Subjects

One hundred and thirty-eight male Wistar rats (Harlan, Indianapolis, IL) weighing 200–225g at the start of the experiment were individually housed in square plastic hanging cages (8 × 9 × 8 cm). The animals were housed in a temperature and humidity controlled room with a 14:10 light/dark cycle, with water available ad libitum. Animals were food restricted throughout the experiment to maintain at least 90% of their free feeding body weight. All procedures were approved by the University of Michigan Committee on the Use and Care of Animals (UCUCA).

Apparatus

Behavioral training, testing, and drug administration took place in 16 operant chambers measuring 22 × 18 × 13 cm (Med Associates, St. Albans, VT, USA) located inside larger sound-attenuating chambers. For the sustained attention task, each operant chamber was equipped with a panel consisting of one light (2.8 W), two retractable levers, and a food pellet dispenser, all located on the front wall. A house light (2.8 W) was located close to the ceiling of the back wall. The floor of the chamber consisted of 19 stainless steel rods (4 mm in diameter) spaced 1.5 cm apart (center-to-center). For the drug self-administration procedure, each operant chamber had two nose poke holes equipped with cue lights. A tone (2900 Hz) was also available inside of the chamber. The floor of the chamber consisted of a solid piece of black plastic placed over the stainless steel rods. Med-PC for Windows software (v. 1.1, Med Associates) controlled all signal presentation, lever operation, pellet delivery, drug delivery, tone presentation and data collection via a Pentium PC.

Sustained Attention Task Training

For a detailed description of sustained attention procedures see McGaughy & Sarter (1995). The task required four phases of training. Phase I. All animals were shaped to lever press for food (45-mg banana flavored pellet, Bioserv, Frenchtown, NJ) utilizing a fixed ratio 1 (FR1) schedule such that one lever press resulted in the delivery of one food pellet. During this stage both of the levers were extended into the box at the beginning of each session and remained extended for the duration of the session. Training continued until the animals had at least three days of 120 reinforced lever presses. Phase II. After reaching this criterion, rats were then shaped to discriminate between signals (illumination of the central signal light for a period of 1 s) and non-signals (no illumination). For this training phase the start of each individual trial was indicated by the extension of both levers into the box. Response on one lever was reinforced if it followed a signal, while response on the other lever was reinforced if it followed a non-signal. The assignment of levers was counter-balanced across subjects to minimize side-bias. To increase the salience of the signal light and facilitate acquisition of the appropriate response, the house light was not illuminated during this training step. Signal and non-signal trials were presented pseudo-randomly with a mean intertrial interval (ITI) of 9±3 s. Four responses were possible during this second phase of training: hit, defined as the correct response to a signal; miss, the incorrect response to a signal; correct rejection, the correct response to a non-signal; and false alarm, the erroneous response to a non-signal (i.e., a claim for a signal following a non-signal; see Figure 1 for schematic). If the animal responded incorrectly, the previous trial was repeated up to three times. If a rat continued to respond incorrectly, it received a forced-choice trial in which only the correct lever was extended into the box while the signal was presented. Forced-choice trials also served to impede the generation of a side bias. Trials in which the rats failed to emit a response within 4 s of lever insertions were counted as omissions. Animals reached criterion for this portion of training when they responded correctly to 65% of the signal and non-signal trials for five consecutive days. Phase III. For the third stage of training the 1.0 s signal was replaced with three different signal lengths of 500, 50, and 25 ms, equally distributed across the session. Following correct responding to 65% of the 500 ms and non-signal trials, the final stage of training began. Phase IV. During the final stage of training, the house light was illuminated; this is a critical step in the acquisition of the sustained attention task as it forces animals to limit competitive behaviors and to closely monitor the intelligence panel in order to detect signal events. The final task consisted of three blocks of 54 trials, with nine trials of each signal length and 27 non-signal trials. The signal and non-signal trials were pseudo-randomized. Animals reached criterion when their performance became asymptotic at a level of at least 70% hits on the longest (500 ms) signal length trials, 70% correct rejections on the non-signal trials, and less than 30% omissions.

Surgical Procedures

Individual animals reached performance criterion on the sustained attention task at different rates, and therefore cocaine self-administration and subsequent attention testing occurred at different times for each animal. After reaching criterion on the attention task, animals were anaesthetized with ketamine and xylazine anesthesia (77:1.5 mg/ml, intraperitoneal [IP], at 0.1 mL/100g of body weight) and a silicone catheter (Plastics One, Roanoke, VA) was inserted into the right jugular vein and passed subcutaneously to exit from the animals’ back (see Caine, et al, 1993). Animals were allowed to recover from surgery for a minimum of 3 days prior to drug administration. Catheters were flushed daily with 0.1 mL of gentamicin (50 mg/kg, in 0.9% sterile bacteriostatic saline).

Cocaine Self-Administration

Rats were transported from their home cage to an operant chamber 6 days a week for 4 weeks, where they were allowed to nose-poke for cocaine (0.4 mg/kg/infusion in 50 µL of saline administered over 1.6 s) on a continuous reinforcement schedule (FR1) with a time-out of 20 s. A training session commenced with the illumination of the “active” nose-poke hole stimulus light. Responding in this hole resulted in drug delivery. Responding in the other nose-poke hole, designated inactive, had no consequences. Rats were initially trained during daily one hour sessions for a total of six days. Animals that did not acquire stable self-administration behavior (at least 5 infusions each day for 3 consecutive days) were removed from the study. After the initial 6-day training period animals were divided into two groups: a long access group (LgA) and a short access group (ShA). These groups were balanced according to both the amount of drug they administered during the first week of training and their performance during attention training. Animals in the ShA group continued to receive one-hour test sessions, whereas animals in the LgA group received six-hour test sessions for an additional 16–17 sessions. A third group of rats (no-drug control group) received sham surgery and were transported each day to a novel test room where they were placed into Plexiglas chambers, similar to the operant chambers.

Sustained Attention Testing

One day following four weeks of drug self-administration (or transport in the no-drug control groups), animals were retested on the sustained attention task (n=12–16). Animals were tested again two weeks after the last self-administration session, followed by daily retraining for 1 week following the test on day 14.

Sustained Attention Testing

Following the completion of self-administration, animals in the No Drug, ShA, and LgA groups were divided into two more groups. One group (n=5–6) was tested on the attention task 1 day following the last self-administration session and another independent group (n=6–8) was tested 30 days following the last self-administration session.

Tissue Collection

Three days following their respective sustained attention testing session (at days 4 & 33 following the last self-administration session) all animals were decapitated and their brains removed and frozen in isopentane (−40 to −50°C) on dry ice. The brains were then stored at −80°C. Coronal brain sections (10 µm) were cut on a cryostat at 200 µm intervals, thaw mounted on Superfrost/Plus slides (4 sections per slide, Fisher Scientific, Pittsburgh, PA) and stored at −80°C until further processing.

In Situ Hybridization

For detailed methodology of the in situ hybridization techniques used see Kabbaj et al. (2000). Following fixation, sections were hybridized with ³⁵S-labeled cRNA probes produced using standard in vitro transcription methodology. Prefrontal cortex sections were processed for in situ hybridization of dopamine D1 and D2 receptor mRNA (480-mer). The probes were diluted in hybridization buffer and slides were coverslipped and incubated overnight at 55°C. Following rinses and dehydration, slides were exposed to x-ray film (Kodak Biomax-MR, Eastman Kodak, Rochester, NY) for 2 weeks. The specificity of the hybridization signal was confirmed with sense probe controls (data not shown).

Quantification

Autoradiographs were digitized using a Scanmaker 1000XL scanner (Microtek, Carson, CA) and the magnitude of the signal of the radioactive probes was determined using Scion Image (Scion Corporation, Frederick, MD). A macro was used to automatically determine signal above background in each brain region (written by Dr. S. Campeau, University of Colorado, Boulder, CO). A background value was obtained from a white matter region such as the corpus callosum in each section and only values exceeding the background by 3.5 times the standard deviation were included in the analysis. Results are expressed as the mean integrated density of signal pixels divided by the total number of pixels in the selected region. The person quantifying was blind to group assignments. Anatomical regions were identified and subdivided for densitometric analysis according to the stereotaxic atlas of Paxinos and Watson (Paxinos and Watson, 1998). More specifically, the medial prefrontal cortex included the cingulate, prelimbic, and infralimbic subregions of the frontal cortex at 2.7mm anterior from bregma, as defined by Paxinos and Watson (1998). The orbital frontal cortex included the medial, lateral, and ventral subregions, at 4.2mm anterior to bregma, as defined by Paxinos and Watson (1999).

Tissue Collection and Sample Preparation

Three or thirty days following their final self-administration session animals were decapitated and their brains removed. The entire prefrontal cortex was dissected, fast frozen on dry ice and kept at −80°C until further processing. Frozen brain tissue was homogenized (1:10, wt/vol) in cold 50 mM Tris-HCl buffer, pH 6.8, containing 2% SDS, 1 mM EDTA, and various protease inhibitors as previously reported (Garcia-Fuster et al., 2007). Aliquots of total homogenate were mixed with equal volumes of electrophoresis loading buffer, denatured and stored at −20°C until use. Protein concentrations were determined by the BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL, USA).

Western Blot Analysis

For western blot analysis, 33 µg of each protein sample (determined to be within the linear range for immunobloting of D2DR) was separated by electrophoresis on 10% acrylamide/bisacrylamide minigels and transferred electrophoretically to nitrocellulose membranes. The membranes were subsequently blocked for 1 hour at room temperature in phosphate buffered saline (PBS, in mM: 137 NaCl, 2.68 KCl, 10.14 Na₂HPO₄, 1.76 KH₂PO₄) containing 0.2% Tween-20, 0.5% bovine serum albumin (BSA), and 5% non-fat dry milk. Incubation with the primary antibodies (mouse anti-dopamine D2 receptor, 1:400 dilution, Santa Cruz Biotechnology, Inc., Santa Cruz, CA; mouse anti-dopamine D1 receptor, 1:1000 dilution, Santa Cruz Biotechnology Inc, Santa Cruz; mouse anti-β-actin, 1:10,000 dilution, Sigma-Adrich, St. Louis, MO) was performed overnight at 4°C. After extensive washing in PBS the membranes were incubated with the secondary antibody, horseradish peroxidase-linked anti-mouse IgG, (1:5,000 dilution) in blocking solution at room temperature for 1 h. Immunoreactivity of target proteins was detected with the ECL Western Blot Detection system (Amersham International, Buckinghamshire, UK) and visualized by exposure to Hyperfilm ECL film (Amersham) for 60 s to 60 min. The autoradiograms were then quantified by densitometric scanning (IOD) using Image J software. Each protein sample was run on 3–4 gels and an average value for each animal was calculated. The amount of target protein in brain samples of treated rats was compared to an internal control sample in the same gel composed of prefrontal cortices from 3 control rats that had not received any treatment. This internal control was run 3 times on each gel and an average was calculated for comparison with other samples on that gel. Finally, percent changes in immunoreactivity with respect to internal controls (100%) were calculated for each sample in each of the various gels. In all the experiments the content of b-actin, a cytoskeletal protein not altered in brain by cocaine treatments (Imam, et al, 2005), was quantified as a loading control.

Cocaine Self-Administration

The number of infusions for the first hour of each session and for the entirety of the session was determined for each day of self-administration. Two-way repeated measures ANOVAs were performed with escalation session (1–16) as the repeated variable, drug group (ShA, LgA) as the independent variable and first hour or total infusions as the dependent variable. For all analyses, alpha was set at 0.05.

Sustained Attention

For each sustained attention session, the number of hits, misses, correct rejections, false alarms, and omissions was calculated. To examine each animal’s accuracy on the task, the relative number of hits [hits/(hits + misses)] for each signal length, and the relative number of correct rejections [correct rejections/(correct rejections + false alarms)] were calculated. In addition the vigilance index (VI), an overall measure of sustained attention performance, was calculated using the relative number of hits (h) and false alarms (fa) with the following formula: VI= (h-fa) / [2(h+fa)−(h+fa)²]. This index is similar to the sensitivity index (Frey and Colliver, 1973) except that omitted trials are excluded from the calculation. The VI provides a sensitive measure of overall performance, taking into account both trial types; VI varies between 1 and −1, with 1 indicating that all responses were hits and correct rejections, 0 that responses were randomly distributed over the four response types (hits, misses, false alarms, correct rejections), and −1 that all responses were misses and false alarms. Performance on each of the trial types (signal vs. non-signal) was also examined to reveal the specific responses contributing to the overall deficits. Two-way ANOVAs were performed with withdrawal day (1 or 30) and drug group (ND, ShA, LgA) as the independent variables and VI or the relative number of hits or correct rejections as the dependent variables. When significant main effects or interactions were revealed, individual group comparisons were made using either additional ANOVAs, or Fisher’s post-hoc tests.

Gene Expression

The levels of mRNA in the experimental groups were expressed as a percent of the mean value for the control group. Group differences in gene expression were assessed using two-way ANOVAs with withdrawal time (4 or 33 days) and drug group (ND, ShA, LgA) as the independent variables and percent of control mRNA levels as the dependent variable. Fisher’s post-hoc comparisons were conducted when main effects or interactions were present.

Western Blot Analysis

The levels of D1 and D2 receptor protein were expressed as a percent of internal control values for each individual gel (see above). Group differences in protein expression were assessed using a two-way ANOVA with withdrawal time (3 or 30 days) and drug group (ND, ShA, LgA) as the independent variables and percent of control protein levels as the dependent variable.

Experiment I: Within-Subjects Study

Experiment II. Between-subjects study

Experiment III. Dopamine Receptor Protein in Medial Prefrontal Cortex

Effects of past experience with self-administered cocaine on cognitive function

The day after the discontinuation of cocaine self-administration, during acute withdrawal, animals that had extended access to self-administered cocaine (LgA) had a profound deficit on the sustained attention task, as indicated by a large decrease in the vigilance index (VI), and by impairments on both signal and non-signal trials. In contrast, animals that had limited access to cocaine (SgA) showed no deficits. With time (within 14 days) performance on signal trials recovered in the LgA group. However, the deficit on non-signal trials persisted for at least 30 days. Experimenter-administered cocaine has been reported to produce persistent cognitive deficits in both rats (Schoenbaum, et al, 2004; Burke, et al, 2006) and monkeys (Jentsch, et al, 2002), but other than the present study, and a recent study by Calu and colleagues (2007) showing persistent effects of self-administered cocaine on reversal learning, most studies utilizing cocaine self-administration procedures have not found evidence of persistent cognitive deficits.

It is not clear why evidence of persistent deficits in cognitive function following cocaine self-administration have been elusive. However, most previous studies involved limited access procedures and it is possible that these do not produce some of the changes in brain and behavior characteristic of addiction. Indeed, accumulating evidence suggests that extended access to cocaine has a greater likelihood of producing a number of symptoms of addiction, relative to limited access, one of which is the tendency to escalate drug intake (Ahmed and Koob, 1998; Paterson and Markou, 2003; Deroche-Gamonet, et al, 2004; Vanderschuren and Everitt, 2004; Ferrario, et al, 2005). However, in one previous study rats were allowed to self-administer a maximum of 150 injections of cocaine over 8 hours and these animals achieved a level of intake comparable to the LgA group here, but did not show persistent deficits on a 5-choice serial reaction time task (2005a). There are at least two possible reasons for this. One, the self-administration procedure used by Dalley et al. (2005a) may not have resulted in an escalation of drug intake or other changes in brain and behavior characteristic of addiction. However, a more likely reason is that in the 5-choice serial reaction time task all trials are signal trials, in that animals are required to report the location of a brief visual stimulus. We also saw only a transient deficit on signal trials, and in this sense the results reported here are consistent with those reported by Dalley and his colleagues (2005a; 2005b).

In the present study there was a persistent deficit only on non-signal trials, and therefore, it is important to consider how the cognitive demands of the task used here differ as a function of trial type, and what this might tell us about the nature of the deficit. First, it is important to emphasize that there were no group differences in omissions or response latencies in any of the experiments, suggesting that the animals were equally motivated to perform and did not suffer from obvious sensorimotor deficits. In contrast, in studies using the 5-choice serial reaction time task drug-treated animals did show a significant increase in omissions and response latencies, complicating interpretation of the nature of their deficit (2005a; 2005b). In addition, after the initial withdrawal period LgA animals’ signal trial performance was normal suggesting that the deficits are not attributable to a failure to retrieve the rules governing the responses in the two types of trials. Nevertheless, on non-signal trials the LgA animals generated an abnormally high number of false alarms, reporting a signal had occurred when it had not. Although more work is required to determine the exact nature of this deficit, the behavioral and cognitive operations underlying performance during non-signal trials certainly differ from those governing signal trials, and/or tasks that involve exclusively signal trials. On signal trials the correct response is prompted by the presence of a visual stimulus and animals typically select the correct lever side and await lever insertion to respond. On non-signal trials, however, animals must first probe their memory as to whether or not a signal occurred before initiating an action. This may not only be more demanding in terms of taxing memory, but it also represents a more ambiguous situation, as the decision that a signal did not occur would be expected to be associated with high levels of uncertainty and therefore a reduced response criterion. Finally, performance on this task also taxes cognitive flexibility, which is required to shift constantly between the processing of the conflicting response rules governing the unpredictable occurrence of trials with or without signal events. Therefore, we hypothesize that prior cocaine use may have produced a persistent impairment in cognitive flexibility, which is required to respond correctly in trials involving high levels of uncertainty about signal status (see also Yu & Dayan 2005), while leaving the ability to respond correctly to a signal intact.

Effects of past experience with self-administered cocaine on brain dopamine

Perhaps the most striking finding in the present study was that extended (but not limited) access to self-administered cocaine decreased dopamine D2 (but not D1) receptor mRNA expression in both the medial and orbital prefrontal cortices and this effect persisted for at least 34 days after the last self-administration session. Furthermore, there was a small but statistically significant decrease in levels of D2 receptor protein in the prefrontal cortex of LgA but not ShA animals at both points in time after the last exposure to cocaine. There has been relatively little research on the long-term effects of cocaine on D2 receptors in the frontal cortex. However, Nogueira et al. (2006) recently reported that experimenter-administered cocaine produces a persistent (2–4 weeks) decrease in, “D2-mediated regulation of cortical excitability” (p. 12308), which may be related to the alterations in D2 receptor mRNA and protein described here, and to the well-documented “hypofrontality” seen in cocaine addicts (Bolla, et al, 2003; Bolla, et al, 2004) and in animals treated repeatedly with cocaine (Sun and Rebec, 2005; Beveridge, et al, 2006; Sun and Rebec, 2006). The decreases in D2 receptor mRNA in the orbitofrontal cortex are particularly relevant in light of data demonstrating persistent deficits in reversal learning following cocaine administration, a task thought to be dependent on the orbitofrontal cortex (Schoenbaum, et al, 2004; Calu, et al, 2007).

These data suggest, therefore, that self-administered cocaine is capable of producing a marked and persistent dysregulation of dopamine function in the prefrontal cortex, which may represent another manifestation of marked frontal cortical dysfunction associated with cocaine addiction. Given that this effect was only seen in animals that received extended access to cocaine it is possible that dysregulation of dopamine D2 receptor signaling contributes to the cognitive deficit, which was also only seen in the LgA group. The integrity of D2 receptor function is closely associated with the ability to manipulate acutely relevant information in prefrontal regions (Wang et al. 2004) and inhibition of D2 receptor activation in both humans and animals, either by antagonist administration or genetic manipulation, results in a decrease in the acquisition of working memory task rules (Glickstein, et al, 2005), attentional set-shifting (Tost, et al, 2006), and spatial working memory (Von Huben, et al, 2006; Rinaldi, et al, 2007). This is consistent with the idea that cocaine impaired cognitive flexibility primarily by disrupting prefrontal D2 receptor function. Finally, we found no effect of cocaine self-administration experience on D1 mRNA expression in the prefrontal cortex, consistent with the recent report that experimenter-administered cocaine has no effect on D1 receptor sensitivity in this region (Nogueira, et al, 2006).

Although the decrease in D2 mRNA in medial prefrontal cortex was large (~70%), the decrease in protein was much more modest (~10%). Discrepancies between mRNA and protein levels are not uncommon (Mansour, et al, 1990; Mansour, et al, 1991) and may be due to a number of different factors. For example, D2 receptor mRNA is localized primarily in the cell bodies of prefrontal neurons but D2 receptor protein is present in both presynaptic and postsynaptic elements, and therefore the population of neurons sampled by the two measures may be quite different. More research is needed to determine if the decreases seen in the current study are primarily due to changes in a specific cell type. It is also possible that rather than reflecting a cell type specificity, the dissociation between mRNA and protein is due to alterations in the translation of mRNA into protein. Nevertheless, despite the difference in the magnitude of the effect of cocaine on mRNA versus protein, both were decreased, indicating a potential persistent dysregulation of D2 function in the prefrontal cortex of animals that had extended access to self-administered cocaine.