects (Burrows et al. 2011; see Waddington et al. 2005 for further discussion of
implications of strain differences in CNS KO models).
6.2 Integration of Novel Gene Manipulation Strategies
Review of single gene mutant and G 9 E studies over the past few years, which
have increasingly involved the application of conditional transgenic technologies
and alternative gene manipulation strategies, demonstrates the power of examining
the effect of dynamic genetic manipulations throughout development, followed by
multidomain behavioural, cellular, and molecular phenotyping. Further integration
of new strategies to modulate gene expression in a phenotypic and regional manner
will facilitate the development of more accurate in vivo disease models based
on available pathophysiological data for disease states (Garbett et al. 2010).
Table 1 Mutant mouse phenotypes relating to dopaminergic and glutamatergic mechanisms implicated in schizophrenia
Anxiety Novelty- Cognition Sociability Psychopharmacology
induced
Working Attention PPI Social Social Social Increased sensitivity Increased Responsivity to
activity
memory memorya aggression interactionb to psychostimulantsc sensitivity antipsychotics
to stress
Dopamine mutation
D1 Receptor – : ; – = – – – = – –
KO
D2 Receptor – ; ; – = – – – :/; – –
KO
D2L KO – = – – = – – – = – ;
D2 Receptor – – – – – – – – – – –
Susceptibility Genes for Schizophrenia
OE
D3 Receptor – – ; – – – – – – – –
KO
DAT KO – : ; – ; – : ; – – –
COMT Het : = =/: – = = : = – – –
COMT KO :/; = : – = = = = ; : –
COMT-Val Tg ; = ; ; = – – – : ; –
AKT1 KO – = – – ; – – – : – –
GSK-3 KO – – – – – – – – – – –
DVL-1 KO – = – – ;/= = – ; – – –
Glutamate mutation
NR1 KO – : – – ; ; ; ; ; – =/;
NR1 S897 – – – – ; – – ; – – –
NR1 PND : : ; – ; ; – – – – –
GluRe1 KO – : – – – – – – – – :
GlyT1 cKO = – = : = – – – ;/= – –
(continued)
235
Table 1 (continued)
236

induced
activity Working Attention PPI Social Social Social Increased sensitivity Increased Responsivity to
memory memorya aggression interactionb to psychostimulantsc sensitivity antipsychotics
to stress
DAO KO ; ;/: – – =/: – – – ;/: – –
DSR KO – : – – = = – = – – –
GLAST KO – : – – – – – ; : – :
SynGAP KO – : ; – ; ; – = ; – –
AMPA GluR1 ; : – – ; – – – ; : :
KO
mGluR4 KO – – = – ; – – ; : – –
mGluR5 KO – : – – ; – – – : – :
VGluT1 Het – – – – – – – – – : –
VGluT2 Het – – – – – – ; : – – –
:, increased relative to wildtype; ;, decreased relative to wildtype; =, no difference;/, separates different findings; -, not reported. Abbreviations: PPI prepulse
inhibition, KO knockout mutants, Het heterozygous mutants, OE over-expression, DAT dopamine transporter, COMT Catechol-O-methyltransferase, Tg transgenic
mice, GSK-3 glycogen synthase kinase 3, NR1 NMDA receptor 1, NR1 PND NMDA receptor postnatal deletion, GluRe1 NR1-associated NR2A subunit, GlyT1 cKO
Glycine transporter 1 conditional knockout, DAO D-amino acid oxidase, DSR D-serine, GLAST glutamate and aspartate transporter, mGluR metabotopic glutamate
receptor, VGluT vesicular glutamate transporter. a Includes social novelty preference and social recognition tests. b Includes investigation of stationary mouse within a
cage and free social interaction tests. c Includes dopamine-enhancing agents, NMDA receptor antagonists
C. M. P. O’Tuathaigh et al.
Table 2 Mutant mouse phenotypes relating to genes associated with risk for schizophrenia
induced
Working Attention PPI Social Social Social Increased sensitivity Increased Responsivity to
activity
memory novelty aggression interactiona to psychostimulantsb sensitivity antipsychotics
preference to stress
Dysbindin sdy =/: ;/=/: ; – ;/=/: – – ; – : –
DISC1 deletion ;/=/: : – ;/=/: – – ; – : – ;
DISC1 Q31 L – = – – – – – ; – : –
DISC1 L100P – : ; ; ; – – – – = :
DISC1-C Tg – – ; – – – – ; – : –
DISC1-DN = : = – = – – = ; : ;
Susceptibility Genes for Schizophrenia
DISC1-hu Tg = : ; – = = ; ; – – –
DISC1-(tr) Tg – – – ; – – – – – – –
NRG1–TM Het = : = – ; ; : = : : –
NRG1 III Het – = ; – ; – – – – – –
NRG1 Ig Het – = – ; – – – – – – :
NRG1 EGF Het – : – – = – – – – – –
NRG1 (I) Tg – : – – = – : ; – – –
NRG1 (I-Thy1) Tg = = – – ; – – – – – –
ErbB4 Het – =/: ; – – – – – – – –
ErbB4 PV – : ; – ; – – – – – –
:, increased relative to wildtype; ;, decreased relative to wildtype; =, no difference; /, separates different findings; -, not reported; PPI prepulse inhibition, DAO
D-amino acid oxidase, DISC1 disrupted-in-schizophrenia-1, DISC1 C Tg DISC1 C-terminal fragment transgenic mice, DISC1-DN DISC1 dominant-negative mice,
DISC1-hu Tg DISC1 human form transgenic mice, DISC1-(tr) truncated DISC1 transgenic mice, NRG1-TM neuregulin 1 transmembrane domain, Het heterozygous
mutants, NRG1-EGF neuregulin1 epidermal growth factor domain, NRG1 (I) neuregulin1 type I over-expressing mice, NRG1 (I-Thy1) Tg neuregulin1 type 1 (Thy1
promoter driven) over-expressing mice, ErbB4 PV mice with ablation of parvalbumin-positive ErbB4. a Includes investigation of stationary mouse within a cage and
free social interaction tests. b includes dopamine-enhancing agents, NMDA receptor antagonists
237
In addition to inducible and conditional systems, the in utero gene transfer tech-
nique, with expression and/or shRNA constructs for RNAi, may prove a promising
methodological innovation by enabling the selection of target cell populations for
genetic modulation, while the introduction of inducible expression constructs will
allow better temporal control. Furthermore, experimental designs for future studies
on mutant models of psychosis would be improved, where appropriate, by the
incorporation of a rescue strategy aimed at restoring or compensating for the loss-
of-gene function; this would assist in providing mechanistic insight into the role of
the gene in mediating schizophrenia-related phenotypes and the development of
novel antipsychotic targets. By using the technical advantage of in utero transfer,
in which expression of more than one gene can be modified at one time by
cotransfection of expression and/or RNAi constructs (Shu et al. 2004; Young-
Pearse et al. 2007), it should prove possible to test the synergistic or other epistatic
effects of multiple genetic factors.
Evidence from the emerging field of molecular epigenomics suggests a putative
deficit in microRNA processing in schizophrenia. MicroRNAs are small, non-
coding RNAs that are believed to target at least a third of protein coding genes and
are associated with the regulation of mRNA levels for multiple genes in the human
cerebral cortex (Farh et al. 2005; Zhang and Su 2008). Dysregulation of miRNA
expression may account for some of the neurodevelopmental aspects of psychiatric
disorders and the difficulty in identifying individual causative genes (Dinan 2010;
Miller and Wahlestedt 2010). For example, disruption of miRNA biogenesis, as a
result of conditional deletion of DiGeorge syndrome critical region gene 8
(Dgcr8), has been associated with the expression of behavioural and structural
brain endophenotypes related to schizophrenia (Stark et al. 2008). Increased study
of the role of miRNAs in regulating phenotypic expression in existing mutant
models has the potential to provide further insight into the mechanisms by which
genes may contribute to specific aspects of the schizophrenia phenotype.
6.3 Examination of Gene 3 Gene Interactions in Schizophrenia
Assuming a polygenic basis for schizophrenia, neither partial nor complete loss-
of-function or over-expression of any single gene in mice is likely to produce an
animal model with construct validity for the disease; simultaneous dysregulation
of several susceptibility genes likely better reflects the genetic contribution to risk
for developing the disorder, emphasising the importance of generating and phe-
notypically characterising compound heterozygous mutants. In these animals,
partial loss- or gain-of-function in several components could perhaps mimic more
precisely the etiopathologic mechanisms, as well as the pathophysiological
features of schizophrenia. Evidence from cellular and molecular studies conducted
till date provides an important framework to understand how several common
susceptibility genes and/or multiple, rare CNVs might converge functionally onto
common pathways that may reflect the etiopathobiology of schizophrenia.
For example, there is evidence to connect NRG1, AKT, and DISC1 in a common
pathway that may regulate neurodevelopment and contribute to susceptibility to
schizophrenia (Kim et al. 2009) Future characterisation of mice with simultaneous
dysregulation of several risk genes may provide novel insights into whether those
risk genes may contribute to unique phenotypes and/or whether they might con-
tribute in a synergistic manner with other risk genes to the expression of schizo-
phrenia-related endophenotypes.
7 Conclusions
Increasingly, the evidence from mutant studies is showing that targeted and
transient ontogenetic challenges of certain spatial and temporal specificity may be
sufficient to invoke a disease process (Thompson and Levitt 2010). Additionally,
recent technical advances allowing manipulation of a single gene in a regionally or
temporally specific manner has provide the opportunity to assess how specific risk
genes might interact with environmental factors to produced schizophrenia-rele-
vant phenotypes. Clearly, the construct validity of any genetic model is determined
by the degree to which the mouse gene mutation successfully mimics the disease-
associated gene variant or mutation, as well as the available arsenal of accurate
and reliable phenotyping instruments. This is particularly the case for a heterog-
enous and/or polygenic disorder such as schizophrenia; however, single and
compound gene mutant models have enduring potential to inform on the contri-
bution of a given risk gene or networks of genes to the expression of schizo-
phrenia-related endophenotypes (Tables 1, 2).
Acknowledgments The authors’ studies are supported by Science Foundation Ireland Principal
Investigator grant 07/IN.1/B960 and Health Research Board of Ireland Postdoctoral Fellowship
PD/2007/20.
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Genetic Models of Sensorimotor Gating:
Relevance to Neuropsychiatric Disorders
Susan B. Powell, Martin Weber and Mark A. Geyer
Abstract Sensorimotor gating, or the ability of a sensory event to suppress a

motor response, can be measured operationally via prepulse inhibition (PPI) of
the startle response. PPI is deficient in schizophrenia patients as well as other
neuropsychiatric disorders, can be measured across species, and has been used
widely as a translational tool in preclinical neuropharmacological and genetic
research. First developed to assess drug effects in pharmacological and develop-
mental models, PPI has become one of the standard behavioral measures in genetic
models of schizophrenia and other neuropsychiatric disorders that exhibit PPI
deficits. In this chapter we review the literature on genetic models of sensorimotor
gating and discuss the utility of PPI as a tool in phenotyping mutant mouse models.
We highlight the approaches to genetic mouse models of neuropsychiatric disease,
discuss some of the important caveats to these approaches, and provide a com-
prehensive table covering the more recent genetic models that have evaluated PPI.

Keywords Prepulse inhibition Startle Mouse models Schizophrenia Genetic
Mutant
S. B. Powell (&) M. A. Geyer

Department of Psychiatry, University of California San Diego,
9500 Gilman Dr, La Jolla, CA 92093-0804, USA
e-mail: sbpowell@ucsd.edu
M. A. Geyer
e-mail: mgeyer@ucsd.edu
S. B. Powell M. A. Geyer
Research Service, VA San Diego Healthcare System,
San Diego, CA, USA
M. Weber
Department of Neuroscience, Genentech Inc, 1 DNA Way,
South San Francisco, CA 94080-4990, USA
e-mail: weber.martin@gene.com

DOI: 10.1007/7854_2011_195
252 S. B. Powell et al.
Contents
1 Introduction: Definitions and Measures of Sensorimotor Gating.................................... 252

2 Utility of Prepulse Inhibition Measures in Genetic Models of Schizophrenia............... 254
2.1 Relationship of PPI to Psychiatric Symptoms, Neurocognitive Measures,
and Overall Function ................................................................................................ 254
2.2 PPI as an Indicator of Neural Processes and a Pharmacological Screen............... 255
2.3 PPI as a Tool to Evaluate Gene–Environment Interactions ................................... 256
2.4 Evaluating the Role of a Susceptibility Gene in Pathology ................................... 262
2.5 Methodological Considerations with Specific Relevance to Genetic Models
of Sensorimotor Gating ............................................................................................ 275
3 Approaches to Genetic Models of Sensorimotor Gating ................................................. 276
3.1 Overview of Molecular Genetic Techniques........................................................... 276
3.2 Models Based on Hypotheses Regarding the Pathophysiology of Disease ........... 277
3.3 Genetic Mouse Models as Pharmacological Tools ................................................. 295
3.4 Genetic Models of Candidate Genes for Neuropsychiatric Diseases ..................... 296
3.5 Phenotype-Based Models Revealing the Function of Genes .................................. 298
4 Discussion .......................................................................................................................... 301
References................................................................................................................................ 303
1 Introduction: Definitions and Measures

of Sensorimotor Gating
Sensorimotor gating refers to the regulation of sensory information as it is

transmitted to motor output systems. When sensory information is processed
centrally, it requires some degree of filtering or ‘‘gating’’ prior to accessing and
impinging upon motor output. While this process has long been observed at
multiple levels of biology, the synaptic and cellular mechanism of sensorimotor
gating (Frost et al. 2003; Nusbaum and Contreras 2004; Rose and Scott 2003) as
well as the functional implications (Braff 2010, 2011; Swerdlow et al. 2008) are
starting to be elucidated. One form of sensorimotor gating that is widely studied in
humans and animals is prepulse inhibition (PPI) of startle. PPI is a form of startle
plasticity in which presentation of a weak stimulus (prepulse) preceding an intense
startling stimulus (pulse) by 30–500 ms inhibits the startle response (Graham
1975; Hoffman and Ison 1980). The fundamental mechanism underlying this
inhibition is thought to resemble the normal process of filtering incoming sensory
stimuli (Geyer and Braff 1987). PPI levels may indicate the current integrity of
sensorimotor gating mechanisms, providing an operational measure of sensori-
motor gating. In humans, startle is measured from the eye blink response through
electromyographic recordings of the orbicularis oculi muscle (Fridlund and
Cacioppo 1986) and typically involves startle to acoustic stimuli, although tactile
stimuli have been used as well (Braff et al. 1992; Kumari et al. 2003; Neuner et al.
2010; Swerdlow et al. 2001b). PPI deficits are observed in schizophrenia patients
[for review see (Braff et al. 2001; Swerdlow et al. 2008)], their unaffected first
Genetic Models of Sensorimotor Gating 253
degree relatives (Cadenhead et al. 2000), and patients with schizotypal personality
disorder (Cadenhead et al. 1993). Additionally, several other neuropsychiatric
disorders are associated with decreased PPI, including Obsessive–Compulsive
Disorder (Swerdlow et al. 1993), Tourette’s syndrome (Swerdlow et al. 2001b),
Huntington’s disease (Swerdlow et al. 1995), manic bipolar patients (Perry et al.
2001), Panic Disorder (Ludewig et al. 2002), Fragile X syndrome (Frankland et al.
2004; Hessl et al. 2009), and adults with autism (Perry et al. 2007). Although the
core symptoms of these disorders are diverse, a feature common to all of them is
deficient gating, with a gating deficit predominating in the cognitive sphere in
some disorders and in the sensory or motor domains in others. Thus, deficient
gating has been reported across a variety of neuropsychiatric disorders, with PPI
deficits in schizophrenia patients being the best characterized and the most widely
replicated (Braff et al. 2001; Kumari et al. 2008; Ludewig et al. 2003; Mackeprang
et al. 2002; Swerdlow et al. 2008).
Sensorimotor gating abnormalities, as measured by PPI, are being used as an
endophenotype in genetic studies of schizophrenia (Braff et al. 2007; Greenwood
et al. 2011), and meet the criteria outlined for a viable endophenotype (Turetsky
et al. 2007). With an increased focus on observable and measurable behaviors as
rational approaches to genetic studies of heterogeneous neuropsychiatric diseases
such as schizophrenia (Gottesman and Gould 2003), large-scale genetic studies are
examining neurophysiological measures such as PPI, P50 auditory evoked sup-
pression, antisaccade eye movement, mismatch negativity, and P300 event-related
potential (Turetsky et al. 2007). Recent studies suggest that PPI is heritable
(Hasenkamp et al. 2010) and associated with polymorphisms in the CHRNA3
gene (Petrovsky et al. 2010), neuregulin 1 (Roussos et al. 2011), and COMT
(Giakoumaki et al. 2008; Quednow et al. 2008; Roussos et al. 2008). The use of
PPI as an endophenotype in genetic studies of schizophrenia, combined with the
observation that PPI has a strong genetic component in mice (Francis et al. 2003),
indicates that PPI may be a useful behavioral phenotype to consider in genetic
mouse models related to neuropsychiatric disease, particularly schizophrenia.
While there are certainly many other symptoms, behavioral traits, and neuro-
physiological deficits observed across the heterogeneous group of patients with
schizophrenia, PPI appears to be a viable endophenotype for human genetic
studies and thus a reasonable approach to investigate in genetic animal models.
Additionally, considering that PPI measures a fundamental component of infor-
mation processing and is observable across many species, it is a useful endpoint
with which to understand the more general impact of specific genes on neurobe-
havioral function and on the neural substrates underlying this function.
Mutant mouse models related to schizophrenia have been based primarily on
the pathophysiology of schizophrenia, the known effects of antipsychotic drugs,
and candidate genes for schizophrenia. In this review, we provide an overview of
PPI in genetic mouse models, concentrating on the time period since the Swerdlow
et al. (2008) review. We discuss the contribution and usefulness of PPI as a
phenotype in the context of genetic mouse models and speculate about the sig-
nificance of PPI deficits and future directions for the field. There have been
previous reviews on candidate genes for schizophrenia (Arguello and Gogos 2010;
O’Tuathaigh et al. 2007), and reviews focusing specifically on PPI, which sum-
marized studies of strain differences, genetic mutants, and the pharmacology of
PPI in mice (Geyer et al. 2002; Powell et al. 2009; Swerdlow et al. 2008). In this
review, we highlight the approaches to genetic mouse models of neuropsychiatric
disease, discuss some of the important caveats to these approaches, and provide a
comprehensive table covering the genetic models that have evaluated PPI since the
Swerdlow et al. (2008) review. To that end, we discuss PPI as a phenotype in
genetic mouse models generated to address hypotheses regarding the pathophys-
iology of neuropsychiatric disease, candidate genes, and basic proteins involved in
neural development or synaptic function. We also discuss the usefulness of PPI in
phenotype-driven approaches in which a PPI phenotype could lead to ‘‘bottom up’’
approaches of identifying novel genes of relevance to PPI and/or neuropsychiatric
disease [i.e. hypothesis-generating; or phenotype-genotype approaches (Jacobson
and Cryan 2010)].
2 Utility of Prepulse Inhibition Measures in Genetic Models

of Schizophrenia
Mutant mouse models of neuropsychiatric disease have targeted genes involved in

the pathophysiology of the disease, candidate or susceptibility genes thought to be
involved in the etiology of the disease, or genes involved in basic physiological
processes. Because many of the target genes overlap at the functional level across
different disorders (e.g. proteins involved in basic processes of neurodevelop-
ment), manipulation of these genes may have relevance to many neuropsychiatric
disorders. Thus, it is important to determine the relevant behavioral tests to best
understand the underlying brain abnormalities resulting from genetic alterations of
proteins. Second, it is critical to develop animal models to screen novel thera-
peutics for specific domains of function. In this context, PPI has emerged as a
useful behavior with which to assess the integrity of basic neural circuits in genetic
mouse models and to screen for pharmacological agents, particularly in the context
of identifying treatments for schizophrenia.
2.1 Relationship of PPI to Psychiatric Symptoms,

Neurocognitive Measures, and Overall Function
Attempts to relate PPI deficits to the positive, negative, and cognitive symptoms of
schizophrenia have yielded mixed results (Thaker 2007). PPI negatively correlates
with thought disorder (Meincke et al. 2004; Perry and Braff 1994; Perry et al.
1999) and distractibility (Karper et al. 1996) in schizophrenia. PPI increases
observed with risperidone treatment correlated with improvements in PANSS

general psychopathology subscale scores (Martinez-Gras et al. 2009) in schizo-
phrenia patients. In a well-powered study with over 300 subjects, PPI did not
correlate with cognitive measures using traditional ‘‘pen and paper’’ tests
[i.e. Wisconsin card sorting task (WCST), California verbal learning task, etc.], but
did correlate positively with global assessment of function (GAF) and Independent
Living scales (Swerdlow et al. 2006). There have been some studies demonstrating
a relationship between cognitive constructs and PPI levels. For example, con-
verging evidence indicates that PPI is correlated with strategy formation and
execution time in the Cambridge Neuropsychological Test Automated Battery
(CANTAB) in healthy controls (Bitsios et al. 2006; Csomor et al. 2008;
Giakoumaki et al. 2006), a finding that should be examined further in patient
populations. The cognitive neuroscience treatment research to improve cognition
in Schizophrenia (CNTRICS) program funded by the National institute of mental
health considered PPI to provide a measure of the cognitive construct of ‘‘gain
control’’ as a specific aspect of the perceptual abnormalities seen in patients with
schizophrenia (Green et al. 2009). The series of CNTRICS workshops concluded
that PPI may have utility as a biomarker for use in proof of concept studies of
potential treatments for the cognitive deficits in schizophrenia that are not ame-
liorated by existing antipsychotic drugs. For the purpose of evaluating genetic
mouse models of neuropsychiatric disease, the more useful comparison to make is
not between PPI and specific symptoms of schizophrenia, but rather the rela-
tionship between a gene and the observable dependent measure, i.e. PPI. The
approach of using endophenotypes in genetic studies has greatly strengthened the
ability to conduct cross-species translational studies by providing specific
observables for study in experimental animals [reviewed in (Geyer and Markou
2002; Gould and Gottesman 2006) Waddington, chapter in this book]. Useful
endophenotypes in this context are measures that are observed in humans and can
be measured in animals.
2.2 PPI as an Indicator of Neural Processes

and a Pharmacological Screen
A PPI deficit in a genetic mutant could indicate that the gene may be involved in
the neural circuitry known to modulate PPI [e.g. cortical, limbic, striatal
(Swerdlow et al. 2001a)]; in other words it could function as a ‘‘surrogate measure
for neural processes’’ as Swerdlow et al. (2008) suggest. While a PPI deficit per se
is not indicative of altered striatal or limbic circuitry, the presence of the deficit
may suggest that these brain regions are affected by the genetic manipulation and
provide a reasonable starting place for further hypothesis testing regarding the
neurobiological implications of the genetic manipulation. Additionally, mutant
mouse models offer the opportunity to screen putative antipsychotics that may
involve a novel target and avoid the problems of receptor tautology inherent in
many pharmacological studies [e.g. dopamine agonist-induced disruption blocked
by a dopamine antagonist; as discussed in Powell et al. (2009). Using mutant mice
to screen for putative antipsychotics may provide a means to develop novel drug
targets not achieved with current pharmacological disruptions of PPI (see Table 1
for examples).
2.3 PPI as a Tool to Evaluate Gene–Environment Interactions
Studies of gene–environment interactions may be particularly informative for

neuropsychiatric diseases, most of which likely involve a genetic susceptibility
combined with environmental factors (e.g. stress) to observe the full manifes-
tation of the disease (Gottesman 1991) (see also Sen and Karg, Gross and
Carola chapters in this book). Three ways in which genetics and environmental
manipulations have been utilized in genetic mouse models are: (1) using a
mutant [e.g. knockout (KO)] to delineate the physiological mechanism of an
environmental manipulation; (2) rescuing a phenotype in a mutant with an
environmental manipulation; or (3) potentiating or unmasking a phenotype in a
genetic mutant with an environmental manipulation. There are a few examples
in which PPI has been a useful endpoint with which to assess gene–environ-
ment interactions in mouse models. For example, PPI deficits associated with
maternal immune activation (MIA) with PolyI:C during mid-gestation, which
typically leads to deficits in PPI in adult offspring (Meyer et al. 2005; Shi et al.
2003), are blocked in interleukin (IL)-6 KO dams (Smith et al. 2007). Thus,
PPI in a genetic mutant (IL-6 KO mice) was used to determine the mechanism
for the effects of an environmental manipulation (immune activation) on brain
development. An example of a PPI phenotype being ‘‘rescued’’ in a KO mouse
comes from studies in Phospholipase C –b1 KO mice, in which PPI deficits
and locomotor hyperactivity were attenuated in KO mice by environmental
enrichment or clozapine (McOmish et al. 2008) (Table 1). More recently, and
perhaps most important to etiological models of neuropsychiatric disease, there
have been several studies examining the ‘‘two-hit’’ approach (Eells et al. 2006;
Ibi et al. 2010). For example, nuclear receptor null Nurr1 heterozygous mice,
which display reduced mesocortical and mesolimbic dopamine (Eells et al.
2002), showed reduced PPI after postnatal isolation rearing, an effect that was
not observed with either isolation rearing or genotype alone (Eells et al. 2006).
This study provides a good example of the utility of PPI in gene–environment
models relevant to schizophrenia, specifically those designed to test the
‘‘two-hit’’ hypothesis for the etiology of schizophrenia. It should be kept in
mind, however, that many studies assessing gene–environment effects are
evaluating additive effects of two manipulations and must be interpreted with
caution.
Table 1 Genetically engineered model organisms based on genes related to: vulnerability for schizophrenia
References Mouse strain, sex Model description/background/ Basal PPI Effects of drugs or Effects of (presumed) antipsychotics/other
rationale manipulations typically treatments
used to induce PPI-deficits
DISC1
Hikida et al. (2007) DN-DISC1 TG, M DISC1 is a potential SZ ;PPI in TG versus WT at
susceptibility gene lowest PP intensity
only
Ibi et al. (2010) DN-DISC1 TG, combined Two-hit model of SZ, genetic [PPI & [PA in TG versus
with neonatal PolyI:C susceptibility combined with WT
5 mg/kg or saline environmental insult (immune
(PND2-6), M & F activation)
Niwa et al. (2010) DISC1 KD via in utero DISC1 plays important role in brain ;PPI & [PA in KD versus CLO: :PPI & [PA in KD; [PPI & [PA in
gene transfer (E14), M development. In utero gene WT at P56, [PPI at WT
Genetic Models of Sensorimotor Gating
&F transfer allows for analysis of P28, effect slightly

DISC1 function during less in F compared to
development and impact on M
adult brain function
Lipina et al. (2010) DISC1L100P point Missense mutation in exon 2 of ;PPI & ;PA in mutant AMP (0.5 mg/kg) ;PPI in HAL: :PPI in mutant (:PPI in WT at PP16);
mutation DISC1 at L100P in mice versus WT mutant but [PPI in ;PA in WT, ÆPA in mutant
WT
Lipina et al. (2011) DISC1L100P point Missense mutation in exon 2 of ;PPI in DISC1L100P TDZD-8 (GSK-3 inhibitor): :PPI & [PA in
mutation (combined DISC1 at L100P in mice; mutant vs. WT; [PPI DISC1L100P mutant; [PPI & [PA in
with GSK3 mutant) DISC1L100P mice crossed with in DISC1L100P x WT
Glycogen synthase kinase-3 GSK-3a HET
(GSK-3), a serine/threonine
protein kinase that interacts
with the N-terminal region of
DISC1
(continued)
257
Table 1 (continued)
258
Reelin
Barr et al. (2008) Reelin KO, M Reelin levels in brains of SZ are [(unimodal acoustic) PPI,
reduced but ;crossmodal PPI at
100 ms PP intervals,
PPP at PP intervals B
60 ms, ;PA
habituation & :PA at
100–110 dB(A)
intensity for +/-
versus WT
Neuregulin
Chen et al. (2008) Type III NRG1 mutants, M NRG1 may represent a SZ ;PPI & [PA in +/- versus Chronic NIC (:PPI in +/-; [PPI in WT)
susceptibility gene. Involved in WT
cell-cell interaction; signals via
erbB TK
Duffy et al. (2008) EGF-like domain NRG1 The EGF-like domain of NRG1 is [PPI in -/- versus WT MK801 (;PPI in -/-
mutants, M sufficient for NRG’s activity versus WT); AMPH
(;PPI in -/-and WT;
no significant
differences between
-/- & WT)
1-Tg
Deakin et al. (2009) NRG1Type Type I NRG1 increased in Hpc and ;PPI & :PA in Tg versus
PFC of SZ patients WT
Barros et al. (2009) ERBB2/B4-CNS-specific NRG1 and ERBB4 are candidate ;PPI in KO versus WT (M CLO :PPI in KO; [PPI in WT
KO, M & F genes for SZ only; [PPI in F); CLO : spine density
PA data not shown
(continued)
S. B. Powell et al.
Table 1 (continued)
Wen et al. (2010) PV-Cre; ErbB4 KO In adulthood NRG1 and ErbB4 may ;PPI & [PA in -/- DIAZ: :PPI in -/- mice; [PPI in WT; PA
regulate neurotransmission. versus WT data not shown
Evidence that ErbB4 is located
at the postsynaptic density of
excitatory neurons, likely on
GABA interneurons
Kato et al. (2010) NRG1 TG5, TG7, M & F Postmortem studies indicate [PPI & [PA in TG versus
increased NRG1 in SZ brain. WT
Transgenic mutants to examine
hyper NRG1 signaling
COMT
Papaleo et al. (2008) COMT KO, COMT Val-Tg Polymorphisms in COMT gene [PPI in -/- or TG versus
reported in SZ; mice WT; :PA in -/-
overexpress human COMT-Val versus WT, ;PA in TG
variant versus WT
Tammimäki et al. (2010) S-COMT KO, M & F Two isoforms of COMT, S-COMT [PPI & :PA in -/-
and MB-COMT, differentially versus WT (although
contribute to DA function in the lack of :PA with : dB
frontal cortex in WT mice)
G-alpha
Kelly et al. (2009) Forebrain-specific Galpha :mRNA G-protein subunit Galpha ;PPI & [PA in +/- versus HAL: :PPI in OE; [PPI in WT
OE, M, F levels, genetically linked to SZ WT; developmental or Rolipram (:cAMP levels): :PPI in OE;
adult expression [PPI in WT
[PPI; ;PPI correlates
with ;CTX & HPC
cAMP levels
(continued)
259
Table 1 (continued)
260
G72/G30
Otte et al. (2009) G72Tg (G72/G30 G72/G30 gene locus implicated in ;PPI & [PA in Tg versus HAL :PPI in Tg; [PPI in WT (slight :PPI at
‘‘humanized’’ BAC SZ, BD, and panic disorder WT low PP intensity in WT); PA data not
transgenic) reported
SMARCA
Koga et al. (2009) SMARCA2 KO, M & F SMARCA encodes BRM, a ;PPI in -/- versus WT;
chromatin remodeling multi- PA data not shown
protein complex; highly
expressed in differentiating
neurons; SNP in gene found in
SZ (this paper)
Neurexin
Etherton et al. (2009) Neurexin-1a KO, M & F CNVs for neurexin-1 a identified in ;PPI in -/- versus WT;
SZ & Autism. Neurexins bind :PA to stimuli 80–100
to neuroligins and function as dB in -/- versus WT
synaptic recognition molecules
TCF4
Brzózka et al. (2010) TCF4 Tg, M TCF4, a helix-loop-helix ;PPI & [PA in Tg versus
transcription factor, identified WT
in several GWAS studies as
susceptibility gene for SZ
(continued)
S. B. Powell et al.
Table 1 (continued)
AKT1
Chen and Lai (2011) AKT1 KO, M & F AKT1 has been associated with F: ;PPI & [PA in -/- RAC: [PPI & [PA in -/- or WT (F only)
schizophrenia in genetic studies versus WT; PA CLOZ: [PPI & [PA in -/- or WT (F
as well as postmortem brain M: [PPI & [PA in only)
analyses. AKT1 signal -/- versus WT 8-OH-DPAT: [PPI in -/- versus WT
transduction pathway important (attenuation weak; no genotype 9 drug
in diverse biological functions interaction)
SB216763: [PPI in -/- versus WT
(attenuation weak; no genotype 9 drug
interaction)
Abbreviations: 5-HT serotonin, AAV adeno-associated virus, Ab amyloid b- peptide, ACE angiotensin converting enzyme, ACH acetylcholine (receptor), AD Alzheimer’s disease, ADX adrenalectomy, AIL
advanced intercross line, AMP amphetamine, AMY amygdala, APD antipsychotic drug, APP amyloid precursor protein, ARIP aripiprazole, APO apomorphine, AT angiotensin, BAC bacterial artificial
chromosome, BACE b-site APP cleaving enzyme, BD bipolar disorder, BG background, CaMKIV calcium-calmodulin-dependent protein kinease IV, cAMP cyclic adenosine monophosphate, Ckr chakragati,
CLO clozapine, CNS central nervous system, COC cocaine, COMT catechol-O-methyltransferase, CORT corticosterone, CRF corticotropin releasing factor, CTR controls, CTX cortex, DA dopamine
(receptor), DAT dopamine transporter, dB decibel, DCC deleted in colorectal cancer, DIAZ diazepam, DIZ dizocilpine, DN dominant-negative, DDO D-aspartate oxidase, E embryonic day, EE
environmental enrichment, EGF epidermal growth factor, ENU N-ethyl-N-nitrosourea, f frontal, F female, FABP fatty acid binding protein, FGF fibroblast growth factor, Fmr1 fragile 9 mental retardation
1 gene, FMRP fragile 9 mental retardation protein, FXS fragile 9 Syndrome, GLAST glutamate and aspartate transporter, GLU glutamate, GluR glutamate receptor, GLUT glucose transporter, GR
glucocorticoid receptor, GRIP glutamate receptor interacting protein, GSK glycogen synthase kinase, GWAS genome wide association studies, HAL haloperidol, HD Huntington’s disease, HPC hippo-
campus, IC imprinted cluster, IL interleukin, ISI interstimulus interval, KI knock-in, KO knock-out, M male, m metabotropic, MDB methyl-CpG binding protein, mo month, NIC nicotine, MPEP 2-methyl-6-
(phenylethylyn)-pyridine hydrocholoride, METH metamphetamine, Mgat N-acetylglucosaminyltransferase, NAA N-acetyl-aspartate, NAAG N-acetylalpha L-aspartyl-L-glutamate, NAC nucleus accumbens,
NCAM neural cell adhesion molecule, NIS nisoxetine, nNOS neuronal nitric oxide synthase, NO nitric oxide, NPS neuropeptide S (receptor), NPY neuropeptide Y, NR NMDA receptor subunit, NRG
neuregulin, NRL neuroligin, ns not significant(ly), NSE neuron-specific enolase, NT neurotensin, OE overexpressor, OXO oxotremorine, PA magnitude of response to pulse alone, PACAP pituitary
adenylate-cyclase-activating polypeptide, PD Parkinson’s disease, PDE phosphodiesterase, PET-1 plasmocytoma expressed transcript-1, PND postnatal day, PLC phospholipase C, PNS prenatal stress, poly
I:C polyinosinic: polycytidylic acid, PP prepulse, PPI prepulse inhibition of startle, PPP prepulse potentiation, PS1 presinilin1, PWS Prader–Willi syndrome, QTL quantitative trait locus, QUET quetiapine,
RAC raclopride, RAGE receptor for advanced glycation end-products, RasGAP Ras GTPase-activating protein, RE renin-enhancer, RIS risperidone, SCOP scopolamine, SI social isolation, SNP single
nucleotide polymorphism, SOCS suppressor of cytokine signaling; SREB superconserved receptor expressed in brain; STR striatum, SYN synapsin, SynGAP synaptic GTP-ase-activating protein, SZ
schizophrenia, TAAR trace amine-associated receptor, TG transgenic, TGF-b transforming growth factor beta, TK typosine kinase, TMS transcranial magnetic stimulation, V1b vasopressin receptor 1b, WT
wild-type
; decreased, : increased, [ unchanged, -/- homozygous mice, +/- heterozygous mice
261
2.4 Evaluating the Role of a Susceptibility Gene in Pathology
When evaluating the role of a susceptibility gene implicated in any neuropsy-

chiatric disease, it is important to consider what criteria should be placed on a
genetic/etiological model. In the present context, it is relevant to consider whether
or not deficient PPI is a necessary phenotype with which to evaluate the usefulness
of a targeted gene deletion of potential relevance to the given neuropsychiatric
condition under study (e.g. schizophrenia). Using schizophrenia as an example, the
failure to see a PPI deficit in a mouse model may indicate a ‘‘false negative’’
particularly if other key behaviors relevant to schizophrenia are observed
(e.g. deficient social interaction, disruptions in attentional set shifting). The lack of
a PPI deficit in this case does not indicate that the genetic model is not of relevance
to schizophrenia. There are several examples provided in Table 1 in which a PPI
deficit was not present in a mutant mouse but other behavioral differences such as
deficits in memory, social interaction, or set shifting were apparent in the mice.
The likelihood of being able to reproduce all aspects of a heterogeneous disease in
another species with a genetic mutation (most often a single gene deletion) is very
rare if not impossible (see Jones et al. (2008); Powell et al. (2009) for discussion).
For that matter, it is not the case that all aspects of schizophrenia are observed in
each patient carrying the diagnosis. Rather, support for a model should be based on
the convergence of data from multiple sources [e.g. many animal models, human
genetic studies, etc. (Jones et al. 2008)]. Thus, no one phenotype should be
considered as being either necessary or sufficient to support a model for a neu-
ropsychiatric disease, particularly since the distributions of the behavioral measure
often overlap between healthy volunteers and patients, as is the case with PPI and
schizophrenia (Swerdlow et al. 2008). Thus an animal model should not be
rejected based on ‘‘normal’’ PPI. For example, GLAST KO mice lacking the
glutamate and aspartate transporter do not show any deficits in PPI but do show
deficits in social approach, nest building, and pairwise discrimination learning
(Karlsson et al. 2009). Along the same lines, there is the possibility that a PPI
deficit in a mutant mouse model could represent a ‘‘false positive’’, in which a
PPI phenotype may be suggestive of an association between that gene or pathway
and disease and no such association is found. As we have argued previously, the
PPI phenotype should be interpreted as meaning that the given genetic manipu-
lation may be involved in the regulation of PPI expression and caution that
PPI phenotypes should not be automatically associated with a specific disease,
e.g. schizophrenia (Powell et al. 2009). Interestingly, there are a few examples
where KO or transgenic mice actually had increased PPI relative to wild-type
controls. One such example is the FMR1 KO mice (Paylor et al. 2008; Thomas
et al. 2011a, b; but see de Vrij et al. 2008; Table 2). It remains unclear how such
findings should be interpreted, but the possibility that the loss of the gene may lead
to gain of function should be considered.
Table 2 Genetically engineered model organisms based on genes related to: neurotransmitters, neuropeptides, and orphan receptors
References Mouse Strain, Sex Model description/ background/ Basal PPI Effects of drugs or manipulations Effects of (presumed) antipsychotics/other
rationale typically used to induce PPI- treatments
deficits
A. DOPAMINE
DA receptors
Doherty et al. (2008) DA D1, D2 & D3 Abnormalities in DA signaling in [PPI in D1, D2 & D3 -/- COC ([PPI in D1 -/-; ;PPI in D2 RAC; SCH23390 (a D1 antagonist) both ;COC-
KO, M, F SZ patients in part have led to versus WT; :PA in D1 -/-, D3 -/- & WT mice; but: induced PPI deficits in WT; [PA in WT)
the DA hypothesis for SZ -/- versus WT; [PA in attenuated ;PPI in D2-/-
D2 (M only); ;PA in D2 versus WT and more
-/- (F only) & D3 -/- pronounced ;PPI in D3 -/-
versus WT & in D3 (F) versus WT; [PA in WT
versus D3 (M) -/-
Young et al. (2011b) DA D4 KO, M Alterations in D4R may contribute [PPI & [PA in -/- or +/-
to cognitive and attentional versus WT

deficits in neuropsychiatric
disorders.
DAT
Powell et al. (2008) DAT KO, (F) M A hyper-DA state and DAT M: ;PPI & [PA in -/- versus M: CLO (PPI in -/- normalized to WT levels, but
abnormalities have been WT; F: inconsistent ;PPI [PPI in -/- & WT versus VEH) ;PA (main
implicated in SZ in -/- versus WT effect); QUET (:PPI & [PA in -/- & [PPI &
;PA in WT)
Other Dopamine related
Grant et al. (2007) DCC KO, M (PPI DCC mice are netrin 1 receptor ;PPI (yet ns) and :PA (trend [AMP ([PPI in +/-, but ;PPI
studies) KO mice. Netrin 1 is strongly only) in +/- versus +/+ in +/+)
expressed on and may affect
development of DA neurons
(continued)
263
Table 2 (continued)
264
deficits
B. GLUTAMATE
NR1
Duncan et al. (2010) NR1 hypomorph Developmental effects of NR1 ;PPI & :PA in hypomorph LY382884, kainic acid antagonist, ;PA in
(NR1 neo/neo), hypomorph on other Glu versus WT hypomorphs & [PA in WT; :PPI in
receptors could contribute to hypomorphs & WT
the SZ-like phenotype.
Evidence for alterations in
kainic acid receptors in SZ.
Belforte et al. (2010) Early postnatal NMDAR hypofunction, Early postnatal NR1
NR1 reduction specifically in GABA reduction: ;PPI & [PA
in GABA interneurons, may contribute in KO versus Ctrl lines
interneurons to SZ pathophysiology. NR1 (Flox-A & Cre), in
(Ppp1r2- is an obligatory subunit of isolated & group-housed
cre +/-; NMDAR. mice
NR1loxP/loxP- Adult NR1 reduction:
line A [KO]), [PPI & [PA
M
AMPA
Sagata et al. (2010) GluR4 KO, M & F GluR4 subunit of AMPA receptor ;PPI & ;PA in -/- versus WT
involved in synaptic plasticity;
genetic association between
GluR4 and SZ
mGLU
Gray et al. (2009) mGlu5 KO, M, F mGlu5 receptor interacts with ;PPI in -/- versus WT; [PA Chronic (8 wks) CLO (:PPI in -/- & [PPI in WT);
NMDA receptor and has been in -/- versus WT :NMDA receptors with CLO treatment
implicated in behavioral
endophenotypes of SZ
(continued)
S. B. Powell et al.
Table 2 (continued)
deficits
Fendt et al. (2010) mGlu8 KO, M mGlu8 is a presynaptic receptor [PPI and ;PA in -/- versus PCP: ;PPI in -/- and WT; [PA in
which regulates Glu release; WT -/- or WT
may be important for
neuropsychiatric disorders
with altered Glu function (e.g.
SZ).
Chen et al. (2010) mGlu5 KO, M & F Evidence of NMDA hypofunction ;PPI & [PA in -/- versus WT Sarcosine: :PPI in -/-; [PPI in WT; [PA
in mGlu5 KO mice; mGlu5 in -/- or WT
KO mice may be useful as LY379268: [PPI & [PA in -/- or WT
novel screen of putative N-acetylcysteine: :PPI in -/- & WT; :PPI in
glutamatergic antipsychotics. -/- not blocked with LY341495

Vesicular glutamate transporter
Wallén-Mackenzie VGLUT2f/f;CKII/Cre Forebrain glutamate plays an ;PPI & ;PA in VGLUT2f/ PCP: ;PPI in WT; [PPI in ARIP (no-drug versus post-ARIP): :PPI in KO;
f;CKII/Cre
et al. (2009) (preadolescent important role in many versus WT VGLUT2f/f;CKII/Cre (no drug [PPI in WT; PA (no data shown)
forebrain- sensory and cognitive versus post-PCP); PA (no data
specific KO), functions relevant to shown)
M&F psychiatric disease;
downstream changes in DA
(this study)
(continued)
265
Table 2 (continued)
266
deficits
Glycine
Benneyworth et al. SR KO, M D-serine is an agonist and NAG an [PPI & [PA in SR -/- versus PCP: ;PPI in SR -/-, GCP2 +/-, &
(2011) GCP2 HET, M antagonist at the glycine WT; [PPI & [PA in WT; inverted U-shaped dose
modulatory site on NMDA GCP2 +/- versus WT response function on PA: high
receptors. Serine racemate dose (6 mg/kg) [PA in WT,
(SR) and glutamic acid :PA in SR -/-; 3 mg/kg dose
decarboxylase 2 (GCP2) :PA in WT, [PA in GCP2 +/-
regulate D-serine and NAG, AMPH: ;PPI in Sr-/-, GCP2
respectively. +/-, & WT; U-shaped dose
response function on PA; no
difference in SR -/-, GCP2 +/-,
& WT
Other glutamate related
Karlsson et al. (2009) GLAST (EAAT1) Glutamate dysfunction associated [PPI in -/- or +/- versus WT;
KO, M, F with SZ; glutamate transporter ;PA in -/- versus +/- &
may be useful WT
pharmacological target for SZ.
Guo et al. (2009) SynGAP +/- gene SynGAP is a RasGAP and ;PPI and :PA in +/- versus CLO: [PPI in +/- or WT; ;PA in +/- and WT
deletion NMDAR-associated protein; WT
interacts with NMDA
receptors
Han et al. (2009) GCPII +/-, M & F Glutamate carboxypeptidase II [PPI & [PA in +/- versus
(GCPII) hydrolyzes NAAG WT
into glutamate and NAA
Wang et al. (2009) Norbin KO Norbin interacts with mGluR5 ;PPI & [PA in -/- versus WT
receptors and positively
regulates mGluR5 signaling.
(continued)
S. B. Powell et al.
Table 2 (continued)
deficits
C. SEROTONIN (5-HT)
Semenova et al. 5-HT7 KO, M Antipsychotics have high affinity [PPI across PP intensities PCP ;PPI in WT, attenuated in -/-;
(2008) for 5-HT7 receptors. and ISI, [PA Apo and Amp ;PPI in WT &
-/-; [PA with Apo, Amp, PCP
Shanahan et al. (2009) 5-HTT KO, F OCD patients have PPI deficits; [PPI in -/-, +/- versus WT RU24969 (5HT1B agonist) ;PPI in
gain of function 5-HTT alleles WT, [PPI in -/-, intermediate
associated with OCD effect in +/-
Schaefer et al. (2009) Pet-1 KO, M &F Pet-1 is a transcription factor :PA & [PPI in -/- versus WT
restricted to 5-HT neurons.
Gene deletion results in loss of
5-HT.
Hill et al. (2011) 5HT2C KO 5HT and BDNF interact; :BDNF [PPI & [PA in -/- versus
levels 5HT2C KO mice (this WT
study)
Groenink et al. (2011) 5HT1A KO, M 5HT1A gene may interact with [PPI & [PA in -/- versus 129S6: Maternal separation ;PPI
(129S6, Swiss adverse life events. WT & :PA in -/- & WT
Webster [SW] SW: Maternal separation
backgrounds) [PPI & [PA in -/- & WT
van den Buuse et al. 5HT1A KO, M & F 5HT1A receptor may mediate [PPI & [PA in -/- versus MDMA: ;PPI in -/- at lower doses
(2011a) effects of MDMA on PPI WT than WT at 100 ms ISI (M
only); ;PA in -/- & WT
van den Buuse et al. 5HT1A KO, M & F 5HT1A receptor may interact with [PPI & [PA in -/- versus APO: ;PPI & [PA in -/- & WT
(2011b) neurotransmitter systems WT
involved in SZ
(continued)
267
Table 2 (continued)
268
deficits
D. ACETHYLCHOLINE (ACh)
Nicotinic
Darvas et al. (2009) LYPD6 Tg LYPD6 modulates nicotinic ACh :PPI in Tg versus WT; [PA
receptors; LYPD6 Tg show in Tg versus WT
enhanced nicotinic tone
Young et al. (2011a) a7-nAChR KO, M a7-nAChR is being investigated as [PPI in -/-, +/-, and WT;
&F a pro-cognitive target for SZ; :PA in -/- versus +/- &
comparison of KO mice WT;
across several cognitive tests
Muscarinic
Turner et al. (2010) M2, M4 KO, M M2 and M4 muscarinic [PPI in M2 or M4 -/- versus
acetylcholine receptors differ C57BL/6 N mice; [PA
in tissue distribution and in M2 -/- versus M4 -/-;
physiology; may play a role in :PA in M2 -/- versus
sleep and arousal C57; [PA in M4 -/-
versus C57 (store bought
mice; not littermates)
(continued)
S. B. Powell et al.
Table 2 (continued)
deficits
Thomsen et al. (2010) M1, M4 KO, F Muscarinic receptors may be F: ;PPI & [PA in M1-/-/ SCOP: ;PPI in WT & M4-/- mice Xanomeline: blocked SCOP-induce ;PPI in WT &
M1KO/ related to pathophysiology of M4-/- versus WT; [PPI (F) & slight ;PPI in M1-/-/ M1-/-, but not in M4-/- mice; data in M1-/-/
M4KO,M,F SZ, and muscarinic agonists & [PA in M1 -/- versus M4-/- (M); [PPI in M1-/- (F) M4-/- difficult to interpret because [PPI with
have been proposed as targets WT; [PPI & :PA in M4 or M1-/-/M4-/- (F) SCOP; blocked SCOP-induced :PA in M1-/-
for antipsychotics and -/-WT; :PA in all groups; M1-/-/ & M1-/-/M4-/-; [PA in SCOP-treated M4-/-
cognition enhancers M: [PPI & [PA in M4-/- (F) and M4-/- (F) more OXO (versus SCOP): blocked SCOP-induced
M1-/-/M4-/- versus WT sensitive to :PA ;PPI in all genotypes (although SCOP effect
not significant in M1-/-/M4-/-); trend for
reversal of SCOP-induced :PA
OXO (alone): ;PPI in WT & :PPI in M1-/-/
M4-/-; ;PA in WT & M1-/-M4-/- (which may

lead to variability in PPI)
CLO: :PPI in M1-/-/M4-/- at doses with
[PPI in WT; ;PPI in WT & M1-/-; [PPI in
M4-/-; ;PA in WT & M1-/-; [PA in M1-/-/
M4-/- & M4-/-
HAL: :PPI in WT & M1-/-/M4-/-; [PA in
WT or M1-/-/M4-/-
Vesicular ACh Transporter
(VAChT)
Schmid et al. (2011) VAChT KD, M Cholinergic transmission [PPI & [PA in -/- versus
associated with cognitive WT; ; between day HAB
function and startle behavior. in -/- versus WT
VAChT KD mice have
reduced cholinergic tone.
(continued)
269
Table 2 (continued)
270
deficits
E. (NEURO)PEPTIDES
Angiotensin/Renin
Martin et al. (2008) RE KO, NSE-AT1 AT/Renin may be involved in [PPI (RE -/- & NSE-AT1 APO, AMP & DIZ (all ;PA (RE
TG, M brain (DA) signaling. ACE TG vs. WT); ;PA (RE KO vs. WT; main effect
levels were reported to be KO (trend)) & [PA in across treatment conditions);
altered in SZ patients. REKO NSE-AT1 versus WT ;PPI in RE -/-, NSE-AT1 TG
have reduced renin signaling; & WT (no genotype x drug
NSE-AT1 TG have over- interactions); APO (;PA in
expression of AT1 receptors. RE -/-, NSE-AT1 TG & WT);
AMP (:PA in RE -/- & WT
(trend), [PA in NSE-AT1 TG
& WT;) DIZ (:PA in RE -/- &
WT; more pronounced in RE
-/-, :PA (trend) in NSE-AT1
TG & WT;
Neurotensin
Feifel et al. (2010a) NT1 KO, M & F Neurotensin (NT) may play a role [PPI in -/- versus WT; [PA AMP ;PPI & [PA in -/- & WT; PD149163, NT1 receptor agonist, :PPI in WT,
in SZ pathophysiology. NT in -/- versus WT MK801 ;PPI; MK801 :PA [PPI in -/-;
levels decreased in CSF from (low dose only; more
SZ patients, and APDs pronounced in WT than -/-)
increase NT levels.
Feifel et al. (2010b) NT2 KO, M & F NT2 receptors highly expressed in :PPI & ;PA in -/- versus WT
brain but not well studied (both sexes)
(continued)
S. B. Powell et al.
Table 2 (continued)
deficits
Oliveros et al. (2010) NT1 KO & NT2 NT receptors may play a role in [PPI & [PA in NT1 -/-, AMP: ;PPI in NT2 -/- & WT, CLO: blocks AMP-induced ;PPI in WT & NT2 -/-
KO, M psychotomimetic and NT2 -/-, & WT [PPI in NT1 -/-; ;PA in WT, ; blocks DIZ-induced ;PPI in WT, does not
antipsychotic effects of drugs [PA in NT1 -/- or NT2 -/- block DIZ-induced ;PPI in NT2 -/-
DIZ: ;PPI in NT2 -/- & WT, NT69L: blocks AMP-induced ;PPI in WT &
[PPI in NT1 -/- NT2 -/-; blocks DIZ-induced ;PPI in NT2 -/-,
does not block DIZ-induced ;PPI in WT;
;PA in WT & NT2-/-
CRF
Groenink et al. (2008) CRF-OE, M Stress physiology, particularly ;PPI in CRF-OE versus WT CORT implant: [PPI in CRF-OE CRF1 antagonists CP154,526 & DMP695
CRF and glucocorticoids, at lower PP intensities; or WT mice. normalized PPI deficit in CRF-OE mice; GR
implicated in neuropsychiatric [PA in CRF-OE versus antagonists and ADX did not; CP154,526
disorders WT ;PA
Oxytocin
Caldwell et al. (2009) Oxt KO, M, F Oxytocin has shown antipsychotic [PPI & [PA in -/- versus PCP: Exacerbated ;PPI in -/-
potential in animal models of WT versus WT, [PA; Apo &
SZ and in some preliminary Amp: ;PPI in -/- & WT; [PA
clinical trials
Arginine Vasopressin
Egashira et al. (2009) V1aR KO, V1bR Arginine vasopressin (AVP), a ;PPI in V1aR & V1bR -/-
KO diuretic peptide with receptors versus WT; :PA in V1aR
widely expressed in the CNS, & V1bR -/- versus WT
has been implicated in
neuropsychiatric disorders.
Neuropeptide Y
Karl et al. (2010a) Y2 KO, M & F NPY levels altered in SZ patients. M: :PPI & [PA in -/- versus MK801: ;PPI & :PA in -/- & WT
NPY Y2 receptor expressed in WT AMP: ;PPI & :PA in -/- &
HPC and AMY F: [PPI in -/- versus WT WT
(continued)
271
Table 2 (continued)
272
deficits
Karl et al. (2010b) Y1 KO, M Decreased Y1 receptor expression [PPI, ;PA, & ;HAB in -/- MK801: ;PPI & :PA in -/- & WT
in lymphocytes of SZ patients versus WT AMP: ;PPI & [PA in -/- &
WT
(effects are ISI & PP
dependent)
Neuropeptide S
Zhu et al. (2010) NPSR1 KO, M & F NPSR1 is a G protein coupled M: ;PA & [PPI in -/- versus
receptor located in brain WT;
regions involved in anxiety F: [PA & [PPI in -/-
and learning and memory, versus WT
whose endogenous ligand is
NPS. NPSR1 SNPs identified
in panic disorder.
Fendt et al. (2011) NPSR KO, M NPS implicated in regulation of [PPI in -/- or +/- versus WT; HAL: :PPI & ;PA in -/-, +/-, & WT
emotional behavior ;PA in -/- & +/- versus
WT
PACAP
Hashimoto et al. PACAP KO, M PACAP affects neurotransmission; ;PPI in -/- versus WT RIS (:PPI in -/-; [ in WT)
(2007) potential SZ susceptibility
gene
Ishihama et al. (2010) PACAP KO, M Genetic susceptibility plus ;PPI in -/- versus WT (PA SI: ;PPI & [PA in -/- & WT EE: ;PPI in -/- & WT
environmental factors may data not shown)
contribute to SZ
(continued)
S. B. Powell et al.
Table 2 (continued)
deficits
Relaxin-3
Smith et al. (2009) Relaxin-3 KO, M & Relaxin-3 modulates feeding, [PPI in -/- versus WT; PA
F stress response, exploratory data not reported
behavior, and arousal
F. ORPHAN RECEPTORS
Matsumoto et al. SREB2 KO, SREB (= GPR85) highly ;PPI & [PA in TG versus
(2008) SREB2 TG, M conserved GPCR; expressed WT; [PPI & [PA in -/-
in brain regions of high versus WT
plasticity; overtransmission of
2 SNPs in SZ (this study)
Logue et al. (2009) GPR88 KO, M & F GPR88 is highly expressed in brain ;PPI in -/- versus WT; [PA HAL: :PPI in -/-, [PPI in WT; ;PA in -/- & WT
regions implicated in SZ and in -/- versus WT RISP: :PPI in -/- & WT; ;PA in -/- & WT
regulates DA function (this
paper).
Kim et al. (2010) TAK1 KO, M TAK1 is a nuclear orphan receptor ;PPI, ;PA, ;HAP in -/-
that regulates transcription; versus WT
TAK1 deletion delays
migration of cerebellar
granular neurons
(continued)
273
Table 2 (continued)
274
deficits
G. TRACE AMINES
Karmacharya et al. TAAR1 KO, M Trace amines are very similar to [PPI in -/- versus WT (PA CLO: :PPI in WT, [PPI in -/-
(2011) biogenic amines and are data not shown)
implicated in neuropsychiatric
disease; trace amine receptors
may mediate effects of
antipsychotic drugs
chromosome, BACE b-site APP cleaving enzyme, BD bipolar disorder, BG background, CaMKIV calcium-calmodulin-dependent protein kinease IV, cAMP cyclic adenosine monophosphate, Ckr
chakragati, CLO clozapine, CNS central nervous system, COC cocaine, COMT catechol-O-methyltransferase, CORT corticosterone, CRF corticotropin releasing factor, CTR controls, CTX cortex, DA
dopamine (receptor), DAT dopamine transporter, dB decibel, DCC deleted in colorectal cancer, DIAZ diazepam, DIZ dizocilpine, DN dominant-negative, DDO D-aspartate oxidase, E embryonic day, EE
wild-type
S. B. Powell et al.
2.5 Methodological Considerations with Specific Relevance

to Genetic Models of Sensorimotor Gating
For a detailed description of the methods of acoustic startle and PPI in mice
see Geyer and Dulawa (2003). When conducting an initial analysis of startle
and PPI in a genetic mutant, several considerations should be made when
evaluating PPI. Typically, startle sessions involve variable prepulse intensities
(e.g. 3, 6, 12 dB above background), which should produce an incremental
increase in PPI with increasing prepulse intensities. Of course any evaluation of
a PPI phenotype should be considered in the context of a thorough assessment
of physical and sensory abnormalities (e.g. hearing loss), as pointed out in
Geyer et al. (2002). In a study examining strain differences in hearing and PPI,
Willot et al. (1994) showed a relationship between PPI levels and hearing
impairments. Hearing loss is also exacerbated by age, particularly in specific
strains of mice such as C57s, which experience high frequency hearing loss
with age (Willott et al. 1994). One way to avoid relying solely on auditory
processing is to assess multimodal PPI using light as a prepulse and/or airpuffs
as a startling stimulus (Brody et al. 2004; Young et al. 2010). This multi-
sensory approach can avoid potential confounds of hearing loss and still enable
the evaluation of PPI. It should be noted, however, that some degree of noise is
often produced with the delivery of airpuff stimuli, therefore, data on airpuff-
tactile startle should be interpreted with this consideration in mind. Other tactile
stimuli, such as mild footshock could provide an alternative approach to this
issue. Another way to provide a gross measure of hearing in genetic mutants is
to incorporate a ‘‘startle threshold’’ block into the session, in which increasing
decibels of acoustic stimuli are presented to measure startle magnitude across
these intensities (Brody et al. 2004). The threshold at which the mouse begins
to startle can then be evaluated. Additionally, differences in baseline startle
magnitude can confound effects on PPI. Although dissociations between startle
and PPI have been reported in mice (Brody et al. 2004) and rats (Sipes and
Geyer 1994), large differences in baseline startle magnitude can complicate
interpretations of changes in PPI. If large differences in baseline startle are
observed, startle magnitude can be ‘‘matched’’ either by using a range of startle
stimuli (e.g. 110, 120 dB) paired with the prepulse stimuli or doing a post hoc
analysis of only those animals across genotypes that have comparable levels of
startle. We have included results on startle magnitude [pulse alone (PA)] in
Tables 1–5 when the data were provided and indicated studies that did not
report data on startle magnitude. A good example of testing over a wide range
of startle amplitudes can be found in Savonenko et al. (2009) in their studies of
EP2 KO mice. The background strain on which genetic mutants are made
should be considered as well. For a discussion of the issue surrounding
background strain the reader is referred to Crawley (2007) and Geyer et al.
(2002).
3 Approaches to Genetic Models of Sensorimotor Gating
3.1 Overview of Molecular Genetic Techniques
Broadly defined, behavioral genetic approaches can be divided into two main
approaches either beginning with the gene of interest or the behavior of interest
(Jacobson and Cryan 2010). Gene-based or ‘‘reverse genetics’’ approaches begin
with the targeted gene being manipulated in the animal and the resultant behavior
evaluated. Phenotype-based or ‘‘forward genetics’’ approaches begin with the
targeted behavioral phenotype (trait) and then involve subsequent genetic analysis
of the trait. Most genetic studies of sensorimotor gating have focused on gene-
based approaches, and as such, these approaches are discussed in more detail than
phenotype-based approaches, which are discussed more extensively in Tarantino
and Eisener-Doman (2011). The goals for any genetic approach can be very dif-
ferent—some approaches seek to understand the more global role of a specific
gene in a particular phenotype related to a disease, while others may be designed
to elucidate the role of a particular gene in a cellular process, in neural circuit
abnormalities, or in brain development. Studies representing all of these goals are
well represented in the literature on genetic models of sensorimotor gating (see
Tables 1–5). The techniques used to generate molecular genetic models using the
gene-based (reverse genetic) approach include constitutive gene deletion (knock-
out), insertion of exogenous DNA into the genome (transgenic), and insertion of
gene at a particular locus via homologous recombination (knockin) [reviewed in
(Crawley 2007; Tecott and Wehner 2001)]. Conditional genetic manipulations,
which restrict the expression of a targeted gene either temporally or regionally, are
useful in avoiding complications of in utero lethality or compensatory brain
changes, or when specific hypotheses exist regarding the developmental expres-
sion of a gene or the specific neuroanatomical function of the gene. Examples
of conditional genetic manipulations include the Cre-LoxP system in which the
targeted gene is floxed with lox-p sites. When combined with Cre recombinase, the
floxed gene is expressed, giving regional, cell-specific, or temporal control over
expression or deletion of targeted gene (van der Neut 1997; Wang 2009).
Restriction to specific brain regions can be achieved through the use of specific
promoters used to drive expression. For example, the CaMKIIa promoter restricts
expression of a genetic mutation to the forebrain (Mayford et al. 1996). Additional
examples of tools to achieve temporal control over gene function involve the
Tet-On and Tet-Off systems. With these systems, the target gene is linked to either
the tetracycline-controlled transactivator (tTA) or the reverse tTA (rtTA) and can
be either turned ‘‘on’’ with doxycycline administered (tTA; Tet-On) or turned
‘‘off’’ in the presence of doxycycline (rtTA; Tet-Off) (Mansuy and Bujard 2000).
Thus, by combining some of these approaches, conditional and inducible gene
expression in mouse brain can be achieved. Additionally, insertion of large seg-
ments of DNA into the genome can be achieved through the use of bacterial
artificial chromosomes (BAC transgenics) (Heintz 2001). The BAC transgenic
technique is a useful approach when a large segment of DNA has been implicated
in a disease (e.g. 22q11.2 microdeletion or microduplications). For example, BAC
transgenic technology was used to generate two lines of transgenic mice
expressing different genes located on the 22q11.2 segment of the chromosome.
Interestingly, mice overexpressing Prodh and Vpreb2 showed increased PPI, while
mice overexpressing Zdhhc8, Ranbp1, Htf9c, T10, Arvcf, COMT showed no
differences in PPI (Stark et al. 2009). More recent techniques, such as in utero gene
transfer, are being developed to modulate the expression of genes during specific
stages of embryonic development (Niwa et al. 2010).
‘‘Phenotype-based’’ approaches to behavioral genetics include ‘‘forward genetic
screens’’ such as mutagenesis via radiation or chemical means [e.g. ENU muta-
genesis; (Tarantino and Bucan 2000) and quantitative trait loci (QTL) studies on
crosses of inbred mouse strains with distinct phenotypes or on mice or rats
selectively bred for PPI levels (Hitzemann et al. 2008; Tarantino and Eisener-
Doman 2011). Thus far, most of the PPI mutants identified through ENU muta-
genesis screens have had some amount of hearing loss, and thus the specificity of
the PPI ‘‘phenotype’’ was most likely confounded by deafness (Lisa Tarantino,
personal communication). Other molecular genetic tools such as siRNA, in which
double stranded RNA homologous to the targeted gene is made and then inserted
into the cell to block expression of that gene, and viral transfection, in which genes
are inserted into viral vectors and injected into the nucleus of cells, are additional
techniques used in neuroscience research assessing sensorimotor gating, but will
not be discussed in this chapter. Due to the increasingly large number of genetic
models generated in which PPI was evaluated, we will provide some examples in
the text of different genetic approaches undertaken to study sensorimotor gating,
but the reader is referred to Tables 1–5 for a comprehensive review of the recent
genetic models assessing PPI.
3.2 Models Based on Hypotheses Regarding the Pathophysiology

of Disease
Genetic models of sensorimotor gating deficits were based initially on the

hypothesized pathophysiology of schizophrenia or the known mechanism of action
of both antipsychotic and psychotomimetic drugs [e.g. amphetamine, PCP]. Thus,
early genetic mutants focused primarily on dopamine (Ralph et al. 2001) and
glutamate (Brody and Geyer 2004; Duncan et al. 2004) neurotransmitters and
receptors (e.g. dopamine-related genes, glutamate-related genes). Many of these
observed effects [e.g. PPI deficits in dopamine transporter KO mice (Ralph et al.
2001)] were not surprising based on the extensive pharmacology of PPI deficits in
rodents, but nevertheless the genetic models provided proof of concept that con-
stitutive gene deletion could produce dramatic functional effects on sensorimotor
gating in adult animals. PPI deficits in dopamine transporter knockout mice
strengthened the converging evidence that dopamine hyperfunction played an
important role in sensorimotor gating and potentially the pathophysiology of

schizophrenia. Similarly, PPI deficits in metabotropic glutamate mutants (Brody
et al. 2003; Brody and Geyer 2004; Gray et al. 2009; Kinney et al. 2003) have
stimulated interest in these receptors as targets for drug development (Cleva and
Olive 2011). Genetic approaches to pathophysiology of neuropsychiatric disease
are becoming increasingly sophisticated and many of these models incorporate
assessments of PPI as a relevant behavioral phenotype (Tables 3, 4). Jumping off
from the original observation that N-methyl-D-aspartate (NMDA) NR1 hypo-
morphs show PPI deficits in addition to other behavioral abnormalities (Duncan
et al. 2004), several genetic mutants have been made based on reduced NMDA
receptor expression, including NMDA subtype-specific mutants (reviewed in
Powell et al. (2009)) and downstream signaling proteins (Table 3).
The converging GABA-glutamate theory of schizophrenia stems from two lines
of evidence (Coyle 2004). First, the glutamate hypothesis of schizophrenia is
derived from evidence that acute administration of phencyclidine (PCP), a non-
competitive NMDA antagonist, produces schizophrenia-like symptoms in healthy
humans (Javitt 2004; Javitt and Zukin 1991). Extending such observations, several
experimental studies have utilized another NMDA antagonist, ketamine, to
induce a model psychosis in normal volunteers (Abel et al. 2003; Krystal et al.
1994; Oranje et al. 2002; van Berckel et al. 1998) and to exacerbate symptoms in
patients with schizophrenia (Malhotra et al. 1997a, b; Krystal et al. 1994, 2003).
Additionally, studies on postmortem brain tissue from schizophrenia patients show
altered GABAergic interneuron function, particularly in parvalbumin (PV) inter-
neurons (Benes and Berretta 2001; Lewis et al. 2005). Evidence that NMDA
hypofunction specifically on GABA interneurons plays a role in the pathogenesis
of schizophrenia comes from several lines of preclinical research. First, NMDA
antagonists increase the firing rate of pyramidal cells and increase prefrontal
glutamate release (Jackson et al. 2004; Moghaddam et al. 1997), suggesting that
NMDA antagonists are preferentially blocking inhibitory interneurons. Second,
preclinical studies suggest that GABA interneurons are more sensitive to NMDA
antagonists than other neuronal subtypes such as pyramidal neurons (Grunze et al.
1996). Third, repeated exposure to NMDA antagonists such as ketamine decreases
PV and GAD67 expression (Behrens et al. 2007). Based on these converging lines
of evidence, Belforte et al. (2010) used the Cre-LoxP system to engineer mice with
deletion of NR1 specifically in GABAergic interneurons (Table 1). Their elegant
set of studies showed that reduction in NR1 early in postnatal development pro-
duced deficits in PPI, but that adult NR1 reduction had no effect on PPI. These
studies corroborate previous studies reporting PPI deficits with constitutive NR1
reduction throughout the brain (Duncan et al. 2004). There have been several other
genetic mutants created to test hypotheses regarding glutamate signaling (see
Table 4). For example, mice with pre-adolescent forebrain-specific deletions of the
vesicular glutamate transporter VGLUT2f/f;CKII/Cre show PPI deficits, which are
attenuated with the antipsychotic drug aripiprazole (Wallén-Mackenzie et al.
2009). Genetic mutants of specific proteins involved in NMDA receptor signaling
such as mice heterozygous for SynGAP gene deletion also show PPI deficits in
Table 3 Genetically engineered model organisms based on genes related to: miscellaneous biological processes
References Mouse strain, sex Model description/background/rationale Basal PPI Effects of drugs or manipulations typically Effects of (presumed) antipsychotics/other
used to induce PPI-deficits treatments
Second messengers
Siuciak et al. PDE4B KO, M PDE4 catalyzes the degradation of cAMP. ;PPI & :PA in -/- versus
(2008) Widely expressed in brain WT (;PPI not
dependent on :PA)
McOmish PLC-b1 KO PLC-b1 is a rate-limiting enzyme involved ;PPI & PA (-/- vs. WT) EE (main effects): :PPI & ;PA (trend)
et al. in the intracellular signaling cascade versus standard housing); EE 9
(2008) linked to several metabotropic genotype 9 PP intensity effect on PPI
receptors implicated in SZ; PLC-b1 (:PPI in -/-); EE 9 genotoype effect
reduced in SZ brain on PA (;PA in WT); HAL (no rescue
of PPI deficit of -/- vs. VEH treated
WT; [PA); CLO (main effect :PPI;
genotype 9 CLO 9 PP intensity effect:

rescue of PPI deficit of -/- vs. VEH
treated WT; main effect: ;PA vs. VEH;
genotype 9 CLO; more pronounced
;PA in WT)
Koh et al. PLC-b1 KO ;PPI & trend for ;PA (-/- HAL: normalized PPI in -/-, no effect in
(2008) vs. WT) WT; [PA
Growth factors
Ransome SOCS-2 TG SOCS-2 affects growth hormone signaling [PPI in TG versus WT
et al. & potentially hippocampal
(2008) neurogenesis
Scearce- Fgf17 KO, M & F Fgf17 is involved in region-specific [PPI & [PA in -/-
Levie development of the rodent fCTX versus +/- or WT
et al.
(2008)
(continued)
279
Table 3 (continued)
280
Ohgake et al. Mdk KO, M & F Midkine involved in neurodevelopment and ;PPI in -/- versus WT, HAL: :PPI in -/-; [PPI in WT
(2009) adult neuroplasticity; abnormal serum +/- intermediate CLO: :PPI in -/-; [PPI in WT
levels in SZ and AD; KO produces (adult); [PPI in -/- Overall ;PA with APD; CLO (3 mg/
;DA and DAR in striatum (this paper) versus WT (4 wk old); kg) ;PA in WT
[PA in -/-, +/-,
WT (adult, 4wk)
Sun et al. Forebrain-specific SMAD4 is an intracellular transducer of ;PPI & [PA in -/-
(2010) SMAD4 KO, TGF-b signaling; TGF-b signaling versus WT
M important for neurodevelopment and
shown to be abnormal in SZ
Nguyen et al. PDGFR-b f/f;Nestin Platelet derived growth factor (PDGF) is ;PPI & [PA in -/-
Cre+
(2011) KO, M important for embryogenesis and CNS versus WT
(neuron- development. PDFGR-b has been
specific associated with SZ and ASD
PDGFR KO)
Protein kinase
Bersudsky GSK3b KO, M GSK3b levels were reported to be reduced [PPI & [PA in +/-
et al. in SZ patients. Li inhibits GSK3b in versus WT
(2008) vitro
Kaidanovich- GSK3a KO, M & F GSK3 implicated in several neuronal :PPI & [PA in -/-
Beilin signaling pathways versus WT
et al.
(2009)
Takao et al. CaMKIV KO, M CaMKIV is a protein kinase that activates [PPI & [PA in -/-
(2010) transcription factor CREB and thus versus WT
may play a role in synaptic plasticity
(continued)
S. B. Powell et al.
Table 3 (continued)
Siuta et al. Rictor f/f;Nestin Cre+ Rictor is a component of mTOR2C, the ;PPI & [PA in -/- NIS: (:PPI in -/-; no data in WT; PA data
(2010) KO (neuron- kinase responsible for phosphorylation versus WT not shown)
specific Rictor of Akt. Akt deficiencies associated CLO: ([PPI in -/-; PA data not
KO) with SZ shown)
Proteases
Horii et al. Neuropsin KO, M The serine protease neuropsin may be [PPI & PA in -/- versus
(2008) involved in neuronal plasticity/ WT
degeneration
Savonenko BACE1 KO, M BACE1 is critical for APP cleavage/ ;PPI in -/- & [PPI in CLO: (amelioration of PPI deficits in -/-;
et al. amyloid b production. May also play a +/- versus WT; [PA [PPI in +/- & WT)
(2008) role in SZ via proteolysis of NRG & in -/- & +/- versus
axon myelination WT; :PA latency in

-/- versus +/- & WT
Synaptic proteins
Dyck et al. SYN II KO SYN II, a vesicle-linked phosphoprotein ;PPI (-/- relative to +/-
(2007) plays a role in neuronal development & & WT)
transmitter release; hypothesized to
contribute to the etiology of SZ
Dyck et al. SYN II KO, M & F ;SYNII in mPFC of SZ patients; :SYNII ;PPI in -/- versus +/-
(2009) levels with chronic APD and WT; ;PA in -/-
versus +/- and WT;
lack of HAB in -/-
(continued)
281
Table 3 (continued)
282
Savonenko EP2 KO, F EP2 is a prostaglandin receptor involved in ;PPI in -/- versus WT;
et al. activity-dependent synaptic plasticity [PA in -/- versus
(2009) WT
Oliver and SNAP-25 Bdr SNARE protein SNAP-25 important for ;PPI & [PA in Bdr mutant CLO: :PPI in BDR/NS & BDR/PNS; [PPI
Davies (Blind drunk synaptic function and neurotransmitter versus WT, ;PPI in WT/NS or WT/PNS
(2009) mutant), release; SNAP-25 genomic region exacerbated by PNS; HAL: :PPI in Bdr/PNS at 86dB PP
SNAP-25 +/-, linked to SZ [PPI & [PA in +/- only; [PPI in WT/NS, Bdr/NS, or WT/
combined with versus WT, no effect PNS
prenatal stress of PNS in +/-
(PNS), M
Blundell et al. Rim1a KO, M Presynaptic proteins involved in Rim1a: ;PPI &[PA in
(2010) Rab3A KO, M neurotransmitter release and associated -/- versus WT
Syt1R233Q with SZ in genetic studies Rab3A: ;PPI & [PA
(point in -/-versus WT
mutation) KI, Syt1R233Q: ;PPI &
M&F [PA in KI versus WT
Rim1aS413A Rim1aS413A: [PPI &
KI, M [PA in KI versus WT
Calcium Signaling
Matsuo et al. RyR3 KO Ryanodine receptor 3 is an intracellular ;PPI in -/- versus WT at
(2009) calcium release channel preferentially 78 dB PP + 110 dB
expressed in HPC and STR and startle pulse only;
involved in synaptic transmission and [PA in -/- versus
plasticity. RyR3 forms a signaling WT
complex with calcineurin, a potential
susceptibility gene for SZ and BD
(continued)
S. B. Powell et al.
Table 3 (continued)
Nakagawasai Cav2.2 KO, M N-type calcium channels are important for ;PPI & [PA in -/-
et al. neurotransmitter release, brain versus WT
(2010) development, and activity-depdendent
plasticity
Potassium channel signaling
Kapfhamer Ppp2r5d KD, M & KCNQ2 gene encodes an M-type potassium Ppp2r5dGt: ;PPI & [PA
et al. F channel subunit and may be a substrate in GT/+ versus WT
(2010) GSK3b KD, M for GSK3b; PP2A regulates GSK3b; GSK3b: ;PPI & [PA
KCNQ2 KD, GSK3b and M-type potassium in +/- versus WT
M&F channels may be linked to SZ KCNQ2: ;PPI & [PA
KD created via in +/- versus WT
gene trap (GT),

reduced
expression by
50%
Immune activation
Smith et al. IL6 KO Maternal immune activation (here Maternal poly I:C injection.
(2007) simulated via poly I:C injection) has Effects in offspring: [PPI in -/- but
been implied in the pathogenesis of SZ ;PPI in WT
Asp et al. Tap1 KO mice Prenatal influenza infection associated with No direct comparisons Influenza infection on PND 3 or 4: ;PPI in
(2010) increased SZ risk. Transporter between -/- and WT adult -/- mice (during ISI block),
associated with antigen processing 1 mice. C57BL6/J WT trend for :PA in infected mice in
mice have reduced expression of MHC mice purchased from startle threshold block; [PPI & [PA
class I Jackson Laboratories in infected versus non-infected WT
C57 mice
(continued)
283
Table 3 (continued)
284
López-Ramos TgIE96, M Mice expressing pseudorabies virus ;PPI & [PA in Tg versus
et al. immediate-early protein IE180 WT (C57BL/6J WT
(2010) (TgIE96) have cerebellar abnormalities mice purchased from
Jackson Laboratories)
Glucose metabolism
Schmidt et al. GLUT3 (Slc2a3) GLUT is widely expressed in brain. Role in ;PPI at lower PP intensities
(2008) KO, M neuronal glucose homeostasis and & :PA in +/- versus
likely role in diverse behaviors WT
Neuromodulatory functions
Errico et al. DDO KO, M The enzyme DDO degrades D-aspartate, an [PPI & [PA in -/- AMP ([PPI in DDO -/-; ;PPI in WT) Chronic D-aspartate ([PPI & PA)
(2008) amino acid with elusive function in the versus WT DIZ (;PPI in -/- & WT, more antagonized AMP & DIZ-induced ;PPI
CNS. A neuromodulatory function at pronounced in WT) in WT (not tested in -/-)
NMDA receptors has been implied
Tanda et al. nNOS KO, M nNOS synthesizes NO from L-arginine; [PPI & [PA in -/- SKF81297 (DA D1 agonist): ;PPI & [PA
(2009) involved in many intracellular versus WT in -/-; [PPI & ;PA in WT
signaling processes; ;nNOS expression
in STR and HPC of SZ;
polymorphisms of nNOS associated
with SZ
Altered myelination
Tanaka et al. PLP1(tg/-) Mylein and oligodendrocyte dysfunction ;PPI in Tg versus WT at
(2009) may contribute to schizophrenia higher PP intensity
pathogenesis. Myelin proteolipid (78dB), PA data not
protein (plp1) decreased in brains of SZ shown
patients
(continued)
S. B. Powell et al.
Table 3 (continued)
Spine morphogenesis
Cahill et al. KALRN KO Kalrinin, a guanine-nucleotide exchange ;PPI in -/- versus WT;
(2009) factor (GEF) for Rac-like GTPases, PA data not shown
regulates spine morphogenesis.
KALRN -/- have decreased spine
density (this paper)
Oxidative stress
Benoit et al. QR2 KO, M QR2 enhances production of quinine and [PPI in -/- versus WT;
(2010) reactive oxygen species (ROS) and is PA data not shown
upregulated in cognitively impaired
aged rats; polymorphism in QR2
promoter region weakly associated

with SZ
Cole et al. GCLM KO, M GCLM is rate-limiting enzyme in [PPI & [PA in -/-
(2011) glutathione (GSH) synthesis; GSH versus WT
defends against oxidative stress
Metallothionines
Koumura MT3 KO, M Metallothionines (MTs) bind zinc and other ;PPI & [PA in -/-
et al. metals and are neuroprotective in some versus WT
(2009) models
Neurodevelopmental genes (other)
Willi et al. Nogo-A KO, M Nogo-A inhibits growth during ;PPI & [PA in -/-
(2010) development and in adulthood and thus versus WT
may be involved in neurodev and
neural plasticity; some evidence of
pathology in SZ
(continued)
285
Table 3 (continued)
286
Takata et al. XBP1 KO, M XBP1 gene encodes transcription factor in :PPI & [PA in +/- versus
(2010) the ER unfolded protein response; WT
involved in brain development and
potentially pathogenesis of bipolar
disorder and depression
Glycoproteins
Sakatani et al. RAGE KO, M RAGE is a receptor for multiple ligands :PPI in -/- versus WT
(2009) including Ab, HMGB1, and S100B, :PA in -/- versus
which contribute to the pathology of WT at low sound
AD, epilepsy, and ischemia pressure levels (70-
100 dB); ;PA in -/-
versus WT at 120 dB
Fukuda et al. Fut8 KO, M a1,6 fucosyltransferase (Fut8)2 transfers ;PPI & [PA in -/- After restraint stress: ;PPI & [PA in +/-
(2011) fucose from a GDP-fucose to position 6 versus +/- & WT versus WT
of N-acetylglucosamine to form N-
linked oligosaccharides of
glycoproteins. Fucosylated
glycoproteins widely distributed in
brain
(continued)
S. B. Powell et al.
Table 3 (continued)
Sex hormones
Chavez et al. ArKO, M, F Gender differences in the age of onset, [PPI in -/- versus WT AMP ;PPI in -/- & WT (;PPI slightly
(2009) severity, and response to treatment in attenuated in F -/-), ;PA in -/- &
SZ. Second increase in SZ incidence in WT
older age for women when estrogen Apo: ;PPI in -/- & WT (;PPI
levels decline. Thus, estrogen may be slightly attenuated in F -/-), ; PA in
neuroprotective against development of -/- & WT
SZ. Aromatase (Ar) converts MK801: ;PPI & :PA in -/- & WT
androgens to estrogens
wild-type
287
Table 4 Genetically engineered model organisms (ca. 07/01/2007-06/28/2011) based on genes related to: models for specific disorders
288
References Mouse strain, sex Model description/background/rationale Basal PPI Effects of drugs or Effects of (presumed) antipsychotics/other
manipulations treatments
typically used to
induce PPI-
deficits
Alzheimer’s disease
Dejaegere Alpha 1B/C c- c-secretases have been implicated in the cleavage ;PPI (most pronounced at high PA HAL: (:PPI & [PA in -/- & WT) CLO: (:PPI
et al. secretase KO of membrane proteins incl. APP, & NRG (a intensities) & [PA in -/- in -/-; [PPI in WT; [PA in -/- & WT)
(2008) SZ vulnerability gene) & in NOTCH versus WT
signaling
Gruart et al. APP, PS1, APP+PS1 Neuropathological changes in brain regions ;PPI in 18 mo old WT, APP KO,
(2008) TG, M regulating PPI may occur in AD models & PS1 KO & APP+PS1 TG versus
may affect PPI 3 mo old WT. Most pronounced
;PPI in 18 mo APP+PS1 TG.
PA data not shown
Tsujimura KF-1 KO, M KF-1 gene increased in AD patients; localized in :PPI in -/- versus WT; ;PA in
et al. hippocampus, cerebellum; modulates protein -/- versus WT
(2008) levels as a ubiquitin ligase; increased in
frontal ctx after chronic antidepressant
treatment, electroconvulsive therapy, and
TMS
Takeuchi P301S Tg, M Tau, a microtubule-associated protein, can cause :PPI & ;PA at 120 dB only in Tg
et al. cell death and is associated with AD and other versus WT
(2011) neurodegenerative diseases. FTDP17
(frontotemporal dementia and PD linked to
chromosome 17) is linked to a point mutation
in tau (P301S)
(continued)
S. B. Powell et al.
Table 4 (continued)
typically used to
induce PPI-
deficits
Autism
Allan et al. MDB1 KO, M & F Via DNA methylation, MDB mediate epigenetic ;PPI & [PA in -/- versus WT
(2008) gene regulation, which has been linked to
neurodevelopmental disorders, including
autism
Chadman NL3 KI, M & F Neuroligin-3 (NL3) is a cell adhesion molecule [PPI in KI versus WT; ;PA in KI
et al. involved in synapse development & versus WT
(2008) implicated in autism; ‘‘knock in’’ of point
mutation identified in autism

Radyushkin NL3 KO, M Point mutations in neuroligin-3 are associated with [PPI & [PA in -/- versus WT
et al. autism
(2009)
Moy et al. NRCAM KO, M & F NRCAM identified as a candidate gene for autism; ;PPI in -/- versus WT (M only;
(2009) involved in cell-cell interactions during brain [PPI in F); [PA in -/- versus
development and thus affect axonal guidance WT
and neural circuit development
DeLorey et al. GABRB3 KO, M & F GABRB3 gene associated with autism [PPI (F) & :PPI (M) in +/- versus
(2011) WT; ;PA (M & F) in +/- versus
WT
Mejias et al. GRIP1/2 DKO (double GRIP1 scaffolding protein that interacts with ;PPI in -/- versus WT mice
(2011) KO, Grip1 flox/ GLU2/3 via PDZ domains 4-6. SNP in PDZ4-
KO, Grip2 KO/ 6 domain and 5 missense variants identified in
KO) ASD (this paper)
(continued)
289
Table 4 (continued)
290
typically used to
induce PPI-
deficits
Fragile X-syndrome (FXS)
deVrij et al. FMR1 KO FXS involves mental retardation & is caused by ;PPI at most PP intensities in -/- MPEP (mGluR5 antagonist; :PPI in -/- & WT)
(2008) the absence of FMRP. PPI is reduced in FXS versus WT
patients
Paylor et al. FMR1 KO, FMR1 KO :PPI & ;PA (trend) in -/- versus Addition of human FMR1 gene normalized, i.e.
(2008) with a yeast WT ;PPI & :PA in FMR1 KO to WT levels, &
artificial ;PPI & [PA in WT
chromosome
containing human
FMR1 gene, M
Baker et al. FMR1 KO, M & F Background strain may affect behavioral :PPI & ;PA in -/- versus WT (both
(2010) phenotype in FMR1 KO mice; bred to albino sexes)
C57BL/6J-Tyr(c-Brd) background
Levenga et al. FMR1 KO, M FMRP involved in translation of mRNA at the ;PPI in -/- versus WT; PA data not AFQ056, mGluR5 antagonist: :PPI in -/-;
(2011) synapse, downstream from mGlu5 signaling reported [PPI in WT
(startle measured via eyeblink)
Veeraragavan FMR1 KO, M Increased M1 muscarinic signaling in FMR1 KO [PPI & [PA in -/- versus WT Dicyclomine: [PPI & [PA in -/- or WT
et al. mice
(2011)
Thomas et al. mG1.FMR1 (mGlu1 FMR1 may increase signaling through Group1 :PPI & ;PA in FMR1-/- &
(2011a) HZ + FMR1 KO); mGluRs (mGlu1, mGlu5) mG5.FMR1 versus WT; :PPI &
mG5.FMR1 ;PA in FMR1-/- &
(mGlu5 HZ + mG1.FMR1 versus WT
FMR1 KO);
FMR1 KO
(continued)
S. B. Powell et al.
Table 4 (continued)
typically used to
induce PPI-
deficits
Thomas et al. FMR1 KO, M & F Group 1 mGluRs may be pharmacological target ;PA & [PPI (trend for predicted :) JNJ (mGlu1
(2011b) in Fragile X in -/- versus WT antagonist):
;PPI in WT,
[PPI in -/-;
[PA in -/-
& WT
MPEP
(mGlu5
antagonist):
[PPI & [PA
in -/- & WT
Huntington’s disease
Menalled R6/2 TG, R6/2B TG, HD results from a known genetic CAG repeat R6/2: ;PPI in TG versus WT
et al. YAC128 TG, mutation; ;PPI in HD patients; HD mutant beginning at 12 wks; ;PA
(2009) YAC128B TG, mice show varying degrees of neuropathology R6/2B: ;PPI in TG versus WT
BACHD TG, and behavioral abnormalities; comparison BACHD: ;PPI beginning at 24
HDHQ111 KI, M & controlling for many factors including sex, wks; [PA
F background strain, husbandry, and handling YAC128B: ;PPI at 54 wks; [PA
YAC128: [PPI; [PA
HDHQ111 KI: [PPI; [PA
Brooks et al. Hdh(CAG)150 KI, M & F Longitudinal characterization of KI mouse ;PPI & ;PA in KI versus WT
(2010) carrying 150 CAG repeats on the mouse Htt
locus
Brooks et al. R6/1 TG, M & F R6/1 TG mouse contains *115 CAG repeats on ;PPI & ;PA in TG versus WT
(2011) exon 1 of the HD gene. Characterization of
R6/1 TG mice on C57BL/6 background is
needed
291
(continued)
Table 4 (continued)
292
typically used to
induce PPI-
deficits
Pietropaolo R6/1 TG, M & F Investigate neuropsychiatric symptoms in pre- [PPI & [PA in TG versus WT
et al. motor stage of pathology
(2011)
Hearing disorders
Allen et al. Kv1.1 KO PPI in rodents is a powerful means to identify ;PPI (for long gaps only)
(2008) hearing deficits. The role of Kv1.1 channels
in PPI gap detection paradigms was assessed
22q11 deletion & duplication syndromes
Suzuki et al. SEPT5 (Septin5) KO, 22q11.2 microdeletions result in cognitive & :PPI & [PA in -/- versus WT
(2009b) M behavioral abnormalities and are associated
with SZ and autism; SEPT5 gene located on
200kb region spanning the deletion
Stark et al. BAC Tg-1: 22q11.2 microduplications are also associated Tg-1: :PPI & [PA in Tg-1 versus
(2009) Overexpress with behavioral problems and learning WT
Prodh & Vpreb2 disabilities; gain of function mutations can Tg-2: [PPI & [PA in Tg-2
BAC Tg-2: also be informative for the deletion syndrome versus WT
overexpress
Zdhhc8, Ranbp1,
Htf9c, T10, Arvcf,
& COMT
(continued)
S. B. Powell et al.
Table 4 (continued)
typically used to
induce PPI-
deficits
Suzuki et al. BAC Tg, M: 22q11.2 is a hotspot for CNVs. 97% of children [PPI & [PA in Tg versus WT
(2009a) overexpress with 22q11.2 duplications show cognitive
190 kb segment of deficits
22q11.2 including
TXNRD2,
COMT, ARVCF
Down syndrome
Ortiz-Abalia TG DyrK1A DyrK1A overexpression has been implicated in ;PPI at higher PP intensities in TG Intrastriatal infusion of AAV iRNA against
et al. overexpressors, M Down Syndrome versus WT (trend only); PA not Dyrk1A: PP intensity-dependent :PPI in TG
(2008) shown treated with functional versus scrambled
viral vectors & pre versus post-treatment
with functional vector; PA not shown
Glycosilation IIa congenital disorders
Soleimani Mgat5 KO, M & F Mgat5 is involed in glycosylation of cell surface [PPI & PA in -/- versus WT; :PA
et al. glycoproteins, including receptors & in M versus F (main effect)
(2008) transporters. Abundant in the CNS
(continued)
293
Table 4 (continued)
294
typically used to
induce PPI-
deficits
Prader-Willi syndrome
Relkovic et al. PWS-IC (+/-), M & F Prader-Willi Syndrome (PWS) ;PPI & :PA in +/- versus WT in M
(2010) neurodevelopmental disorder resulting from & F (only at P105 in M)
deletion of paternally expressed imprinted
genes of chromosome 15q11-q13, a region
also implicated in autism and SZ
environmental enrichment, EGF epidermal growth factor, ENU N-ethyl-N-nitrosourea, f frontal, F female, FABP fatty acid binding protein, FGF fibroblast growth factor, Fmr1 fragile 9 mental retardation 1
gene, FMRP fragile 9 mental retardation protein, FXS fragile 9 Syndrome, GLAST glutamate and aspartate transporter, GLU glutamate, GluR glutamate receptor, GLUT glucose transporter, GR
neuregulin, NRL neuroligin, ns not significant(ly), NSE neuron-specific enolase, NT neurotensin, OE overexpressor, OXO oxotremorine, PA magnitude of response to pulse alone, PACAP pituitary adenylate-
cyclase-activating polypeptide, PD Parkinson’s disease, PDE phosphodiesterase, PET-1 plasmocytoma expressed transcript-1, PND postnatal day, PLC phospholipase C, PNS prenatal stress, poly I:C
polyinosinic: polycytidylic acid, PP prepulse, PPI prepulse inhibition of startle, PPP prepulse potentiation, PS1 presinilin1, PWS Prader–Willi syndrome, QTL quantitative trait locus, QUET quetiapine,
wild-type
S. B. Powell et al.
addition to other behavioral abnormalities (Guo et al. 2009). For a review of other
neurotransmitter-related genetic mutants [e.g. acetylcholine, serotonin, dopamine
(see Table 4)].
In addition to classic neurotransmitters, the important role of neuropeptides in
neuropsychiatric disease is becoming more widely appreciated. PPI has been
assessed in genetic mutants for many neuropeptides including angiotensin, neu-
rotensin, corticotropin releasing factor (CRF) overexpression, oxytocin, arginine
vasopressin, neuropeptide Y, PACAP, and Relaxin-3 (Table 4). Stress physiology,
particularly CRF and glucocorticoids, has been implicated in neuropsychiatric
disease. Mice overexpressing a CRF transgene exhibit deficits in PPI (at low
prepulse intensities), which are normalized by CRF1 antagonists, whereas glu-
cocorticoid receptor antagonists did not reverse the PPI deficit (Groenink et al.
2008). Moreover, several genetic mutants have been created to determine the role
of specific proteins in brain processes, including second messenger signaling,
synaptic proteins, synaptic vesicles, protein kinases, etc. (Table 3). While the
direct link for many of these proteins has not necessarily been established for
specific neuropsychiatric diseases, a thorough behavioral evaluation of these mice
including an assessment of PPI can help elucidate the functional implications of
the target protein. For example, gene deletion of PDE4B, which catalyzes the
degradation of cAMP and is widely expressed in brain, is associated with PPI
deficits (Siuciak et al. 2008). Decreased presynaptic proteins such as synapto-
physin, SNAP-25, and complexin II have been observed in postmortem brains of
schizophrenia patients (Harrison and Weinberger 2005). These data, combined
with evidence of problems with neuronal migration and abnormal neuronal pro-
cesses, have led investigators to conceptualize schizophrenia as a disease of
functional ‘‘dysconnectivity’’ (Friston and Frith 1995; McGlashan and Hoffman
2000; Weinberger et al. 1992) or a ‘‘disorder of the synapse’’ (Frankle et al. 2003;
Mirnics et al. 2001) reviewed in (Harrison and Weinberger 2005). Thus many genetic
mutants have been created for specific synaptic proteins (Table 3). For example,
SYNII, a vesicle-linked phosphoprotein that plays a role in neuronal development
and neurotransmitter release (Cesca et al. 2010), is genetically associated with
schizophrenia, decreased in brain of schizophrenia patients, and increased with
chronic antipsychotic drug treatment (Chen et al. 2004; Chong et al. 2002). SYNII
KO mice exhibit decreased PPI, providing more evidence for the essential role of
SYNII in synaptic function and behavior (Dyck et al. 2007, 2009). Similarly, a
mutation of SNAP-25, a SNARE protein that is integral to synaptic function and
neurotransmitter release, is associated with PPI deficits (Oliver and Davies 2009).
3.3 Genetic Mouse Models as Pharmacological Tools
Because many of the early molecular genetic approaches focused on neurotrans-

mitter receptor genes, several of these mutants have also been used to delineate
the receptor mechanisms of drugs that disrupt PPI in order to expand our
understanding of the neurotransmitter systems and neural circuitry underlying

deficient PPI (Doherty et al. 2008; Dulawa et al. 1997; Ralph et al. 1999; van den
Buuse et al. 2011a) (Table 4). Since most of these genetic mutants were built on
rat pharmacology, we must keep in mind several important species differences
between the pharmacology of mouse versus rat PPI (e.g. differential effects
of dopamine D1 and D2 receptors, 5-HT agonists, etc.; Powell et al. 2009).
Pharmacological disruptions of PPI and their reversal with antipsychotics have
been better characterized in rats than in mice, although the literature on PPI
pharmacology in mice is rapidly increasing (Swerdlow et al. 2008). In order to use
mutant mouse models to examine the receptor mechanisms for alterations in PPI,
more complete dose response studies are warranted in mice. Nevertheless, drugs
that both impair and improve PPI continue to be investigated in genetic mutants.
This approach is particularly useful in determining the functional role of a receptor
(e.g. does the gene deletion alter the response to a psychotomimetic drug) and in
determining the receptor mechanisms for a drug effect when either receptor-
selective drugs are not available or when novel drugs are being evaluated. For
example, amphetamine disrupts PPI in WT mice, but not in mice with gene
deletions of DDO, an enzyme that degrades D-aspartate (Errico et al. 2008),
suggesting a functional role of DDO in PPI disruptions. Interestingly, oxytocin KO
mice show an increased sensitivity to the PPI-disruptive effects of PCP (Caldwell
et al. 2009), suggesting that endogenous oxytocin may protect against PCP-
induced disruptions of PPI, a finding that supports the clinical data showing
putative antipsychotic properties of oxytocin (Feifel et al. 2010a). A similar
approach was used to show protective properties of nNOS, which synthesizes NO
from L-arginine, against PPI-induced disruptions with the dopamine D1 agonist
SKF81297 (Tanda et al. 2009).
3.4 Genetic Models of Candidate Genes for Neuropsychiatric

Diseases
As the field of neuropsychiatric genetics has grown, several gene targets have been
identified consistently for particular disorders and have thus been modified in
mouse models through gene deletion, the addition of a transgene, etc. When
evaluating candidate genes for mouse models, one should consider that many
single nucleotide polymorphisms (SNPs) identified in genetic screens for neuro-
psychiatric disease involve mutations in introns of genes. Thus, it is very important
to consider (1) whether or not functional mutations in the gene have been
identified before embarking on mutant mouse models, and (2) the impact of
species–specific alterations in gene function in the mutant mouse (Low and Hardy
2007). Table 1 summarizes genetic mouse models of vulnerability genes for
schizophrenia, and Table 2 summarizes genetic models of other neuropsychiatric
disorders in which PPI deficits have been observed. Two approaches in human
studies have increased our understanding of the genetics of sensorimotor gating

(1) assessing the relationship between polymorphisms in a target gene (e.g.
NRG1, COMT) and PPI levels in healthy volunteers and (2) endophenotype-
based genetic studies of schizophrenia (e.g. Consortium on the Genetics of
Schizophrenia; COGS). For example, recent studies suggest that PPI is associ-
ated with polymorphisms in CHRNA3 (Petrovsky et al. 2010), neuregulin 1
(Roussos et al. 2011), and COMT (Giakoumaki et al. 2008; Quednow et al.
2008; Roussos et al. 2008) genes. Some of these same genes (e.g. NRG1,
COMT) are also associated with prepulse inhibition in the COGS data set
(Greenwood et al. 2011).
Several different mutants for neuregulin 1 isoforms have been created with
varying effects on behavior and neuroanatomy (reviewed in Duffy et al. 2008;
O’Tuathaigh et al. 2007). Overall, the PPI phenotype in NRG1 mutants has been
inconsistent (O’Tuathaigh et al. 2011, 2009). The Type III NRG1 heterozygote
mouse has shown abnormalities in multiple neuroanatomical and behavioral
endpoints relevant to schizophrenia, including PPI deficits that are improved with
chronic nicotine administration (Chen et al. 2008). CNS-specific ERBB2/B4 KO
mice exhibit PPI deficits, which are attenuated by the antipsychotic drug clozapine
(Barros et al. 2009). Conditional knockouts with better temporal and regional
specificity have allowed for evaluation of more specific roles of NRG1 and its
receptors. For example, deletion of ErbB4 selectively in PV interneurons produces
PPI deficits, suggesting a functional interaction between NRG1 and the integrity of
PV interneurons (Wen et al. 2010).
The COMT Val allele is associated with reduced P300 and P50 ERPs (Gallinat
et al. 2003; Golimbet et al. 2006; Lu et al. 2007) and reduced PPI (Quednow et al.
2008; Roussos et al. 2008), presumably due to decreased dopamine function in the
prefrontal cortex. There have been several mice created to examine the effects of
COMT gene on schizophrenia relevant phenotypes, COMT KO mice (Gogos et al.
1998; Papaleo et al. 2008; Yavich et al. 2007), COMT Tg mice that overexpress
the human COMT-Val polymorphism (Papaleo et al. 2008), and S-COMT isoform
KO (Tammimäki et al. 2010) Thus far, none of the COMT mouse models have
shown robust deficits in PPI (Table 1), although other behaviors probing prefrontal
cortex such as the attentional set shifting task (ASST) are altered in the COMT-Val
TG mice (Papaleo et al. 2008).
The disrupted in Schizophrenia 1 (DISC1) gene has shown association with
schizophrenia and behavioral and neuroanatomical biomarkers for schizophrenia
(for review see Mackie et al. 2007; Porteous et al. 2006). DISC1 is associated with
schizophrenia neuropathology (Cannon et al. 2005; Callicott et al. 2005), abnormal
P300 ERP (Blackwood et al. 2001), and neurocognitive function (e.g. learning and
memory; Burdick et al. 2005; Cannon et al. 2005; Hennah et al. 2005). The data on
the dominant negative DISC1 models have reported both decreased PPI (Hikida
et al. 2007) and no change in PPI (Ibi et al. 2010); whereas, the DISC1L100P
mutations have shown more consistent decreases in PPI (Lipina et al. 2011; Lipina
et al. 2010). The new technology of in utero gene transfer, allows for the
knockdown of DISC1 into the mouse brain during early embryonic development.
Mice with in utero knockdown of DISC1 show reduced PPI when tested in
adulthood (Niwa et al. 2010). As we have discussed previously, modeling the
human DISC1 mutation in mice is difficult because most of the models have
assumed that the mutation in neuropsychiatric disease is due to the formation of a
truncated DISC1 protein (Powell et al. 2009). Another gene, Boymaw, is also
disrupted on chromosome 11 in the Scottish family. Two fusion transcripts are
generated between DISC1 and Boymaw genes in the translocation carriers in the
Scottish schizophrenia family (Zhou et al. 2008). Thus, expressing the two fusion
transcripts may be a better strategy for creating mutant DISC1 mice for the
study of neuropsychiatric disorders. The increasing identification of copy number
variants (CNVs) associated with schizophrenia and large-scale GWAS studies
identifying novel candidate genes will lead to even greater potential to create new
mouse models (see Tables for examples). Additionally, PPI is being used more
widely to characterize mutant mouse models of candidate genes for other neuro-
psychiatric disorders such as autism and Huntington’s Disease (Table 2).
3.5 Phenotype-Based Models Revealing the Function of Genes
PPI deficits have also been used in phenotype-to-genotype approaches, or ‘‘for-

ward genetic’’ screens such as ENU mutagenesis, QTL on strain crosses, and QTL
with selective breeding. For example, using F2 mice from a C57BL/6 x C3h/HE
cross, QTL identified 6 loci for PPI, including the gene FABP7, which has been
linked to NMDA receptor function (Watanabe et al. 2007). In a study comparing
A/J, C57Bl6/J, and congenic crosses thereof (recombinant congenic mouse strains)
(Torkamanzehi et al. 2008) found some common markers for startle but not PPI
compared to previous studies (Joober et al. 2002). Selective breeding for high and
low levels of PPI has identified QTLs on chromosomes 11 and 16 in low PPI
versus high PPI mouse lines (Hitzemann et al. 2008; Schwabe et al. 2007). An
ENU mutagenesis screen produced 2 PPI mutants (Cook et al. 2007). One major
problem with these approaches is that many of the PPI mutants, particularly with
ENU mutagenesis, will likely be deaf or have some degree of hearing loss. In the
(Torkamanzehi et al. 2008) study on crosses of A/J, C57Bl6/J and congenic
crosses, light prepulses and tactile startle pulses were used to measure PPI to avoid
this potential confound (but airpuff startle also has an acoustic component to it).
The field of molecular genetics continues to produce new mutant mouse models
with unknown effects on the central nervous system. Many of these mutants have
behavioral abnormalities that have been observed anecdotally. Phenotypic char-
acterization of mice with mutations of genes heretofore not known to be relevant
to CNS disease (some examples found in Tables 1–5) may reveal novel genes for
further study of neuropsychiatric genetics. Although these approaches were gene-
based, some have the capacity to identify novel genes for a particular disease,
which is why we have included these examples here. Two examples of the way a
novel gene of relevance to psychiatric conditions can be discovered through the
Table 5 Phenotype-based approaches
References Mouse strain, sex Model description/ Basal PPI Effects of drugs or manipulations typically Effects of (presumed)
background/rationale used to induce PPI-deficits antipsychotics/other
treatments
QTL approaches
Cook et al. ENU-mutagenesis- Genome-wide screening 2 PPI mutants with ;PPI
(2007) induced variants for neurobehavioral
phenotypes, including
PPI
Watanabe F2 mice from Screening for PPI and 6 loci for PPI, 6 for startle and four for startle
et al. C57BL/6 9 startle phenotypes latency were identified, incl. FABP7
(2007) C3H/HE crosses, (linked to NMDAR function (;PPI & ;PA
M, & F latency)
Hitzemann C57BL/6J, DBA/2J, Selective breeding for [PPI & PA in M versus F and no correlation METH (Line 9 dose interaction. ;PPI in HAL(:PPI; main effect of
et al. BALBc/J and high and low PPI between PPI & PA initially. Breeding line high PPI line, but [PPI in low PPI line dose only, no line 9
(2008) LP/J crosses, M 9 generation effects on PPI (low vs. high at highest METH dose). DIZ (;PPI; dose interaction)
&F PPI line) after five generations. No line 9 main effect of dose only, no line 9
generation effects on PA, but :PA in low dose interaction)
PPI line versus ;PA in high PPI line (main
effect only). QTL on chromosomes 11 &
16 in low PPI versus high PPI line
Torkamanzehi A/J, C57Bl6/J and Screening for PPI and PA Comparable PPI, but not PA in parental strains.
et al. congenic crosses phenotypes using Significant variation for congenic strains
(2008) thereof, M light prepulses and for PA for both BG and for PPI in C57Bl6
tactile startle pulses BG strains. Common markers with
acoustic PA, but not acoustic PPI from
previous study (Joober et al. 2002)
Samocha et al. LG/J x SM/J F2 cross PPI and PA phenotyping QTLs found at chromosome 12 for all PP
(2010) & F34 advanced followed by QTL in intensities; QTLs for PP6 & PP12 on
intercross line F2 generation and chromosome 7; QTLs on chromosomes 7
(AIL), M, F genotyped F34 line at & 17 for startle response; QTLs on
3000 SNP markers chromosome 4 for startle habituation; AIL
mice reduced size of some QTLs to less
than 5 cm
(continued)
299
Table 5 (continued)
300
References Mouse strain, sex Model description/ Basal PPI Effects of drugs or manipulations typically Effects of (presumed)
background/rationale used to induce PPI-deficits antipsychotics/other
treatments
Insertional mutagenesis
Verma et al. Ckr mouse, M, F Ckr serendipidous ;PPI (modest) at all PP intensities in Ckr versus CLO (:PPI in Ckr; [PPI in
(2008) mutation resulted in (+/- & WT); [PA in Ckr versus -/+ or WT; RIS ([PPI in Ckr
mouse with abnormal WT & WT), HAL ([PPI in
circling behavior Ckr & WT); PA data
not shown
Abbreviations: 5-HT serotonin, AAV adeno-associated virus, Ab amyloid b- peptide, ACE angiotensin converting enzyme, ACH acetylcholine (receptor), AD Alzheimer’s disease, ADX
adrenalectomy, AIL advanced intercross line, AMP amphetamine, AMY amygdala, APD antipsychotic drug, APP amyloid precursor protein, ARIP aripiprazole, APO apomorphine, AT
angiotensin, BAC bacterial artificial chromosome, BACE b-site APP cleaving enzyme, BD bipolar disorder, BG background, CaMKIV calcium-calmodulin-dependent protein kinease IV, cAMP
cyclic adenosine monophosphate, Ckr chakragati, CLO clozapine, CNS central nervous system, COC cocaine, COMT catechol-O-methyltransferase, CORT corticosterone, CRF corticotropin
releasing factor, CTR controls, CTX cortex, DA dopamine (receptor), DAT dopamine transporter, dB decibel, DCC deleted in colorectal cancer, DIAZ diazepam, DIZ dizocilpine, DN dominant-
negative, DDO D-aspartate oxidase, E embryonic day, EE environmental enrichment, EGF epidermal growth factor, ENU N-ethyl-N-nitrosourea, f frontal, F female, FABP fatty acid binding
protein, FGF fibroblast growth factor, Fmr1 fragile 9 mental retardation 1 gene, FMRP fragile 9 mental retardation protein, FXS fragile 9 Syndrome, GLAST glutamate and aspartate
transporter, GLU glutamate, GluR glutamate receptor, GLUT glucose transporter, GR glucocorticoid receptor, GRIP glutamate receptor interacting protein, GSK glycogen synthase kinase,
GWAS genome wide association studies, HAL haloperidol, HD Huntington’s disease, HPC hippocampus, IC imprinted cluster, IL interleukin, ISI interstimulus interval, KI knock-in, KO knock-
out, M male, m metabotropic, MDB methyl-CpG binding protein, mo month, NIC nicotine, MPEP 2-methyl-6-(phenylethylyn)-pyridine hydrocholoride, METH metamphetamine, Mgat N-
acetylglucosaminyltransferase, NAA N-acetyl-aspartate, NAAG N-acetylalpha L-aspartyl-L-glutamate, NAC nucleus accumbens, NCAM neural cell adhesion molecule, NIS nisoxetine, nNOS
neuronal nitric oxide synthase, NO nitric oxide, NPS neuropeptide S (receptor), NPY neuropeptide Y, NR NMDA receptor subunit, NRG neuregulin, NRL neuroligin, ns not significant(ly), NSE
neuron-specific enolase, NT neurotensin, OE overexpressor, OXO oxotremorine, PA magnitude of response to pulse alone, PACAP pituitary adenylate-cyclase-activating polypeptide, PD
Parkinson’s disease, PDE phosphodiesterase, PET-1 plasmocytoma expressed transcript-1, PND postnatal day, PLC phospholipase C, PNS prenatal stress, poly I:C polyinosinic: polycytidylic
acid, PP prepulse, PPI prepulse inhibition of startle, PPP prepulse potentiation, PS1 presinilin1, PWS Prader–Willi syndrome, QTL quantitative trait locus, QUET quetiapine, RAC raclopride,
RAGE receptor for advanced glycation end-products, RasGAP Ras GTPase-activating protein, RE renin-enhancer, RIS risperidone, SCOP scopolamine, SI social isolation, SNP single nucleotide
polymorphism, SOCS suppressor of cytokine signaling; SREB superconserved receptor expressed in brain; STR striatum, SYN synapsin, SynGAP synaptic GTP-ase-activating protein, SZ
schizophrenia, TAAR trace amine-associated receptor, TG transgenic, TGF-b transforming growth factor beta, TK typosine kinase, TMS transcranial magnetic stimulation, V1b vasopressin
receptor 1b, WT wild-type
S. B. Powell et al.
creation of a mutant mouse are the SP4 and the SREB2 genes. SP4 is a member of
the Sp1 family of transcription factors. Hypomorphic Sp4 mice showed vacuoli-
zation in the hippocampus, age-dependent decrease in neurotrophin-3 expression
in the dentate granule cells, and robust deficits in both PPI and contextual memory
(Zhou et al. 2004). These studies revealed a novel Sp4 pathway that is important
for hippocampal development and essential to many behaviors, including PPI,
relevant to schizophrenia (Zhou et al. 2004). Zhou et al. (2009) have gone on to
examine the role of the human SP4 gene in schizophrenia and bipolar disorder.
Several SNPs from the human SP4 gene are found to associate with both bipolar
disorder and schizophrenia in both Caucasian and Chinese samples. This work
represents an example in which a PPI phenotype, in combination with other
behavioral abnormalities, suggested the association of this gene with neuropsy-
chiatric disorders. A similar finding was also observed with SREB2 Tg mice,
which display PPI deficits. Follow-up genetic studies in schizophrenia found a
genetic association between SREB2 and schizophrenia (Matsumoto et al. 2008).
4 Discussion
Mutant mouse models of schizophrenia provide a unique way to assess the

function of a susceptibility gene, test hypotheses about the pathophysiology of
disease, address receptor mechanisms of drugs, and generate hypotheses about the
function of relatively unknown genes. In this review, we provided a comprehen-
sive overview of PPI deficits in genetic models since July, 2007 and elaborate on
specific examples where appropriate. As illustrated in Tables 1–5, perhaps the
most notable advances in genetic approaches to sensorimotor gating over the last
few years have come from candidate genes for schizophrenia and other neuro-
psychiatric diseases exhibiting PPI deficits, genes involved in basic synaptic
processes and receptor signaling, and conditional genetic approaches that help in
understanding the dynamic function of specific genes both regionally and tem-
porally. In particular, since our last reviews (Powell et al. 2009; Swerdlow et al.
2008), many genetic mutants of proteins involved in synaptic plasticity, second
messenger systems, calcium signaling, etc. have been developed. In this case, PPI
is used as a functional measure of sensorimotor processing in models of basic brain
development. Whether or not these cellular processes turn out to be relevant
for a specific disease such as schizophrenia remains to be determined. Thus far,
most models are still in the ‘‘characterization’’ phase—testing multiple behavioral/
cognitive constructs that are deficient across neuropsychiatric disorders (e.g.
sensorimotor gating, attention, social interaction, learning, and memory, etc.).
Few models, however, have shown any predictive power for drug development.
Perhaps this new wave of mutants based on susceptibility genes, synaptic function,
or brain development, as opposed to genetic manipulation based on mechanism of
action of antipsychotics or psychotomimetics, will be better models to lead the
field forward in medication development for mental illness. As Moore (2010)
argues, too many models that are not based on either ‘‘etiologic or pathogenic’’
theories, may result in too many ‘‘false positives’’ in drug screens. Rather, refined
models that more closely mimic the etiological risk factors and/or neuropathology
of disease (e.g. schizophrenia) may generate more predictive models for drug
development (Moore 2010). While this approach might be very useful if the goal is
to use the mutant mice for drug development, we have illustrated the benefits of
careful behavioral characterization in genetic models of basic brain processes as
well. As mentioned above, it is unlikely that all aspects of a heterogeneous disease
will be recapitulated in another species with a genetic mutation. Investigators must
rely on convergence of behavioral and neuroanatomical or neurochemical data in a
given mutant mouse to support clinical data on the link between the candidate gene
and disease. No single phenotype such as PPI should be considered as being either
necessary or sufficient to substantiate a model as having relevance to neuropsy-
chiatric disease.
Animal studies addressing neuropsychiatric disease would benefit greatly from
more neurobiologically based biomarkers for these disorders. Such an approach
has been taken in the CNTRICS initiative, in an explicit attempt to incorporate
more cognitive neuroscience-based testing into treatment trials of putative cog-
nitive therapies for schizophrenia. Along the same lines, genetic studies of
schizophrenia have focused on psychophysiological endophenotypes such as PPI
instead of the broader, more heterogeneous diagnosis of schizophrenia. In fact,
many laboratories are now using PPI as an endophenotype in genetic studies of
schizophrenia (Braff et al. 2007; Greenwood et al. 2007; Greenwood et al. 2011;
Hokyo et al. 2010). Some of these genetic studies have generated further support
for a genetic contribution to PPI (Greenwood et al. 2007, 2011), and other studies
have suggested that genetic variants in COMT (Quednow et al. 2008; Roussos
et al. 2008), CHRNA3 (Petrovsky et al. 2010), and neuregulin 1 (Roussos et al.
2011) directly affect PPI levels (as reviewed above). Thus, as human studies
materialize with more neurobiologically defined behavioral measures, the ability
to translate these measures or ‘‘endophenotypes’’ into animal models should
improve dramatically. PPI in genetic models offers a behavioral endpoint that has
shown predictive validity in rat pharmacological models, cross-species homology
with the same measure in humans, and alterations in response to genetic manip-
ulations implicated in the pathogenesis of schizophrenia. While these mutant
mouse models are not without shortcomings, they offer some of the best attempts
at etiological models that are possible in rodents. Merging the genetic etiological
models with a second hit approach may strengthen some of the mutant mouse
models. Hence, consideration for other factors, such as the importance of envi-
ronmental risk factors (e.g. prenatal infection) and the role of epigenetics (e.g.
DNA methylation) in the etiology of schizophrenia, should also be incorporated
with genetic models.
Acknowledgments This work was supported by grants from the National Institute of Mental
Health (R01MH042228, R01MH052885, R01MH091407) and by the Veterans Affairs VISN 22
Mental Illness Research, Education, and Clinical Center.
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Part II
Clinical Behavioral Genetics
Applications of Second Generation
Sequencing Technologies in Complex
Disorders
Mònica Bayés, Simon Heath and Ivo Glynne Gut
Abstract Second generation sequencing (2ndGS) technologies generate unprec-

edented amounts of sequence data very rapidly and at relatively limited costs,
allowing the sequence of a human genome to be completed in a few weeks. The
principle is on the basis of generating millions of relatively short reads from
amplified single DNA fragments using iterative cycles of nucleotide extensions.
However, the data generated on this scale present new challenges in interpretation,
data analysis and data management. 2ndGS technologies are becoming widespread
and are profoundly impacting biomedical research. Common applications include
whole-genome sequencing, target resequencing, characterization of structural and
copy number variation, profiling epigenetic modifications, transcriptome
sequencing and identification of infectious agents. New methodologies and
instruments that will enable to sequence the complete human genome in less than a
day at a cost of less than $1,000 are currently in development.
Keywords Sequencing technologies Bioinformatics Personalized genomics
Contents
1 Sequencing Technologies.................................................................................................. 322

1.1 Pyrosequencing (454-Roche-IonTorrent) ................................................................. 324
1.2 Sequencing with Reversible Terminators (Illumina)............................................... 325
1.3 Sequencing by Ligation (Applied Biosystems) ....................................................... 325
1.4 Single-Molecule Sequencing (Pacific Biosciences) ................................................ 326
M. Bayés (&) S. Heath I. G. Gut

Centro Nacional de Análisis Genómico, C/Baldiri Reixac 4,
08028 Barcelona, Spain
e-mail: mbayes@pcb.ub.es

DOI: 10.1007/7854_2011_196
322 M. Bayés et al.
1.5 Novel Technologies in Development....................................................................... 326

1.6 Comparison of Cost, Throughput and Accuracy..................................................... 327
2 Informatics and Bioinformatics for Second Generation Sequencing .............................. 328
2.1 Data Storage.............................................................................................................. 329
2.2 Checks of Sequence Quality and Integrity .............................................................. 329
2.3 Sequence Alignment to the Reference Genome...................................................... 330
2.4 Single-Nucleotide Variant Calling ........................................................................... 331
3 Application of Second Generation Sequencing Technologies......................................... 332
3.1 Whole-Genome Sequencing ..................................................................................... 332
3.2 Targeted Sequencing ................................................................................................ 334
3.3 Characterizing Structural and Copy Number Variation.......................................... 337
3.4 Profiling Epigenetic Modifications .......................................................................... 338
3.5 Transcriptome Sequencing ....................................................................................... 339
3.6 Identification of Infectious Agents .......................................................................... 340
References................................................................................................................................ 340
1 Sequencing Technologies
In 1977 two very different methods were presented for directly sequencing DNA:
the Maxam–Gilbert chemical cleavage method (Maxam and Gilbert 1977) and the
Sanger chain-termination method (Sanger et al. 1977). The Sanger sequencing
method relies on base-specific termination of the growing chain during DNA
synthesis caused by the incorporation of dideoxy-nucleotides (ddNTPs) that lack
the hydroxyl group necessary for DNA chain elongation. The labeled DNA
fragments are then separated according to their length on a gel matrix. Sanger and
Gilbert shared the Nobel prize in 1980. Sanger’s method became widely used first
in slab-gel implementations with radioactive detection and later in capillary
electrophoretic systems using fluorescent-labeled ddNTPs. It revolutionized the
genetics field and eventually led to the sequencing of the human (Lander et al.
2001; Venter et al. 2001) and many other genomes. However, the effort to
sequence the human genome required many hundreds of automated capillary
sequencers over a ten-year period and cost over $10 M. At the present time,
automated capillary sequencing instruments with up to 384 capillaries can generate
around 1 Mbase of sequence every 24 h.
In 2005 there was a paradigm shift in DNA sequencing with the introduction of
the first second generation sequencing (2ndGS) technologies (for a review see
Ansorge 2009). While Sanger sequencing relies on the individual analysis of DNA
fragments (one fragment per capillary), second generation sequencers are able to
analyze millions of DNA templates in parallel (Fig. 1, Table 1). This is achieved
first by placing single DNA molecules into individual and miniaturized reaction
vessels for amplification. In emulsion PCR, template DNA molecules are indi-
vidually captured on beads, and compartmentalized and clonally amplified in
droplets within an oil emulsion (Diehl et al. 2006). An alternative approach is to
spread out the DNA fragments on a planar surface and generate clonally amplified
Applications of Second Generation Sequencing Technologies 323
Genomic DNA
Fragmentation
Adapter ligation
Clonal amplification
Emulsion PCR Solid-phase amplification
DNA
Sequencing polymerase
primer
Sequencing
chemistry
Imaging
Conversion
to sequence
Fig. 1 Schematic representation of the principles of 2ndGS technologies. Genomic DNA (gray)
is randomly sheared and ligated to universal adapter sequences (black). Clonal amplification of
the fragments is achieved either in droplets within an oil emulsion (emulsion PCR; left) or on the
surface of a solid substrate (solid-phase amplification; right). The sequencing process consists of
iterative cycles of enzyme-driven nucleotide extension and imaging-based data acquisition
Table 1 Comparison of sequencing platforms

ABI3730xl GS FLX HiSeq2000 5500xl PacBio RS
(Applied (454, Roche) (Solexa, Illumina) SOLiD (Life (Pacific
Byosistems) Technologies) Biosciences)
Amplification PCR/ Emulsion PCR Solid-phase Emulsion None
approach cloning amplification PCR
Sequencing Sanger Pyrosequencing Reversible dye Ligation
chemistry method terminators based
sequencing Single-molecule
real-time
sequencing
Reads per run 1,536 1M 3,000 Ma 2,400 Ma NA
Bases per read 850 bp 700 bp 2 9 100 bp 2 9 60 bp 1,000 bp
Output per run 1.3 Mb 700 Mb 600 Gba 300 Gba NA
Time per run 1.5 days 1 day 11 days 7 days \1 day
a
From two flowcells or slides
templates in situ (Bentley et al. 2008). Once the clonal amplification of the original
templates is completed, millions of DNA ‘‘polymerase clones’’ or ‘‘polonies’’ are
subjected to a sequencing chemistry in a microfluidic device coupled with a high-
resolution imaging system. For each DNA ‘‘polony’’, the resulting images are
converted into sequence reads.
1.1 Pyrosequencing (454-Roche-IonTorrent)
The Roche/454 system (www.454.com) was the first 2ndGS system released to the
market. The templates are amplified by emulsion PCR and the sequences are
obtained by iterative pyrosequencing (Magulies et al. 2005). Pyrosequencing
(Ronaghi et al. 1996) is a sequencing-by-synthesis method that relies on an
enzymatic cascade in which the release of pyrophosphate during the incorporation
of a nucleotide drives a chemiluminescent reaction that can be detected with a
high-resolution charge-coupled device (CCD) camera. It uses regular deoxy-
nucleotides that are flowed sequentially in a known order. The amount of light
emitted is proportional to the number of base incorporations. Because dNTPs are
used, homopolymers in the template DNA are the main source of errors due to the
difficulty to unambiguously assign the number of bases incorporated. The current
454/Roche platform provides reads that are several hundred bases, more than any
other 2ndGS technology. Roche markets two different systems, the Genome
Sequencer (GS) FLX instrument that can generate about 0.7 Gbase of sequence per
run, and the GS Junior that produces about 35 Mbases (Table 1).
Recently Ion Torrent introduced an instrument that uses the same underlying
methods as the Roche system for clonal amplification and parallel sequencing but
with a different detection method (www.iontorrent.com). Rather than detecting

chemiluminescence generated through an enzymatic cascade, the Ion Torrent
system measures directly the pH change caused by the release of a positively
charged hydrogen ion during each base incorporation. The system uses semicon-
ductor technology that converts chemical information into digital, without any
optics. The specification that currently is being provided moves this instrument
into the realm of the Roche FLX junior.
1.2 Sequencing with Reversible Terminators (Illumina)
The first Solexa sequencing platform (Genome Analyzer 1G) was commercialized
in late 2006 and Illumina acquired the company soon after the launch of this
instrument (www.illumina.com). In this technology templates are amplified in near
vicinity by solid-surface bridge amplification using a microfluidic cluster station
(Bentley et al. 2008) (Fig. 1). Sequencing proceeds on the same surface using four
reversible terminator nucleotides each labeled with a different fluorescent dye. The
identity of each incorporated nucleotide is determined by recording the fluores-
cence with a CCD camera. After each imaging cycle the fluorescent moiety is
cleaved from the base and the 30 end is regenerated to enable next nucleotide
incorporation. The technology has undergone several technical and chemical
upgrades to give read lengths over 100 bases. The new version of the Illumina
instrument, the HiSeq2000, can run two slides simultaneously and generate over
600 Gbases of sequence in 11 days of operation (Table 1). This equates to
30x coverage of six human genomes in a single run.
1.3 Sequencing by Ligation (Applied Biosystems)
Applied Biosystems introduced the SOLiD system in late 2007 (http://www.

appliedbiosystems.com/absite/us/en/home/applications-technologies/solid-next-
generation-sequencing.html). As for the Roche/454 system, this system uses
emulsion PCR for clonal amplification. The key distinguishing feature of this
platform is the sequencing chemistry that uses a DNA ligase rather than a DNA
polymerase (for a review see Ansorge 2009). A set of four fluorescently labeled
octamers whose fourth and fifth bases are encoded by the attached fluorescent
group are hybridized to the template. The probes that match the templates are then
ligated to a primer oligonucleotide in a template-directed reaction. The fluorescent
readout identifies the fixed bases. A cleavage reaction removes the last four bases
with the fluorescent tag and hybridization and ligation cycles are repeated to
determine in successive cycles bases 9 and 10, 14 and 15 and so on. After com-
pletion of a defined number of cycles, a new primer is annealed that is offset from
the first primer by one base. The ligation procedure is repeated to give the
sequence of bases 3 and 4, 8 and 9, and 13 and 14. The same procedure is repeated
for the two-base, three-base and four-base offset primers, which completes the
sequence. Because in the whole process each base is interrogated twice, the SOLiD
technology shows the greatest accuracy. The latest version of the SOLiD instru-
ment, 5500xl, can generate 60 base reads and process two slides simultaneously to
produce more than 300 Gbases of sequence in a 7-day run (Table 1).
All of the three 2ndGS systems described above can be used with paired-end
protocols that enable the sequence to be obtained from the two extremities of the
templates (for a review see Holt and Jones 2008). Paired-end protocols partially
compensate for the shortcoming of short read lengths of these systems compared to
Sanger sequencing by providing linking information. Illumina offers a paired-end
protocol to read 2 9 100 bases at a distance of up to 600 bases and a mate-pair
procedure in which the insert size can be up to 5 kbases.
1.4 Single-Molecule Sequencing (Pacific Biosciences)
A new system from Pacific Biosciences was introduced in 2011 (www.

pacificbiosciences.com). It is considered the first of a third generation of DNA
sequencing instruments because it can sequence single DNA fragments without the
need for PCR amplification. Single molecules of DNA polymerase are immobi-
lized at the bottom of nanometer scale aperture chambers called zero-mode
waveguides (ZMWs) (Korlach et al. 2010). The template-directed primer exten-
sion reaction with nucleotides labeled with fluorescence at the end of the phos-
phate moiety is monitored in real time. The zero-mode wave guides restrict the
observation to the volume containing the DNA polymerase. This dramatically
reduces the perturbation due to background fluorescence of the labeled nucleotides
that are not involved in the nucleotide incorporation reaction. Because the indi-
vidual base incorporation error rate is quite high, DNA molecules are circularized
and read several times to generate a consensus sequence. Reads of several kbases
in length have been achieved by the developers, although total throughput of the
system is still in the order of Mbases.
1.5 Novel Technologies in Development
There are several companies and academic groups working in the development of
new methodologies and instruments to enable sequencing of the complete human
genome in less than a day at a cost of less than $1,000 (for a review see Schadt
et al. 2010). Oxford Nanopore Technologies is developing a nanopore-based
sequencing platform (www.nanoporetech.com). The principle of the method is
the detection of nucleotides as they are driven by an electrophoretic field pass
through an a–hemolysin membrane nanopore that is coupled with an exonuclease.
The change in the membrane potential at the nanopore is used to determine the
identity of the nucleotide passing through. The system offers the potential to
identify individual nucleotide modifications including 5-methylcytosine.
The unprecedented resolution of the new generation of sequencing technologies
will allow high-throughput single cell DNA and RNA sequencing in the near
future. The underlying concept is that a target sequence of DNA is generated in all
of the cells of a histological section simultaneously. Several studies have already
shown the feasibility of sequencing the transcriptome of individual cells with
current instruments using whole-genome or transcriptome amplification proce-
dures (Tang et al. 2010).
Current sequencing methods do not provide long-range haplotype information
than can be very important for applications such as disease mapping or population
history analysis. Molecular procedures for the determination of haplotypes will
become more important when the methods for the determination of simple
sequence motifs are well established. There are several approaches to stretch and
immobilize long lengths of single genomic DNA molecules on a surface, allowing
molecular sequencing tests to be carried out in situ that will provide haplotype
information.
1.6 Comparison of Cost, Throughput and Accuracy
The three second generation platforms described in this chapter have their own
advantages and disadvantages. Currently the HiSeq2000 and 5500xl SOLiD
platforms have the higher sequencing capacity, 25–50 Gbases per day, while the
GS FLX can generate up to 1 Gbase per day (Table 1). In addition, the reagent
cost per Gbase on the GS FLX is more than two orders of magnitude higher than
on the other platforms. However, these numbers have to be taken with caution
because the rapid increase in throughput at constant reagent consumption has
raised the importance of other cost components such as instrument depreciation,
labor and data management that need to be considered.
The GS FLX system provides intermediate read length, with an average of 700
bases. In contrast, the 5500xl SOLiD and HiSeq2000 platforms read many more
DNA molecules in parallel (up to 3000 million) but have shorter read lengths (up
to 100 bp) (Table 1). This would be a severe limitation if the companies were not
offering paired-end reads, which facilitate their mapping onto the reference
sequence.
Error rates largely dictate the read length limits of different sequencing plat-
forms. However, the different systems are difficult to compare in terms of accuracy
because base quality values are not comparable across platforms. Raw accuracies
of [99.94, [99 and [98.5% are reported in 5500xl SOLiD, GS FLX and
HiSeq2000 specifications, respectively. In the GS FLX platform, the most com-
mon errors are insertions and deletions in homopolymer segments. By contrast, the
5500xl SOLiD and HiSeq2000 instruments have a very distinct error profile, with
the vast majority of the sequencing errors being base substitutions. The Life-
Technologies two-base encoding system shows the highest individual base call
accuracy, although it needs to be pointed out that the method uses the reference
sequence to make corrections to the raw base calls. At present, the error distri-
bution of third generation technologies based on single-molecule sequencing is
much higher than that of PCR-based methods.
As with genotyping technologies, it is essential to use the right 2ndGS instru-
ment for the application in hand. For applications requiring long reads, such as
metagenomic sequencing, and de novo sequencing of genomes, GS FLX is the
technology of choice. The long reads guarantee best possible assembly of the data.
The 5500xl SOLiD and HiSeq2000 systems work better for tag counting appli-
cations such as mRNA and smallRNA profiling and identification of DNA–protein
binding sites, because they provide very high number of individual reads with just
enough sequence to map them to the reference genome. Because of the lower
reagent cost, HiSeq 2000 and 5500xl SOLiD are also the ideal solution for rese-
quencing of genomes or exomes where reference sequences are available. The use
of the SOLiD platform with the very accurate two-base encoding system is rec-
ommended for low coverage projects and for detecting rare variants and somatic
mutations. In some cases, the use of various 2ndGS platforms is recommended to
take the advantages of each technology: error profile, read length, paired-end
reads, number of tags, etc.
2 Informatics and Bioinformatics for Second Generation

Sequencing
The use of 2ndGS technologies that produce hundreds of millions of short reads
brings with it a collection of informatics and bioinformatics challenges. The most
immediate issue is that of data storage, with even small projects needing tens of
terabytes of storage, and sequencing centers having storage requirements in the
petabyte range. The next concerns the assessment of the quality of the produced
sequence data. Each sequencing platform produces its own quality metrics, but it is
often advisable to add additional quality checks to check the consistency of the
results. The final challenge that will be addressed in this chapter is that of the
primary data analysis, which will generally include assembly and/or alignment of
the individual sequence reads followed by subsequent analyses that will depend on
the particular sequencing experiment being performed. A survey of all the analyses
used for the different types of sequencing experiments that have been described
using 2ndGS technologies is beyond the scope of this chapter, but a short
description of variant calling for the case of the resequencing of genomic data will
be given. A list of available 2ndGS software has not been included as such lists
become quickly out of date with the development of new packages and the
improvement of existing packages. Up-to-date information on analysis packages
can be found from online forums such as SEQanswers (http://www.seqanswers.

com), while the focus of this chapter is on the important features that such
packages should possess, allowing the reader to make their own selection.
2.1 Data Storage
The principal data produced by the sequencers are the sequence reads and asso-
ciated quality measures, which alone can require a large amount of storage space.
For example, WGS of a human genome at 309 coverage will require almost
100 Gbases of sequence. The common data formats used for this type of data
require 2 bytes per base (1 for the base and 1 for the quality) with some extra to
store the read identity, so 100 Gbases of sequence will require [200 Gbytes of
storage, although this can be reduced to \50 Gbytes using data compression
techniques. The sequence reads, however, are only a part of the story, and to get
full picture of the storage requirements it is necessary to look at the complete
analysis pipeline from the initial data produced by the sequencers (depending on
the platform these could be the sequence reads or could be unprocessed data such
as images) to the final products of the pipeline (i.e., called variants). Each stage of
the pipeline will require space to calculate and store the results of the analysis, and
a working space of at last several Tbytes will typically be required to work on
WGS data from higher organisms.
An important decision to be made that has a large impact on the total storage
requirements is how long the results files from different stages of the pipeline
should be kept. Many of the stages in the analysis pipeline (particularly the early
stages) involve data reduction and some consequent data loss, so that it is often not
possible to take the results of a particular analysis stage and recreate the results of
an earlier stage. A balance must therefore be struck between the cost of recreating
the data from a particular stage if necessary, and the costs of storing them.
2.2 Checks of Sequence Quality and Integrity
Current 2ndGS platforms provide measures of sequencing quality for each

sequenced base. Base calling is generally done by calculating the probability that
the true base at a given read position is A, C, G or T, and selecting the most likely
base at each position. A common measure of quality is based on the negative of the
log ratio of the probability of the most likely base to that of the next most likely
base. A low quality score indicates that the probability of the called base being
incorrect is high, whereas a high quality score indicates that the probability of a
false call is low (e.g., an Illumina quality score of 30 means that the probability
that this base is called incorrect is 0.01%).
It is important to keep account of the quality scores, both as a measure of
the overall quality of the sequencing run, and for downstream analyses.
For example, a useful metric to assess the overall quality of a sequencing run is the
proportion of sequenced bases that have quality scores that exceed a certain
threshold. For downstream analyses there are two basic approaches to use quality
scores: (a) reads with low quality bases can be trimmed so that all remaining bases
are high quality and (b) the analyses explicitly take account of the quality scores
by weighing bases according to their qualities, with low quality bases being down
weighted and so having less effect on the analyses.
In addition to errors in the base calling, another potential source of problems is
sample mix-ups, leading to the sample that is being analyzed not being what is
expected. Such mix-ups can occur at many points in the process, for example at the
collection facility, during library preparation, at the moment the samples are
placed on the sequencer or during the analyses themselves. It is difficult to com-
pletely eliminate the possibility of sample mix-ups, but a combination of good
laboratory practice and analysis checks can both greatly reduce the chances of
mix-ups and detect the majority of mix-ups that do occur. For example, a common
practice is to perform systematically SNP genotyping on the same samples that are
sequenced using one of the genome-wide SNP array platforms. This provides a set
of reference genotypes that can be checked against genotypes predicted from the
sequence data (Koboldt et al. 2010).
2.3 Sequence Alignment to the Reference Genome
For species for which a good reference sequence exists, the first analysis to be
performed after base calling and initial quality control is to align the short
sequence reads to the reference. When 2ndGS technologies were first developed,
existing sequence aligners were not adapted to handle efficiently the very large
number of short reads. In recent years a large number of aligners have been
developed explicitly for short sequence reads, and now aligning hundreds of
Gbases of sequence for WGS studies is a matter of hours rather than days or weeks
(Li and Homer 2010; Koboldt et al. 2010). The aim of this section is not to give an
exhaustive survey of available alignment software but rather to discuss the features
that a good aligner should have, that may itself depend on the type of analysis that
is required. All aligners report for each read the number of matches that have been
found with an indication of the differences between the read and the reference at
the match position. To reduce the number of possible matches, aligners only
consider matches that are close to the reference, although how ‘close’ is defined
can vary between aligners, and can often be adjusted by the user. A typical
definition of close would be where the read differs from the reference at n or less
positions, possibly only considering high quality bases in the comparison with the
reference. Important considerations when choosing an aligner are whether the
search for matches is exhaustive (i.e., is there a guarantee that all close matches
will be found), what the behavior is with reads that map to repetitive regions (are
all matches returned in these cases, or a subset of matches or a single match
selected using some criterion), whether the aligner can find gapped or split
alignments (discussed below), and the computational requirements. For variant
calling (discussed below) it is important that only uniquely mapping reads are used
otherwise there is a high risk of false calls being made, but for different analyses
(i.e. of CNVs), nonuniquely mapping reads give important information and should
be made available by the aligner.
The alignment of reads from paired-end libraries requires a post-processing step
after alignment where the potential matches from both ends of the same fragment are
examined to see if a consistent pair of matches exists where the orientation and
distance of the two ends is as expected given the library construction method. In this
way a nonuniquely mapping read can be ‘rescued’ if the corresponding pair is itself
uniquely mapped. Paired-end reads are useful for the detection of structural variation
as large deletions/insertions or translocations and inversions will create sets of
paired-reads with incoherent matches (i.e., with the wrong distance between the pairs
or with the two ends mapping in the wrong orientation or on different chromosomes).
This approach is powerful for larger structural variants in the order of several
hundred base pairs or larger, but for smaller variants the distortion of the distance
between paired-reads is too small to allow detection by this method. Smaller
insertion/deletion variants can be detected using gapped and split alignments, if the
aligner can generate these. Gapped alignments allow a short insertion/deletion
(typically \30 bp to be present) within the reads, while split alignments are a
generalization of gapped alignments where an arbitrary gap can exist in a read,
with the two extremities of the same read being potentially mapped to different
chromosomes. The use of gapped/split alignments along with paired-end reads
allows the detection of deletions across the entire scale of the genome from single
bases up to entire chromosomes. Other types of structural rearrangements can also
be detected across most of the range, although with current technologies there is a
gap between very short features (\30 bp) and features [*200 bp where rear-
rangements cannot be reliably detected, although increasing read lengths should
reduce this gap in the near future. It should also be mentioned here that gapped/
split alignments are also very useful in the mapping of reads from the transcrip-
tome on to the genomic reference, allowing the mapping of exon/intron boundaries
(Trapnell et al. 2009; Bryant et al. 2010).
2.4 Single-Nucleotide Variant Calling
The principle of Single-Nucleotide Variant (SNV) calling is straightforward; at

each position the number of reads of each base is counted, and the genotype at the
position is called using the base counts. All SNV callers work very similarly; if
only one base is seen then the most likely genotype is homozygous for the
observed base, and if a more or less equal number of two different bases are seen
then a heterozygous call will be the most likely. However, different callers do
differ in the details of how ambiguous situations are dealt with, and how the
confidence for the call is calculated. Simple callers use heuristic filters to deter-
mine thresholds for calling different genotypes, while more sophisticated callers
use explicit statistical models that allow statements to be made about the precision
of the estimation, and that permit the incorporation of prior information such as the
base quality scores and knowledge about the frequency and location of known
variants (Li et al. 2009; Koboldt et al. 2009).
3 Application of Second Generation Sequencing Technologies
Current high-throughput sequencing technologies are becoming widespread and

allow even individual research groups to generate unprecedented amounts of
sequence data very rapidly and at relatively limited costs. They enable new lines of
experimental investigation in basic and clinical research (Voelkerding et al. 2009;
Mardis 2009). In the near future, the cost of genome sequencing will be greatly
reduced and will make personalized genomics for medical purposes a close reality.
The next sections describe current applications of 2ndGS technologies in the
medical field. With the development of 2ndGS technologies, searching for changes
in DNA sequence, copy number, structure, methylation status and expression
profile can be accomplished with a single platform.
3.1 Whole-Genome Sequencing
Today, 2ndGS technologies are being applied to sequence complete human gen-
omes in order to unravel the complexity of the normal and disease genomes in
terms of single-nucleotide variants (SNVs), small insertions and deletions (indels),
structural variants (SVs) and copy number variations (CNVs). The number of
individual genomes that has been publicly announced to be sequenced is rapidly
increasing. However, it is difficult to identify variants contributing to disease from
individual sequences (Fig. 2b). As for the Genome-Wide Association Studies
(GWAS) performed with SNP arrays, sample size needs to be very large to
establish genotype-phenotype relationships for most disorders.
There are several ongoing large-scale projects that aim to sequence the whole
genome of several hundreds or even thousands of individuals. The 1,000 Genome
Project (www.1000genomes.org) aims to sequence the genomes of a large number
of people to provide a comprehensive catalog of human genetic variation,
including rare variants (present in \5% in the population) that are thought to play
a major role in the genetic basis of complex diseases and that are not
readily screened by current genotyping technologies. The International Cancer
Genome Consortium (ICGC, www.icgc.org) has been organized as an interna-
tional effort to obtain a comprehensive description of genomic, transcriptomic and
epigenomic changes in 50 different tumor types (The International Cancer Genome
Consortium 2010).
(a) De novo whole genome sequencing
(b) Whole genome resequencing
(c) Targeted resequencing
(d) Characterization of structural and copy number variation
(e) Profiling epigenetic modifications
(f) Transcriptome sequencing
Fig. 2 Examples of the sequencing reads obtained in different applications. When appropriate
the genome reference sequence (Ref) is shown above, with exons marked with different colors.
Sequence variants in the reads (SNVs and indels) are shown in red (b, c). Paired-end reads
mapping to different chromosomal locations are shown in d. Positions displaying complete
methylation (C), no methylation (T) and partial methylation (T or C) after bisulfite treatment are
depicted in blue (e)
Application of 2ndGS technologies to tumor samples has already yielded sig-

nificant insights. Several landmark studies have determined the complete DNA
sequences of clinical tumor samples or cell lines, and compared these to normal
tissues from the same individual. In this way, genome-wide molecular profiles of
cancer have been obtained for individuals with acute myeloid leukemia, lung
cancer, glioblastoma, malignant melanoma and breast cancer revealing a large
range of mutation loads, ranging from 10 somatic missense mutations in leukemia
genomes to more than 100 mutations in tumors subjected to substantial exogenous
mutagenic exposures such as lung and skin cancer (Robison 2010). Results
obtained so far support the hypothesis that there is extensive genetic heterogeneity
within a given tumor type, with a large number of infrequently mutated genes and
a few genes with mutations of high incidence (Stratton et al. 2009). In addition,
resequencing complete cancer genomes from multiple samples from the same
patient allows the identification of changes that occur during cancer progression
and will possibly lead to improved drug therapies (Ding et al. 2010).
Whole genome sequencing (WGS) has the potential to identify mutations
located outside the protein-coding regions of genes, including putative deep
intronic mutations or pathogenic variants in regulatory regions that are not being
addressed by other methods. All the genomes sequenced so far have yielded
similar results in terms of SNVs: around 3 million SNVs per genome of which
around 0.5 million are novel SNVs not present in dbSNP. However, because of the
difficulties of establishing the pathological relevance of these variants, the analysis
of WGS data has been focused on the identification of a subset of variants located
in protein-coding regions and large SVs. Comprehensive mutation databases such
as COSMIC (Catalog of Somatic Mutations in Cancer, www.sanger.ac.uk/
genetics/CGP/cosmic) will substantially facilitate the interpretation of findings.
Reduced costs are causing a rise in WGS. Two recent studies have applied
WGS for genetic diagnosis in two patients with Charcot-Marie-Tooth disease and
metachondromatosis (Lupski et al. 2010; Sobreira et al. 2010). Some companies
and Institutions, such as Complete Genomics (www.completegenomics.com),
Illumina (www.illumina.com) and the Beijing Genomics Institute
(www.genomics.cn), offer individual genome sequencing services to researchers
and also directly to consumers. However, WGS is still not affordable for large
genetic epidemiologic studies that require large sample sizes to find out genome-
wide significant associations. WGS of carefully selected individuals that are at the
extreme ends of a trait distribution sequencing selected families with multiple
affected individuals or the use of pooled samples are appealing alternatives.
3.2 Targeted Sequencing
Because whole-genome sequencing is still an expensive endeavor, many

researchers need to focus their analysis on specific genomic regions, specific genes
or the whole exome to approach disease genetics (Fig. 2c). 2ndGS can be coupled
Array with capture probes Genomic DNA Array with capture probes
Sequencing library Cleaved probes
Target DNA selection by

hybridization to capture probes
Target DNA elution
2nd generation sequencing
Fig. 3 Strategies for selecting specific regions of interest. Genomic DNA (gray, with regions of
interest in red) is randomly sheared and ligated to universal adapter sequences (black). The
fragments of interest are then captured by hybridization to target-specific probes either on a
microarray surface (left) or in solution (right). The hybrid-selected enriched output library is
eluted and sequenced
with DNA capturing or enrichment methods to resequence targeted regions of

interest (Fig. 3).
Hybridization-based methods have been developed to capture specific genomic
regions using a set of long oligonucleotide probes that are complementary to the
regions of interest. Randomly fragmented, denatured genomic DNA is hybridized
to the oligonucleotide probes that are immobilized on a solid surface or used
in solution. The bound DNA is recovered and processed for sequencing.
After sequencing, the percentage of mappable reads that target to the region of
interest is typically [60%, although this is highly variable between regions
(Mamanova et al. 2010). Robust and reproducible protocols are currently being
worked out in order to minimize variations in capture uniformity across regions.
3.2.1 Sequencing Specific Genes
Some Mendelian disorders are known to be caused by mutations in tens or hun-

dreds of different genes. For these diseases, it is desirable to sequence a panel of
candidate genes at high depth of coverage to identify the causal mutations in a
particular family. PCR coupled with 2ndGS can be used to screen for mutations in
disease-causing genes but it is unpractical if the number of exons to analyze is very
large. High-throughput PCR systems that are based on microfluidics, such as the
ones developed by RainDance Technologies (www.raindancetechnologies.com) or
Fluidigm (www.fluidigm.com), partially circumvent the limited multiplex capa-
bility and high costs of traditional PCR. Alternatively, hybridization-based cap-
turing procedures can be used to enrich the sample for the genes of interest. As an
example, Vasta et al. (2009) developed an assay for the diagnosis of mitochondrial
disorders that allows the simultaneous capture and sequencing of the entire
mitochondrial genome and the exons of 362 nuclear gene encoding mitochondrial
proteins. Other groups have designed similar multigene-resequencing assays for
disorders that exhibit a high degree of locus and allelic heterogeneity such as
breast/ovarian cancer (Morgan et al. 2010; Walsh et al. 2010) and familial
hypertrophic cardiomyopathy (Dames et al. 2010).
At some point in the future these methods are likely to replace some of the
genetic disease diagnostic products that are on the market today. In addition to
reductions in cost and time, the digital nature of 2ndGS-based methods will allow
for the detection of somatic variants that are present in a subpopulation of cells and
that may indicate the presence of a preclinical disease.
3.2.2 Sequencing Exomes
Most Mendelian disorders are caused by mutations in protein-coding regions that

represent a small part (up to 2%) of the human genome. Given the elevated cost of
WGS at high coverage, exome sequencing (sequencing the collection of all human
exons) has emerged as an alternative screening strategy to find variants underlying
a mendelian disease. Companies such as Agilent (www.agilent.com) and Nim-
blegen (www.nimblegen.com) have developed user-ready products that enable to
capture the human exome very cost-effectively (Mamanova et al. 2010).
These exome-capture methods coupled with 2ndGS technologies are extremely
valuable for finding mutations that cause rare Mendelian disorders using small
family pedigrees that are not tractable to linkage analysis. The approach has
successfully been applied to familial pancreatic cancer (Jones et al. 2009), Miller
syndrome (Ng et al. 2010) and other rare monogenic diseases. A typical project of
this type involves sequencing a few individuals with highly selected clinical
phenotypes, identifying all variants with respect to the reference sequence, filtering
out common variation present in public SNV databases and selecting and vali-
dating the putative pathogenic variants shared by the affected individuals.
In the near future, exome sequencing will probably trigger the discovery of non-
recurring variants with large effects on complex phenotypes such as schizophrenia,
autism, amyotrophic lateral sclerosis and X-linked mental retardation (McCarthy
2009).
3.2.3 Sequencing Large Genomic Regions
Several GWAS in complex disorders have identified regions of the genome that
are significantly associated with the risk of the disease (www.genome.gov/
26525384). However, the majority of these reported associations involve variants
that lie outside known genes, with no obvious functional consequences, and it
remains a challenge to pinpoint the true causative variants.
Using targeted capture and 2ndGS methods, large contiguous stretches of
genome (up to few Mbases) identified through GWAS can be sequenced to help
refine association signals and find the biologically causative mutation among these
correlated variants. Following this approach, Yeager et al. (2008) resequenced the
gene-poor 8q24 region that is associated to breast, prostate and colorectal cancer
and generated a detailed map of common and rare genetic variation across the
region. Hopefully, this kind of data together with the development of assays that
test directly the effect of the newly discovered variants will shed some light on the
biologically relevant variants that predispose to complex disorders.
In a similar way, other groups have used targeted capture methods to quickly
and cost-effectively sequence the genomic intervals identified by linkage analysis
in rare Mendelian disorders (Brkanac et al. 2009; Volpi et al. 2010 and Nikopoulos
et al. 2010). In both applications, a major limitation of this approach is that in all
hybridization-based capture methods, repetitive regions are masked during the
probe design process leaving only the unique fraction of the genomes represented.
3.3 Characterizing Structural and Copy Number Variation
Like SNVs, CNVs and SVs may account for the modulation of many complex
phenotypic traits and disease susceptibility in humans. 2ndGS has been quickly
applied to obtain single-base resolution data on CNVs and SVs partially sup-
planting array Comparative Genome Hybridization (aCGH) and SNP genotyping
arrays.
An adequate capture of SVs can be obtained with the analysis of sequence reads
from both ends of contiguous DNA fragments that are few hundred base pairs long
(paired-reads) or from captured distal ends of larger fragments (mate pairs). Long
mate paired-reads are more sensitive for detecting large events, although they have
a broader size distribution and therefore have less resolution for breakpoint
localization. The analysis of the separation distance of the paired-reads with
respect to the reference genome enables the identification of genomic insertions
and deletions. In addition and unlike microarrays, 2ndGS can also provide data on
copy neutral variations such as translocations and inversions by searching for read
pairs with ends mapping to different genomic locations or in opposite orientation,
respectively (Fig. 2). The formation of chimeric clones during the library prepa-
ration and the sequencing errors resulting in incorrectly mapped reads are the
major limitations (Medvedev et al. 2009).
In a massively parallel sequencing experiment with several millions of reads,
the number of sequences aligning to a genomic region is proportional to the
number of times this region appears in the genome. Therefore, CNVs can be
detected at high sensitivity by determining the depth of coverage across the
genome after correcting for differences in GC content and uniqueness across the
genome. Reports on WGS of individual genomes have identified several thousands
of CNVs which is in several fold more than the numbers that were reported in
array-based studies (Ku et al. 2010).
However, paired-end WGS may be not meaningful for those patients in which
cytogenetic techniques have previously localized the disease-associated chromo-
some rearrangement. In these cases, sequencing of sorted derivative chromosomes or
microdissected chromosomal regions has proven to be a feasible approach for the
characterization of chromosomal breakpoints (Chen et al. 2008; Weise et al. 2010).
3.4 Profiling Epigenetic Modifications
Eukaryote genomes carry relatively stable chemical marks that are added to either
DNA or its packing histones. These epigenetic changes are known to play an
important role in the regulation of gene expression and to contribute to the
understanding of some cancers, congenital anomaly syndromes and other complex
non-neoplastic disorders, including some metabolic and cardiovascular diseases.
2ndGS using slightly modified sample preparation protocols allows for an unbi-
ased exploration of DNA methylation and histone modifications and has sub-
stantially accelerated epigenetic research.
Standard methodology for the detection of cytosine methylation, the most
commonly studied epigenetic modification, is based on bisulfate treatment that
converts the cytosine residues to uracils while leaving 50 -methylcytosines
unchanged. Bisulfite Sequencing (BS-Seq) combines bisulfite treatment with
2ndGS technology, providing a digital measure of the frequency that a cytosine is
methylated (Lister and Ecker 2009) (Fig. 2e). Other methods such as reduced
representation BS sequencing or MeDIP sequencing allow saving some costs by
targeting only CpG-rich or highly methylated regions, respectively. In the near
future, novel single-molecule sequencing approaches, such as the ones being
developed by Pacific Biosciences (www.pacificbiosciences.com) and Oxford
Nanopore Technologies (www.nanoporetech.com), will directly detect 5-methyl-

cytosine (mC) and 5-hydroxymethylcytosine (hmC) without prior bisulfite treat-
ment thus enabling comprehensive epigenome mapping.
Post-translational covalent modifications of histone tails, including methylation,
acetylation and phosphorylation, can be characterized by chromatin immunopre-
cipitation with an antibody that specifically recognizes the histone variant and
characterization of the bound DNA by massive parallel sequencing (ChIP-Seq)
(Park 2008). Follow-on bioinformatic analysis enables the genome-wide charac-
terization of the binding sites of the histone of interest with high precision and
provides new insights into how eukaryotic genomes are organized.
Several consortium projects such as the Human Epigenome Project (HEP,
www.epigenome.org) and the Encyclopedia of DNA Elements (ENCODE) aim to
produce reference epigenome maps for a variety of human cells. Lister et al. (2009)
provided the first complete methylation map, also called the methylome, of two human
cell types and interpreted this data in relation to mRNA expression levels. Several other
groups are currently using 2ndGS technologies to study the influence of cytosine
methylation and histone modifications in developmental stages and disease states.
3.5 Transcriptome Sequencing
2ndGS technologies have been proven to be very useful for sequencing RNA
(RNA-Seq) and are currently replacing microarray-based methods. They not only
provide a digital overview of gene expression profiles but also enable the char-
acterization of splice variants, antisense transcription, fusion genes, allele-specific
expression, RNA editing and other forms of sequence variation in the transcribed
portion of genes (Fig. 2f).
In RNA-Seq, the expression level of a gene is deduced from the total number of
reads each gene sequence appears and normalized by the length of the gene. Low
abundant transcripts that would escape detection by microarray-based methods can
be detected by increasing sequencing depth. In this regard, short read sequencing
technologies provide much deeper coverage at lower cost although they may be
problematic when characterizing novel exon junctions (Morozova et al. 2009).
Aberrant transcriptional events resulting from chromosomal rearrangements
play a causative role in many human cancers. In recent years several studies have
successfully used paired-end RNA-Seq to detect fusion transcripts in cancer at
single-nucleotide resolution (Maher et al. 2009; Zhao et al. 2009). In addition,
sequencing the transcriptome is an alternative to exome sequencing for identifying
expressed genetic variants when performed in the appropriate tissue type (Cirulli
et al. 2010). For example, a recurrent somatic mutation in FOXL2 gene has been
found in granulosa-cell tumors using this approach (Shah et al. 2009).
A related application of 2ndGS technologies is the discovery and profiling of
non-coding RNAs (ncRNA) in order to elucidate their role in health and disease.
ncRNAs are small RNA molecules involved in post-transcriptional regulation of
gene expression that have been suggested to modulate parameters such as disease
onset, severity or progression. The short length of ncRNAs makes 2ndGS plat-
forms ideal for ncRNA characterization on a genome-wide basis, as illustrated by a
recent study by Uziel et al. (2009) that demonstrated how miRNA overexpression
collaborates with the Sonic Hedgehog pathway in medulloblastoma. Similarly,
other groups have reported the involvement of specific microRNAs in the etiology
of ovarian and breast cancer that could be used as biomarkers for tumor classifi-
cation and prognosis (Wyman et al. 2009; Nygaard et al. 2009).
3.6 Identification of Infectious Agents
2ndGS has also been used for the rapid identification of pathogens in chronic as
well as acute disease replacing conventional Sanger sequencing methods. This is
typically accomplished by extracting all nucleic acids from the sample of interest,
sequencing the complex DNA or RNA mixture and then assembling the non-
human sequence reads derived from the infectious agents. In 2008, a new arena
virus and a new polyomavirus were associated with febrile illness after visceral
organ transplantation and Merkel cell carcinoma, respectively, by sequencing
RNA from affected tissues (Palacios et al. 2008; Feng et al. 2008).
For known pathogens, the high sensitivity of 2ndGS has proven to be ideal to
detect rare variants that might confer to the pathogen some resistance to treatment
or other virulence markers. Given the high throughput of second GS technologies,
hundreds of samples individually tagged with a molecular barcode can be analyzed
simultaneously. This approach has been used to identify drug-resistant HIV strains
in patients at the earliest stages (Hofmann et al. 2007).
The Human Microbiome project (http://nihroadmap.nih.gov/hmp/) aims to
analyze the entire collection of microbes (microbiome) that are present in the inner
and outer surfaces of our body, including the skin, the oral cavity, the vagina and
the intestine, and analyze its role in human health and disease. It uses a metage-
nomic approach that allows the study of native microbial or viral communities
with no initial cloning steps combined with the pyrosequencing technology that
provides with long ([300 bp) and highly accurate reads ([99.5%) (MacLean et al.
2009). In the near future, sequencing the microbiome at intermediate stages from
health to disease will greatly increase our understanding of some human conditions
such as obesity and cancer. In addition, WGS of the host genomes will allow us to
investigate the genetic basis of susceptibility to microbial infection.
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Rare Genomic Deletions and Duplications
and their Role in Neurodevelopmental
Disorders
Joseph T. Glessner, John J.M. Connolly and Hakon Hakonarson
Abstract Copy number variations (CNVs) are deletions and duplications of DNA
sequences that vary in length from a few base pairs to several million. While
these structural variations are often benign, they can disrupt vital biological
functions and result in disease. CNVs have been identified as causal in a number
of neurodevelopmental disorders (NDs), including but not limited to, autism,
attention-deficit/hyperactivity disorder (ADHD), and schizophrenia. Here, we
examine CNV research into these disorders, and discuss relevant methodological
considerations. By identifying specific rare deletions and duplications, we may be
better able to determine the etiology of neurodevelopmental disorders and identify
appropriate treatments.

Keywords Copy number variation Neurodevelopmental disorder Autism

Schizophrenia Attention deficit hyperactivity disorder
J. T. Glessner J. J.M. Connolly H. Hakonarson (&)

Center for Applied Genomics,
The Children’s Hospital of Philadelphia,
Philadelphia, PA 19104, USA
e-mail: hakonarson@email.chop.edu
H. Hakonarson
Division of Genetics,
The Children’s Hospital of Philadelphia,
H. Hakonarson
Department of Pediatrics,
University of Pennsylvania School of Medicine,

DOI: 10.1007/7854_2011_179
346 J. T. Glessner et al.
Contents
1 Introduction........................................................................................................................ 346
2 Methodological Considerations......................................................................................... 346
2.1 CNV Detection and Validation ................................................................................ 346
2.2 Interpretation ............................................................................................................. 350
3 Large Constitutional Deletions and Duplications in Neurodevelopmental Disease ....... 351
4 Common Neurodevelopmental Disorders......................................................................... 353
4.1 Autism ....................................................................................................................... 354
4.2 Schizophrenia............................................................................................................ 354
4.3 ADHD ....................................................................................................................... 355
5 Conclusion ......................................................................................................................... 356
References................................................................................................................................ 357
1 Introduction
NDs are present in approximately 6% of children (Polanczyk et al. 2007;

Newschaffer et al. 2007; Arajärvi et al. 2005), require lengthy evaluation, can be
difficult to treat, and contribute significantly to overall disease burden and healthcare
costs. While the causes of NDs remain largely unknown, recent research efforts have
identified CNVs as potentially important pathogenic factors. In the past several
years, techniques used to identify CNVs have advanced considerably, making it
easier to identify structural variations. Two common techniques for detecting CNVs
include 1) using microarray assays from single nucleotide polymorphism (SNP)
genotypes and 2) using comparative genomic hybridization (CGH) arrays based on
intensity data to identify contiguous probes that signal CNV events. The improved
sophistication of whole-genome discovery approaches necessitates a renewed
emphasis on rigorous statistical oversight and validation methods. In the section
below, we discuss some of the challenges present in studying CNVs, while later we
focus on these structural variations in relation to specific NDs.
2 Methodological Considerations
2.1 CNV Detection and Validation
Identifying CNVs using high-density microarrays requires high-quality genotype

signals. Factors that help validate the genome signal include increasing the pro-
portion of SNPs in genotype regions, minimizing the standard deviations of
intensity signals, monitoring the number of CNVs putatively associated with given
loci (a high number of CNVs may indicate erroneous identification), controlling
population substructures, and validating calls by using an alternate method (e.g.
quantitative PCR or multiplex ligation-dependent probe amplification (MLPA)).
Rare Genomic Deletions and Duplications 347
Table 1 Deletions and duplications associated with neurodevelopmental disorders

Locus CNV Disease Gene
Type
22q11.2 Del/Dup DiGeorge syndrome and velocardiofacial TBX1+
syndrome
17p13.3 Del Miller–Dieker syndrome LIS1 +
17q11.2 Del/Dup Neurofibromatosis type 1 NF1 +
Xq22 Del/Dup Pelizaeus–Merzbacher disease PLP1
15q11–q13 Del Prader–Willi syndrome/Angelman syndrome UBE3A +
15q11–q13 Dup Autism UBE3A +
17p11.2 Del Smith–Magenis syndrome RAI1 +
17p11.2 Dup Potocki–Lupski syndrome RAI1 +
7q11.23 Del/Dup Williams syndrome (WS) many
11q14.2–14.3 Del Attention-Deficit/Hyperactivity Disorder GRM5
3p26.1 Del Attention-Deficit/Hyperactivity Disorder GRM7
22q13 Del Autism SHANK3
Xp22.33 Del Autism NLGN4
16p11.2 Del Autism many
4q28.3 Del Autism PCDH10
3q24 Del Autism NHE9
15q11–q13 Dup Autism many
2p16.3 Del Autism NRXN1
3p26.3 Del/Dup Autism CNTN4
22q11.21 Dup Autism TBX1+
6q26 Del Autism PARK2
1q25.2 Dup Autism RFWD2, PAPPA2
7p14.3 Dup Autism AK057321
3q13.33 Dup Autism FBXO40, GOLGB1
2p24.3 Dup Autism AK123120
3p26.2 Del Autism UNQ3037
10q23.2 Del Autism GRID1
3q26.31 Dup Autism NLGN1
4q31.21 Dup Autism GYPELOC441046
3q13.3 Dup Bipolar disorder GSK3B
Xq28 Del Rett syndrome MECP2
2q34 Del Schizophrenia ERBB4
5p13.3 Del Schizophrenia SLC1A3
2q31.2 Del Schizophrenia RAPGEF4
12q24 Dup Schizophrenia CIT
16q22.1 Del Schizophrenia PDPR
16p11.2 Dup Schizophrenia QPRT, DOC2A,
TBX6+
22q11.21 Del Schizophrenia COMT, TBX1+
9q34.3 Del Schizophrenia CACNA1B
10q11.21 Del Schizophrenia RET
4p16.1 Del Schizophrenia WDR1
18q12.3 Del Schizophrenia RIT2, PIK3C3
3p26.2 Del Schizophrenia SUMF1
Xp22.33 Del Tourette’s syndrome NLGN4
Another important validation measure is to limit signal intensity waves—gen-

ome-wide artifacts of signal intensities that alternately yield inflated or deflated
signals (Marioni et al. 2007). Typically, these correlate with genomic content (total
guanines (Gs) and cytosines (Cs) per total of the four bases) and require the impo-
sition of wave-correction models. In a study of IlluminaÒ’s BeadStudio software, we
previously demonstrated that approximately 10% of DNA samples from commercial
cell line repositories show visually discernable wavy patterns (Diskin et al. 2008).
Because CNVs are typically inferred from gains and losses of intensity signals, the
imposition of wave-correction models are critical for accurate detection.
CNV detection algorithms, including the methods and options they implement,
are still under development and debate—although PennCNV (Wang et al. 2007)
and BirdSuite (Korn et al. 2008) have gained popularity. Similarly, while Illu-
minaÒ (San Diego, CA) (Steemers and Gunderson, 2007) and AffymetrixÒ (Santa
Clara, CA) (Fodor et al. 1991) genome-wide arrays are widely used, the actual
array content is constantly evolving, and new versions and probe designs make the
task of integrating datasets a difficult one (Fig. 1). The CNV signal can vary
depending on the type of source sample used (e.g. cell lines or saliva), batch
effects, equipment variation, reagent variation, and the approach taken to signal
normalization. All of these factors can introduce disparities that need to be
modeled and statistically corrected.
Some individuals with associated CNVs may not show any notable phenotypic
variation. This indicates a threshold effect or a possible second-hit modifying
mutation. The second hit is extremely hard to determine given the rarity of the initial
CNV, which limits the sample size. At rare CNV frequencies, small numbers of
controls with a given CNV quickly dilute significance tests versus CNVs not detected
in a large control cohort. It should also be noted that differences in the number of
statistical tests being performed on CNV regions mean that classical multiple-test
statistical corrections or Bonferroni corrections are difficult to adapt to rare CNV
association. A more practical alternative is to use the significance observed in control-
excess as a model for the null distribution. This model allows us to accept CNV
regions where case-excess is of greater significance as surpassing the null distribution.
Related to this are issues of pleiotropy and/or variable expressivity of the
underlying genes. More dense continuous coverage is needed to confidently call
CNVs, culminating in genome-sequencing technologies. Individual SNPs and their
underlying probe sequences have different efficiencies in differentiating modes of
CNVs. Enrichment for clearly differentiating probes is essential for SNP arrays
with strong CNV detection utility. Nevertheless, rare CNVs may be so rare that
individual labs evaluating their own dataset may lack the requisite power to reach
confident association. Collaboration and data sharing are important to boosting
marginally significant signals. Collaboration is becoming more widespread based
on collegial agreements and a trend among funding agencies to stipulate that raw
data be deposited in the public domain.
The development of public datasets such as the rapidly growing dbGaP has the
added benefit of facilitating replication studies, which are critical to validation.
Existing CNV data can also be integrated into the major public databases.
(a) FISH (b) qPCR
(c) Genome-wide Array
Fig. 1 CNV detection methods and data. A Two-color FISH shows large CNV, deletion shown.
B Based on a control value, a ratio of observed to control for specific loci signal CNV. C SNP
arrays or aCGH assay contiguous SNPs or CN probes are bound to the genome allowing for
detection of variations from normal diploid
Genome-wide association remains focused on SNP genotypes by the current

definition, but the underlying principles can be incorporated into copy number
variations. The central databases for disease association results such as Online
Mendelian Inheritance in Man (OMIM) and NHGRI: A Catalog of Published
Genome-Wide Association Studies largely exclude CNV association results.
While the CNV event is certainly more difficult to represent, a central consensus
region SNP ID could be very analogous to reporting significant SNPs. Single loci
reporting is complicated by factors that include varying confidence of specific
calls, association, and marginal success in replication (due to rarity of individual
variants). Because of the lack of CNV entries in disease association resources,
such a query becomes a large primary literature search. An alternative is to present
a gene network of related function or interaction of around 100 genes. Further
abstract is the idea that psychiatric patients have a CNV burden in genic regions,
meaning there are more large rare CNVs in psychiatric patients than controls.
The primary catalog for CNVs is currently the Database of Genomic Variants
(projects.tcag.ca/variation/), which is also integrated into the UCSC browser.
There are key caveats for interpreting this data. Firstly, a variety of array platforms
and CNV-calling algorithms are used. Secondly, the subjects are part of studies
which typically focus on a specific phenotype. By virtue of being de-identified for
research purposes, they do not have a complete phenotype to apply to other disease
studies. Thirdly, acquiring the source sample to validate the CNV with the array
and calling algorithm from an alternate laboratory is not straightforward and is
often unsuccessful. Since 2004, papers describing genome-wide maps of copy
number variation have revealed that CNVs and segmental duplications are very
abundant features of the human genome.
Given the massive amounts of CNVs that can be detected and specifically assayed,
collections of CNV-specific markers such as copy number polymorphisms (CNPs)
can be developed. Similar to SNPs, CNPs are assumed to be common in above 1%
population frequency. Although the hypothesis of common disease explained by
common variants makes intuitive sense, mounting support for common disease rare
variants in the form of CNVs are being found associated with NDs.
2.2 Interpretation
When CNVs are observed in a parent but not in a child, this may indicate an error
in the CNV call. The converse is not necessarily the case, and de novo CNVs have
been strongly associated with a number of NDs (e.g. Sebat et al. 2007). De novo
CNVs may be particularly informative for pronounced NDs such as brain mal-
formations, where the parent is often annotated as healthy. De novo mutations may
also be of special relevance to heterogeneous NDs such as autism and ADHD,
where etiologies are complex and a specific disease label can have very different
phenotypes and combinations (Geschwind and Levitt 2007). Further, CNVs may
have prospective diagnostic value in differentiating behaviorally similar NDs. In
certain cases, the parent may have an attenuated ND or an additional protective
variant, which has obvious implications for treatment. Rare private mutations in
certain families are expected from a population genetics perspective without
detrimental effect. This necessitates observation of recurrent rare CNVs in unre-
lated individuals from diverse geographic and ethnic populations, which can be
detected by different array technologies.
Proximal to CNV regions, highly homologous low-copy-number repeats (LCRs)
are frequently observed, which can predispose to recombination between non-allelic
LCRs (non-allelic homologous recombination) (Britten and Davidson, 1976; Lupski
1998). Evolution and genome condensation occur through various mechanisms,
including chromosome splicing of highly similar sequences known as homologous
recombination (HR). In somatic cells, HR is needed to repair extreme DNA damage
such as double strand breaks (DSB). If spliced incorrectly, CNVs and genomic
instability can result. An intermediate state is formed between two DNA strands,
which proceeds by crossover (two-way sharing, meiosis, and DSB) or gene
conversion (one-way sharing, DSB). The human genome has much segmental
duplication, which provides similar sequences for HR to occur. HR may expand
gene families with diverse functions but also leads to more mistakes. Segmental
duplications can masquerade as allelic sequences during meiosis and lead to
erroneous splicing, termed non-allelic homologous recombination. Gene conversion
can result in inserts of non-expressed poor function elements into homologous
expressed genes. Segmental duplications are also referred to as low-copy repeats.
Non-homologous end-joining (NHEJ) (Moore and Haber, 1996), fork stalling, and
template switching (FoSTeS) (Lee et al. 2007) can result in less typical CNV generation.
The widespread and, in some regions, high prevalence of CNVs is somewhat
counterintuitive for such an aberration from the classical genetics of a maternal
and paternal diploid chromosome set. However, it is likely that CNVs may con-
tribute to evolutionary mechanisms, whereby gene families with homologous
sequences are extended into complementary functions over time. Perhaps CNVs
involving genes responsible for behavior, cognition, and psychological state are
most active in humans representing a successful expansion of gene functions based
on ancestral duplications.
3 Large Constitutional Deletions and Duplications

in Neurodevelopmental Disease
Early studies with chromosomal karyotypes and fluorescent in situ hybridization

(FISH) were limited in their ability to detect only the largest CNVs, but increased
resolution of SNP microarrays and DNA sequencing technologies have made
smaller CNVs far easier to identify. Large CNVs inevitably have a greater like-
lihood of disrupting crucial functional elements but examples exist of small
targeted CNV that can cause phenotype variation. Certain CNVs can give rise to
very specific phenotypes while others may have variable expressivity. Some genes
may have complementary function to other genes such that deletion of one gene
homolog does not result in significant phenotypic variation.
Perhaps the most familiar CNV linked with ND involvement is trisomy 21 or

Down syndrome, which results from the duplication of an entire chromosome 21
(Lejeune et al. 1959). Symptoms include distinct facial features, as well as delayed
mental and social development. The CNV variant is highly penetrant since the
duplication is directly associated with a specific phenotype.
Williams syndrome is associated with a deletion of 7q11.23 and is characterized
by a distinctive facial appearance, visuospatial deficits, learning disorders, ADHD,
and hypersociability (Francke 1999; Fishman et al. 2010) (Table 1).
Deletion and duplication of 15q11–q13 have been described with variable
expressivity based on maternal or paternal inheritance. Maternal 15q11–q13
deletion results in Angelman syndrome (AS), which is characterized by devel-
opmental delay, sleep disturbance, seizures, jerky movements (especially
hand-flapping), frequent laughter or smiling, and a happy demeanor (Greenberg
et al. 1991). AS is caused by the under-expression of UBE3A, whose protein
product E6-AP is involved in the transfer of ubiquitin molecules for degradation
(Kishino et al. 1997; Matsuura et al. 1997). Paternal deletion of 15q11–13 causes
Prader–Willi syndrome (PWS), which is characterized by hypotonia in infancy,
hypogonadotrophic hypogonadism, facial dysmorphism, obesity, short stature, and
cognitive and behavioral impairments (Cassidy, Dykens, and Williams, 2000).
The microdeletions associated with PWS all share a 4.3 kb deleted region, which
includes the promoter region, first exon, and part of the first intron of the
SNRPN gene (Rodriguez-Jato et al. 2005). A number of phenotypes have also
been associated with duplication in the 15q11–13 region. Hypotonia, language
and motor delay, epilepsy, facial dysmorphism, and cognitive and learning
impairments are key features of 15q duplication syndrome. Maternal 15q11–q13
duplication results in autism, while paternal duplication often is of no effect or
slight developmental delay (Gillberg et al. 1991). These same features are asso-
ciated with 15q trisomy (Chamberlain and Lalande, 2010).
Miller–Dieker syndrome (MLS) is associated with deletion of 17p13.3
impacting the LIS1 gene (Dobyns et al. 1993). Features of MLS include lissen-
cephal (smooth brain), mental retardation, and facial dysmorphism (Schiff et al.
2010). Other than brain malformations, many patients also present epilepsy,
spastic behavior, and shortened life expectancy. Larger deletions incorporating
proximal genes result in more severe phenotypes, indicating the involvement of
long range regulation or epistatic interaction between genes.
Another example is the 22q11.2 deletion that causes a multi-systemic disorder.
Many patients with the deletion have a learning disorder, congenital heart defects,
a malformed palate, mild facial abnormalities, schizophrenia, and sensitivity to
infections (Liu et al. 2002). The 22q11.2 deletion syndrome (also known as
velocardiofacial syndrome or DiGeorge syndrome) is caused by a microdeletion in
chromosome 22. The phenotype is variable and can include facial dysmorphism,
cardiovascular abnormalities, short stature, cognitive and behavioral impair-
ments, and a high risk of schizophrenia (Karayiorgou et al. 2010). Genes in the
22q11.2 region include COMT (Shashi et al. 2006) and TBX1 (Paylor et al.
2006). Duplication of 22q11.2 also has variable clinical presentations, including
developmental delay, dysmorphic facial features, autism, and cognitive and

behavioral impairments (Portnoï 2009). Additional structural variation of this
locus includes (11;22) translocation causing the der(22) syndrome and (2) the
formation of a supernumerary chromosome resulting in Cat-Eye syndrome. Eight
different LCRs are located in proximal 22q, and are presumably susceptible to
homologous recombination events and promote nonallelic homologous recombi-
nation (Shaikh et al. 2001). The 22q13 deletion causes Phelan–McDermid syn-
drome, characterized by pervasive developmental disorders, speech delay, and
hypotonia (McDermid and Morrow, 2002).
Rett syndrome (RTT), which is found almost exclusively in females, is asso-
ciated with deletion of Xq28 including the MECP2 gene. Duplications of MECP2
were found in males exhibiting severe intellectual disability, and autistic features
(Ramocki et al. 2010). MECP2 appears to be the singularly important gene that is
impacted by the Xq28 CNV.
Pelizaeus–Merzbacher disease is associated with PLP1 duplication and disease
is characterized by coordination, motor abilities, and intellectual function deteri-
oration, which were described as early as 1885 (Pelizaeus 1885). Xq26.2–q27.1
duplication impacting the SOX3 gene was identified in females in a family with
language impairments that included stuttering and dyslalia, short height, and
dysmorphic features. Deletions on chromosome X may be due to submicroscopic
X-to-autosome translocation, Alu–Alu recombination, and non-homologous end-
joining (Inoue et al. 2002).
Thus, large CNVs are high confidence calls that can be detected on different
resolution arrays. These large CNVs often encompass many genes and in turn
cause multi-systemic syndromes rather than singular features. As such, they are
straightforward to detect with available technology but in the majority of cases the
actual critical gene(s) or variant(s) within the CNV, or the micro level instability,
remains to be elucidated.
4 Common Neurodevelopmental Disorders
Taken together, multiple rare CNVs have been implicated in the pathogenesis of
NDs. However, a few common variant models have also been implicated in pre-
disposing to several of the NDs, including autism and schizophrenia (Wang et al.
2009; International Schizophrenia Consortium 2009). While common SNP geno-
types have been implicated to a greater degree in inflammatory and metabolic
disorders, such as type 1 diabetes, inflammatory bowel disease and type 2 diabetes,
it is conceivable that with greater sample sizes that many more common SNP
variants will be uncovered that predispose to ND susceptibility. In addition,
common CNVs are also hypothetically possible but current methods often under
call their true frequency. Thus, rare CNVs have been most successful in NDs
lending support to a common disease-rare variant model that is disparate from the
more typical common disease-common variant model, previously implicated by
many (Schork et al. 2009).
4.1 Autism
A potentially efficient approach to identifying CNVs in neurodevelopmental dis-

orders is to target functional gene clusters of associated loci. Previously reported
ASD candidate genes, such as NRXN1 (Kim et al. 2008) and CNTN4 (Roohi et al.
2008; Fernandez et al. 2008) have been independently replicated by Glessner et al.,
Glessner et al. 2009. We also identified several new susceptibility genes encoding
neuronal cell-adhesion molecules, including NLGN1 and ASTN2, which were
enriched with CNVs in ASD cases. Moreover, CNVs within or surrounding genes
in ubiquitin pathways—UBE3A, PARK2, RFWD2, and FBXO40—were observed
in cases but not in controls. Duplications 55 kilobases upstream of complementary
DNA AK123120 were identified (Glessner et al. 2009). Using a *550,000 SNP
array, we observed an average, 15.5 CNV calls for each individual, with a similar
frequency observed in cases and controls.
Recently, Pinto et al. (2010) identified inherited and de novo CNVs associated
with 25 genes that were enriched in a large ASD sample. These include SHANK2,
SYNGAP1, DLGAP2, and the DDV53-PTCHD1 locus. Interestingly, the group also
found that individuals with ASDs have neither significantly more, nor significantly
larger, CNVs than matched controls. However, they are more likely to have CNVs
in genic regions, particularly in loci previously associated with ASDs and/or
intellectual disability. This is important because it replicates our previous finding
that CNV frequencies are comparable for cases and controls. This underscores the
point that individuals with NDs may not be any more prone to duplications or
deletions per se, but more importantly to CNVs in specific coding regions. The
localization of CNVs at genes linked with intellect is also theoretically significant
in that it suggests a putative link between the two (Skuse 2007). CNVs impacting
DPP6, DPP10, and PCDH9 were also associated with ASDs (Marshall et al.
2008). CNVs on 16p11.2 and genotype association on 5p15 (SEMA5A/TAS2R1)
were also found in autism (Weiss et al. 2008; Weiss and Arking 2009).
4.2 Schizophrenia
We recently used 1.7 million probes to perform a whole-genome CNV analysis on

a cohort of 977 schizophrenia cases and 2,000 controls, validating positive findings
in an independent cohort of 758 schizophrenia cases and 1,485 controls (Glessner
et al. 2010). The gene ontology synaptic transmission family of genes was notably
enriched for CNVs in the cases (P = 1.5 9 10-7). Among these, CACNA1B and
DOC2A, both calcium-signaling genes responsible for neuronal excitation, were
deleted in 16 cases and duplicated in 10 cases, respectively. In addition, RET and
RIT2, both ras-related genes important for neural crest development, were sig-
nificantly affected by CNVs. RET deletion was exclusive to seven cases, and RIT2
deletions were overrepresented in the schizophrenia cases. Similar to the ASD
studies above, rare CNVs were not overrepresented in cases versus controls. This
is largely consistent with the literature, though (Walsh et al. 2008) found novel
large rare deletions and duplications of genes observed in 15% of cases versus 5%
of controls. A study of CNVs in Chinese schizophrenia patients detected no sig-
nificant difference in rare CNVs between cases and controls (Shi et al. 2008).
Another study of 1,013 cases and 1,084 controls of European ancestry also failed
to find more rare CNVs [100 kb in cases or enrichment for neurodevelopmental
pathways (Need et al. 2009).
Previous studies have associated various CNVs with schizophrenia, including
deletions of 22q11.2 (Liu et al. 2002), NRXN1 (Kirov et al. 2007), APBA2 (Kirov
et al. 2007), and CNTNAP2 (Friedman et al. 2008). However, each of these CNVs
is rare and they account for a relatively small proportion of the overall genetic risk
in schizophrenia. Large rare CNVs impacting many different genes enriched in
neurodevelopmental pathways have been reported elsewhere (Walsh et al. 2008;
International Schizophrenia Consortium 2008; Stefansson et al. 2008). Specific
loci exhibiting runs of homozygosity (ROHs) have also been associated with
schizophrenia (Lencz et al. 2007), and de novo CNVs (P = 7.8 9 10-4) were
recently observed in sporadic schizophrenia cases in comparison with controls
(Xu et al. 2008). Comparison of genomic findings in schizophrenia and autism has
suggested a diametric etiology (Crespi et al. 2010).
4.3 ADHD
Attention-deficit/hyperactivity disorder (ADHD), the most common neuropsychi-

atric disorder in children, with a 5.2% worldwide prevalence, causes significant
academic, behavioral, and social impairment throughout the life span (Polanczyk
et al. 2007). The phenotype consists of extreme manifestations of continuous traits,
including hyperactivity, impulsivity, and dysfunction in executive and self-regu-
latory skills that involve working memory, temporal organization, planning,
organizing, maintaining focus, effort, and motivation (Elia and Devoto 2007).
Genome-wide association studies of ADHD including CNVs have been few
despite high prevalence.
Specific genetic factors underlying risk for ADHD remain elusive. To assess the
role of structural variation in ADHD, we previously identified 222 inherited copy
number variations (CNVs) within 335 ADHD patients and their parents that were
not detected in 2,026 unrelated healthy individuals. (Elia et al. 2010). Again, no
excess CNVs, either deletions or duplications, were found in the ADHD cohort
relative to controls. Inherited rare CNVs impacted candidate genes of autism,
schizophrenia, and Tourette syndrome, including A2BP1, AUTS2, CNTNAP2, and
IMMP2L. The ADHD CNVs enriched in cases were related to psychological
and neurological functions, including learning, behavior, synaptic transmission,
and central nervous system development. A deletion within the glutamate receptor
gene, GRM5, was found in an affected parent and all three affected offspring.
GRM7 deletion was also observed in cases and not in controls (Elia et al., 2010).
Rare inherited structural variations play an important role in ADHD development
and indicate a set of putative candidate genes for further study in the etiology of
ADHD.
5 Conclusion
In summary, both common and rare variants have been implicated in NDs. While
considerable progress has been made over the past few years in unveiling the
genetic causes of NDs, genome-wide studies explain roughly 10–15% of the
heritability of NDs. A recent simulation study suggests that some of the ‘missing’
heritability might be accountable for synthetic associations—a term used to
describe the relationship between a (common) causal variant and a (rarer) non-
causal variant. Dickson et al. (2010) simulated output from a genome-wide
association study (GWAS), where some individuals had rare variants (0.5–2% of
the population) that likely caused a disease. They found that the GWAS technique
erroneously identified the synthetically associated variant as causal, essentially
obscuring the signal from the rarer ‘‘at risk’’ variant. In other words, synthetic
associations infer that many people share a variant that confers some small risk of
disease. In fact, only a few of these individuals share a rarer variant that carries a
much higher risk.
CNV studies, which are sensitive to specific rare deletions and duplications,
provide a potential route around this problem. However, as noted above, this
approach is relatively insensitive to second-hit variants because of the limits of
sample size. A combination of the two approaches is arguably the most attractive.
The explosion of GWAS publications in the past several years has generated
numerous loci putatively associated with NDs. Perhaps the most important repli-
cation that can be made without the need for genotyping multitudes of additional
samples is in functional gene clusters of associated loci. This also boosts confi-
dence in association signals of rare CNVs, which singularly may have arisen by
chance. The CNVs were recently identified at CNTN4 and NRXN1 have well-
established associations with autism (Glessner et al. 2009).
Some of the missing heritability should also disappear as arrays with increas-
ingly powerful resolutions are developed. Although much progress has been made
to this end, further resolution of data is needed to gain confidence in CNV calls,
and to establish clear breakpoints. Forty-two million probes have already been
achieved with genome-tiling set of multiple arrays (Conrad et al. 2010). The best
resolution, of course, is sequencing the genome for every contiguous base, which
has also been investigated (Kidd et al. 2008).
Many of the issues allied to structural studies will doubtless be resolved by
technological advances. Nevertheless, additional complexity in the CNV theme
exists in mosaicism, translocations, and inversions. Neither translocations nor
inversions can be detected with a SNP array, and clone paired-end sequencing
(fosmid) and sequence-assembly comparison are the only genome-wide technol-

ogies that can detect these structural variants (excepting targeted methods such as
FISH and Southern blotting). Other challenges are organizational, and these
include issues of data-integration, sharing, and management, all of which have
been touched-on above.
With the ability to recognize sequence-level variation already a diagnostic
reality, the necessity of quickly dissociating benign and disease-causing changes
will become more pressing. Rett syndrome, a ND on the autism spectrum, is one
example of a specific microdeletion associated with a specific phenotype. Similar
associations are likely to emerge from the CNV field over the coming years.
Similarly, family based studies may help to differentiate de novo and inherited
forms, and may ultimately help identify protective variants.
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Toward a Mechanistic Understanding
of How Variability in Neurobiology
Shapes Individual Differences in Behavior
Ryan Bogdan, Justin M. Carré and Ahmad R. Hariri
Abstract Research has begun to identify how variability in brain function

contributes to individual differences in complex behavioral traits. Examining
variability in molecular signaling pathways with emerging and established meth-
odologies such as pharmacologic fMRI, multimodal PET/fMRI, and hormonal
assays are beginning to provide a mechanistic understanding of how individual
differences in brain function arise. Against this background, functional genetic
polymorphisms are being utilized to understand the origins of variability in sig-
naling pathways as well as to efficiently model how such emergent variability
impacts behaviorally relevant brain function and health outcomes. This chapter
provides an overview of a research strategy that integrates these complimentary
levels of analysis; existing empirical data is used to illustrate the effectiveness
of this approach in illuminating the mechanistic neurobiology of individual dif-
ferences in complex behavioral traits. This chapter also discusses how such efforts
can contribute to the identification of predictive risk markers that interact with
unique environmental factors to precipitate psychopathology.
R. Bogdan (&) J. M. Carré A. R. Hariri

Laboratory of NeuroGenetics, Department of Psychology & Neuroscience,
Institute for Genome Sciences & Policy, Duke University,
417 Chapel Drive, Durham, NC 27708, USA
e-mail: bogdan.ryan@gmail.com
J. M. Carré
Laboratory of Social Neuroendocrinology, Department of Psychology,
Wayne State University, Detroit, MI 48202, USA
e-mail: justin@carrelab.com
A. R. Hariri
e-mail: ahmad@haririlab.com

DOI: 10.1007/7854_2011_182
Published Online: 23 March 2012
362 R. Bogdan et al.

Keywords Neurogenetics Amygdala Striatum Threat Reward Aggression
Stress
Contents
1 Introduction........................................................................................................................ 362
2 Trait Anxiety, the Amygdala and Serotonin .................................................................... 364
3 Aggression, the Amygdala and Testosterone ................................................................... 368
4 Impulsivity, the Ventral Striatum and Dopamine ............................................................ 371
5 Endocannabinoids, Threat- and Reward-Related Brain Functions.................................. 373
6 Stress, the HPA Axis and the Mineralocorticoid Receptor ............................................. 377
7 Summary and Future Directions ....................................................................................... 380
References................................................................................................................................ 384
1 Introduction
Individual differences in trait affect, personality and temperament critically shape

complex human behaviors, successfully navigating social interactions and
overcoming challenges from our ever changing environments. Such individual
differences may also serve as important predictors of vulnerability to psychopa-
thology including depression, anxiety, addiction, and antisocial personality disor-
der, especially upon exposure to environmental adversity. Accordingly, identifying
the biological mechanisms which give rise to trait individual differences affords
unique opportunity to develop a deeper understanding of complex human behaviors,
disease liability and treatment. Having established multiple modal neural processes
supporting specific aspects of complex social behavior, research has now begun to
reveal the neural substrates of inter-individual variability in these and related
constructs. Moreover, recent studies have established that these neural substrates
represent temporally stable and reliable indices of brain function. Thus, much like
their behavioral counterparts, brain function represents an enduring, trait-like
phenomenon, which in and of themselves may serve as important markers of
individual differences as well as disease liability and pathophysiology.
As research continues to illustrate the predictive relationship between brain
activation and trait-like behaviors (e.g., increased amygdala reactivity predicts trait
anxiety), an important next step is to systematically identify the underlying
mechanisms driving variability in brain circuit function. In this regard, neuroim-
aging studies employing pharmacologic challenge paradigms, principally targeting
monoamine neurotransmission and hormonal systems, have revealed that even
subtle alterations in dopaminergic, noradrenergic, serotonergic and neuroendo-
crine signaling can have profound impact on the functional response of brain
circuitries supporting affect, personality and temperament. Similarly, multimodal
neuroimaging approaches have provided evidence for directionally specific
Toward a Mechanistic Understanding 363
relationships between key components of monoaminergic signaling cascades,

assessed with radiotracer positron emission tomography (PET), and brain function,
assessed with BOLD fMRI. Collectively, pharmacological challenge neuroimag-
ing and multimodal PET/fMRI are revealing how variability in behaviorally
relevant brain activation emerges as a function of underlying variability in key
brain signaling pathways (e.g., increased serotonin signaling predicting increased
amygdala reactivity). The next logical step is to identify the sources of inter-
individual variability in these key neurochemical signaling mechanisms.
In the modern era of human molecular genetics, one step is firmly planted in
the direction of identifying common variation in the genes that influence the
functioning or availability of components in these pathways. As DNA sequence
variation across individuals represents the ultimate wellspring of variability in
emergent molecular, neurobiological and related behavioral processes, under-
standing the relationships between genes, brain and behavior is important for
establishing a mechanistic foundation for individual differences in behavior and
related psychiatric disease. Moreover, such genetic polymorphisms can be readily
identified from DNA collected via cells from individual blood or even saliva
samples using relatively well-tolerated, inexpensive and standardized laboratory
protocols. Once collected and isolated, an individual’s DNA can be amplified
repeatedly providing an almost endless reservoir of material for genotyping of
additional candidate polymorphisms as they are identified. When a precise cascade
of related neurobiological and behavioral effects are clearly established, common
polymorphisms can represent incredibly powerful predictive markers of such
emergent properties that are more readily accessible (e.g., samples can be collected
in doctor’s offices), applicable (e.g., even newborns can be genotyped) and
economical (e.g., costing only tens of dollars per sample in comparison to the
hundreds and even thousands required for fMRI and PET) than their technological
counterparts in neuroimaging and neuropharmacology. Of course, arriving at this
ultimate reduction requires intensive and expansive efforts wherein all these
technologies as well as epidemiological and clinical studies are first brought
to bear on explicating the detailed biological mechanisms mediating individual
differences in trait behaviors and related risk for neuropsychiatric disease.
In the last 5 years, significant progress has been made in describing the con-
tributions of multiple common genetic polymorphisms to individual differences in
complex behavioral phenotypes and disease liability—in particular, by identifying
effects of functional genetic variation on the neural processes that mediate
behavioral responses to environmental challenge (Caspi and Moffitt 2006; Hariri
and Holmes 2006). The current chapter will review how the integration of
psychology, neuroimaging, neuroendocrinology, neuropharmacology and molec-
ular genetics can work toward the ultimate goal of understanding the detailed
mechanisms mediating individual differences in human behavior and, in turn,
establish predictive markers of disease vulnerability. The vast potential of such an
integrated approach will be highlighted by reviewing recent studies whose col-
lective results demonstrate that common sequence variation in human genes that
bias key components of molecular signaling cascades results in altered brain
circuit function that mediates individual differences in complex behavioral traits

such as temperamental anxiety, aggression, stress responsiveness and impulsivity.
With their increased utilization and continued expansion each level of analysis in
this integrative strategy—brain circuit function, neural signaling cascades and
molecular genetics—also has the potential to uniquely illuminate clinically rele-
vant information that can be used in efforts to devise individually tailored treat-
ment regimes and establish predictive disease markers. In lieu of further describing
a general framework, five specific examples will be used to illustrate the effec-
tiveness of this integrated strategy to parse biological mechanisms mediating
individual differences in complex behaviors.
Multiple mechanisms involving de novo biosynthesis, vesicular release, active
reuptake, metabolic degradation as well as a myriad of both pre- and post-synaptic
receptors contribute to the regulation of neurotransmission and its subsequent
modulation of brain function. To illustrate the powerful capacity of functional
genetic polymorphisms to model emergent variability in signaling pathways, the
five exemplars below focus on different critical node in regulating the magnitude
of neurotransmission, namely autoregulatory negative feedback, active synaptic
reuptake, post-synaptic receptor binding, intracellular receptor binding, and
enzymatic degradation. In the first example, individual differences in trait anxiety
will be mapped onto threat-related amygdala reactivity. Variability in amygdala
reactivity will, in turn, be mapped to serotonin signaling. Finally, variability in
serotonin signaling will be mapped to a common functional polymorphism
impacting the capacity for negative feedback inhibition of serotonergic neurons in
the midbrain. In the second example, a similar relationship will be described
between variability in aggression, amygdala reactivity, testosterone signaling and a
variable number of tandem repeats in the androgen receptor. The third example
describes variability in impulsivity, reward-related ventral striatum reactivity,
dopamine signaling and a polymorphism impacting synaptic clearance of striatal
dopamine. In the fourth example, a common polymorphism affecting the enzy-
matic degradation of endocannabinoids will be linked to divergent effects on
threat-related amygdala reactivity and reward-related ventral striatum reactivity.
In the fifth and last example, variability in stress-responsiveness and hypotha-
lamic–pituitary–adrenal axis function will be linked to a missense polymorphism
affecting the mechanistic action of the mineralocorticoid receptor.
2 Trait Anxiety, the Amygdala and Serotonin
The experience of anxiety is commonplace amongst both human and non-human

primates as well as other highly social animals. In the context of social interac-
tions, especially within delimited social hierarchies consisting of dominant and
subordinate individuals, anxiety serves to shape appropriate and often opposing
responses to precipitating events such as competition for limited resources (e.g.,
food, water, reproductive partners). Sensitivity to potentially threatening social
cues (e.g., affective facial expressions) varies considerably between individuals

and represents a core component of commonly employed constructs representing
trait anxiety. Individuals with high trait anxiety exhibit a propensity to more
frequently appraise situations as more threatening than do others and are generally
more sensitive to social cues including those representing both explicit and
implicit threat (e.g., angry and fearful facial expressions). In turn, these individuals
are at increased risk for developing psychopathology characterized by abnormal
social and emotional behaviors such as depression and often precipitated by
exposure to chronic or severe stressors. Examining the neural correlates of indi-
vidual variability in dispositional temperament such as trait anxiety represents an
important step in understanding key socioemotional behaviors as well as an
effective means of elucidating pathophysiological processes contributing to related
disordered states.
Converging evidence from animal and human studies clearly demonstrates that
the amygdala is centrally involved in mediating both physiological (e.g., auto-
nomic reactivity) and behavioral (e.g., reallocation of attentional resources) effects
that allow an individual to respond adaptively to varied environmental and social
challenges (LeDoux 2000). A large corpus of human neuroimaging research
reveals that the amygdala is robustly engaged by varied biologically salient
stimuli, most notably emotional facial expressions especially those representing
threat. However, individuals differ appreciably in the magnitude of amygdala
activation on exposure to emotionally expressive facial expressions, and these
individual differences appear to be stable over time (Johnstone et al. 2005; Manuck
et al. 2007). Thus, they may contribute to the emergence of stable differences in
temperament such as trait anxiety.
Recent neuroimaging studies have reported positive relationships between the
magnitude of amygdala reactivity to affective, especially threatening, stimuli and
inter-individual variability in indices of trait (Dickie and Armony 2008; Etkin et al.
2004; Haas et al. 2007; Killgore and Yurgelun-Todd 2005; Most et al. 2006;
Ray et al. 2005) and also state anxiety (Bishop et al. 2004; Somerville et al. 2004).
In one study, Stein et al. (2007) report that high trait anxiety is associated with
greater amygdala reactivity not only to angry and fearful but also happy facial
expressions. Consistent with this pattern of normal variability, various mood and
anxiety disorders (e.g., unipolar and bipolar depression, generalized anxiety disor-
der, social phobia) have been linked with greater amygdala responses to facial
expressions depicting fear and anger, as well as sadness and disgust, and, more
variably, to emotionally neutral facial expressions (Cooney et al. 2006; Evans et al.
2008; Phan et al. 2006; Phillips et al. 2003; Stein et al. 2002; Whalen et al. 2002).
Such findings demonstrate that anxiety-related psychopathology is associated with a
heightened amygdala response to diverse affective stimuli. More importantly, in the
absence of such disorders, variability in the magnitude of threat-related amygdala
reactivity is an important predictor of individual differences in trait anxiety.
Having first established a predictive link between amygdala reactivity and trait
anxiety, factors that drive such behaviorally relevant variability in brain function
can be now be identified in the broader context of detailing the biological
mechanisms mediating individual differences in temperamental anxiety.

Converging preclinical and clinical evidence indicates that amygdala functioning
is sensitive to the effects of central serotonin (Sadikot and Parent 1990), whose
principle forebrain innervation is provided by the midbrain dorsal raphe nuclei
(DRN). Available data from animal studies indicate that relative increases in local
5-HT result in potentiation of amygdala activation and associated behavioral
phenomenon, such as fear conditioning (Amat et al. 1998, 2004; Burghardt et al.
2004, 2007; Forster et al. 2006; Maier and Watkins 2005). As advanced in the
introduction of this chapter, recent neuroimaging studies using multimodal PET/
fMRI or pharmacological challenge BOLD fMRI have provided direct evidence
for parallel effects of 5-HT in humans. Specifically, in vivo PET has revealed that
decreased endogenous capacity for local 5-HT reuptake (Rhodes et al. 2007) is
associated with relatively increased amygdala reactivity. Acute IV administration
of a selective serotonin reuptake inhibitor, which reduces capacity for 5-HT
reuptake, during BOLD fMRI is likewise associated with not only increased
amygdala reactivity but also decreased habituation of amygdala reactivity over
time (Bigos et al. 2008). These data clearly indicate that variability in the regu-
lation of 5-HT signaling is an important source of individual differences in
amygdala reactivity.
Crucial among components regulating 5-HT neurotransmission and its sub-
sequent modulation of brain function is activation of somatodendritic 5-HT1A
autoreceptors, which mediate negative feedback on DRN neurons resulting in
decreased 5-HT release at postsynaptic targets in the forebrain (Sharp et al. 2007).
Using multimodal PET/fMRI, we previously reported that the density of 5-HT1A
autoreceptors accounts for 30–44% of variability in amygdala reactivity in healthy
adults (Fisher et al. 2006), confirming the important role of 5-HT1A autoreceptors
in modulating the activity of serotonergic target regions. Given the critical role of
5-HT1A autoreceptors in regulating 5-HT signaling and its resulting influence on
the functioning of major brain targets, such as the amygdala, as well as complex
behavioral processes (Cowen et al. 1994; Hansenne et al. 2002; Lesch and
Gutknecht 2004), it is important to identify sources of emergent variability in
5-HT1A function.
Common sequence variation in the human 5-HT1A gene (HTR1A) represents
one potential source of such inter-individual variability. Recently, a relatively
frequent single nucleotide polymorphism, C(-1019)G, in the promoter region of
HTR1A was demonstrated to impact transcriptional regulation of the gene through
altered binding of the transcription factors. Specifically, the -1019G allele
abolishes or impairs transcriptional repression of the promoter and, as a conse-
quence, is associated with increased 5-HT1A expression (Lemonde et al. 2003), a
phenomenon that appears to be specific to autoreceptors (Czesak et al. 2006).
Consistent with this finding, in vivo human PET has revealed specifically
increased 5-HT1A autoreceptor density in both healthy adults and depressed
patients carrying the -1019G allele (Parsey et al. 2006). However, a similar effect
was not observed in an earlier PET study (David et al. 2005). Regardless, the in
vitro effects of the HTR1A -1019G allele and the more general relationship
Fig. 1 Trait anxiety is indirectly predicted by HTR1A genotype (rs6295) through amygdala
reactivity (adapted from Fakra et al 2009; Hariri 2009). Lines are labeled with unstandardized
path coefficients and standard errors in parentheses. Bolded coefficients outside of the lines
represent values from the trimmed model. Unbolded coefficients presented internally represent
values from the full model with all paths included. Significant indirect effects of HTR1A genotype
on trait anxiety were observed (ab = -1.60, SE = 0.73, P \ 0.05) while direct effects were
nonsignificant and dropped from the model. E1 and e2 represent the residual variances not
explained by variables included in the model. *P \ 0.05, **P \ 0.01
documented between increased 5-HT1A autoreceptor density and decreased

amygdala reactivity (Fisher et al. 2006) suggest that this common functional
genetic variation may contribute significantly to the emergence of inter-individual
variability in serotonin signaling which, in turn, biases amygdala reactivity.
Consistent with the existing data (i.e., increased 5-HT1A autoreceptors leading
to increased negative feedback inhibition of DRN and decreased 5-HT release), we
recently demonstrated that the HTR1A -1019G allele is associated with signifi-
cantly decreased threat-related amygdala reactivity (Fakra et al. 2009). In addition,
we found that HTR1A genotype effects on trait anxiety were mediated through its
impact on threat-related amygdala reactivity, which presumably reflects the
genotypes modulation of postsynaptic 5-HT release. Specifically, while path
models revealed no significant direct genotype effect on trait anxiety they dem-
onstrated that HTR1A C(-1019)G and amygdala reactivity indirectly predicted a
significant proportion (9.2%) of individual differences in trait anxiety through their
respective indirect and direct paths (Fig. 1). The data from this study is remarkably
consistent with that reported for other common functional polymorphisms also
associated with relatively increased 5-HT signaling, most notably the 5-HTTLPR
short allele (Hariri et al. 2002b; Munafo et al. 2008) and MAOA low-activity
alleles (Meyer-Lindenberg et al. 2006). More importantly, these findings represent
an important step in this avenue of research by providing empirical documentation
for the basic premise that genetic variation in neural signaling cascades indirectly
impact emergent behavioral processes by biasing the response of underlying neural

circuitries (Hariri et al. 2006b; Hariri and Weinberger 2003).
3 Aggression, the Amygdala and Testosterone
Aggression, defined as any behavior directed toward the goal of harming or

injuring another living being (Baron and Richardson 1994), has a major negative
impact on society. For instance, the World Health Organization estimated that
over 500 individuals between the ages of 10 and 29 die every day as a result of
inter-personal conflict while countless more suffer psychologically and physically
from aggression-related events. Nevertheless, despite these negative conse-
quences, the use (or threat) of aggression can be beneficial under certain conditions
(e.g., athletic competition, self-defense and establishment of status hierarchies).
Two factors contributing to the expression of aggressive behavior are the
pursuit of a desired goal (e.g., money, territory, status, mates) and interpersonal
provocation. Accordingly, researchers have typically classified aggressive
behavior as either proactive or reactive. Proactive aggression, also referred to as
instrumental aggression, occurs in the absence of direct provocation, does not
involve physiological arousal, and is a goal-oriented behavior aimed at the
acquisition of a valued resource (Dodge and Coi 1987). On the other hand, reactive
aggression is a defensive response to perceived or actual provocation and is
characterized by anger, impulsivity, affective instability and high levels of phys-
iological arousal (Dodge and Coi 1987). Many cases of proactive aggression are
highlighted in the media (e.g., assassinations, serial murders); however, reactive
aggression likely accounts for most societal problems (Nelson and Trainor 2007).
Thus, in the current section, we will focus primarily on putative neurobiological
mechanisms underlying reactive aggression.
Non-human animal and human neuroscience research indicates that aggressive
behavior is regulated by several inter-connected nodes of a ‘social behavior net-
work’ including the hypothalamus, amygdala, bed nucleus of the stria terminalis,
lateral septum, periaquaductal gray, and orbitofrontal cortex (see Nelson and
Trainor 2007; Davidson et al. 2000 for reviews). In humans, a number of studies
converge to implicate amygdala hyper-reactivity and/or reduced amygdala-
orbitofrontal cortex (OFC) coupling in response to social threat among individuals
prone to anger and reactive aggression (see Siever 2008 for review). For example,
in a PET study with criminal offenders, Raine et al. (1997) reported that affective
murderers (i.e., reactively aggressive inmates) demonstrated increased glucose
metabolism in subcortical structures (including the amygdala) and decreased
glucose metabolism in the prefrontal cortex. Subsequent imaging studies found
differences in neural responses to threat among individuals characterized by
reactive aggressive behavior. For instance, Coccaro et al. (2007) reported that
adults diagnosed with intermittent explosive disorder displayed amygdala hyper-
reactivity and decreased amygdala-OFC coupling to angry facial expressions.
Patients with borderline personality disorder, who are prone to engage in reactive
aggression, demonstrate relatively increased amygdala reactivity to facial
expressions depicting threat (Mauchnik and Schmahl 2010) and decreased
amygdala-PFC coupling (New et al. 2007). Finally, spousal abusers characterized
by reactive (but not proactive) aggression display heightened amygdala reactivity
and also demonstrate attentional biases for aggressive words (Lee et al. 2008;
Chan et al. 2010).
Studies in non-clinical samples indicate that even normal variation in constructs
linked to aggressive behavior maps onto variability in threat-related neural
responses. For instance, Beaver et al. (2008) reported that individual differences in
approach motivation, a construct linked to reactive aggression (Harmon-Jones
2003), were positively correlated with amygdala reactivity to angry facial
expressions. Other research has found that individual differences in approach
motivation were associated with decreased ventral ACC-amygdala coupling dur-
ing processing of angry facial expressions (Passamonti et al. 2008). Given the
important role of highly interconnected prefrontal regions (e.g., ventral ACC,
OFC) in mediating top-down regulation of amygdala driven emotional reactivity
(see Davidson et al. 2000 for review), such decreased functional coupling may, in
part, explain the positive link between approach motivation and aggressive
behavior. Also, individual differences in trait anger are positively correlated with
amygdala reactivity to angry faces, but only among men with relatively elevated
trait anxiety scores (Carré et al. in press-a). Finally, in a more direct test of the
hypothesis that amygdala reactivity to facial cues of threat represents a neuro-
biological marker for reactive aggression, we have found that variation in self-
reported physical aggression is positively correlated with amygdala reactivity to
neutral and angry facial expression in two independent samples of healthy men
(Carré et al. in press-b). Together, these findings converge to suggest that amyg-
dala hyper-reactivity and/or decreased amygdala-OFC coupling during processing
of threat-related stimuli may represent a distinct neural signature for one’s pro-
pensity to engage in reactive aggression (Siever 2008). It is important to note that
individuals characterized by callous-unemotional traits and proactive aggression
(e.g., conduct disorder, psychopathy) display amygdala and OFC hypo-reactivity
to facial signals of threat (see Blair 2010 for review).
As described above, it is important to consider the underlying molecular sub-
strates that give rise to individual variation in threat-related amygdala reactivity
and amygdala-OFC coupling. Testosterone, the end-product of the hypothalamic
pituitary gonadal axis, is one prime candidate. The physiological effects of
testosterone occur mainly through binding to intra-cellular steroid hormone
receptors (i.e., androgen and estrogen receptors) to ultimately influence gene
transcription. Importantly, androgen (and estrogen) receptors are abundantly
located in the amygdala and interconnected limbic structures involved in medi-
ating aggressive behavior (see Newman 1999; Simon 2002 for reviews). Thus,
through stimulation of steroid hormone receptors and subsequent modulation of
cell function (see Adkins-Regan 2005 for review) testosterone can influence the
functioning of neural circuits implicated in the expression of human aggression.
Recent functional neuroimaging studies have detailed some of the neural

structures that are sensitive to individual differences in testosterone concentrations.
In particular, individual differences in baseline testosterone concentrations are
positively correlated with amygdala reactivity to facial expressions signaling threat
(Derntl et al. 2009; Manuck et al. 2010) and negatively correlated with OFC
reactivity to provocation (Mehta and Beer 2010). Consistent with these correla-
tional studies, pharmacologic challenge experiments indicate that acutely raising
testosterone concentrations causes an increase in amygdala reactivity and a
decrease in amygdala-OFC connectivity in response to facial signals of threat
(Hermans et al. 2008; van Wingen et al. 2008, 2010). These findings suggest that
the association between acute fluctuations in testosterone and reactive aggression
in men (Carré and McCormick 2008; Carré et al. 2009, 2010) may be due to the
influence of testosterone on neural processing of threat (e.g., angry faces or
provocation), which may ultimately bias aggressive behavior during social
challenges.
As discussed above, many of the physiological effects associated with testos-
terone are mediated by activation of intra-cellular androgen receptors. Specifically,
when activated by testosterone, androgen receptors (AR) migrate to the cell
nucleus where they regulate gene transcription by activating hormone response
elements (HRE) located within gene regulatory sequences. Importantly, the
transcription potential of the androgen receptor varies with the expansion of a
polyglutamine stretch in the N-terminal domain of the AR protein, as encoded by a
trinucleotide (CAG) repeat polymorphism in exon 1 of the X chromosome-linked
AR gene (Zitzmann and Nieschlag 2003). Specifically, in vitro work indicates that
the transactivation potential of the androgen receptor declines in relation to an
increase in the number of CAG repeats (Chamberlain et al. 1994), and that
androgen receptor concentrations decline with an increasing number of CAG
repeats (Choong et al. 1998).
Recent evidence indicates that the number of CAG repeats correlate negatively
with testosterone responses to social interactions with attractive women (Roney
et al. 2009). In other words, men with fewer AR CAG repeats demonstrate a more
robust neuroendocrine response to potential mates, suggesting that this androgen
receptor polymorphism may influence the efficiency with which an individual may
mount an endocrine response to social interactions. Furthermore, genetic studies
have found that high testosterone men with fewer CAG repeats are more
aggressive (Rajender et al. 2008; Vermeesch et al. 2010). Finally, Manuck et al.
(2010) found that individual differences in CAG repeat length were negatively
correlated with ventral amygdala reactivity to facial expressions depicting threat,
particularly among men with relatively high baseline testosterone concentrations.
Thus, variation in the number of AR CAG repeats modulates testosterone
responses to social interactions and amygdala reactivity to facial signals of threat.
Collectively, these findings provide support for the idea that heightened
amygdala reactivity to social threat (e.g., provocation and/or facial signals of
threat) may represent a neurobiological mechanism through which androgens
modulate human aggressive behavior. Pharmacologic challenge experiments in
which aggressive behavior is measured directly during fMRI are needed to confirm
the causal role of testosterone in modulating human aggressive and threat-related
neural responses.
4 Impulsivity, the Ventral Striatum and Dopamine
Discounting future outcomes underlies much of human decision making and

figures prominently in several overlapping psychological constructs such as self-
regulation, impulse-control, delay of gratification and intertemporal choice
(Manuck et al. 2003). Moreover, individuals who strongly prefer immediate over
deferred rewards of larger nominal value are often generally impulsive or lacking
in self-control and at risk for addictive disorders such as pathological gambling,
cigarette smoking and drug and alcohol abuse (Alessi and Petry 2003; Bickel
et al. 1999; Kirby et al. 1999; Madden et al. 1997). In experimental research on
intertemporal choice, discounting of future rewards or delay discounting (DD) is a
well-characterized behavioral measure of preference for immediate over delayed
rewards and provides an index of impulsive tendencies in humans (Green and
Myerson 2004). Behavioral tests used to derive estimates of DD commonly ask
participants to choose between multiple immediate rewards that vary in value and
a constant, larger reward available after varying intervals of delay. In such tasks,
rates of discounting often differ appreciably and consistently among individuals
(Simpson and Vuchinich 2000). Thus, DD represents a potentially important
psychometric index of individual differences in present versus future-oriented
tendencies.
Similar to the research on trait anxiety and amygdala reactivity explication of
the underlying neural processes that give rise to such inter-individual variability
has the potential to allow for a more comprehensive understanding of the mech-
anisms leading not only to normal variability in such behaviors but also the
pathophysiology of addiction and related disorders. Through reciprocal cortical
and subcortical connections, the nucleus accumbens (NAcc) and, more broadly,
the ventral striatum (VS), contribute to the motivational salience of stimuli and
abet appetitive or reward-dependent behaviors (Berridge and Robinson 2003).
Activity of the VS increases in response to both the anticipation and receipt of
rewarding stimuli including primary (e.g., food) and secondary (e.g., money)
reinforcers (O’Doherty 2004). Moreover, in addiction, craving and compulsive
drug seeking as well as sensitivity to drug cues are associated with dysregulated
increases in VS activity (Kalivas and Volkow 2005). Because the response of the
VS involves an immediate response to rewards, the magnitude of VS activity
may contribute to individual differences in a relative preference for immediate,
compared to delayed, rewards.
Using BOLD fMRI, we have demonstrated that the magnitude of VS reactivity
predicts individual differences in a simple laboratory measure of DD (Hariri et al.
2006a). Specifically, analyses revealed that individual differences in DD correlate
positively with magnitude of VS activation in response to both positive and

negative feedback as well as with differential reward-related VS activation in
response to positive compared with negative feedback. Consistent with the strong
general correlation between DD and traditional self-report measures of impulsivity
(De Wit et al. 2004, 2007), we have also found that reward-related VS reactivity is
positively correlated with scores from the Barratt Impulsiveness Scale (Forbes
et al. 2009). Collectively, our results suggest that increased self-reported impul-
sivity as well as the preference for smaller immediate over larger delayed rewards
reflect both a relatively indiscriminate and hyper-reactive VS circuitry. Similar
variability in VS function has also been associated with more complex measures of
incentive-based decision making (Knutson et al. 2007). Moreover, dysregulation
of the VS contributes to addiction, perhaps by affecting impulsive decision making
(Kalivas and Volkow 2005). As such, inter-individual variability in VS reactivity
to reward-related stimuli likely contributes to the emergence of differences in the
intermediate behavioral risk factors for, as well as the clinical expression of,
addiction. Identifying variability in neural signaling pathways that contributes to
individual differences in VS function offers additional traction in the search for
underlying biological mechanisms.
Dopamine modulation of neuronal activity, especially in the VS (i.e., meso-
limbic system), serves as a nexus for the expression of DA signaling at the level of
reward-related behaviors (Cardinal et al. 2004; Kelley 2004). Functioning of the
DA system has been linked to normal individual differences in reward-related
traits (Depue et al. 1994), and disorders involving enhanced reward-seeking, such
as addiction, have been hypothesized to reflect maladaptive alterations of this
mesolimbic reward system (Hyman et al. 2006; Volkow et al. 1999). Multimodal
and pharmacological neuroimaging studies of DA effects on brain function again
offer a unique opportunity to more directly evaluate underlying molecular mech-
anisms regulating this circuitry. A recent in vivo human study reported a direct
relationship between striatal DA synthesis, assessed with PET and brain activity,
assessed with BOLD fMRI (Siessmeier et al. 2006). Acute increase of DA release
via oral amphetamine has also been linked with relatively increased extent of
BOLD fMRI assessed VS activity (Menon et al. 2007). More generally, acute
pharmacologic increase of DA in both healthy volunteers (Hariri et al. 2002a) and
patients with Parkinson’s disease (Tessitore et al. 2002) results in relatively
increased BOLD fMRI assessed activity in closely related limbic brain regions,
namely the amygdala. Given the importance of DA in modulating this behaviorally
relevant neural circuitry, identifying factors that determine inter-individual
variability in DA signaling and its related impact on the reactivity of the VS will
facilitate our understanding of the neurobiological mechanisms governing
reward-related behaviors and augment efforts to improve the treatment and even
prevention of pathological behaviors such as drug abuse and addiction.
We have explored the role of altered DA signaling, resulting from a common
functional polymorphism impacting active synaptic reuptake in the striatum, in
determining inter-individual variability in reward-related VS reactivity and corre-
lated variability in behavioral impulsivity. Consistent with the research on serotonin
signaling, amygdala reactivity and trait anxiety, the selection of our candidate
polymorphism was driven by available in vitro and/or in vivo assays demonstrating
significant impact of the variant on aspects of biological function related to DA
neurotransmission and not on available data from association studies with behav-
ioral (e.g., impulsivity) or clinical (e.g., alcoholism) phenotypes. While association
studies are necessary for understanding the ultimate contribution of genetic poly-
morphisms to variability in behavioral and clinical phenomena, they do not readily
allow for inferences regarding polymorphic effects on gene or protein function.
Such inferences are instrumental for the development of biologically plausible and
tractable hypotheses regarding the impact of genetic variation on inter-individual
variability in brain function and associated behaviors such as those pursued in our
current work (Hariri et al. 2006b; Hariri and Weinberger 2003).
The dopamine transporter is responsible for the active clearance of synaptic DA
and, thus, plays a critical role in regulating the duration of postsynaptic DA
signaling, especially in the striatum (Sesack et al. 1998). Accumulating evidence
indicates that a 40-base pair variable number of tandem repeats (VNTR) poly-
morphism in the 30 untranslated region of the DAT gene (SLC6A3) impacts the
expression and availability of DAT (Bannon et al. 2001). Although a genotype
effect has not been consistently observed across all studies (Martinez et al. 2001;
Michelhaugh et al. 2001; Mill et al. 2005; van Dyck et al. 2005), several suggest
that in comparison to the 9-repeat allele, the 10-repeat is associated with relatively
increased levels of DAT both in vivo (Cheon et al. 2005; Heinz et al. 2000) and in
vitro (Mill et al. 2002; Van Ness et al. 2005). We hypothesized that there would
be relatively greater VS reactivity associated with the 9-repeat allele, which is
linked with reduced DAT expression and presumably greater striatal synaptic DA,
in comparison with the 10-repeat allele. Consistent with our hypothesis, the DAT1
9-repeat allele was associated with relatively greater VS reactivity and accounted
for nearly 12% of the inter-individual variability. In contrast, genetic variation
directly affecting DA signaling only in the prefrontal cortex (i.e., COMT
Val158Met) was not associated with variability in VS reactivity. These results
highlight an important role for a genetic polymorphism affecting striatal DA
neurotransmission in mediating inter-individual differences in reward-related VS
reactivity. They further suggest that altered VS reactivity may represent a key
neurobiological pathway through which these polymorphisms contribute to vari-
ability in behavioral impulsivity and related risk for substance use disorders.
5 Endocannabinoids, Threat- and Reward-Related Brain

Functions
Modern neuroscience methodologies have greatly advanced our understanding of

the intrinsic mechanisms mediating and regulating endogenous cannabinoid or
endocannabinoid (eCB) signaling in the CNS (Piomelli 2003). Such eCB signaling
has emerged as a potent modulator of neural circuitries mediating both basic
physiological (Calignano et al. 1998; Meng et al. 1998) and advanced behavioral
responses (Maldonado et al. 2006; Scherma et al. 2008; Viveros et al. 2005).
Experimental manipulation of these mechanisms has revealed significant behav-
ioral effects, especially in threat- and reward-related domains, which are generally
consistent with the effects of Cannabis intoxication, which are largely driven by
the constituent chemical D9-tetrahydrocannabinol (Robson 2005). The elucidation
of molecular mechanisms regulating eCB signaling, akin to that for serotonin and
dopamine, has motivated attempts to understand its possible contribution to the
emergence of variability in brain circuit function and related individual differences
in behavioral attributes (e.g., anxious or impulsive temperament) associated with
increased risk for psychiatric disorders.
After their biosynthesis from arachidonic acid, eCBs such as anandamide
(AEA) and 2-arachidonoylglycerol (2-AG) typically modulate synaptic neuro-
transmission through stimulation of CB1, the principal CNS cannabinoid receptor
widely expressed on multiple neuronal subtypes and their distributed circuitries.
In turn, the duration and intensity of eCB signaling, especially for AEA, is regu-
lated by two complementary mechanisms: enzymatic degradation via fatty acid
amide hydrolase (Cravatt et al. 1996) and active synaptic clearance via the AEA
transporter (Piomelli et al. 1999). The psychotropic and THC-like effects of AEA,
however, appear to be coupled with fatty acid amide hydrolase (FAAH), but not
AEA transporter function (Solinas et al. 2007). Thus, FAAH, an integral membrane
enzyme, may uniquely regulate behaviorally relevant eCB signaling by mediating
the hydrolytic breakdown of AEA into arachidonic acid and ethanolamine.
Again, common genetic variation (i.e., polymorphisms) affecting the functioning
of components involved in eCB neurotransmission (e.g., AEA, CB1, FAAH) may
represent a significant potential source of inter-individual variability in eCB sig-
naling that mediates emergent differences in emotion- and reward-related behaviors
(Onaivi et al. 2002). Because of its critical role in regulating the signaling duration
and intensity of AEA (Cravatt et al. 1996), and its selective contribution to the
psychotropic effects of AEA (Solinas et al. 2007), we have recently examined the
neurobiological and behavioral effects of a common functional nonsynonymous
SNP resulting in the conversion of a conserved proline residue to threonine (P129T)
in the amino acid sequence of FAAH (Hariri et al. 2009). In vitro, FAAH 385A is
associated with normal catalytic properties, but reduced cellular expression of
FAAH, possibly through enhanced sensitivity to proteolytic degradation (Chiang
et al. 2004; Sipe et al. 2002). Moreover, the C385A is the only common mutation in
FAAH (Flanagan et al. 2006) and the 385A, which putatively augments AEA sig-
naling via decreased enzymatic degradation, has been associated with reward-
related pathologies including street drug use and problem drug/alcohol abuse, as
well as being overweight and obese (Flanagan et al. 2006; Sipe et al. 2002).
In animal models, both pharmacologic and genetic disruption of FAAH function
result in decreased anxiety-like behaviors, as well as increased consumption and
preference for ethanol (Basavarajappa et al. 2006; Blednov et al. 2007; Kathuria
et al. 2003; Moreira et al. 2008; Solinas et al. 2007). Moreover, a recent pharma-
cologic fMRI study in human subjects has reported that acute oral administration of
(c)
(a) (b)
(f)
(d) (e)
Fig. 2 Effects of FAAH genotype (rs324420) on threat- and reward-related brain activation
(adapted from Hariri et al. 2009; Hariri 2009). Statistical parametric maps displaying the
correlation between threat-related amygdala reactivity and trait anxiety in a FAAH 385A carriers
and b C385 homozygotes. c Plots of the correlation between threat-related amygdala reactivity
and trait anxiety according to FAAH C385 genotype. Statistical parametric maps displaying the
correlation between reward-related ventral striatal (VS) reactivity and delay discounting in d
FAAH 385A carriers and e C385 homozygotes (no significant correlation observed). f Plots of the
correlation between reward-related VS reactivity and delay discounting according to FAAH
C385A genotype. FAAH: fatty acid amide hydrolase
THC is associated with reduced amygdala reactivity to threat-related facial

expressions of emotion (Phan et al. 2008). Consistent with these effects, we
hypothesized that the FAAH 385A would be associated with relatively decreased
threat-related amygdala reactivity, but increased reward-related reactivity in
the VS. Analyses revealed that carriers of the FAAH 385A, associated with reduced
enzyme expression and, presumably, increased AEA signaling, have decreased
threat-related amygdala reactivity. In contrast, carriers of the FAAH 385A exhib-
ited increased reward-related VS reactivity in comparison to C385 homozygotes.
Moreover, divergent effects of FAAH C385A genotype on brain function were
manifest in a consistent manner at the level of brain-behavior relationships (Fig. 2).
Relative to C385 homozygotes, FAAH 385A carriers showed a diminished rela-
tionship between amygdala reactivity and trait anxiety. In contrast, 385A carriers
exhibited a markedly increased relationship between VS reactivity and delay dis-
counting, a behavioral index of impulsivity and reward-sensitivity.
It is important to note that there were no direct associations between FAAH

genotype and behavioral phenotypes (i.e., anxiety or impulsivity) in this study, a
common occurrence when working with relatively small samples, possibly
reflecting the minimal effect the proximal biological impact associated with any
genotype has on any distal behavioral phenotype (Hariri et al. 2006b; Hariri and
Weinberger 2003), as well as the importance of environmental stressors in
unmasking genetically driven effects on behavior (Caspi and Moffitt 2006).
However, there were robust differences in the relationships between regional brain
function and complex behaviors as a function of FAAH C385A genotype. These
observed brain-behavior patterns may reflect the influence FAAH C385A associ-
ated differences in endogenous eCB tone on stimulus-driven neural circuit function
mediating complex behavioral processes. Relatively higher levels of AEA in the
amygdala of FAAH 385A carriers may reduce the responsivity of this structure to
salient input (possibly through CB1-mediated potentiation of local GABAergic
interneurons) and, as a consequence, lead to reduced anxiety-like behaviors pre-
dicted by amygdala function. In contrast, higher levels of AEA may increase the
responsivity of the VS in FAAH 385A carriers (possibly through CB1-mediated
increased dopamine release and potentiation of VS neuron activity) leading to
increased reward-sensitivity predicted by VS function. Support for this speculation
exists in studies reporting a failure of restraint stress to effect changes in amygdala
activation in knockouts lacking FAAH or animals treated with FAAH inhibitors
(Patel et al. 2005), and increased food-intake as a result of local FAAH inhibition
in the nucleus accumbens (Sorice-Gomez et al. 2007). Thus, the endogenous state
of eCB signaling associated with either constitutive genetic variation such as the
FAAH C385A or acute pharmacologic manipulation likely biases the responsivity
of neural circuits to behaviorally relevant information and their subsequent reg-
ulation of complex behaviors.
Decreased threat-related amygdala reactivity and associated trait anxiety may
contribute to the emergence of pathologies such as addiction and obesity, previ-
ously associated with the FAAH 385A (Flanagan et al. 2006; Sipe et al. 2002;
Tyndale et al. 2007), by reducing the sensitivity of these individuals to potential
environmental threat or harm. In fact, blunted amygdala reactivity has been
reported in individuals at high familial risk for alcoholism and this has been
interpreted as possibly contributing to decreased threat-sensitivity and subse-
quently increased risk-taking behaviors in these genetically predisposed individ-
uals (Glahn et al. 2007). An increase in reward-related VS reactivity and
associated impulsivity (e.g., steeper discounting of future, relative to immediate
rewards) may likewise contribute to disinhibitory psychopathologies through
heightened reward-sensitivity and impulsive decision making. Studies in addicted
patients have generally reported a sensitization of the neural circuitry for reward,
including the VS (Kalivas and Volkow 2005). And, increased behavioral impul-
sivity and reward-sensitivity are significant risk factors for addiction (de Wit and
Richards 2004). Thus, through divergent effects on both threat- and reward-related
brain functions, the influence of FAAH C385A on eCB signaling may have a
compound and accelerated effect on risk for related pathologies.
6 Stress, the HPA Axis and the Mineralocorticoid Receptor
All organisms strive to maintain homeostasis by regulating physiological states

within a dynamic equilibrium. Stress, the perception of inadequate coping
resources in the context of environmental demands appraised as threatening, is a
common experience that disrupts homeostasis, and triggers a biological-behavioral
stress response to promote adaptation/survival. While stress can promote adaptive
coping to environmental challenges by recruiting necessary resources, it is
also associated with many adverse physical and mental health conditions such
as cardiovascular disease, depression, post-traumatic stress disorder (PTSD) and
immune system dysfunction (Cohen et al. 2007; McEwen and Gianaros 2010).
Importantly, there is tremendous variability in which environmental demands
individuals perceive as stressful as well the extent of physiological and psycho-
logical response to these demands (Dickerson and Kemeny 2004; Kudielka et al
2009). Because this variability is associated with stress-related physical and
mental health outcomes and differences in related neural activation (Marques et al.
2009; Yehuda 2002), identifying the factors that contribute to variability in stress
reactivity is an important step for understanding the etiology of stress-related
disorders as well as limiting the sequelae of stressful experiences.
A wealth of research has established that stress reactivity is centrally regulated
by the hypothalamic–pituitary–adrenal (HPA) axis (for reviews see de Kloet
et al. 2005; Ulrich-Lai and Herman 2009). Briefly, pathways from the medial
prefrontal cortex, hippocampus, amygdala and brainstem involved in the behav-
ioral, neuroendocrine, autonomic and immune responses to stress converge within
the paraventricular nucleus of the hypothalamus to regulate the release of corti-
cotropin releasing hormone (CRH) in response to perceived stress. CRH triggers
the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary
gland, which upon binding to receptors in the adrenal gland, stimulates cortisol
release. As part of a negative feedback loop, increases in cortisol inhibit CRH and
ACTH release from the hypothalamus and anterior pituitary gland, respectively. At
all levels in the cascade, HPA axis function varies widely across individuals and is
relatively stable over time (Fox et al. 2006; Kudielka et al. 2009; Márquez et al.
2005) suggesting that trait-like variation in HPA axis function may contribute to
stable differences in responses to stressors.
Rodent knockout and non-human and human pharmacologic challenge studies
have demonstrated that disruption at all nodes of the HPA axis (e.g., hypothala-
mus, pituitary, adrenal gland) can induce anxiety and depressive-like behavior
(e.g., Kolber and Muglia 2009; Marques et al. 2009; Müller et al. 2002). Moreover,
nearly 50 years of research has strongly linked abnormal function at all levels of
the HPA axis to stress-related psychopathology (Gibbons and McHugh 1962;
Marquis et al. 2009; Yehuda 2002). For instance, depression is characterized by
elevated CRH, ACTH and cortisol as well as a disrupted negative feedback loop
whereby cortisol does not effectively inhibit CRH and ACTH (van Praag et al.
2004). PTSD is associated with elevated CRH but diminished ACTH and cortisol
as well as an enhanced cortisol-induced inhibition of CRH and ACTH (Yehuda

2002). Furthermore, HPA dysregulation is associated with a number of risk factors
for psychopathology such as childhood maltreatment (Tarullo and Gunnar 2006)
and low social support (Abercrombie et al. 2004).
More recently, and consistent with earlier non-human animal work (Rodrigues
et al. 2009; Ulrich-Lai and Herman 2009), translational neuroimaging research
suggests that HPA axis function is associated with structural and functional dif-
ferences in brain regions, such as the hippocampus, amygdala, basal ganglia and
prefrontal cortex, that are relevant to stress-related psychopathology (for review
see Pruessner et al. 2010; McEwen and Gianaros 2010). For example, Urry et al.
(2006) show that heightened amygdala and reduced ventromedial prefrontal cortex
activation during the regulation of negative affect is associated with dysregulated
HPA axis function. Moreover, prefrontal glucose metabolism, an index of neuronal
activity, predicts variability in HPA axis function (Jahn et al. 2010; Kern et al.
2008), and lesions to these brain regions result in HPA axis dysregulation
(Buchanon et al. 2010).
Given the firm association between HPA axis function and stress-related psy-
chopathology and related neural circuitry, factors that shape the responsiveness of
this system can be studied with the ultimate goal of identifying mechanisms
underlying individual differences in response to stress. As mentioned above, in
addition to numerous regulatory mechanisms inside and outside of the HPA axis,
cortisol is a major regulator of HPA axis function and related neural systems.
In support of a causal relationship between cortisol and stress-related psychopa-
thology, 20% of patients prescribed chronic high doses of hydrocortisone
(a synthetic form of cortisol), develop psychopathology including depression,
mania and psychosis. In addition, while 75% report some psychiatric symptoms
these disappear following treatment cessation (see Marques et al. 2009).
Cortisol operates through a binary corticosteroid receptor system, binding to
both the high affinity mineralocorticoid receptor (MR) and low affinity gluco-
corticoid receptor (GR) which are widely co-expressed in limbic neurons including
those in the amygdala (de Kloet et al. 2005; Joëls et al. 2008). These intracellular
receptors function as transcriptional regulators whereby binding can alter the
expression of 70–100 genes (de Kloet et al. 2005), which likely has widespread
consequences for neural activation and stress responsiveness. Recent research
provides support for the existence of long-hypothesized membrane-bound MR and
GR, although the mechanism(s) through which these effects occur remain unclear
(Bartholome et al. 2004; Karst et al. 2006; Joëls et al. 2008).
Because of its low affinity, the GR is only extensively occupied in the wake of
large spikes in cortisol such as those associated with stressors or circadian
rhythms. One of the primary functions of the GR is to normalize brain activity
following the extinction of a stressor and to inhibit continued HPA axis response.
On the other hand, because of its high affinity, the MR is almost always occupied
and non-human animal research suggests that it is necessary for a stable excitatory
tone in the hippocampus which inhibits the HPA axis under basal and stressful
circumstances (Reul et al. 2000). In addition to tonically inhibiting HPA axis
response, MR binding is relevant to behavioral stress response functions including

the appraisal of novel situations as well as the selection of appropriate responses to
deal with challenge (de Kloet et al. 2005; Joëls et al. 2008). Thus, it is believed
that the MR is prominently involved in basal stress system regulation and the onset
of a stress response while the GR primarily functions to terminate an initial stress
reaction. As such, the MR is an ideal candidate to contribute to differences in stress
reactivity.
In mice, MR knockout or antagonism increases basal and stress-evoked HPA
axis activity (Gass et al. 2001) and worsens response to antidepressant medication
(DeRijk et al. 2008). Conversely, enhanced MR expression is associated with
reduced depressive- and anxiety-like behaviors and corticosterone (the rodent
homolog of cortisol) secretion under stressful and basal conditions (Mitra et al.
2009; Rozeboom et al. 2007). Consistent with these mechanisms, antidepressant
medications increase MR expression (Brady et al. 1991). Collectively, these
findings suggest that reduced MR function may contribute to elevated HPA axis
activity at baseline and in dysregulated responses to stressors. Thus, given the
prominent role of the MR in stress-responsiveness, stress-related behavior and
psychopathology, it is important to identify sources of variability in MR function.
A missense isoleucine(Iso)/valine(Val) polymorphism (rs5522) located in
exon 2 of the MR gene (NR3C2) occurs in approximately 10% of the Caucasian
population. Importantly, in vitro studies show that the Val allele is associated
with reduced cortisol, but not aldosterone, binding and reduced cortisol-induced
transactivation (De Rijk et al. 2006; Arai et al. 2003). Thus, the MR Val allele
may promote elevated HPA axis activity under basal conditions and in response
to small stressors due to reduced cortisol-MR binding and subsequently
decreased inhibition of the HPA axis. The Val allele may promote the devel-
opment of HPA axis dysregulation, not only through reduced inhibition of HPA
axis responsiveness but also because of reduced MR expression typically elicited
by cortisol.
Consistent with these functional associations and speculations, the MR Val
allele has been linked to heightened endocrine and autonomic responses to acute
stress and stress perception. Specifically, the Val allele is associated with elevated
cortisol and heart rate responses to acute social stress (DeRijk et al. 2006), skin
conductance responses evoked by acute physical stress (threat-of-shock, Bogdan
et al. 2010), perceptions of the aversiveness of shock levels (Bogdan et al. 2010)
and waking cortisol levels (van Leeuwen et al. 2010). Furthermore, relative to Iso
homozygotes, youth Val carriers have heightened threat-related amygdala reac-
tivity, particularly in the context of low emotional neglect (Bogdan et al. in press).
Collectively these finding suggest that the functional effect of the Val allele does
result in enhanced stress responsiveness and perception. Furthermore, the Val
allele has been associated with enhanced reward learning under basal conditions,
but a vulnerability to stress-induced reward learning deficits (Bogdan et al. 2010)
as well as depressive symptoms (Kuningas et al. 2007). Thus, it appears that the
heightened stress responsiveness characteristic of the Val allele may translate to
important behaviorally relevant differences related to psychopathology.
Taken together, individual differences in HPA axis function are predictive of

stress-related physical and mental health disturbance in what appears to be a causal
relationship. In addition to work showing that environmental experience (e.g.,
childhood maltreatment) can have long-lasting and even epigenetic effects
(McGowan et al. 2009) on HPA axis system function, emerging research has
shown that common polymorphisms including that in the MR gene detailed here as
well as in the genes for GR (DeRIjk et al. 2008), CRHR1 (Binder 2009; Binder and
Nemeroff 2010; Thode et al. unpublished observation) and FKBP5 (REF) influ-
ence variability in HPA axis function, stress sensitivity and psychopathology.
Given the association of both environmental and genetic factors with individual
differences in HPA axis function, this system may be a particularly fruitful area to
further pursue gene-by-environment interactions research.
7 Summary and Future Directions
As detailed above, multimodal techniques assessing brain function, have begun to

identify how variability in neural substrates associated with processing specific
forms of information contribute to emergent individual differences in stable and
enduring aspects of human behaviors such as personality and temperament. In
parallel, the application of pharmacologic fMRI, multimodal PET/fMRI, and
neuroendocrinology is allowing for an understanding of how variability in specific
molecular signaling pathways influences individual differences in behaviorally
relevant brain function. Moreover, information on DNA sequence variation in
humans and related identification of functional genetic polymorphisms is now
being utilized to understand the biological origins of variability in component
processes of molecular signaling pathways as well as to efficiently model how such
emergent variability impacts behaviorally relevant brain function. Such ongoing
efforts to understand the detailed mechanisms that mediate individual differences
in complex behavioral traits and related psychopathology at the level of brain
circuit function, molecular signaling pathways and functional genetic polymor-
phisms have the potential to inform clinically relevant issues and provide guiding
principles for the development of more effective and individually tailored treat-
ment regimes. In addition, the elucidation of such mechanisms, especially those
mapped to functional genetic polymorphisms, can lead to identification of pre-
dictive risk markers that interact with unique environmental factors to precipitate
psychopathology.
While the five examples highlighted in this chapter are evidence for the
potential of an informed and integrated research strategy to identify the neurobi-
ology of individual differences in complex behavioral traits and their related
clinical endpoints, much work is left to be done. First, to allow for tractable
experimental designs and testable hypotheses in existing samples, the studies
highlighted above have focused on the effects of a single signaling pathway on
behaviorally relevant brain circuitry. Of course, it is very clear that there are
numerous complex interactions between signaling pathways and that more than
one pathway contributes to the regulation of any brain circuitry. For example, we
know that DA plays an important role in modulating amygdala function and
anxiety (Hariri et al. 2002a; Tessitore et al. 2002), and that 5-HT can influence
reward-related brain circuitry and impulsivity (Manuck et al. 1998). However,
existing studies lack the power and sophistication to model such complex inter-
actions while effectively controlling for other important modulatory factors (e.g.,
age, gender, stress exposure) in the context of BOLD fMRI, pharmacologic fMRI
or multimodal PET/fMRI protocols. To do so, we must aggressively expand the
scale and scope of our studies to include hundreds and, preferably, thousands of
subjects. This will afford opportunities to effectively examine interactions between
signaling pathways (e.g., 5-HT and DA) on brain function and behavior through
modeling of multiple functional polymorphisms (e.g., HTR1A -1019 and DAT1),
and examine the effects of genetically driven variation in signaling pathways on
multiple behaviorally relevant brain circuitries.
A second important consideration is that existing studies have been largely
conducted in ethnically and racially homogenous populations. Thus, the observed
effects may not generalize to other populations. This is especially true of studies
utilizing functional genetic polymorphisms because the potential effect of any
single genetic variant on a complex biological and behavioral phenotype is likely
to be small against the background of the approximately 20,000–25,000 human
genes and the multitude of other neurobiologically relevant functional variants
they likely harbor. In fact, we have already seen that the well-replicated effects of a
common functional polymorphism affecting 5-HT signaling on amygdala reac-
tivity in Caucasian subjects may be reversed in those of Asian ancestry (Lee and
Ham 2008; Munafo et al. 2008). Importantly, our most recent studies have
experimentally controlled for occult genetic stratification independent of self-
reported race or ethnicity as well as the independence of the target genotype from
other functional polymorphisms impacting the brain functions under study. While
such efforts allow for the attribution of emergent variability in brain and behavior
to the candidate variant of interest and not to other possible polymorphisms or
more general differences between genotype groups in genetic background, it is
important to explicitly test the independence of functional polymorphisms through
rigorous statistical modeling in larger samples and also to test the validity of any
associations derived in one sample population (e.g., Caucasian) to populations
with different genetic backgrounds (e.g., Asian or African).
A third important consideration for the future of this research is the need to
conduct large-scale prospective studies beginning in childhood to determine any
developmental shifts in neurogenetic pathways mediating individual differences in
behavior as well as their predictive utility in identifying risk for psychopathology
as a function of environmental or other stressors. All of the studies described
above and most of the studies available in the literature as a whole have been
conducted in adults carefully screened for the absence of psychopathology.
Because of this, these findings identify mechanisms contributing to variability in
the normative range of behavior only. The utility of these markers of individual
differences in behavior be they neural, molecular or genetic in predicting vul-

nerability to psychopathology is unclear. Such predictive utility is ideally tested
through prospective studies beginning with premorbid populations that account for
the moderating effects of environmental stress in the emergence of clinical dis-
order over time (Caspi and Moffitt 2006; Viding et al. 2006).
A fourth issue is the need to further integrate pharmacologic challenge protocols
with multimodal PET/fMRI to determine if variability in molecular components of
signaling pathways mediate effects of specific neurotransmitters or neuromodula-
tors on individual differences in behaviorally relevant brain circuit function. For
example, despite the remarkable convergence of findings implicating variability in
eCB signaling in threat- and reward-related brain functions, the exact nature of the
downstream signaling pathways through which FAAH C385A may modulate neu-
ronal and neural circuit function cannot be determined from the available results.
FAAH catalyses the hydrolysis of other biologically active endogenous fatty acid
amides (e.g., oleamide and oleoylethanolamide), which impact threat- and reward-
related behaviors independently of AEA (Wei et al. 2007; LoVerme et al. 2005).
Although, FAAH has high selectivity for AEA (Desarnaud et al. 1995) the effects of
FAAH C385A cannot be specifically linked to AEA neurotransmission without
additional data. If the neural and behavioral effects of FAAH C385A are mediated by
genotype-driven differential availability of AEA, then these effects should be sen-
sitive to manipulation of CB1 receptors. An interesting test of this putative mech-
anism would be to examine the impact of CB1 antagonists, such as rimonabant, on
neural phenotypes associated with FAAH C385A genotype using pharmacologic
fMRI. The availability of a PET radiotracer for CB1 (Burns et al. 2007) also allows
for the determination of any FAAH C385A effects on endogenous receptor con-
centrations. If this polymorphism biases brain function through AEA stimulation of
CB1, then antagonism of the receptor should eliminate the divergent effects on
amygdala and VS reactivity documented here. Any genotype related alterations in
AEA concentrations may also be reflected in relative up- or down-regulation of CB1
receptors assayed via PET. If CB1 antagonism fails to abolish the differential effects
of FAAH C385A on brain function or if there are no differences in CB1 concen-
trations based on the genotype, then the existing effects are likely mediated by non-
eCB fatty acid amides. In addition to testing this mechanistic hypothesis with
pharmacologic fMRI and multimodal PET/fMRI, future studies with substantially
increased sample sizes can model allele load effects of FAAH 385A, as well as
potential FAAH interactions with functional genetic polymorphisms affecting other
components of eCB neurotransmission (Chakrabarti et al. 2006).
Fifth, in light of evidence for environmental modulation (Tarullo and Gunnar
2006), gene-by-environment interactions (Caspi and Moffitt 2006) and epigenetic
regulation (MacGowan et al. 2009), it is important for future research to not only
assess genetic variation, but also to optimize assessments available to assess the
objective and subjective impact of positive and negative environmental variables.
Examples of this include interview-based methods to assess the objective and sub-
jective impact of stressful life events and prospective study designs to examine the
influence of stress over the course of study assessments (e.g., the Life Events and
Difficulties Schedule; Brown and Harris 1978; Monroe 2008; Williamson et al.
2003). Furthermore, in light of theoretical arguments that genotypes associated with
adversity may reflect plasticity to the environment and not just vulnerability (e.g.,
Belsky et al. 2009; Manuck 2010), it is critical for these assessments to also assess
positive effects of the environment such as social support (e.g., Hyde et al. 2011).
Finally, there is tremendous potential in developing large databases (again
preferably thousands of subjects) with detailed measures of behavioral traits,
neuroimaging-based measures of multiple brain circuitries and extensive geno-
typing. One of the most exciting applications of molecular genetics is in identi-
fying novel biological pathways contributing to the emergence of complex traits
(Gibson and Goldstein 2007; McCarthy et al. 2008). The continued refinement of a
detailed map of sequence variation across the entire human genome (i.e., SNPs
that ‘‘tag’’ every gene) and production of technologies supporting efficient high-
throughput identification of such variation in individuals have dramatically
accelerated the discovery of genes involved in the emergence of complex disease
processes (Fellay et al. 2007; Link et al. 2008) as well as normal variability in
continuous traits (Lettre et al. 2008). Many of the genes identified in such studies
have illuminated novel pathways not previously implicated in these processes or
traits, spurring intensive efforts to understand the potential biological effects of the
proteins produced by these genes. As such, these ‘‘genome-wide’’ screens repre-
sent an opportunity to leap forward beyond the available pool of candidate mol-
ecules and pathways in parsing the mechanisms of complex biological processes.
Because neuroimaging-based measures of brain function reveal key mechanisms
involved in the emergence of individual differences in behavioral traits and are
closer to the biological effects of functional genetic polymorphisms, they are ideal
substrates for genome-wide screens. For example, BOLD fMRI estimates of
amygdala reactivity predicting variability in trait anxiety can be used as the
continuous trait in a genome-wide screen. Any significant associations that emerge
between genetic variation and amygdala reactivity may confirm existing rela-
tionships (e.g., the importance of genes biasing 5-HT signaling) or, more impor-
tantly, reveal unexpected candidate molecules or pathways (e.g., a gene producing
a molecule that is expressed in the brain and may function in second-messenger
signaling cascades). Once identified and, ideally, replicated in large-scale
databases that effectively address confounds common to genome-wide screens
(e.g., controlling for multiple comparisons resulting from testing the association
of a phenotype with hundreds of thousands or millions of SNPs), the impact of
variation in novel genes associated with amygdala reactivity can be explored at
each level of the biological cascade leading to trait anxiety (i.e., be fed back into
the discovery loop outlined in the introduction). In addition to exponentially
improving our understanding of neurobiological pathways leading to individual
differences in complex behavioral traits these efforts may lead to the discovery of
novel therapeutic strategies targeting related disease processes.
Acknowledgment This manuscript is largely based on an earlier publication in the Annual

Review of Neuroscience (Hariri 2009).
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Behavioural Genetics of Childhood
Disorders
Christine M. Freitag, Philip Asherson and Johannes Hebebrand
Abstract After a general introduction into genetic risk factors for child psychi-
atric disorders, four specific child psychiatric disorders with a strong genetic
component, namely, Autism Spectrum Disorders, Attention Deficit / Hyperactivity
Disorder, Nocturnal Enuresis, and obesity, are discussed in detail. Recent evidence
of linkage, candidate gene, and genome-wide association studies are presented.
This chapter ends with a prospectus on further research needs.

Keywords Autism spectrum disorders Attention deficit/hyperactivity disorder

Nocturnal enuresis Obesity BMI Linkage Association Genetic
C. M. Freitag (&)
Department of Child and Adolescent Psychiatry,
Psychosomatics and Psychotherapy, Goethe-Universität Frankfurt am Main,
Deutschordenstraße 50, 60528 Frankfurt am Main, Germany
e-mail: C.Freitag@em.uni-frankfurt.de
P. Asherson
MRC Social, Genetic and Developmental Psychiatry Centre,
Institute of Psychiatry, London, UK
e-mail: philip.asherson@kcl.ac.uk
J. Hebebrand
Department of Child and Adolescent Psychiatry,
University of Duisburg-Essen, Essen, Germany
e-mail: johannes.hebebrand@uni-due.de

DOI: 10.1007/7854_2011_178
Springer-Verlag Berlin Heidelberg 2012
Published Online: 1 March 2012
396 C. M. Freitag et al.
Contents
1 Introduction........................................................................................................................ 396
2 Selected Child Psychiatric Disorders with a Strong Genetic Component ...................... 401
2.1 Autism Spectrum Disorders ..................................................................................... 401
2.2 Attention-Deficit/Hyperactivity Disorder................................................................. 405
2.3 Nocturnal Enuresis.................................................................................................... 411
2.4 Obesity ...................................................................................................................... 412
3 Prospectus .......................................................................................................................... 415
References................................................................................................................................ 415
1 Introduction
Over the past 50 years, substantial progress has been made in linking specific
behavioural and psychiatric phenotypes to chromosomal aberrations or genetic
variation at the DNA level. Prerequisites of this development were significant
conceptual, methodological and technical advances in both molecular and statis-
tical genetics as well as in phenotypic assessment.
Advances in cytogenetics allowed the identification of specific syndromes based
on quantitative chromosomal imbalances of complete chromosomes, as in trisomy
21 in 1959, and of chromosomal regions, as in the cri du chat syndrome due to
monosomy 5p, in 1963. Approximately five megabases represent the resolution
limits of banding techniques; fluorescent in situ hybridisation (FISH) allows
detection of deletions as small as 1.5 Mb. Recently, microarray-based comparative
genomic hybridisation (array CGH) has been introduced as a complementary
method with even higher resolution (Miller et al. 2008).
Micro-deletions can result in syndromes with distinct behavioural features such as
the Williams-Beuren Syndrome (7q11.23), Prader-Willi syndrome (paternal 15q11),
Angelman syndrome (maternal 15q11), 16p11.2 deletion, the velocardiofacial/
DiGeorge syndrome (22q11) and 22q13.3 deletion syndrome (Xiang et al. 2010).
Because a single gene on average encompasses 10–15 (kb), the phenotypes of micro-
deletion syndromes result from the loss of a number of genes (partial monosomy).
Corresponding to micro-deletions, several micro-duplication syndromes exist,
e.g., corresponding to the Williams-Beuren Syndrome region at 7q11.23, and the
chromosome 16p11.2-duplication (Shen et al. 2010). The numerous findings of copy
number variations (CNVs) in recent whole genome studies on child psychiatric
disorders, as Attention-Deficit/Hyperactivity Disorder (ADHD) or Autism Spectrum
Disorders (ASD) (see below) emphasises the relevance of small cytogenetic findings
for psychiatric disorders.
Quantitative imbalances can functionally result in over expression (e.g., trisomy
or partial trisomy) or under expression (monosomy or partial monosomy) of those
genes located on a chromosome or within a specific chromosomal region. Due to the
high proportion of genes expressed in the central nervous system, e.g., *80% in mice
(Lein et al. 2007), brain function is almost always perturbed in such disorders.
Behavioral Genetics of Childhood Disorders 397
Findings in Down syndrome have revealed that of the approximately 200 genes
on chromosome 21, only a subset is responsible for the characteristic phenotype.
One well-known example is the early onset of dementia in subjects with Down
syndrome, which can partially be attributed to the over expression of the amyloid-
ß precursor protein gene (APP) located at 21q21-22. In addition to the quantitative
imbalance, the Down syndrome phenotype is affected by allelic variation; for
example, differences in length of a tetranucleotide repeat in intron 7 of the APP
locus explain substantial variation in age at onset of dementia in subjects with
trisomy 21 (Margallo-Lana et al. 2004).
The first large scale and systematic genotype–phenotype studies with a primary
behavioural, psychological and psychiatric focus centred on sex chromosome
(gonosome) disorders. Long-term follow-up of their development into adulthood
revealed that these disorders are characterised by subtle neuropsychiatric and
neuropsychological symptoms such as an IQ distribution shifted slightly to the left
and elevated rates of attention problems, speech and reading difficulties and
reduced impulse-control. Nevertheless, the dissection of a highly specific behav-
ioural phenotype associated with any one of these sex chromosome disorders was
not possible (Hebebrand 1990; Walzer 1985).
The ability to detect variation at the DNA level, e.g., mutations, formed the
basis for the successful elucidation of the molecular mechanisms underlying many
monogenetic disorders, which can entail more or less specific behavioural phe-
notypes. Examples are shown in Table 1. Frequently, such variation is detectable
in exons of genes underlying monogenetic disorders and simplistically either
entails that the respective gene product is structurally altered or not formed at all.
For example, missense mutations entail the substitution of the regular amino acid
at a specific position of the protein with another; this alteration of the amino acid
sequence of the respective protein can have functional implications at the levels of
the cell, tissue and organism. Two monogenetic disorders, which can cause autism,
tuberous sclerosis (TSC1-/TSC2-gene) and fragile X syndrome, have been studied
thoroughly, and due to the elucidation of the cellular pathways affected by a
lack of hamartin or tuberin (tuberous sclerosis) or the FMR1 proteine (fragile X
syndrome), new treatment options have been developed which are currently tested
in randomised controlled trials (Ehninger et al. 2009; Hagerman et al. 2009).
The majority of DSM-IV TR psychiatric disorders are most likely complex
implying that they only infrequently result from single gene mutations. Instead, it is
assumed that several gene variants interact in a complex manner with environmental
factors to produce the phenotype (Kendler 2005). Complex disorders typically show
higher concordance rates in monozygotic than dizygotic twins; concordance rates in
monozygotic twins are typically below 1 implying that environmental factors play a
role in the manifestation of the disorder. Family studies have shown that complex
psychiatric disorders are characterised by elevated recurrence risks in first and second
degree relatives which are below those expected for classical monogenetic dominant or
recessive traits. Moreover, for the complex psychiatric disorders a steep decline
in recurrence risks is observable between first and second degree family members.
In third degree relatives recurrence risks are usually only minimally elevated above
population based rates of the respective disorder (Hill et al. 2006; Knoblauch 2007).
398
Table 1 Examples of the influence of single gene disorders on cognition and behaviour
Monogenic Mode of inheritance Gene Cognitive phenotype Behavioural phenotype
disorder
Lesch-Nyhan X-chromosomal Hypoxanthine guanine Mental retardation Self-mutilative biting of fingers and lips
syndrome recessive phosphoribosyltransferase
(HPRT)
Tuberous Autosomal dominant Tuberous sclerosis-1 and -2 Learning difficulties, low Behavioural problems, autism, autism spectrum
sclerosis (TSC1/-2) IQ, mental retardation disorder
Fragile X X-chromosomal, triplet Fragile X mental retardation Mental retardation, Autism spectrum disorder, social phobia, ADHD
syndrome repeat expansion protein (FMR1)
Leptin deficiency Autosomal recessive Leptin (lep) Diminished perception of food reward, decreased
response to satiety signals, hyperphagia
Chorea Autosomal dominant, Huntingtin Dementia Depression, schizophrenia, personality change,
Huntington triplet repeat obsessive–compulsive behaviour
expansion
Phenylketonuria Autosomal recessive Phenylalanine hydroxylase Mental retardation Autism
(PAH)
Wilson disease Autosomal recessive Membrane copper-transport Psychiatric symptoms (personality changes,
protein (ATP7B) depression, psychosis)
Neurofibromatosis Autosomal dominant Neurofibromin Mental retardation ADHD
C. M. Freitag et al.
Table 2 Heritability estimates of selected behaviours and developmental milestones

Assessments Heritability estimates
Behaviours
Dieting EAT 42%
Body dissatisfaction EDI 52%
Drive for thinness EDI 44%
Disinhibition of eating TFEQ 40%
Restrained eating TFEQ 28%
Hunger TFEQ 28%
Obsessive compulsive behaviour CBCL 45–58%
Developmental milestones
Motor Crawling, sitting 0–90%
Development Standing, walking
Female Male
Expressive language ‘‘vocabulary’’ MCD-I-R 8% 20%
‘‘Two-word-combination-use’’ MCD-I-R 28% 10%
EAT= Eating Attitudes Test; EDI= Eating Disorder Inventory; MCD-I-R= MacArthur
Communicative Development Inventories-Short Form; CBCL= Child Behaviour Checklist;
TFEQ= Three Factor Eating
Table adapted from Hebebrand et al. (2010)
Within child and adolescent psychiatry, most twin, family and adoption studies
have been carried out for attention-deficit/hyperactivity disorder (ADHD) (Freitag
et al. 2010a). ADHD is also noteworthy because large population-based twin
studies have analysed quantitative dimensions of the disorder; complex gene-
environment twin studies have also been performed. For many behavioural traits
and developmental milestones heritability estimates based on categorical or
dimensional (quantitative) data indicate that overall approximately half of the
variance is explained by genetic factors (Plomin 2005), the other half by the
environment (Table 2). For many behavioural traits the environmental influences
are predominantly unique effects that explain differences for familial traits; with
some exceptions, such as conduct problems, where the familial environment also
plays an important role. It is worth noting that in the absence of direct evidence for
the impact of the familial environment on a trait, there may still be a role for such
influences through gene–environment interactions, which are part of the heritable
component in genetic model fitting studies.
For most psychiatric disorders, which are usually assessed categorically,
genetic factors have also been shown to play an important role (Table 3).
Heritability estimates typically exceed 0.5. ADHD and autistic disorders have been
shown to be one of the most highly heritable child and adolescent psychiatric
disorders (Asherson et al. 2005; Freitag et al. 2010a, b; Hebebrand et al. 2010).
Knowledge of the magnitude of the genetic basis of a particular disorder is
valuable for interpreting psychiatric findings within a patient’s family and for
probing for specific disorders in relatives of the index patient.
Until recently, candidate gene and linkage studies dominated the attempts to
uncover genetic variation underlying such complex disorders. However, viewed in
Table 3 Heritability estimates of selected psychiatric disorders

Disorder Heritability estimates Reference
PDD 70–90% Freitag (2007)
Enuresis 67–70% Von Gontard et al. (2001)
Conduct Disorder 53% Gelhorn et al. (2005)
OCD 47% Bolton et al. (2007)
Anxiety Disorders 30–40% Eley et al. (2003)
ADHD 70–80% Faraone et al. (2005)
Anorexia Nervosa 48–88% Hinney et al. (2010)
Bulimia Nervosa 28–83% Hinney et al. (2010)
Schizophrenia 73–90% Sullivan et al. (2003)
Bipolar Disorder 85% McGuffin et al. (2003)
Major Depression 31–42% Sullivan et al. (2000)
Table adapted from Hebebrand et al. (2010)
OCD= Obsessive Compulsive Disorder; PDD= Pervasive Developmental Disorders (including
autistic disorder, Asperger disorder, disintegrative disorder and PDD Not Otherwise Specified);
ADHD= Attention-Deficit and Hyperactivity Disorder
retrospect it can be concluded that these large scaled efforts were largely unsuc-
cessful; progress in the molecular dissection of complex psychiatric phenotypes
proved to be exceedingly slow for a period of over 20 years (Burmeister et al. 2008).
Candidate gene studies could only be performed for those genes for which an a priori
hypothesis existed as to their relevance for the respective disorder; obviously for
each disorder this represented only a very limited number in light of the totally
known number of human genes. In addition, candidate gene studies in different
psychiatric disorders frequently focussed on the same set of genes of a particular
neurotransmitter system, such as dopamine and serotonin transporters and receptors.
In other words, the candidate gene studies reflected the paucity of hypotheses as to
the underlying pathways involved in complex psychiatric disorders.
In 2006, the first genome-wide association studies (GWAS) based on DNA chip
technology were introduced (Hardy et al. 2009). Whereas it is still too early to judge
the total insight that this novel technology will provide into the pathogenesis of
complex disorders, we can nevertheless already conclude that GWAS have entailed a
paradigm shift, thus justifying the nomination as ‘breakthrough of the year’ by
Science magazine in 2007 (Pennisi 2007). For many complex somatic and neuro-
psychiatric disorders, novel genes have been detected which provide initial insights
into frequently unknown pathways involved in the respective disorders. For many
disorders, different groups pooled their data to come up with several thousand cases
and controls, such numbers had almost never been analysed in the pre-GWAS era.
A major finding has been that the effect sizes of validated trait or disease-related
SNPs are in most cases extremely small. According to a recent synopsis (Hindorff
et al. 2009) the median odds ratio was 1.33 with an interquartile range of 1.2–1.61,
although more recent evidence for very large-scale studies of traits such as height and
body mass index in samples of 100,000–200,000 individuals, indicate effect sizes
equivalent to odds ratios of 1.1 or less, with hundreds or risk alleles involved
(Speliotes et al. 2010).
Developmental aspects represent a key feature of child and adolescent psychi-

atry. The unfolding of gene expression provides virtually all of the information
necessary to guide the orderly succession of events underlying the development of
any organism and the central nervous system in particular. A behavioural trait or the
symptoms of any given mental disorder are more uniform for a specific develop-
mental stage than across all of infancy, childhood and adolescence; in addition,
comorbidity is dependent on developmental stage. Many traits and disorders must
be viewed in the context of brain development, elucidation of the underlying
molecular mechanisms will contribute to the identification of genes involved in
normal development of the central nervous system and its function. Dyslexia genes,
which are involved in global brain-development processes such as neural migration
and axonal guidance represent just one such example (Scerri et al. 2010).
Specific disorders run their course during specific developmental phases.
Examples include nocturnal enuresis which at age 7 affects approximately 10%
and at age 18 only 1% (von Gontard et al. 2001). The reduction of hyperactivity in
ADHD during adolescence is another example (Kessler et al. 2005). Both anorexia
and bulimia nervosa rarely start in childhood and only infrequently persist beyond
30 years of age (Holtkamp et al. 2005); and in Tourette’s disorder both comorbid
disorders and the development of the tics show age-related patterns. It appears
probable that alterations in expression levels of specific genes partially account for
disorder specific manifestation ages and the symptom development over time.
2 Selected Child Psychiatric Disorders with a Strong Genetic

Component
Genetic findings in four exemplary child psychiatric disorders, ASD, ADHD,

nocturnal enuresis (NE) and obesity will be presented in more detail in the second
part of this chapter.
2.1 Autism Spectrum Disorders
ASD are characterised by social interaction and communication difficulties as well

as stereotyped behaviour and special interests. ASD show a heritability of around
80–90% (Freitag 2007; Lichtenstein et al. 2010). Linkage has been found in at least
two independent studies in regions 2q21–33, 3q25–27, 3p25, 4q32, 6q14–21,
7q22, 7q31–36, 11p12–13, 17q11–21 (Alarcon et al. 2008; Duvall et al. 2007;
Freitag 2007; Ma et al. 2007; Schellenberg et al. 2006; Szatmari et al. 2007).
A meta-analysis confirmed the region 7q22–32, and reported suggestive evidence
for linkage to 10p12–q11.1 and 17p11.2–q12 (Trikalinos et al. 2006).
Following linkage studies, genes in replicated linkage regions have been
screened for mutations or common variants increasing the risk for ASDs. In the
following overview on candidate gene association studies, only replicated findings

of genes in linked regions will be presented.
Chromosome 2
Different common variants in the mitochondrial aspartate/glutamate carrier

(SLC25A12) gene located on 2q24 were associated with ASD in several studies
(Freitag 2007; Silverman et al. 2008).
Chromosome 3
Oxytocin plays a crucial role in social cognition and behaviour (Donaldson

et al. 2008) and the oxytocin receptor gene (OXTR) is located under the linkage
peak on 3p25. Several single nucleotide polymorphism (SNP) alleles and haplo-
types of OXTR were associated with ASD (Jacob et al. 2007; Lerer et al. 2008;
Wu et al. 2005).
Chromosome 6
Three studies found evidence for association of different SNP genotypes

or alleles in the Glutamate receptor 6 (GluR6) gene under the linkage peak on
chromosome 6 (Freitag 2007).
Chromosome 7
Most candidate genes assessed in ASD are located on chromosome 7q22–36, as

this area is best replicated from linkage studies. Mutations or variants in the
following candidate genes could not clearly be replicated as risk factors for ASD
(Freitag 2007): FOXP2 (a gene which was mutated in a severe monogenic form of
speech and language impairment in one family) (Fisher et al. 1998), RELN
(neuronal migration, formation of cortical layers, synaptogenesis), PTPRZ1
(highly expressed during embryogenesis), NRCAM, WNT2 and HOXA1 (hindbrain
development in mouse).
The RELN (Reelin) gene codes for a signalling protein that plays a crucial
role in neuronal migration, formation of cortical layers and synaptogenesis.
Three studies supported the involvement of a trinucleotide repeat polymorphism
in the 50 UTR region in Reelin in ASD (Persico et al. 2001; Serajee et al. 2006;
Skaar et al. 2005), whereas five other, at least similarly powered, studies did not
(Bonora et al. 2003; Devlin et al. 2004; Krebs et al. 2002; Li et al. 2004; Zhang
et al. 2002).
Different rare and common, possibly functional variants in LAMB1 were
associated with ASD in two studies (Bonora et al. 2005; Freitag 2007; Hutcheson
et al. 2004). LAMB1 encodes the b1 chain of laminin, which is an important
glycoprotein promoting neuronal migration and neurite outgrowth in the devel-
oping nervous system.
Different alleles of two SNPs in the Engrailed 2 (EN2)-gene on chromosome

7q36 were associated with ASD in several independent samples (Brune et al. 2008;
Freitag 2007; Wang et al. 2008; Yang et al. 2008). EN2 is a homeobox tran-
scription factor which plays a role during the development of cerebellar and
brainstem functions.
Common variants as well as rare mutations in the contactin-associated protein-
like 2 (CNTNAP2), a member of the neurexin superfamily involved in cell-
adhesion and neuronal migration (Alarcon et al. 2008; Arking et al. 2008;
Bakkaloglu et al. 2008) also increased the risk for ASD in several independent
samples. Gene-expression analyses in the developing human brain identified
CNTNAP2 as enriched in circuits important for language development.
The gene encoding the pleiotropic MET receptor tyrosine kinase plays a role in
brain development and gastrointestinal repair. As some individuals with ASD
suffer from gastrointestinal symptoms (GIS), this gene was specifically assessed in
individuals with ASD and GIS. A functional promotor variant, several other SNP
genotypes or alleles as well as rare mutations were associated with ASD in several
independent samples, predominantly in individuals with GIS (Campbell et al.
2006, 2009; Sousa et al. 2009). In a post-mortem brain protein expression analysis,
ASD individuals showed lower levels of the MET protein compared to controls
(Campbell et al. 2007).
Chromosome 17
Due to findings of platelet hyperserotonemia in children with autism and their

first-degree relatives, common variants in the Serotonin-transporter gene
(SLC6A4) were assessed by several studies (Freitag 2007). A recent meta-analysis,
however, did not report an effect of 5-HTTLPR and STin2 alleles on ASD risk
(Huang et al. 2008).
In addition to association of common variants, co-occurrence of ASD and
single gene disorders has been observed for a long time. The most prevalent single
gene disorders in ASD are tuberous sclerosis (TSC1/TSC2; around 1%) and fragile
X syndrome (around 3–5%). More rare (1%), but medically treatable single
gene disorders are phenylketonuria (PKU) and Smith-Lemli-Opitz (SLO) syn-
drome caused by mutations in 7-dehydrocholestrol reductase (DHCR7) (Baieli
et al. 2003; Freitag 2007; Sikora et al. 2006). The rate of ASD in these disorders
also is increased, but ASD is not observed in all individuals carrying the mutation:
fragile X syndrome ca. 25% (males), tuberous sclerosis ca. 20%; SLO ca. 50%;
PKU ca. 10% (Abrahams et al. 2008). Mutation screening and subsequent asso-
ciation studies have elucidated other rare causes of (most likely) single gene
disorders in individuals with ASD. Despite rare positive linkage findings for loci
on the X-chromosome, several variants in genes on the X-chromosome were
assessed for association with ASD, as the sex distribution in ASD is markedly
skewed (male:female = 4:1). Two neuroligin genes on Xq13 and Xp22 were
screened for mutations in several studies. Neuroligins are essential components of
synaptogenesis. Despite the findings of several non-conservative mutations in
single families in NLGN3 and NLGN4 these could not be replicated in larger
samples of individuals with ASD (Freitag 2007). Similarly, two mutations iden-
tified in the ribosomal protein gene RPL10 on Xq28 were not replicated in a
subsequent study (Gong et al. 2009; Klauck et al. 2006).
For a long-time suspected causes of heterogeneity, ASD are chromosomal
abnormalities which can be observed by standard karyotyping (Vorstman et al.
2006b). With a rate of approximately 1%, the most prevalent cytogenetic abnor-
malities are observed on chromosome 15q11-13 (Depienne et al. 2009; Freitag
2007) in most cases maternally, but also paternally inherited duplications. Further,
relatively frequent findings are deletions of chromosome 2q37, chromosome 7q31
and deletions or duplications of chromosome 22q13. In addition, Klinefelter
Syndrome (XXY) as well as duplications of the Williams-Beuren Syndrome region
7q11.23 and deletions of 22q11 (Velo-cardio-facial syndrome) are associated with
increased autistic traits (Berg et al. 2007; van Rijn et al. 2008; Vorstman et al.
2006a).
Due to technological advances, it is now possible to also assess small cyto-
genetic abnormalities (‘copy number variations’ CNVs) not detected by standard
karyotyping (Henrichsen et al. 2009). Recent publications show that some–espe-
cially rare–CNVs are observed more frequently in ASD patients compared to
control subjects (Bucan et al. 2009; Glessner et al. 2009; Kumar et al. 2008;
Marshall et al. 2008; Mefford et al. 2008; Pinto et al. 2010; Sebat et al. 2007;
Weiss et al. 2008). Similar to the cytogenetic findings obtained by standard
karyotyping, most CNVs represent rare, unique events rather than representing
recurrent deletions or duplications. Replicated rare CNVs from genome-wide
studies, which were observed more frequently in ASD compared to control
individuals, are located on the following chromosomes: 1q21, 2p16.3 (NRXN1),
3p25–26 (CNTN4), 7q36.2 (DPP6), 15q11–13 (UBE3A, OR4M2, OR4N4); 16p11.2
(MAPK3, MAZ, DOC2A, SEZ6L2, HIRIP3, IL6); 22q11.2; X (DDX51-PTCHD1;
maternally inherited). Some of these CNVs were observed also more frequently in
individuals with mental retardation or schizophrenia than in controls. ASD specific
CNVs were not exclusively observed in ASD individuals with specific dysmorphic
features or mental retardation but were also present in high functioning patients with
autism with only minor dysmorphology (van der Zwaag et al. 2009).
Molecular genetic studies in ASD have come a long way from the early linkage
studies, which aimed at describing a few loci and subsequently finding one or a
few genes of major effect relevant for all cases of ASD. It has now become clear
that ASD are heterogeneous disorders, caused by several rare—most likely—
monogenetic disorders (as fragile X syndrome, mutations in TSC1/TSC2, LAMB1,
CNTNAP2, PTEN, DHCR7, SHANK3, NLGN3/4 or RPL10). In addition, ‘con-
tiguous gene syndromes’ are likely causes of ASD, as the overall rate of rare
CNVs and large chromosomal deletions, duplications and translocations is
increased in individuals with ASD compared to controls.
Common variants, on the other hand, may shape the phenotype or eventually
may lead to the disorder by interacting with rare mutations or rare CNVs.
A mechanism like this has been shown for PTEN haploinsufficient individuals.
The serotonin-transporter gene SLC6A4 has been discussed as both an ASD sus-
ceptibility gene and a second-site modifier in ASD (Bartlett et al. 2005; Hessl et al.
2008). A study in PTEN haploinsufficient mice (Page et al. 2009) demonstrated
that the phenotypes of these mice were modified in an additive fashion by SLC6A4
haploinsufficiency. In addition, the role of PTEN in the maintenance of genomic
stability (Shen et al. 2007; Stiles 2009) makes it likely that PTEN haploinsuffi-
ciency may increase the probability of a secondary modifying event, such as a
copy number variation in a chromosomal region relevant to ASD. Common
variants also might increase the risk for autistic traits in the general population as
well as for less severe autistic disorders as Asperger Syndrome or PDD-NOS.
From results of current genetic findings in ASD, it is likely that mutations or
common variants in genes coding for gene products involved in (1) cell–cell
interaction and synaptic function, including development of dendritic spines, (2)
neuronal migration and growth or (3) excitatory and inhibitory neurotransmission
are causes of ASD. The pathway influencing cell–cell interaction and synaptic
function includes NRXNs, NLNGs, CNTN3/4, CNTNAP2 and SHANK3. In addi-
tion, the FMR protein, which is missing in fragile X syndrome, modulates den-
dritic spine formation and synaptic plasticity by inhibiting mGluR1/5-mediated
dendritic protein synthesis (Hagerman et al. 2009). Neuronal migration and growth
are influenced by gene products of LAMB1, EN2 or the MET receptor tyrosine
kinase gene. The mTor/PI3-kinase (PI3 K) pathway involves PTEN, TSC1/2 and
several other genes, which were observed in rare CNVs in individuals with ASD
(Cusco et al. 2009). It strongly influences (neuronal) cell growth. Gene products
influencing the regulation of excitatory and inhibitory neurotransmission are
GABA and glutamate receptors. In addition, disbalance of excitatory and inhibi-
tory neurotransmission was also observed in fragile X syndrome. Clearly, this list
of possibly involved pathways is not exhaustive, and other mechanisms or path-
ways may emerge as results of further studies will be published.
2.2 Attention-Deficit/Hyperactivity Disorder
ADHD is characterised by age-inappropriate hyperactivity, impulsivity and attention

problems (American Psychiatric Association 2000). The disorder usually starts in
early childhood and persists into adults in around two-thirds of cases (Faraone et al.
2006). It is thought to be caused by the interplay of genetic and environmental risk
factors and shows a heritability of 60–80% (Freitag et al. 2010a). A recent meta-
analysis on twin studies in ADHD has reported additive and dominant genetic as well
as non-shared environmental effects in ADHD (Burt 2009), a finding, which was
discussed and challenged by a comment, emphasising the role of appropriate data
transformation and analysis as well as the limitations of models obtained on twins
reared together, possibly underestimating common environmental effects obtained
from twin studies on ADHD (Wood 2010). Nevertheless, based on current data
common (familial) environment is not thought to play an important role in ADHD.
The genetic effects on the development of ADHD throughout the life span are
not well understood. The heritability of self-rated ADHD symptoms in adults is far
lower than that reported for children and adolescents, based on parent and teacher
reports; being around 30% (Boomsma et al. 2010) compared to the average
heritability in children of around 76% (Faraone et al. 2005). This may be a
measurement artefact, based on self versus informant ratings, and confounding
with adult onset disorder that might mimic some of the symptoms of ADHD, or
might reflect a growing impact of unique environmental effects on the long-term
course of ADHD into adulthood. Further work is needed to clarify this important
question.
In terms of the genetic architecture, a segregation study resulted in a model
implicating non-Mendelian major gene effects with low penetrance in ADHD
(Maher et al. 1999). However, these findings are partly in contrast to the obser-
vation of twin studies, which point to a model implying multiple common genetic
variants of small effect in the aetiology of the disorder (Levy et al. 1997).
Results of hypothesis free molecular genetic studies are currently supportive of
both models, too, indicating clear genetic heterogeneity in ADHD. Linkage studies
elicited several loci likely containing rare genetic variants of major effect only
present in single large families, but also common variants of smaller effect present
across several families (Romanos et al. 2008). In addition, genome-wide associ-
ation studies indicate a very limited number of common variants of small effect to
date (Franke et al. 2009; McCarthy et al. 2008) and rare copy number variants that
confer greater (moderate) levels of risk (Elia et al. 2010; Williams et al. 2010).
We therefore conclude that on the basis of current data, both types of genetic
effects likely play a role with evidence for both common risk alleles of minor
effect, and variants that are individually rare in the population but have a larger
impact on risk for the disorder. As we shall see much more work is needed to
clarify the balance between these two types of genetic effects and it remains
feasible that at least some of the risk for ADHD is conferred by many individually
rare alleles of minor effect; which would be extremely difficult to detect.
Another question that can be addressed by quantitative genetic approaches is
the aetiological relationship between inattention and hyperactivity-impulsivity.
Twin studies that have investigated this have found that these are separable traits
with only partially overlapping genetic influences. While the correlation between
the two domains is largely accounted for by shared genetic effects there are in
additional unique genetic effects acting on each of the two domains (McLoughlin
et al. 2007). Genetic investigation of the two domains would therefore be expected
to provide some evidence for unique as well as shared aetiological pathways.
In support of this general conclusion we see differential relationships between
each of the two domains and comorbid traits. One example is reading disability
(RD) and inattention where the correlation between ADHD and RD was found to
be largely driven by genetic factors not shared with hyperactivity-impulsivity
(Paloyelis et al. 2010). A contrasting example is a recent study of ADHD
and oppositional behaviour that found very highly phenotypic and genetic
correlations with hyperactivity-impulsivity but much lower with inattention
(Wood et al. 2009). These data suggested that in middle childhood ADHD and
conduct problems may represent the same underlying liability, distinct from the
inattentive domain.
Twin studies have found that there are many other examples of overlapping
genetic influences between ADHD symptoms and comorbid disorders and traits
including autism spectrum disorder (Ronald et al. 2008), motor coordination
(Francks et al. 2003), conduct disorder (Thapar et al. 2001) and depression (Cole
et al. 2009) among others. These studies demonstrate the complexity of shared
and unique genetic and other aetiological influences among many mental health
disorders and comorbid traits. At a basic level further work using longitudinal
twin designs can further delineate the causal relationships between ADHD and
co-occurring traits which include pleiotropic effects (multiple effects of individual
sets of genes), mediating effects (brain functions or behaviours that mediate
genetic effects on ADHD) and risk models (one disorder leading to another).
Dopaminergic Candidate Gene Studies
Molecular genetic studies on ADHD started with candidate gene association

studies in the mid 1990s with the first two reported associations between repeat
length polymorphisms in the dopamine D4 receptor (DRD4) (LaHoste et al. 1996;
Swanson et al. 1998a, b) and dopamine transporter (DAT1) genes (Cook et al.
1995). Subsequently association was reported with a microsatellite marker near to
the dopamine D5 receptor gene (DRD5) (Lowe et al. 2004). Since then there have
been numerous further studies with relatively few independent replications.
However, meta-analysis of available data reports strong evidence for the associ-
ation of dopamine system genes, in particular with the 7-repeat allele of a variable
number tandem repeat (VNTR) polymorphism within DRD4 and the microsatellite
which lies upstream of DRD5. These two associations are significant because they
are as yet the only genetic association findings to reach genome-wide levels of
significance, in the meta-analytic study of Li et al. (2006), which is generally
accepted as the gold standard for determining that a genetic association is true.
The recommended level of significance, in the region of 5 9 10-8 (Dudbridge
et al. 2008) is important because it means that in a systematic screen of the genome
for association, these findings would occur less than 5% of the time by chance
alone; whereas conventional levels of significance such as 0.05 would be detected
by chance every 20th selection of a random independent genetic polymorphism, of
which there are hundreds of thousands across the genome. We can therefore be
confident that these two findings are truly associated with ADHD.
The other genetic association that is often cited in the previous literature is
between the 10-repeat allele of a VNTR within the 30 -UTR of the dopamine
transporter gene (DAT1). However, the evidence for this association from meta-
analytic studies is far from convincing with three negative reports (Li et al. 2006;
Maher et al. 2002; Purper-Ouakil et al. 2005) while three other reports found only
weak evidence of association (Faraone et al. 2005; Gizer et al. 2009; Yang et al.
2007) far below the stringent criteria reached by DRD4 and DRD5. Whether DAT1
is associated with ADHD therefore remains a contentious question, yet there is

evidence for heterogeneity across the datasets included in the meta-analytic studies
suggesting that there may be identifiable sources for differences in the strength of
the association across the various studies (Gizer et al. 2009; Li et al. 2006).
One potential source of error in the analysis of DAT1 comes from undetected
genotyping errors that cause apparent over transmission of common alleles when
using the transmission disequilibrium test (TDT). Mitchell and colleagues found
that in studies reporting a positive association using the TDT, 87% identified an
association with alleles that were present at a frequency of [50%, whereas in
case–control studies, the proportion of common allele associations was 40%
(Mitchell et al. 2003). This highly significant difference could only be explained by
systematic genotype error related to the method, especially with high frequency
alleles such as the DAT1 10-repeat which occurs on around 70% of chromosomes
from people of European ancestry.
There are, however, more interesting sources of heterogeneity for DAT1. First,
recent studies of ADHD in adults find evidence for association with the 9-repeat
rather than the more common 10-repeat allele, suggesting that the effects of DAT1
may differ depending on the developmental age, indicating a role for DAT1 in the
modulation of symptoms at different ages, rather than the aetiology of ADHD
(Franke et al. 2008). Second, the DAT1 VNTR has been found to interact with
measures of the pre-natal environment including maternal use of tobacco during
pregnancy on oppositional and hyperactive-impulsive symptoms (Becker et al.
2008; Kahn et al. 2003) and maternal use of alcohol during pregnancy (Brookes
et al. 2006). Interestingly, recent research has shown that when genetic factors are
controlled for studies of the association mothers smoking during pregnancy
and ADHD, the association appears to be accounted for mainly genetic factors,
suggesting that the environmental measure may be correlated with maternal genes
(Thapar et al. 2009); suggesting that the observed interactions with DAT1 may be
explained by gene–gene interactions.
Third, there is evidence that two or more haplotypes of DAT1 may be involved
in the association with ADHD, indicating that more than one genetic variant is
likely to be involved. Further, the known VNTR may not be the primary functional
variant but may tag other genetic variants; and there appears to be more than
associated region with the gene (Asherson et al. 2007; Brookes et al. 2006, 2008).
Strength of the association would then depend on the relationships between the
various functional genetic variants involved, which may differ across sample
populations. Fourth, there is evidence that the association with genetic variants
across DAT1 may be specific to non-comorbid ADHD and is not found in children
with ADHD plus conduct disorder (Zhou et al. 2008a).
Other Candidate Gene Association Studies
There have now been numerous candidate gene association studies in ADHD
and these are best summarised in the recent review and meta-analysis from Gizer
et al. (2009). They conducted a systematic review of the association literature with
the aim of identifying the most consistent findings in ADHD research to date.
They further tested for heterogeneity across studies because other genes, like
DAT1, might show significant sources of variability due to identifiable moderating
factors. Overall they concluded that the following genes were significantly asso-
ciated with ADHD: DAT1, DRD4, DRD5, 5HTT (serotonin transporter), HTR1B
(serotonin 1B receptor) and SNAP-25 (synaptosomal associated protein); and the
following genes showed significant evidence of heterogeneity: DAT1, DRD4,
DRD5, DBH, ADRA2A (adrenergic 2A receptor), 5HTT, TPH2 (Tryptophan
hydroxylase 2), MAOA (monoamine oxidase A) and SNAP25. For the candidate
genes showing heterogeneity future studies could usefully investigate the potential
moderating factors that lead to variability in effect size across studies.
Despite significant progress, the total estimated impact of the most replicated
candidate gene findings is around 3.3% of the phenotypic variance of ADHD;
accounting for 4.3% of the estimated average heritability of ADHD of 76%
(Kuntsi et al. 2006). Further work is clearly needed to explain the rest of the
genetic influences on ADHD.
Genome-Wide Association Studies (GWAS)
Other approaches to the identification of novel genes for ADHD include

genome-wide association studies which have the potential to detect entirely novel
associations where there is no previous a priory hypothesis. Given that we know
so little about the function of the brain and how molecular processes lead to sus-
ceptibility for ADHD, it makes sense for aetiological research to focus efforts on
empirical approaches that systematically screen the entire genome for associations.
The first GWAS study of ADHD investigated 438,784 SNPs in 958 combined
type ADHD proband–parent trios. No genes of moderate to large effect were iden-
tified and no findings passed genome-wide levels of significance (Neale et al. 2008).
However, when a set of 51 candidate genes were investigated, there was significant
evidence at the group level that positive association signals were emerging from the
selected SNPs, implicating mainly dopamine, noradrenalin and serotonin neuro-
transmitter genes. Similar findings were subsequently reported in a study that
combined genome-wide association data from several studies, with a total sample
size of 2,064 trios, 896 cases and 2,455 controls (Neale et al. 2010), confirming the
important role of the traditional neurotransmitter systems genes in ADHD.
Overall, the total number of samples analysed in GWAS studies of ADHD to
date is far too small and the disappointing findings to date are not unexpected.
Further work is required to obtain GWAS information on much larger sets of
samples for ADHD. There are, however, potentially interesting new findings that
emerge from the GWAS studies of ADHD (Franke et al. 2009). Of particular
interest is the Cadherin gene (CDH13) which was found to be associated with
ADHD in two out of five GWAS studies (Lasky-Su et al. 2008; Lesch et al. 2008)
and lies within the only region that reached genome-wide significance in a meta-
analysis of linkage studies of ADHD (Zhou et al. 2008b). Furthermore CDH13 is
among the most consistent findings on a wide range variety of phenotypes related
to drug abuse and dependence (Uhl et al. 2008). This finding and other hints from
GWAS indicate that genes involved in cell division, cell adhesion, neuronal
migration and neuronal plasticity may also confer risk for ADHD (Franke et al.
2009).
Overall there is a long way to go to delineate the specific genetic factors
that explain the high heritability of the disorder. This is, however, a common
phenomenon in common disorders research and several possible explanations for
the so-called ‘dark-matter’ of (missing) heritability has been put forward—dark
matter in the sense that we know it exists can detect its influences but simply
cannot see it (Manolio et al. 2009). Potential reasons include numerous genes of
very small effect, genetic heterogeneity with risk conferred by many different
genes and variants within genes, higher order interactions between genes and with
environment and aetiological heterogeneity (Manolio et al. 2009).
Copy Number Variations
An important contribution to ADHD risk is rare copy number variants (CNVs)

or other types of rare genetic variation. Recent data suggested that in a few cases
CNVs may exert a major influence on risk for ADHD and that this risk overlaps
with other neurodevelopmental disorders, including schizophrenia and autism
(Elia et al. 2010). These initial findings have now been confirmed in one of the
most robust studies of ADHD genetics to data. Thapar and colleagues (Williams
et al. 2010) investigated a sample of 410 children with ADHD and 1,156 ethically
matched controls. The analysis focused on large CNVs greater than 500,000 base
pairs in size, that are robust to call from genome-wide SNP arrays, and further
validated all identified CNVs using a separate technology. Furthermore, findings
were evaluated in a further sample of 825 patients with ADHD from Iceland and
35,243 controls. There were several key findings from this paper. First, the overall
burden of risk was increased, with 16% of ADHD cases carrying CNVs, compared
to 8% of controls, giving an overall twofold risk. Second, the increased risk was
particularly striking for a subgroup of ADHD cases with intellectual disability:
42%, giving an approximate sixfold risk compared to the control population.
Third, that the burden of risk remained significant, but much lower, in the group
with no intellectual impairment, around 1.7-fold risk. Fourth, there was a clear
excess on chromosome 16p13.11, identifying a specific region. Finally, the
identified CNVs were significantly enriched for loci previously reported for
schizophrenia and autism.
Linkage Analysis and Genes of Major Effect
There is evidence that genes of major effect might lead to ADHD in a few rare
families. Furthermore, variation in some of these genes may have more general
effects on ADHD. There are two main examples.
The first is a gene called latrophilin 3 (LPHN3) that is located within a
linkage region on chromosome 4q13.2. This region was first identified in a large
multi-generational pedigrees from the Paisa population in Columbia (Arcos-Burgos

et al. 2004). Subsequent fine-mapping narrowed the region. Subsequently genetic
association analysis in worldwide samples totalling 2,627 ADHD cases and 2,531
controls confirmed the association of LPHN3 with an average odds ratio of 12,
although not yet at genome-wide levels of significance (Arcos-Burgos et al. 2010).
Since then further replication was reported in a sample of adults with ADHD, which
is interesting because the original extended pedigrees used to identify the linked
regions included both children and adults with ADHD (Ribases et al. 2010).
The second example comes may represent a private mutation found in one or a
few families in Germany (Lesch et al. 2010). To detect micro-deletions and micro-
duplications, they carried out a genome-wide screen for copy number variations in
a sample of 99 children and adolescents with severe ADHD. Among several genes
containing CNVs in ADHD, they identified the gene encoding neuropeptide Y
which was included within a 3 megabase region on chromosome 7p15.2–15.3.
Investigation of other family members found evidence that the duplication
co-segregated with ADHD.
Conclusion
Overall current molecular genetic studies of ADHD find evidence for both
frequent and rare variants that influence the risk for ADHD. The level of evidence
is sufficient to confirm that identity of some of the genes and gene regions
involved, although the data only explains a very small amount of the total genetic
variation that impacts on risk for ADHD. Future studies will need to include very
much larger sample sizes and take advantage of the improving technology to
screen for both common and rare variants. The specific findings confirm the old
neurotransmitter hypotheses of ADHD, but also extend the focus to many other
neurobiological processes that impact on brain development and brain function.
2.3 Nocturnal Enuresis
Nocturnal enuresis is characterised by bed wetting after age 5 years old in

otherwise healthy children after exclusion of urinary tract infections, malforma-
tions, other organic causes or intellectual disability (Von Gontard et al. 1997).
Population based twin studies have resulted in heritability estimates of around 70%
for NE (Hublin et al. 1998). Despite this relatively large heritability, there is a
scarcity of research into NE. Linkage studies have predominantly been performed
in large families with autosomal dominant inheritance pattern replicating loci on
4p16.1, 12q24.2, 13q13–q14.3, and 22q11.21 (Arnell et al. 1997; Eiberg et al.
1995, 2001; Eiberg 1995, 1998; Loeys et al. 2002; Von Gontard et al. 1999).
Hardly any fine-mapping or candidate gene association studies were performed.
Given that nocturnal enuresis can be a persistent disorder more research should be
done to elucidate its genetic basis, e.g., by sequencing the replicated segregating
loci in the respective families. Interestingly, a recent study reported higher rates of
nocturnal enuresis in individuals with schizophrenia, and individuals with enuresis
showed a worse performance on two frontal lobe cognitive tests compared to
individuals without (Hyde et al. 2008).
2.4 Obesity
Numerous twin, adoption and family studies have allowed for the calculation of
heritability estimates for body mass index (BMI), which typically range from 0.4
to 0.7; twin studies have consistently led to higher estimates. Based on complex
statistical modelling single family studies have nevertheless also led to estimates
in the range of 0.7 (Maes et al. 1997). A recent review of 13 longitudinal studies
found a strong genetic continuity in BMI from early childhood to onset of
adulthood (Silventoinen and Kaprio 2009). There was also evidence for an effect
of a common environment on the tracking of BMI during childhood; in adults the
influence of the non-shared environment substantially exceeded that of the shared
environment.
Monogenic Obesity
The cloning and initial detection of mutations in the mouse and human
(Montague et al. 1997; Zhang et al. 1994) leptin gene marked the beginning of the
large-scale elucidation of DNA variation underlying inter-individual differences in
body weight. Currently, several types of monogenic forms of obesity have been
elucidated. All of the respective mutations are rare, some have only been reported
in single cases worldwide; the genes are all expressed in the hypothalamus. The
elucidation of such mutations and the successful treatment of leptin deficient
extremely obese (Farooqi et al. 1999) individuals has firmly established that the
behavioural phenotype hyperphagia/overeating and as a consequence obesity can
be caused by mutations in specific genes.
Several of the detected novel monogenic forms of obesity encompass other
clinically recognisable features. For instance, mutations in the leptin gene entail
hypothalamic hypogonadism, other endocrinological abnormalities and immune
system dysfunction (Hinney et al. 2008). In the original report of the clinical
features of patients with mutations in the pro-opiomelanocortin gene (POMC)
adrenal insufficiency and red hair were characteristic features in addition to
extreme obesity (Krude et al. 1998). Developmental delay was shown to co-occur
with severe obesity in a patient with a mutation in the neurotrophin receptor TrkB
(Yeo et al. 2004).
The detection of mutations in the melanocortin-4 receptor gene (MC4R)
(Hinney et al. 1999; Vaisse et al. 1998; Yeo et al. 1998) marked a turning point in
the sense that for the first time the molecular basis of a subgroup of patients with
idiopathic obesity was identified. The detection of the association between MC4R
mutations and obesity is also noteworthy because the combined frequency of all
functionally relevant mutations is in the range of 1–6% among extremely obese
children and adolescents (Hinney et al. 2003).
Two family-based studies have attempted to quantify the effect of functionally
relevant MC4R mutations (Dempfle et al. 2004; Stutzmann et al. 2008) by com-
paring BMI of mutation and wild-type carriers of relatives ascertained via the
index cases. According to Dempfle et al. (2004) and Stutzmann et al. (2008) adult
male carriers were 4 and 4.3 kg/m2 heavier than their male relatives with the
wildtype genotype. In females the mean effect size of MC4R mutations is larger;
the corresponding values were 9.5 and 8.7 kg/m2.
In the pre-GWAS era, the MC4R was the first gene shown to harbour a common
variant with a small effect size (Geller et al. 2004). The association of the minor
allele, coding for Ile103, with leanness was confirmed in subsequent larger
meta-analyses with up to almost 124,000 probands (Heid et al. 2008; Speliotes
et al. 2010; Wang et al. 2010a; Young et al. 2007). Roughly 3–6% of different
populations are heterozygous for this variant, which according to German popu-
lation-based data for adults entails a 0.5 kg/m2 reduction in mean BMI. This would
qualify this SNP as the one with the strongest effect size of all currently known
common variants including the SNPs in intron 1 of the fat mass and obesity
associated gene (FTO) (Frayling et al. 2007). A second coding MC4R variant also
protects from obesity (Stutzmann et al. 2007). The MC4R is thus currently unique
in that it harbours variants of considerably different effect sizes both within and
outside the coding region whose minor alleles both increase and decrease BMI.
There are over 90 known coding variants in the MC4R that lead to a reduced
receptor function seemingly support the common disease—rare variant hypothesis,
according to which a substantial proportion of the predisposition to complex
disorders is due to rare variants (Bodmer et al. 2008). Indeed, it seems likely to
assume that other such loci exist in the genome that cannot readily be picked up
via linkage or association analyses due to locus heterogeneity and the low overall
combined rate of such mutations at a specific gene locus.
Polygenic Obesity
The results of genome-wide association studies (GWAS) indicate that the

common disease—common variant hypothesis is potentially also important. Prior
to the most recent meta-analysis of GWAS, ten loci with genome-wide signifi-
cance defined via a p-value \5 9 10-8 had been identified (FTO, MC4R,
TMEM18, GNPDA2, BDNF, NEGR1, SH2B1, ETV5, MTCH2 and KCTD15;
Hinney et al. 2009; Willer et al. 2009). The most recent and up-to-date worldwide
largest meta-analysis of the GIANT (Genetic Investigation of ANthropmetric
Traits) Consortium included 46 studies in total encompassing 123,865 individuals
of European ancestry (Speliotes et al. 2010). In a joint analysis of the original data
and the confirmatory study encompassing data of up to almost 1,25,000 individ-
uals, a total of 32 SNPs revealed p-values \5 9 10-8.
The per allele change in BMI ranged from 0.06 (MTCH2, KCTD15, PTBP2,
RPL27A, NUDT3) to 0.39 (FTO) kg/m2; a total of 10 SNPs showed per allele
changes\0.1 kg/m2 equivalent to less than 324 and 289 g in males and females of
average heights (1.8 and 1.7 m). Five SNPs with the lowest per allele change of
0.06 kg/m2 each account for approximately one half of the aforementioned
weights of individuals of average height.
The 32 identified variants explained a mere 1.5% of the BMI variance; this
roughly corresponds to 3% of the genetic variance based on an estimated
BMI-heritability of 0.5. Speliotes et al. (2010) estimated that there are approxi-
mately an additional 200 loci (95% CI: 98–350) with similar effect sizes as the
detected 32, which together would account for roughly 3.5% of the variation in
BMI or 7% of the genetic variation.
Sub-analyses based on children and adolescents revealed a substantial overlap
with the genetic predisposition to adult obesity. Directionally consistent effects
were found for 23 of the 32 SNPs. Common variants detected in adults are
seemingly important throughout different developmental stages. In accordance
with the low heritability estimates for BMI in early life, a significant effect of the
FTO genotypes was not detectable in children prior to age 4 in two birth cohort
studies (Rzehak et al. 2010).
Because most obesity genes are expressed in the central nervous system, the
role of the brain in the regulation of body weight is seemingly substantiated.
However, the high proportion of all genes expressed centrally needs to be
considered prior to drawing firm conclusions. Pathway-based analyses based on
the genes identified via the association signals revealed evidence of enrichment for
pathways involved in the platelet-derived growth factor signalling, translation
elongation, hormone or nuclear hormone receptor binding, homeobox transcrip-
tion, regulation of cellular metabolism, neurogenesis and neuron differentiation,
protein phosphorylation and numerous other pathways related to growth, metab-
olism, immune and neuronal processes (Speliotes et al. 2010).
The Role of Copy Number Variants in Obesity
Willer et al. (2009) reported genome-wide significant association with BMI of a

SNP capturing a common 45-kb deletion near NEGR1 (neuronal growth regulator
1 precursor gene). Recently, another common CNV (chromosome 10p11.22)
was shown to be associated with BMI (Sha et al. 2009) in a Chinese sample (unad-
justed p-value 0.011). In contrast to the common CNV with relevance for BMI
reported above, three recent reports (Bochukova et al. 2010; Walters et al. 2010;
Wang et al. 2010b) showed the importance of rare large CNVs for body weight
regulation. Two studies (Bochukova et al. 2010; Walters et al. 2010) depicted a
genomic region on chromosome 16p11.2 which harbours highly penetrant
micro-deletions (about 500 kb) associated to (extreme) obesity. This region had
previously been associated with autism and mental retardation (Bochukova et al.
2010; Walters et al. 2010; Weiss et al. 2008); in fact, some of the obese patients
analysed by Farooqi and co-workers (Bochukova et al. 2010) additionally had
developmental disorders. However, Walters and co-workers found the association

of these microdeletions and obesity also in individuals ascertained only for the
obese phenotype, suggesting a possible direct association of the deletions at 16p11.2
with obesity, distinct from their implications for cognition.
Wang et al. (2010b) found that CNVs larger than 1 Mb were over-represented
in obese cases versus normal weight controls (non-significant odds ratio of 1.5)
with a stronger effect for larger CNVs (above 2 Mb). As the effect was more
pronounced for CNVs disrupting genes, these genes should be further analysed
(Wang et al. 2010b).
3 Prospectus
The genetics of four childhood conditions was elaborated in more detail in this
chapter. Other common psychiatric disorders of childhood, adolescence and
adulthood also show a heritability [50%, especially anxiety disorders. Gene–
environmental effects are likely for this condition, as it is also the case for ADHD
and obesity.
Research over the last 10 years in the genetic of child psychiatric disorders has
resulted in many promising findings, but also has shown that elucidating the
genetic background of psychiatric disorders is challenging and will require new
technologies. With sequencing approaches, methylation pattern assessment, and
functional studies, new and clinically relevant results are to be expected and will
ultimately result in more individualised and targeted treatment options.
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B Neuropsychiatr Genet 8:1392–1398
Schizophrenia Genes: On the Matter
of Their Convergence
Dan Rujescu
Abstract Schizophrenia is a common mental disorder, affecting 0.5–1% of

the population. The mode of inheritance is complex and non-Mendelian with a
high heritability of ca. 65–80%. Given this complexity, until most recently it was
difficult to identify disease genes. But fortunately this has changed. Due to new
technologies the last few years have brought highest interest in human genetics of
complex diseases. The knowledge resulting from the availability of the complete
sequence of the human genome, the systematic identification of single nucleotide
polymorphisms (SNPs) throughout the genome, and the development of parallel
genotyping technology (microarrays) established the conditions that brought about
the current successful time in our ability to probe the genome for identifying
disease genes. All these studies showed up new avenues for the biology of
common complex diseases and yielded a multitude of genes showing strong
association with complex diseases.
Keywords Schizophrenia Genetics CNV Copy number variant GWA

Polymorphism
Contents
1 Schizophrenia..................................................................................................................... 430
2 Structural Genetic Variants ............................................................................................... 430
3 Common Genetic Variants................................................................................................ 434
References................................................................................................................................ 436
D. Rujescu (&)
Department of Psychiatry,
Ludwig-Maximilians-University,
Nußbaumstr. 7, 80336 München, Germany
e-mail: Dan.Rujescu@med.uni-muenchen.de

DOI: 10.1007/7854_2011_183
430 D. Rujescu
1 Schizophrenia
A major challenge in medicine is to understand genetic, molecular and cellular

mechanisms underlying common mental disorders including schizophrenia, which
involve complicated genetic and environmental determinants. Schizophrenia is a
common mental disorder, affecting 0.5–1% of the population. Schizophrenia
mostly presents with several episodes and tends to become chronic. Approximately
30% of patients with schizophrenia require support throughout their lives. Roughly
50% will have lifelong disabilities and social problems. Its direct costs in western
countries range between 1.6 and 2.6% of total health care expenditures, and is the
seventh most costly medical illness to western societies. Active psychosis has been
ranked the third most-disabling condition after quadriplegia and dementia (Ustün
et al. 1999), and life expectancy is reduced by ten to twelve years, due to increased
physical health problems and a high suicide rate. There is evidence for a strong
genetic component in the etiology of schizophrenia, as demonstrated by family,
twin and adoption studies. The relative contribution of genetic factors has been
estimated to be ca. 80% (Cardno et al. 1999). The mode of inheritance is complex
and non-Mendelian (Ross et al. 2006). Given this complexity, together with
imprecise and differing definitions of phenotype, until most recently it was noto-
riously difficult to identify genes involved in this chronic disabling brain disease.
2 Structural Genetic Variants
Several approaches have been used to find causative genetic variants including
linkage studies, candidate gene or genome wide association studies as well as
studies on structural genetic variants. It is not a new assumption that structural
genetic variants including deletions or duplications may play a role in
schizophrenia. However, the assumption that rare genetic variants with large
effects may account for a significant number of schizophrenia cases has been
somehow neglected. This occurred despite the fact that rare variants with large
effects on schizophrenia risk are already well established, with the 22q11 deletion
syndrome and DISC1 being among the more prominent findings.
David St Clair et al. (1990) identified a balanced 1q43:11q14 translocation
associated with major mental illness in a large pedigree showing several psychi-
atric phenotypes associated with this translocation (St Clair et al. 1990). Within
this translocation a disrupted gene (DISC1) was identified which is now an
important risk gene for psychosis. Although the mutation is very rare, its discovery
boosted a series of studies on the potential pathobiology underlying psychosis.
Twenty years of intensive work went by and much research has been undertaken
to define the biological function of the DISC1 protein and to further understand
how it contributes to the pathogenesis of schizophrenia. There is evidence for
Schizophrenia Genes 431
a role in multiple cell functions including cAMP signalling, centrosomal and

microtubule-based functions, kenesin-mediated intracellular transport and neurite
extension which have led to its description as a ‘hub protein’ for both the
developing and adult brain (for review see Gill et al. 2009; Muir et al. 2008).
Interestingly, DISC1 expression is regulated by beta-site amyloid precursor protein
cleaving enzyme-1-neuregulin cascade (Seshari et al. 2010), implicating another
potential schizophrenia risk gene, neuregulin 1 (Stefansson et al. 2003) and its
receptors in the same schizophrenia risk pathway as DISC1 (for review see Gill
et al. 2010). DISC1 has multiple identified protein interaction partners that high-
light pathologically relevant molecular pathways. Amongst these are proteins
involved in neuronal migration (e.g. APP, Dixdc1, LIS1, NDE1, NDEL1), neural
progenitor proliferation (GSK3b), neurosignalling (Girdin, GSK3b, PDE4) and
synaptic function (Kal7, TNIK). Furthermore, emerging evidence of genetic
association (NDEL1, PCM1, PDE4B) and copy number variation (NDE1) impli-
cate several DISC1-binding partners as risk factors for schizophrenia (Bradshaw
and Porteous 2010).
A microdeletion at 22q11.2 has been repeatedly linked to schizophrenia
although it is responsible for the Velo-Cardio-Facial Syndrome (VCFS) also known
as DiGeorge syndrome. The first connections starting when Shprintzen et al. (1992)
reported schizophrenia-like psychotic symptoms in VCFS affected adolescents and
young adults, beside other characteristic symptoms like different physiognomy or
cardiac defects. Several follow up studies occurred and provided hope that this
deletion could serve as a model for schizophrenia (Karayiorgou and Gogos 2004).
A series of animal model and cell culture studies started. One major focus was
on the genes lying in this deleted region (for review see Gothelf et al. 2008;
Karayiorgou and Gogos 2004). One of the genes within this region is the COMT
(Catechol-O-methyl transferase) gene which encodes for the COMT enzyme
responsible for the degradation of dopamine, especially in the prefrontal cortex.
Although there is evidence for a contribution of COMT to neuropsychological and
neurophysiological domains, no clear picture is seen for a major contribution of
COMT in schizophrenia per se as demonstrated in a recent meta-analysis by
Okochi et al. (2009) in 13,088 cases and 16,531 controls. Most probably COMT
seems to be more generally involved in neuropsychological and neurophysiological
functions including executive functioning, working memory, fluid intelligence and
attentional control (for review see Dickinson and Elvevåg 2009).
Other genes which were intensively studied within the 22q11.2 region are e.g.
TBX1 and GNB1L. It has been shown that prepulse inhibition deficits in Df1/+
mice are caused by haploinsufficiency of these two genes TBX1 and GNB1L
(Paylor et al. 2006). In humans, sequence variation in GNB1L was associated with
gene expression and psychosis in VCFS (Williams et al. 2008). Furthermore,
mouse models of 22q11.2 deletion syndrome suggest that diminished dosage of
certain 22q11 genes disrupts neurogenesis and cortical development (Meechan
et al. 2009). Interestingly, Maynard et al. (2008) studied six genes in the 1.5 Mb of
the 22q11.2 region which encode mitochondrial proteins (Prodh, Slc25a1, Txnrd2,
T10, Zdhhc8). All six genes are expressed in the brain, and maximal expression
432 D. Rujescu
coincides with peak forebrain synaptogenesis shortly after birth. Furthermore, their
protein products are associated with brain mitochondria, including those in syn-
aptic terminals. The authors concluded that 22q11 deletion may alter metabolic
properties of cortical mitochondria during early post-natal life, and that several
22q11 mitochondrial genes, particularly during early post-natal cortical develop-
ment, may disrupt neuronal metabolism or synaptic signalling which seems highly
interesting for the pathophysiology of schizophrenia (Maynard et al. 2008).
These two regions were the most prominent large structural variants associated
with schizophrenia until recently. However, the rapid technological development
which paved the way for genome wide association studies provided new unex-
pected possibilities for the detection of shorter structural variants, namely for copy
number variants (CNVs). The first report on submicroscopic micodeletions or
microduplications in schizophrenia was provided by Walsh et al. (2008) and was
the starting point for the revival of structural aberrations in schizophrenia. Novel
deletions and duplications of genes were present in 5% of controls versus 15% of
cases and 20% of young onset cases. These mutations disrupted genes dispro-
portionately more often from signalling networks controlling neurodevelopment,
including neuregulin and glutamate pathways. Although the sample size for the
detection of these rare events was small, Walsh et al. (2008) suggested that
multiple, individually rare mutations altering genes in neurodevelopmental path-
ways contribute to schizophrenia.
The first well powered study on CNVs associating with schizophrenia was
performed by the SGENE+ consortium. A population based sample was used to
identify de novo CNVs by analysing 9,878 transmissions from parents to offspring
(Stefansson et al. 2008). The 66 de novo CNVs identified were tested for asso-
ciation in a sample of 1,433 schizophrenia cases and 33,250 controls. Three
deletions at 1q21.1, 15q11.2 and 15q13.3 showing nominal association with
schizophrenia in the first sample were followed up in a second sample of 3,285
cases and 7,951 controls and all three deletions were significantly associated with
schizophrenia and related psychoses in the combined sample. Interestingly, two of
these regions (1q21.1 and 15q13.3) could be replicated independently by
the International Schizophrenia Consortium in 3,391 patients with schizophrenia
and 3,181 controls (ISC 2008). The third (15q11.2) association has also been
replicated independently by Kirov et al. (2009a). These studies have brought
highest interest in human genetics of complex diseases and encouraged other
groups to analyse CNVs.
Another main finding in schizophrenia is that deletions within the neurexin 1
gene (NRXN1; 2p16.3) are associated with schizophrenia (Kirov et al. 2008;
Walsh et al. 2008). The SGENE+ consortium examined Neurexin 1 for CNVs in
2,977 schizophrenia patients and 33,746 controls from seven European populations
using microarray data. The association analysis was restricted to CNVs that disrupt
exons. These were significantly associated with a high odds ratio (OR 8.97),
showing that Neurexin 1 deletions disrupting exons confer risk of schizophrenia
(Rujescu et al. 2009). A meta-analysis which included 8,789 cases and 42,054
controls provided further evidence for the involvement of Neurexin 1 in Schizophrenia.
An excess of deletions [100 kb in patients (0.19 vs. 0.04%, p = 0.000013,

OR = 4.78) with higher ORs for deletions[100 kb that disrupt exons (p = 0.000037,
OR = 7.44) was detected (Kirov et al. 2009b).
Interestingly, these microdeletions are also found in patients with a broad
overlapping spectrum of other neurodevelopmental phenotypes like autism (Sebat
et al. 2007; Abrahams and Geschwind 2008; Weiss et al. 2008), mental retardation
(de Vries et al. 2005; Sharp et al. 2006; Murthy et al. 2007; Mefford et al. 2008),
epilepsy (Sharp et al. 2008) and further diseases (for review see (Cook and Scherer
2008; Ramocki and Zoghbi 2008; Slavotinek 2008). Therefore, a genomic region
on chromosome 16p13.1, which has been implicated in childhood-onset devel-
opmental disorders, was studied in schizophrenia by the SGENE consortium
(Ingason et al. 2011a). Deletions were detected in 0.12% of cases and 0.04% of
controls (p [ 0.05). Furthermore, also duplications were present in 0.30% of cases
versus 0.09% of controls (p = 0.007). The region can be divided into three
intervals defined by flanking low copy repeats. Duplications spanning intervals I
and II showed the most significant (p = 0.00010) association with schizophrenia.
The age of onset in duplication and deletion carriers among cases ranged from 12
to 35 years, and the majority were males with a family history of psychiatric
disorders. In a single Icelandic family, a duplication spanning intervals I and II was
present in two cases of schizophrenia, and individual cases of alcoholism, attention
deficit hyperactivity disorder and dyslexia (Ingason et al. 2011a).
Given that also duplications seem to be involved in the risk of schizophrenia,
another large study on copy number variants was conducted studying maternally
derived 15q11–q13 duplication overlapping the Prader-Willi/Angelman syndrome
critical region (Ingason et al. 2011b). In a discovery sample of 22 schizophrenia
patients with a very early onset of illness (10–15 years of age), one duplication
was observed in a patient. Based on this, 7,582 patients with schizophrenia or
schizoaffective disorder and 41,370 comparison subjects without known psychi-
atric illness were screened for copy number variants at 15q11–q13. Duplications
were found in further four patients and five comparison subjects. All four patients
had maternally derived duplications (0.05%), while only three of the five com-
parison duplications were maternally derived (0.007%), resulting in a significant
excess of maternally derived duplications in case patients (OR = 7.3). This excess
is compatible with earlier observations that risk for psychosis in people with
Prader–Willi syndrome caused by maternal uniparental disomy is much higher
than in those caused by deletion of the paternal chromosome. These findings
suggest that the presence of two maternal copies of a fragment of chromosome
15q11.2–q13.1 that overlaps with the Prader–Willi/Angelman syndrome critical
region may be a rare risk factor for schizophrenia and other psychoses. Given that
maternal duplications of this region are among the most consistent cytogenetic
observations in autism, the findings provide further support for a shared genetic
etiology between autism and psychosis (Ingason et al. 2011b).
Actually first whole-genome sequencing efforts are under way and raise hope
that further rare variants will be found. A recent study based on whole-genome
DNA sequencing data from 185 human genomes mapped over 22,000 deletions
434 D. Rujescu
and 6,000 additional CNVs. Over half of them could be mapped to nucleotide
resolution, which facilitates analysing their origin and functional impact. This
study shows that CNVs are abundant in humans, differing from other forms of
variation in extent, origin and functional impact (Milles et al. 2011). Therefore it is
very possible that based on these new findings, rare new genomic disorders will be
defined and that our understanding of neurodevelopmental disorders including
schizophrenia will hopefully be highly enlarged.
3 Common Genetic Variants
Also common polymorphisms (SNPs) were analysed using genome wide associ-
ation studies. The first study of this type included 178 cases and 144 controls and
was performed by Lencz et al. (2007). The best associated SNP out of 500,000
SNPs was located in CSF2RA (colony stimulating factor, receptor 2 alpha).
A second genome wide association (GWA) study did not achieved genome wide
significance Sullivan et al. (2008). Both studies were clearly underpowered to
provide conclusive result. Therefore, O’Donovan (2008) performed an initial
GWAS on a much larger sample including 479 cases and 2,937 controls. Loci
surpassing p \ 10-E5 were followed up in 6,829 cases and 9,897 controls. Of 12
of these loci, 3 had strong independent support and the overall pattern of repli-
cation was unlikely to occur by chance. The evidence for association for the top
SNP in the ZNF804A gene strengthened when the affected phenotype included
bipolar disorder (O’Donovan et al. 2008). Interestingly, this result was replicated
independently in the Irish Case Control Study of Schizophrenia (ICCSS) sample
(Riley et al. 2010) as well as in an independent sample of 5,164 schizophrenia
cases and 20,709 controls (Steinberg et al. 2011). A fine-mapping, replication and
meta-analysis study of 18,945 schizophrenia and schizoaffective disorder patients,
21,274 schizophrenia plus bipolar disorder cases and 38,675 controls showed
further evidence for this SNP (Williams et al. 2010). Additionally, there is evi-
dence for the involvement of this SNP in neuropsychological phenotypes. Walters
et al. (2010) investigated whether the identified risk allele of the SNP rs1344706 is
associated with variations in neuropsychological performance in patients and
controls. Patients with DSM-IV diagnosed schizophrenia and healthy controls
from independent samples of Irish (n = 297 cases and n = 165 controls) and
German (n = 251 cases and n = 1472 controls) nationality were included. In the
Irish samples ZNF804A genotype was associated with differences in episodic and
working memory in patients but not controls. These findings replicated in the same
direction in the German sample. Furthermore, in both samples, when patients with
lower IQ were excluded the association between ZNF804A and schizophrenia
strengthened (Walters et al. 2010). Taken together, the ZNF804A gene is one of
the most promising genes for schizophrenia to date.
The largest GWA study to date on schizophrenia was led by Stefansson et al.
(2009). 2,663 schizophrenia cases and 13,498 controls from eight European
locations were studied within the SGENE+ consortium. Findings from the top
1,500 markers were combined with results for these markers from both the
International Schizophrenia Consortium (ISC 2,602 cases/2,885 controls) and the
European–American portion of the Molecular Genetics of Schizophrenia Con-
sortium (MGS 2,687 cases/2,656 controls). The best markers were followed up in
5,013 cases and 15,559 controls from four sets of additional samples from Europe.
This approach could identify three novel schizophrenia loci: Neurogranin
(NRGN), TCF4 and the HLA region (Stefansson et al. 2009). Interestingly, the
HLA associations were also found in the large GWAS by the International
Schizophrenia Consortium ISC (2009) studying 3,322 European individuals with
schizophrenia and 3,587 controls as well as by (Shi et al. 2009) suggesting further
evidence for the inflammation theory of schizophrenia. The second gene, TCF4
(transcription cell factor 4), is of high interest too as the Pitt–Hopkins Syndrome’s
gene was identified by unrelated groups by comparative genomic hybridization
(CGH) in 2007 showing that the haploinsufficiency of the TCF4 gene (18q21.2) is
due to an autosomal dominant de novo mutation, which is considered to be
causative (for review see Taddeucci et al. 2010). Finally, NRGN (neurogranin), the
third gene is highly promising. It is a calmodulin-binding protein expressed
exclusively in the brain. It is the main postsynaptic protein regulating the avail-
ability of calmodulin, binding to it in the absence of calcium (Stefansson et al.
2009). Neurogranin belongs to the calpacitin family and contains an IQ domain
that interacts with the Ca2+ free form of calmodulin (apo-calmodulin). Depending
on the intracellular Ca2+ concentration, neurogranin releases calmodulin, so that it
can bind Ca2+ and activate downstream signalling molecules. Calmodulin fur-
thermore binds different proteins including CaMKII, the calcium/calmodulin-
dependent protein kinase II gamma (Hayashi 2009). Zhong et al. (2009) show that
neurogranin is concentrated in dendritic spines and that the number of neurogranin
molecules available determines the efficiency of calmodulin signalling in the
synapse and the strength of AMPA receptor transmission (Zhong et al. 2009).
An exogenous overexpression of neurogranin is sufficient to trigger a Ca2+ signal
that normally induces Long Term Potentiation (LTP) which is crucial for learning
and memory. This neurogranin-induced synaptic potentiation and LTP share
common signalling mechanisms. Like LTP, neurogranin-induced potentiation
requires the activity of NMDA receptors and CaMKII. Furthermore, in such
neurons that overexpress neurogranin and where the AMPA receptor response is
therefore already potentiated, LTP can no longer be electrophysiologically
induced. Taken together, these observations in turn imply the involvement of
neurogranin in the signalling pathway of LTP (Hayashi 2009).
Further large genome wide association studies on schizophrenia are currently
under way and will hopefully add new relevant knowledge on schizophrenia.
Based on this, functional studies including cell and animal models will be
necessary. There is new hope that these new avenues will help understanding the
neurobiology of schizophrenia in more depth leading to the development of new
innovative diagnostic tools and therapies.
436 D. Rujescu
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Gene 3 Environment Interaction Models
in Psychiatric Genetics
Katja Karg and Srijan Sen
Abstract Gene–environment (G 9 E) interaction research is an emerging area in

psychiatry, with the number of G 9 E studies growing rapidly in the past two
decades. This article aims to give a comprehensive introduction to the field, with
an emphasis on central theoretical and practical problems that are worth consid-
ering before conducting a G 9 E interaction study. On the theoretical side, we
discuss two fundamental, but controversial questions about (1) the validity of
statistical models for biological interaction and (2) the utility of G 9 E research
for psychiatric genetics. On the practical side, we focus on study characteristics
that potentially influence the outcome of G 9 E interaction studies and discuss
strengths and pitfalls of different study designs, including recent approaches like
Genome–Environment Wide Interaction Studies (GEWIS). Finally, we discuss
recent developments in G 9 E interaction research on the most heavily investi-
gated example in psychiatric genetics, the interaction between a serotonin trans-
porter gene promoter variant (5-HTTLPR) and stress on depression.
Keywords Genomic Stress Behavior Serotonin
Contents
1 Introduction........................................................................................................................ 442
1.1 What is a G 9 E Interaction? .................................................................................. 442
1.2 Other Forms of Gene–Environment Co-Action:
Gene–Environment Correlations .............................................................................. 444
2 Theoretical Considerations for G 9 E Interaction Studies ............................................. 444
2.1 Can We Model G 9 E Interaction in Statistics? .................................................... 444
K. Karg S. Sen (&)

University of Michigan, Ann Arbor, MI, USA
e-mail: srijan@umich.edu

DOI: 10.1007/7854_2011_184
442 K. Karg and S. Sen
2.2 Is G 9 E Interaction Research Worth the Effort? .................................................. 446

3 Practical Considerations for G 9 E Interaction Studies.................................................. 447
3.1 Methodological Issues in G 9 E Interaction Research........................................... 447
3.2 Assessment of Environmental Exposure and Disorder Status ................................ 449
3.3 Study Designs ........................................................................................................... 450
3.4 Wide Interaction Studies .......................................................................................... 453
4 Empirical Evidence for G 9 E Interaction in Psychiatric Genetics ............................... 453
5 Future Directions ............................................................................................................... 456
6 Summary ............................................................................................................................ 456
References................................................................................................................................ 457
1 Introduction
One of the oldest and most enduring questions in psychiatry is whether mental illness is
caused by nature (genes) or nurture (environment). Decades of epidemiology studies
have tried to answer this question through twin and adoption studies. These studies
have demonstrated a moderate genetic component for some disorders (depression and
alcohol dependence) and a high genetic component for others (schizophrenia and
autism). The relatively high heritability of psychiatric disorders has prompted
investigators to look deeply for direct connections between genes and mental illness.
Over the past 20 years, thousands of studies have been performed assessing the direct
relationship between genes and mental illness in the form of candidate gene associ-
ation studies, linkage studies and more recently, genome-wide association studies
(GWAS). Despite the intense effort, very few direct genetic effects have been iden-
tified (Moffitt et al. 2005; Rutter et al. 2006). Therefore, researchers have increasingly
directed their attention to the investigation of interactions between genes and envi-
ronment, a possibility that has traditionally been understudied in behavioral and
psychiatric genetics (Caspi 1998; Scarr 1992). In contrast, G 9 E interactions have
been demonstrated consistently in other branches of medicine (van Os et al. 2008).
Hence, G 9 E interaction research is an emerging discipline in psychiatric genetics
with growing numbers of novices in need of a comprehensive introduction to the field.
In this chapter we aim to give such an introduction, starting with a detailed definition of
G 9 E interaction. We then discuss two fundamental, but controversial theoretical
questions about the validity of statistical models for biological interaction and the
utility of G 9 E interaction research for the field of psychiatric genetics. Finally, we
discuss practical aspects of studying G 9 E interactions, with an emphasis on study
designs and assessment methods that may affect the success of G 9 E interaction
studies, and present relevant examples from the field.
1.1 What is a G 3 E Interaction?
The term ‘‘G 9 E interaction’’ stems from regression models that seek to divide
the population variance for disorder risk into environmental and genetic parts.
Effects of these factors that are independent from one another are called
Gene 9 Environment Interaction Models in Psychiatric Genetics 443
Fig. 1 Illustration of main and interaction effects of genes and environmental exposure on
disorder risk. Solid line Genotype A, dashed line Genotype B. a Genetic main effect
b Environmental main effect c Additive effect of genes and environmental exposure
d Quantitative interaction effect e Qualitative interaction effect
main effects. The main effect of either the genetic or the environmental factor can
explain the variance for the disorder entirely (Fig. 1a, b) or both factors can coact and
explain the variance additively, operating independently alongside each other
(Fig. 1c). Consider a child with a retinoblastoma, a malignant tumor of the retina
caused by an inherited mutation in one allele of the tumor suppressor gene Rb1. If the
patients’ unaffected eye gets injured through an accident and the eyesight of this
patient becomes worse, the genetic and the environmental factor operate together on
the same outcome (eyesight), but are not involved in the same biological pathway and
fully independent factors. In contrast, in an interaction effect, the factors depend from
each other (Fig. 1d, e). In biological terms, such a G 9 E interaction effect occurs
when the effect of exposure to an environmental factor on the disorder status depends
on the person’s genotype (Moffitt et al. 2006). In other words, a G 9 E interaction is
defined by differences of genotypes in susceptibility to environmental exposure
(Kendler and Eaves 1986). For example, our patient with retinoblastoma has an
impaired DNA repair system causing her to be markedly more susceptible to UV
light compared to an individual without the mutation. By exposure to UV light,
tumors develop and worsen the patient’s eyesight. Thus, the effect of the exposure to
the environmental factor (radiation) on the outcome (eyesight) depends on the per-
son’s genotype, constituting an example for G 9 E interaction. G 9 E interactions
can be quantitative, i. e. the exposure to the environmental pathogen increases the
disorder risk for all genotypes, but to different extends (Fig. 1d) or they can
be qualitative, i.e. the exposure to the environmental factor increases the risk for one
genotype, but decreases it for another (Fig. 1e) (Ottman 1990). With respect to our
previous example, a qualitative interaction would occur if UV radiation decreases the
risk for retinoblastoma for one genotype, whereas it would increase it for another.
1.2 Other Forms of Gene–Environment Co-Action:

Gene–Environment Correlations
Genes and environmental factors can co-act in different ways, and not all of them
are G 9 E interactions [see (Moffitt et al. 2006) for details]. Gene–environment
correlations (rGE) are of particular importance, because they can produce false-
positive findings in G 9 E interaction research. rGE can occur when a person’s
genotype influences her probability of exposure to environmental risks (Plomin
et al. 1977; Rutter and Silberg 2002). Several mechanisms have been proposed to
drive rGE (Plomin et al. 1977; Jaffee and Price 2007). In active rGE an individual
actively selects her environment according to her (genetically influenced) traits
and behaviors. For instance, an individual characteristically risk-seeking and
impulsive may be much more prone to risk environments than a cautious indi-
vidual. The presence of rGE has been demonstrated through twin and adoption
studies for a wide range of factors, including the occurrence of life events, such as
divorce, job loss and serious accidents (Rutter et al. 2006; Rutter and Silberg
2002). The common nature of rGE underscores the danger in the independence
assumption of genotype and environment in G 9 E interaction research. This
assumption can be a major problem for some study designs, in particular case-only
studies (Jaffee and Price 2007) (further details below).
2 Theoretical Considerations for G 3 E Interaction Studies
There are two fundamental, theoretical questions about G 9 E interaction studies

that are currently the subject of considerable debate in the literature: (1) Whether
the current state of our knowledge about the neurobiology underlying psychiatric
disorders allows us to explore G 9 E interactions in a meaningful way;
(2) Whether the expected benefits derived from this research are important enough
to justify the considerable resources that these studies require. We address both
questions here and try to accurately represent the two opposing camps in the
discussion.
2.1 Can We Model G 3 E Interaction in Statistics?
Although the biological definition of G 9 E interaction is straightforward, its

implementation into statistics is far less clear. Two models are commonly used, the
additive and the multiplicative model. The additive model constitutes a G 9 E
interaction when the disorder risk if exposed to both the risk gene (G) and the risk
environment (E) differs from the sum of the risks if exposed only to G or to E. In
biological terms, this is equivalent to the deviation from a simple additive main
Table 1 Illustration of the additive and multiplicative model in statistical G 9 E interaction

testing
E- E+
(a)
G- 2 5
G+ 3 10 (6)
Condition for G 9 E interaction Example 1 Example 2
(b)
Additive model Risk (G+, E+) =1 10 =1 3+5-2 6 =1 3+5-2
Risk (G+, E-) ? Risk (G 9 E present) (G 9 E absent)
(G-, E+) - Risk (G-, E-)
Multiplicative model Risk (G+, E+) =1 Risk 10 =1 3 9 5 6 =1 3 9 5
(G+, E-) 9 Risk (G-, E+) (G 9 E present) (G 9 E present)
In Table a, two numerical examples for disorder risk depending on the absence (-) or presence
(+) of exposure to the genetic risk factor (G) and environmental risk factor (E) are given, differing
only in the (G+, E+) field. Table b illustrates the statistical problem associated with G 9 E
interaction testing: Whereas example 1 leads to the consistent positive result for G 9 E inter-
action across the additive and the multiplicative model, the models yield conflicting results for
example 2
1
Statistical significance of the deviation needs to be tested
effects model. This model is used for continuous outcomes, such as depression
scores. The multiplicative model constitutes a G 9 E interaction when the disorder
risk if exposed to both G and E differs from the product of the risks if exposed only
to G or to E. This is used for categorical outcomes, e.g. diagnosis of depres-
sion with the two categories ‘‘depressed’’ and ‘‘non-depressed’’. The biological
meaning of a multiplicative model is hard to grasp and most researchers argue that
the additive model better reflects biological concepts (Rutter et al. 2009). The
problem is that in some cases, a study result might deviate significantly from a
multiplicative model, but not from an additive model, and vice versa (Kendler and
Gardner 2010) (Table 1a, b). This is particularly problematic as continuous out-
comes can be converted to categorical outcomes by setting an arbitrary threshold.
Given sufficient statistical power, this threshold can be chosen so that either of
both models indicate a significant interaction effect. Some researchers argue that
this model-dependency renders positive G 9 E interaction findings arbitrary
(Zammit et al. 2010a) and testing for interactions across multiple models is
therefore ‘‘no different from trawling through many statistical tests looking for
those that are significant’’ (Kendler and Gardner 2010). Therefore, the statistical
model to be tested should be carefully selected a priori, based on biological
background considerations, and thresholds for categorical data should be set before
the analysis. Unfortunately, our current knowledge about neurological pathways is
very limited, and, as a result, it is still unclear which statistical model is appro-
priate (Thompson 1991). This situation has caused some leaders to conclude that
we might be unable to move back and forth between statistical and biological
interaction models (Kendler and Gardner 2010). The debate remains controversial
(Rutter et al. 2006; Zammit et al. 2010a; Caspi and Moffitt 2006; Munafo et al.
2009). One way that investigators have used to circumvent this statistical problem
is utilizing new study designs such as case-only or exposed-only designs. These
designs do not rely on testing statistical interactions, but directly test differences in
exposure rate (case-only design) or in disorder status (exposed-only design)
between genotype groups. To date, these designs have mostly been applied in
psychiatric G 9 E interaction research to investigate the interaction between a
serotonin transporter gene promoter variant (5-HTTLPR) and stress on the risk of
depression (Caspi et al. 2003), with mostly positive results (Karg et al. 2010).
2.2 Is G 3 E Interaction Research Worth the Effort?
There are three primary arguments for why the identification of G 9 E interaction
effects will substantially advance the field. First, they can help identify new
genetic and environmental main effects associated with psychiatric disorders
(Kraft et al. 2007). Some risk genes and environments might be masked from
detection in scans for direct genes-to-disorder or environment-to-disorder associ-
ations because of genotype-specific environmental effects on the disorder status
due to G 9 E interactions. Second, knowledge about the interaction effect of gene
and environment on a psychiatric disorder might enhance the identification of the
biological pathway underlying the interaction by revealing the genetic and envi-
ronmental factors involved and thus channel neuroscience studies in a productive
direction (Caspi and Moffitt 2006). Third, G 9 E interaction findings may have
clinical relevance and drive the development toward personalized medicine or
individual lifestyle recommendations based on the genetic profile (Dempfle et al.
2008; Uher and McGuffin 2007). They could explain differences in response to
pharmacological and psychological treatments by differences in the susceptibility
of genotypes to environmental factors. Individuals with high-susceptibility geno-
types could be identified and prevented from suffering exposure to the relevant
environmental pathogens.
Several researchers have criticized this optimistic view, pointing out that the
G 9 E interaction effects identified to date are small, with odds ratios generally
between 0.67 and 1.5 (Manolio et al. 2008), limiting the potential influence of G 9 E
interaction on advances in psychiatric genetics and clinical practice (Zammit et al.
2010a, b; Hunter et al. 2008). In particular, the power for finding main effects
might only marginally increase by including G 9 E interaction effects in the
statistical model (Munafo et al. 2009). In addition, G 9 E findings might help
identify the underlying biological pathway only through the detection of qualita-
tive G 9 E interactions, a case known to be rare in epidemiology (Thompson
1991). Thus, there is an ongoing debate about the benefit of G 9 E interaction
research and the considerable amounts of resources spent in the field (Kendler and
Gardner 2010; Uher and McGuffin 2007; Zammit et al. 2010b).
3 Practical Considerations for G 3 E Interaction Studies
Investigating G 9 E interactions is challenging. For each participating subject,

detailed information from three distinct domains is needed: (1) genotype,
(2) environmental exposure, (3) psychiatric disorder status. Fortunately, it has
become increasingly inexpensive to reliably determine the genotypes of large
numbers of subjects due to improved molecular genetic techniques. Gathering
valid information in the domains of environmental exposure and disorder status,
however, remains expensive and time consuming. This mismatch has led to an
increasing number of studies where a huge sample of subjects is genotyped but the
quality of phenotype information is comparatively poor. Further, researchers have
taken advantage of declined genotyping costs by adding genotype data to studies
originally not designed for G 9 E interaction research (Caspi et al. 2010). Here we
give a brief overview on the consequences of these trends and the other meth-
odological issues associated with G 9 E interaction research. We will present
different study design approaches, each with particular advantages and limitations
as well as examples from the psychiatric genetics literature (Table 2). For further
detailed information on G 9 E interaction testing see (Caspi and Moffitt 2006;
Kendler and Gardner 2010 and Rutter 2002). Complementary research guidelines
can be found in (Moffitt et al. 2005, 2006).
3.1 Methodological Issues in G 3 E Interaction Research
Three major methodological confounding issues are important to consider in

planning G 9 E interaction research: Selection bias, population stratification and
recall bias. Selection bias can occur when cases and controls are not drawn from
the same underlying population, resulting in erroneous conclusions about associ-
ations between genotype, environmental exposure and disorder risk (Hunter 2005).
For example, gene–environment correlations can arise in a situation where the
presence of a genotype group is correlated with exposure to a particular risk
environment. This can result in an overrepresentation of cases with this genotype
and therefore steer the study outcome toward false-positive findings regarding
differences between cases and controls. Population stratification is the presence of
a systematic difference in allele frequencies between subpopulations in a popu-
lation possibly due to different ancestry (Hunter 2005). Specifically, populations
differ with regard to allele frequencies at loci throughout the genome. If these
populations also differ in their prevalence of the disorder of interest, spurious
associations can be found between this disorder and genetic loci that neither affect
the relevant disorder nor are linked to a causative loci. Fortunately, methods have
been developed to control for stratification, using unlinked genetic markers to
identify and correct for population structure (Cardon and Palmer 2003). These
genomic control methods should be utilized in modern day G 9 E studies.
448
Table 2 Study designs in G 9 E interaction

Design Description Advantages Disadvantages
Family-based Twin study Comparison of disorder frequency No genetic data required; reduced High costs and efforts
designs between twin pairs in different selection and stratification bias
environments
Trio design Comparison of expected genes in Increased power; High costs and efforts
cases to possibly transmitted reduced selection and
genes from both parents, stratified stratification bias
by case’s environment
Sib design Case-control design with unaffected Increased power; High costs and efforts
relative as control reduced selection and
stratification bias
Traditional Prospective Comparison of disorder frequency Reduced selection and High costs and efforts;
population-based cohort across groups defined by genotype stratification bias; reduced time-consuming;
designs and environment; exposure assessed recall bias; high-quality Low, possibly biased
previous to diagnosis measurement for environmental follow-up rates
exposure
Cross-sectional Like prospective cohort, but exposure Reduced selection and stratification Increased recall bias
assessed simultaneously with bias; more cost-efficient
diagnosis compared to prospective
cohort design
Retrospective Comparison of genotype frequencies Increased power and more Increased selection and
case-control and exposure between cases cost-efficient compared to stratification
and controls cohort designs bias; increased recall bias
Novel population- Prospective Comparison of cases with matched Combined advantages of prospective –
based designs nested non-affected cohort members cohort and retrospective
case-control case-control designs
Case-only Comparison of exposure across Increased power compared to High risk of bias due to
groups defined by genotype case-control; cost-efficient confounding with rGE
Exposed-only Comparison of genotype frequencies Cost-efficient Risk of bias due to confounding
across exposed individuals with rGE; increased
grouped by genotype selection bias
K. Karg and S. Sen
The third major problem in G 9 E interaction research is recall bias. Recall bias
occurs when subjects cannot accurately recall past events or when particular events
become more or less important in retrospect than when they occurred. In partic-
ular, patients often overcount potential environmental causes for their disorder,
a phenomenon termed mood-congruent memory revision (Joormann et al. 2009;
Schwarz and Clore 1983). Recall bias tends to become greater with the greater
length of time between the environmental exposure and its report. However, this
retrospective forgetting is often selective and its magnitude and character differs
between affected and unaffected individuals (Monroe 2008). The difficulties in
overcoming the problem of recall bias in retrospective studies provide the impetus
for specific novel study designs that we will discuss in later sections.
3.2 Assessment of Environmental Exposure and Disorder Status
An important, but underappreciated factor affecting the power of G 9 E studies is

the assessment method for environmental exposure (Caspi et al. 2010). Poor
measurement quality has been correlated with negative findings (Uher and
McGuffin 2007, 2010). Simulation studies have demonstrated that in G 9 E
interaction studies, moderate decreases in the measurement accuracy of the
environmental variable can result in a 20-fold reduction in statistical power to
detect interaction (Moffitt et al. 2005). In line with this simulation result, in a
recent meta-analysis on studies investigating the moderating effect of a serotonin
transporter gene polymorphism (5-HTTLPR) on the relationship between stressful
life events and depression, we found that studies that utilized more intensive stress
assessment methods, such as in-person interviews, were more likely to detect an
effect than studies that utilized self-report questionnaires (we will discuss the set of
5-HTTLPR-stress studies in more detail in Sect. 4). One reason for these findings
is likely that the effect of measurement error, such as recall bias, is more pro-
nounced in self-report questionnaires than in personal interviews because trained
interviewers can counteract poor recall by using appropriate techniques such as life
event calendars and memory enhancement (Caspi et al. 2010). Self-report event
checklists have been shown to result in more imprecise information (Monroe
2008). Objective measurements may also be superior to self-report questionnaires
because they minimize the effects of recall bias by focusing objective information.
Further, the objective stressor design reduces between-subject heterogeneity by the
use of clearly operationalized and objectively identifiable environmental factors,
resulting in an increase of internal validity (Caspi et al. 2010). These findings
underscore the importance of choosing assessment methods for G 9 E studies
carefully. The use of several independent measurements such as self-report,
diagnostic interview or informant reports are excellent possibility to increase the
accuracy of assessment (see Caspi et al. (2003), for a good example). A similar set
of methodological considerations apply to the assessment of disorder status.
In comparison to many systemic disorders, psychiatric disorders are difficult to
diagnose, relying on arbitrary thresholds on symptom severity scales (Eaton et al.

2007). For instance, a wide range of threshold scores (12–23) have been suggested
for diagnosing depression with the commonly used Beck Depression Inventory
(Nuevo et al. 2009). While commonly used diagnostic instruments for many
psychiatric disorders (such as depression, alcohol and drug use disorder) have
acceptable measurement characteristics, others perform poorly (e.g. panic disor-
der, obsessive–compulsive disorder, bipolar disorder and schizophrenia), with
particularly poor sensitivity (40%) and specificity (89%) for schizophrenia (Eaton
et al. 2007).
3.3 Study Designs
G 9 E interaction study designs can broadly be categorized into family-based

designs and population-based designs. Both designs have particular strengths
and limitations regarding the methodological issues described above (Table 2).
Family-based studies generally assess whether there is a greater than expected
transmission of specific alleles to affected family members (Ewens and Spielman
1995). The specific family-based study designs include twin studies (Ottman
1994), trio designs with an affected individual and both parents (Schaid 1999;
Witte et al. 1999), and sib designs with one affected and one unaffected sibling or
relative (Gauderman et al. 1999). If the frequency of transmission differs between
exposed and non-exposed cases, a G 9 E interaction is present (Schaid 1999). The
main advantages of family-based designs is a per subject increase in power
compared to population-based designs, and robustness against population strati-
fication. However, family-based designs have some major drawbacks that have
limited their use. One is that it is often harder to recruit an adequate number of
sibling or twin pairs than unrelated subjects, and the unavailability of living
parents can limit the scope of trio studies (Hunter 2005). Further, newer genomic
control methods can robustly control for stratification, rendering the primary
advantage of family-based methods less useful. Therefore, in most cases of G 9 E
interaction research, population-based designs are used.
In contrast to the family-based design, design studies generally draw from a set
of unrelated subjects. These studies differ according to how these subjects are
selected. Subjects can be can be drawn from a cohort [cohort study design (Collins
2004)], selected and matched as cases and controls [case-control design (Yang and
Khoury 1997)], drawn from affected individuals only [case-only design (Khoury
and Flanders 1996)], or from individuals exposed to the environmental risk factor
only [exposed-only design (Moffitt et al. 2006)].
Cohort study design. In cohort study designs, the sample studied should
accurately represent the target population in terms of genotype, exposure rate and
disorder status. Information can be assessed either once (cross-sectional design) or
repeatedly over time (prospective/longitudinal design). When analyzing the data,
subjects can be assigned to groups according to their genotype and their exposure
rate (e.g., genotype A with low environmental exposure vs. genotype B with low
environmental exposure), and disorder frequencies can be compared between these
groups. If high follow-up rates are obtained, the prospective cohort design can
provide high-quality data because it efficiently handles the three major methodo-
logical issues facing G 9 E studies: it minimizes selection bias, because the
disorder usually occurs after subjects are selected (Yang and Khoury 1997),
it minimizes population stratification by sampling from a defined cohort and it
reduces recall bias to a minimum if the baseline information is assessed early in
life of the cohort and when it can be followed several times over years (Hunter
2005).
Three of the most important findings in psychiatric G 9 E interaction research
were produced by utilizing through a study a prospective cohort study design, the
Dunedin Multidisciplinary Health and Development Study (Dunedin Longitudinal
Study) (Caspi et al. 2002, 2003, 2005). The Dunedin Longitudinal Study inves-
tigated a large birth cohort of 1,037 children born in 1972–73 in Dunedin,
New Zealand. The cohort was first assessed at age three and since then followed up
every two years for two decades (Silva 1990). Data from this cohort demonstrated
significant G 9 E interaction effects on violent behavior (Caspi et al. 2002),
depression (Caspi et al. 2003) and adult psychosis (Caspi et al. 2005). These
landmark studies provide evidence supporting the strength and accuracy of the
prospective cohort design.
The downside of this study design is the long time frame necessary to conduct
these studies. For instance, the Dunedin Longitudinal Study was started 30 years
before the first G 9 E interaction finding was published. In addition, large samples
are needed because the environmental exposure and/or the disorder might be at
low prevalence in the cohort (Hunter 2005). As a result, many investigators opt for
quicker and less expensive designs. The cross-sectional modification of the cohort
study assesses cohort information only once. Although this design loses some of
the advantages of a prospective study, the cost and time frame necessary to carry
out the study is often more feasible.
Retrospective case-control. Another inexpensive and popular alternative to the
prospective cohort design is the retrospective case-control design. Here, affected
subjects with the disorder are selected and matched with subjects who do not have
the disorder (‘controls’). This procedure allows for the controlled sampling of
subjects with disorder and/or environmental exposure, yielding the advantage of
increased power compared to cohort studies (McClelland and Judd 1993). Infor-
mation about past exposure is gathered and the exposure rates and genotype
frequencies are compared between cases and controls. Due to the selection and
matching process, this design is particularly prone to selection bias and population
stratification, especially when the source population for controls is hard to define
(Hunter 2005). The prospective-nested case-control design is a more sophisticated
study design that addresses these methodological problems by selecting cases and
controls from a predetermined longitudinal cohort. As cases and controls stem
from the same cohort, confounding from selection bias and population stratifica-
tion is avoided. In addition, recall bias is eliminated because exposure is assessed
before the diagnose. Compared to a full cohort approach, this design offers sub-
stantial reductions in costs and efforts.
Case-only. Recently, investigators have proposed a study design that eliminates
the use of control subjects (Khoury and Flanders 1996; Piegorsch et al. 1994).
In the case-only design, affected subjects are selected from the population and
grouped according to their genotype and then compared for their exposure rates.
In the presence of G 9 E interaction, some genotypes are more susceptible to the
environmental pathogen than others, resulting in an overrepresentation of subjects
with environmental exposure in this genotype group. Therefore, differential
distributions of exposure rates across genotype groups can be interpreted as a G 9 E
interaction effect. As an example, Mandelli et al. (2006) utilized the case-only
design to investigate the interaction effect of 5-HTTLPR and stress on depression.
They studied a sample of 686 patients diagnosed for major depression or bipolar
disorder and classified them into six groups according to their genotype and the
presence or absence of environmental exposure to life stress in the year before
depression onset. On comparing the proportion of the sample exposed between
each genotype group, they found higher proportions of previously exposed
subjects in the genotype groups carrying the short allele. The authors interpreted
this finding as evidence for higher stress susceptibility of short allele carriers.
However, this conclusion has to be viewed with some caution because the case-
only design is prone to confounding. Differential distributions in exposure rates
across genotypes can also emerge through G-E correlation, with specific genotypes
being more likely to be exposed to the environmental factor than others (Khoury
and Flanders 1996). In this study, it is possible that short allele carriers are more
prone to experience stressful situations and that this causes their overrepresenta-
tion in the exposed group. The only safe way to rule out this potential bias is
through the verification of the underlying assumption of gene–environment
independency. Therefore, the case-only design should be used only if the inde-
pendency assumption is verified or for exploratory studies (Albert et al. 2001).
Exposed-only. A related, but subtly different approach that has become
increasingly popular is the exposed-only design. Here, subjects exposed to the
same environmental factor are selected, grouped according to their genotype and
compared for their disorder status. In the presence of G 9 E interaction, disorder
frequencies should be higher in the genotype group with higher susceptibility to
the environmental exposure. However, as we discussed concerning the case-only
design, this conclusion is only valid in the absence of G-E correlation. An example
might illustrate this problem. A recent study utilized an exposed-only design to
explore a moderating effect of the FKBP5 (FK506 binding protein 5) gene on the
relationship between severe injury and peritraumatic dissociation (Koenen et al.
2005). Peritraumatic dissociation is a evolutionary conserved response to life-
threatening events and a risk factor for the development of post-traumatic stress
disorder (Ozer et al. 2003). The study sample consisted of 46 severely injured
hospitalized children who were genotyped and compared for their peritraumatic
dissociation scores with logistic regression analysis. The study revealed a signif-
icant G 9 E interaction effect of FKBP5 genotype and severe injury on the
development of peritraumatic dissociation. However, this finding could have arisen

through rGE, with one genotype group particularly prone to risk-seeking and
therefore more likely to suffer severe injury and corresponding peritraumatic
dissociation. This could lead to the erroneous conclusion that this genotype is more
susceptible to peritraumatic dissociation than others. In this study, however, injury
severity was taken into account in the statistical analysis, rendering a false-positive
result due to rGE less likely. Another elegant way to guard against bias due to rGE
is exact matching for exposure across participants (Moffitt et al. 2006). This allows
investigators to bypass the model-dependency problem. Hence, the problem of rGE
in exposed-only designs is much easier to handle than in case-only designs where
additional empirical evidence is needed. The exposed-only design is thus an
attractive cost-efficient design that can be used to test G 9 E interaction for can-
didate genes as well as for the discovery of unknown risk genes (Moffitt et al. 2006).
3.4 Wide Interaction Studies
With the advent of genome-wide association studies (GWAS) it is now possible to

genotype up to one million SNPs for each participant, allowing investigators to
scan the entire genome for relevant genes without prior hypothesis. While most
GWAS to date have explored direct associations, groups have begun to modify
GWAS to include assessment of environmental variables in order to conduct
Gene–environment wide interaction studies (GEWIS) (Khoury and Wacholder
2009). GEWIS allow us to investigate several candidate pathways at once at
relatively low costs and hold the promise to identify new possible G 9 E inter-
actions. The greatest challenge for GEWIS involves finding a balance between
dismissing true findings through stringent correction for multiple testing and
reporting false-positive results (Sebastiani et al. 2005). Without any prior
hypothesis it is hard to distinguish false from true positives, especially as inter-
action effects in complex traits such as mental disorders are supposed to be small.
However, systematic approaches to the problem are emerging (Onkamo and
Toivonen 2006; Wacholder et al. 2004). Despite the great remaining conceptual
challenges, GEWIS paired with thorough phenotyping holds promise in producing
advances in the field of G 9 E interaction research.
4 Empirical Evidence for G 3 E Interaction

in Psychiatric Genetics
G 9 E interactions in psychiatric genetics have been reported for various disorders

such as depression, attention deficit/hyperactivity disorder (ADHD), schizophrenia,
obesity and substance use disorders (Table 3). The identified environmental
pathogens range from prenatal factors such as maternal smoking (Kahn et al. 2003)
Table 3 Selected G 9 E interaction findings in psychiatric genetics

Gene Risk environment Disorder Original finding
SLC6A4 Stressful life events Depression Caspi et al. (2003)
SLC6A4 Childhood maltreatment Depression Caspi et al. (2003)
SLC6A4 Mother’s expressed emotion ADHD Sonuga-Barke et al.
(2009)
SLC6A4 Early life stress Alcohol abuse Olsson et al. (2005)
MAOA Childhood maltreatment Antisocial personality; Caspi et al. (2002)
Conduct disorder
DRD4 Priming alcohol doses Alcohol craving Hutchison et al. (2002a)
DRD4 Smoking cues Tobacco craving Hutchison et al. (2002b)
DAT1 Prenatal maternal smoking ADHD Kahn et al. (2003)
DAT1 Prenatal maternal use of alcohol ADHD Brookes et al. (2006)
DAT1 Season of birth ADHD Seeger et al. (2004)
DAT1 Psychosocial adversity in ADHD Laucht et al. (2007)
childhood
DAT1 Mother’s expressed emotion ADHD Sonuga-Barke et al.
(2009)
DAT1 Institutional deprivation ADHD Stevens et al. (2009)
COMT Cannabis use in adolescence Adult psychosis Caspi et al. (2005)
COMT Low birth weight ADHD Thapar et al. (2005)
COMT Stress Psychosis van Winkel et al. (2008)
CRHR1 Stress Alcohol abuse Blomeyer et al. (2008)
CRHR1 Childhood trauma Mood and anxiety Bradley et al. (2008)
disorders
FTO Physical inactivity Obesity Andreasen et al. (2008)
FKBP5 Acute injury Psychological Koenen et al. (2005)
dissociation
FKBP5 Childhood abuse Mood and anxiety Binder et al. (2008)
disorders
or maternal alcohol use (Brookes et al. 2006) to factors relevant at birth [e.g. season
of birth (Seeger et al. 2004), birth weight (Thapar et al. 2005)] and early devel-
opment [e.g. childhood maltreatment (Caspi et al. 2003), childhood trauma
(Bradley et al. 2008)] to factors affecting adolescence [e.g. cannabis use (Caspi
et al. 2005)] and adulthood [e.g. stress (Blomeyer et al. 2008), physical inactivity
(Andreasen et al. 2008)]. However, track record of replications has often been poor,
casting doubt on the validity of these findings (Thomas 2010). Nonreplication can
be due to false-negative results, false-positive results or true heterogeneity between
studies. False-negative results in psychiatry studies are most often caused by
insufficient power, either due to a small sample size or suboptimal phenotyping or
genotyping quality. False-positive results can often result from multiple testing and
population stratification. True heterogeneity occurs if the interaction exists in some
populations studied or with some environmental factors studied but not with others.
Here, we present the most heavily investigated example in psychiatric G 9 E
interaction research, a G 9 E interaction between a polymorphism in the promoter
region of the serotonin transporter gene (5-HTTLPR) and both adult stressful life
events and childhood maltreatment on the risk of depression (Caspi et al. 2003). We
will discuss conflicting results between studies exploring this interaction and
potential reasons for the conflict.
The original study exploring this interaction utilized a prospective-longitudinal
cohort design with almost 1,000 children and found that individuals homozygous
or heterozygous for the low-expressing short variant of 5-HTTLPR are more
susceptible to depression after stress than individuals homozygous for the alternate
long variant. The same pattern was found for childhood maltreatment. This study
caused a great deal of excitement in G 9 E interaction research and encouraged
further research on this issue. To date, there have been 55 follow-up studies with
some confirming the original finding, some finding evidence of higher stress
susceptibility of individuals with the alternate long allele, and others finding no
interaction effect at all (Karg et al. 2010). This inconsistency might be due to the
heterogeneity of studies in many relevant aspects. First, studies exploring the
relationship between 5-HTTLPR, stress and depression have utilized very different
research designs, including longitudinal, cross-sectional, case-control, case-only,
exposure-only and family-based designs. Second, studies have measured many
different depression phenotypes using diverse assessment strategies, including
clinical interviews and self-report checklists, and diverse depression scales, vari-
ously yielding both categorical and continuous outcome measures. Third, studies
have investigated an extraordinarily varied set of stressors with various assessment
methods. For instance, stressors counted in different studies for stressful life events
ranged from becoming homeless, and the death of a parent or spouse to growing up
in a household with siblings who quarreled or as the child of a father in an
unskilled occupation. Other studies used more specific, but highly diverse stressors
such as stroke survival, hurricane exposure, bullying victimization or childhood
maltreatment. To clarify this confusion, three meta-analyses have been carried out
to date. The first two (Uher and McGuffin 2007; Risch et al. 2009) concluded that
there was no evidence supporting the presence of an interaction. However, these
analyses investigated only small subsamples of all 55 studies due to methodo-
logical constraints. The latest meta-analysis (Karg et al. 2010) included all relevant
studies and detected stressor type (stressful life events, childhood maltreatment,
and specific medical conditions) and stress assessment method (questionnaire,
interview, objective) as two critical sources for variability in study outcomes.
In particular, studies with childhood maltreatment or specific medical conditions
as environmental stressor were more likely to find a significant G 9 E effect than
studies with broader defined stressful life events, as were studies with objective or
interview assessment methods for environmental stressors. This again supports the
assumption that measurement quality can affect results in G 9 E research.
Since this original study, further evidence from various fields has emerged
(Caspi et al. 2010). First, several empirical studies link the short 5-HTTLPR
variant to stress-sensitive phenotypes such as post-traumatic stress disorder
(Xie et al. 2009), post-trauma suicide (Roy et al. 2007), stress-related sleep
disturbance (Brummett et al. 2007) and anxiety (Stein et al. 2008). Second,
a multitude of neuroimaging studies confirmed increased and faster amygdala
reactivity following threat in carriers of the short allele e.g. (Furman et al. 2010;
Heinz et al. 2005) and linked it to specific brain anatomy characteristics
e.g. (Pacheco et al. 2009; Pezawas et al. 2005). Third, Rhesus macaques carrying
the short variant exhibit greater anxiety-related behaviors in response to adverse
rearing conditions compared to their conspecific with the long alternate (Barr et al.
2004; Spinelli et al. 2007). Fourth, in addition to 5-HTT knockout mice, 5-HTT
knockout rats showed increased anxiety levels in response to stress (Homberg et al.
2007). Taken together, these outcomes across a wide variety of techniques, models
and species as well as the numerous positive G 9 E studies robustly demonstrate
the interaction effect between stress and 5-HTTLPR genotype on depression and
are to date the most intriguing finding of G 9 E interaction in psychiatric genetics.
5 Future Directions
Although much progress has been made in the past two decades, many questions in
G 9 E interaction research in psychiatric genetics remain open. New, more
carefully conducted epidemiological studies could shed light on these questions.
Another major step for clarification is the identification of the biological mecha-
nisms underlying interaction effects. Not much is known about how environmental
factors can interact with a person’s genotype and her nervous system to moderate
the disorder risk. Therefore, joining forces with neuroscience is an important step
in making progress in the field (Caspi and Moffitt 2006). Many epidemiological
studies on G 9 E interaction in psychiatric genetics were motivated by findings of
neuroscience research and positive epidemiological findings, in turn, can stimulate
new studies in neuroscience. The interaction between 5-HTTLPR and life stress on
depression provides an example where neuroscience studies can illuminate the
black box between genes, environment and disorder (Merikangas and Risch 2003)
and confirm and explain epidemiological findings. Another fruitful approach for
advances in the understanding G 9 E interaction might be the collaboration with
epigenetic research. Many environmental risk factors operate early in develop-
ment, and fine-tuning of neuronal pathways is known to be affected by environ-
mental factors (Abdolmaleky et al. 2004). If these epigenetic modifications depend
on the person’s genotype, a plausible mechanism is constituted for G 9 E inter-
action in psychiatric genetics. Epigenetic studies for psychiatric disorders are still
in their infancy, and new exciting insights in the interplay of genes and environ-
ment on the development of mental disorders are to be awaited.
6 Summary
Although the fundamental questions about the validity of statistical models for
biological interaction and the utility of G 9 E interaction findings for advances in
psychiatric genetics are still highly debated, novel study designs such as case-only
and exposed-only designs can overcome at least some of the statistical concerns.
Study designs differ broadly in their strengths and limitations regarding selection
bias, population stratification and recall bias. Previously undetermined study
characteristics that might additionally affect the outcome of G 9 E interaction
studies are the assessment methods for environmental exposure and disorder sta-
tus, as shown for the G 9 E interaction effect between the serotonin transporter
promoter variant and stress on depression. New insights into the interplay between
genes and environment on the development of mental disorders may emerge
through more carefully conducted G 9 E interaction studies as well as through
collaboration with neuroscience and epigenetic research.
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Behavioral Genetics of Affective
and Anxiety Disorders
Katharina Domschke and Andreas Reif
Abstract As shown by clinical genetic studies, affective and anxiety disorders are
complex genetic disorders with genetic and environmental factors interactively
determining their respective pathomechanism. Advances in molecular genetic
techniques including linkage studies, association studies, and genome-wide asso-
ciation studies allow for the detailed dissection of the genetic influence on the
development of these disorders. Besides the molecular genetic investigation of
categorical entities according to standardized diagnostic criteria, intermediate
phenotypes comprising neurobiological or neuropsychological traits (e.g., neuro-
nal correlates of emotional processing) that are linked to the disease of interest and
that are heritable, have been proposed to be closer to the underlying genotype than
the overall disease phenotype. These intermediate phenotypes are dimensional and
more precisely defined than the categorical disease phenotype, and therefore have
attracted much interest in the genetic investigation of affective and anxiety dis-
orders. Given the complex genetic nature of affective and anxiety disorders with an
interaction of multiple risk genes and environmental influences, the interplay of
genetic factors with environmental factors is investigated by means of gene-
environment interaction (GxE) studies. Pharmacogenetic studies aid in the dis-
section of the genetically influenced heterogeneity of psychotropic drug response
and may contribute to the development of a more individualized treatment of
K. Domschke (&) A. Reif

Department of Psychiatry, University of Würzburg,
Füchsleinstrasse 15, D-97080, Würzburg, Germany
e-mail: domschke_k@klinik.uni-wuerzburg.de
A. Reif
e-mail: reif_a@klinik.uni-wuerzburg.de

DOI: 10.1007/7854_2011_185
464 K. Domschke and A. Reif
affective and anxiety disorders. Finally, there is some evidence for genetic factors
potentially shared between affective and anxiety disorders pointing to a possible
overlapping phenotype between anxiety disorders and depression.
Keywords Intermediate phenotype GWAS CNV Gene-environment

interaction (GxE) Pharmacogenetics
Contents
1 Affective Disorders ........................................................................................................... 464

1.1 Clinical Genetics (Family, Twin, Adoption Studies) .............................................. 465
1.2 Molecular Genetics (Linkage, Association, GWAS, CNV Analysis) .................... 466
1.3 Pharmacogenetics...................................................................................................... 478
2 Anxiety Disorders.............................................................................................................. 480
2.1 Clinical Genetics....................................................................................................... 480
2.2 Molecular Genetics ................................................................................................... 481
2.3 Genetics of Intermediate Phenotypes of Anxiety Disorders................................... 483
2.4 Gene-Environment Interaction ................................................................................. 484
2.5 Pharmacogenetics...................................................................................................... 484
3 Overlapping Phenotypes.................................................................................................... 484
4 Outlook .............................................................................................................................. 486
References................................................................................................................................ 487
1 Affective Disorders
The group of affective disorders comprises both major depressive disorder (MDD;
unipolar depression) with various subtypes as well as bipolar disorder (BPD). The
latter displays by changes between (hypo-)manic and depressive phases, with
intermittent euthymic phases, while the course of MDD is characterized by
depression and euthymia. As patients rarely develop their first manic phase only
years after their first depression, they might well be initially mischaracterized as
MDD patients (so-called ‘‘hidden bipolars’’), which is a challenge for genetic
studies on affective disorders. To overcome this problem, several indicators for the
presence of BPD in depression have been suggested, e.g. subthreshold hypomanic
symptoms (Angst et al. 2010; Fiedorowicz et al. 2011). This however has not been
incorporated in current genetic studies and thus one should always consider that
MDD studies might well include a substantial amount of ‘‘hidden bipolar’’
patients, obfuscating MDD-specific findings. Furthermore, in the following section
more recent approaches such as genome-wide association studies (GWAS) and
copy-number variant (CNV) analyses are reviewed, as there are already plenty of
scholarly review articles on linkage and association studies. These issues will
therefore be touched upon more briefly; with respect to intermediate phenotype
and gene x environment (GxE) studies in affective disorders, the reader is referred
Behavioral Genetics of Affective and Anxiety Disorders 465
to more specialized reviews for the sake of space, as this extensive topic is beyond
the scope of this article.
1.1 Clinical Genetics (Family, Twin, Adoption Studies)
There is ample evidence that BPD is a highly genetic condition featuring an

estimated heritability of 0.75 as evidenced by numerous clinical genetic studies.
The risk for a bipolar patient to have a bipolar first-degree relative is increased
about tenfold, and the risk to have an MDD relative is even higher (10- to 15-fold).
The largest study to date, that investigated more than two million Swedish nuclear
families (Lichtenstein et al. 2009), demonstrated a heritability of bipolar disorder
of 59%. Furthermore, relatives of bipolar patients also had a two to fourfold
increased risk to suffer from schizophrenia. Environmental influences were mainly
due to non-shared environment. In line with these data, twin studies also argue
for a heritability ranging from 59 to 87% (for an overview see Shih et al. 2004).
As little as two adoption studies on bipolar disorder have been carried out to date
(Mendlewicz and Rainer 1977; Wender et al. 1986), also argued for a genetic
cause of the disorder. An excellent overview on clinical genetic studies in bipolar
disorder can be obtained from Smoller and Finn (2003).
On the other hand, the heritability of MDD is comparatively lower (estimated to
be around 0.37 in the most comprehensive review and meta-analysis available to
date; Sullivan et al. 2000) and environmental influences (unique, but not shared
environment) are considered to play a more important role as compared to BPD.
This is also reflected in twin and adoption studies. However, especially older
studies do not discriminate between bipolar and unipolar depression and hence
explicit data on unipolar depression is in fact quite limited; only five family and six
twin studies, but no adoption study meet the stringent inclusion criteria in a
comprehensive analysis (Sullivan et al. 2000). Across the five family studies, the
summary odds ratio for MDD in first relatives of MDD patients was 2.84 and
significant. Interestingly, the odds ratio increased when only considering controls
which have been screened for absence of psychiatric disorders. Two of the three
reported twin studies argued for a substantial genetic component of MDD, and
finally, the twin studies including more than 21,000 individuals yielded a herita-
bility of 37% and an influence of individual-specific environmental effects of 63%.
No large differences in heritability indices were found between community and
clinical studies, and the influence of shared environment was negligible. Hence,
taken together, there is a clear genetic liability towards MDD although it is much
smaller as compared to BPD—which has also been taken into account when
reviewing studies where both conditions were not carefully treated separately.
Furthermore, it is evident for both MDD as well as BPD that these disorders do not
follow a strict Mendelian pattern of inheritance, but rather are complex genetic in
nature featuring polygenic and oligogenic models (‘‘common variant, common
disease’’ model), but probably also—for a part of the patients—highly penetrant

risk genes (‘‘multiple rare variant, common disease’’ model, see below).
1.2 Molecular Genetics (Linkage, Association, GWAS,

CNV Analysis)
1.2.1 Linkage Studies
More than 40 linkage scans for BPD have been published to date which generated
plenty of disparate findings. However, quite a number of loci meeting significance
criteria were described by two or more groups: 3p12-14, 4p16, 4q31-35, 5q31-33,
6q16/21-25, 8q21-24, 10q25-26, 11p15.5, 12q23-24, 13q14-32, 18p11, 18q21-22
20q13, 21q21-22, 22q11-12, and Xq24-28. Confirmed positional candidate genes
however are yet to emerge from these studies. An initially highly promising
linkage peak on chromosome 11p15 is considered meanwhile to be due to type I
error (Egeland et al. 1987; Berrettini 2001). Also, other loci which have been
initially promising could subsequently not be confirmed. A paradigmatic case in
this respect is for e.g. the 12q23-24 locus (Dawson et al. 1995; Ewald et al. 1998,
2002; Green et al. 2005), which is noteworthy due to the co-segregation of Darier’s
disease with BPD (Maziade et al. 2001). It has been shown that, in some families
suffering from both disorders, the BPD locus lies outside of the Darier’s disease
causing ATP2A2 gene, yet is in linkage disequilibrium with this variant (Jones
et al. 2002). Hence, there might indeed be intermediately penetrant variants in this
region which are exclusive to only a few families and which are lost in noise when
combining many families, or cases, respectively. Similarly, a functional mutation
in the gene encoding the brain-specific tryptophan hydroxylase 2 (TPH2) has
been described which segregates with BPD in three families (Cichon et al. 2008;
Grigoroiu-Serbanescu et al. 2008). Findings like these argue for a ‘‘common
disease, multiple rare variant’’ model (McCarthy et al. 2008) and underscore the
clinical and genetic heterogeneity of BPD. This however does not argue against
the concurrent existence of a ‘‘common disease, common variant model’’. As both
models most likely are present in clinical samples, this additional level of com-
plexity further hampers the identification of BPD risk genes.
Different meta-analyses found the strongest evidence for BPD susceptibility
loci on 13q and 22q (Badner and Gershon 2002; 1228 patients from 353 families),
or 9p22.3-21.1, 10q11.21-22.1 and 14q24.1-32.12 (948 to 2437 patients; Segurado
et al. 2003). The latter study used the rank-based genome scan (GSAM) method
which, together with sample heterogeneity, might account for the different findings
as compared to the first study. Finally, in a combined analysis, 6q21-q25 and 8q24
showed genome-wide significance (5179 patients from 1067 families; McQueen
et al. 2005). Again, the underlying genes have not yet been identified. Further
scholarly reviews on this topic have been provided by Schulze and McMahon
(2003), Serretti and Mandelli (2008), Barnett and Smoller (2009), and Craddock
und Sklar (2009).
Also in MDD, numerous linkage scans were carried out (reviewed e.g. by
Lohoff (2010)) yet did not point to clear regions of susceptibility, as expected from
the lower heritability rate of MDD as compared to BPD. No meta-analysis has
been performed to date, which is surprising given the recent efforts to uncover the
genetic basis of MDD and hence there is a clear need for further research. There is
no meaningful overlap of linkage peaks between studies, although it is noteworthy
that two linkage signals have previously been implicated in BPD: one on chro-
mosome 18q (Camp et al. 2005) and the locus mentioned above on 12q23-24
(McGuffin et al. 2005; Abkevich et al. 2003). Therefore, this region appears to be a
promising region for affective disorders, yet most likely carries more than just one
risk gene.
1.2.2 Association Studies
With regard to candidate gene studies, many genes were shown to be associated
with BPD, but none of them has been established as a specific BPD susceptibility
gene. Among the best replicated genes are DAOA/G72 (which was associated in a
case–control study, but not in a meta-analyses; Muller et al. 2011; Shi et al. 2008),
BDNF (again, meta-analyses provided differing data: Kanazawa et al. 2007; Fan
and Sklar 2008), DISC1, NRG1, ARNTL/CLOCK, FAT, and GSK3B (Barnett and
Smoller 2009; Serretti and Mandelli 2008; Luykx et al. 2010). Not surprisingly,
many genes encoding for components of neurotransmitter pathways have been
tested for an association with BPD (such as SLC6A3, HTR2A, TPH2, MAOA,
COMT, DRD1, and SLC6A4). Coming from GWAS on schizophrenia, the risk
gene ZNF804A was demonstrated not to be specific for this disorder, but rather
was also associated with BPD (O’Donovan et al. 2008; Williams et al. 2011;
Steinberg et al. 2011). The same was true for the GWAS schizophrenia risk loci
around the MHC region and NRGN (Williams et al. 2011) and other genes which
initially have been described as schizophrenia risk genes: the above-mentioned
DISC1 and DAOA/G72 genes, but also NRG1, DTNB1, and NPAS3 (Huang et al.
2010; Pickard et al. 2009).
Most of the association studies published to date suffer from the drawback of
small sample sizes and lack of replication, and hence, the combination of large,
well described international samples (as done in the Psychiatric GWAS consor-
tium [PGC]) is paramount especially in the search for risk variants assuming a
‘‘common variant, common disease’’ model. Furthermore, meta-analytic treatment
of existing data might shed some light on the contribution of suggested BPD risk
genes. Such have been performed on considerably sized samples on only a few
genes. The gene encoding for methylenetetrahydrofolate reductase (MTHFR) has
been tested for an analysis with mood disorders several times. While the first study
on 1222 MDD patients yielded negative results (Gaysina et al. 2008), the latest
analysis comprising 9648 cases (MDD, BPD and schizophrenia combined) yielded
again a significant yet unspecific association of MTHFR with mood disorders (best
OR = 1.26; Peerbooms et al. 2011). This is in line with a positive meta-analysis
on BPD by Gilbody et al. (2007), but in discrepancy to three further meta-analyses
on MTHFR in BPD (Zintzaras 2006, 1415 cases; Chen et al. 2009, 1260 cases;
Cohen-Woods et al. 2010, 2584 cases). Given that the original genotyping data
which was presented in the Cohen-Woods study (n = 897 BPD patients) was
negative, but not included in the Peerbooms study, which demonstrated a signif-
icant effect only when all mood disorders were combined, the role of MTHFR in
affective disorders seems to be rather unspecific and small.
A meta-analysis on all mood disorders found borderline evidence for an
association of the dopamine receptor 2 (DRD2) Taq1 polymorphism (which in fact
localizes to the neighboring gene ANKK1) with affective disorders, yet two more
SNPs in DRD2 proved to be negative in much larger data sets so that there is only
weak evidence for this gene being associated with BPD or MDD (Zou et al. 2010).
The gene for catechol-O-methyltransferase (COMT) that degrades dopamine,
features a well described functional polymorphism resulting in a Val to Met
transition and which has been shown to be linked to BPD in a meta-analysis on
2944 cases (Zhang et al. 2009b), although this seemed to be more pronounced in
Asian populations. Obviously, also genes coding for components of the serotonin
system were subjected to meta-analyses, which often were combined with genuine
genotyping efforts. A functional SNP in the promoter region of the serotonin
receptor gene HTR1A (rs6295) was demonstrated to be significantly associated
with BPD (1148 cases, Kishi et al. 2011). In the MAOA gene, three polymor-
phisms (sample sizes mostly [1000 cases) were meta-analyzed and the main
finding was an association of an intronic CA repeat with BPD in Caucasians
(Fan et al. 2010). The SLC6A4 promoter polymorphism (5-HTTLPR), which has
mainly been studied in MDD GxE (see below) was also included in several meta-
analyses that conclusively demonstrated a small yet significant association of the
short variant with BPD (Lasky-Su et al. 2005; Cho et al. 2005, 1712 cases). Two
meta-analyses focused on the TPH1 gene (Chen et al. 2008, 2011) and in unison
came to the conclusion that TPH1 is not associated with MDD (2340 and 1812
patients, respectively), but with BPD (1951 and 2083 cases). Given that in the
brain only the TPH2 isoform of tryptophan hydroxylase is expressed, this finding
is rather surprising; however, as the foetal brain depends on maternal 5-HT pro-
duction which is accomplished by placental TPH1 the observed association might
in fact be true but rather due to maternal and not case genotype as has been
described for rare TPH1 mutations in ADHD (Halmoy et al. 2010).
The lower heritability of MDD as compared to BPD notwithstanding, plenty of
case–control association studies have been published thereon as well. A scholarly
overview on candidate gene studies is provided by Lohoff (2010). In order to
separate the wheat from the chaff, replication is key and meta-analytic aggregation
of data is a possible route to success. Accordingly, a thorough meta-analysis on the
data available until June 2007 examined 183 papers on 393 polymorphisms
(Lopez-Leon et al. 2008). Twenty-two of these polymorphisms have been tested in
at least three studies and were thus subjected to further meta-analysis. Here,
significant association was demonstrated for the genes APOE2 (827 cases), GNB3
(375 cases), MTHFR (875 cases), SLC6A4 (3752 cases), and SLC6A3 (as little as
151 cases). Negative results were obtained for ACE, BDNF, COMT, DRD3,
GABRA3, HTR1A, HTR1B, HTR2A, HTR2C, MAOA, SLC6A2, and TPH1.
Further meta-analyses which have been published before this study were per-
formed on ACE, DRD4, HTR2A, MTHFR, SLC6A4, and TH; positive findings
were obtained for DRD4 (Lopez Leon et al. 2005; 917 cases). In the years fol-
lowing the meta-analysis by Lopez Leon, only few other meta-analytic studies
have been published including those on MTHFR and TPH1 cited above. Fur-
thermore, Franke and associates recently conducted a meta-analysis on the func-
tional BDNF Val66Met polymorphism. As BDNF has been implicated both in the
pathogenesis of depression as well as the mechanism of action of anti-depressant
treatment (Duman and Monteggia 2006), it is an obvious candidate gene and
accordingly was shown to be associated with depression in this meta-analysis of
2812 cases, although the association is sex-specific and only detectable in males
(Verhagen et al. 2010).
The largest body of evidence, and by far the largest sample sizes, exists for the
gene encoding the serotonin transporter (SLC6A4). Following the seminal finding
by Caspi et al. (2003) that environmental influences interact with SLC6A4
genotype to increase the risk toward depression, emphasis has been put on studies
aimed to test such GxE interactions adding a further level of complexity. Fol-
lowing positive meta-analyses confirming a main gene effect of SLC6A4 in
depression (Furlong et al. 1998; Lopez-Leon et al. 2008; Clarke et al. 2010), also
meta-analyses on GxE interaction studies yielded support for the notion that
SLC6A4 has a role in the etiology of depression (Karg et al. 2011). As Karg and
Sen elaborate in depth on this topic in this book, the reader is referred to their
contribution as well as the review articles by Uher and McGuffin (2008, 2010).
Also, genes encoding components of the cortisol pathway have proven to be
interesting candidates for GxE in MDD. The glucocorticoid receptor-regulating
co-chaperone FKBP5 has first been associated with recurrence of depression and
response to antidepressant treatment in 2004 (Binder et al. 2004). This has later
been replicated (Lekman et al. 2008) and FKBP5 was shown to interact with
HTR2A and GRIK4 in moderating the response to antidepressant treatment
(Horstmann et al. 2010). Most interestingly, FKBP5 has been shown to interact
with severity of childhood abuse on later-life PTSD symptoms (Binder et al. 2008)
which however might be confined to African Americans (Xie et al. 2010). One of
the involved SNPs (rs1360780) was later replicated to interact with childhood
maltreatment to affect depression measures (BDI-II) in adult life (Appel et al.
2011). This SNP also displayed a main gene effect on suicidal events in depressive
adolescents (Brent et al. 2010), a categorical diagnosis of depression (Lekman
et al. 2008) and general depressive symptoms (Lavebratt et al. 2010; Zobel et al.
2010; Velders et al. 2011) and interestingly is associated with decreased cortisol
levels (Velders et al. 2011) providing a possible pathophysiological mechanism for
the association data. Also, rs1360780 went along with smaller right hippocampal
volume in patients with depression (Zobel et al. 2010). Taken together, there is
good evidence (Binder 2009) that risk genotypes in FKBP5 (especially rs1360780)
interact with early life adversity on later life depression, possibly by long-term
adaptive changes of the HPA axis and subsequent morphological changes of the
hippocampus increasing vulnerability to disease. In line with these findings, it was
also shown that rs110402 in the corticotropin-releasing hormone receptor
(CRHR1) interacted with child abuse to increase the risk toward later life
depression (Bradley et al. 2008), most interestingly in interaction with the sero-
tonin transporter risk genotype mentioned above (Ressler et al. 2010).
It seems to be a common phenomenon that candidate genes rarely replicate
when tested for in GWAS (see below) data sets. A thorough study on MDD
candidate genes (Bosker et al. 2011) tested 57 genes in the Genetic Association
Information Network (GAIN) MDD sample (n = 1862 cases). From 93 selected
candidate SNPs, only 18 were present on the array, and a further 47 were imputed.
Of those, only five (including an SNP in NPY) were associated in the GAIN
sample, all with p[0.03. When candidates were tested on a gene-based level,
analyzing 4870 SNPs, the TNF and NET genes yielded suggestive evidence.
In general, heavy use of imputation might have introduced a further source of
noise in this particular study. Likewise, when candidate genes were targeted in
other individual GWAS, rarely more than expected by chance replicated.
Taken together, numerous association studies on MDD and BPD—actually too
many to mention in this overview—hitherto only presented few convincing find-
ings. Amongst them, associations of affective disorders with MTHFR, BDNF, and
SLC6A4 seem to be robust. A length variant in the latter gene shows solid evi-
dence for GxE effect; FKBP5 is another promising candidate for the moderating
effects of early life stress regarding depression in adult life. Lessons that can be
learned from the plethora of false-positive findings to date are that large and well-
characterized samples have to be accrued, with careful evaluation of life events
and the assessment of biological measures such as neuroimaging endophenotypes
or therapy response.
1.2.3 Genome-Wide Association Studies
Along with autism, attention-deficit hyperactivity disorder (ADHD) and schizo-

phrenia affective disorder comprise the core disorders of the Psychiatric GWAS
Consortium (Sullivan 2010; Psychiatric GWAS Consortium [PGC] 2009), and at
the time of writing, more than 12,000 cases of BPD and 14,000 cases of MDD are
available within the PGC, with 20,000 more cases each expected to be included
over the next 2 to 3 years. While these numbers may at first sound impressive, one
has to consider that GWAS are the method of choice to pick common risk alleles
conveying only small individual risk. Other complex-genetic traits might provide
some clues for appropriate sample sizes: it took almost a quarter million people to
detect and respectively confirm 32 risk variants for body mass index, explaining as
little as 2–4% of genetic variance, and it was estimated that another 284 variants
would carry comparable effect sizes and together would explain 6–11% of the
genetic variation. The authors assumed that further 730,000 individuals would
have to be genotyped to uncover 95% of these variants (Speliotes et al. 2010).
Likewise, a recent study on body height (Lango Allen et al. 2010) examined
180,000 subjects and found 180 loci explaining 16% of phenotypic variants.
As body weight and height are for sure somewhat easier to determine than
depression, which is a heterogeneous condition from the start, one can easily see
the obstacles one has to face when dealing with these kinds of studies. Having said
this, and thereby also lowering the bar of expectations somehow, the above-men-
tioned studies are also encouraging and can be seen as proof-of-principle: GWAS
can detect novel pathways and provide meaningful results, and thus larger scale
studies should be encouraged in order to identify the molecular determinants of
affective disorders, as previous linkage and association studies fell short in con-
clusively delineating these. Concluding these introductory remarks, the recent
debate on the missing—or, rather, hidden—heritability has to be mentioned. The
discussion whether the major endogenous psychoses are due to the sum of multiple
common alleles with small individual effects (e.g. Purcell et al. 2009) or due to many
rare variants, also including copy number variants (CNV), and causing ‘‘synthetic
associations’’ (Dickson et al. 2010) and resulting in phenocopies (Gershon et al.
2011) is held lively and far from being resolved. These authors’ personal view is that
both models might exist—which however complicates matters even more.
BPD
GWA studies on BPD, which has a higher heritability and presumably less
GxE effects as compared to depression, have (probably due to these facts)
provided stronger findings than MDD GWAS. The following section elaborates
on the most interesting findings from BPD GWAS at the time of writing
(04/2011) with focus on replicated risk genes and pathways, while issues like
population admixture, microarray technology, and statistical comments are not
further commented upon.
In one of the first published GWAS on BPD, Baum et al. (2008a) reported
genome-wide significance of rs10120253 in intron 1 of diacylglycerol kinase eta
(DGKH) in a German and US American population using a pooling approach. The
gene product of DGKH metabolizes diacylglycerol (DAG), which is produced
upon cleavage of PIP2 into IP3 and DAG by phospholipase C. DAG, in turn,
activates protein kinase C which phosphorylates a variety of proteins including
Dishevelled, an inhibitor of GSK3b (which itself has been considered an out-
standing candidate gene for BPD due to several lines of molecular genetic evi-
dence; Luykx et al. 2010). Furthermore, DGKH knockdown in HeLa cells
impaired the MEK/ERK pathway, while overexpression activated the pathway
(Yasuda et al. 2009). DGKH might therefore be involved in crucial pathways for
psychiatric disorders and especially the mechanism of action of lithium. However,
replication of DGKH failed in three studies on BPD and/or lithium response,
respectively (Manchia et al. 2009; Tesli et al. 2009; Takata et al. 2011) while four
other studies were ambiguous or positive (Baum et al. 2008b; Ollila et al. 2009;
Squassina et al. 2009; Zeng et al. 2011). A recent study provided evidence for
an association of a DGKH risk haplotype with MDD, BPD, and adult ADHD (Weber
et al. 2011). Meta-analyses on Caucasian (Weber et al. 2011) as well as Asian (Takata
et al. 2011) samples however demonstrated that DGKH is significantly associated
with BPD. Furthermore, increased expression of DGKH in BPD (Moya et al. 2010)
was demonstrated in human post-mortem tissue, so that DGKH represents one of the
most promising candidate genes for BPD to date. Other candidates from the Baum
et al. GWAS include NXN, VGCNL1, DFNB31, and SORCS2, the latter two of
which were replicated in a later study (Ollila et al. 2009).
The UK Wellcome Trust Case Control Consortium (WTCCC 2007) aims at the
investigation of several complex genetic disorders with high prevalence. As evi-
dent from the first glance on the Manhattan plots in this paper, there are no
‘‘skyscraping’’ BPD risk SNPs as compared to very clear signals in cardiovascular
or metabolic disorders. The WTCCC BPD GWAS provided genome-wide evi-
dence for a non-gene marker next to PALB2, NDUFAB1, and DCTN5; other
signals were observed for KCNC2, GABRB1, GRM7, and SYN3, all of which are
in pathways previously implicated in BPD. Shortly after the WTCCC report, Sklar
et al. reported on the STEP-UCL study and provided significant findings for
MYO5B, TSPAN8, CDH7, and EGFR (Sklar et al. 2008). Some of those genes
were attempted to replicate using a targeted approach; both TSPAN8 (Scholz et al.
2010) and CDH7 (Soronen et al. 2010) were confirmed in doing so. When the
significant signals from the WTCCC and Baum data sets were tested in Sklar’s
STEP-BD/UCL sample, negative findings were observed for DGKH and PALB2,
however, this analysis provided further support for CACNA1C and DFNB31
arguing for the rationale to combine large data sets. Accordingly, the fourth
GWAS study, ED-DUB-STEP2 (Ferreira et al. 2008), investigated another 1000
patients and included meta-analytic treatment of the WTCCC and STEP-UCL data
sets (total n = 4387 cases). In doing so, the holy grail of genome-wide significance
was reached for markers in two genes: CACNA1C (alpha-1 subunit of a voltage
dependent calcium channel) and ANK3 (ankyrin 3). Other interesting candidate
genes from this study include SYNE1, SPRED1, CMTM8 (which interacts with
EGFR), NPAS3 (which has previously been suggested to be associated with
schizophrenia and bipolar disorder; Pickard et al. 2009), and ARNT2. In a sub-
sequent meta-analysis including two samples from Nordic countries, the breast
cancer risk genes PALB2 and BRCA2 were followed up in these as well as the
WTCCC and STEP-UCL/ED-DUB-STEP2 samples (total case n = 5547). In doing
so, variants in both genes were shown to be associated with BPD (Tesli et al.
2010). Not surprisingly nevertheless, CACNA1C and ANK3 drew most attention
in follow-up studies.
For both ANK3 and CACNA1C, it is noteworthy that replication attempts not
only provided evidence that these genes are associated with BPD; rather, they were
demonstrated to be associated with a broad range of disorders across diagnostic
boundaries arguing for a more unspecific role of these genes in psychiatric dis-
orders. For example, ANK3 was not only replicated in BPD (Lee et al. 2010;
Schulze et al. 2009; Scott et al. 2009; Smith et al. 2009), but also associated with
schizophrenia (Athanasiu et al. 2010). Likewise, CACNA1C was again found to be
associated with BPD (Keers et al. 2009), but also with schizophrenia (Green et al.
2009; Moskvina et al. 2009; Nyegaard et al. 2010), MDD (Green et al. 2009), and
psychopathological features (e.g., agitation) therein (Casamassima et al. 2010).
Neuroimaging studies demonstrated an effect of the CACNA1C risk variant
rs1006737 on brain structure (Franke et al. 2010; Kempton et al. 2009) as well as
function (Erk et al. 2010; Krug et al. 2010; Wessa et al. 2010).
In the last 2 years, several other GWAS and meta-analyses on BPD have been
published. A small study from Japan (Hattori et al. 2009), which applied a two-stage
design, provided nominal although not corrected significance for markers within
AUTS2 (previously implicated in autism), SNAP25 (which is a schizophrenia and
ADHD candidate gene), PLXNA2 (which has been found in schizophrenia and
anxiety GWAS) and CSMD1, which was already one of the candidates from the Baum
et al. study (Baum et al. 2008a). No other top hits from previous BPD GWAS however
were replicated. In Han Chinese patients suffering from BPD type I, Lee et al. (2010)
likewise did not provide findings on the genome-wide level, yet interestingly
also found suggestive association of BPD with SNPs in another voltage-dependent
calcium channel subunit, CACNB2 (other highly significant SNPs were located in
KCTD12, SP8, and ST8SIA2) pointing again to calcium signalling having a role in
BPD. Targeted investigation of previously identified GWAS candidate genes yielded
a p = 10-5 for an SNP near ANK3, yet no other gene has been replicated.
Scott and colleagues combined two GWAS studies from the US, Canada, and
UK (the NIMH/Pritzker and GSK GWAS) and analyzed them separately as well as
in conjunction with the WTCCC study (Scott et al. 2009). In doing so, no genome-
wide significant finding was observed; yet three regions with a p around 10-7 were
reported encompassing the genes MCTP1 (which encodes a high-affinity calcium
binding protein which is highly expressed in the brain), ITIH1 and GLN3.
Furthermore, CTNNA2 was amongst the top hits which also gained support from
other hypothesis-free approaches in psychiatric disorders such as ADHD (Lesch
et al. 2008). Neither DGKH, ANK3 nor CACNA1C were confirmed in this
GWAS, however the latter yielded convincing support upon a fixed-effects meta-
analysis including the Ferreira and Schulze studies. In 2009, Kelsoe and associates
reported on two GWAS examining US Americans of European and African
ancestry (Smith et al. 2009), respectively, with a combined n = 1346 BPD cases.
Again, no genome-wide significant findings emerged and interestingly, significant
findings were discrepant for each subsample. One of the promising top hits in this
study is NTRK2, as this gene which encodes a neurotrophin receptor has been
implicated in mood disorders previously. When previous GWAS risk genes
were tested for, ANK3 yielded further support, while CACNA1C was negative.
It should be noted that this sample overlaps with the one tested by Baum et al.;
generally, the sample overlap between different studies will become the rule rather
than the exception due to the need for international cooperation and large sample
sizes. Furthermore, a small GWAS from Norway (n = 194), which however
was followed up in a larger Icelandic sample (Djurovic et al. 2010), provided
suggestive evidence for several interesting candidate genes (e.g. GUCY1B2,
SHANK, and CNTNAP5), none of which however was amongst the top hits in
previous studies. The largest study to date has employed a two-stage design using
a discovery cohort of 682 BPD patients and carried over the top 48 SNPs to
replication samples; SNPs surviving this procedure were subjected to a meta-
analysis with previous BPD GWAS data sets (Cichon et al. 2011). The total case
number investigated in this study was thus n = 8441 BPD patients. In doing so, the
neurocan (NCAN) gene was identified as a susceptibility factor for BPD with the
best SNP yielding a p = 2.1 9 10-9, i.e. genome-wide significance.
In conclusion, GWAS on BPD and subsequent meta-analysis provided evidence
that BPD shares risk variants with schizophrenia, MDD, and ADHD; furthermore,
calcium and GABA signalling pathways were repeatedly found to be associated
with disease, along with genes modifying neuronal plasticity. At the time of
writing, CACNA1C, ANK3, and DGKH can be considered the risk genes with the
most compelling body of evidence. Accordingly, those are scrutinized more
thoroughly and first functional studies already provided evidence for changes in
brain function in risk allele carriers.
MDD
While the prevalence of MDD is five to tenfold higher as compared to BPD, its
heritability is lower and presumably heterogeneity is even higher. These issues
complicate GWAS on this phenotype, probably explaining the lack of genome-
wide findings despite the fact that studies on MDD and schizophrenia feature the
largest of all disorders analyzed in the PGC.
The NIH sponsored Genetic Association Information Network (GAIN) studies
also featured major depression and these studies were amongst the first published
GWAS on MDD (Sullivan et al. 2009). Discovery sample patients came from two
Dutch longitudinal studies (NESDA and NTR, combined n [1700). The top 25
SNPs featured four SNPs in the PCLO gene, which encodes for a subunit of the
presynaptic vesicle fusion complex, although none of them met the criteria for
genome-wide significance. Considerable overlap was noted for the mood disorder
candidate genes CACNA1C, ANK3, GRM7, and DGKH. While PCLO did not
clearly replicate in the Sullivan et al. study, a later reanalysis questioned this initial
notion and argued for an association of a non-synonymous coding SNP with MDD
in the very same replication cohorts (Bochdanovits et al. 2009). Furthermore, a
later population-based study demonstrated an association of PCLO rs2522833 with
depressive disorders (Hek et al. 2010), which also held true when a meta-analysis
of all published data was conducted especially when only population-based studies
were considered (p = 1.9 9 10-9). Most interestingly, in a hypothesis-free
approach, PCLO was demonstrated to be differentially expressed and associated
with BPD again questioning the diagnostic specificity of GWAS candidate genes
(Choi et al. 2011). However, meta-analysis of an MDD and a BPD GWAS did not
support a role of PCLO in BPD (Liu et al. 2011).
In a medium-sized GWAS from Germany (Rietschel et al. 2010), there was
a suggestive finding for HOMER1 which replicated in an independent sample.
In this study, the authors also conducted a genomic imaging study and demonstrated
decreased dorsolateral prefrontal cortex activation in the n-back task as well as
decreased anterior cingulate cortex activation upon anticipation of a monetary

reward in risk allele carriers. Especially the latter might be related to anhedonic
behavior, one of the key features of depression. Most interestingly, also CACNA1B
was amongst the highest ranked genes, again implicating calcium signalling in
affective disorders. A larger (n = 1636 cases) UK-based GWAS argued for the BICC1
gene in MDD (almost needless to say, genome-wide significance was missed);
however, it was not replicated in samples from Munich and Lausanne. When all three
studies were treated by meta-analysis, the schizophrenia/autism candidate gene
NLGN1 was amongst the most promising findings with a pcombined = 8.5 9 10-6
(Lewis et al. 2010). When the STAR*D study was analyzed for the phenotype MDD
(n = 1,221; Shyn et al. 2011), no genome-wide significant findings also emerged.
Promising or previously implicated candidate genes—however, all at a p[10-5—
included ANKRD46, CTNND2, and CSMD3. Another recent GWAS focused on
recurrent early onset MDD, as defined by an onset before the age of 31 (GenRED, n =
1020; Shi et al. 2011). Nested candidate gene analysis yielded the lowest p value in
CACNA1C; as is common for all mood disorder GWAS, there were no findings
meeting the genome-wide significance threshold, but several highly suggestive
findings with the top hit in a brain-expressed transcript of unknown function. Other
interesting candidates include GDNF, SP4, STIM1, KCNQ1, VAMP4, and CSMD1.
Most noteworthy, the SP4 signal (which almost entirely came from female subjects)
became stronger when the GenRED sample was treated meta-analytically with the
STAR*D and GAIN studies (total ncases = 3,957; Shyn et al. 2011). This meta-
analysis yielded better, although still only suggestive significance levels and also
argued for an association of the GRM7 gene. While SP4 encodes a transcription
factor orchestrating gene networks implicated in affective disorders (and most
notably, as mentioned above, the SP8 transcription factor has been found in a BPD
GWAS), GRM7 which encodes a glutamate receptor was not only of suggestive
significance in the WTCCC BPD GWAS, but also amongst the top hits of another
MDD GWAS (Muglia et al. 2010). This study was performed in two European
samples from Southern Bavaria and Lausanne (total n[1,500), yet also did not result
in genome-wide significant findings or meaningful overlap of top SNPs between both
samples. The authors computed a meta-analysis of both samples as well, along with
the implementation of gene-wide tests. This interesting method yielded several genes
which also survived a correction procedure, the genes with the lowest p-values being
SMG7 and NFKB1. Candidate genes from previous studies however did not replicate
in this analysis, apart from the glutamate receptor gene GRM7. A similar approach
(i.e., discovery GWAS followed by meta-analysis and gene-based tests) was taken by
the largest MDD GWAS to date, the MDD2000 + study (Wray et al. 2012). Here, a
total of[2400 cases were examined and a meta-analysis was conducted by including
the GAIN sample and the UK-based study reported by Lewis et al. (total n[5700
cases). Suggestive findings (p\10-5) include NOS1AP, ADCY3, and the schizo-
phrenia/autism risk gene CNTNAP2 (the latter in males only). The adenylate cyclase
ADCY3 gene was ranked second in the gene-based test, and also the gene encoding
galanin (which was previously shown to be associated with antidepressant treatment
response and disease severity in MDD and anxiety disorders; Unschuld et al. 2010)
was amongst the top ten hits in this analysis. From the pre-selected candidate genes,
IL10, OPRM1 (being a candidate stemming from the GAIN MDD GWAS), HTT,
HTR1B, GRIN1, and the apparently pleiotropic risk gene CACNA1C were associ-
ated with disease. Meta-analysis did not yield significant findings and in particular
did not support PCLO as a risk gene for MDD.
Trait depression, as assessed with the NEO-PI personality questionnaire (where
‘‘depression’’ is a subscale of the Neuroticism domain) was assessed in two GWAS
in the general population from Sardinia and the US (combined n = 4811; Terr-
acciano et al. 2010). The top hit, at a p = 6 9 10-7, was an intronic SNP in the
RORA gene. Two other noteworthy high-ranking candidates include the glutamate
receptor gene GRM8, which hitherto has mainly been associated with cognitive
phenotypes, and CDH13, which has been identified in an ADHD linkage scan
meta-analysis (Zhou et al. 2008) as well as GWAS on ADHD (Lesch et al. 2008)
and substance use disorders.
Cross-Disorder Analyses
In order to yield larger samples, several meta-analyses have been conducted.
The largest meta-analysis combined the WTCCC, STEP-BD, NIMH-BD, and the
German BPD sample, as well as the GAIN-MDD GWAS on MDD. The total
number of cases exceeded 6600, compared against [9000 controls (McMahon
et al. 2010). An inherent problem with this kind of study is the use of different
genotyping platforms, diagnostic heterogeneity, as well as ethnic heterogeneity;
to minimize these limitations, only subjects of European descent have been
analyzed. In doing so, six SNPs which were located in the PBRM1 gene met the
criteria for genome-wide significance. The best SNP was also significant in the
replication sample, yielding a final p = 1.7 9 10-9. When the ED-DUB-STEP2
GWAS was analyzed together with an MDD GWAS (Liu et al. 2011), CAC-
NA1C SNPs passed the hurdle of genome-wide significance while ANK3 was
not supported in the meta-analysis, probably suggesting that this gene is more
specific to BPD. SYNE1 was one of the candidates where the significance level
actually increased upon meta-analysis, and which interestingly also turned up in
the primary PGC BPD GWAS meta-analysis. Not surprisingly, also schizo-
phrenia and BPD were treated meta-analytically (Wang et al. 2010). In this
study, meta-analysis provided evidence for the genes ASTN2 and CNTNAP2,
both of which have been implicated in ADHD, as well as the GABA receptors
GABRR1 and GABRR2. When the three large US American GWAS on psy-
chiatric disorders and treatment efficacy—namely, STEP-BD (BPD), CATIE
(schizophrenia), and STAR*D (MDD) were analyzed jointly (yielding a total
ncases[3000; (Huang et al. 2010), one locus met the criteria for being genome-
wide significant (near the ADM gene, and apparently being specific for bipolar II
disorder). A total of 24 more SNPs reached the defined Omnibus GWAS Test
Threshold; however, more than half of them were imputed. Promising candidates
are again CTNND2, SP8, ODZ4, and NPAS3.
An alternative rationale is to search for risk variants influencing phenotypic
features of mood disorders. Suitable phenotypes include, for example, therapy
response (see below) or suicidal ideation. Accordingly, the STEP-BP, WTCCC,

UCL, and STAR*D studies were evaluated with respect to the latter (Perlis et al.
2010). None of the 11 loci which were identified in the discovery cohorts however
replicated. Also, candidate genes which were selected according to previous data
(such as HTR1A or TPH2) did not yield convincing evidence. Meta-analysis of all
samples argued for an involvement of SORBS1 and PRKCE, a gene with some
a priori biological evidence. Analysis of STAR*D alone, where 90 out of 1953
patients developed treatment-emergent suicidal ideation (Laje et al. 2009),
revealed a highly significant association of an SNP in PAPLN and suggestive
association of an IL28RA SNP. Additive effects with previous risk alleles for
treatment-associated suicidal ideation in the GRIK2 and GRIA3 genes were
observed. However, paucity of psychometric data on the suicidal patients along
with the very limited sample size bears the high chance of a type-I error. Similar
analyses in GENDEP (total n = 706, thereof n = 244 with treatment-associated
suicidal ideation under treatment with either escitalopram or nortriptyline; Perroud
et al. 2010) provided some evidence for the genes GDA, KCNIP4, and ELP3 to be
associated with escitalopram-associated suicidal ideation. Nested candidate-driven
approaches did not yield significant results. A major concern regarding these
studies is whether or not treatment-associated suicidal ideation is genetic at all and
whether these studies are homogeneous—the striking differences in the percentage
of suicidal ideation casts some doubts on this assumption.
Analysis of the complete PGC data set, comprising 12,000 BPD cases and
52,000 controls, yielded 21 SNPs with a corrected p\0.05, the best candidate
genes being CACNA1C, ODZ4, and two regions of chromosome 11 and 12. As
also suggested from earlier cross-disorder analyses demonstrating a significant
overlap of common risk variants for BPD and schizophrenia (including also
CACNA1C, as well as another voltage-dependent calcium channel and a member
of the diacylglycerol kinase family [DGKI]; Moskvina et al. 2009), the latest
cross-disorder analysis of the PGC yielded strong evidence for an association of
CACNA1C with endogenous psychoses as evidenced by p = 8.45 9 10-9 when
BPD and schizophrenia samples were combined. In these analyses, it became
also evident that BPD risk genes were highly predictive for schizophrenia,
and vice versa. On the other hand, BPD neither predicted MDD nor did MDD
predict BPD or schizophrenia. When all three disorders were pooled together (total
ncases[25,000), six genes met the criteria for genome-wide significance: ITIH3, the
HLA/HIST cluster on chromosome 6p21-p22, CACNA1C, TCF4, NT5C2/
CNNM2, and IFI44/ELTD1. In MDD alone, the situation is much more frustrat-
ing: when combining more than 11,000 cases from the GAIN, GenRED, GSK,
mdd2000, MPIP, RADIANT, STAR*D, and NGFN Germany studies, only two
SNPs (in the genes NVL and GPHN, which is a highly interesting candidate) came
near the level of genome-wide significance. Lower heritability in conjunction with
increased heterogeneity might explain the scarcity of solid findings in MDD as
compared to BPD.
1.2.4 Copy Number Variations
Currently, there is increasing interest about the role of deletions/duplications of

large chunks of the genome (copy number variations, CNVs) in psychiatric dis-
orders. Especially large ([100 kb) and rare CNV harboring many different genes
seem to occur more often in schizophrenia, mental retardation, and autism—
although there is no diagnostic specificity and some CNVs might underlie any of
these three conditions. In any case, these CNVs are rare and can only account for a
small percentage of cases, again arguing for a ‘‘common disease, multiple rare
variant’’ model. In bipolar disorder, there are only two studies to date which
yielded conflicting findings. While the first study (Zhang et al. 2009a; 1001
patients) demonstrated an excess of large ([100 kb) and rare CNVs in BPD,
Grozeva et al. (2010) did not find an increased rate of large and rare CNVs in
almost 1700 patients suffering from BPD. Hence, even larger samples are needed
to unequivocally evaluate the contribution of CNVs in BPD, although from the
present data it seems to be clear that at least large and rare CNVs can only account
for a very small fraction of BPD cases, if at all.
1.3 Pharmacogenetics
In the treatment of depression, antidepressive pharmacotherapeutic agents have

proven to be highly effective for a large proportion of patients. However, two
major problems have to be faced: (1) treatment resistance: 30–40% of all patients
fail to respond sufficiently to the initial treatment (Fava and Davidson 1996) and
(2) treatment intolerance: There is a considerable rate of ‘‘treatment emergent
adverse effects’’ associated with antidepressive pharmacotherapy such as hypo-
tension, weight gain, anticholinergic effects, antidepressant-induced mania, or
sleep disturbance, which leads to discontinuation of treatment in about 10% of the
cases (MacGillivray et al. 2003).
Among multiple reasons underlying non-response to antidepressive pharma-
cotherapy or differential development of treatment emergent adverse effects under
antidepressants, it has been suggested that psychotropic drug response may be
heritable with first-degree relative pairs being significantly concordant for anti-
depressant treatment response (Pare et al. 1971; O’Reilly et al. 1994; Franchini
et al. 1998). Pharmacogenetic studies allowing for the detailed dissection of the
genetically influenced heterogeneity of psychotropic drug response have revealed
several risk genes on a pharmacokinetic as well as on a pharmacodynamic level to
drive antidepressant treatment response. On a pharmacokinetic level, variation in
the CYP2D6 gene resulting either in poor metabolizers (PM; 7% of the Caucasian
population) or in rapid (RM) or even ultrarapid metabolizers (UM; 3% of the
Caucasian population) has been reported to be associated with response to tricyclic
antidepressants and SSRIs, particularly paroxetine (cf. Kirchheiner et al. 2004).
On a pharmacodynamic level, association of response to antidepressants has been
observed with variation in candidate genes of depression, especially those involved

in the serotonergic system (e.g. SLC6A4, HTR1A, HTR2A, MAOA, TPH1 (for a
review see Kato and Serretti 2010; Serretti et al. 2005). Additionally, there is first
evidence for differential genetic effects on treatment response specifically in
melancholic depression (Baune et al. 2008), depression with comorbid anxiety
(Domschke et al. 2008a, 2010) or in gender subgroups (Domschke et al. 2008c).
The identification of genetic risk factors for antidepressant treatment response as
known to date tremendously helps in better understanding the mechanism of action
of antidepressants. These rapidly growing molecular genetic findings might
nourish further biochemical, physiological, or pharmacological studies and even-
tually lead to a personalized medicine with an individually tailored antidepressive
pharmacotherapy according to genotype reducing the patients’ suffering and
lowering healthcare costs at the same time.
Most of the MDD GWAS were embedded in efficacy studies; thus not sur-
prisingly, treatment response GWAS were reported frequently. It should be noted
that treatment response of course encompasses a plethora of diverse factors
ranging from adherence to medication to exogenous pharmacokinetic influences,
adding further noise to already noisy genetic data. The first report was on the
German MARS trial and also included an independent German sample as well as
the STAR*D study (Ising et al. 2009). Obviously, not only diagnostic but also
treatment heterogeneity has to be taken into account in the interpretation. The best
signal, which however was not significant on the genome-wide level, came from
an SNP in the 50 region of CDH17; when the 338 best SNPs from the German
samples were tested in the STAR*D sample, 46 were associated at the nominal
level of significance. Amongst them, interestingly, was HOMER1 (see above).
When the level 1 participants of STAR*D, which received citalopram, were
analyzed separately (743 remitters versus 608 non-remitters; Garriock and
Hamilton 2009a, b), as little as three SNPs were associated with response on the
p\1 9 10-5 level. The most interesting finding from this study probably is
ARNTL, which is also a member of the PAS superfamily and related to NPAS3.
In 2010, the GWAS data of the multicentre European GENDEP study was pub-
lished (n = 811 cases, treated with either nortriptyline or escitalopram; Uher et al.
2010). Analysis of the complete sample did not provide meaningful signals, while
analysis of either compound alone pointed to the IL11, UST, and RGL1 genes.
Genotype by drug interaction analyses interestingly implicated a region 11 kb
downstream of NOL4, which was also one of the four top regions in the STAR*D
study. As both studies were published in parallel, this was not mentioned in either
paper, yet can be considered a true independent replication.
As compared to MDD, pharmacogenomics studies in BPD are sparser. As
treatment response to lithium, the gold standard drug treatment in BPD, is familial,
lithium treatment response studies seem to be most worthwhile; as only ca. 40% of
all BPD patients can be considered clear lithium responders, and as treatment
might go along with considerable side effects in the case of non-response, data
on genetic prediction of lithium response would directly translate into the
clinical routine. Several case–control association studies comparing responders to
non-responders have suggested a variety of risk genes such as the SLC6A4 (seven
studies, mixed findings) and other hypothesis-driven targets mainly of neuro-
transmitter pathways, intracellular signal transduction pathways involved in the
mechanism of action of lithium, and circadian clock genes. GSK3B and CREB1
are amongst the candidates with the best empirical support and also good face
validity and hence should be further tested, as evidence is far from being solid.
Samples were often rather small and again, most genes lack replication. Scholarly
overviews on published association studies can be obtained from McCarthy et al.
(2010) and Smith et al. (2010). Although not specifically designed for this ques-
tion, the STEP-BD trial tested for lithium response as well by means of a GWA
study, which was replicated in a second, independent sample (n = 458, and n = 359
bipolar I or II patients, respectively). Not surprisingly, there were no genome-wide
significant findings, however five SNPs associated in the STEP-BD cohort repli-
cated in the second sample including a polymorphism within the GRIA2 gene
(Perlis et al. 2009). To specifically search for lithium response genes, the
ConLiGen consortium (Schulze et al. 2010) has gathered more than 1200 lithium
treated BPD patients whose treatment response has been evaluated using the Alda
scale. Genome-wide genotyping has been accomplished and initial data are
expected for the second half of 2011.
2 Anxiety Disorders
2.1 Clinical Genetics
Panic disorder has been found to be highly familial with an up to three to fivefold
increased prevalence of the disorder in first-degree relatives of patients with panic
disorder (Horwath et al. 1995; Maier et al. 1993; Hettema et al. 2001). In relatives
of the subgroup of patients with panic disorder and suffocation anxiety, an even
higher familiarity has been discerned (Horwath et al. 1997). Furthermore, famil-
iarity of panic disorder seems to depend on the age of onset in the index patient
with an onset before the age of 20 years predicting a 17-fold increased risk of
panic disorder in first-degree relatives (Goldstein et al. 1997). Also for generalized
anxiety disorder and specific phobias a significant familial aggregation was
reported (Hettema et al. 2001; Marks and Herst 1970).
Twin studies have identified up to 2–3 times higher concordance rates for panic
disorder in monozygotic as compared to dizygotic twins (Skre et al. 1993), with an
even higher concordance rate for the subgroup of patients with carbon dioxide-
sensitive panic disorder (Bellodi et al. 1998). According to a comprehensive meta-
analysis, the contribution of genetic factors has been calculated to be as high as up
to 48%, with the remaining 52% being attributable to individual environmental
factors. Generalized anxiety disorder has been estimated to have a heritability of
about 32%, while the common heritability of phobias was reported to be about
30%, with highest estimates for agoraphobia (67%), blood-injection-phobia (59%),

and social phobia (51%). The heritability of posttraumatic stress disorder was
reported to be about 20–30% (Kendler et al. 1999; Hettema et al. 2001; Segman
and Shalev 2003). Finally, several studies point towards overlapping genetic risk
factors for panic disorder and agoraphobia or other phobias, respectively (Kendler
et al. 1995; Mosing et al. 2009; Tsuang et al. 2004). In panic disorder and other
anxiety disorders, segregation analyses failed to identify a mode of inheritance
according to Mendelian patterns, which points to a complex genetic inheritance
with an interaction of multiple ‘‘vulnerability’’ or ‘‘risk genes’’, each with only a
minor individual influence (‘‘oligo- or polygenic model’’), and environmental
influences (Vieland et al. 1996).
2.2 Molecular Genetics
2.2.1 Linkage Studies
In panic disorder, linkage studies have yielded a variety of potential risk loci on
chromosomes 1p, 4q, 7p, 9q, 11p, 15q, und 20p (Crowe et al. 1987, 2001; Knowles
et al. 1998; Gelernter et al. 2001; Hamilton et al. 2003; Thorgeirsson et al. 2003;
Fyer et al. 2006; Kaabi et al. 2006). In subgroups of patients with panic disorder
with comorbid bipolar disorder or kidney/bladder dysfunction, respectively, risk
loci on chromosomes 2, 12, 13, and 18 or 13 and 22, respectively, have been
described (MacKinnon et al. 1998; Logue et al. 2009; Weissman et al. 2000;
Hamilton et al. 2003). In social or specific phobia, linkage studies have excluded a
major influence of HTR2A and SLC6A4 loci, with however, some evidence for
potential risk loci on chromosomes 16q and 14p (Stein et al. 1998; Gelernter et al.
2003, 2004).
2.2.2 Association Studies
In panic disorder, a variety of association studies has been published so far. Most
studies have investigated variation in classical candidate genes for panic disorder
as suggested by animal models (e.g. knock-out mice), challenge experiments (e.g.
cholecystokinin [CCK] challenge, caffeine challenge), or pharmacological obser-
vations (e.g. clinical efficacy of selective serotonin reuptake inhibitors (SSRIs),
monoamine oxidase (MAO) inhibitors). Most significant evidence has been yiel-
ded for association of variants in the CCKBR (Kennedy et al. 1999; Hösing et al.
2004), MAOA, particularly in female patients (Deckert et al. 1999; Samochowiec
et al. 2004; Maron et al. 2005b), COMT (again restricted to female patients,
Hamilton et al. 2002; Domschke et al. 2004; Woo et al. 2002; Woo et al. 2004;
Domschke et al. 2007; Zintzaras and Sakelaridis 2007), HTR1A (Rothe et al.
2004; Huang et al. 2004) and ADORA2A (Deckert et al. 1998; Hamilton et al.
2004a; Hohoff et al. 2010). Furthermore, there is some evidence for several other
potential risk variants to be involved in the pathogenesis of panic disorder such as
polymorphisms in HTR2A (Inada et al. 2003; Rothe et al. 2004; Maron et al.
2005a; Unschuld et al. 2007; Yoon et al. 2008), SLC6A4 (Ohara et al. 1998;
Maron et al. 2005a, b; Strug et al. 2010; but: Deckert et al. 1997; Hamilton et al.
1999; Blaya et al. 2007), TPH2 (Maron et al. 2007; Kim et al. 2009; but: Mössner
et al. 2006), NET (Lee et al. 2005; but: Sand et al. 2002a), CCK (Wang et al. 1998;
Hattori et al. 2001), ACE (Olsson et al. 2004; Erhardt et al. 2008; Bandelow et al.
2010; but: Shimizu et al. 2004), the transcription factor CREM (Domschke et al.
2003; Hamilton et al. 2004b), ‘regulator of G-protein signaling’ (RGS2, RGS7)
(Leygraf et al. 2006, Smoller et al. 2008b; Hohoff et al. 2009b) and several
hormone receptors (Sand et al. 2002b; Ho et al. 2004; Keck et al. 2008; Hodges
et al. 2009). However, since most of these studies either did not withstand repli-
cation in independent samples or still warrant replication, these results have to be
considered preliminary. The role of the GABA-ergic system in panic disorder
remains to be further elucidated on a molecular genetic level with only little
evidence so far for the glutamate decarboxylase (GAD) or GABA receptors and
transporters, respectively (Crowe et al. 1997; Sand et al. 2000; Hettema et al.
2006; Nakamura et al. 2006; Kobayashi et al. 2007; Thoeringer et al. 2007, 2009;
Unschuld et al. 2009). Recently, besides the classic neurotransmitter systems much
attention has been paid to the role of neuropeptides in the mediation of anxiety.
Significant association of anxiety or panic disorder in particular have been reported
for variants in genes for galanin (Unschuld et al. 2008), the neuropeptide Y (NPY)
system (Domschke et al. 2008b) and the neuropeptide S receptor (NPSR)
(Domschke et al. 2011). Finally, there is preliminary support for possible inter-
active effects of several genetics variants in the mediation of the genetic risk for
panic disorder, e.g. for HTR1A and COMT (Freitag et al. 2006). For social phobia
and generalized anxiety disorder, association has been reported with variation in
the dopamine transporter (SLC6A3) gene (Rowe et al. 1998), while DRD2 variants
seem to play a role in the pathogenesis of posttraumatic stress disorder (Segman
and Shalev 2003). Further associations were observed for COMT in specific
phobias (McGrath et al. 2004), for HTR2A in social phobia (Lochner et al. 2007)
and MAOA in generalized anxiety disorder (Tadic et al. 2003).
In summary, consistent with findings from clinical genetic and linkage studies,
molecular genetic association studies point to a complex genetic etiology of
anxiety disorders with an additive or rather interactive effect of multiple risk
variants.
2.2.3 Genome-Wide Association Studies
The first genome-wide association study in panic disorder in a Japanese sample

yielded evidence for several markers in genes, which have not been implicated in
the pathogenesis of anxiety before (PKP1, PLEKHG1, TMEM16B, CALCOCO1,
SDK2, and CLU) (Otowa et al. 2009). However, these findings could not be
replicated in a follow-up GWAS by the same group (Otowa et al. 2010). Another
genome-wide association study in three German samples points to a potential role
of the TMEM132D gene in the pathogenesis of panic disorder (Erhardt et al.
2011). Currently, another large GWAS on a homogenous sample from Germany is
under analysis (Reif et al., in preparation).
2.3 Genetics of Intermediate Phenotypes of Anxiety Disorders
Dimensional markers such as neuroticism, anxiety sensitivity, state or trait anxiety

or behavioral inhibition have been proposed as valid intermediate phenotypes of
anxiety disorders with aggregation in families and elevated concordance rates in
monozygotic twins pointing to a significant heritability (e.g. Rosenbaum et al.
1991; Maier et al. 1992; Stein et al. 1999). Linkage studies have discerned risk loci
on chromosomes 8, 18, 20, and 21 for harm avoidance (e.g. Cloninger et al. 1998),
and association studies have reported a potential role of genetic variation of e.g.
SLC6A4 for harm avoidance and neuroticism (Lesch et al. 1996) and the corti-
cotropin releasing hormone (CRH) for behavioral inhibition (Smoller et al. 2003,
2005), respectively.
Besides neuropsychological markers, more recently, neurobiological traits have
been investigated as intermediate phenotypes of anxiety disorders. Here, signifi-
cant association was observed, for e.g. increased sympathetic activity and
ADORA2A as well as COMT gene variation (Hohoff et al. 2009a; Kang et al.
2010), blushing propensity in social phobia with SLC6A4 variation (Domschke
et al. 2009), an increased startle response with COMT and SLC6A4 variants (e.g.
Montag et al. 2008; Brocke et al. 2006), as well as CO2-sensitivity to panic attacks
with again SLC6A4 variation (Schmidt et al. 2000; Schruers et al. 2011).
Another very promising intermediate phenotype of mental disorders in general
and affective and anxiety disorders in particular are neuronal activation correlates
of emotional processing as captured by functional imaging techniques such as
magnetic resonance imaging (fMRI). In panic disorder, first imaging genetics
findings may indicate a distorted corticolimbic interaction depending on variants
of the COMT and HTR1A (Domschke et al. 2006, 2008d). In patients with social
phobia, polymorphisms in SLC6A4 and TPH were found to be associated with
increased amygdala excitability (Furmark et al. 2004, 2008, 2009). Finally,
markers spanning RGS2 were reported to be associated with childhood behavioral
inhibition and with increased limbic activation during emotion processing
(Smoller et al. 2008b). In summary, these first imaging genetics findings in panic
disorder, social phobia, and anxiety-related traits may indicate that—depending
on variants of COMT, SLC6A4, HTR1A, and RGS2—patients with anxiety dis-
orders are prone to impaired cerebral processing of anxiety-related stimuli
in cortical regions known to play a crucial role in the evaluation of emotional
stimuli and determination of salient events (for a review see Domschke and
Dannlowski 2010).
2.4 Gene-Environment Interaction
In contrast to a multitude of gene-environment studies (GxE) available in

depression (see 1.2.2., e.g. SLC6A4 and FKBP5), with respect to anxiety disorders
as a categorical nosological entity or anxiety-related traits, to the best of our
knowledge only few GxE studies have been performed yet (for a review see
Klauke et al. 2010). No associations were found between 5-HTTLPR, childhood
emotional abuse, and neuroticism (Antypa and Van der Does 2010). An exemplary
GxE study with respect to anxiety-related traits has been published by Stein et al.
(2008), who observed a significant interaction between levels of childhood mal-
treatment and the less active 5-HTTLPR S allele on anxiety sensitivity as mea-
sured by the anxiety sensitivity index (ASI). 5-HTTLPR S and LG haplotypes were
furthermore reported to be associated with increased anxiety in interaction with
daily stressors (Gunthert et al. 2007). Conversely, assessing 5-HTTLPR genotype
and environmental adversity at birth (family adversity) and at 19 years of age
(stressful life events), Laucht et al. (2009) found an interactive effect of more
active 5-HTTLPR LL genotype and high family adversity on anxiety disorders.
Other studies have identified association of the ADORA2A with increased anxiety
after caffeine administration in healthy volunteers, demonstrating that a panic
disorder risk gene might drive the sensitivity to an environmental stimulus and,
therefore, the vulnerability to anxiety (Alsene et al. 2003; Childs et al. 2008).
2.5 Pharmacogenetics
In anxiety disorders, so far only three exemplary studies have investigated the
impact of genetic variants on response to a pharmacological treatment regime.
Two groups reported significant association of 5-HTTLPR with response to SSRI
treatment in panic disorder as well as in generalized anxiety disorder (Perna et al.
2005; Stein et al. 2006). Furthermore, SSRI treatment in panic disorder might in
part be driven by variation in HTR1A (Yevtushenko et al. 2010).
3 Overlapping Phenotypes
Depression and anxiety are highly comorbid with up to 60% of patients with
depression also displaying anxiety (Leckman et al. 1983) and about 58% of those
patients actually meeting DSM criteria for anxiety disorders (see review by
Lydiard 1991; e.g. de Graaf et al. 2002; Kessler et al. 1996; Zimmerman et al.
2002). Comorbidity of affective and anxiety disorders has a significant impact on
the course and treatment of the respective leading disease with a more chronic
course and a significantly detrimental effect on treatment response (e.g. Clayton
et al. 1991; Liebowitz 1993; Lydiard 1991). Patterns of occurrence allow for both
affective and anxiety disorders preceding the respective other disease.
Besides either simultaneous or sequential true comorbidity of anxiety disorders
and major depression there is a continuous debate about a possible overlapping
phenotype between anxiety disorders and depression. The clinical phenotype of
‘‘anxious depression’’ (Overall et al. 1966; Overall and Zisook 1980) capturing
major depression with subthreshold anxious features has been suggested to con-
stitute a diagnostic entity of its own requiring specific diagnostic and therapeutic
attention (see Levine et al. 2001; Lydiard and Brawman-Mintzer 1998; Silverstone
and von Studnitz 2003). Dual action drugs acting as reuptake inhibitors on both
transporters (serotonin and norepinephrine reuptake inhibitors, SNRI) have been
suggested to be superior to SSRI only or tricyclic antidepressants TCA in the
treatment of anxious depression (Rudolph et al. 1998; Silverstone and Ravindran
1999). While three large meta-analyses discerned similar response rates to anti-
depressant treatment in highly anxious and less anxious patients with major
depression (Levine et al. 2001; Nelson et al. 2009; Papakostas et al. 2008; see
Nelson 2008), there is accumulating evidence for anxious features of depression
potentially complicating the course of antidepressant treatment (e.g. Altamura
et al. 2004; Domschke et al. 2010; Fava et al. 2008; Joffe et al. 1993; see review by
Bagby et al. 2002).
Apart from the individual genetic risk for affective and anxiety disorders, both
disease entities also exhibit a common familial risk (as reviewed by Middeldorp
et al. 2005). There is evidence from twin studies that depression and general
anxiety disorder, panic disorder, and post-traumatic stress disorder share a con-
siderable proportion of their genetic risk (Kendler et al. 1992, 2007; Kendler 1996;
Roy et al. 1995). Consistently, molecular genetic studies have yielded evidence for
specific genetic loci that may generally influence susceptibility across the anxiety-
depression spectrum, e.g. on chromosome 18q (cf. Camp et al. 2005; Hettema
2008). In particular, the combined clinical phenotype of anxious depression has
been suggested to constitute a specific subtype with an increased familial risk of
depression (Clayton et al. 1990, 1991), which points to a possibly increased her-
itability of anxious depression with a specific set of genetic risk factors mediating
the vulnerability for the development of anxious depression. First imaging and
pharmacogenetic studies in anxious depression have implied CNR1, NPY, and
SLC6A4 to confer parts of antidepressant treatment response particularly in the
clinical phenotype of anxious depression, potentially via a dysfunctional cortico-
limbic interaction underlying distorted emotional processing (e.g. Baffa et al.
2010; Domschke et al. 2008a; 2010).
These molecular and imaging genetic findings of overlapping genetic variants
as well as common brain networks of emotional processing partly driving both
clinical phenotypes of anxiety and affective disorders point to similar neurobio-
logical mechanisms underlying these disorders and therefore possibly a common
clinical sub-phenotype shared by anxiety and affective disorders. Particularly,
the clinical phenotype of ‘‘anxious depression’’ might thus possibly constitute a
diagnostic entity of its own requiring specific diagnostic and therapeutic attention
(cf. Lydiard and Brawman-Mintzer 1998; Silverstone and von Studnitz 2003).
So, back from bench to bedside, genetic and imaging studies might inspire a
re-evaluation and refinement of DSM-IV categorized nosological concepts of
depression and anxiety. Alternatively, the current and still emerging body of
knowledge in the field of neurobiological research in anxiety and depression might
have even more far-reaching consequences in the future by challenging the DSM
concept in itself in favor of a more neurobiologically oriented taxonomy of mental
disorders. As suggested by Smoller et al. (2008a), genetic and imaging research
revealing etiological mechanisms of mental disorders might infer a novel noso-
logical concept based on pathogenesis more than phenomenology. To date,
however, despite first essential steps having been made, neurobiological knowl-
edge about the pathomechanism of depression and anxiety has still not progressed
far enough to provide a reliable and valid fundament for diagnostic decisions in
daily clinical practice. So, in summary the presently known vulnerability genes
and patterns of affective and anxiety disorders are slowly beginning to challenge
the DSM-defined nosological boundaries and might have the potential to evolve
into a valuable tool to more precisely delineate the diagnostic system of mental
disorders in the future.
4 Outlook
Future research with respect to the genetic dissection of affective and anxiety
disorders will have to comprise technical as well as clinical aspects. On a
molecular genetic level, more comprehensive analyses such as tagging SNP
approaches, haplotype analyses, as well as the investigation of epistasis of several
genes constituting relevant biochemical pathways or cascades are warranted. Here,
novel genomic techniques such as duplication/deletion analysis using genotyping
arrays and next-generation sequencing of the whole exome or genome for point
mutation identification might have a large impact on risk gene identification.
Furthermore, it will be of utmost importance to analyze the functional conse-
quences of the associated genetic variants and thereby gain more knowledge about
the pathomechanism of the disease of interest. Additionally, there is a need for
more detailed gene-environment interaction studies potentially also in a genome-
wide fashion (cf. Poulton et al. 2008; Thomas 2010) in order to disentangle the
interactive effect of genetic and environmental factors conferring risk or resilience,
respectively, to affective and anxiety disorders. In this respect, epigenetic studies
investigating e.g. DNA methylation or histone modifications regulating gene
activity will tremendously contribute to the elucidation of the interplay between
environmental and genetic factors in the pathogenesis of affective and anxiety
disorders (cf. for bipolar disorder and schizophrenia: Abdolmaleky et al. 2006,
2008).
Besides the more technical aspects as detailed above, future research in the
genetics of affective and anxiety disorders will greatly benefit from clinical
considerations. Given that—apart from very few results—most linkage and

association findings either did not withstand replication in independent samples or
still warrant replication and given that genome-wide association studies in affec-
tive as well as in anxiety disorders so far fell short of expectations regarding
replicating previous candidate genes or generating novel hypotheses, one possible
reason might be the great neuropsychological and neurobiological heterogeneity of
the investigated phenotypes of categorical nosological entities as defined by DSM-
or ICD-criteria. Thus, besides the recruitment of even larger sample sizes, a more
precise definition of the clinical phenotype will be key. In the latter respect, the
approach of investigating intermediate phenotypes of affective and anxiety dis-
orders will have to be intensified with the search for novel depression- and/or
anxiety-related neurophysiological, biochemical, endocrinological, neuroana-
tomical, cognitive, or neuropsychological endophenotypes (cf. for major depres-
sion: Hasler et al. 2004) and their analysis with respect to their genetic basis.
In summary, to date there is some support for several risk genes contributing to
the development of affective and anxiety disorders or their intermediate pheno-
types and some light has been shed on gene-environment interactions contributing
to the disease risk. However, so far the identified genetic risk factors are of no
diagnostic or predictive value, which will only change if the entirety of all genetic
risk factors interdependent with environmental factors is identified, which is not
foreseeable in the near future. Nevertheless, the increasing elucidation of genetic
risk factors tremendously helps in better understanding the pathophysiology of
affective and anxiety disorders and might nourish the development of innovative
pharmacotherapeutic substances in the treatment of these diseases (e.g. Domschke
and Zwanzger 2008), preferably in an individually tailored manner according to
genotype.
Acknowledgments This paper was supported by the DFG (Grant RE1632/1-1,/1-3 and/5 to AR,
KFO 125 TP4 to AR, DE357/4-1 to AR, SFB TRR 58 C2 and Z2 to AR and KD, respectively;
RTG 1252, to AR) and the BMBF (Panik-Net Subproject 6, to AR). We thank A. Alttoa for
valuable discussions and her kind help in the preparation of the manuscript.
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Behavioural Genetics of the Serotonin
Transporter
K. Haddley, V. J. Bubb, G. Breen, U. M. Parades-Esquivel

and J. P. Quinn
Abstract The serotonin transporter is a key regulator of the bioavailability of

serotonin and therefore any modulation in the expression or action of the trans-
porter would be expected to have consequences on behaviour. The transporter has
therefore become a target for pharmaceutical intervention in behavioural and
mood disorders. The search for polymorphic variants in the transporter that would
associate with neurological disorders has been extensive but has become focused
on two domains which are both termed variable number tandem repeat (VNTR)
polymorphisms. Both of these VNTRs are in non-coding DNA and therefore
proposed to be mechanistically involved in a disorder through their ability to
modulate transcriptional or post-transcriptional regulation of the transporter. The
most extensively studied is in the promoter and is a bi-allelic insertion/deletion
found in the 50 promoter region of the gene 1.2 kb upstream of the transcriptional
start site. This VNTR, termed, 5-HTTLPR was initially identified as two variants
containing either, 14 (short/deletion) or 16 (long/insertion) copies of a 22 bp
repeat. A second widely studied VNTR found in the non-coding region of the
transporter is located within intron 2 and comprises 9, 10 or 12 copies of a
16–17 bp repeat termed, STin2.9, STin2.10 and STin2.12, respectively. These
VNTR polymorphisms have been associated with a range of behavioural and
psychiatric disorders including depression, OCD, anxiety and schizophrenia,
K. Haddley V. J. Bubb J. P. Quinn (&)

Department of Clinical and Molecular Pharmacology,
Institute of Translation Medicine, University of Liverpool,
Liverpool, Merseyside L69 3BX, UK
e-mail: jquinn@liv.ac.uk
G. Breen U. M. Parades-Esquivel
Social, Genetic and Developmental Psychiatric Centre (SGDP MRC),
Institute of Psychiatry, King’s College London, DeCrespigny Park,
London SE5 8AF, UK

DOI: 10.1007/7854_2011_186
504 K. Haddley et al.
however often the lack of reproducibility in different cohorts has led to debate on
the actual association of the polymorphisms with this extensive range of
neurological conditions. Here we review these two polymorphic VNTRs in depth
and relate that to pharmaceutical response, their ability to regulate differential
transporter expression, their core involvement in gene-environment interaction and
their genetic association with specific disorders.

Keywords Mood disorders Genetic polymorphism Serotonin Serotonin

transporter Variable number tandem repeat Gene regulation Monoaminergic
signaling
Contents
1 Introduction...................................................................................................................... 504
2 Investigating Polymorphic Variation .............................................................................. 506
3 Serotonin Transporter: Variable Number Tandem Repeats and Clinical
Association Studies. The Linked Polymorphic Region (5-HTT LPR) ......................... 507
4 The Serotonin Transporter Intron 2 VNTR (STin2 VNTR).......................................... 512
5 Pharmacogenetics of the 5-HTT Polymorphisms and Selective Serotonin
Reuptake Inhibitor (SSRI) Response.............................................................................. 515
6 Gene 9 Environment (G 9 E) Interaction Role in the SLC6A4 Polymorphism ........ 515
7 Haplotype Analysis of the SLC6A4 Polymorphisms, Gene 9 Gene (G 9 G)
and Gene 9 Gene 9 Environment (G 9 G 9 E) Interactions..................................... 516
8 Nuclear Imaging of SLC6A4 Polymorphic Variants in Affective Disorders ............... 517
9 Effects of 5-HTTLPR on Serotonin Signalling Systems (MRI) ................................... 518
10 Effects of 5HTTLPR on Serotonin Transporter Binding .............................................. 520
11 Effect of the Serotonin Transporter Polymorphisms on Functional Gene Expression... 522
12 Lessons from the 5-HTT Polymorphisms and Their Application to Other
Gene Polymorphisms....................................................................................................... 524
13 Summary.......................................................................................................................... 524
References................................................................................................................................ 526
1 Introduction
The monoaminergic signaling system is a key component in maintaining

physiological control over many aspects of human health including emotional
behaviour and psychological states. One of the major players in this system is the
neuromodulator, serotonin (5-HT). Disturbances in the 5-HT system have been
implicated in the pathophysiology of many CNS-related disorders such as anxiety
(Lesch et al. 2003), depression (Ressler and Nemeroff 2000), Obsessive
Compulsive Disorder (OCD) (Bengel et al. 1999), aggression and antisocial
behaviour (Lesch and Merschdorf 2000), addiction (Kreek et al. 2005a, b) and
suicide (Arango et al. 2002). The level of neuromodulatory 5-HT present in the
synaptic cleft and available for action at cell surface serotonin receptors is
Behavioural Genetics of the Serotonin Transporter 505
dependent on reuptake by the serotonin transporter (5-HTT) that belongs to the

Na+/Cl- dependant solute-carrier family of transporters and is encoded for by the
SLC6A4 gene. In the CNS, 5-HT and 5-HTT are mostly localised on the perisy-
naptic membrane of neurons of the raphe nuclei whose processes innervate many
areas of the brain thought to be involved in cognition and behaviour regulation
(McLaughlin et al. 1996). Since this transporter regulates the spatiotemporal
fine-tuning of 5-HT signalling and is paramount in the processes controlling
many aspects of mood and behaviour, it is naturally a major target for several
antidepressants, drugs of abuse and potent neurotoxins and is a major focus in the
investigation of several affective disorders.
Genetic variability between individuals comes in many guises including single
nucleotide polymorphisms (SNPs), which may be coding or non-coding, and
micro- or mini-satellites. Micro-satellites such as simple tandem repeats exist
as di, tri or tetra nucleotide repeats, whilst mini-satellites consist of 10–100
nucleotides repeated several times in tandem bordered by unique DNA sequences.
Mini-satellites are more stable than micro-satellites and are believed to be present
in the genome due to unequal crossover (non-allelic homologous recombination)
or unequal sister chromatid exchange. Mini-satellites frequently exist as a multi-
allelic polymorphism due to variation in the number of the tandem repeat units
(reviewed in Haddley et al. 2008). Here we will focus on a subclass of mini-
satellite known as a variable number tandem repeat (VNTR). VNTRs show some
degree of degeneration where one repeat may be slightly different from the next
but overall the core consensus sequence is maintained (Jeffreys et al. 1985;
Naslund et al. 2005). The majority of VNTRs are found in non-coding regions of
the genome and many are found at higher density in gene enriched areas compared
to non-genic regions. VNTRs have the potential to act as transcriptional regulatory
domains as they have a number of sequence specific DNA motifs within the repeat
that can act as transcription factor binding sites. They may display evolutionary
conservation between humans and non-human primates but are often not found in
lower mammals (Lesch et al. 1997; Soeby et al. 2005). The SLC6A4 gene locus
presents a number of polymorphisms within non-coding regions and it has been
shown that the allelic variation in these VNTRs can function in a tissue-specific
and stimulus-inducible manner, in vitro and in vivo, to fine-tune gene expression
(Lesch et al. 1996; Heils et al. 1996; MacKenzie and Quinn 1999; Hariri et al.
2002; Hranilovic et al. 2004; Roberts et al. 2007). This fine tuning may be cor-
related, mechanistically, not only with normal physiological function and variation
between individuals, but also with a predisposition to behavioural disorders by
altering neurotransmitter signaling in response to challenges and stress. Further-
more, if stimulus-inducible expression varies with a specific disorder-associated
polymorphism then that may have similar implications in the response of an
individual to therapeutics (Fiskerstrand et al. 1999; Lovejoy et al. 2003; Klenova
et al. 2004; Roberts et al. 2007). In addition, VNTRs can participate in regulation
of gene expression at other levels. For example, it has been suggested that VNTRs
located in untranslated (UTR) regions may play an important role in mRNA
stability, which can also affect levels of transcript available in the cellular
Fig. 1 Schematic representation of the 5-HTTLPR, STin2VNTR and rs25531/2 SNP in the
SLC6A4 gene
environment (Nakamura et al. 1998; Zamorano et al. 2006). Further, VNTRs

located in exons are known to affect protein folding and have been associated with
differential levels of mature RNA availability in the cytoplasm (Nakamura et al.
1998). More recently the role of VNTRs in the epigenetic regulation of response to
challenges has been investigated (Olsson et al. 2010; Kinnally et al. 2010; Ali et al.
2010b; Vasiliou et al. 2011; Van Ijzendoorn et al. 2010). We have previously
reviewed clinical correlation and function with VNTR genotype in the SLC6A4
gene including the linked polymorphic region (5-HTTLPR) found in the promoter
region of the gene and the STin2 VNTR found in intron 2, also sometimes referred
to as intron 3, Fig. 1 (Haddley et al. 2008). Here we include more recent findings
and create a picture of how these correlations stand to date.
2 Investigating Polymorphic Variation
Beside genome wide association studies, which link a specific polymorphism to a

particular disorder, there are a number of applications both in vitro and in vivo, by
which the putative molecular function of a polymorphism can be assessed. In vitro
techniques that can be applied to investigate gene expression under basal condi-
tions and in response to challenge e.g., drugs (cocaine, citralopram) or transcrip-
tion factors (CTCF) include: (1) Production of polymorphism-reporter gene
constructs to examine the ability of a polymorphism to alter gene expression in
clonal or primary cell lines (Klenova et al. 2004; Guindalini et al. 2006; Roberts
et al. 2007; Ali et al. 2010a, b; Vasiliou et al. 2011); (2) Quantitative or real-time
PCR in lymphoblast cell lines of known genotype to determine relative quantities
of gene expression as a function of a specific genotype (Hranilovic et al. 2004); (3)
Allelic expression imbalance (AEI) that can determine the relative abundance of
transcript derived in an allele-specific manner (Lim et al. 2006; Martin et al. 2007).
In vivo techniques that can be applied to examine allele-specific changes include:
(1) Chromatin immunoprecipitation (ChIP) in a cell line to examine transcription
factor-DNA binding and associated changes in histone modifications over a
specific polymorphism (Vasiliou et al. 2011). This can also be performed genome-
wide using Geneseq arrays; (2) The generation of ‘‘humanised’’ transgenic mice, in
Fig. 2 a Sequence of the

5-HTTLPR. The deletion
in the the short variant
is represented in grey.
b Sequence of the
STin2VNTR. * Denotes
repeats absent from both the
nine and ten copy variants
and ** denotes a repeat
missing in the nine copy
variant
which a VNTR driving marker gene expression for example, b-galactosidase, may
be transiently expressed to identify tissues in which the VNTR may be active
in response to certain challenges or at particular developmental stages
(Mackenzie and Quinn 1999); (3) Formaldehyde assisted identification of regu-
latory elements (FAIRE) that identifies genome-wide open regions of chromatin
allowing assessment of changes in chromatin structure over polymorphic regions
in response to challenge (Esquivel et al. 2011); (4) DNA methylation that identifies
regions which are not accessible to regulation by transcription factors; (5) Nuclear
imaging including single-photon emission computed tomography (SPECT) and
positron emission tomography (PET) that allow resolution of events at a molecular
level (for example transporter/receptor binding potentials and density) and func-
tional magnetic resonance imaging (fMRI) that allows analysis of a whole system
activity and connectivity approach between brain regions (see Sect. 6).
Used in combination these techniques provide a powerful armoury for delin-
eating the functional mechanism of action of polymorphisms with the observed
clinical associations for such polymorphisms.
3 Serotonin Transporter: Variable Number Tandem Repeats

and Clinical Association Studies. The Linked Polymorphic
Region (5-HTT LPR)
The 5-HTT LPR is a biallelic insertion/deletion found in the 50 promoter region of

SLC6A4 approximately 1.4 kb upstream of the transcription start site (Heils et al.
1995, 1996). Initially the LPR was identified as an allelic variant comprising 14
(short) or 16 (long) copies of a 22–23 bp repeat, Fig. 2a. More recently, Nakamura
et al. verified the existence of subgroups within these variants to uncover as many
as 14 allelic variations of this locus. Most studies focus on the more common
biallelic 14 (s) and 16 (l) alleles of the LPR, however, since the discovery of a
functional SNP (rs25531) within the long variant of the LPR (an A to G substi-
tution) this is now referred to as the triallelic LPR variant. Inclusion of this SNP
(lg) has conferred clinical properties normally associated with the s-allele onto the
l-allele (Nakamura et al. 2000; Hu et al. 2005; Kraft et al. 2005; Wendland et al.
2006). However, no significant variation was observed between la and lg variants
in a study employing the use of AEI (Martin et al. 2007). The distribution of these
genotypes varies, with individuals Hm for the s-allele appearing at lower or similar
frequencies to those Hm for the l-allele in the Caucasian population (Heils et al.
1995, 1996). However, allelic frequencies vary among different ethnic groups.
The frequency of the alleles for the Caucasian population has been shown to be
27.4–28.3% (l/l), 43.4–51.0% (s/l) and 21.6–28.3% (s/s), whilst that of Asian
population has been shown to be 4.2–10.0% (l/l), 30.0–39.2% (s/l) and
55.6–60.0% (s/s) (Smits et al. 2004).
We have previously discussed the clinical and functional associations of this
variant with a number of affective disorders and behaviour traits (Table 1) and levels
of gene expression in vitro and in vivo (Table 2) (extensively reviewed in Haddley
et al. 2008). In general the l-allele of 5-HTTLPR has been associated with increased
levels of reporter gene activity, mRNA abundance and transporter binding. We will
now extrapolate on these associations using more recent data and meta-analyses.
Early and promising associations were made between 5-HTT polymorphisms
and a number of affective disorders with the Hm s-allele believed to be predisposing
to affective disorders. However, these initial findings were not always consistent and
even the result of meta-analysis found some of these association to be lacking in
power. More recent investigations have sought to clarify the specifics of these
associations including more detailed parameters such as age, population, environ-
ment and co-morbidities, as well as the mechanisms by which these variants may
mediate their effect. Recent meta-analysis found a significant association of the Hm
s-allele genotype with an increased risk of major depressive disorder (MDD) in
Caucasians but not in Asians (Kiyohara and Yoshimasu 2010). In agreement with
the latter, when studied in a Thai population no association could be made between
5-HTTLPR genotype and MDD (Tencomnao and Wongpiyabovorn 2010). A recent
investigation in a Northern Swedish population could not find any association of the
5-HTTLPR with bipolar depression (Alaerts et al. 2009).
Overall the picture remains unclear on the exact role of the LPR polymorphism
risk associations particularly in Asian populations, where the s-allele is present at a
higher frequency than the l-allele, but taking into account other factors, especially
gene x environment (G 9 E) interactions and treatment response, may go some
way to clarifying current understanding.
Homozygosity for the s-allele has conferred threefold higher odds of a patient
suffering from post-stroke depression (PSD) compared to those with one or more
l-allele (Kohen et al. 2008). Similarly, in a Chinese population, homozygosity for
the s-allele has been associated with a higher incidence of PSD (Fang et al. 2011).
The mechanism(s) by which these alleles may confer susceptibility to mood
Table 1 A sample of previously reported clinical associations for the 5-HTT LPR polymorphism
Author Disorder Cohort/ethnicity Predisposing
genotype
5-HTTLPR genotype positively associated with affective disorder
Cervilla et al. (2006) Depression European (Spanish) s/s
Zalsman et al. (2006) Depression Caucasian (European) s/s
Collier et al. (1996a) Unipolar depression European s/s
Hauser et al. (2003) Unipolar depression European s/s
Collier et al. (1996a) Bipolar depression European s/s
Cho et al. (2005)a Bipolar depression Mixed s/s
Lotrich and Pollock Bipolar depression – s/s
(2004)a
Lasky-Su et al. (2005)a Bipolar depression Mixed s/s
Hauser et al. (2003) Bipolar depression European s/s
Lotrich and Pollock Major depressive – s/s
(2004)a disorder
Lesch et al. (1996) Anxiety Caucasian (German) s/s
Mazzanti et al. (1998) Anxiety European (Finnish) s/s
Katsuragi et al. (1999) Anxiety Japanese s/s
Du et al. (2000) Anxiety Caucasian s/s
(predominantly)
Melke et al. (2001) Anxiety Caucasian s/s
Feinn et al. (2005)a Substance abuse Mixed s
(alcohol)
Caspi et al. (2003) Suicide Caucasian (non-Maori) s/s
Bengel et al. (1999) OCD Caucasian l/l
Malhotra et al. (1998) Psychosis/ Mixed (N.American) l/l
hallucinations
Dubertret et al. (2005) Schizophrenia Mixed I
5-HTTLPR genotype not positively associated with affective disorder
Lasky-Su et al. (2005)a Unipolar depression Mixed
Mendlewicz et al. (2004) Unipolar depression European
Mansour et al. (2005) Bipolar depression Caucasian (N.America)
Meira-Lima et al. (2005) Bipolar depression Brazilian
Mendlewicz et al. (2004) Bipolar depression European
Ospina-Duque et al. Bipolar depression Columbian
(2000)
Arango et al. (2003) Major depressive –
disorder
Frisch et al. (1999) Major depressive European (Jewish)
disorder
Middledorp et al. (2007) Anxiety European (Dutch)
Deary et al. (1999) Anxiety British
Mendlewicz et al. (2004) Suicide European
Arango et al. (2003) Suicide –
Geijer et al. (2000) Suicide Caucasian (European)
Serretti et al. (2002) Major Psychosis European
Dickel et al. (2007) OCD Mixed
(continued)
Table 1 (continued)
genotype
Rao et al. (1998) Schizophrenia Caucasian (N.
American)
Rao et al. (1998) Schizophrenia African-American
Rao et al. (1998) Schizophrenia Caucasian (Swedish)
Where cohort states European or British, ethnicity was either mixed or not stated
a
meta-analysis
disorders in unknown, however, Gillihan et al. (2010) suggested that individuals

with an s-allele display greater amygdala activity during intentional mood regula-
tion and suggested that hyperactivity of the amygdala during mood recovery may be
a potential mechanism by which the s-allele could increase the risk of depression. In
addition, the chronicity of depression has been associated with homozygosity for the
l-allele in a Korean population who showed a higher rate of chronicity, but not
recurrent tendency, compared to those with at least one s-allele (Myung et al. 2010).
The observation that genotype can affect amygdala response would imply a role
for the polymorphism in mood and anxiety disorders and higher anxiety scores have
been shown to be significantly associated with s-allele or lg-allele homozygosity in a
Turkish population concomitant with improved academic performance (Calapoglu
et al. 2011). Interestingly, it has also been demonstrated that these anxiety-related
associations can have an influence as early on as foetal developmental. Maternal
anxiety during pregnancy and negative emotionality in early infancy was significant
in infants carrying one or more copies of the s-allele (Pluess et al. 2011). Allelic
variation at the LPR has also been shown to influence mother-offspring bonding;
mothers with an s- or lg-allele showed higher sensitivity and greater attachment
during mother-infant interactions (Mileva-Seitz et al. 2011).
The importance of tighter definition of sub-group classification can be seen in two
recent studies investigating genotype interactions and OCD, for example, meta-
analysis suggests that the I-allele may be associated with childhood-onset OCD and
OCD in Caucasians (Bloch et al. 2008). A further study found little effect of the
triallelic LPR in OCD in a mostly male population and the authors suggest that
further replication in a larger cohort with an equal number of female subjects might
be more predictive of risk associations with OCD (Tibrewal et al. 2010).
The s-allele of the 5-HTTLPR has also shown considerable associations in rela-
tion to alcohol dependence (McHugh et al. 2010; Wang et al. 2011), consumption in
adolescents (Van der Zwaluw et al. 2010) and relapse in recovering alcoholics (Pinto
et al. 2008). Furthermore, the risk association of the LPR with substance abuse has
been demonstrated to display age-dependant bias: prior to the age of 15 individuals
Hm for the s-allele did not show any difference to those with an l-allele, however, post
15 years of age they showed higher tobacco use and in early adulthood they were
more active alcohol, drug and tobacco users (Merenakk et al. 2011).
A recent multi-centre study paired with meta-analysis failed to detect any
association with the 5-HTTLPR polymorphism and attention deficit hyperactivity
Table 2 A sample of previously reported functional effects for the 5-HTT LPR polymorphism
Author Model Experimental detail Gene expression bias
Heils et al. (1996) Placental choriocarcinoma cell line (JAr) VNTR within the promoter (reporter) l[s
Lesch et al. (1996) Lymphoblast cell lines VNTR with the promoter (reporter) l[s
Sakai et al. (2002) Immortalised serotonergic raphe neurons Isolated VNTR (reporter) l=s
(RN46A)
Cos-7 cell lines
PC12 cell lines
Mortensen et al. (1999a, b) Placental choriocarcinoma cell line (JAr) 1.8 kb fragment upstream to the cap site l=s
including VNTR and a novel internal
379 bp fragment (reporter)
Mortensen et al. (1999a, b) Immortalised serotonergic raphe neurons 1.8 kb fragment upstream to the cap site l[s
(RN46A) including VNTR and a novel internal
Behavioural Genetics of the Serotonin Transporter
379 bp fragment (reporter)

Lesch et al. (1996) Lymphoblast cell lines mRNA quantification l/l [ l/s or s/s
Hranilovic et al. (2000) mRNA quantification l/l [ l/s or s/s
Bradley et al. (2005) mRNA quantification l/l [ l/s or s/s
Lim et al. (2006) Human pons tissue post mortem mRNA quantification l=s
Lesch et al. (1996) Lymphoblast cell lines 5-HT binding l/l [ l/s or s/s
Lesch et al. (1996) Lymphoblast cell lines 5-HT uptake rate l/l [ l/s or s/s
Greenberg et al. (1999) Blood platelets from healthy controls 5-HT uptake rate l/l [ l/s or s/s
Hanna et al. (1998) Blood platelets OCD patients 5-HT uptake rate l/l [ l/s or s/s
Nobile et al. (1999) Blood platelets from depressed patients 5-HT uptake rate l/l [ l/s or s/s
511
disorder (ADHD) in a Norwegian sample (Landaas et al. 2010). Other studies have
also shown a lack of association in schizophrenia, neurocognition or core psy-
chotic symptoms (Konneker et al. 2010), migraine in Europeans and Asians
(Schurks et al. 2010a) and bulima nervousa (Lee and Lin 2010). Recent associa-
tions have been made between the presence of an s-allele and insomnia (Deuschle
et al. 2010) and anorexia nervosa (Lee and Lin 2010).
4 The Serotonin Transporter Intron 2 VNTR (STin2 VNTR)
The STin2VNTR comprises 9, 10 or 12 copies of a 16-17 bp repeat giving rise to

six possible genotypes 9/9, 10/10, 12/12, 9/10, 9/12 and 10/12 based on copy
number alone without the added complexity of SNPs within each VNTR domain,
Fig. 2B (Lesch et al. 1994). We have previously discussed the clinical and
functional associations of this variant with a number of affective disorders and
behaviour traits (Table 3) and levels of gene expression in vitro and in vivo
(Table 4) (extensively reviewed in Haddley et al. 2008).
Clinical associations with the STin2VNTR have been less prevalent than
those of the 5-HTTLPR over the last 5 years. In contrast to the 5-HTTLPR
polymorphism, a significant effect of the STin2VNTR genotype on susceptibility
to MDD was seen with the 10-allele in a Chinese Han population (Pan et al. 2009).
In addition, individuals with the 9/12 or 12/12 genotype displayed a fourfold
higher incidence of PSD compared with those Hm for the 10-allele (Kohen et al.
2008). Furthermore, chronicity and recurrent tendency of depression were not
associated with STin2VNTR (Myung et al. 2010). Meta-analysis in a migraine
population of European descent found a protective effect in the absence of the
12-allele (Schurks et al. 2010b).
Individually the serotonin polymorphisms have various effects across affective
disorders and populations and more often than not results are conflicting or no
associations can be made. However, when taken in combination with other poly-
morphisms both intra- and intergenic or when considering haplotypes, distinct
associations have been made, for example, in a recent migraine study the
STin2VNTR genotype had no significant effect, however when taken as a haplotype
in conjunction with a serotonin receptor SNP, which also showed no solo association,
a positive correlation was seen with the two polymorphisms having a synergistic
influence on susceptibility to migraine (Joshi et al. 2010). Many haplotype associ-
ations for affective disorders are now being considered investigating not only
interactions between the serotonin transporter LPR and VNTR but also other SNPs
within the SLC6A4 gene locus and between other genes such as the dopamine
receptor 4 (DRD4), serotonin precursor genes, and brain derived neurotrophic factor
gene (BDNF). Such haplotype associations are seen with other gene synergies in
which individual polymorphisms have no association with a particular condition
whereas when addressed together they do demonstrate significant association
(Bellivier et al. 1998; Wendland et al. 2007; Miyajima et al. 2008; Hiio et al. 2011).
Table 3 A sample of previously reported clinical associations for the STin2VNTR

polymorphism
genotype
StlN2 genotype positively associated with an affective disorder
Ogilvie et al. (1996) Major depression British 9
Battersby et al. (1996) Uni/bipolar depression British 9
Evans et al. (1997) Anxiety British 9
Ogilvie et al. (1998) Migraine with aura British 9/9
Hranilovic et al. Suicide Croatian/southern Slavic 10/10
(2003)
Jernej et al. (2004) Suicide Croatian/southern Slavic 10/10
Gutierrez et al. Major depression with Caucasians (Spanish) 10
(1998b) melancholia
Collier et al. (1996b) Bipolar depression Caucasian (English, European 12
and other)
Kirov et al. (1999) Bipolar depression Caucasian (British) 12
Ohara et al. (1999) Anxiety + OCD Asian 12
Baca-Garcia et al. OCD Caucasians (Spanish) 12
(2007)
Liu et al. (1999) Schizophrenia – 12
Hranilovic et al. Schizophrenia Croatian/southern Slavic 12
(2000)
Kaiser et al. (2001) Schizophrenia Caucasian (German) 12
Ogilvie et al. (1998) Migraine without aura British 12/12
StlN2 genotype not associated with an affective disorder
Furlong et al. (1998)a Unipolar depression Caucasian (English and
European)
Collier et al. (1996b)a Unipolar depression Caucasian (English, European
and other)
Furlong et al. (1998)a Bipolar depression Caucasian (English and
European)
Stober et al. (1996) Uni/Bipolar depression Caucasian (German)
Bellivier et al. (1998) Bipolar depression Caucasian (French)
Kunugi et al. (1996)a Uni/Bipolar depression Japanese
Lasky-Su et al. Uni/Bipolar depression Mixed
(2005)a
Lotrich and Pollock Bipolar and major –
(2004)a depression
Gutierrez et al. Bipolar depression Caucasians (Spanish)
(1998a)
Hoehe et al. (1998) Bipolar and major Western European
depression
Rees et al. (1997) Bipolar and major Caucasian (British)
depression
Anguelova et al. Suicide –
(2003)a
(Collier et al. 1996b) Schizophrenia Caucasian (English, European
and other)
Where cohort states European or British, ethnicity was either mixed or not stated
a
Meta-analysis
514
Table 4 A sample of previously reported functional effects for the STin2VNTR polymorphism
Author Model Experimental detail Gene expression
bias
Fiskerstrand et al. (1999) Embryonic stem cells VNTR in isolation cloned into a reporter vector showed 12 [ 10
responses following differentiation
Lovejoy et al. (2003) Murine embryonic stem cells Trimeric oligonucleotides composed of individual repeat
elements cloned into reporter vector showed varied
responses following differentiation.
Lovejoy et al. (2003) JAr human clonal cell line VNTR in isolation 9 [ 12 [ 10
Klenova et al. (2004) Monkey Cos 7 clonal cells Human Differential expression and regulation by transcription factors 12[10[9 9 [ 10 [
HEK293 clonal cells CTCFand YB1 12
MacKenzie and Quinn Transient transgenic mice Differential expression driven by the human StlN2VNTR 10 or
(1999) 12 variant supporting LacZ expression
Hranilovic et al. (2003) Lymphoblasts Allelic expression imbalance/semi-quantitative real time PCR 12/12 [10
Roberts et al. (2007) Stably transfected human JAr clonal cell Differential binding of CTCF and YB-1 transcription factors to
line. Human HEK293T clonal cells the allelic variants in response to lithium chloride
Kaiser et al. (2002) Human blood platelets The maximum rate of 5-HT uptake by platelets was unaffected 12/12 \ 9/10
although the affinity of uptake varied as a function of
genotype
K. Haddley et al.
5 Pharmacogenetics of the 5-HTT Polymorphisms and Selective

Serotonin Reuptake Inhibitor (SSRI) Response
There is vast variability in the response of patients to antidepressant treatment which is

believed to be in part due to heritable characteristics. This would suggest that poly-
morphisms that may affect the expression of ‘‘mood-related genes’’ may also account
for variation in treatment response. A number of studies have investigated such var-
iability in treatment response as a function of genotype particularly in relation to 5-HT
specific reuptake inhibitors (SSRIs) e.g., citralopram, escitalopram, paroxetine and
fluoxetine. The SSRIs selectively block the 5-HTT maintaining the concentration of
5-HT in the synaptic cleft. There are a myriad of published papers that analyse the
effect of the 5-HTT polymorphisms on antidepressant response and tolerance in
patients with affective disorders and in particular MDD (extensively reviewed in Keers
and Aitchison 2011 and Porcelli et al. 2011). Initially in MDD patients a better
response to SSRIs was associated with those individuals with an l-allele in the
5-HTTLPR (Serretti et al. 2007) however, a recent meta-analysis of 5408 participants
found no correlation between 5-HTTLPR genotype and drug response (Taylor et al.
2010). In the recent STAR*D analysis an association was found between adverse
events to citralopram and the triallelic lg-allele (Hu et al. 2007). Other studies have also
pointed to 5-HTT triallelic variant affecting not only tolerance to antidepressants but
also interaction with other polymorphisms such as the STin2VNTR (Kraft et al. 2005;
Smeraldi et al. 2006). It has also been reported that genotype and response associations
may be dependent on (1) the minor allele frequencies between populations because a
significant association between genotype (l-allele) and treatment response was seen
only in non-Hispanic Caucasians (Mrazek et al. 2009), (2) gender differences (Keers
and Aitchison 2010) and (3) environment (stress) (Keers et al. 2011).
Fewer studies have assessed the effect of the STin2VNTR genotype however having
a 10/12 genotype has been associated with a poor response and tolerance (Kim et al.
2000; Peters et al. 2004; Mrazek et al. 2009; Min et al. 2009) although these obser-
vations were not replicated in other studies (Ito et al. 2002; Ham et al. 2005; Smits et al.
2007). Finally, the recent review by Keers and Atchison, highlights the difficulties in
coming to a standard agreement on the effect of polymorphisms due to the heteroge-
neity between studies but also the probability that an amalgamation of small genotypic
effects could be responsible for variation in response and that gene x environment
interactions must also be considered in these analyses (Keers and Aitchison 2010).
6 Gene 3 Environment (G 3 E) Interaction Role

in the SLC6A4 Polymorphism
Personality traits are suggested to be generated by a complex interaction of envi-

ronmental and genetic factors. Various studies suggest that the effect of the SLC6A4
polymorphisms might only be unmasked following specific environmental cues, for
example SLC6A4 Knockout (KO) mice displayed more fearful behaviour compared
to wild-type (WT) only in response to stress (Murphy et al. 2001; Wellman et al.
2007). The potential interaction between the 5-HTTLPR and early experiences was
demonstrated in non-human primates; rhesus macaques with an s-variant analogous
to that of human s-allele, displayed decreased serotonergic function but only in
animals reared under stressful conditions (Bennett et al. 2002). Furthermore, peer-
reared females with the s-variant were more vulnerable to alcoholism and showed
more aggressive behaviour whereas those reared by their mothers were not differ-
entiated by genotype (Champoux et al. 2002; Barr et al. 2003, 2004). Furthermore, in
agreement with a seminal study by Caspi et al. demonstrating a greater risk of
depression and suicide in those individuals Hm for the s-allele dependant on the
number of past stressful life events and childhood maltreatment, it has been found
that individuals in a Spanish population Hm for the s-allele acquired a higher risk of
depression after considerably fewer stressful life episodes (Caspi et al. 2003; Cervilla
et al. 2007). Moreover, in an adult twin study, individuals Hm for the s-allele dis-
played increased sensitivity of depressogenic effects of stressful-life events than
those with one copy of the l-allele (Kendler et al. 2005).
Recently, a potential role for the 5-HTTLPR polymorphisms has been inves-
tigated in post-traumatic stress disorder (PTSD) as a function of a gene 9 envi-
ronment interaction. Having one or more copies of the s-allele predisposed
individuals who had suffered both childhood adversity and adult traumatic events
to develop lifetime PTSD (Xie et al. 2009). A similar effect has also been seen in
patients with a copy of the la-allele with more than 60% of carriers developing
PTSD following three or more stressful life events (Grabe et al. 2009). At first,-
these results would seem conflicting, however, other environmental factors may
need to be considered, for example the s-allele has been associated with decreased
risk of PTSD in low-risk environments (low crime/unemployment rates) but
increased risk of PTSD in high-risk environments (Koenen et al. 2009).
These studies emphasise the significant impact of the environment and early
experiences on genetic polymorphisms and suggest that with a particular genotype
an individual may be more vulnerable to environmental stress and have higher
tendency to develop psychiatric disorders, depending on their genotype. The
interaction between SLC6A4 polymorphisms and stressful life events is striking
and one that has stood up to scrutiny particularly in longitudinal studies (reviewed
in Uher and McGuffin 2008).
7 Haplotype Analysis of the SLC6A4 Polymorphisms,

Gene 3 Gene (G 3 G) and Gene 3 Gene 3 Environment
(G 3 G 3 E) Interactions
Greater associations have been made between genotype and phenotype now
that focus has begun to shift towards investigating polymorphisms in gene-gene
interactions or as haplotypes rather than as standalone entities. According to the
international HapMap project the SLC6A4 gene exists as two haplotype blocks.
A recent study in a Chinese Han population found that when assessed alone no
association could be made between the STin2VNTR and schizophrenia, however,
significant association was found when the locus was studied as a haplotype in
conjunction with a number of tagging SNPs, identifying two haplotypes with positive
association (Lin et al. 2009). Similar results were also found in a North European
population, in that schizophrenia was associated with a haplotype block but not with
single loci (Zaboli et al. 2008). Furthermore, haplotype analysis has revealed that
having the l-allele and the STin2VNTR 10-allele predisposes Russian females, but
not males to suicide (Gaysina et al. 2006). In a Turkish population predisposition
towards suicidal behaviour was not observed for either the 5-HTTLPR or
STin2VNTR polymorphisms, however when combined it was found that those with
an s/10 or l/12 genotype were predisposed to suicidal behaviour (Akar et al. 2010).
Moreover, in a study of German alcoholics the sa/10 and lg/12 haplotypes were more
prevalent in type 2 alcoholics compared with controls (Reese et al. 2010).
Polymorphic interactions are not limited to those in-cis and evidence is emerging
for in-trans interaction between genes as predisposing factors for disease. A recent
investigation by Lorenzi et al. (2010) found that in an Italian sample of patients with
Alzhiemers linked dementia that s-allele homozygosity was particularly abundant and
the 5-HTTLPR synergistically interacted with a polymorphism in the saitohin gene
(Q7R) to increase susceptibility. In addition, interactions between the Hm s-allele
5-HTTLPR genotype and polymorphisms within the cannabinoid receptor 1 locus
have been associated with increased anxiety scores in Caucasian Hungarians (Lazary
et al. 2009). Finally, Armbruster et al. found a significant interaction between the
5-HTTLPR and DRD4 polymorphisms in response to stress in a German population
with individuals Hm for the 5-HTTLPR la-allele and one copy of the DRD4 7R allele
having a lower cortisol response compared to other genotypes (Armbruster et al. 2009).
In addition to two-way interactions many studies have identified three-way
interactions, so called G 9 G 9 E interactions. Ressler et al. conducted an analysis
of SLC6A4 interaction with corticotropin-releasing hormone haplotypes as a
function of child abuse in an African-American cohort. They found that the
5-HTTLPR s-allele interacted with CRHR1 haplotypes and child abuse to predict
current depressive symptoms (Ressler et al. 2010). Similarly, Conway et al. (2010)
found that in individuals Hm for the catechol-o-methyltransferase (COMT) val158
mutation, a Hm 5-HTTLPR l-allele genotype exerted a protective effect in
adolescents in response to stress-related depression.
8 Nuclear Imaging of SLC6A4 Polymorphic Variants

in Affective Disorders
Over the last decade advances in in vivo imaging techniques have enabled further
investigation into the in vitro and genome-wide associations made between
polymorphic variation, changes in brain activity and affective disorders, in the
hope of clarifying not only whether these associations are accurate but also in the
hope of determining biological endophenotypes associated with genotype. Such
investigations have included examining differences in brain structure, brain
metabolism, transporter and receptor-ligand binding and the signaling interactions
between brain regions.
The three most widely used techniques in brain imaging for affective disorders
are SPECT, positron emission tomography (PET) and fMRI. MRI is used to give a
whole system overview of brain activity, whereas SPECT and PET provide res-
olution of events at a molecular level allowing the investigation of binding of a
radiolabelled ligand to its target receptor or transporter. MRI is rapid but gives low
resolution and is generally used as a functional measure of activity. Both SPECT
and PET are used to determine binding potential, target density or distribution, and
volume ratios (Innis et al. 2007). The most commonly used ligands for assessing
binding potentials and distribution for the monoamine transporters are summarised
in Table 5. Many studies concentrating on affective disorders and polymorphisms
using these techniques analyse multiple brain regions, whereas others focus on
specific regions for example the midbrain or striatum which are both rich in
serotonin or the amygdala and limbic systems which are central regions involved
in the control of mood, anxiety and emotion. For this reason and in combination
with the different techniques available there is still conflict over the role of
polymorphisms and their associations however a clearer picture is emerging. The
results of these investigations, with a focus on the serotonin LPR are discussed
below.
9 Effects of 5-HTTLPR on Serotonin Signalling Systems (MRI)
Functional MRI studies have been undertaken to determine the effects of the
5-HTTLPR polymorphism on brain organisation and structure in brain regions
associated with affective disorders. Healthy individuals with an s or lg-allele
displayed reduced grey matter volumes, when compared to those with a la-allele,
suggesting that the polymorphism plays a role in development of brain structure.
Interestingly, the same study found that patients with depression suffered from
reduced bilateral hippocampal volumes and this was particularly pronounced in
those who were Hm for the la-allele, compared to those who were lg/lg, lg/s or s/s
suggesting that individuals Hm for the la-allele are more susceptible to morpho-
logical changes during depressive episodes (Frodl et al. 2004, 2008a, b). A further
study assessed hippocampal volumes in an elderly cohort in a similar manner but
on the basis of the age of onset of depression. This study determined that those
individuals Hm for the l-allele and with late-onset depression had significantly
smaller hippocampal volumes than those with early-onset depression or healthy
controls. In addition they found that in individuals Hm for the s-allele early onset
of depression was associated with smaller hippocampal volumes suggesting that
genotype and age interact to affect the timing of depression (Taylor et al. 2005).
Tracers used in nuclear imaging studies of the 5-HTT
Ligand A.K.A Selectivity Application
[123l](-)-2b-Carbomethoxy-3b-(4-iodophenyl) [123I]b-CIT Cocaine analogue DAT1 SPECT
tropane 5-HTT
NET
[l-123]-N-omega-fluoropropyl-2beta -carbomethoxy- [1231] FP-CIT [123I]-b-CIT analogue DAT1 SPECT
3beta -(4-iodophenyl)nortropane 5-HTT
NET
Behavioural Genetics of the Serotonin Transporter
[11C]3-amino-4-[2-[(di(methyl)amino)methyl]phenyl] [11C]DASB 5-HTT PET

sulfanylbenzonitrile
[llC](+)-6b-(4-Methylthiophenyl)-l,2,3,5,6a, [11C]McN 5652 5-HTT PET
10b-hexahydropyrrolo[2,l -a] isoquinoline
[F-18]fluoro-2-deoxy-D-glucose FDG Glucose metabolism PET
519
Functional MRI has been used by both Hariri et al. and Heinz et al. in healthy
individuals to demonstrate that those with at least one s-allele where found to have
stronger amygdala reactivity and stronger amygdala-prefrontal cortex coupling in
response to aversive pictures or uncertain contexts, which could be considered
stressful (Hariri et al. 2006; Heinz et al. 2005, 2007). Furthermore, in individuals
exposed to fearful faces those Hm for the s-allele showed greater activity in the
right fusiform gyrus (FG), and greater positive functional connectivity between
right amygdala and FG and between right FG and right ventrolateral prefrontal
cortex, compared to those with other genotypes (Surguladze et al. 2008). These
replications are a good indicator that the 5-HTTLPR polymorphism can contribute
to differential hyper-responsiveness of the amygdala, the control centre for emo-
tions, and go some way towards defining a mechanism for the clinical and in vitro
observations that the s-allele is associated with a predisposition to anxiety-
depression spectrum disorders. It has also been suggested that these findings may
also be key in other psychiatric disorders because those Hm for the l-allele dis-
played what could be considered ‘‘hypo-responsiveness’’ a trait that may be
important where a lack of emotion is a key factor e.g., psychopathy (Surguladze
et al. 2008; reviewed in Glenn 2011). The effect of 5-HTTLPR genotype on
connectiveness between the ventral anterior cingulate cortex and the amygdala has
also been examined as this pathway is hyporesponsive in patients with bipolar
disorder. When patients with bipolar depression or healthy controls were presented
with happy and fearful facial imagery those with bipolar disorder showed
decreased activity in this pathway as expected. Furthermore, in healthy individuals
and in those with bipolar depression Hm for the s-allele, this pathway was found to
be hypoactive compared to those with an l-allele (Shah et al. 2009). Taken together
these data strongly suggest an effect of the 5-HTTLPR polymorphism, in particular
the s-allele, on amygdala responsiveness and connectivity although the molecular
mechanisms behind these differences cannot be delineated using MRI.
10 Effects of 5HTTLPR on Serotonin Transporter Binding
A SPECT study in 22 individuals, 14 of which were alcoholics assessed the effect

of the 5-HTTLPR polymorphisms on transporter-ligand binding using [123I]b-
CIT. In healthy individuals Hm for the l-allele the binding capacity of the 5-HTT
in the midbrain was almost twice that of those with an s-allele. By contrast, the
same study noted a modest reduction in binding in alcoholics Hm for the l-allele
compared to those with an s-allele (Heinz et al. 2000). These initial results in
healthy individuals were not confirmed by further SPECT studies using [I-123]b-
CIT that showed no differences in binding on consideration of genotype (Jacobsen
et al. 2000; Willeit et al. 2001). In a further study the predominant findings were
opposed to those of Heinz et al. in that these subjects Hm for the s-allele displayed
significantly higher ligand-binding potential compared to any l-allele containing
genotypes (Van Dyck et al. 2004), however, in this assessment all s-allele
homozygotes were much younger than non-s-allele homozygotes and it should be

noted that decreased ligand-binding in nuclear imagining has been associated with
increasing age (Pirker et al. 2000). Supporting a lack of function in vivo seen with
SPECT, quantitative autoradiography in the prefrontal cortex of post-mortem
brains of suicide victims also failed to show any correlation between 5-HTT
binding and genotype (Mann et al. 2000).
Decreased levels of 5-HTT binding have been observed across a number
of brain regions in a PET study using [C-11]DASB (Selvaraj et al. 2011) or
[11C]McN 5652 (Parsey et al. 2006a) in depressed individuals. The activity of
serotonin re-uptake and metabolism in the context of LPR polymorphisms was
assessed in healthy individuals using PET with fludeoxyglucose F18 (FDG). The
results of this study showed genotype dependant differences in serotonin metab-
olism across the brain. In particular, individuals Hm for the s-allele (n = 6)
showed higher activity in the limbic system, prefrontal and temporal cortices,
whilst in those Hm for the l-allele (n = 6) higher activity was noted in bilateral
frontal structures. Overall the authors suggest that a more widespread effect could
be the foundation for individuals Hm for the s-allele being more susceptible to
anxiety-depression spectrum disorders (Graff-Guerrero et al. 2005).
Interestingly, high-resolution PET FDG imaging showed that monkeys with the
s-allele of the LPR variant displayed increased brain activity in orbitofrontal
cortical regions, amygdala and the bed nucleus of the stria terminalis in response to
stress (relocation to a foreign environment) which provides further support that the
s-allele may confer a susceptibility to stress by mediating aberrant activity in
limbic structures (Kalin et al. 2008).
Similar to investigations using SPECT imaging, correlation between genotype
and binding has also been negative in PET studies utilising the 5HTT-selective
radioligand [11C]McN 5652 (Parsey et al. 2006b; Shioe et al. 2003). However,
PET studies utilising the more sensitive and preferable 5HTT-selective tracer,
[11C]DASB, have demonstrated positive correlations between genotype and
binding capacity. These studies analysed the triallelic variants of the 5-HTTLPR
(la, lg, s). Replicable studies found that, when compared to non-la-allele homo-
zygotes, those Hm for the la-allele demonstrated higher 5HTT-ligand binding
capacity in brain regions associated with high transporter expression in the puta-
men, caudate and the midbrain (Kalbitzer et al. 2009; Praschak-Rieder et al. 2007;
Reimold et al. 2007, respectively). It is worth noting that the study of Reimold
et al. found a significant effect of genotype on 5-HTT binding in the midbrain but
no effect in the amygdala or thalamus, further stressing the importance of exper-
imental design in assessing genotype effects, as these effects are likely to be tissue-
specific or context dependant.
In contrast, in a study using the selective 5-HTT ligand, [11C]DASB, in
alcoholic subjects and healthy controls, Martinez et al. (2009) did not find any
significant association between genotype and 5-HTT binding across a number of
brain regions.
The majority of genotypic effects examined for 5-HTT have been in the mid-
brain but little evidence exists for other regions. Since the origin of serotonergic
projections is the midbrain this may point to an important affect of polymorphism

during development which may have an impact on the circuitry projections to
other brain regions which may affect signaling in later life and be the basis for the
effect of a transporter polymorphism. It follows that a lack of polymorphism-
dependant effects on binding potential in limbic areas may not necessarily be seen
in adults despite differential metabolic activity being observed by FDG PET.
It is particularly interesting to note that a number of studies have found dif-
ferential effects on ligand-binding dependant on the season of the year at which
the experiments where performed with higher binding in healthy controls noted in
the autumn and winter months in both PET (Praschak-Rieder et al. 2008) and
SPECT (Ruhe et al. 2009) scanning.
11 Effect of the Serotonin Transporter Polymorphisms

on Functional Gene Expression
The regulation of expression of a given gene can vary between individuals because
of epigenetics and polymorphic variation in regulatory domains where for example,
transcription factors may bind. Many studies have shown that cis-regulatory loci
(regulatory polymorphisms) in promoters and other non-coding regions of a gene, in
addition to transcription factors (trans-acting modulators) regulate allele-specific
expression (Heils et al. 1996; Yan 2002; Bray et al. 2003; Lo et al. 2003; Klenova
et al. 2004; Roberts et al. 2007). In the absence of these cis-regulatory domains,
paternal and maternal alleles of a gene are equally expressed, unless one allele is
imprinted. However, when an individual is heterozygous for a cis-regulatory
domain, mRNA expression level may vary from each allele; termed differential
allelic gene expression. Different combinations of these polymorphisms within our
genome create the ‘genetic fingerprint’ that contributes to determining phenotypic
diversity and leads in part to individuality amongst us. Nearly three decades ago,
King and Wilson pointed out that changes in the mechanisms controlling the gene
expression, rather than the DNA sequence itself account for the morphological,
behavioural and cognitive differences between human beings and other primates
(King and Wilson 1975). Therefore, it is suggested that the person we are is not
solely determined by our genes but how we control their expression, hence drugs
such as cocaine vary our behaviour in part by modulating gene expression.
We and others have previously demonstrated the transcriptional capacity of the
VNTRs to support tissue-specific and stimulus inducible gene expression (Table 4)
(reviewed in Haddley et al. 2008). We further extended our studies to investigate
the possible combinatorial interaction of these polymorphisms with one another in
regulating gene expression and recently demonstrated that the 5-HTTLPR and
STin2VNTR can act in concert, in the context of a transient or stable reporter gene
assay in human 5-HTT-expressing JAr cells and primary rodent neurons, to
demonstrate differential reporter gene expression which was not a simplistic
additive effect of the individual domains (Ali et al. 2010a).
One characteristic feature of psychiatric disorders, particularly depression, is the

long-lasting nature of the disease and the slow response to treatment. Recent psy-
chiatric research suggests that these long-lasting changes in gene expression might
be due to modulation of the epigenetic mechanisms regulating neuronal gene
expression. A better understanding of how epigenetic changes associate with psy-
chiatric disorders, polymorphims and drug addiction will help in the understanding
of the neurobiology of these diseases. A study by Tsankova et al. revealed robust and
long lasting histone modifications associated with the promoter of the BDNF gene in
the hippocampus of a mouse model of depression. Interestingly, the negative-histone
modifications marks were present for 4 weeks following ending of defeat-stress and
were not reversed by anti-depressant treatment, indicating the stability of these
‘acquired marks’, the anti-depressant drugs exerted their effect by establishing
positive-histone marks; methylation of H3K4 and acetylation of histone H3 across
the promoter (Tsankova et al. 2004). Similarly, research suggests that drugs of abuse
and drug-associated cues or stress, exert long lasting changes in gene expression
through changes in epigenetic mechanisms (Levine et al. 2005; Kumar et al. 2005;
Renthal et al. 2007). Transcription factors play an early role in regulating gene
expression and histone modifications and one mechanism by which drugs of
abuse and antipsychotic drugs can exert their effect on chromatin modification is via
modulating intracellular signalling cascades, for example, cocaine elicits phos-
phorylation of the cAMP response element (CREB) initiating a transcriptional
DNA-binding cascade that results in histone modifications leading to further
recruitment of transcriptional activators (Carlezon et al. 1998). Acute cocaine
treatment was also found to increase H4 histone acetylation in cfos and fosb pro-
moters within 30 min which disappeared by 3 h (Kumar et al. 2005). Cocaine was
also found to regulate the specific histone methyl-transferases and demethylases
(Maze et al. 2010).
The SLC6A4 gene is an important modulator of addictive behaviour (Hall et al.
2004), and studies have demonstrated the role of the serotonergic system in cocaine
addiction (Cabrera-Vera et al. 2000; Uhl et al. 2002). Little et al. (1998) suggested
that SLC6A4 expression could be regulated in a region-specific pattern and that this
could be affected by the genotype of the LPR variant. Although performed in post-
mortem material, this would be consistent with the action of these domains as tissue-
specific or stimulus-induced regulators (Haddley et al. 2008; Ali et al. 2010a).
We hypothesised that cocaine could differentially modulate epigenetic markers on
the SLC6A4 gene based on the genotype of the polymorphisms. We focused on the
transcription factor CTCF, which has been implicated in epigenetic remodelling
(Ishihara et al. 2006), and which we had previously characterised as a regulator of
both the 5-HTTLPR and STin2VNTR (Klenova et al. 2004; Roberts et al. 2007;
Ali et al. 2010a). The identification of CTCF suggested epigenetic changes opera-
tional at these loci might also involve MeCP2, known to bind to methylated DNA,
which we confirmed occurs at the LPR domain. Specifically, we demonstrated that
exposure of JAr cells to acute cocaine treatment resulted in CTCF binding only to the
l-allele of the 5-HTTLPR whereas MeCP2 was observed to bind only to the s-allele
and to recruit the histone deacetylase complex (HDAC) to this allele. These changes
correlated with an increase in the association of positive histone marks and RNA
polymerase II binding within the SLC6A4 promoter. Similarly, we have also
demonstrated that the mood stabiliser lithium chloride alters the binding of CTCF to
the STin2VNTR dependent on genotype (Roberts et al. 2007). These observations
suggest a potent mechanism by which genotype can effect gene regulation and
indirectly the level of 5-HTT protein present by altering the transcription factor
complexes bound by allelic variants under basal conditions and in response to stimuli
and may provide further insight into the mechanism(s) behind genotypic influence on
susceptibility to affective disorders.
12 Lessons from the 5-HTT Polymorphisms and Their

Application to Other Gene Polymorphisms
We have focused in this chapter on two of the many polymorphisms present in the
SLC6A4 gene there are many others including SNPs and other mini-satellites present
and more are being identified as deep sequencing of the human genome takes place.
All genes contain what could be considered functional SNPs and these are not only
present in protein coding regions. SLC6A4 is obviously not the only gene involved in
the plethora of psychosis and affective disorders and many other genes contain
functional and disorder-associated polymorphisms. For example, the widely studied
DAT1 (SLC6A3) gene from the same family as the 5-HTT contains VNTRs in non-
coding regions that have been associated with a range of disorders including ADHD
and drug addiction (Guindalini et al. 2006; Yang et al. 2007). The SLC6A3 poly-
morphisms have thus been investigated with as much vigour as the SLC6A4 and the
conclusions of such studies have been similar with association both negatively and
positively correlated with disorders, conflicting imaging studies and differential
frequency distribution of alleles in different populations. Nevertheless, molecular
models have shown that these polymorphisms support differential gene expression
and the lessons that can be learnt from studies with one group of polymorphisms can
be applied to other polymorphisms for example the SLC6A4 and SLC6A3 VNTRs
share similar transcription factor binding sites within their sequences implying that
they may be functional on similar pathways and in response to similar challenges
such as stress. The same can be said for many other VNTRs within other neuronal
genes such as those in the dopamine R4 receptor (DRD4), monoamine oxidase A
(MAOA), neuronal restrictive silencer factor (NRSF) and N-methyl-D-aspartic acid
(NMDA) glutamate receptor (GRIN1).
13 Summary
After almost two decades of research the importance of the link between individual
genetic polymorphisms as standalone agents and their relation to disease suscepti-
bility remains somewhat controversial and tenuous for many polymorphisms. Initial
twin studies pointed towards a genetic component for many psychiatric disorders
making the polymorphisms a likely candidate. Early indications linking the serotonin
transporter polymorphisms to a range of psychiatric disorders including depression,
OCD, anxiety, schizophrenia and treatment response while at first positive have
fallen under recent scrutiny in the light of many negative associations. Amongst most
researchers, however, there is little doubt that these functional polymorphisms do
have a role to play in the aetiology of psychiatric disorders but the key to unlocking
these roles is more complex than a simple ‘one polymorphism equals one disorder
model’. It has been apparent for a long time that the same polymorphism occurs
at different frequencies within distinct populations and that an ‘at risk’ allele in a
Caucasian population of European descent will not be an at risk allele for the same
disorder in, for example, a Chinese Han population. This points to a more complex
network of other factors being involved and recent investigations have begun to
expand on this idea looking not only for single polymorphism associations but
examining haplotype associations where one polymorphism may only exert an effect
in the presence of a distinct polymorphism in the same or another gene, as has been
shown on many occasions for the BDNF Val66Met polymorphism (Miyajima et al.
2008; Wells et al. 2010; Hiio et al. 2011). In addition to haplotype analysis it is also
apparent that G 9 G 9 E interactions play as big a role as the polymorphisms
themselves. A polymorphism can be a marker or predictor for susceptibility but its
effect may only become apparent under certain circumstances; it may only have an
adverse (or in some cases positive) effect within a specific context for example
following stressful-life events (Caspi et al. 2003).
Advances in sequencing and array technologies are allowing rapid and cost
effective analysis of the thousands of human polymorphisms that exist allowing
large scale mapping projects worldwide in different populations such as the 1,000
man genome project and the international HAPMAP project. Genome wide
association studies are being supported by functional genome wide studies such as
ChIP-Geneseq and FAIRE which expand current knowledge on how polymor-
phisms may regulate gene expression at a molecular level by interaction with
transcription factors and by epigenetic remodelling. Perhaps most useful in human
functional studies are the advances made in nuclear medicine. Imagining is cer-
tainly an effective tool for assessing genotype effects on binding and possible
function although like all methods compounding factors must also be considered
and not just those inherent in the experimental design e.g., tracer sensitivity or
brain region under study. Environmental effects on endogenous transporter
expression must also be taken into account for example, underlying pathologies,
seasonal changes, age and lifestyle, as these may mask any underlying mecha-
nisms apparent due to polymorphic variation. A large proportion of imaging
studies failed to detect any effect of a polymorphism on transporter function, not
solely in the serotonin transporter but also in other monoaminergic transporters
such as the dopamine transporter. This could be due to the heterogeneity of such
studies, as of yet there are still no clear parameters in place that make comparing
studies wholly effective. Studies have, however, found differences in brain region
volumes, connectivity and activity and it has been suggested by Keers and
Atchinson, that polymorphisms may be involved in indirect effects such as

skewing the hypothalamic-pituitary-adrenal (HPA) axis, the neuroendocrine sys-
tem that mediates mood and which is severely affected in psychiatric disorders, or
by affecting neurogenesis, which when suppressed can lead to dysfunction in the
HPA axis (Schloesser et al. 2009). Further imaging studies of whole brain function
in conjunction with haplotype analysis and epigenetic investigations may help to
further elucidate indirect effects of polymorphisms that until recently have been
neglected. There is still much to be uncovered and understood about how genetic
variation can affect an individual’s mental state and capacity, and how small
changes in genetic sequence can have major effects on complex systems. Within
the next 10 years it is hoped that with the rapid expansion in post-genomic
analysis, functional assessments and more concerted effort on implementing a
degree of homogeneity between association studies that the true clinical value of
polymorphisms as markers of disease and markers and targets of therapeutic
response will be revealed.
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Genetic Factors Modulating the Response
to Stimulant Drugs in Humans
Amy B. Hart, Harriet de Wit and Abraham A. Palmer
Abstract Individuals vary in their responses to stimulant drugs, and several lines
of evidence suggest that the basis for this variation is at least partially genetic in
origin. Association studies have examined the effects of polymorphisms in specific
genes on acute and chronic responses to stimulant drugs. Several of these genetic
polymorphisms are also associated with other psychiatric dimensions and disor-
ders. This chapter examines the evidence for genetic associations between the
genes that have been most carefully examined for their influence on the response
to stimulant drugs.

Keywords Stimulants Inter-individual variation Drug response Candidate

gene Genetic association Genetic polymorphism
Contents
1 Introduction...................................................................................................................... 538
2 Twin Studies of Stimulant Drug Phenotypes ................................................................. 539
3 Association Studies ......................................................................................................... 540
4 Monoamine Transporters................................................................................................. 541
5 Neurotransmitter Receptors............................................................................................. 558
A. B. Hart A. A. Palmer (&)

Department of Human Genetics, University of Chicago,
920 E 58th St. CLSC-507D, Chicago, IL 60637, USA
e-mail: aap@uchicago.edu
H. de Wit A. A. Palmer
Department of Psychiatry and Behavioral Neuroscience,
University of Chicago, Chicago, IL 60637, USA

DOI: 10.1007/7854_2011_187
538 A. B. Hart et al.
6 Biosynthetic Enzymes ..................................................................................................... 561

7 Miscellaneous .................................................................................................................. 564
8 Exploratory Studies ......................................................................................................... 566
9 Genome-Wide Association Studies ................................................................................ 566
10 Closing Remarks ............................................................................................................. 566
References................................................................................................................................ 567
1 Introduction
A growing body of evidence suggests that genetic variation underlies inter-indi-

vidual variability in the response to drugs, including stimulant drugs. Stimulant
drugs produce behavioral and subjective effects that include increased vigilance
and attention, and feelings of energy and euphoria (Lamb and Henningfield 1994;
Martin et al. 1971; Sevak et al. 2009). However, there is substantial individual
variation in these responses (Brown et al. 1978; de Wit et al. 1986; Holdstock and
de Wit 2001), and some of this variation has been shown to be heritable (Crabbe
et al. 1983; Nurnberger et al. 1982). Genetic variation in the response to stimulants
may contribute to the potential to develop drug abuse (Fergusson et al. 2003;
Haertzen et al. 1983), and may also be relevant for therapeutic uses of stimulants.
In this review we will focus on five prototypic stimulant drugs that have been
studied most intensively: amphetamine, methamphetamine, methylphenidate,
cocaine, and bupropion. All of these drugs inhibit the reuptake of dopamine,
norepinephrine, and serotonin, thereby increasing the levels of these monoamine
neurotransmitters in the synaptic cleft. These drugs differ in their potency, synaptic
actions, degree of reuptake blockade, pharmacokinetic properties and their spec-
ificity and actions on other neurotransmitter systems. Some of the drugs (e.g.,
amphetamine) also cause the reuptake transporters to work in reverse, leading to
non-impulse dependent release of neurotransmitters. Additionally, some stimulants
inhibit the monoamine oxidase enzymes (MAO-A and MAO-B), thus preventing
the degradation of monoamines (Seiden et al. 1993). Finally, there is evidence that
stimulant drugs disrupt the function of the vesicular monoamine transporter type 2
(VMAT2), which transports monoamine neurotransmitters from stores in the
cytoplasm to synaptic vesicles (Uhl et al. 2000). Genetic variation in these genes
as well as in their up- or down-stream neighbors make them likely candidates to
contribute to inter-individual variation in the response to stimulant drugs.
We will focus on two types of genetic studies in this review, twin and asso-
ciation studies. Twin studies estimate the heritability of traits, whereas association
studies examine specific polymorphisms in relation to a phenotype. We will not
include family-based linkage studies of drug abuse or animal studies on genetic
determinants of stimulant effects, both which have been carefully reviewed
elsewhere (Phillips et al. 2008; Kreek et al. 2005; Uhl 2006). We will also not
discuss the stimulant caffeine, because it acts by distinct neurochemical mecha-
nisms and because we have recently reviewed the genetics of caffeine elsewhere
Genetic Factors Modulating the Response to Stimulant Drugs in Humans 539
(Yang et al. 2010). We will discuss two types of association studies: candidate
gene studies and genome-wide association studies. Candidate gene studies draw on
prior pharmacological knowledge of how stimulants affect the brain to select
‘candidate’ genes that are likely to be the source of genetic differences. In these
studies, polymorphisms within or near the candidate gene are tested to see if they
are statistically associated with relevant phenotypes. Phenotypes might include
measures of acute response, patterns of drug use or therapeutic response in
patients, or clinical diagnoses of drug dependence or abuse. Other studies use a
genome-wide association (GWAS) approach to examine many or most of the
common polymorphisms in the genome; these studies do not depend on prior
hypotheses about which genes might be important.
In all of the studies discussed: twin, candidate gene association, and genome-
wide association, we will address two types of genetic polymorphisms: single
nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs).
SNPs are sites at which a single nucleotide differs among individuals within a
population. SNPs can either occur in the coding sequence of a gene and thus alter
amino acid sequence (termed non-synonymous) or, more commonly, they may be
outside the coding sequence and alter gene regulation. In both cases, a SNP may
alter a biological function itself (coding or gene expression), or be linked to
another polymorphism that is functionally significant. VNTRs are polymorphisms
in which a variable number of short repetitive sequences (tandem repeats) are
present at a given locus; as with SNPs they may have direct functional conse-
quences or may be linked to some other functionally significant polymorphism.
We will attempt to synthesize genetic studies of acute, sub-chronic, and chronic
administration of stimulant drugs in this review. First, we will discuss overall
heritability of stimulant drug-related phenotypes based on twin studies. Next, we
will discuss candidate gene and genome-wide association studies that implicate
specific genes in modulating responses. Last, we will highlight the key conclusions
and identify future directions for study.
2 Twin Studies of Stimulant Drug Phenotypes
Two early twin studies provide strong evidence for the heritability of acute
responses to stimulant drugs (Nurnberger et al. 1982; Crabbe et al. 1983). Twin
studies estimate heritability by comparing the concordance rate between mono-
zygotic twins, who share a familial environment and all genes, to dizygotic twins,
who share the same environment but only half of their genes. Typically, biometric
modeling is used to explain variability due to genetic or environmental effects.
Heritability estimates can range from 0 (no variation contributed by genetic
sources) to 1 (all variation contributed by genetic sources). Nurnberger et al.
(1982) administered d-amphetamine intravenously (0.3 mg/kg) to 13 pairs of
monozygotic twins and 3 pairs of dizygotic twins and measured physiological and
subjective effects of the drug. Responses to the drug in monozygotic twins were
highly concordant for a subjective measure of excitation, as well as growth

hormone and prolactin release, suggesting a large genetic component underlying
these traits. Consistent with this, Crabbe et al. (1983) administered 10 mg
d-amphetamine sulfate to six pairs of monozygotic twins and found less variation
in physiological and subjective responses within pairs than between pairs, which
suggests these traits are heritable.
More recently, twin studies have examined the heritability of lifetime stimulant
use, dependence, and abuse using liability threshold model fitting (Kendler et al.
1999, 2003, 2000). In their most recent study, Kendler et al. (2005) estimated
heritability for lifetime use of stimulant drugs excluding cocaine to be 0.42, and
heritability for lifetime use of cocaine to be 0.70. They also found substantial
familial environmental contributions to the variance (i.e., 0.20) for lifetime use of
other stimulant drugs, but not for cocaine use. Specific environmental effects,
which are distinguished from shared familial environmental effects, were esti-
mated at 0.38 for other stimulant use and 0.30 for cocaine use. In addition to this
study, a study of male twin war veterans yielded similar results (Tsuang et al.
1996). That study estimated the heritability of stimulant abuse (including cocaine
abuse) based on DSM-III-R criteria (American Psychiatric Association 1987) to be
0.44, but with 0.49 of the variance contributed by specific environmental effects,
and no contribution of familial environment in the best-fit model. Although this
was a slightly higher estimate of heritability than by Kendler et al. (2005),
both studies suggest a fairly large contribution of genetic factors to heritability of
stimulant drug abuse.
3 Association Studies
Genetic determinants of response to acute, sub-chronic, and chronic administration

of stimulant drugs have been examined using association studies in several dif-
ferent populations. We have conducted a series of association studies of the acute
responses to amphetamine (e.g., Lott et al. 2005; Dlugos et al. 2010). In this
double-blind, placebo-controlled study, healthy young adults (ranging from
n = 99 to n = 152, depending on the analysis) received oral doses of d-amphet-
amine (10 and 20 mg) and placebo over the course of three different sessions.
Measures of subjective, cardiovascular and behavioral responses were obtained at
regular intervals. The subjective measures included the Profile of Mood States
(POMS; McNair et al. 1971), Drug Effects Questionnaire (DEQ; Johanson and
Uhlenhuth 1980) and Addiction Resource Center Inventory (ARCI; Martin et al.
1971). In children diagnosed with ADHD, methylphenidate is typically adminis-
tered daily or several times a day in a therapeutic context, and the outcome
measure is usually therapeutic response as measured by the Clinical Global
Impression-Severity scale (CGI-S; Guy 1976) and the ADHD Rating Scale-IV
(ARS; DuPaul et al. 1998). In both studies of healthy adults and studies of children
diagnosed with ADHD, neuroimaging has also been used to examine variation
in brain responses to acute amphetamine and methylphenidate administration.

These include techniques such as single photon emission computerized tomogra-
phy (SPECT), functional magnetic resonance imaging (fMRI) and positron
emission tomography (PET) to assess the binding of specific ligands (Warwick
2004; Cabeza and Nyberg 2000).
Sub-chronic (i.e., \8 weeks) or chronic ([8 weeks) stimulant responses have
been measured in patients receiving methylphenidate for ADHD or bupropion for
smoking cessation. Other studies have examined the phenotypes of dependence,
abuse, and consequences of abuse such as drug-induced psychosis. Although it is
not possible to accurately determine the doses in studies of active drug abusers, it
is safe to assume that they involve higher doses than are found in acute or ther-
apeutic studies. In addition, drug users commonly self-administer drugs via
injection, inhalation (smoking) or intranasal routes, where the acute laboratory
studies and therapeutic studies most commonly involve oral administration.
Studies of responses to stimulant drugs have focused on a small number of
candidate genes that have also been associated with other psychiatric phenotypes.
Many of these genes are the direct targets of stimulant drugs or other known
psychoactive agents. Table 1 lists these studies and they are also discussed in the
following sections. Genes that have been less well studied both in relation to
stimulant drugs and other psychiatric phenotypes are only briefly mentioned in the
‘‘Exploratory Studies’’ section and summarized in Table 2.
4 Monoamine Transporters
SLC6A3
The dopamine transporter (SLC6A3; DAT1; DAT) is a direct target of stimulant

drugs and thus a logical candidate gene for studies or stimulant sensitivity.
Polymorphisms in this gene have been extensively studied in relation to stimulant
drug responses and susceptibility to ADHD (reviewed in Banaschewski et al.
2010). In particular, attention has focused on a 40 bp variable nucleotide tandem
repeat (VNTR) located in the 30 -untranslated region (UTR) of the gene, with a
range of 3–11 repeats. The two most common alleles have either 9, or more
commonly, 10 repeats (Vandenbergh et al. 1992). The 10-repeat allele has been
associated with both higher and lower expression of the dopamine transporter in
the brain (Fuke et al. 2001; van de Giessen et al. 2009; Van Dyck et al. 2005). The
evidence described below suggests that polymorphisms in SLC6A3 play an
important role in responses to stimulant drugs, although the direction of the
association is inconsistent across studies.
In analyses examining associations of amphetamine response with the SLC6A3
30 -UTR VNTR, we found that individuals homozygous for the 9-repeat allele
showed decreased responses to amphetamine at 20 mg, compared to heterozygous
(9/10) and homozygous (10/10) individuals (Lott et al. 2005). Using a larger
Table 1 Candidate genes associated with responses to stimulant drugs and associated SNPs
542
Strong
Candidate
Genes
Gene name Locus Administration Study Phenotype Number Ethnicity Group Dose Associated Notes
symbol type polymorphisms
Dopamine SLC6A3; DAT1; Chronic Bellgrove Methylphenidate 43 Irish Children with – 30 -UTR VNTR 10-repeat allele
transporter DAT et al. therapeutic ADHD associated
(2005) response with better
treatment
response
Acute Cheon et al. Methylphenidate 11 Korean Children with Up to 0.7 30 -UTR VNTR 10/10 associated
(2005) therapeutic ADHD mg/kg/day with poor
response treatment
response
Chronic Gelernter et al. Cocaine-induced 58 Caucasian, Cocaine users – 30 -UTR VNTR 9-repeat allele
(1994) paranoia African– associated
American with increased
risk
Acute Hamidovic Amphetamine subjective 152 Caucasian Healthy Placebo, rs460000 C/C increased
et al. response volunteers 10 mg, Euphoria and
(2010a) 20 mg Stimulation at
10 and 20 mg
Acute Kooij et al. Methylphenidate 42 – Adults with Placebo, 0.5 mg/ 30 -UTR VNTR 10/10 associated
(2008) therapeutic ADHD kg/day by with poor
response week 1, response
0.75 mg/kg/
day by
week 2, up
to 1.0 mg/
kg/day by
week 3
(continued)
A. B. Hart et al.
Table 1 (continued)
Strong
Candidate
Genes
Sub-chronic Joober et al. Methylphenidate 159 – Children with Placebo, 0.5 mg/ 30 -UTR VNTR 9/9 associated with
(2007) therapeutic ADHD kg/day poor treatment
response response
Acute Lott et al. Amphetamine subjective 101 67 Caucasian, 20 Healthy volunteers Placebo, 10 mg, 30 -UTR VNTR 9/9 associated
(2005) response African– 20 mg with poor
American, response at
12 Asian, 2 20 mg
unknown
Acute Purper-Ouakil Methylphenidate 141 132 Caucasian; 8 Children with 10–60 mg/day 30 -UTR VNTR 10/10 associated
et al. therapeutic Other ADHD with poor
(2008) response treatment
response
Acute Rohde et al. Methylphenidate 8 – Children with 0.35–0.7 mg/kg 30 -UTR VNTR 10/10 associated
(2003) therapeutic ADHD (male) with higher
response cerebral blood
flow
Sub-chronic Stein et al. Methylphenidate 47 – Children with Placebo, 18 mg, 30 -UTR VNTR 9/9 associated with
(2005) therapeutic ADHD 36 mg, poor response
response 54 mg
Chronic Ujike et al. Methamphetamine- 124 cases 160 Japanese Methamphetamine – 30 -UTR VNTR Possession of allele
(2003) induced psychosis controls dependence/ with 8 = 9 or
psychosis fewer repeats
patients associated
with
Genetic Factors Modulating the Response to Stimulant Drugs in Humans
prolonged
psychosis
(continued)
543
Table 1 (continued)
544
Strong
Candidate
Genes
Serotonin SLC6A4; Acute Lott et al. Amphetamine 101 67 Caucasian, Healthy volunteers Placebo, 10 mg, 5-HTTLPR & L/L and 12/12
transporter 5-HTT (2006) subjective 20 20 mg Intron 2 genotype
response African– VNTR combination
American, haplotype associated
12 Asian, 2 with weak
unknown subjective
response
Chronic Ezaki et al. Methamphetamine- 166 cases Japanese Methamphetamine- – 5-HTTLPR Frequency of S
(2008) induced 197 induced allele higher
psychosis controls psychosis in patients
patients with
prolonged
psychosis
Sub-chronic Thakur et al. Methylphenidate 157 – Children with Placebo, 0.5 mg/ 5-HTTLPR LG/LG associated
(2010) therapeutic ADHD kg/day triallelic with best
response treatment
response
(continued)
A. B. Hart et al.
Table 1 (continued)
Strong
Candidate
Genes
Norepinephrine SLC6A2; Acute Dlugos et al. Amphetamine 99 65 Caucasian, Healthy Placebo, 10 mg, rs36017, rs47958 rs36017 C/C, G–C–
transporter NET (2007) subjective 20 volunteers 20 mg rs36017- C haplotype
response African– rs2270935- associated with
American, rs47958 increased
12 Asian, 2 haplotype positive mood;
unknown rs47958 C/C
associated with
increased
elation
Acute Dlugos et al. Amphetamine 159 Caucasian Healthy Placebo, 10 mg, rs36017, rs36017 C/C,
(2009a) subjective volunteers 20 mg rs1861647 rs1861647 A/A
response rs36017- associated with
rs10521329- increased
rs3785155 elation and
haplotype vigor; C–C–G
haplotype
associated with
increased vigor
(continued)
545
Table 1 (continued)
546
Strong
Candidate
Genes
Dopamine DRD2 Sub-chronic David et al. Bupropion 722 Caucasian Smokers Placebo, Taq1A A2/A2 3x more
D2 (2007) therapeutic 150 mg/day (rs1800497; likely to
receptor response first 3 days, T/C) abstain from
then 300 smoking after
mg/day treatment; no
effect for
A1/A1, A1/A2
Acute Hamidovic et al. Impulsivity 93 Caucasian Healthy Placebo, 10 mg, rs12364283 rs12364283
(2009) amphetamine volunteers 20 mg A/G, G/G
response increased
reaction time
at 10 mg
Chronic Ujike et al. Methamphetamine- 202 cases 243 Japanese Methamphetamine – Taq1A A2/A2, A1/A2
(2009) induced controls dependence (rs1800497; associated with
psychosis patients T/C) DRD2- prolonged
141 Ins/Del methamphetamine-
induced
psychosis
Del/Del,
Ins/Del
associated
with rapid onset
(continued)
A. B. Hart et al.
Table 1 (continued)
Strong
Candidate
Genes
Dopamine DRD4 Acute Lee et al. Amphetamine 100 66 Caucasian, 20 Healthy Placebo, Exon III VNTR Single 7-repeat allele
D4 (unpublished subjective African– volunteers 10 mg, associated with
receptor data) response American, 12 20 mg increased elation,
Asian, 2 friendliness, heart
unknown rate, decreased
dysphoria and
anxiety at 10 and
20 mg
Chronic Chen et al. Methamphetamine 416 cases 435 Taiwanese Methamphetamine – Exon III Higher frequency of
(2004a) abuse controls abusers VNTR 6-repeat allele in
cases
Chronic Hamarman Methylphenidate 45 – Children with – Exon III Subjects with 7-repeat
et al. therapeutic ADHD VNTR allele require higher
(2004) response doses of
methylphenidate
Chronic Li et al. Methamphetamine 416 cases 435 Taiwanese Methamphetamine – Promoter Higher
(2004) abuse controls abusers VNTR ? exon frequency
III of 7-repeat
VNTR allele in
haplotype cases
l-opioid OPRM1 Acute Dlugos et al. Amphetamine 162 Caucasian Healthy volunteers Placebo, rs510769, rs510769 G/G A/G,
receptor (2010) subjective 10 mg, rs2281617 rs2281617 C/C
response 20 mg associated increased
euphoria and
energy at 10 mg
Chronic Ide et al. (2004) Methamphetaime 138 cases 213 Japanese Methamphetamine – A118G (rs1799971) G/G associated with
psychosis controls dependence/ psychosis within
psychosis 3 years of first use
patients
Chronic Ide et al. (2006) Methamphetamine 128 cases 232 Japanese Methamphetamine – IVS2 ? G691C G/G associated with
abuse/ controls dependence/ (rs2075572) methamphetamine
psychosis psychosis abuse and psychosis
patients
547
(continued)
Table 1 (continued)
548
Strong
Candidate
Genes
Adenosine A2A ADORA2A Acute Hohoff et al. Amphetamine 99 65 Caucasian, Healthy volunteers Placebo, 1976C/T rs5751876 & rs3032740
receptor (2005) subjective 20 African– 10 mg, (rs5751876) associated with
response American, 12 20 mg 2592C/T ins increased anxiety at
Asian, 2 (rs3032740) 10 & 20 mg
unknown
Catechol O- COMT Acute Hamidovic et al. Cognitive response 161 Caucasian Healthy volunteers Placebo, val158met Val/Val, Val/Met
methyl (2010b) to 10 mg, (rs4680; performance
transferase amphetamine 20 mg G/A) improves with
amphetamine;
Met/Met no change
Chronic Li et al. (2004) Methamphetamine 416 cases 435 Taiwanese Methamphetamine – val158met Val allele frequency
abuse controls abusers (rs4680; higher in abusers
G/A)
Chronic Lohoff et al. Cocaine dependence 330 African–American Cocaine dependence – val158met Met allele frequency
(2008) patients (rs4680; higher in abusers
G/A)
Acute Mattay et al. Amphetamine brain 123 – Healthy volunteers Placebo, val158met Val/Val performance
(2003) response 0.25 mg/ (rs4680; improves with
kg of G/A) amphetamine; Met/
body Met perform worse
weight with amphetamine
Chronic Suzuki et al. Methamphetamine- 143 cases 200 Japanese Methamphetamine- – val158met Met allele frequency
(2006) induced controls induced (rs4680; higher in cases with
psychosis psychosis G/A) spontaneous relapse
patients
(continued)
A. B. Hart et al.
Table 1 (continued)
Strong
Candidate
Genes
Monoamine MAOA Chronic Nakamura Methamphetamine- 118 cases 199 Japanese Methamphetamine- – MAOA-u VNTR 4-repeat allele associated
oxidase A et al. induced controls induced with prolonged (vs.
(2009) psychosis psychosis transient) psychosis
patients in males
Dopamine beta- DBH Chronic Cubells et al. Cocaine-induced 45 European– Cocaine – Del-A haplotype Frequency higher in cases
hydroxylase (2000) paranoia American dependence/
paranoia
patients
DBH Acute Kalayasiri Cocaine-induced 31 Euopean– Cocaine dependence 0, 8, 16, and -1021C?T T/T genotype associated
et al. paranoia American, patients 32 mg/ (rs1611115) with increased risk
(2007) African– 70 kg for paranoia
American body
weight
Tryptophan TPH2 Acute Manor et al. Methylphenidate 498 – Children with 0.3–1 mg/kg rs1386488- C–G–C–A– A–G–A–C
hydroxylase (2008) therapeutic ADHD rs2220330- ‘‘Yang’’ haplotype
2 response rs1386495- associated with
rs1386494- better response to
rs6582720- methylphenidate
rs1386492-
rs4760814-
rs1386497
haplotype
Fatty acid amide FAAH Acute Dlugos et al. Amphetamine 72 Caucasian Healthy volunteers Placebo, rs3766246, C/C genotype for both
hydrolase (2009b) subjective 10 mg, rs2295633; SNPs associated
response 20 mg rs3766246- with increased

rs324420- arousal and
rs2295633 decreased fatigue
haplotypes at 10 mg
(continued)
549
Table 1 (continued)
550
Strong
Candidate
Genes
Brain-derived BDNF Acute Flanagin et al. Amphetamine 100 66 Caucasian, Healthy Placebo, val66met Val/Val associated
neurotrophic (2006) subjective 20 African– volunteers 10 mg, (rs6265; with higher
factor response American, 12 20 mg G/A) response at
Asian, 2 10 mg
unknown
Casein kinase 1 CSNK1E Acute Veenstra- Amphetamine 101 67 Caucasian, 20 Healthy Placebo, 10 mg, rs135745 (C/G) C/C associated with
epsilon VanderWeele subjective African– volunteers 20 mg greater
et al. (2006) response American, 12 sensitivity at
Asian, 2 10 mg
unknown
Adenosine A2A ADORA2A Acute Hohoff et al. Amphetamine 99 65 Caucasian, 20 Healthy Placebo, 10 mg, 1976C/T rs5751876 &
receptor (2005) subjective African– volunteers 20 mg (rs5751876) rs3032740
response American, 12 2592C/T ins associated
Asian, 2 (rs3032740) with increased
unknown anxiety at 10
& 20 mg
Cytochrome CPY2D6 Chronic Otani et al. Methamphetamine 202 cases 337 Japanese Methamphetamine – CYP2D6*10, *10 and *14
P450 2D6 (2008) dependence controls dependence CYP2D6 alleles at
patients *14 lower
frequency
in cases
(continued)
A. B. Hart et al.
Table 1 (continued)
Strong
Candidate
Genes
Glycoprotein MANEA Chronic Farrer et al. Cocaine-induced 1,612 individuals Euopean–American, Cocaine – rs9387522 and rs9387522 A allele
endo-alpha-1, (2009) paranoia for family-based; African–American dependence/ rs9387522 in associated with
2-mannosidase 1,921 unrelated paranoia all cocaine induce
subjects for case– patients populations; paranoia in all
control replication rs6937479 populations
from
family based
Genes are discussed in the following order: transporters, receptors, biosynthetic enzymes, and miscellaneous
551
Table 2 Association studies of less commonly studies genes with responses to stimulant drugs
552
Possible
candidate
genes
Glutathione GSTM1 Chronic Nakatome Methamphetamine 100 cases Japanese Methamphetamine – Null Null/null
S-transferase et al. abuse 150 users allele associated
Mu 1 (2009) controls with abuse in
females
Glutathione GSTT1 Chronic Nakatome Methamphetamine 100 cases Japanese Methamphetamine – Null allele Higher liablity to abuse
S-transferase et al. abuse 150 users in combination
theta-1 (2009) controls with GSTM1 null
genotype
Glutathione S- GSTP1 Chronic Hashimoto Methamphetamine 189 cases Japanese Methamphetamine- – Ile105Val G/G, A/G associated
transferase P et al. abuse/ 199 induced (rs947894; A/G) with abuse and
(2004) psychosis controls psychosis psychosis
patients
Prodynorphin PDYN Chronic Nomura Methamphetamine 143 cases Japanese Methamphetamine – Promoter VNTR 3- and 4-repeat alleles
et al. dependence 206 dependence associated with
(2006) controls patients increased risk
Prokineticin PROKR2 Chronic Kishi et al. Methamphetamine 199 cases Japanese Methamphetamine – rs3746684, rs6085086, Individual SNPs and
receptor 2 (2010) dependence 337 dependence rs3746684 and G–G–G–C–G
controls patients rs17721321-rs6085086- haplotype
rs3746684-rs3746682- associated with
rs4815787 haplotype dependence
Solute carrier SLC22A3 Chronic Aoyama Methamphetamine 213 cases Japanese Methamphetamine- – rs509707-rs4709426 Associated with
family 22 et al. dependence 443 induced haplotype polysubstance
member 3 (2006) controls psychosis abuse
patients
Glycine GLYT1 Chronic Morita Methamphetamine 204 cases Japanese Methamphetamine – IVS3 ? 411C [ T T/T and C/T genotypes
transporter et al. dependence/ 210 users (rs2486001); associated with
(2008) psychosis controls rs2486001-rs2248829 methamphetamine
haplotype use disorder
Alpha-synuclein SNCA Chronic Kobayashi Methamphetamine 170 cases Japanese Methamphetamine – rs1372520, rs3756063, Association only in
et al. dependence/ 161 dependence/ rs3756059 females
(2004) psychosis controls psychosis
patients
Gamma- GABRG2 Chronic Nishiyama Methamphetamine 178 cases Japanese Methamphetamine – 315 C [ T-1128-99C [ A Increased susceptibility
aminobutyric et al. use disorder 288 use disorder haplotypes to
acid receptor (2005) controls patients methamphetamine
subunit use disorder
gamma-2
(continued)
A. B. Hart et al.
Table 2 (continued)
Possible
candidate
genes
Dystrobrevin- DTNBP1 Chronic Kishimoto Methamphetamine- 197 cases Japanese Methamphetamine- – rs3213207, rs2619538; rs3213207 G allele,
binding et al. induced 243 induced rs2619539- rs3213207 T allele
protein 1 (2008a) psychosis controls psychosis rs3213207- at higher frequency
patients rs2619538 in cases
haplotype
Frizzled-3 FZD3 Chronic Kishimoto Methamphetamine- 288 cases Japanese Methamphetamine- – rs2241802-rs2323019- G–A–T–G and A–G–C–
et al. induced 240 induced rs352203-rs880481 A haplotypes
(2008b) psychosis controls psychosis haplotype negative risk
patients factors for
psychosis
D-amino acid G72 Chronic Kotaka Methamphetamine- 209 cases Japanese Methamphetamine- – rs778293 (A/G); rs778293 G/G
oxidase et al. induced 291 induced rs3916965- associated with
activator (2009) psychosis controls psychosis rs2391191 (G-A) Methamphetamine-
patients and rs947267- induced psychosis
rs1421292 (T–T)
Glutamate GRM2 Chronic Tsunoka Methamphetamine- 196 cases Japanese Methamphetamine – rs3821829–rs12487957– C–C–A, C–T–T
receptor, et al. induced 802 dependence/ rs4687771 haplotypes
metabotropic 2 (2010) psychosis controls psychosis haplotype associated with
patients Methamphetamine-
induced psychosis
NAD(P)H NQO2 Chronic Ohgake Methamphetamine- 191 cases Japanese Methamphetamine – Promoter indel Del/Del associated with
dehydrogenase, et al. induced 207 dependence/ prolonged
quinone 2 (2005) psychosis controls psychosis psychosis
patients
PRKCA-binding PICK1 Chronic Matsuzawa Methamphetamine- 208 cases Japanese Methamphetamine – rs713729, rs2076369 rs713729
protein et al. induced 218 abusers methamphetamine
(2007) psychosis controls abuse; rs713729
and rs2076369
spontaneous
relapse of
psychosis
(continued)
553
Table 2 (continued)
554
Possible
candidate
genes
Superoxide SOD2 Chronic Nakamura Methamphetamine- 116 cases Japanese Methamphetamine- – Ala/Val exon Ala/Val associated with
dismutase 2 et al. induced 189 induced 2 (rs4880) prolonged
(2006) psychosis controls psychosis psychosis; Ala allele
patients at higher frequency
Carboxylesterase 1 CES1 Sub-chronic Nemoda Methylphenidate 122 Hungarian Children 10–30 mg Gly143Glu Glu/Glu, Glu/Gly
et al. therapeutic with ADHD (by body (rs71647871) associated with
(2009) response weight) better treatment
twice response
daily (0.22 to
0.95 mg/kg/
day)
The genes here are not as well studied in relation to the phenotypes discussed in this review; they are therefore denoted ‘possible candidate genes’. The associations described have primarily been performed
for methamphetamine use-related phenotypes
A. B. Hart et al.
sample from the same study, Hamidovic et al. (2010a) tested 4 additional SNPs
near the 50 end of SLC6A3 that were not in linkage disequilibrium with the 30 -UTR
VNTR, and found a significant association between the rs460000 C/C genotype
and increased scores on the ARCI Euphoria and Stimulation composite scales after
10 and 20 mg amphetamine administration, when compared to A/A and A/C
individuals. Interestingly, this SNP is in perfect linkage disequilibrium with
rs463379, a SNP that has been associated with increased risk of ADHD (Friedel
et al. 2007), which suggests that variants underlying acute response to amphet-
amine may also underlie disease risk. Overall, these results suggest that both the
30 -UTR VNTR and SNPs in SLC6A3 affect acute response to amphetamine.
The 30 -UTR VNTR polymorphism has also been studied in relation to acute
responses to methylphenidate in children that have been diagnosed with ADHD. In
a pilot study, eight children received acute doses of methylphenidate before
SPECT neuroimaging (Rohde et al. 2003). Children homozygous for the 10-repeat
allele exhibited greater cerebral blood flow, which may reflect higher transporter
activity, in the frontal and basal ganglia brain areas in response to methylphenidate
than children who were not homozygous for the 10-repeat allele.
SLC6A3 has also been the analyzed for its role in modulating therapeutic
responses to sub-chronic (8 weeks) administration of methylphenidate. Similar to
the Rohde et al. (2003) study discussed above, Cheon et al. (2005) used SPECT to
measure dopamine transporter density in several brain regions (basal ganglia, right
basal ganglia, and occipital cortex) of 11 children diagnosed with ADHD treated
with varying doses of methylphenidate, and measured both clinical response and
transporter density. Children with at least one copy of the 9-repeat allele (n = 4)
showed a better therapeutic response to methylphenidate than 10/10 individuals (4
out of 4 responders versus 2 out of 7 responders in the 10/10 group), and signif-
icantly lower dopamine transporter density than 10/10 individuals. This suggests
that 10/10 individuals may require more methylphenidate due to increased dopa-
mine transporter density within the brain.
The association between the 30 -UTR VNTR and response to sub-chronic
methylphenidate has also been examined in a larger cohort of children and
adolescents with ADHD (Purper-Ouakil et al. 2008). Individuals received placebo
and varying doses of methylphenidate until no further clinical improvement or
limiting side effects occurred (mean dose 31.19 mg/day). They were phenotyped
with the ARS, the Stroop test (Stroop 1935), the Trail Making Test (Reitan 1958),
and the Continuous Performance Test (Rosvold et al. 1956). Subjects that were
homozygotes for the 10-repeat allele had significantly lower treatment responses.
However, the final methylphenidate doses reached did not differ across genotype
group. These results suggest that the maximal response to methylphenidate may be
lower in 10/10 homozygotes and cannot be overcome with larger doses. Similarly,
another study of adults with ADHD found that the 10/10 genotype was associated
with non-statistically significant lower therapeutic response to 3 weeks of treat-
ment with methylphenidate as assessed by the CGI-S and ARS (Kooij et al. 2008).
Although the results discussed above suggest that the 10-repeat allele may be
associated with poor treatment response to methylphenidate, other studies have
reported that therapeutic responses are poorer in patients with the 9-repeat allele.
In a double-blind study, Stein et al. (2005) found that children with the 9/9
genotype showed poor response to methylphenidate treatment as measured by
ARS and CGI-S when they received higher (36 and 54 mg) but not lower (placebo
or 18 mg) doses of methylphenidate. These results suggest that individuals with at
least one copy of the 10-repeat allele responded better when given higher doses of
methylphenidate. Joober et al. (2007) also performed a double-blind, placebo-
controlled fixed-dose study, in which children diagnosed with ADHD were treated
with methylphenidate for two weeks (0.5 mg/kg/day) and the effect of 30 -UTR
VNTR was assessed. Similar to the results of Stein et al. (2005), children with the
9/10 and 10/10 genotypes showed significant improvement in ADHD symptoms
following treatment as measured by the CGI-Parents scale when compared to 9/9
children; no difference was observed between the 9/10 and 10/10 groups. The
10-repeat allele was also associated with better treatment response to methyl-
phenidate following longer term treatment in children diagnosed with ADHD
using family-based association testing (Bellgrove et al. 2005). In addition to these
studies, three studies have observed no association between the 30 -UTR VNTR
polymorphism and methylphenidate response (da Silva et al. 2010; McGough et al.
2006; Roman et al. 2001). Therefore, the reported effects of the 30 -UTR VNTR on
treatment response are contradictory and warrant further investigation.
The dopamine transporter has also been studied in relation to stimulant drug
abuse. In these studies, the exact doses of drug are unknown and presumed to be
high relative to laboratory or clinical studies. A case–control study examined
methamphetamine dependence and drug-induced psychosis in a cohort of indi-
viduals with methamphetamine use disorder and psychosis (Ujike et al. 2003). No
associations were found between the four polymorphisms tested and metham-
phetamine dependence or psychosis. However, when the patients with prolonged
psychosis were analyzed separately from those with transient psychosis, there was
a strong association indicating that individuals with nine or fewer repeat alleles of
the 30 -UTR VNTR had prolonged psychosis. The 9-repeat allele was also mar-
ginally associated with increased risk of cocaine-induced paranoia in cocaine
dependent subjects when the frequency of the 9-repeat allele was compared in
individuals with and without psychosis (Gelernter et al. 1994). The reasons for
these associations are unclear, given the lack of information about the doses that
were ingested. The findings may indicate that drug users with the 9-repeat allele of
the 30 -UTR VNTR polymorphism are more sensitive to stimulant-induced
psychotic phenotypes, or, alternatively, they may have ingested higher doses of the
drug to achieve their desired effect, thus increasing their risk for psychosis.
Lastly, the effects of an additional polymorphism in SLC6A3 and cocaine-
related behaviors have also been examined. Guindalini et al. (2006) examined the
SLC6A3 Intron 8 VNTR (Int8 VNTR), which consists of either five or six repeats
and is in moderate to low linkage disequilibrium with the 30 -UTR VNTR
(Asherson et al. 2007), and found that the 6-repeat allele was associated with
cocaine abuse in a case-control study of cocaine abusers. When the 5- and 6-repeat
alleles were cloned into expression vectors and transfected into a dopaminergic
cell line, the 6-repeat allele showed increased SLC6A3 expression following
treatment with various stimuli (including cocaine), while the 5-repeat allele
showed no change in expression. This study, along with Hamidovic et al. (2010a),
suggests that polymorphisms other than the 30 -UTR VNTR may also contribute to
responses to stimulant drugs.
SLC6A4
The serotonin transporter (SLC6A4; 5-HTT) is another direct target of stimulant
drugs, and has been associated with numerous psychiatric phenotypes including
obsessive-compulsive disorder (Bloch et al. 2008), autism (Huang and Santangelo
2008), and depression (Brown and Harris 2008; Kato and Serretti 2008; Risch
et al. 2009). The 5-hydroxytryptamine transporter gene-linked polymorphic region
(5-HTTLPR), which may be the most widely studied polymorphism in all of
psychiatric genetics, has at least two common alleles: a short (S) 14-repeat and a
long (L) 16-repeat allele (Nakamura et al. 2000; Rausch 2005). The 5-HTTLPR S
allele has been associated with reduced gene expression (Hranilovic et al. 2004)
and increased risk for psychiatric phenotypes like depression. The long allele has
been further refined into two alleles (LA and LG) distinguished by an A?G
polymorphism within the first repeat; the LG allele is reported to have equivalent
expression to the S allele (Hu et al. 2006). In additional to 5-HTTLPR, a VNTR in
Intron 2 of SLC6A4 has also been described, which consists of either a 10- or 12-
repeat allele. The 12-repeat allele has been associated with increased gene
expression (Hranilovic et al. 2004).
We have examined both of the SLC6A4 polymorphisms mentioned above to
determine whether they influence acute responses to amphetamine using our
sample of healthy volunteers described in the previous section (Lott et al. 2006).
When these two polymorphisms were analyzed separately, individuals homozy-
gous for the 10-repeat allele of the Intron 2 VNTR showed a stronger euphoric
response. When the polymorphisms were analyzed jointly, no significant associ-
ation was observed, but trends in the predicted directions were observed—subjects
homozygous for the low expressing alleles (S and 10-repeat) had the strongest
responses to amphetamine (for the POMS Anxiety, DEQ Feel Drug, and ARCI
Euphoria scales). These data identify a non-significant trend towards decreased
expression of the serotonin transporter being associated with increased responses
to stimulants.
The serotonin transporter has also been investigated for its role in sub-chronic
methylphenidate treatment response in children diagnosed with ADHD (Thakur
et al. 2010). In a 2 week trial, children received placebo and methylphenidate at
varying doses. Subjects homozygous for the higher expressing LA had the worst
response to placebo but the best response to methylphenidate, heterozygotes were
intermediate, and individuals homozygous for the LS and LG alleles exhibited the
best response to placebo but deterioration with methylphenidate treatment as mea-
sured by the CGI-Parents subscale. Thus, lower expression of SLC6A4 is associated
with stronger euphoric, but weaker therapeutic responses to stimulant drugs.
The 5-HTTLPR polymorphism has also been associated with adverse responses
in methamphetamine abusers. The 5-HTTLPR polymorphism was tested for
association with prolonged methamphetamine-induced psychosis in a case-control
study of methamphetamine abusers (Ezaki et al. 2008), utilizing the sample and
methods described in the previous section (Ujike et al. 2003). SLC6A4 was chosen
as a candidate gene for this phenotype based on previous neuroimaging findings
from the same group showing that methamphetamine abusers had lower serotonin
transporter density than non-abusers (Sekine et al. 2006). The lower expressing
S allele was significantly associated with methamphetamine psychosis, and in
particular with prolonged psychosis. Thus, the S allele (and the LG allele in the one
instance where it was differentiated from the LA allele) appears to be associated
with more intense acute and chronic responses to various different stimulants.
Furthermore, it appears that the lower expressing alleles of both the dopamine and
the serotonin transporters are associated with greater propensity to methamphet-
amine-induced psychosis.
SLC6A2
The norepinephrine transporter (SLC6A2; NET) is a third direct target of stimulant
drugs (Sulzer et al. 2005). Variants in SLC6A2 have been associated with psy-
chiatric phenotypes including depression (Haenisch et al. 2009; Min et al. 2009),
antidepressant response (Min et al. 2009), and panic disorder (Lee et al. 2005).
Using our sample of healthy adults (Dlugos et al. 2007) we found that rs36017 (C/
C genotype) was associated with increased positive mood, as measured by a
composite scale composed of ‘‘elation’’ minus ‘‘depression’’, following amphet-
amine administration (20 mg), while rs47958 (C/C genotype) was associated with
increased elation. Haplotypes containing these SNPs (rs36017-rs10521329-
rs3785155, G–C–C and C–C–A) were also associated with increased positive
mood. A follow-up study with a larger number of individuals replicated the
association between rs36017 and the POMS Elation scale, as well as with the
POMS Vigor scale, and identified a new association between the A/A genotype at
rs1861647 and these scales (Dlugos et al. 2009a). In addition, a different haplotype
than those mentioned above, which was also constructed from rs36017,
rs10521329, and rs3785155 (C–C–G), was associated with increased vigor fol-
lowing amphetamine administration. In sum, these studies suggest that multiple
variants in SLC6A2 influence responses to acute amphetamine administration.
5 Neurotransmitter Receptors
DRD2
The dopamine D2 receptor (DRD2) has been studied in relation to a variety of

psychiatric traits, including schizophrenia (Allen et al. 2008), smoking cessation
(David and Munafò 2008) and impulsivity (Eisenberg et al. 2007; Rodriguez-
Jimenez et al. 2006). In particular, the historically named Taq1A SNP (also known
as rs1800497) has been widely studied. Historical nomenclature describes the A1
(T) and the A2 alleles (C). This polymorphism was initially described as being
located in DRD2, but is now recognized to be located in a neighboring gene,
ANNK1 (Neville et al. 2004). However, this polymorphism appears to influence the
expression of the DRD2 gene and thereby alter DRD2 function. In a study of
DRD2 polymorphisms and gene expression in postmortem human brain tissues,
rs1800497 was not associated with DRD2 expression, but the G allele of another
SNP in DRD2, rs12364283, was found to be associated with enhanced expression
(Zhang et al. 2007).
Variation in DRD2 has been investigated in relation to amphetamine-induced
impulsive behavior. Amphetamine increases behavioral inhibition, and the degree
of this inhibition varies across individuals (de Wit et al. 2000). We examined the
role of several SNP polymorphisms in DRD2 and the effects of amphetamine on
behavioral inhibition utilizing the sample of healthy human subjects that is
described above. Individuals were phenotyped with the stop task (Logan et al.
1984), a measure of behavioral inhibition, and genotyped at 12 SNPs in DRD2 (but
not the Taq1A polymorphism). One SNP, rs12364283, was significantly associated
with better performance on the stop task following 10 mg amphetamine admin-
istration in the G/G and A/G groups. This G allele of this SNP, as discussed earlier,
is associated with increased DRD2 expression (Zhang et al. 2007); increased
DRD2 expression may be related to better task performance (Cropley et al. 2006).
Variation in DRD2 has been investigated for its effect on bupropion treatment
response (David et al. 2007). David et al. (2007) found that smokers with the
A2/A2 genotype at Taq1A who received bupropion (150 mg/day for the first
3 days, then 300 mg/day) were three times more likely to abstain from smoking at
the end of the trial compared to A2/A2 subjects receiving placebo. This difference
was not observed for the other genotypic groups.
Finally, DRD2 has also been investigated for association with methamphet-
amine dependence and methamphetamine-induced psychosis. Šerý et al. (2001)
found no association between polymorphisms in DRD2 and methamphetamine
dependence. Ujike et al. (2009) tested 3 SNPs in DRD2: 141C insertion/deletion
(rs1799732; -/C), Ser311Cys (rs1801028; C/G) and Taq1A, and found that the
Taq1A A2 (C) allele, which was associated with good response to bupropion for
smoking cessation, was associated with prolonged methamphetamine psychosis
(A2/A2, A1/A2 genotypes). Additionally, they found that the Del/Del and Ins/Del
genotypes of 141C insertion allele (rs1799732) were associated with rapid onset of
psychotic symptoms (within 3 years of initial abuse). Therefore, the Taq1A
polymorphism, along with other polymorphisms in DRD2, may influence devel-
opment of stimulant-induced psychosis as well as drug dependence.
DRD4
The dopamine D4 receptor (DRD4) has been associated with smoking behavior
(Laucht et al. 2008), schizophrenia (Shi et al. 2008), novelty seeking (Munafò
et al. 2008) and impulsivity (Munafò et al. 2008), as well as responses to

stimulant drugs. The exon III VNTR polymorphism of DRD4 is the most
commonly studied. This polymorphism has 8 alleles, varying from 2 to 8 and 10
repeats (Lichter et al. 1993). The 7-repeat allele has been associated with ADHD
in some studies (Brookes et al. 2006; Faraone et al. 2001; Li et al. 2006), but not
in all (Johansson et al. 2008). Because of its association with ADHD, the
7-repeat allele has been the focus of most studies involving stimulant drug
responses.
We have investigated the effect of the exon III VNTR polymorphism and acute
subjective and physiological responses to amphetamine in our sample (Lee et al.
2006), and found that individuals with a single copy of the 7-repeat allele expe-
rienced more rewarding effects of the drug, with increased scores on the POMS
Friendliness, POMS Elation, and heart rate scales at 20 mg and on the DEQ More
scale at 10 mg and decreased scores on the ARCI Dysphoria and POMS Anxiety
scales at 20 mg when compared to individuals lacking the 7-repeat allele.
Therefore, the 7-repeat allele may modulate subjective and physiological
responses to acute amphetamine administration.
Variation in DRD4 has also been tested for association with therapeutic
response following sub-chronic methylphenidate administration in children diag-
nosed with ADHD (Hamarman et al. 2004). Children with the 7-repeat allele of the
exon III polymorphism required higher doses of methylphenidate (about two times
more) to achieve the same therapeutic response over a 2-week period as measured
by the CGI-S. Cheon et al. (2006) undertook a similar study and found that the
4-repeat allele was associated with the best treatment responses based on the ARS
and the Kiddie-Schedule for Affective Disorders and Schizophrenia-Present and
Lifetime Version (K-SADS-PL; Kaufman et al. 1997). Only one individual in that
study had the 7-repeat allele, and therefore the previous finding could not be
examined; conversely, the 4-repeat allele was not analyzed in the Hamarman et al.
(2004) study. Therefore, these two sub-chronic studies found associations with
different alleles of the exon III polymorphism and different measures of methyl-
phenidate response.
DRD4 has also been investigated for association with methamphetamine
dependence. Chen et al. (2004a) reported a higher frequency of the 7-repeat exon
III VNTR allele in methamphetamine abusers as compared to controls, although
the association did not reach statistical significance. A subsequent study utilizing
the same population examined another VNTR polymorphism in the promoter
region of DRD4, with a long (240 bp) and short allele (120 bp) (Li et al. 2004).
Although no association between methamphetamine abuse and the promoter
VNTR was found, when the promoter and exon III VNTR polymorphisms were
analyzed as a haplotype, there was a significant association of the 7-repeat allele
exon III VNTR and short promoter VNTR allele with methamphetamine abuse.
Therefore, the 7-repeat allele of the exon III VNTR increases the dose of meth-
ylphenidate needed for therapeutic effects and may also increase the risk for
development of methamphetamine abuse.
OPRM1
The l-opioid receptor, OPRM1, has been investigated for a role in the subjective
response to acute amphetamine. Stimulation of l-opioid receptors by endogenous
beta-endorphins in the ventral tegmental area increases dopamine release
(Spanagel et al. 1992); therefore, variation in OPRM1 may influence dopamine
release and therefore response to stimulants. A commonly studied coding poly-
morphism within OPRM1, Asp40Asn (A118G; rs1799971; A/G), was first
described by Bergen et al. (1997) and has been associated with numerous phe-
notypes, including heroin addiction (Drakenberg et al. 2006), schizophrenia (Šerý
et al. 2010), and naltrexone treatment response (Oroszi et al. 2009). This SNP has
also been shown to alter binding of beta-endorphin to the receptor (Bond et al.
1998). We examined associations between several SNPs (including rs1799971)
and the acute responses to amphetamine on the ARCI Euphoria, Energy and
Stimulation scales (Dlugos et al. 2010), and found that two polymorphisms
(rs510769 G/G, A/G and rs2281617 C/C) were associated with increased euphoric
responses to amphetamine at 10 mg; rs510769 is in strong linkage disequilibrium
with rs1799971. The results indicate that variation in OPRM1 may be related to
variation in positive, euphoric responses to amphetamine.
Rs1799971 has been associated with the duration of methamphetamine-induced
psychosis in a case-control study of individuals with methamphetamine depen-
dence/psychosis; G/G individuals were more likely to become psychotic within
3 years of their first methamphetamine intake (Ide et al. 2004). In a more recent
study using the same population, the previous association did not replicate, but the
G/G genotype of an additional SNP in OPRM1 (IVS2 ? G691C; rs2075572; G/C)
was associated with both methamphetamine dependence and psychosis (Ide et al.
2006). These results suggest the possibility that additional variants underlie
chronic methamphetamine response, although rs1799971 has been the main target
of the literature in investigating acute and chronic stimulant responses.
ADORA2A
The adenosine A2A receptor (ADORA2A) is a major target of caffeine (Daly and
Fredholm 1998) and forms a heterodimer with the dopamine D2 receptor (Fuxe
et al. 2005). We investigated the effect of ADORA2A polymorphisms on the
anxiety response to amphetamine in healthy human subjects (Hohoff et al. 2005)
and found that two polymorphisms (rs5751876; T/T and rs3032740; Tins/Tins),
were associated with increased anxiety at the 10 and 20 mg doses.
6 Biosynthetic Enzymes
COMT
Various enzymes involved in the synthesis and breakdown of neurotransmitters
have also been analyzed for associations with acute response to stimulant drugs.
One major candidate gene is catechol O-methyl transferase (COMT). This gene
codes for an enzyme that preferentially metabolizes dopamine in the prefrontal
cortex, rather than in the limbic and striatal brain regions, where the dopamine
transporter is more important for clearance (Chen et al. 2004b). COMT contains a
val?met coding polymorphism (val158-met; rs4680; G(val)/A(met)) that has been
associated with a variety of phenotypes including personality, cognition, risk for
psychiatric disorders (Tunbridge et al. 2006), and pain sensitivity (Andersen and
Skorpen 2009). The met allele has been associated with 3–4 times lower activity
compared to the val allele (Lachman et al. 1996).
Polymorphisms in COMT have been associated with acute amphetamine
response. In a landmark study (Mattay et al. 2003), healthy volunteers completed
the Wisconsin Card Sorting Test (WCST; Heaton et al. 1993) after placebo or
amphetamine, and underwent fMRI while performing the N-back working memory
task (Kirchner 1958). Amphetamine administration reduced prefrontal cortical
activity at all working memory loads when compared to placebo in individuals
homozygous for the val allele. This was interpreted as a more efficient physiological
response; this reduction was associated with improved reaction time and no
decrease in accuracy on the task. Conversely, amphetamine increased prefrontal
cortical activity in the most difficult part of the working memory task in met
homozygotes. This was interpreted as a reduction in efficiency, because increased
prefrontal cortical activity was associated with increased reaction time and
decreased accuracy. The authors proposed that amphetamine increased dopamine
levels above an optimum level in met/met individuals and thus negatively impacted
cortical function. In contrast, in val/val individuals, amphetamine increased the
lower pre-drug dopamine levels so that they were closer to optimal and thus
enhanced function. The WCST also showed genotypic effects, with val/val subjects
improving following amphetamine administration and met/met individuals per-
forming worse. Taken together, the results of this study suggest that an optimum
level of dopamine in the prefrontal cortex is necessary for efficient prefrontal cortex
function, and that this efficiency is in part mediated by val158-met genotype. Another
study (Hamidovic et al. 2010b) evaluated the effect of val158-met on a processing
speed task (Digit Symbol Substitution Test; Wechsler 1958) and a reaction time test
measuring attention lapses (Deviation from the Mode; de Wit 2009) and reported
that when compared to placebo, amphetamine improved DSST performance in val
homozygotes and heterozygotes, but not in met homozygotes. However, val
homozygotes and heterozygotes exhibited more lapses in attention under placebo
conditions, suggesting that the drug improved a preexisting deficit in one genotypic
group. Taken together, these findings suggest that individuals with the met allele are
less sensitive to the cognitive enhancing effects of stimulant drugs.
COMT has been investigated for association with chronic stimulant drug
phenotypes. The val allele has been associated with polysubstance abuse
(Vandenbergh et al. 1997), and in a later case–control study, Li et al. (2004) found
that the val/val genotype was significantly associated methamphetamine abuse.
In addition, Lohoff et al. (2008) found that cocaine-dependent individuals possessed the
lower activity met allele more often than controls, suggesting that this polymorphism may
play some role in dependence. These results differ from the Vandenbergh et al. (1997) and
Li et al. (2004) studies described above; Lohoff et al. suggest their finding of the met allele
association may also be due to linkage disequilibrium or population stratification.
MAOA
Monoamine oxidase A (MAOA) degrades monoamine neurotransmitters and has
been most commonly associated with aggressive behavior (reviewed in Gri-
gorenko et al. 2010). MAOA contains a 30 bp VNTR polymorphism within its
promoter that consists of 3, 3.5, 4, or 5 repeats (MAOA-u VNTR); the 3.5 and 4
repeat alleles are transcribed more efficiently than the 3- or 5-repeat alleles (Sabol
et al. 1998). Nakamura et al. (2009) tested the MAOA-u VNTR polymorphism for
association with methamphetamine-induced psychosis, and found that in males,
the 4-repeat allele was associated with having prolonged psychosis rather than
transient; this effect was not seen in females.
DBH
Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to nor-
epinephrine (Kaufman and Friedman 1965) and has been associated with ADHD
(reviewed in Banaschewski et al. 2010); additionally, a locus near DBH was
recently associated with smoking cessation (Furberg et al. 2010). Cubells et al.
(2000) investigated the effects of DBH polymorphisms on cocaine-induced para-
noia in patients with cocaine dependence/paranoia, and identified a haplotype found
more frequently in cocaine-dependent individuals reporting paranoia, that was also
associated with low DBH plasma levels in normal subjects (-4784–4803del-
444A?G; del-A haplotype). Individuals from the same group later sequenced DBH
in eight individuals at the phenotypic extremes for DBH plasma levels, and iden-
tified a putative functional polymorphism (-1021C?T; rs1611115; C/T)
accounting for 35–52% of phenotype variance in DBH activity (Zabetian et al.
2001); the T allele was associated with low DBH plasma levels, and found to be in
positive linkage disequilibrium with the del and A alleles associated with low DBH
plasma levels and paranoia in the Cubells et al. (2000) study (Zabetian et al. 2003).
A controlled laboratory study was later conducted in which 31 cocaine-using
volunteers acutely self-administered intravenous doses of the cocaine (0, 8, 16, and
32 mg/70 kg body weight) over four sessions (Kalayasiri et al. 2007). The previ-
ously identified -1021C?T polymorphism was tested for association with
cocaine-induced paranoia, and the low DBH plasma level T/T genotype group
reported higher paranoia when compared to the C/T and C/C groups.
TPH2
Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in the synthesis

of serotonin, and TPH2 polymorphisms have been investigated for association
with depression, suicidal behavior, bipolar disorder (Zhang et al. 2006) and ADHD
(Banaschewski et al. 2010; Brookes et al. 2006). Manor et al. (2008) tested for
association of TPH2 SNPs and acute therapeutic response in children diagnosed
with ADHD, and identified an 8-SNP haplotype (rs1386488, rs2220330,

rs1386495, rs1386494, rs6582720, rs1386492, rs4760814, rs1386497; C–G–C–A–
A–G–A–C) that was significantly associated with better acute therapeutic response
following methylphenidate treatment.
FAAH
Fatty acid amide hydrolase (FAAH) has also been studied in relation to acute
response to stimulants; FAAH degrades several endocannabinoids that bind to
cannabinoid receptors (McKinney and Cravatt 2005). Recently, polymorphisms in
FAAH have been associated with brain response to cannabis (Filbey et al. 2009)
and other phenotypes such as obesity (Engeli 2008). Much of the work associating
variants in FAAH with stimulant response has been done in animal models (e.g.,
Madsen et al. 2006), and has shown that response to stimulants may also be
influenced by the cannabinoid system. We investigated the effect of FAAH poly-
morphisms on subjective responses to acute amphetamine, using the sample of
healthy volunteers that has been previously described (Dlugos et al. 2009b). Two
SNPs, rs3766246 (C/C genotype) and rs2295633 (C/C genotype), were associated
with amphetamine-induced arousal and decreased fatigue at 10 mg but not 20 mg,
supporting the hypothesis that the endocannabinoid system may influence the
acute response to low doses of amphetamine.
7 Miscellaneous
BDNF
Brain-derived neurotrophic factor (BDNF) is a growth factor that is involved in

developmental processes (Hyman et al. 1991) and has been implicated in
depression (Kato and Serretti 2008), smoking behavior (Furberg et al. 2010),
obesity (den Hoed et al. 2010), and body mass index (Shugart et al. 2009). BDNF
contains a val?met substitution (val66-met; rs6265; G/A); the met allele produces
a protein that is improperly secreted from neurons and has been reported to affect
memory and hippocampal function (Egan et al. 2003). We examined the effects of
BDNF val66-met genotype on subjective responses to acute amphetamine (Flana-
gin et al. 2006) and found that individuals homozygous for the val allele possessed
increased feelings of arousal and energy when administered 10 mg amphetamine
relative to the other genotype groups. Therefore, the BDNF val66-met genotype
may also contribute to subjective responses to stimulant drugs.
CSNK1E
Another enzyme showing association with acute stimulant drug responses is
casein kinase 1 epsilon (CSNK1E). CSNK1E encodes an enzyme that phosphor-
ylates dopamine and cAMP-regulated phosphoprotein (DAARP-32; PPP1R1B), a
second messenger that integrates dopaminergic and glutamatergic signaling
(Greengard 2001). We have previously reported that a quantitative trait locus

(QTL) for methamphetamine sensitivity co-maps with an expression QTL for
Csnk1e in mice, suggesting that the latter might cause the former (Palmer et al.
2005). Furthermore, pharmacological inhibition of Csnk1e blocks the locomotor
response to methamphetamine in mice (Bryant et al. 2009) and rats (unpublished
data). Mice with a null allele for Csnk1e exhibit paradoxically higher response to
methamphetamine (unpublished data). We have also investigated the effects of
SNPs in CSNK1E on subjective amphetamine response in humans (Veenstra-
VanderWeele et al. 2006). The C allele of rs135745, which is located in the 30 -
UTR of the gene, was associated with the response to amphetamine at 10 mg (but
not 20 mg) as measured by the DEQ Feel Drug and ARCI Euphoria scales.
Interestingly, other SNPs in CSNK1E have been associated with both heroin
addiction in a case-control study (Levran et al. 2008) and ADHD in a family-based
genome-wide association study (Mick et al. 2010), suggesting another possible
link between ADHD, sensitivity to stimulants and drug abuse.
CYP2D6
Cytochrome P450 2D6 (CYP2D6) is a p450 enzyme that metabolizes metham-
phetamine (Lin et al. 1997). Otani et al. (2008) investigated the effect of CYP2D6
polymorphisms in methamphetamine dependent patients in a case-control study,
and found the lower activity CYP2D6*10 and CYP2D6*14 alleles were under-
represented in patients, suggesting that these alleles are protective against devel-
opment of methamphetamine dependence.
MANEA
An association between rs1133503, located in the 30 -UTR of glycoprotein endo-
alpha-1,2-mannosidase (MANEA), and cocaine-induced paranoia was the strongest
result in a genome-wide scan for variants associated with cocaine dependence and
cocaine-induced paranoia (Yu et al. 2008). In a later study from the same group, 11
SNPs in MANEA were studied further using a family-based approach in two
separate populations (African-–American; AA and European–American; EA).
Nine of these SNPs were at least nominally associated with cocaine-induced
paranoia in the AA population, whereas six of them at least nominally associated
in the EA population (Farrer et al. 2009). Additionally, the rs9400554-rs6937479-
rs9387522 haplotype (T–T–A) was associated with cocaine-induced paranoia in
the pooled AA/EA family-based sample, and the C–A–C haplotype was associated
with decreased risk of cocaine-induced paranoia in the pooled sample. No sig-
nificant associations were found for the cocaine dependence phenotype. Two
separate case–control samples (EA and AA) were used for replication. The indi-
vidual SNP associations from the family-based studies did not replicate in either
population sample. However, two associated haplotypes in the replication study
contained SNPs that were found in associated haplotypes in the family-based
analysis (rs900554 and rs9387522; C–A; T–A); the C–A haplotype was associated
with increased risk of cocaine-induced paranoia and cocaine dependence in EA,
and the T–A haplotype was associated with increased risk of cocaine dependence
in the AA replication sample.
8 Exploratory Studies
A number of association studies have focused on genes that are not as well studied as
those mentioned in the previous section; these more exploratory studies are sum-
marized in Table 2. These studies mostly focus on methamphetamine abuse-related
phenotypes. These genes include CES1 (Nemoda et al. 2009); DTNBP1 (Kishimoto
et al. 2008a); FZD3 (Kishimoto et al. 2008b); G72 (Kotaka et al. 2009); GABRG2
(Nishiyama et al. 2005); GLYT1 (Morita et al. 2008); GRM2 (Tsunoka et al. 2010);
GSTM1 (Nakatome et al. 2009); GSTP1 (Hashimoto et al. 2004); GSTT1 (Nakatome
et al. 2009); NQO2 (Ohgake et al. 2005); PDYN (Nomura et al. 2006); PICK1
(Matsuzawa et al. 2007); PROKR2 (Kishi et al. 2010); SLC22A3 (Aoyama et al.
2006); SNCA (Kobayashi et al. 2004) and SOD2 (Nakamura et al. 2006).
9 Genome-Wide Association Studies
Presently, only three GWAS has been conducted investigating responses to

stimulant drugs. One study investigated the therapeutic response to methylpheni-
date (Mick et al. 2008). Children diagnosed with ADHD (n = 187), received
varying doses of methylphenidate over a 5-week period, and were phenotyped with
the ADHD-RS IV scale and then genotyped at about 300,000 SNPs. No associa-
tions reached genome-wide significance, and the strongest associations were not in
genes examined in any previous candidate gene studies. Two studies have
examined genetic variation underlying stimulant abuse. Uhl et al. (2008) used a
case–control nested study to investigate genetic variants underlying metham-
phetamine dependence, with the goal of identifying genes with an unexpected
accumulation of low p-values, and Yu et al. (2008) took a family-based association
test (FBAT) approach and scanned evenly spaced markers for association with
cocaine dependence and cocaine-induced paranoia. As previously mentioned, the
association between MANEA SNP (rs1133503) and cocaine-induced paranoia was
the most significant result in this study, but after correction for multiple testing this
result was not statistically significant.
10 Closing Remarks
The main focus of this review was to investigate sources of genetic variation
across a range of different phenotypes and prototypic stimulant drugs. Some of
the data are specific to acute or chronic administration and therapeutic or
non-therapeutic responses. Some genes, such as OPRM1, were associated with

both acute subjective amphetamine liking and methamphetamine dependence.
Understanding how these polymorphisms influence various phenotypes has
important implications for the study of drug abuse and for our understanding of the
pathophysiology and treatment of disorders such as ADHD. Interestingly, several
genes discussed here are implicated in both ADHD and ADHD treatment response.
Many of these studies test multiple hypotheses and in some cases underpowered
to do so, therefore there is a need for replication of these results. For example, our
investigation of healthy human subjects has been used to test many different
genetic hypotheses, so that if corrections for multiple testing across all of these
different genes were applied, several results would not be considered statistically
significant. In an effort to examine the reliability of our results we now have
collected almost 400 subjects and are in the process of performing replication
analyses.
There is still much to be learned about the polymorphisms reviewed here. The
SLC6A3 30 -UTR VNTR is particularly illustrative of this, since it has been asso-
ciated with many phenotypes in quite a number of studies, yet the results appear
inconsistent or even contradictory. Resolving these apparent inconsistencies across
studies is an urgent goal to better understand the function of the 30 -UTR VNTR
and its relation to drug response.
There is significant promise in future technologies that will become available
shortly. Genome-wide association studies will remain important in providing un-
biased answers. As whole-genome re-sequencing becomes more readily available,
it will be possible to study rare variants in more sophisticated ways. Copy number
variation is another relatively unexplored type of genetic variation that has been
suggested to be important in pharmacogenomics (Johansson and Ingelman-Sund-
berg 2008). Finally, there are substantial opportunities and challenges in inte-
grating the findings from genetic studies in humans and model organisms (Phillips
et al. 2008).
Acknowledgements This work was supported by NIH grants DA02812, DA027545 and
DA021336.
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The Cognitive Genetics
of Neuropsychiatric Disorders
A. Corvin, G. Donohoe, A. Hargreaves, L. Gallagher and M. Gill
Abstract Classification in psychiatry is heavily dependent on clinical symptoms

and illness course. This ignores the critical role that cognitive problems play in
neuropsychiatric disorders affecting different domains across the lifespan, from
ADHD and autism to schizophrenia and Alzheimers disease. At this point, it is
unclear whether cognitive mechanisms are specific to disorders, whether multiple
processes can contribute to the same disorder, or whether aberrant neural processing
can result in many different phenotypic outcomes. Understanding this would allow us
to better grasp normal as well as pathological brain function. This could inform
diagnostics based on understanding of neurophysiological processes and the con-
sequent development of new therapeutics. Genetics, and the development of geno-
mic research, offers real opportunities to understand the molecular mechanisms
relevant to cognition. This chapter defines and describes the main cognitive phe-
notypes, which are investigated in psychiatric disorders. We review evidence for
their heritability and early progress in the field using cytogenetic, linkage and can-
didate gene-based research methodologies. With high-throughput genomics it is now
possible to explore novel common and rare risk variants for psychiatric disorders and
A. Corvin (&) G. Donohoe A. Hargreaves L. Gallagher M. Gill

Department of Psychiatry, Trinity College Dublin, Dublin, Ireland
e-mail: acorvin@tcd.ie
G. Donohoe
e-mail: donoghug@tcd.ie
A. Hargreaves
e-mail: hargrea@tcd.ie
L. Gallagher
e-mail: lgallagh@tcd.ie
M. Gill
e-mail: mgill@tcd.ie

DOI: 10.1007/7854_2011_188
580 A. Corvin et al.
their role in cognitive function at a genome-wide level. We review the results of early
genomic studies and discuss the novel insights that they are starting to provide.
Finally, we review the analysis of whole-genome DNA sequence data and the
challenges that this will bring for cognitive genomics research.
Keywords Genetics Phenotypes Cognition Genomics
Contents
1 Introduction........................................................................................................................ 580
2 Selection of Cognitive Deficits in Neuropsychiatric Genetics........................................ 581
2.1 Social Cognition and Psychiatric Disorder.............................................................. 582
3 Testing Cognitive Deficits in Neuropsychiatric Genetics ............................................... 588
4 Cytogenetics and Cognitive Phenotypes .......................................................................... 589
4.1 Cytogenetics of Childhood-Onset Neuropsychiatric Disorders .............................. 589
4.2 Cytogenetics of Adult-Onset Neuropsychiatric Disorders ...................................... 591
5 Linkage and Candidate Gene-Based Approached to Cognitive Genetics....................... 593
5.1 Linkage and Candidate Genes Studies for Disorders of Childhood....................... 594
5.2 Linkage and Candidate Genes Studies for Disorders of Adult-Onset.................... 597
6 Application of Genomics Methods ................................................................................... 599
6.1 Genome-Wide Association Studies ........................................................................ 600
6.2 Studies of Structural Genomic Variation in Neuropsychiatric Disorders .............. 603
7 Future Directions: New Phenotypes and New Approaches............................................. 605
References................................................................................................................................ 606
1 Introduction
Classification in psychiatry is heavily dependent on clinical symptoms and illness

course. This ignores the critical role that cognitive problems play in neuropsy-
chiatric disorders affecting different domains across the lifespan, from ADHD and
autism to schizophrenia and Alzheimer’s disease. At this point, it is unclear
whether aberrant cognitive mechanisms are specific to disorders, whether multiple
disturbances in cognitive processes can contribute to the same disorder, or whether
aberrant neural processing can result in many different phenotypic outcomes.
Understanding this would allow us to better grasp normal as well as pathological
brain function. This could inform diagnostics based on understanding of neuro-
physiological processes and the consequent development of new therapeutics.
Genetics, and the development of genomic research, offers real opportunities to
understand the molecular mechanisms relevant to cognition. This chapter defines
and describes the main cognitive phenotypes, which are investigated in psychiatric
disorders. We review evidence for their heritability and early progress in the field
using cytogenetic, linkage and candidate gene-based research methodologies. With
high-throughput genomics it is now possible to explore novel common and rare
risk variants for psychiatric disorders and their role in cognitive function at a
genome-wide level. We review the results of early genomic studies and discuss the
The Cognitive Genetics of Neuropsychiatric Disorders 581
novel insights that they are starting to provide. Finally, we review the analysis of
whole-genome DNA sequence data and the challenges that this will bring for
cognitive genomics research.
2 Selection of Cognitive Deficits in Neuropsychiatric Genetics
Historically, no aspect of cognition (or any human trait for that matter) has
received more attention in terms of its underlying genetics basis than general
cognitive ability or intelligence. While this history has not been without contro-
versy (cf. the eugenics movement), the genetic basis of intelligence has been
studied widely in both the general population and across psychiatric disorders.
A key question in the selection of specific cognitive phenotypes is what extent
performance on a given task is heritable. Differing degrees of relationships within
families, associated with more or less sharing of genetic material (e.g. monozygotic
twins share 100% of genes, dizygotic twins/siblings 50%, and half-siblings 25%)
allow estimation of the proportion of individual differences in performance in a
population at a given time that are due to genetic differences (termed heritability
(h2)). The availability of twin and population-based disease register data has con-
firmed the importance of heritability for many psychiatric disorders. Less infor-
mation has been available to allow interpretation of the heritability of cognitive
deficits within twin samples. Because of this practical sampling constraint, most
epidemiological studies have used a family-based design to investigate cognitive
deficit in healthy relatives of patients. The finding that this group has higher rates of
a particular cognitive deficit is taken as suggestive evidence of heritability.
The total variance in general intelligence that can be attributed to genetic influ-
ences range from 30 to 80%. Broad domains of cognitive ability, such as verbal and
perceptual abilities show similar measures of genetic influence (Posthuma et al.
2001), although the genetic influence on subdomains such as memory tends to be
smaller, due to measurement error and variance, but being highly correlated with
general intelligence, genetic effects overlap. The heritability of general intelligence
increases with age to 70–80% in adulthood. Variations in brain structures such as the
density and the volume of grey and white matter, amygdala, and hippocampus, and
overall brain volume are thought to be endophenotypes for intelligence. Therefore,
genes involved in intelligence might be more closely associated with variations in
brain structure and function than to measures of intelligence.
By comparison, selection of specific cognitive functions for analysis in genetic
studies has been heavily influenced by the kinds of deficits observed within specific
disorders. This selection has also been strongly influenced by the perceived potential
of a cognitive function or process as ‘intermediate phenotypes’ or ‘endophenotypes’
for the disorder. The ‘endophenotype’ concept in psychiatry (described by Gottesman
and Gould 2003) relates to the identification of heritable quantifiable characteristics,
which may be useful targets for genetic studies as they represent some intermediate
stage between genotype and clinical disorder. The authors suggested that the potential
utility of an endophenotype can be judged against a set of criteria:
1. Association with illness in the population

2. Co-segregation of the endophenotype with illness in families
3. Evidence that this is genetically mediated (i.e. heritable)
4. Evidence that the endophenotype is state-independent (i.e. present whether
illness is active or not)
5. That the endophenotype can be measured accurately and reliably
6. The endophenotype is also shared by nonaffected family members of the proband,
this may be advantageous for genetic studies.
Across disorders, deficits in executive function, memory function and attentional
control have each been a particular focus for research. Clinical awareness of these
impairments has increased as it has been established that such deficits are predictive of
psychiatric morbidity. In the case of schizophrenia for example, cognitive deficits are
present from an early stage of the disorder and often predate the emergence of clinical
symptoms (Erlenmeyer-Kimling et al. 2000). They are relatively stable over time and
closely related to functional outcome (Green et al. 2004). This includes deficits in
general cognitive ability (Donohoe et al. 2011b) and specific deficits in working and
episodic memory (Donohoe et al. 2011a) and attentional control (Bellgrove and
Mattingley 2008; Donohoe et al. 2011a). Genetic epidemiological research using
family and twin studies indicates that some of these deficits may themselves have a
substantial genetic component (Goldberg et al. 1990, 1995; Cannon et al. 2000). While
cognitive deficits are somewhat correlated with clinical symptoms (for example,
negative symptoms in schizophrenia) the amount of variance shared by these variables
appears to be small, and cognitive function often emerges as a separate factor from
clinical symptoms in factor analysis (Donohoe and Robertson 2003). Taken together
these data indicate that cognitive phenotypes in schizophrenia are heritable, trait-like
and generally independent of clinical symptomatology.
Mood disorder researchers, investigating bipolar disorder and recurrent depres-
sive disorder have focused less on general cognitive ability and more on memory related
phenotypes (Thomas and Elliott 2009; Frodl et al. 2009). In attention deficit hyperac-
tivity disorder (ADHD), attention has inevitably been a particularly important focus,
both in terms of orienting attention, sustaining attention, and inhibiting processing of
task-irrelevant stimuli (Bellgrove and Mattingley 2008). In autism, impairments in
overall cognitive ability are found in 50% of affected individuals. In higher functioning
individuals deficits in executive functioning (specifically attention orienting, response
inhibition, and set shifting) are reported. The most widely investigated area of
cognition in autism relates to impairments in social cognition. Social cognition is the
ability to process social information, thought to be fundamentally impaired in autism.
2.1 Social Cognition and Psychiatric Disorder
A significant development in cognitive genetic studies of psychiatric disorders has

been the increased focus on the genetics of social cognition that began with
research in autism. Social cognition is the sum of those processes that allow
individuals of the same species (conspecifics) to interact with one another

(Frith and Frith 2007), specifically it refers to the set of skills that allow us to
understand the thoughts and intentions of others and frequently involves the
investigation of social information processing, especially its encoding, storage,
retrieval, and application to social situations. Essentially it depends upon the
exchange of signals, such as speech, facial expression, body posture, and eye gaze
(Frith and Frith 2007). Signals such as these can be socially informative in that
they tell us what someone may be feeling (Vuilleumier and Pourtois 2007), where
they are focusing their attention, and what they are intending to do (Frith and Frith
2006). The ability to process, comprehend and act appropriately to these signals is
tantamount to social success and relies on coordination of several cortical regions,
e.g. dorsomedial and dorsolateral prefrontal cortices, the paracingulate cortex and
the right and left temporoparietal junctions and amygdala (Mitchell 2009). In
several psychiatric disorders this ability is impaired leading to social mispercep-
tions, unexpected reactions to and from the person, and social withdrawal (Green
et al. 2004), resulting in difficulties with maintaining friendships, employment, and
general community functioning (Penn et al. 2008). Alongside these clinical and
outcome goals, there is increasing interest in identifying the neural basis under-
lying social cognitive deficits in psychiatric illness. As such, further research in
this domain is viewed as highly valuable (Green et al. 2004).
Currently, social cognition is investigated both in behavioural terms and in
terms of neuroanatomy. Some of the tests that fall into the former category
include theory of mind (TOM) tests (such as mind in the eyes, Faux pas and
hinting task), the hotel task, multiple errands task, Iowa gambling task, the social
cognitive skills test (SCST), Mayer-Salovey-Caruso emotional intelligence test
(MSCEIT), the Penn emotional recognition task, facial affect recognition, attri-
bution test, and tests of self-control (see Table 1 for task summaries). The latter
category however is most often assessed using magnetic resonance imaging
(MRI) and involves performing simple social cognitive tests such as facial aspect
recognition, and imaging the brain to see what areas are activated during task
performance.
To investigate the utility of these measures of social cognition as psychiatric
‘endophenotypes’ (based on the criteria outlined above) we undertook a review of
the main tests found in the literature. Search included test name, heritability, twin,
sibling, state, independent, co-segregation, brain region, and endophenotype.
Secondary search terms used in the event of the primary terms yielding no results
included family, trait, dependent, gene, psychiatric, schizophrenia, and autism.
A list of the common measures of social cognition reviewed and evidence of their
feasibility as endophenotypes is presented in Table 2. As with neuropsychological
measures most of the evidence of heritability for each of the measures of social
cognition reviewed is derived from evidence that family members of psychiatri-
cally affected probands also show deficits at a level intermediate between cases
and healthy controls. These deficits have been associated with a number of
candidate risk genes for psychiatric disorders, including 5HT2A, oxytocin, and
vasopressin.
584
Table 1 Definition and usage of various social cognitive tests

Test Definition/Usage
Emotion recognition MSCEIT Measures emotional intelligence, namely 4 areas: perceiving emotions, facilitating
thought, understanding emotions, managing emotions
Penn emotional recognition A computer-based test that includes 96 colour photographs of facial expression of
task evoked—or felt—emotions: happy, sad, angry, fearful, disgusted, and nonemotional
or neutral
Facial affect recognition Facial emotion identification test (FEIT) and the facial emotion discrimination test
(FEDT). Both use black and white photographs of facial emotions that are presented
on DVD
Theory of mind TOM A ‘‘Theory of mind’’ (often abbreviated to TOM) is a specific cognitive ability to
understand others as intentional agents, that is, to interpret their minds in terms of
theoretical concepts of intentional states such as beliefs and desires
Mind in the eyes Measure of adult mentalising. The ability to deduce emotion from looking at
photographs of people’s eyes
Faux pas test The faux pas test ascertained the participants’ ability to identify and understand a social
faux pas, and to understand the mental states of the characters (the speaker and the
recipient) in a conversation with a social faux pas
Hinting task Mental state reasoning, inferring. Measures ability of subject to ‘read between the lines’
in a social context
Attribution Attribution test How one assigns causality in a social context
Social perception Social cognitive skills test Focuses on the assessment of social reasoning skills
(SCST)
Iowa gambling task Decision making abilities. Looks at trust and the ability to analyse intentions
Self regulation Self control test (implicit Measure the strength of automatic association between mental representations of
association test (IAT)) objects (concepts) in memory
A. Corvin et al.
Table 2 Suitability of social cognition measures as endophenotypes for schizophrenia
Test First author N Associated with Heritability/ State Co-segregates
illness Present in other independent with illness
family members
Penn emotional Greenwood et al. (2007) 183 nuclear families ascertained through Yes Yes
recog task probands with schizophreni
Eack et al. (2010) 70 first-degree relatives of schizophrenia Yes Yes
probands; 63 healthy controls
Calkins et al. (2010) SZ/Szaff = 610, Relatives = 928, HC = 334 Yes Yes Yes
Gur et al. (2002) 14 patients with schizophrenia and 14 matched Yes
comparison subjects
Bediou et al. (2007) Drug-naive patients with first-episode Yes Yes Yes
schizophrenia (n = 40) and their
unaffected siblings (n = 30) compared
with controls (n = 26)
Facial affect Erol et al. (2010) Patients with schizophrenia (n = 57), their Yes Yes
recognition unaffected biological siblings (n = 58)
and healthy controls (n = 58)
TOM task McKinnon et al. (2010) 14 bipolar, 14 control Yes
The Cognitive Genetics of Neuropsychiatric Disorders
Sabbagh and Seamans (2008) 46 3-year-olds and their parents Yes

Sprong et al. (2007) 29 studies (combined n = 1518) Yes
de Achával et al. (2010) 20 Schizophrenia patients, 20 healthy age- and Yes Yes
gender-matched individuals, 20 unaffected
first-degree relatives of the schizophrenia
patients, and 20 healthy individuals
matched for age and gender
Hinting task Janssen et al. (2003) 43 patients with schizophrenia or Yes Yes Yes
schizoaffective disorder, 41 first degree
non-psychotic relatives and 43 controls
from the general population
Mind in the eyes Losh and Piven (2007) 48 parents of individuals with autism (13 of Yes Yes . Yes
whom were identified as ‘aloof’), and
22 control parents
(continued)
585
Table 2 (continued)
586

family members
Bora et al. (2005) Forty-three euthymic bipolar patients and Yes
30 controls
Faux pas de Achával et al. (2010) 20 Schizophrenia patients 20 healthy age- and Yes Yes
gender-matched individuals, 20 unaffected
first-degree relatives of the schizophrenia
patients, and 20 healthy individuals
Shamay-Tsoory et al. (2009) 19 patients, 20 controls Yes
Attribution test Lau and Eley (2008) Childhood(birth–12 years) (100), School Age Yes Yes Yes
(IPSAQ) (6–12 years) (180), Adolescence
(13–17 years) (200), Adulthood(18 years
plus) (300), Young Adult(18–29 years)
(320)
Janssen et al. (2006) 23 patients with psychosis, 36 first-degree Yes
relatives of patients with psychosis, 31
subjects with subclinical psychotic
experiences and 46 normal controls
Social cognitive Scourfield et al. (1999) Population-based sample of twins aged 5–17 Yes
skills test
(SCST)
Scourfield et al. (2004) A population-based sample of twins aged 5–17 Yes Yes
Coleman et al. (2008) 8 children with ASD and 8 control children Yes
aged 5–13 years
Iowa gambling task Viswanath et al. (2009) 25 unaffected siblings of OCD probands with Yes Yes
familial OCD, and 25 individually
matched healthy controls
(continued)
A. Corvin et al.
Table 2 (continued)
family members
Cavedini et al. (2010) 35 pairs of OCD probands and unaffected first- Yes Yes
degree relatives and 31 pairs of HC
subjects without a known family history of
OCD and their relatives
Implicit association Egloff and Schmukle (2002) 41 introductory psychology students (33 Yes
test women and 8 men)
Sasaki et al. (2010) Individuals scoring High (n = 26) and Low Yes
(n = 18) on Social Anxiety
Glashouwer and de Jong Patients (n = 2,329) and non-clinical controls Yes Yes
(2010) (n = 652)
While many tests show good evidence that poorer performance is both associated with illness and heritable (at least based on assessments in first-degree relatives) both classical twin studies
and studies of co-segregation in families are currently lacking
The Cognitive Genetics of Neuropsychiatric Disorders
587
3 Testing Cognitive Deficits in Neuropsychiatric Genetics
Cognitive neuroscience approaches to investigating both cognitive performance in

the healthy population and cognitive disability associated with psychiatric disor-
ders have included both behavioural and neuroimaging paradigms. Behavioural
measures include traditional neuropsychological paper and pen tasks (e.g. the child
and adult versions of the Wechsler intelligence scales, the Wechsler memory
scales) and computer-based tasks (e.g. various versions of the continuous perfor-
mance task (CPT); the Cambridge automated test battery). The main advantage of
these neuropsychological tests is that they generally have well-established psy-
chometric properties that can be easily administered to large groups of varying
ability, a key requirement for cognitive genetics research. Imaging modalities have
included both functional magnetic resonance imaging (fMRI) during tasks of
memory, attention and—more recently—affective processing, with the high spatial
resolution it offers, and electroencephalogram recordings (EEG), with the high
temporal resolution it provides. While, previously, individual groups tended to
specialise in one main approach, groups working in this area increasingly aim to
integrate findings across modalities. In our own group for example, we have used
high density EEG and structural MRI to follow up on specific associations between
individual genetics variants and neuropsychological test performance, such as
the contribution of abnormal sensory level processing to cognitive performance
deficits (Donohoe et al. 2008).
Across disorders, available family and twin studies generally support the concept
that the cognitive deficits associated with these disorders are themselves inherited.
In the case of schizophrenia, the heritability of a number of specific deficits have been
confirmed by twin studies, including general cognitive ability, working memory, and
episodic memory (Goldberg et al. 1990, 1995; Cannon et al. 2000; Kremen et al.
2006; Toulopoulou et al. 2007, 2010). The heritability of deficits in general cognitive
ability and working memory in particular appear to overlap strongly with the heri-
tability of illness risk in schizophrenia (Toulopoulou et al. 2007) although not to the
point of suggesting an identity between the genetic architecture of schizophrenia and
cognition. In autism research, deficits in general cognitive ability, executive func-
tioning, and processing of socially relevant information (e.g. face recognition) have
consistently been reported (Adolphs et al. 2001). The most consistent familial traits
are language and communication skills, insistence on sameness and non-verbal IQ
(Szatmari et al. 2007). In ADHD, cognitive studies find widespread abnormalities in
children and adults with the disorder, particularly in the executive function domains
of response inhibition/delay aversion and sustained attention (Willcutt et al. 2005)
and in reaction time variability (Klein et al. 2006), one of the most discriminating
between ADHD and controls. Attention, and in particular, sustained attention,
deficits are prominent in ADHD, with unaffected siblings performing better than their
affected siblings but worse than healthy controls (Slaats-Wilemse et al. 2005).
Sustained attention has been examined in preschool twins using the Amsterdam
neuropsychological tasks set with correlations higher for MZ compared to DZ twins
suggesting a heritability of 0.46–0.72 for this measure (Groot et al. 2004). Yet many
proposed endophenotypes in ADHD have not been examined in classical population-
based twin studies. It is not yet clear if these impairments are related or perhaps have
separate aetiological pathways. Kuntsi et al. (2010) examined this in a large sample
of ADHD probands and their siblings using a multivariate familial factor analysis
approach. The results suggest that two familial phenotypes, mean reaction time/
reaction time variability and omission/commission errors on the go/no-go task reflect
85–98 and 13% of the familial variance of ADHD, respectively. The findings for
response time variability reflect recent population twin data (Wood et al. 2010).
4 Cytogenetics and Cognitive Phenotypes
Cytogenetic methods to investigate chromosomes microscopically, initially allowed

gross examination of chromosomes, incorporating staining methods which identi-
fied cytogenetic bands on chromosomes; these evolved to molecular hybridization
methods, known as fluorescent in situ hybridization (FISH). Cytogenetics methods
contribute to localising susceptibility genes by identifying chromosomal abnor-
malities such as deletions or translocations, which segregate with a disorder in
families or are found more commonly in cases than in control populations.
A number of these (described further below) have been identified for childhood
disorders affecting cognitive ability and mental health, including sex chromosome
aneuploidies such as 47XYY, Klinefelters and Turner’s syndrome; fragile X,
Williams’ syndrome, and autism. In adult-onset disorders, two cytogenetic abnor-
malities have provided consistent evidence that the chromosomal regions involved
contribute to disease risk. One is a balanced translocation t(1;11)(q42.1;q14.3) that
co-segregates with schizophrenia within a large Scottish pedigree. The other
involves one of the commonly known chromosomal abnormalities, small interstitial
deletions of chromosome 22q11. The latter cause velo-cardio facial syndrome
(VCFS), which increases the risk of psychosis by at least 20-fold, and also presents
with cranio-facial dysmorphology and congenital heart disease. In carriers of these
cytogenetic abnormalities, both general and specific cognitive effects are associated,
including language deficits (Williams syndrome, autism), specific learning deficits
(e.g. mathematical ability in Turner’s syndrome, Williams syndrome), and memory
deficits (Schizophrenia); these are discussed next.
4.1 Cytogenetics of Childhood-Onset

Neuropsychiatric Disorders
Sex chromosome aneuploidies (SCAs), where there is variation in the number

of sex chromosomes, are the most common chromosomal abnormalities occurring
in 1 per 400 births. The commonest syndromes include the 47XYY syndrome,
Klinefelter syndrome (47XXY), and Turner’s syndrome (XO). Frequently deficits

in speech and language, motor skills, and educational achievement are reported in
relation to all SCAs although in general affected individuals usually live inde-
pendently and the severity of cognitive deficits is not clear (Leggett et al. 2010).
Males with 47XYY frequently present normally and the phenotype may go
undetected. In general in SCAs, the presence of an additional X chromosome is
frequently associated with lowered verbal IQ. Klinefelter syndrome is associated
with executive function deficits (Samango-Sprouse 2010). The absence of an
X chromosome in girls is also associated with cognitive deficits. Turner syndrome
(XO), an SCA affecting girls is characteristically associated with visual-spatial and
executive function deficits and social cognitive impairments (Hong et al. 2009).
Mathematical learning difficulties are prevalent in Turner syndrome which may
relate to deficits in executive function or spatial deficits although this is not clear
(Mazzocco 2009). Non-verbal learning deficits are present in 80% of girls with
Turner syndrome. The syndrome is also associated with social cognitive deficits
such as facial emotion recognition and gaze perception that are also reflected by
reported neuroanatomical abnormalities in the amygdala (Burnett et al. 2010).
William Beurens Syndrome (WBS) is a contiguous gene syndrome arising from a
deletion of approximately 1.5–1.8 Mb affecting 28 genes on chromosome 7q. It is
associated with a characteristic physical phenotype and a cheerful, sociable manner.
Individuals with WBS frequently have intellectual disability but present with
superficially good language ability. They present with poor visuo-spatial ability
(Pober 2010). A microduplication syndrome of the same region has also been
described with somewhat contrasting phenotypic characteristics; intellectual
disability is also reported; speech and language deficits are more common while
visuo-spatial functioning is spared (Merla et al. 2010). Both the duplication and
deletion syndromes are reported to be associated with ADHD and autistic type
deficits; social deficits and aggression are more characteristic of the duplication
syndrome. A clear relationship between specific genes within the affected region and
the cognitive phenotype has not been determined, however, several interesting
candidate genes exist. Frizzled drosophila homologue of 9 (FZD9) is expressed in the
hippocampus and null mouse mutants have defects in memory and learning although
WBS-related phenotypic features are not universally reported (Ranheim et al. 2005).
The Syntaxin 1A (STX1A) gene is a brain-expressed protein implicated in presyn-
aptic vesicle docking. Expression levels of this gene are correlated with intelligence
in WBS (Gao et al. 2010). LIM kinase 1 (LIMK1) is a serine protein kinase pre-
dominantly expressed in the nervous system and implicated in synapse formation
(Scott and Olson 2007). LIMK1 knockout mice have abnormalities in the dendritic
spine morphology and have impaired fear conditioning and spatial learning
(Meng et al. 2002). CAP-Gly domain-containing linker protein 2 (CLIP2) is
expressed widely in the nervous system, including the hippocampus. Knockout mice
have growth deficits, motor coordination deficits and altered synaptic functioning in
the hippocampus (Hoogenraad et al. 2002). Clinically, patients with deletions not
including CLIP2 have reduced visuospatial impairments and less deficits in gross and
fine motor skills (Ferrero et al. 2010).
Fragile X syndrome, is the commonest cause of inherited intellectual disability.

Not specifically a cytogenetic abnormality, it is caused by a trinucleotide repeat
expansion (CGG) in the FMR-1 protein on the X chromosome. The condition is
associated with autistic type difficulties in social interaction, e.g. gaze avoidance,
social anxiety, poor pro-social behaviour, and peer relationships. Decreased pre-
frontal brain activation is reported and reduced frontal and temporal cortical
volumes are reported compared with control subjects or individuals with idio-
syncratic autism (Hoeft et al. 2011). Declines in IQ and adaptive function are also
reported in longitudinal studies with greater declines in males (Fisch et al. 2010).
Declines in central executive and verbal working memory are also reported in later
life in males. Working memory deficits correlate with the length of the CGG
expansion (Cornish et al. 2009). Attentional difficulties, anxiety, aggression, and
mood symptoms are also reported (Boyle and Kaufmann 2010).
Prader-Willi syndrome (PWS) is a rare genetic syndrome associated with
nonexpression of a set of genes on the paternal chromosome 15q11–q13. This may
be due to deletion of the region on the paternal chromosome (70%) or uniparental
disomy (UPD) (25%) involving the maternal chromosome. The region is imprinted
and also associated with Angelman’s syndrome caused by lack of expression of the
UBE3A gene originating on the maternal chromosome. Individuals with PWS have
a characteristic physical phenotype and usually intellectual disability (approxi-
mately 70% of cases), however, they often have strengths in visual perception,
reading, and vocabulary. Strengths in verbal IQ are reported in UPD compared
with carriers of the deletion (Copet et al. 2010). Deficits in auditory processing are
reported as are weaknesses in mathematics, visual, and auditory memory and
auditory attention. Larger deletions in the region tend to be associated with greater
impairments in cognitive ability (Milner et al. 2005). In Angelman syndrome,
deletions are associated with greater cognitive impairment than UPD and cognitive
skills are stronger than motor or language skills with relative strengths in the area
of receptive language (Gentile et al. 2010).
Down syndrome is caused by Trisomy for all or part of chromosome 21.
Associated with learning disability and early onset dementia, the cognitive profile
of individuals with Down syndrome can be heterogeneous, but relative strengths in
receptive language compared with expressive language are reported.
4.2 Cytogenetics of Adult-Onset Neuropsychiatric Disorders
Cytogenetic studies have also implicated both a relatively common deletion of

chromosome 22q11.2 and a complex, rare translocation as increasing risk for
psychotic disorders. The gene disrupted-in-schizophrenia-1 (DISC1) was identified
at a balanced translocation between chromosome one and eleven, which strongly
co-segregates with mental illness in a Scottish family. Although the index case had
a diagnosis of conduct disorder, within the family 18 of 29 (70%) translocation
carriers had a major mental illness (schizophrenia, bipolar disorder or major
depressive disorder), whereas none of 38 non-translocation carriers had such a

diagnosis (Blackwood et al. 2001). Outside this family, there is equivocal sup-
port for involvement of the gene as a risk factor for major mental disorders
(Porteous et al. 2006) and there has been substantial investigation of its role in
neurodevelopment. At a cognitive level, within the affected Scottish family there
was no difference in mean IQ between 12 relatives with the translocation and eight
with a normal karyotype. Despite this, unaffected, as well as affected, translocation
carriers had abnormalities on event-related potential (ERP) P300-typical of
schizophrenia and bipolar disorder-suggesting that specific cognitive processing
deficits may be important.
Using methods (described in Sect. 5), Finnish researchers have identified
linkage between a locus containing the DISC1 gene and impaired working
memory dysfunction (Gasperoni et al. 2003) and visual working memory (Hennah
et al. 2005). Two subsequent studies have reported association between markers at
the DISC1 locus and impairments in verbal learning and memory (Cannon et al.
2005); working memory (Callicott et al. 2005); and reduced hippocampal volumes
(Cannon et al. 2005; Callicott et al. 2005). In assessing the evidence implicating
hippocampal dysfunction in DISC1 it is worth noting that different genetic variants
have been investigated, with potentially different phenotypic effects (e.g. con-
flicting evidence for involvement of a DISC1 SNP and cognitive ageing) but none
of these have been confirmed as functionally causal.
The 22q11.2 deletion syndrome (22q11.2DS; also known as velo-cardio-facial
syndrome (VCFS)) is caused by the most common large micro deletion in the
human genome and has an incidence of one in *4000 live births. The phenotype
is highly variable and can affect multiple organs and tissues, but carriers have a
30-fold increased risk of schizophrenia and an increased rate of other psychiatric
phenotypes including ADHD and autistic spectrum disorders. Many, but not all,
carriers fall into the lower than average IQ (FSIQ 70–75) range or have mild
learning disability. Most studies, particularly in children, report higher scores on
verbal than non-verbal tasks. Investigation of these non-verbal deficits across age
groups indicate impairments in comparisons of magnitude and time duration,
which implicate parietal and frontal circuitry underlying attentional and numerical
cognition (reviewed, Karayiorgou et al. 2010). However, the ability to inhibit
processing of extraneous information is also critical and impaired performance on
inhibition tasks is also reported in affected children. Impairments in other aspects
of inhibition including prepulse inhibition and reduced frontal lobe activation
during the mismatch negativity paradigm have also been reported. These findings
indicate that how information is selected or inhibited in attentional processing is
important in the non-verbal deficits evident in this syndrome. Some brain regions
are either structurally enlarged or reduced in children with 22q11.2DS compared
to healthy controls. There appears to be cortical thinning and reduced cortical
gyrus complexity in frontal and parietal cortices. The few reported functional and
connectivity studies broadly confirm the findings of the neurocognitive tests with
performance on arithmetic and spatial attention tasks correlating with frontal and
parietal connectivity and functional measures.
There are a number of ways in which loss of genes at this locus could contribute
to the expression of these cognitive phenotypes. First, under-expression of a single
gene at this locus could exert a major effect on the phenotype. Second, the effect
could be the result of under-expression of a number of proximally located genes.
Finally, the microdeletion could unmask the effects of one or more recessive
mutations. How these effects can be functionally investigated in an animal (e.g.
mouse model) is beyond the scope of this chapter, but is reviewed in Karayiorgou
et al (2010). The example of 22q11.2DS identified several key issues in cognitive
genetics. Unlike the risk variants reported in the association studies of DISC1,
22q11.2DS is uncommon, so ascertainment of sufficient samples for neurocogni-
tive studies is challenging. Compared to DISC1, we are more informed as to the
potential molecular mechanisms involved making this locus more tractable for
functional studies in model systems. Unlike the DISC1 example, which implicates
one gene, the 22q11.2DS locus potentially involves more than 25 genes, which is
more biologically challenging. In the next section, we consider how advances in
genotyping technology have expanded the range of risk loci available for inves-
tigation in cognitive genetic studies of neuropsychiatric disorders.
5 Linkage and Candidate Gene-Based Approached

to Cognitive Genetics
The identification of common polymorphic genetic markers shared in individuals

within populations was a key step for molecular genetics. This made possible two
new approaches to identify genes or genetic loci, which caused or contributed to
phenotypes or traits. The first, genetic linkage analysis, capitalised on genetic
maps of markers across the genome to investigate large single pedigrees or mul-
tiple families affected with a given disorder. Statistical evidence that specific
markers co-segregated with illness, could be used to map loci linked to this dis-
order. Linkage analysis is most effective when there is a strong correlation
between the phenotype being measured and the inferred genotype at the markers
being tested for co-segregation. Further fine-mapping of linked loci, could then
implicate specific risk genes. This approach was highly successful for Mendelian
disorders, including many with cognitive phenotypes, but less successful for dis-
orders with a more complex genetic aetiology and weaker correlation between
phenotype and inferred genotype.
The second strategy, which could use either case-control or family-based
designs, directly targeted genetic markers at specific genes which were implicated
in a disorder through understanding of the disease process (functional candidate
gene studies) or targeted likely candidates within a region implicated by linkage or
cytogenetic studies (positional candidate genes studies). Association studies are
based on populations instead of pedigrees, and compare frequencies of marker
alleles in affected individuals versus unaffected individuals from the general
population. Challenges in identifying such candidate genes include uncertainty
regarding the biological aetiology of the disorder, and limited study power to
detect common alleles of small effects. This power issue becomes even more
relevant for the genome-wide association studies (GWAS) described in the
Genomics section.
5.1 Linkage and Candidate Genes Studies for Disorders

of Childhood
5.1.1 ADHD
Genetic studies of the ADHD clinical phenotype have followed the traditional
pathway of twin and adoption studies to establish heritability, followed by genetic
linkage studies, association studies based on candidate genes and more recently
genome wide association studies (GWAS) and analysis of rare structural variants
(detailed below).
Results from the handful of published linkage studies using the ADHD diag-
nosis as a phenotype (Fisher et al. 2002; Arcos-Burgos et al. 2004; Asherson et al.
2008; Rommelse et al. 2008), show some degree of overlap for regions on chro-
mosomes 5p, 9q, 16q, and 17p if nominally significant findings are considered;
however, no regions have achieved genome-wide significance using strict criteria.
Attempts have been made to include neurocognitive measures in the linkage
analysis of ADHD to identify quantitative trait loci linked to these traits. This
approach assumes that the neurocognitive impairments in ADHD index a latent
trait, or traits, that overlap, at least in part, with the heritable pathophysiology of
ADHD. Taking this approach, Rommelse et al. (2008) examined candidate
endophenotypes in a genome-wide search for susceptibility loci for ADHD. This
study found strong evidence for linkage to 2q21.1 and 13q12.11 for measures of
motor timing and digit span measures, respectively, incorporating ADHD symp-
toms as covariates. Doyle et al. (2008) identified a region on 3q13 showing sug-
gestive evidence for linkage to several neurocognitive traits and inattention
symptoms in ADHD. None of these studies have produced convincing evidence
for linkage, making the presence of one or a small number of gene variants with a
large effect on a given trait measure unlikely.
Early candidate gene studies focused on a range of candidate genes with some
a priori evidence for a potential role in ADHD pathophysiology. As with the linkage
studies described above, putative ADHD risk variants at candidate genes have been
tested for association against clinical and cognitive variables. Kebir et al. (2009)
reviewed 29 studies examining 10 genes (DRD4, DAT1, COMT, DBH, MAOA,
DRD5, ADRA2A, GRIN2A, BDNF and TPH2) in relation to neuropsychological
traits relevant for ADHD. For DAT1, there are conflicting results in relation to
omission and commission errors, but more consistent findings that increased
reaction time variability (Bellgrove et al. 2005a) and abnormalities in spatial
attention (Bellgrove et al. 2005b) are associated with the ADHD associated
10-repeat variant. Against what might have been expected, several studies (Manor
et al. 2002; Bellgrove et al. 2005a, b, c), reported better performance on tests of
attention in children with the 7-repeat DRD4 variant previously shown to be
associated with ADHD. This is a similar finding to studies in psychoses where the
GWAS identified ZNF804A risk variant (discussed below) may identify a patient
subgroup with relatively spared cognitive performance, suggesting that the DRD4
risk variant indexes a pathophysiological pathway to ADHD not mediated by poor
performance on cognitive measures. The effect of the 7-repeat variant was confined
to children with ADHD and not seen in controls and in a more recent study (Johnson
et al. 2008), spectral analysis of reaction time variability supported the hypothesis
that the association of greater variability was with the absence of the 7-repeat allele
and was also specific to ADHD. Other ADHD candidate genes examined in relation
to cognitive function include the X-linked steroid sulfatase gene where case reports
of deletions have been found in cases with neurodevelopmental disorders associ-
ated with abnormal cognitive function and ADHD (Doherty et al. 2003).
Stergiakouli et al. (2011) showed that ADHD associated risk variants were asso-
ciated with inattentive symptoms and poor performance on verbal IQ and
comprehension subtests in ADHD subjects but not controls.
5.1.2 Autism Spectrum Disorders
A number of linkage regions, replicated in two or more studies have been identified
in autism 2q21–33, 3q25–27, 3p25, 4q32, 6q14–21, 7q22, 7q31–36, 11p12–13,
17q11–21 (reviewed by (Freitag et al. 2010). The chromosome 2q region had
marginally stronger evidence for linkage in individuals with language delays
(Buxbaum et al. 2001). A further study stratifying linkage analyses found nonsig-
nificant evidence of linkage in individuals with IQ [ 70 and those with delayed
language (Liu et al. 2008). Multiple candidate gene studies have been conducted but
none have been reliably replicated. Consequently, we have focused here on genetic
studies in social cognition where a convergence of evidence appears to support the
role of neuropeptides oxytocin and vasopressin.
Nonapeptides and Social Cognition
Oxytocin (OXT) and vasopressin (AVP) are highly conserved neuropeptides with
marked diversity in the regulation of their receptors. Modulated significantly by
sex steroids, they are likely to have sexually dimorphic effects and are therefore
of interest for further investigation in disorders such as autism, which show
marked gender bias (M:F*4:1). Considerable evidence from animal literature
has implicated nonapeptides oxytocin and vasopressin in social behaviour (Insel
2010) relating particularly to pair bonding, maternal care, social recognition, and
response to threat (reviewed by (Donaldson and Young 2008). Administration of
oxytocin to humans is associated with reduced anxiety, alteration in parenting
behaviour, increases in prosocial behaviour (e.g. trust, generosity, altruism and
betrayal aversion), reduction in gaze aversion, improved mentalisation, and

differential amygdala activity in fMRI in response to face perception and
changes in social memory (Skuse and Gallagher 2011). Studies reporting asso-
ciation with OXT and autism are inconsistent and no evidence for association
has emerged from GWAS studies. However, beneficial effects of exogenous
oxytocin on core ASD symptoms have been reported offering the potential
possibility of new therapeutics (Green and Hollander 2010). Vasopressin has
been implicated in aggression, social recognition, and pair bonding. The AVP
receptor 1A gene is highly conserved. It contains genetic variation reported to
influence species-specific differences in pair bonding in animal studies (Wang
and Aragona 2004). Genetic variation in AVPR1A has been investigated in ASD
with variable reports of association (Kim et al. 2002; Wassink et al. 2004;
Yirmiya et al. 2006). One of the variants in humans has demonstrably reduced
expression (Tansey et al. 2011) possibly demonstrating a functional route for
genetic association with the gene in autism. The wide-ranging effects of these
neuropeptides on human social behaviour are perhaps not specific to autism and
may potentially have utility in a wider range of psychiatric disorders with social
cognitive deficits.
5.1.3 Intellectual Disability
Notwithstanding the high heritability of intelligence, little progress has been made
in identifying loci reliably linked or associated with intelligence in normal pop-
ulation samples. There are exceptions, such as the association, predominantly in
older people, between ApoE variants and general cognitive ability, episodic
memory, and executive function, and the weak associations reported with COMT
and BDNF variants accounting, if true for only a very small proportion of the
variance in intelligence. In contrast, several hundred genes are known to be
associated with intellectual disability (Chelly et al. 2006).
Genetic forms of ID are divided into syndromic ID, characterised by associated
clinical, radiological, metabolic or biological features, and non-syndromic ID in
which cognitive impairment represents the only manifestation of the condition.
The distinction might be helpful for clinical purposes, but recent phenotype–
genotype studies are blurring the distinction. Causes of ID are extremely hetero-
geneous and include environmental forms (e.g. premature birth, perinatal brain
ischaemia or foetal alcohol syndrome), disorders due to chromosomal abnormal-
ities (including sub-microscopic copy number variation discussed below) and
conditions due to monogenic causes or dysregulation of imprinted genes. About
50% of cases with moderate or severe ID have a definable cause with a lower
percentage in milder cases. Taken together, the emerging genetic findings in ID are
suggesting a neurobiology around synaptogenesis, synaptic activity, and plasticity
with aberrant length and density of dendritic spines a frequent histological finding.
Genetic defects and biochemical abnormalities have been described in several
pathways that feed into synaptic function including the RhoGTPase signal
transduction pathway; with loss of function at its components PAK3, OPHN1,

TM4SF2, and FMRP leading to LD and the Ras/MAPK transcription signaling
cascade; with the genes NF1, RKS2, CBP, and PAK3 involved in ID. Many of
these genes when mutated in animal models affect learning and memory processes
that require gene transcription and translation of proteins.
5.2 Linkage and Candidate Genes Studies for Disorders

of Adult-Onset
5.2.1 Schizophrenia
A meta-analysis of more than 30 schizophrenia linkage studies by Ng et al.

(2009), suggests the involvement of multiple chromosomal loci in schizophrenia
susceptibility. Few investigations focussed on cognitive phenotypes in linkage
analysis of such families have been reported. One example, reported by Almasy
et al. 2008, investigated 43 families and identified significant linkage to the
chromosome 5q region for the cognitive phenotypes of abstraction and mental
flexibility. A more recent study of 557 sibling pairs of Han Chinese ethnicity
identified association with the 12q24.32 locus and undegraded CPT hit rate (Lien
et al. 2010). Rather than focussing on individual neurocognitive phenotypes,
Hallmayer et al. (2005) identified families with co-segregation of more pervasive
cognitive deficits and identified linkage in these families to chromosome 6p24, a
region that had previously been implicated in schizophrenia risk across multiple
studies (Straub et al. 1996). Individual markers at this locus have also been
associated with deficits in CPT performance in the Han Chinese population
detailed above (Lin et al. 2009). The lack of consistency in measured phenotypes
makes replication, and final interpretation of these results difficult. Very few
studies of this type have been reported for other psychiatric disorders. For
example, in bipolar disorder, where many large-scale linkage analyses have also
been reported the focus has been on dividing families according to clinical rather
than cognitive covariates.
Candidate gene studies of cognitive phenotypes have received much wider
attention. This was prompted by studies of functional variants at two candidate
genes, the catechol-O-methyltransferase (COMT) and brain derived neurotrophic
factor (BDNF) genes. From both animal and human studies it is known that
reduced dopamine in prefrontal cortex is associated with impaired performance
on cognitive testing. Deficits in working memory can be reversed with dopamine
agonists, but both very low and very high levels of dopamine activity are
associated with impaired prefrontal cortex function. The COMT gene appears
to be key to dopamine catabolism in the prefrontal cortex (PFC) and is a
logical candidate for investigation in disorders such as schizophrenia as
well as for studies of cognition. Numerous association studies for neuropsychi-
atric phenotypes have been performed with equivocal results, with several large
meta-analyses failing to find association with schizophrenia (Munafo et al.

2005). In the first cognitive study, Egan et al. (2001) reported that the high-
activity val allele was associated with poorer performance on the wisconsin card
sort test (WCST) and reduced efficiency of physiological response of the dorso-
lateral prefrontal cortex during a working memory task. This finding has
received consistent replication and a meta-analysis of 12 studies supports the
original WCST finding (Barnett et al. 2007). The WCST is a complex problem
solving task with many cognitive components and several authors have tried to
identify simpler tests for specific components of this task involving cognitive
stability and flexibility. Because COMT influences the ratio of activation of
D1/D2 receptors and D4 receptors are known to have an effect on PFC function,
variants in these three genes have also been investigated in cognitive studies of
adults with no clear findings emerging.
BDNF is known to have an important role in learning and regulates activity-
dependent synaptic plasticity necessary for short- and long-term memory storage
(Alonso et al. 2002). Studies of BDNF have focused on a valine (val) to
methionine (met) substitution in the 50 region of the gene, which decreases
BDNF activity-dependent secretion. Association between the variant and
neuropsychiatric clinical phenotypes has been reported, although replication of
these findings has been inconsistent. A meta-analysis of studies across multiple
phenotypes reported that the met allele was associated with risk for eating dis-
orders and schizophrenia and a protective effect for substance-related disorders
(Gratacos et al. 2007). This may have represented a publication bias. On the
basis of BDNF’s known role in hippocampal function, it has been suggested that
met allele carriers may have impaired performance on memory tasks. Supporting
this hypothesis Egan et al. (2003) identified poorer episodic memory perfor-
mance, a disruption in normal hippocampal fMRI findings during a working
memory task and reduced hippocampal levels of a marker for neuronal function
in schizophrenia patients and healthy controls. The same year, Hariri et al.
(2003) showed that met-carriers had reduced hippocampal engagement during
encoding and retrieval of a spatial task and also made more recognition errors on
the task. Subsequently, it was reported that val/met heterozygotes have lower
hippocampal volumes than carriers of the val/val genotype (Pezawas et al. 2004).
Within schizophrenia patients, met-carriers are also reported to have poorer
medial temporal lobe-related performance and correspondingly smaller temporal
and occipital lobar grey matter volume (Ho et al. 2006). Although negative
studies have been reported, most of the available data supports a modest
association between the met allele and reduced cognitive performance.
With the emergence of putative candidate genes from the schizophrenia liter-
ature these were also systematically investigated for cognitive phenotypes. The list
of investigated genes includes Neuregulin-1, Dysbindin, and DAO. Unlike the case
of COMT and BDNF, no clear functional variants have been identified at these
genes and multiple studies have reported different risk variants, alleles, or hap-
lotypes. This makes direct comparison of studies, which have often also examined
different phenotypes, difficult (reviewed in Gill et al. 2010).
5.2.2 Cognitive Ageing and Psychiatric Disorders
Association between an increased risk for Alzheimers disease and the e4 allele of
the apolipoprotein E (ApoE) gene is one of the most robust findings in complex
disorder genetics. Carriers of one copy of this allele are 3–4 times more likely to
develop late-onset Alzheimers disease (LOAD), but carriers of two copies have a
more than 10-fold increase in risk (Farrer et al. 1997). Multiple studies have shown
that APOE e4 is associated with cognitive decline in patients with AD. More
recently it has been demonstrated that this allele has an effect on cognitive per-
formance in non-patient groups as well. Carriers of the risk allele perform sig-
nificantly poorer on tests of episodic memory, global cognitive ability, executive
functioning, and perceptual speed although the effect sizes are small. There was no
difference between carriers and non-carriers for tests of attention, primary mem-
ory, verbal ability, and visuo-spatial skill. For the domains where differences were
detected these differences became more significant with increased age (Wisdom
et al. 2011). These data lead researchers to explore the effects that other candidate
genes may have on cognitive ageing. A study investigating 10 candidate genes
(including BDNF, COMT and DISC1) for cognitive function failed to identify
association with performance and cognitive ageing in the Lothian birth cohort of
over a 1,000 Scottish 70-year old individuals (Houlihan et al. 2009).
6 Application of Genomics Methods
Genome-wide association studies (GWAS), by combining advances in high-

throughput genotyping platforms and understanding of common genetic variation
in populations, allow most common variation in the genome to be tested in a single,
usually case–control experiment. Testing all genes is a powerful hypothesis-free
approach and GWAS have proven remarkably successful at identifying common
risk variants for complex human disease. However, this comes with a significant
multiple testing burden and requires large sample sizes—in the thousands or tens of
thousands—to identify what are typically modest gene effects (Corvin et al. 2009).
In psychiatry as with other medical specialties, this requirement has driven
collaboration, for example, the formation of the psychiatric GWAS consortium,
which is currently performing meta-analysis of GWAS data for schizophrenia,
bipolar disorder, autism, recurrent major depression, and ADHD (Psychiatric
GWAS Consortium Coordinating Committee 2009). For many of these disorders
novel susceptibility loci have been identified (detailed below). GWAS can also
inform on the genetic architecture of psychiatric disorders: identified loci appear in
many cases to increase risk across traditional diagnostic boundaries; a substantial
proportion of schizophrenia and bipolar disorder risk may involve thousands of
overlapping gene variants of small effect (International Schizophrenia Consortium
2009a, b) whereas smaller numbers of variants of large effect appear involved in
autism susceptibility.
GWAS platforms were designed to assay common genetic variation and SNPs
with a population frequency of at least 5%, however, rare or even unique genetic
variants are much more frequent in the human genome. Until now our ability to
test for involvement of this type of variation in human disease ‘the rare variant
common disease hypothesis’ has been very limited. GWAS platforms and custom-
designed microarrays using comparative genomic hybridization (CGH) have
allowed investigation of one class of rare genetic variation, namely, copy number
variation (CNV). This provides some insight into likely challenges for cognitive
research in analysing rare variants: an issue that will become more relevant with
the increasing availability of whole-genome sequence data.
6.1 Genome-Wide Association Studies
6.1.1 GWAS for Adult-Onset Psychiatric Disorders
Schizophrenia
Nine schizophrenia loci have been identified: the zinc finger protein 804A
(ZNF804A) gene on chromosome 2q32; at the major histocompatibility complex
(MHC) region on chromosome 6p21–22; upstream of the neurogranin (NRGN)
gene on chromosome 11q24; at the transcription factor 4 (TCF4) gene on chro-
mosome 18q21; downstream from microRNA miRNA137 on chromosome 1p21.3;
a 0.5 Mb gene-rich region on chromosome 10q24.32; an intronic SNP in the CUB
and sushi multiple domains 1 (CSMD1) gene; and common variants in gene
deserts on chromosomes 2q32.3 and 8p21.3. Additionally, there is substantial
evidence for overlap in particular between schizophrenia and mood disorders, as
the schizophrenia risk variant at ZNF804A has also been implicated in bipolar
disorder (Williams et al. 2011) and the CACNA1C variant, identified in bipolar
disorder has also been implicated in schizophrenia and recurrent major depression
(Green et al. 2010). A logical next step is for cognitive studies to test whether
specific neural mechanisms underlie this susceptibility and its clinical expression.
The psychosis risk variant at gene ZNF804A has received the most attention to
date. Esslinger et al. 2009 investigated the influence of the risk variant (rs1344706)
on cortical activity within, and connectivity between, regions during working
memory (N-back task) and emotional recognition task performance in a sample of
115 healthy controls. Differences in functional connectivity, but not regional
activation, were observed. They reported reduced connectivity in the dorso-lateral
prefrontal cortex (DLPFC) between and within hemispheres, but also increased
connectivity between the hippocampal formation (HF) and the DLPFC, and
between the amygdala and the HF, orbitofrontal cortex and prefrontal cortex. They
have subsequently reported evidence for involvement of the variant in aberrant
brain activation during social information processing using a theory of mind
(TOM) task (Walter et al. 2011) and in state-independent inter-hemispheric
processing (Esslinger et al. 2009). Their interpretation that the risk allele has a
deleterious effect on cognitive performance has been questioned by several more
recent studies. First et al. (2010) found and replicated evidence for better cognitive
performance on working memory and episodic memory tasks—which involve the
DLPFC and HF—in patient carriers of the risk allele. This effect was not present in
controls. In a subsequent study, of a different patient group, the authors found
relatively larger hippocampal volumes in risk allele carriers (Donohoe et al. 2011a,
b). These data suggest that the ZNF804A risk variant may identify a patient sub-
group with relatively spared cognitive performance, but possibly more social
deficits. Although we note that several smaller equivocal studies have also been
reported (Lencz et al. 2010; Balog et al. 2010). Further studies particularly in the
domain of social cognition would be useful.
Of the other identified schizophrenia loci, some are large and implicate many
genes (e.g. the MHC region) and some map to gene deserts which are not obvious
candidates for involvement in cognitive functioning. Of the identified genes,
neurogranin (NRGN) is the most compelling target as it plays an important role in
calcium–calmodulin signaling, is abundantly expressed in hippocampus, and
NRGN knockout mice have severe deficits in hippocampus-dependent tasks.
However, a recent study by Donohoe et al. (2011b) failed to identify a strong
relationship between the risk allele and neuropsychological performance in either
patient or control populations on general cognitive ability, verbal episodic and
working memory, spatial episodic or working memory or attentional control.
Bipolar Disorder
In bipolar disorder the best supported loci are the calcium channel, voltage-
dependent, L type, alpha 1C subunit (CACNA1C), and the ankyrin 3, node of
Ranvier (ANK3) genes (Ferreira et al. 2008). Perceived wisdom is that cognitive
deficits are less prominent in bipolar disorder, although a range of abnormalities
have been reported including altered identification of emotional stimuli (e.g. facial
expression), processing speed, working memory, and impairments in sustained
attention (Arts et al. 2010). Cognitive phenotypes in bipolar disorder may vary
with mood state and have received less attention in family studies (to estimate
heritability) than equivalent studies in schizophrenia. Of the common risk variants
identified in GWAS studies, the CACNA1C gene has received the most attention as
non-synonymous mutations of CACNA1C cause Timothy syndrome, a multi-organ
disorder, which includes cognitive impairments (Splawski et al. 2005). Cacna1c
heterozygous female mice also demonstrate mood-related phenotypes including
reduced risk-taking behaviour and increased anxiety (Dao et al. 2010).
Association studies have been reported in case and control populations between
the risk allele at rs1006737 across different neuropsychological testing paradigms
and imaging studies. The first reported study (Krug et al. 2010) found reduced
semantic verbal fluency with increased activation of the left inferior frontal gyrus
and left precuneus in healthy male subjects who carried the risk variant. The
authors acknowledged that the data to suggest reduced verbal fluency in euthymic
bipolar disorder is limited and their results require independent replication. To date
five imaging studies have been published, with somewhat mixed results. An initial
report of reduced grey matter volume in healthy UK carriers of this risk variant
(Kempton et al. 2009) did not replicate in a much larger German control sample
(Franke et al. 2010). The latter reported association between genetic variation at
the gene and reduced brainstem volume, but this was with different SNPs at the
gene and requires independent replication. Studies using blood-oxygenation level-
dependent (BOLD) functional magnetic resonance imaging (fMRI) have targeted
circuits potentially implicated in bipolar disorder. A study of patients and controls,
by Bigos et al. (2010) implicated circuits putatively involved in bipolar disorder
and schizophrenia. They identified a trend for increased hippocampal activity
during emotional processing and also greater prefrontal cortical activity during a
working memory paradigm, a pattern previously associated with putative schizo-
phrenia risk variants. A smaller study of healthy controls targeted increased limbic
activity as a bipolar disorder phenotype and identified association with increased
amygdala activity in response to reward. Erk S. Meyer-Lindenberg et al. (2010) in
studying brain activation during a declarative memory task identified reduced
bilateral hippocampal activation during episodic memory recall and reduced
coupling between left and right hippocampal regions in 110 healthy subjects.
Alzheimers Disease
Until recently, APOE was the only gene known to increase risk of the common
form of Alzheimer’s disease with late-onset. A number of new susceptibility
variants have been identified by GWAS including novel loci for late-onset
Alzheimers disease (AD) implicating the genes clusterin (CLU), the phospatid-
lyinositol-binding clathrin assembly protein gene (PICALM), the complement
receptor gene (CR1), the bridging integrator 1 gene (BIN1), the ATP-binding
cassette (ABC) transporter gene (ABCA7), MS4A gene, CD2AP, CD33 and
EPHA1 (Hollingworth et al. 2011). These genes appear to be involved in different
processes, with five being linked to immune function; four to cell membrane
endocytosis; and three being involved in lipid processing. As of yet none have
been tested for involvement in specific aspects of cognitive functioning or their
role in cognitive ageing.
6.1.2 GWAS for Childhood-Onset Psychiatric Disorders
Data from GWAS studies to date have not generally supported the linkage regions
previously identified for childhood-onset disorders. A region on chromosome 5p.14
harbouring cadherin genes CDH9 and CDH10 showed evidence for association in
one study (Wang et al. 2009). A SNP at 5p.15 was located close to a taste receptor
gene (TASR1) and a member of the semaphorin family (SEMA5A), the latter
family of genes are implicated in axonal guidance (Weiss et al. 2009). Genome-
wide evidence for association at the MACROD2 gene, a gene of uncertain function,
was detected in a further study (Anney et al. 2010). Stratification in the latter
analysis based on IQ and verbal status did not reveal statistically significant
genome-wide evidence of association.
Results of the PGC consortium ADHD meta-analysis have yet to be reported
and smaller GWAS studies have yet to provide genome-wide significant evidence
of association.
6.2 Studies of Structural Genomic Variation

in Neuropsychiatric Disorders
As recently as 2004, it was discovered that submicroscopic deletions or duplica-

tions involving the gain or loss of entire DNA segments (e.g. from a thousand to
several million bases) are common (Sebat et al. 2004) and may encompass more
than 10% of the average human genome. This technology also made it possible to
test for the involvement of inherited or de novo CNVs in disease. It rapidly became
evident that CNVs play an important role in susceptibility to neurodevelopmental
disorders including autism (Sebat et al. 2009), learning disability, schizophrenia
(Walsh et al. 2008), and ADHD (Williams et al. 2010). For autism, CNVs have
been identified in at least 10% of cases, implicating a large number of novel
genomic loci and risk genes (reviewed Betancur 2011). In schizophrenia, the
seven most established CNVs collectively account for *2–4% of susceptibility
(reviewed Sebat et al. 2009). An excess of CNVs have also been identified
in ADHD including a duplication of chromosome 16p13.11. Data for other
neuropsychiatric disorders is more equivocal, with the exception of bipolar dis-
order where a large study of 1,697 cases and 2,806 controls found no evidence of
either an increased total burden or association with individual CNVs (Grozeva
et al. 2010).
6.2.1 Cognitive Studies of Structural Genomic Variants

and Rare Mutations Implicated in Psychiatric Disorders
The results thus far challenge many preconceptions about the clinical entities
being investigated. The same CNVs are being implicated in different disorders.
For example, in autism although 70% of affected individuals have learning disability,
almost all of the implicated CNVs have also been associated with learning disability.
Many of the CNVs identified in schizophrenia have also been implicated in autism:
the duplication reported in ADHD has been reported across all three disorders, being
most common in ADHD patients with co-morbid learning disability. This diverse
phenotypic expression extends beyond psychiatric phenotypes. The 1q21.1 deletion
reported in schizophrenia (International Schizophrenia Consortium 2009a, b) is now
known to be associated with a broad array of paediatric developmental abnormalities
including autism, but also heart defects and cataracts (Mefford et al. 2008). These
findings suggest that at least a subset of patients with clinical disorders have
underlying rare genomic disorders.
Candidate gene studies of cognitive phenotypes have received much wider
attention. This was prompted by studies of functional variants at two candidate
genes, the catechol-O-methyltransferase (COMT) and brain derived neurotrophic
factor (BDNF) genes. From both animal and human studies it is known that
reduced dopamine in prefrontal cortex is associated with impaired performance on
cognitive testing. Deficits in working memory can be reversed with dopamine
agonists, but both very low and very high levels of dopamine activity are asso-
ciated with impaired prefrontal cortex function. The COMT gene appears to be key
to dopamine catabolism in the prefrontal cortex (PFC) and is a logical candidate
for investigation in disorders such as schizophrenia as well as for studies of
cognition. Numerous association studies for neuropsychiatric phenotypes have
been performed with equivocal results, with several large meta-analyses failing to
find association with schizophrenia (Munafo et al. 2005). In the first cognitive
study, Egan et al. (2001) reported that the high-activity val allele was associated
with poorer performance on the wisconsin card sort test (WCST) and reduced
efficiency of physiological response of the dorso-lateral prefrontal cortex during a
working memory task. This finding has received consistent replication and a meta-
analysis of 12 studies supports the original WCST finding (Barnett et al. 2007).
The WCST is a complex problem solving task with many cognitive components
and several authors have tried to identify simpler tests for specific components of
this task involving cognitive stability and flexibility. Because COMT influences
the ratio of activation of D1/D2 receptors and D4 receptors are known to have an
effect on PFC function, variants in these three genes have also been investigated in
cognitive studies of adults with no clear findings emerging.
Extrapolating from examples of rare genomic disorders that have already
been classified we could suspect that some will share core phenotypic features
(e.g. Williams syndrome and Prader-Willi/Angelman syndrome) but others
(e.g. chromosomal deletions involving 1q21.1 and 22q11.21) may have such a wide
range of phenotypic expression as to encompass several clinical syndromes (Lee and
Scherer 2010). Not all CNVs are causative: some are likely to have more modest
effects on risk and probably interact with other genetic or environmental risk factors.
What does this mean for cognitive studies? We know that some of the
implicated loci can have a profound effect on cognitive functioning leading to
significant general learning disability. Would a general screen of IQ in clinical
populations identify these CNV carriers? Or do some CNVs cause more subtle
cognitive deficits? Plausibly, specific CNVs may impact on, and be extremely
informative about, discrete aspects of cognitive functioning. Performing such
studies is problematic because of the numbers involved.
Lessons can be learned from investigation of chr22q11.21, but many of
the validated CNVs have a frequency of less than one in 500 in case samples,
having large-scale collaboration and common assessment methods will be essen-
tial. Some consensus on batteries of tests is also essential to allow comparison
across CNVs, which may be important in identifying where the phenotypic effects
may be a consequence of involvement of the same molecular mechanism or
pathway. Obvious targets for investigation are loci where a single, or small
number of genes are disrupted as these are currently most tractable for other func-
tional studies. CNVs are often complex and both gain or loss of function at a locus
may need to be considered. For example, mutation of the gene encoding methyl-
CpG-binding protein-2 (MECP2) causes Rett syndrome a neurodevelopmental
disorder almost exclusively found in females, however, duplications or triplications
of the gene are associated with developmental delay or learning disability in males.
7 Future Directions: New Phenotypes and New Approaches
A major development in cognitive genetics disorders has, as with psychiatric

genetics studies, been the move from single gene studies to genome-wide studies
of the genetic architecture of cognition. Examples of these studies are already
published (e.g. Need et al. 2009; Davis et al. 2010) with several more in progress.
These studies are likely to replicate difficulties found in genome-wide association
studies (GWAS) of psychiatric disorder, in particular the low power to detect small
effects in samples. Current estimates (Visscher and Montgomery 2009) suggest the
need for more than 10,000 samples to detect small effects in psychiatric disorders;
power to detect variants with an odds ratio of 1.1–1.2 (the effect size associated
with already identified common variants) are likely to require even larger samples.
If the effect size of ‘cognition’ genes is similar this requires us to plan experiments
on a scale that is far beyond what was traditionally thought of as large. One
example of an attempt to achieve the required scale is the COGENT consortium,
which has to date amassed data on *7,000 neuropsychological phenotyped
healthy participants for the purposes of a genome-wide association study of
general cognitive ability or ‘g’. In advance of the results of these experiments, the
evidence that cognitive phenotypes are unlikely to be much less complex in its
genetic architecture than seen in psychiatric disorders should cause us to expect
meaningful but small advances in our understanding of cognitive genetics.
At the same time, the increased ability to investigate rare genetic effects is
identifying many interesting candidate genes for further cognitive studies. These
may be particularly important as they may each be associated with significant risk.
For studies of CNV carriers, the logistics of performing studies of sufficient statistical
power is challenging, although not insurmountable as we know from the 22q11.2DS
studies. One major question will be whether specific CNVs have distinct cognitive or
clinical effects. This will be addressed by ongoing large studies in Europe where
carriers of these variants are being targeted for neurocognitive and neuroimaging
studies (Meyer-Lindenberg 2010).
This type of analysis will be even more challenging when there are large
data resources available with full genome sequencing information. We already
know that in the human population, many disease genes can show dozens or even
hundreds of independent mutations. Mutations that have more severe functional

consequences (i.e. to the production of the gene product) may be lethal or asso-
ciated with severe phenotypes, such as microcephaly. Research from single gene
cognitive disorders suggests that, depending on the functional consequence of
the mutation, there may be a range of phenotypic outcomes, which include much
more subtle phenotypes (Walsh and Engle 2010). Many of the genes known to be
building blocks for important neurodevelopmental processes are likely to harbour
mutations with these types of genetic effects. This offers a potential framework for
understanding and potentially grouping molecular mechanisms at the level of the
gene, or in pathways based on understanding of molecular neurodevelopment.
The potential to perform cellular and animal studies as well as the ability to return
to families, which carry mutations should offer fascinating insights into the
genomic underpinnings of cognitive function. In performing such studies we must
not lose sight of the fact that the molecular programmes that govern and modify
neurodevelopment are affected by stochastic variation and actively influenced by
the world that surrounds us. To maximise what we can learn about cognitive
genomics we will need to understand the impact of environment.
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Behavioral Genetics of Neurodegenerative
Disorders
Daniela Galimberti and Elio Scarpini
Abstract Alzheimer’s disease (AD) is the most common cause of dementia in the
elderly, and is typically characterized by memory loss. In addition, during the
disease progression, most patients develop behavioural and psychiatric symptoms
of dementia (BPSD). Frontotemporal Lobar Degeneration (FTLD) is the most
frequent neurodegenerative disorder with a presenile onset. It is characterized
mainly by behavioural disturbances, whereas memory is conserved. The two major
neuropathologic hallmarks of AD are extracellular Amyloid beta (Ab) plaques and
intracellular neurofibrillary tangles (NFTs). Conversely, in FTLD the deposition of
tau has been observed in a number of cases, but in several brains there is no
deposition of tau but instead a positivity for ubiquitin. In some families these
diseases are inherited in an autosomal dominant fashion. Genes responsible for
familial AD include the Amyloid Precursor Protein (b-APP), Presenilin 1 (PS1)
and Presenilin 2 (PS2). The majority of mutations in these genes are often asso-
ciated with a very early onset (40–50 years of age). Regarding FTLD, the first
mutations described are located in the Microtubule Associated Protein Tau gene
(MAPT). Tau is a component of microtubules, which represent the internal support
structures for the transport of nutrients, vesicles, mitochondria and chromosomes
within the cell. Mutations in MAPT are associated with an early onset of the
disease (40–50 years), and the clinical phenotype is consistent with Frontotem-
poral Dementia (FTD). Recently, mutations in a second gene, named progranulin
(GRN), have been identified in some families with FTLD. The pathology associ-
ated with these mutations is most frequently characterized by the immunostaining
of TAR DNA Binding Protein 43 (TDP-43), which is a transcription factor.
D. Galimberti (&) E. Scarpini

Department of Neurological Sciences, ‘‘Dino Ferrari’’ Center, University of Milan,
Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico,
Via F. Sforza 35, 20122 Milan, Italy
e-mail: daniela.galimberti@unimi.it

DOI: 10.1007/7854_2011_181
616 D. Galimberti and E. Scarpini
The clinical phenotype associated with GRN mutations is highly heterogeneous,

including FTD, Progressive Aphasia, Corticobasal Syndrome, and AD. Age at
disease onset is variable, ranging from 45 to 85 years of age. The majority of cases
of AD and FTLD are however sporadic, and likely several genetic and environ-
mental factors contribute to their development. Concerning AD, it is known that
the presence of the e4 allele of the Apolipoprotein E gene is a susceptibility factor,
increasing the risk of about 4 fold. A number of additional genetic factors,
including cytokines, chemokines, Nitric Oxide Synthases, contribute to the sus-
ceptibility for the disease. Some of them also influence the risk to develop FTLD.
Variability in serotonin transporter gene could influence the development of
BPSD. In this chapter, current knowledge on molecular mechanisms at the basis of
AD and FTLD, as well as the role of genetics, will be presented and discussed.

Keywords Dementia Frontotemporal lobar degeneration (FTLD) Behavioural
disturbances
Alzheimer’s disease (AD) Behavioural and psychological
symptoms of dementia (BPSD)
Contents
1 Alzheimer’s Disease and Frontotemporal Lobar Degeneration....................................... 616

2 Familial AD ....................................................................................................................... 617
3 Sporadic AD ...................................................................................................................... 618
3.1 Genes Influencing the Risk to Develop AD............................................................ 618
3.2 Genes Influencing the Risk to Develop BPSD in AD Patients.............................. 622
4 Familial FTLD................................................................................................................... 623
5 Sporadic FTLD.................................................................................................................. 626
References................................................................................................................................ 626
1 Alzheimer’s Disease and Frontotemporal Lobar

Degeneration
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly,
with a prevalence of 5% after 65 years of age. The disease was originally described
by Alois Alzheimer and Gaetano Perusini in 1906, and it is clinically characterized
by a progressive cognitive impairment, including impaired judgment, decision-
making and orientation, often accompanied, in later stages, by behavioural and
psychiatric symptoms of dementia (BPSD), including agitation, hallucinations and
delusions, as well as language impairment. Loss of noradrenergic and serotonergic
neurons contributes to the emergence of BPSD. Conversely, loss of cholinergic
neurons is the major contributor to the cognitive impairment of AD (Palmer 1996).
The two major neuropathologic hallmarks of AD are extracellular beta-amyloid
(Ab) plaques and intracellular neurofibrillary tangles (NFTs). The production of
Behavioral Genetics of Neurodegenerative Disorders 617
Ab, which represents a crucial step in AD pathogenesis, is the result of the

cleavage of a bigger precursor, named Amyloid precursor protein (APP), which is
over-expressed in AD (Griffin 2006). Ab forms highly insoluble and proteolysis
resistant fibrils known as ‘‘senile plaques’’.
Neurofibrillary tangles are composed of the tau protein. In healthy controls, tau
is a component of microtubules, which are the internal support structures for the
transport of nutrients, vesicles, mitochondria and chromosomes within the cell.
Microtubules also stabilize the growing axons, which are necessary for the
development and growth of neurites (Griffin 2006). In AD, tau protein is abnor-
mally hyperphosphorylated and forms insoluble fibrils, which originate deposits
within the cell.
Frontotemporal lobar degeneration (FTLD) occurs most often in the presenile
period, and age at onset is typically 45–65 years, with a mean in the 50 s. Dis-
tinctive features in FTLD concern behaviour, including disinhibition, loss of social
awareness, overeating and impulsiveness. Despite profound behavioural changes,
memory is relatively spared (Hou et al. 2004). Conversely to AD, which is more
frequent in women, FTLD has an equal distribution among men and women. The
current consensus criteria (Neary et al. 1998) identify three clinical syndromes:
Frontotemporal Dementia (FTD), Progressive nonfluent Aphasia (PA) and
Semantic Dementia (SD), which reflect the clinical heterogeneity of FTLD.
Frontotemporal dementia is characterized by behavioural abnormalities, whereas
PA is associated with progressive loss of speech, with hesitant, nonfluent speech
output (Scarpini et al. 2006), and SD is associated with loss of knowledge about
words and objects (Neary et al. 1998). This variability is determined by the relative
involvement of the frontal and temporal lobes, as well as by the involvement of
right and left hemispheres (Rosen et al. 2002).
Despite the majority of AD and FTLD are sporadic and likely caused by the
interaction between genetic and environmental factors, so far it was observed that
clinically typical AD and FTLD can cluster in families and be inherited in an
autosomal dominant fashion, suggesting a genetic cause.
2 Familial AD
Autosomal dominant AD forms are characterized by mutations in three genes:

Amyloid Precursor Protein (b-APP; Goate et al. 1991), Presenilin 1 (PS1;
Sherrington et al. 1995) and Presenilin 2 (PS2; Levy-Lahad et al. 1995).
In 1987, a region of linkage with AD was reported on the long arm of chro-
mosome 21, which encompassed a region harboring the b-APP gene, a com-
pelling candidate for AD (Tanzi et al. 1987). The gene is located at chromosome
21q21.22 and encodes for a transmembrane protein that is normally processed into
amyloid fragments. In 1991, the first missense mutation in b-APP was reported
(Goate et al. 1991). Since then, 32 different mutations have been described in the
b-APP gene in 89 families (http://molgen-www.uia.ac.be). All these mutations
cause amino acid changes in putative sites for the cleavage of the protein, thus
altering the APP processing, such that more pathological Ab42 is produced (Hardy
and Selkoe 2002). Interestingly, the chromosome 21, in which b-APP resides, is
triplicated in Down syndrome and most of the cases manifest also AD by the age
of 50. Post-mortem analyses of Down’s patients who die young show diffuse intra-
neuronal deposits of Ab, suggesting that its deposition is an early event in cog-
nitive decline. The recent discovery of an extra copy of the b-APP gene in
familial AD (Rovelet-Lecrux et al. 2006) provides further support that increased
Ab production can cause the disease.
The other two genes causing familial AD are PS1 (14q24.3) and PS2 (1q31-
q42). Presenilins represent a central component of c-secretase, the enzyme
responsible for originating Ab from the C-terminal fragment of the APP protein.
Mutations in presenilins also alter APP cleavage, leading to an increased pro-
duction of Ab42. So far, 179 mutations in PS1 have been identified and 14
additional mutations have been found in the homologous gene PS2 (http://molgen-
www.uia.ac.be).
Most variants in PS1 are missense mutations resulting in single amino-acid
substitutions. Some are more complex, for example, small deletions or splice
mutations. The most severe mutation in PS1 is a donor–acceptor splice mutation that
causes a two-aminoacid substitution and an in-frame deletion of exon 9. However,
the biochemical consequences of these mutations for c-secretase assembly seem to
be limited (Bentahir et al. 2006; Steiner et al. 1999). All these clinical mutations are
likely to cause a specific gain of toxic function for PS1, determined by an increase of
the ratio between Ab42 and Ab40 amyloid peptides, thus indicating that presenilins
might modify the way in which c-secretase cuts APP.
Mutations in presenilins occur in the catalytic subunit of the protease respon-
sible for determining the length of Ab peptides therefore generating toxic Ab
fragments. However, presenilins have also non-proteolytic functions (Baki et al.
2004; Huppert et al. 2005), the disruption of which might also contribute to
familial AD pathogenesis.
Despite several carriers develop the disease early (40–50 years of age) with a
typical AD phenotype, in some cases patients carrying the same mutation develop
signs and symptoms resembling FTD instead of AD (Bruni et al. 2010). In addi-
tion, other mutations are associated with myoclonus, seizures, bilateral spasticity,
parkinsonian features or ataxia (Larner and Doran 2009).
3 Sporadic AD
3.1 Genes Influencing the Risk to Develop AD
Risk genes are likely to be numerous, displaying intricate patterns of interaction

with each other as well as with non-genetic variables, and-unlike classical Men-
delian (‘‘simplex’’) disorders-exhibit no simple mode of inheritance. Mainly due to
this reason, the genetics of sporadic AD has been labeled ‘‘complex’’ (Bertram and
Tanzi 2005).
The gene mainly related to the sporadic forms of AD is the Apolipoprotein E
(APOE) (Corder et al. 1993), which is located at chromosome 19q13.32 and was
initially identified by linkage analysis (Pericack-Vance et al. 1991). The rela-
tionship between APOE and AD has been confirmed in more than 100 studies
conducted in different populations. The gene has three different alleles, APOE*2,
APOE*3 and APOE*4. The APOE*4 allele is the variant associated with AD.
Longitudinal studies in Caucasian populations have shown that carriers for one
APOE*4 allele have a two-fold increase in the risk for AD (Raber et al. 2004). The
risk increases in homozygous for the APOE*4 allele, and this allelic variant is also
associated with an earlier onset of the disease.
Several linkage studies have been performed, giving rise to additional candidate
susceptibility loci at chromosomes 1, 4, 6, 9, 10, 12 and 19. In particular, prom-
ising loci have been found at chromosome 9 and 10 (Grupe et al. 2006; Li et al.
2006). Recently, a wide genome analysis identified variants at CLU (which
encodes clusterin or ApoJ) on chromosome 8 and PICALM in chromosome 11
associated with AD (Harold et al. 2009). Data on CLU were replicated in an
independent study, which, in addition, demonstrated that CR1, encoding the
complement component (3b/4b) receptor 1and locate on chromosome 1, is asso-
ciated with AD (Lambert et al. 2009).
Also, a large number of candidate genes studies have been performed in order
to search a robust risk factor for the sporadic form of the disease. Several studies
were mainly focused in genes clearly involved in the pathogenesis of AD such as
genes encoding for inflammatory molecules or involved in the oxidative stress
cascade, both considered major factors in AD pathology. One of the strongest
evidence of the role played by genetic variants in inflammatory molecules to
increase the risk of AD involves the Interleukin-1 (IL1) complex, which map at
chromosome 2q14-21 and includes IL1-a, IL1-b, and IL1R antagonist protein
(IL-1Ra), all of which have a number of polymorphisms found to be associated
with AD in several case-controls studies carried out in different populations
(Du et al. 2000; Grimaldi et al. 2000; Papassotiropoulous et al. 1999). Several
polymorphisms in IL-6, which is a potent inflammatory cytokine but has also
regulatory functions, have been investigated so far. The IL6 gene is located at
chromosome 7p21 and polymorphisms exist in the -174 promoter region and in
the region of a variable number of tandem repeats (VNTR), which is located in the
30 untraslated region. Both of them have been found associated with AD in case–
controls studies (Licastro et al. 2003; Nicoll et al. 2000). Investigation of Tumor
Necrosis Factor-a (TNFa) polymorphisms was initiated because genome screening
suggested a putative association of AD with a region on chromosome 6p21.3,
which lies within 20 centimorgans of the TNFa gene. Furthermore, other
polymorphisms located in the promoter region of TNFa have been associated with
autoimmune and inflammatory diseases (Collins et al. 2000).
As with TNFa, investigations of the role of a-2macroglobulin (A2M) were
initiated as a result of screening studies of the genome. In this case, linkage was
found in the region of chromosome 1p, where A2 M and its low-density lipoprotein
receptor are found. Blacker et al. (1998) tested for association of polymorphisms
with AD showing a strong involvement of this gene in its pathogenesis.
Moreover, polymorphisms in chemokines have been investigated with regard of
susceptibility of AD. Monocyte Chemoattractant Protein-1 (MCP-1) and RANTES
genes have been widely screened in different neurodegenerative diseases (Huerta
et al. 2004). The distribution of the A-2518G variant was determined in different
AD populations with concordant results (Fenoglio et al. 2004; Combarros et al.
2004a) showing no evidence for association of this variant in AD compared with
controls. Moreover, Fenoglio et al. (2004) found a significant increase of MCP-1
serum levels in AD carrying at least one G polymorphic allele. Therefore, the
A-2518G polymorphism does not seem to be a risk factor for the development of
AD, but its presence correlates with higher levels of serum MCP-1.
RANTES promoter polymorphism -403 A/G, found to be associated with
several autoimmune diseases, was examined in AD population, failing to find
significant differences between patients and controls (Huerta et al. 2004).
CCR2 and CCR5 genes, encoding for the receptors of MCP-1 and RANTES
respectively, have been screened for association with AD. The most promising
variants involve a conservative change of a valine with an isoleucine at codon 64
of CCR2 (CCR2-64I) and a 32-bp deletion in the coding region of CCR5
(CCR5D32), which leads to the expression of a non-functional receptor.
A decreased frequency and an absence of homozygous for the polymorphism
CCR2-64I were found in AD, thus suggesting a protective effect of the poly-
morphic allele on the occurrence of the disease (Galimberti et al. 2004); con-
versely, no different distribution of the CCR5D32 deletion in patients compared
with controls were shown (Galimberti et al. 2004; Combarros et al. 2004b).
A mutation scanning of the Interferon-c-induced protein 10 (IP-10) gene coding
region has been performed in AD patients searching for new variants. The analysis
demonstrated the presence of two previously reported polymorphisms in exon 4 (G/
C and T/C), which are in complete linkage disequilibrium, as well as a novel rare one
in exon 2 (C/T). Subsequently these SNPs have been tested in a wide case–control
study but no differences in haplotype distribution were found (Venturelli et al. 2006).
Other genes under investigation are related to oxidative stress, a process closely
involved in AD pathogenesis. In this regard, genes coding for the nitric oxide
synthase (NOS) complex have been screened. The common polymorphism con-
sisting in a T/C transition (T-786C) in NOS3, previously reported to be associated
with vascular pathologies, has been tested in AD, but no significant differences
with controls were found. Nevertheless, expression of NOS3 in PBMC either from
patients or controls seems to be influenced by the presence of the C polymorphic
allele, and is likely to be dose dependent, being mostly evident in homozygous for
the polymorphic variant. The influence of the polymorphism on NOS3 expression
rate supports the hypothesis of a beneficial effect exerted in AD by contributing to
lower oxidative damage (Venturelli et al. 2005).
An additional variant in NOS3 gene has been extensively investigated in AD
patients, although the results are still controversial. It is a common polymorphism
consisting in a single base change (G894T), which results in an aminoacid sub-

stitution at position 298 of NOS3 (Glu298Asp). Dahiyat et al. (1999) determined the
frequency of the Glu298Asp variant in a two-stage case–control study, showing that
homozygous for the wild-type allele were more frequent in late onset AD. How-
ever, studies in other populations failed to replicate these results (Crawford et al.
2000; Monastero et al. 2003; Sánchez-Guerra et al. 2001; Tedde et al. 2002).
More recently Guidi et al. (2005) correlated this variant with total plasma
homocysteine (tHcy) levels in AD patients and controls, demonstrating that the
Glu/Glu genotype is correlated with higher levels of tHcy, which represent a known
risk factor for AD (Seshadri et al. 2002), and its frequency was increased in AD
patients (Guidi et al. 2005). Thus, the mechanism by which this genotype contributes
to increase the risk in developing AD could be mediated by an increase of tHcy.
However, NOS-1 is the isoform most abundantly expressed in the brain. Recent
genetic analyses demonstrated that the double mutant genotype of the synonymous
C276T polymorphism in exon 29 of the NOS1 gene represents a risk factor for the
development of early onset AD (Galimberti et al. 2005), whereas the dinucleotide
polymorphism in the 30 UTR of NOS1 is not associated with AD (Liou et al. 2002).
To date, the promoter region of NOS1, located approximately 200 kb upstream of
these polymorphism, has not been investigated for susceptibility to AD. Due to this
reason and to further explore a possible association of NOS1 polymorphisms with
AD, the distribution of a functional polymorphisms and a VNTR was analyzed in a
case-control study (Galimberti et al. 2008). The functional variant considered is
located in exon 1c, which is one of the nine alternative first exons (named 1a–1i),
resulting in NOS1 transcripts with different 50 -untraslated regions (Wang et al.
1999). Three SNPs have been identified in exon 1c, but only the G-84A variant
displays a functional effect, as the A allele decreases the transcription levels by
30% in in vitro models (Saur et al. 2004). Regarding exon 1f, a VNTR poly-
morphism has been recently reported in its putative promoter region, termed NOS1
Ex1f-VNTR. This VNTR is highly polymorphic and consists of different numbers
of dinucleotides (B-Q), which, according to their bimodal distribution, have been
dichotomized in short (B-J) and long (K-Q) alleles for association studies. Both
Ex1c G-84A and Ex1f-VNTR are associated with psychosis and prefrontal func-
tioning in a population of patients with schizophrenia (Reif et al. 2006). Notably,
both Ex1c and Ex1f transcripts are found in the hippocampus and the frontal
cerebral cortex, i.e. brain regions implicated in the pathogenesis of schizophrenia
as well as AD. The presence of the short (S) allele of NOS1 Ex1f-VNTR represents
a risk factor for the development of AD. The effect is cumulative, as in S/S carriers
the risk is doubled. Most interestingly, the effect of this allele is likely to be gender
specific, as it was found in females only. In addition, the S allele was shown
to interact with the APOE*4 allele both in males and females, increasing the risk
to develop AD by more than 10 fold (Galimberti et al. 2008). Thus, NOS1 seems to
be a risk factor for AD, but only in female population. This could be explained by
a possible interaction with other genes or with additional environmental factors
present in females but not males.
3.2 Genes Influencing the Risk to Develop BPSD

in AD Patients
Serotonin (5-hydroxytryptamine, 5-HT) is involved in a range of behaviors and

psychiatric processes, including mood, aggression, impulsivity and anxiety.
Several studies have implicated 5-HT dysfunctions in the pathogenesis of
psychiatric diseases, such as depression, obsessive–compulsive disorder and
schizophrenia. Nevertheless, 5-HT play a role also in the development of BPSD in
patients with AD (Cross 1990). Postmortem studies of brain from patients with
AD showed decreased levels of 5-HT, 5-HT receptors and 5-HT transporter
(5-HTT) (Reinikaininen et al. 1990). The 5-HTT plays a central role in the fine-
tuning of 5-HT neurotransmission by determining the duration and amount of
5-HT present in the synaptic cleft. Its encoding gene, named SLC6A4, contains
several polymorphisms influencing its expression, including a functional poly-
morphism, named serotonin-transporter linked-polymorphic region (5-HTTLPR),
which is located upstream of the gene promoter and displays length variations of
14 (short type) or 16 (long type) repeats of a 20- to 23-base pair (bp) element and
a 5-HTT VNTR (5-HTTVNTR) in the second intron, that contains 9, 10 or 12
copies of a 17-bp element (Lesch et al. 1994). These polymorphisms can influence
transcriptional activity of the SLC6A4 and ultimately the level of 5-HTT
transporters available.
Several studies have found a relationship between 5-HTTLPR and VNTR in
various psychiatric conditions, including personality traits (Lotrick et al. 2001).
Regarding BPSD in patients with AD, the long variant of 5-HTTLPR has displayed
association with aggressive behaviour and psychosis ind 2 out of 5 studies (Assal
et al. 2004; Ha et al. 2005; Rocchi et al. 2003; Sukonick et al. 2001; Sweet et al.
2001). No association with 5-HTTVNTR has been shown in patients with AD and
BPSD (Assal et al. 2004; Ueki et al. 2007). Nevertheless, a significant association
was found between the presence of the 5-HTTVNTR allele 10 and BPSD or
aggressiveness (Ueki et al. 2007). Conversely, no influence of 5-HTTLPR
polymorphism on BPSD in AD was shown (Albani et al. 2009).
Recently, in a 3-year follow-up study, Angelucci et al. evaluated the association
of 5-HT receptor 5-HT2a 102T/C SNP with psychotic symptom severity and
response to treatment with atypical antipsychotics (risperidone, olanzapine and
quetiapine) in 80 patients with mild AD, showing a significant difference in the
frequency and severity of delusions, measured through the Neuropsychiatric
Inventory (NPI), with patients carrying the TT genotype most delusional during the
follow-up period. Moreover, patients carrying the T allele were resistant to
treatment with antipsychotics (Angelucci et al. 2009).
Lastly, dopamine D3 receptor (DRD3) has been investigated in AD patients, as
it is present in the limbic system, which is thought to regulate affect and cognition.
No effect of genetic variability in DRD3 on the risk to develop AD was shown.
However, a significant association was found between the presence of the DRD3
glycine allele and paranoid and delusional ideation (Sato et al. 2009).
4 Familial FTLD
Frontotemporal Lobar Degeneration is a heterogeneous disease characterized by a

strong genetic component in its aetiology as up to 40% of patients report a family
history of the disease in at least one extra family member (Snowden et al. 2002).
In 1994 an autosomal dominantly inherited form of FTD with parkinsonism was
linked to chromosome 17q21.2 (Wilhelmsen et al. 1994). Subsequently, other
familial forms of FTD were found to be linked to the same region, resulting in the
denomination ‘‘frontotemporal dementia and parkinsonism linked to chromosome
17’’ (FTDP-17) for this class of diseases.
In 1998, MAPT gene on chromosome 17q21, which encodes the microtubule
associated protein tau was described as the cause of the disease in these families
(Hutton et al. 1998; Poorkaj et al. 1998; Spillantini et al. 1998). So far, 44 different
mutations in the MAPT gene have been described in 132 families (http://molgen-
www.uia.ac.be). MAPT mutations are either non-synonimous or deletions, or silent
mutations in the coding region, or intronic mutations located close to the splice-
donor site of the intron after the alternatively spliced exon 10 (Rademakers et al.
2004). Mutations are mainly clustered in exons 9–13, except for two identified
mutations in exon 1 (Rademakers et al. 2002). As regards possible effects on
MAPT mutations, different mechanisms are involved, depending on the type and
location of the mutation. Many of them disturb the normal splicing balance,
producing altered ratios of the different isoforms. A number of mutations promote
the aggregation of tau protein, whereas others enhance tau phosphorylation
(Goedert and Jakes 2005).
However, after the discovery for MAPT as causal gene for FTDP-17, there were
still numerous families with autosomal dominant FTLD genetically linked to the
same region of chr17q21 that contains MAPT but in which no pathogenic muta-
tions had been identified, despite extensive analysis of this gene (Lendon et al.
1998; Rosso et al. 2001; van der Zee et al. 2006). The neuropathology phenotype
in these families was similar to the microvacuolar-type observed in a large pro-
portion of idiopathic FTD cases with ubiquitin immunoreactive neuronal inclu-
sions. Moreover, clinically, the disease in these families was consistent with
diagnostic criteria for FTLD (Neary et al. 1998). Sequence analysis of the whole
MAPT region failed to find a mutation and tau protein appeared normal in these
families. Moreover the minimal region containing the disease gene for this group
of families was approximately 6.2 Mb in physical distance. This region defined by
markers D17S1787 and D17S806 is particularly gene rich, containing around 180
genes. Collectively, these data strongly argued against MAPT and pointed to
another gene. Systematic candidate gene sequencing of all remaining genes within
the minimal candidate region was performed and after sequencing 80 genes,
including those prioritized on known function, the first mutation in progranulin
gene (GRN) was identified. It consists in a 4-bp insertion of CTGC between coding
nucleotides 90 and 91, causing a frameshift and premature termination in pro-
granulin (C31LfsX34) (Baker et al. 2006). These results have been contemporarily
replicated by Cruts et al. (2006), who analyzed other families with a FTLD-U
disease without MAPT pathology, finding a mutation five base pairs into the intron
following the first non coding exon of the GRN gene (IVS0 ? 5G-C). This is
predicted to prevent splicing out of the intron 0, leading the mRNA to be retained
within the nucleus and subjected to nuclear degradation (Cruts et al. 2006).
At present there is no obvious mechanistic link between the mutations in MAPT
and GRN, currently assuming that their proximity on chromosome 17 is simply a
coincidence. Progranulin is known by several different names including granulin,
acrogranin, epithelin precursor, proepithelin and prostate cancer (PC) cell derived
growth factor (He and Bateman 2003). The protein is encoded by a single gene on
chromosome 17q21, which produces a 593 amino acid, cysteine rich protein with a
predicted molecular weight of 68.5 kDa. The full-length protein is subjected to
proteolysis by elastase and this process is regulated by a secretory leukocyte
protease inhibitor (SLPI) (Zhu et al. 2002). Progranulin and the various granulin
peptides are implicated in a range of biological functions including development,
wound repair and inflammation by activating signaling cascades that control cell
cycle progression and cell motility (He and Bateman 2003). Excess progranulin
appears to promote tumor formation and hence can act as a cell survival signal.
Despite the increasing literature on the function of progranulin, its role in neuronal
function and survival remains unclear. In the human brain, GRN is expressed in
neurons but significantly is also highly expressed in activated microglia (Baker
et al. 2006), with the result that GRN expression is increased in many neurode-
generative diseases.
Since the original identification of null-mutations in FTLD in 2006, 68 different
mutations spanning most exons have been reported so far (http://www.molgen.
ua.ac.be/). Interestingly, the majority of mutations identified create functional null
alleles, causing premature termination of the GRN coding sequence. This leads to
the degradation of the mutant RNA by nonsense mediated decay, creating a null
allele (Baker et al. 2006; Cruts et al. 2006). The presence of a null mutation causes
a partial loss of functional progranulin protein, which in turn leads eventually to
neurodegeneration (haploinsufficency mechanism), although how loss of GRN
causes neuronal cell death remains unclear. Estimates of the frequency of GRN
mutations in typical FTD patient populations suggests that they account for about
5–10% of all FTD cases, although numbers vary markedly depending on the nature
of the populations considered (Cruts et al. 2006; Gass et al. 2006; Snowden et al.
2006).
Neuropathology analysis revealed that ubiquitin immunoreactive neuronal
cytoplasmatic and intranuclear inclusions were present in all cases with FTDP-17,
where pathological findings were available (Mackenzie et al. 2006). Furthermore,
soon after the identification of mutations in GRN, biochemical analyses demon-
strated that truncated and hyperphosphorylated isoforms of the TAR-DNA binding
protein (TDP-43) are major components of the ubiquitin-positive inclusions in
families with GRN mutations as well as in idiopathic FTD and a proportion of
Amyotrophic Lateral sclerosis (ALS) cases (Neumann et al. 2006). TDP43 is a

ubiquitously expressed and highly conserved nuclear protein that can act as a
transcription repressor, an activator of exon skipping or a scaffold for nuclear
bodies through interactions with survival motor neuron protein. Under patholog-
ical conditions, TDP-43 has been shown to relocate from the neuronal nucleus to
the cytoplasm, a consequence of which may be the loss of TDP-43 nuclear
functions (Neumann et al. 2006). The mechanism by which loss of progranulin
leads to TDP-43 accumulation and whether this is necessary for neurodegeneration
in this group of diseases is still to be clarified.
In conclusion, the function of progranulin in the brain is currently unclear and
why loss of this protein leads to a neurodegenerative diseases in mid-life remains
to be established, and its possible role as regulator of a repair activity in the central
nervous system, as it is well known to happen in periphery, remains a challenge for
science. The gene encoding for TDP-43, named TARDBP, has been extensively
studied and a number of mutations found in its C-terminal glycine rich region.
Unexpectedly, the clinical phenotype of carries was ALS, and aggregates made of
TDP-43 have been described in brain and spinal cord of such patients (see Pesiridis
et al. 2009 for review).
A recently published collaborative study (Yu et al. 2010) analyzed GRN in a
population of 434 patients with FTLD, including FTD, PA, SD, FTD/ALS, FTD/
Motor Neuron Disease (MND), Corticobasal Degeneration (CBD), Progressive
Supranuclear Palsy (PSP). Fifty-eight variants were identified, including 24
pathogenic variants. The frequency of GRN mutations was 6.9% of all FTLD-
spectrum cases, 21,4% of cases with a pathological diagnosis of FTLD-U, 16%
of FILD-spectrum cases with a family history of a similar neurodegenerative
disease, and 56.2 of cases of FTLD-U with a family history. Clinical information
were available for 31 GRN mutation-positive patients from 28 different families.
The most common clinical diagnosis was FTD (n = 24); 3 patients were diag-
nosed with PA, 3 with AD and 1 with CBD. The majority of GRN mutations
Introduced a premature termination codon, suggesting that their corresponding
mRNA will be degraded through nonsense mediated decay, supporting the
hypothesis that most GRN mutations create functional null allele (Yu et al.
2010).
An additional gene shown to cause familial FTLD is named CHMP2B (charged
multivescicular body protein 2B), and is part of the endosomal ESCRTIII-complex
(Skibinski et al. 2005). Four different mutations in this gene have been so far
described in 4 families (http://www.molgen.ua.ac.be/), making CHMP2B a rare
genetic cause of FTLD.
Lastly, the first evidence of linkage with chromosome 9q21-22 comes from a
study carried out in families with FTD/MND (Hosler et al. 2000). Despite the
evidence of linkage to chr9q21-22 in several additional FTD-MND families
(Le Ber et al. 2009; Morita et al. 2006; Vance et al. 2006), the gene responsible for
the disease in this locus has yet to be identified.
5 Sporadic FTLD
The best well-known risk factor for late onset SAD, Apo E4, has also been con-
sidered as a risk factor for sporadic FTLD. A number of studies suggested an
association between FTLD and APOE*4 allele (Bernardi et al. 2006; Fabre et al.
2001; Farrer et al. 1995; Gustafson et al. 1997; Helisalmi et al. 1996; Stevens et al.
1997). Other Authors however, did not replicate these data (Geschwind et al. 1998;
Riemenschneider et al. 2002; Short et al. 2002). Recent findings demonstrated an
association between the APOE*4 allele and FTLD in males, but not females
(Srinivasan et al. 2006), possibly explaining the discrepancies previously reported.
An increased frequency of the APOE*4 allele was described in patients with SD
compared to those with FTD and PA (Short et al. 2002).
Concerning the APOE*2 allele in the development of FTLD, heterogeneous data
have been obtained in different populations. Bernardi et al. (2006) showed a pro-
tective effect of this allele towards FTLD, whereas other Authors failed to do so
(Engelborghs et al. 2003; Riemenschneider et al. 2002; Short et al. 2002; Srinivasan
et al. 2006). Despite these results, a recent meta-analysis comprising a total of 364
FTD patients and 2671 controls demonstrated an increased susceptibility to FTD in
APOE*2 carriers (Verpillat et al. 2002).
Besides pathogenic mutations, several polymorphisms have been reported to
date, both in MAPT and GRN. An association between Progressive Supranuclear
Palsy (PSP) and a dinucleotide repeat polymorphism in the intron between MAPT
exons 9 and 10 was described in 1997 (Conrad et al. 1997). The alleles at this locus
carry 11–15 repeats. Subsequently, two common MAPT haplotypes, named H1 and
H2, were identified (Baker et al. 1999). They differ in nucleotide sequence and
intron size, but are identical at the amino acid level. Homozygosity of the more
common allele H1 predisposes to PSP and Corticobasal Degeneration (CBD), but
not to AD or Pick Disease (Baker et al. 1999; Di Maria et al. 2000).
Regarding GRN, an association of a SNP located in the promoter and an
increased risk to develop FTLD in patients who did not carry causal mutations has
recent been demonstrated (Galimberti et al. 2010).
In addition, a known polymorphism in MCP-1 (A-2518G) has been shown to
exert a protective effects towards the development of FTLD (Galimberti et al.
2009), whereas NOS3 G894T (Glu298Asp) and NOS1 C276T SNPs likely
Increases the risk to develop FTLD (Venturelli et al. 2008, 2009).
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Index
1q21.1 / 15q11.2 / 15q13.3, 432 Autism, 12, 289, 345, 354

22q11, 292, 293 Autism spectrum disorders, 395, 401
22q11.2, 431
5HT, 267
5-HTT knockout mice, 62 B
B. glutamate, 264
Backcross, 32
A Background strain, 275
Acethylcholine, 268 Behavior, 441, 442, 444, 451, 456
ACh Transporter, 269 Behavioral despair, 63
Acoustic startle, 13 Behavioural and psychological, 616
Advanced Intercross Lines, 36 Behavioural disturbances, 615, 616
Aggression, 76, 368–370 Bioinformatics, 328
AKT1, 218, 261 Biomarker, 255
Alzheimer’s disease (AD), 10, 615, 616 BMI, 395, 412–414
Alzheimer’s, 288 Brain-derived neurotrophic factor, 66
AMPA, 67, 264, 435 Bupropion, 538
Amphetamine, 538
Amygdala, 65, 364–370
Amyloid-b precursor protein (APP), 11 C
Androgen receptor, 364, 370 CA1, 66
Angiotensin/Renin, 270 Calcium Signaling, 282
Animal models, 68, 140–142, 157, 171 CaMKII, 435
Antisense oligonucleotides, 6 Candidate gene studies, 539
Anxiety, 11, 12, 64, 140–145, 148, 157–171, Candidate Genes, 296
364–367 Catechol-O-Methyltransferase, 217
Apolipoprotein E (ApoE), 11 Chromosome Substitution Strains, 33
Approaches, 276 Chronic social defeat, 67
Arabidopsis, 6 CNV, 464, 466, 471, 478
Association, 395, 400, 402, 403, 405–409, Cocaine, 538
411–415 Cognition, 189–193, 195–197, 199–201, 203,
Attention deficit/hyperactivity disorder, 395, 205, 580, 584, 582–585, 588, 590, 592,
396, 399, 405 595–598, 600, 601, 604, 605
Attention-deficit/hyperactivity disorder, 345, Cognitive constructs, 255
355 Collaborative Cross, 39

DOI: 10.1007/978-3-642-27859-4
634 Index
C (cont.) Frontal cortex, 67

COMT, 259, 297, 431 Frontotemporal lobar degeneration (FTLD),
Congenic Strains, 35 616, 617
Consomic Lines, 33 Fruit fly, 4
Copy number variants (CNVs), 432
Copy number variations, 223, 228, 345
Cre/loxP, 8 G
CRF, 271, 295 GABA, 106
Cross-species, 255 GABA-A receptors, 66
c-secretase, 11 GABA-glutamate, 278
CSF2RA, 434 Gal4-UAS, 8
CS-US association, 15 Gating deficit, 253
Gene 9 environment interaction, 209, 210, 232
Gene expression, 6
D Gene regulation, 504, 524
Dementia, 616, 617, 623 Gene, 66
Depression, 11, 60, 140–148, 151–157, 159, Gene-environment interaction (GxE), 464,
160, 162–164, 166 484, 486, 487
Development, 63 Gene–environment, 256
Developmental disorders, 433 Genetic component, 430
DISC1, 257, 297, 298, 430 Genetic polymorphism, 504, 516, 524
Disrupted-in-schizophrenia-1 (DISC-1), 223 Genetic, 276, 395–397, 399, 401, 404–415
DNA microarrays, 6 Genetics, 297, 580–582, 588, 589, 591, 593,
Dopamine Receptors, 215 599, 605
Dopamine Transporter, 216, 373 Genome-wide association studies, 539
Dopamine, 263, 364, 371–373, 374, 376 Genomic, 441, 447, 450, 580, 581, 594,
Down syndrome, 293 599, 606
Drug of Abuse, 230 Glucose, 284
Drug targets, 256 Glutamate transporter, 266
D-Serine, 221 Glutamate, 189, 190, 195, 196, 198, 199–206,
dsRNAs, 7 219–223, 265
Dysbindin, 225 Glutamatergic, 189, 190, 195, 198, 199,
201, 207
Glycine, 220, 266
E Glycoprotein, 286
Elevated plus maze, 65 Growth factors, 279
Embryonic stem cells, 8 GSK-3, 218
Emotionality, 189, 191–193, 195, 197, 199, GWAS, 464, 466, 467, 470–476, 479, 483
201–203, 205, 207
Endocannabinoid, 364, 373
Endophenotype, 253, 302 H
ENU, 43 Haplotype association mapping, 41
Environment, 66 Hearing loss, 275
Ethyl-nitroso-urea (ENU), 9 Hearing, 292
Heatshock, 8
Heritable, 253
F Heterogeneous Stocks, 37
Fatty acide amide hydrolase, 374, 375 Hippocampus, 16, 65
Fear, 63 Homologous recombination in embryonic
Flat worm, 4 stem cells, 8
FMR1, 290, 291 Hormones, 287
Forward genetic screens, 277 Huntington’s, 291
Forward genetic, 6, 27 Hypothalamic-Pituitary-Adrenal
Fragile X-syndrome, 290 Axis, 364
Index 635
I Neurodevelopmental disorders, 345

Immune activation, 283 Neurodevelopmental, 285, 294
Impulsivity, 364, 368, 371–373, 375, Neurogranin (NRGN) / TCF4 / HLA, 435
376, 381 Neuropeptide S, 272
Inbred Strains, 27 Neuropeptide Y, 271
Influenza infection, 283 Neuropeptides, 109, 263, 270, 295
Intercross, 32 Neurotensin, 270
Inter-individual variability, 538 Neurotransmitters, 263
Intermediate phenotype, 464, 483, 487 Nicotinic, 268
NMDA, 219, 222, 265, 278, 435
NMDAR, 264
K NMDAR Signalling Complex, 222
Knock out technology, 8 nNOS, 284
Nocturnal enuresis, 395, 401, 411, 412
NRG1, 258, 297
L NRXN1, 432
Life stress events, 66 Nucleotide polymorphisms, 539
Linkage studies / candidate gene / genome
wide association studies, 430
Linkage, 395, 399, 401–404, 406, 409, 410, O
411, 413 Obesity, 395, 396, 401, 412–415
Obsessive compulsive disorders, 11
Open field, 65
M Orphan receptors, 263, 273
Maternal care, 63 Outbred Mice, 39
Metabotropic Glutamate Receptors, 222 Oxidative stress, 285
Methamphetamine, 538 Oxytocin, 271
Methodological Considerations, 275
Methylphenidate, 538
mGLU, 264 P
mGluRs, 291 PACAP, 272
microRNA, 7 Parkinson’s Disease, 10
Mineralocorticoid receptor, 362, 364, Pathophysiology, 277
377, 378 PDE4, 279
Model, 301, 302 PDE4B, 295
Monoaminergic signaling, 504, 525 Personalized genomics, 332
Mood disorders, 60, 504 Pharmacogenetics, 464, 478, 484
Morpholinos, 6 Pharmacological, 295
Mouse Phenome Project, 41 Phenotype, 210–214, 217, 219, 221–228,
Mouse, 61 230–239, 298, 299
Muscarinic, 268 Phenotypes, 580–583, 585, 589, 592–594, 597,
Mutagenesis, 43, 298 598, 601, 603–606
Mutant model, 210–214, 223, 229, 230, 234, Phobias, 11
238, 239 Pitt-Hopkins-Syndrome, 435
Mutant mouse models, 254 Potassium channel signaling, 283
Mylein, 284 PPI, 302
Prader-Willi/Angelman syndrome, 433
Prepulse inhibition, 13, 252
N Presenilins, 11
Neural circuitry, 255 Presynaptic proteins, 282, 295
Neuregulin, 226, 258 Protein kinase, 280
Neurexin, 260 Psychiatric symptoms, 254
636 Index
P (cont.) Stimulant drugs, 538

Psychosocial stress, 65 Stress, 11, 189–207, 364, 376–382, 441, 446,
Psychotic illness, 210, 211 449, 452, 454–457
Stress exposure, 60
Stress risk factor, 66
Q Stressed, 191, 192, 194
QTL Mapping, 28 Stressful, 190, 199
QTL, 27, 298, 299 Stressor, 190, 191, 194, 203
Quantitative reverse transcriptase polymerase Structural genetic variants, 430
chain reaction (q-RT-PCR), 6 Susceptibility gene, 210, 233, 238, 262
Synaptic proteins, 281
R
Recombinant Inbred Lines, 29 T
Reelin, 258 Tau, 10
Relaxin, 273 Tauopathies, 10
Retroviruses, 7 TBX1 / GNB1L, 431
Reverse genetic, 6 TCF4, 260
Reward, 364, 371–376, 379, 381, 382 Testosterone, 368–370
RNA-interference, or RNAi, 7 Tetracycline transactivator, 8
Tetraploidy, 9
Tilling, 6
S Trace amines, 274
Schizophrenia, 12, 210–223, 234, 237, 238, Trait, 81
239, 345, 354, 429 Transgenic technologies, 7
Screen, 255 Translational, 255
Sea slug, 4 Transposons, 7
Selective breeding, 140, 141, 143, 161 Twin studies, 538
Sensorimotor gating, 252 Tyrosine hydroxylase, 10
Sequencing technologies, 322
Serotonin transporter, 60, 504, 505, 507, 512,
520, 522, 525 V
Serotonin turnover, 67 Variable number tandem repeat, 504, 505,
Serotonin, 66, 81, 84, 267, 364–367, 441, 446, 507, 539
449, 454, 457 Vasopressin, 271
Single Ventral striatum, 364, 371
SMARCA2, 260 VIVO-morpholino, 7
SNPs, 296 Vulnerability, 257, 296
SP4, 301
Spatial learning, 16
Spectrum disorders, 12 Z
Spine, 284 Zinc finger nucleases, 8
Startle magnitude, 275 ZNF804A, 434
Startle, 252
States, 81

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Behavioral Neurogenetics

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Current Topics in Behavioral Neurosciences

For further volumes:

Current Topics in Behavioral Neurosciences provides critical and comprehensive

ISSN 1866-3370 ISSN 1866-3389 (electronic)

Library of Congress Control Number: 2012936979

Ó Springer-Verlag Berlin Heidelberg 2012

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

These chapters, either individually or as a whole, provide a broad overview of

Cork, Ireland John F. Cryan

Part I Preclinical Behavioral Genetics

Using Zebrafish to Unravel the Genetics of Complex

Forward Genetic Approaches to Understanding

Mouse Models of the 5-HTTLPR 3 Stress Risk Factor

Behavioral and Pharmacogenetics of Aggressive Behavior . . . . . . . . . 73

Selectively Bred Rodents as Models of Depression and Anxiety . . . . . . 139

Stress-Induced Deficits in Cognition and Emotionality:

Susceptibility Genes for Schizophrenia: Mutant Models,

Genetic Models of Sensorimotor Gating: Relevance

Part II Clinical Behavioral Genetics

Applications of Second Generation Sequencing Technologies

Rare Genomic Deletions and Duplications

Toward a Mechanistic Understanding of How Variability

Behavioural Genetics of Childhood Disorders. . . . . . . . . . . . . . . . . . . 395

Schizophrenia Genes: On the Matter of Their Convergence . . . . . . . . 429

Gene 3 Environment Interaction Models

Behavioral Genetics of Affective and Anxiety Disorders . . . . . . . . . . . 463

Behavioural Genetics of the Serotonin Transporter . . . . . . . . . . . . . . 503

Genetic Factors Modulating the Response to Stimulant

The Cognitive Genetics of Neuropsychiatric Disorders . . . . . . . . . . . . 579

Behavioral Genetics of Neurodegenerative Disorders . . . . . . . . . . . . . 615

Abstract The zebrafish has been prominently utilized in developmental biology

Curr Topics Behav Neurosci (2012) 12: 3–24 3

1 Zebrafish, a Novel Research Tool with Some Advantages............................................. 4

1 Zebrafish, a Novel Research Tool with Some Advantages

2 Zebrafish, the Favorite of Geneticists

3 Reverse Genetic Tools

In addition to constitutive transgene expression, inducible expression systems

4 Forward Genetic Tools

Unlike reverse genetic approaches, forward genetics has provided decades of

Parkinson’s disease is the second most prevalent human neurodegenerative dis-

6 Tauopathy and Alzheimer’s Disease

Another class of neurodegenerative diseases is the tauopathies. These diseases are

7 The Anxiety ‘‘Cluster’’

8 Autism Spectrum Disorders

Schizophrenia is a neurodevelopmental disorder that often manifests first during

10 Expanding Our Horizon: The Need for Sophisticated

11 Learning and Memory

demonstrated that computer animated images of zebrafish can serve as a positive

stimulus/no-stimulus schedules (random versus fixed ratio, increasing stringency

12 Fear and Anxiety

Acknowledgments Supported by NIH/NIAAA (USA) and NSERC (Canada).

Amsterdam A, Hopkins N (2006) Mutagenesis strategies in zebrafish for identifying genes

Lisa M. Tarantino and Amy F. Eisener-Dorman

Keywords Mouse Genetics Behavior Mapping QTL Mutagenesis

1 The Importance of Phenotype............................................................................................. 27

L. M. Tarantino (&) A. F. Eisener-Dorman

Curr Topics Behav Neurosci (2012) 12: 25–58 25

4.1 Recombinant Inbred Lines ......................................................................................... 29

function. However, studying induced mutants exclusively fails to account for