MATERIALS AND METHODS

Materials. Long–Evans rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Phospho-MAPK and MAPK antibodies were obtained from New England Biolabs (Beverly, MA). Anti-active c-Jun N-terminal protein kinase (JNK) antibodies and anti-rabbit HRP-conjugated secondary antibodies were purchased from Promega (Madison, WI) and Sigma (St. Louis, MO), respectively. Cy3-conjugated streptavidin was obtained from Jackson Immunochemicals (West Grove, PA), PD098059 was purchased from Biomol (Plymouth Meeting, PA), and SB203580 was purchased from Calbiochem (San Diego, CA).

Intrahippocampal infusion and drug preparation. All protocols involving the use of animals are in compliance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Male Long–Evans rats (250–300 gm) were anesthetized with 400 mg/kg chloral hydrate and implanted with bilateral guide cannulas aimed at the dorsal hippocampus (anteroposterior, −3.3 mm; lateral, ± 2.0 mm from bregma; and ventral, −1.5 mm from the dura). The rats were allowed to recover from surgery in their home cages for 10–12 d. During infusion, the injection cannulas extended 1.5 mm beyond the tips of the guides, yielding a total depth of 3.0 mm below the dura. Stock solutions of PD098059 was prepared by dissolving in DMSO. Before infusion, PD098059 was diluted in sterile saline yielding either a 2 mg/ml solution in 40% DMSO or a 0.2 mg/ml solution in 4% DMSO. SB203580 was prepared by dissolving in DMSO and was diluted in sterile saline to a concentration of 0.75 mg/ml in 10% DMSO. Vehicle-treated animals were infused with saline and an amount of DMSO corresponding to that used to dissolve the drug (4 or 40% DMSO for 0.2 and 2.0 μg of PD098059, respectively, and 10% DMSO for SB203580). All injections (1 μl/side of either drug or vehicle) were performed in freely moving animals at a rate of 0.25 μl/min using a dual syringe infusion pump (Stoelting).

Behavioral training. All behavioral training was performed by an experimenter who was kept blind to the treatment schedule. All training was completed in a single day. For post-training infusion experiments, naive animals were trained in the hidden platform version of the Morris water maze task (Morris et al., 1982, 1986; Dixon et al., 1994) with an intertrial interval (iti) of 4 min until they could locate the platform three consecutive times in 15 sec. Animals that failed to reach this criterion by trial 12 were eliminated from the study. Each trial was initiated by placing the animal in one of four randomly chosen locations facing the wall of the tank. Animals were allowed to search for the hidden platform for a period of 60 sec. If an animal failed to find the platform, it was placed there by the experimenter. Animals were allowed to remain on the platform for a period of 30 sec before being returned to their home cages. For pretraining infusion experiments, naive animals were given a recovery period of 20 min in their home cages before the start of behavioral training. The training protocol consisted of 16 trials with an iti of 20 sec, as previously described by Guzowski and McGaugh (1997).

Retention testing. All behavioral testing was performed by an experimenter who was kept blind to the treatment schedule. Animals were tested for retention by placing them in the tank facing the wall at a point opposite the location of the hidden platform, as previously described by Guzowski and McGaugh (1997). Animals were given 60 sec to locate the hidden platform. If an animal failed to find the platform, it was placed there by the experimenter. Animals were allowed to remain on the platform for a period of 30 sec before being returned to their home cages. A 5 min iti was used between each of the three retention trials. For experiments with SB203580, 0.2 μg of PD098059, or delayed infusion of PD098059, retention was assessed by a transfer test in which the hidden platform was removed from the maze, and animals were allowed to search for a period of 60 sec. Movement within the maze was monitored using a video camera linked to a tracking software (Chromotrack; San Diego Instruments). The time to platform was calculated as the latency for each animal to cross the site at which the hidden platform was located during training. Using the tracking software, swimming speed was calculated by dividing the cumulative total distance (in centimeters) traversed in each zone by the cumulative dwell time (Hagiwara et al., 1993).

Visible platform task. After the completion of behavioral testing, animals were tested for visual acuity using a visible platform version of the Morris water maze. In this task, the water level was decreased so that the top of the platform was even with the water surface. A large visual cue (abstract sign connected to a metal rod) was attached to the platform to increase its visibility. Animals were initially placed on the platform for 30 sec to familiarize them with the cue. Each rat was given three trials in which they were allowed to search for the platform for a period of 60 sec. No animal failed to find the platform during this time period. The rat’s starting position remained constant throughout the visible testing, but the platform location was randomized between trials to overcome any residual preference for the previous location of the hidden platform. The latencies to reach the visible platform for the three trials was averaged to obtain each animal’s visual acuity score.

Histology. Animals were killed by chloral hydrate (1 gm/kg) overdose and transcardially perfused with 150 ml heparinized (0.1% v/v) PBS followed by 150 ml PBS containing 4% paraformaldehyde and 15% picric acid. Brains were removed and post-fixed in perfusant overnight. Brains were then cryoprotected with 30% sucrose in fixative solution, briefly dried, embedded in OCT (Myers Laboratory), and sectioned on a cryostat into 40-μm-thick sections. Every sixth section throughout the hippocampus was mounted on gelatin-subbed slides and stained with cresyl violet. The location of the site of infusion was evaluated and recorded for each animal.

Immunohistochemistry. Animals were trained for phospho-MAPK immunohistochemistry, as described for the post-training infusion experiments. Control animals were given either one training trial or 10 ± 1 trials in which the platform was moved to a new random location after each trial. At the appropriate time points, animals were killed, and the hippocampal tissues were quickly removed. Tissues were fixed in ice-cold 4% paraformaldehyde and 15% picric acid for at least 16 hr and then cryoprotected with 30% sucrose in PBS. Tissues were briefly dried to remove any surface liquid and embedded in OCT (Myers Laboratory). Forty-micrometer-thick sections were prepared on a cryostat. Free-floating slices were incubated overnight in 1 μg/ml primary antibodies in PBS containing 2% bovine serum albumin (BSA) and 2.5% normal goat serum, as described previously (Moore et al., 1996). The immunoreactivity was visualized using a biotinylated secondary antibody and a streptavidin–Cy3 complex, as recommended by the vendor. For double-labeling experiments, the binding of a neuron-specific antibody (NeuN; Chemicon, Temecula, CA) was detected using an FITC-conjugated secondary antibody. Sections were mounted on microscope slides and coverslipped under 50% glycerol in 0.25 mTris–HCl, pH 9.5. Fluorescence was protected using paraphenylenediamine (1 mg/ml in mounting media) and observed using a UV microscope with the appropriate filter set (Axiophot). The number of immunopositive cells for each animal was calculated as an average of three randomly chosen 1350 × 900 μm areas from three different slices counted by two independent blind observers. The total number of cells in these areas was counted after staining sections with cresyl violet. The results for each group were obtained from five animals.

Confocal microscopy. Nuclear signal for phospho-MAPK was assessed using a Zeiss krypton–argon laser confocal microscope. Digitized images were captured by a computer using Zeiss LSM software. Nuclear immunoreactivity was quantified by comparing the average pixel intensity of a defined area within the nucleus with a size-matched area of the cytoplasm for each individual neuron. The data were compiled from five randomly chosen fields.

Preparation of protein extracts. At the appropriate time points, animals were killed, and the hippocampal tissues were quickly removed as described in the immunohistochemistry section. The tissues were homogenized in 10 volumes of 10 mm Tris–HCl, pH 7.4, 1 mm EGTA, 1 mm EDTA, 0.5 mm DTT, 0.5 mm PMSF, 10 μg/ml leupeptin, and phosphatase inhibitors (2 mm NaF, 2 mmNaPP_i, 5 μm microcystin-LR, and 1 mm Na₃VO₄). After 20 strokes in a motor-driven Teflon-glass homogenizer, a portion of each sample was aliquoted and immediately frozen at −80°C. The rest of the homogenate was aliquoted and centrifuged at 15,000 × gfor 30 min at 4°C. The pelletized material and supernatant solutions were separated and stored at −80°C until needed.

Western blots. The amount of protein in each sample was measured using a MicroBCA assay (Pierce, Rockford, IL) using BSA as the standard. Samples were prepared by boiling in sample buffer, and equal amounts of protein were resolved in a 10% Tris–Tricine SDS-PAGE gel and transferred to an Immobilon-P (Millipore, Bedford, MA) membrane using a semidry transfer apparatus (Millipore). Membranes were blocked overnight in 5% BSA in PBS and incubated with 0.2 μg/ml the primary antibodies for 3 hr at room temperature. Membranes were then given five 10 min washes in TBST (10 mm Tris–HCl, pH 7.9, 150 mm NaCl, and 0.05% Tween 20). Immunoreactivity was assessed by a HRP-conjugated secondary antibody and chemiluminescence, which allowed the production of multiple fluorographs with different lengths of exposure. The quantification of the immunoreactive bands was performed using a Bio-Rad (Hercules, CA) model GS-670 imaging densitometer. The data represents the average of four independent experiments. Before reprobing, blots were stripped by two 10 min washes in a buffer containing 62 mm Tris–HCl, pH 6.8, 2% SDS, and 100 mm β-mercaptoethanol at 50°C. The membranes were then washed extensively with TBST and reblocked overnight in 2% BSA.

Kinase assays. Calcium–calmodulin-dependent protein kinase (CaMK) activity was measured using a synthetic peptide substrate (autocamtide-3; KKALHRQETVDAL), as described by Moore et al. (1996). Phosphorylation reactions were initiated by adding 3 μg of protein to a 20 μl mixture, resulting in the following final concentrations: 10 mm HEPES, pH 7.4, 0.5 mm DTT, 20 μm substrate peptide, 50 μm ATP, 2.0 mm CaCl₂, 2 μm calmodulin, 5 mm MgCl₂, and 2 μCi of [γ-³²P]ATP (3000 Ci/mmol). Control reactions for background determination were prepared simultaneously by omitting the substrate peptide. After a 1 min incubation at 30°C, the reactions were terminated by spotting 15 μl of the reaction mixture on P-81 phosphocellulose filters. The filters were washed three times, 10 min each, in 75 mm phosphoric acid, rinsed with ethanol, and dried under air flow. The radioactivity on the phosphorylated peptide substrate was quantitated in a scintillation counter by the Cerenkov method.

cAMP-dependent protein kinase A (PKA) assays using Kemptide (LRRASLG) as a substrate were performed as described previously (Roberson and Sweatt, 1996). The assay mixture consisted of 37.5 mm MES, pH 6.0, 15 mm Mg acetate, 30 μm ATP, 2 μCi of [γ-³²P] ATP (3000 Ci/mmol), 0.2 mm IBMX, 20 μm chlorophenylthiol cAMP, and 300 μmKemptide. The phosphorylation reaction was initiated by adding 5 μg of protein. Control reactions to determine the background phosphorylation were performed simultaneously by omitting the substrate peptide. The reactions were performed at 30°C for 5 min and terminated by spotting 15 μl of reaction mixture on P81 phosphocellulose filters as described above.

Protein kinase C (PKC) activity was measured using the specific substrate peptide (AAAKIQASFRGHMAR) as described previously (Klann et al., 1993). The assay mixture consisted of 20 mm HEPES, pH 7.4, 10 mm MgCl₂, 2 mmNaPPi, 0.1 mm PMSF, 100 μm ATP, 1 mm CaCl₂, 320 μg/ml phosphatidylserine, 30 μg/ml diacylglycerol, 2 μCi of [γ-³²P] ATP, and 20 μm substrate peptide. The reaction mixture was incubated with 3 μg of protein extract for 1 min at 30°C. The reactions were spotted on P81 paper and processed as described above.

Statistical analysis. Statistical significance was determined by an ANOVA followed by appropriate post hocanalysis or by a two-tailed Student’s t test for unpaired variables. Data were considered significant at p ≤ 0.05. Statistical analyses were performed using either the integrated optical densities (Western blots), cell counts (as percentage of total cells for immunohistochemistry), counts per minute per milligram of protein (kinase assays), or latencies (behavior).

RESULTS

DISCUSSION

The results presented in this report revealed three novel findings relevant to spatial memory. First, behavioral training in a spatial memory task is accompanied by increased number of phospho-MAPK-positive cells in the CA1/CA2 subfield, but not the dentate gyrus or CA3 subfield, of the hippocampus. Second, this enhanced phosphorylation of MAPK after training was restricted to the dorsal hippocampus. Finally,in vivo inhibition of MAPK phosphorylation impairs long-term, but not short-term, spatial memory. These findings demonstrate a critical role for a MAPK-mediated cellular event in the CA1/CA2 subfield of the dorsal hippocampus in long-term spatial memory.

Immunohistochemistry for phospho-MAPK indicated that animals not exposed to the Morris water maze show MAPK phosphorylation in ∼5% of neurons in the CA1/CA2 subfield. Control groups (one trial or randomized platform) did not present any increase in the percentage of phospho-MAPK-immunopositive neurons. After training, however, this number increased to ∼12% (Fig. 1f), indicating that MAPK activation is specific to the learning component of the task. The observation that increased MAPK phosphorylation is restricted to only CA1/CA2 neurons after behavioral training raises the hypothesis that this subfield is of critical importance in long-term spatial memory storage. This hypothesis is further supported by previous studies that correlated spatial memory with LTP in the various hippocampal subfields using genetically modified mice (Tsien et al., 1996; Chen and Tonegawa, 1997). It has been reported that mice with impaired CA1 LTP, but not mice lacking CA3 or dentate gyrus LTP, show spatial memory deficits (Huang et al., 1995; Nosten-Bertrand et al., 1996). Taken together, our finding of a training-associated activation of MAPK in only the CA1/CA2 neurons, combined with the correlation of LTP and spatial memory in this subfield, provides evidence that the different subfields may be serving different functions during long-term memory storage. In addition, based on the proposed role of MAPK in growth and differentiation, this suggests that the CA1/CA2 subfield in particular may be the site for hippocampal plasticity associated with long-term spatial memory.

Previous lesion studies, as well as electrophysiological studies, have indicated that the dorsal hippocampus, in contrast to the ventral hippocampus, may participate in the storage of spatial memory (Jung et al., 1994; Moser et al., 1995; Colombo et al., 1998). For example,Moser et al. (1993) have reported that selective lesions of the dorsal hippocampus impair performance in the Morris water maze task. In contrast, lesion of the ventral side did not produce any observable deficits. Consistent with the proposed role of the dorsal hippocampus in the Morris water maze task, the enhanced MAPK phosphorylation we observed was restricted to only this segment. This increase in MAPK activation was found only after training of animals to criterion and not after training in which the spatial learning component was eliminated (Fig. 1f). “Place cells”, cells whose activities correlate with the position of the animal, are believed to play a fundamental role in spatial navigation and are more common in the dorsal hippocampus for both rodents and monkeys (Jung et al., 1994;Colombo et al., 1998). Neuronal activity in response to nonspatial stimuli is found to be evenly distributed throughout the hippocampus. It is not known at present if the neurons that are positive for phosphorylated MAPK are place cells.

To establish a causal role for MAPK activity in spatial memory, we infused PD098059 and assessed its affect on behavioral performance. This inhibitor was found to decrease the phosphorylation of MAPK when compared with simultaneously infused vehicle controls. However, the phosphorylation of a related MAPK family member, SAPK (or JNK), was unaffected. Moreover, the activities of protein kinase A, calcium-calmodulin-dependent protein kinase and protein kinase C were also unchanged as a result of PD098059 infusion (Fig. 3b). However, this study cannot rule out the possibility that PD098059 may be nonspecifically inhibiting other kinases that were not directly examined or that diffusion of the inhibitor during homogenization reduced its efficacy. When PD098059 was infused before behavioral training, immunohistochemical analysis showed a decrease in the number of phospho-MAPK-immunoreactive cells (Fig. 3c). This infusion protocol did not affect acquisition as compared with vehicle-infused animals (Fig. 4a). This indicates that MAPK may not play a role in either hippocampus-dependent learning or short-term memory. However, long-term spatial memory was found to be significantly impaired in the PD098059-infused animals when tested 48 hr after training (Fig. 4a). This difference in performance was not caused by any deficits in motor function or visual acuity because their performance in subsequent retraining trials was not significantly different from the vehicle-infused controls.

The involvement of MAPK activity in long-term memory was further substantiated by the post-training infusion of PD098059. As predicted by the time course for MAPK activation, immediate infusion, but not delayed infusion, of PD098059 after behavioral training blocked long-term memory. Our data indicates that there is no increase in the number of phospho-MAPK-immunopositive cells after a single training trial. Only multiple training trials resulted in increased number of phospho-MAPK-immunopositive cells. This suggests that MAPK activity, as well as its translocation, accumulates with repeated training. This is consistent with previous findings by Martin et al. (1997), in culturedAplysia neurons, which demonstrated that repeated exposure to serotonin caused an accumulation of nuclear MAPK immunoreactivity. This accumulation was necessary to overcome a “threshold” needed for downstream gene expression. This threshold phenomenon suggests that even small changes in MAPK activity above or below this value would have profound effects on memory storage. Therefore, immediate infusion of PD098059 after training may decrease MAPK activity below this threshold value, resulting in insufficient downstream gene induction and attenuation of long-term memory. In addition, the half-life of active MAPK inside the nucleus of hippocampal neurons may be short, which would require constant rephosphorylation by MEK to have the needed cumulative effect. Similarly, the pretraining infusion of PD098059, even if the drug was only effective through part of the training protocol, would affect accumulation of activated MAPK sufficiently to impair gene expression and long-term memory formation.

Studies of MAPK phosphorylation in Hermissendaphotoreceptors after classical conditioning experiments inAplysia sensory–motor neuron cocultures, odor learning, and LTP in hippocampal slices have suggested that activation of the MAPK cascade is necessary for long-term plasticity (English and Sweatt, 1997; Martin et al., 1997; Berman et al., 1998; Crow et al., 1998). Recently, Atkins et al. (1998), using a fear-conditioning paradigm, reported that MAPK is required for associative learning. Both the behavioral protocols used in their study (contextual and cued-contextual conditioning) caused robust increases in MAPK phosphorylation in the hippocampus (shown by Western blotting of homogenized tissue). However, their protocol for contextual conditioning (where no tone was present during training, also called foreground conditioning) has been shown to be independent of hippocampal function (Phillips and LeDoux, 1994). Therefore, the increases in MAPK phosphorylation they observed may not be learning-related because it occurred in both hippocampal-dependent and hippocampal-independent tasks.

Our experiments show that MAPK-mediated changes occurring in the CA1/CA2 subfield of the dorsal hippocampus of a behaving animal are critical for long-term spatial memory formation and provide a clear launching point for further investigation into the molecular and genomic cascades involved in long-term memory. It has been reported that CREB “knock-outs”, as well as rats treated with antisense oligonucleotides to downregulate CREB in the dorsal hippocampus, have impaired long-term spatial memory (Bourtchuladze et al., 1994;Guzowski and McGaugh, 1997). Several protein kinases (PKA, Akt, CaMK IV, MAPK via p90^rsk) have been shown to be able to activate CREB via phosphorylation of ser¹³³ (Yamamoto et al., 1988; Sun et al., 1994;Bito et al., 1996; Xing et al., 1996). Thus, it is possible that the activation of MAPK we observed after behavioral training may mediate its effects via CREB activation and the expression of CRE sequence-containing genes. Alternatively, MAPK can induce expression of downstream genes necessary for long-term memory, independent of CREB by phosphorylating other transcription factors such as Elk-1 (Whitmarsh and Davis, 1996). Future experiments will help distinguish between these possibilities.