Beninger, Richard J.a b d; Beuk, Jonathana; Banasikowski, Tomek J.a; van Adel, Michaelb; Boivin, Gregory A.b; Reynolds, James N.a c
Schizophrenia is a debilitating psychiatric disorder characterized by positive, negative, and cognitive symptoms. This diversity of symptoms implicates dysfunction of various neuronal systems (Javitt, 2007). Thus, there is an urgent need to understand the causes of schizophrenia to improve treatment and prevent the disease.
Schizophrenia is associated with dysfunctional dopamine neurotransmission. Support for the dopamine hypothesis of schizophrenia stems from the findings that most effective antipsychotic medications are dopamine receptor antagonists (Carlsson, 1988; Kapur et al., 2000), schizophrenic patients exhibit an upregulation of dopamine receptors (Abi-Dargham et al., 2000), psychomotor stimulants that increase dopaminergic transmission such as amphetamine or cocaine are psychotogenic (Snyder, 1972), and susceptibility genes for schizophrenia code for proteins involved in dopamine signaling (Riley and Kendler, 2006). The mechanisms underlying the putative changes in dopamine function remain to be fully elucidated.
The glutamate hypothesis of schizophrenia states that positive and negative symptoms may be the result of hypofunctioning glutamate neurons in corticolimbic structures (Olney et al., 1991). This hypothesis originated from the observation that subanesthetic doses of noncompetitive antagonists at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamatergic receptors, such as phencyclidine (PCP), produce cognitive-behavioural effects mimicking some symptoms of schizophrenia (Luby et al., 1962; Javitt, 2007). Moreover, PCP exacerbates psychotic episodes in schizophrenic patients (Morris et al., 2005). Glutamate hypofunction might lead to subcortical dopamine hyperfunction associated with schizophrenia, linking the two hypotheses (Carlsson et al., 2001).
Repeated use of PCP in humans leads to persistent symptoms that resemble many of the positive, negative, and cognitive impairments seen in patients with schizophrenia (Jentsch and Roth, 1999). Moreover monkeys treated with PCP twice a day for 14 days showed impairment on a cognitive object retrieval task and decreased dopamine utilization 7 days after the last drug administration, showing enduring cognitive dysfunction (Jentsch et al., 1997a, b).
Rodents treated with PCP show behavioral and neurochemical alterations that resemble some of the changes seen in human schizophrenic patients (Morris et al., 2005). Although acute administration of PCP activates the frontal cortex, delayed effects include a decrease in frontal cortex activity measured by regional glucose metabolism (Gao et al., 1993). A PCP treatment model that produces a number of behavioral changes similar to those characterized in schizophrenia is subchronic treatment followed by a washout period (Jentsch and Taylor, 2000; Morris et al., 2005). For example, Jentsch et al. (1998) administered PCP (5.0 mg/kg two times per day for 7 days) and found that 7 days after the final drug administration rats showed an increased motor response to amphetamine, modeling the enhanced sensitivity to amphetamine witnessed in schizophrenia. Jentsch et al. (1997a, b) also reported that PCP administration (10 mg/kg daily for 14 days) followed by a 2-day washout period produced memory impairment in a delayed alternation task. Using similar subchronic treatment protocols, others have shown that PCP leads to impairments on operant reversal learning (Abdul-Monim et al., 2006, 2007; McLean et al., 2009a, b), novel object recognition (Fujita et al., 2008; Hagiwara et al., 2008; Hashimoto et al., 2008a, b; McLean et al., 2009a), and conditional discrimination tasks (Dunn and Killcross, 2006). All these tasks depend on working memory; subchronic PCP-induced impairments mimic working memory deficits observed in schizophrenia. People with schizophrenia are also impaired in set-shifting tasks and rats treated subchronically with PCP show similar impairments in an equivalent animal task (Rodefer et al., 2005, 2008; McLean et al., 2008). A number of these studies showed that impairments were reversed by antipsychotic drugs. Results suggest that subchronic PCP treatment leads to working memory impairments (e.g. set shifting) and increased sensitivity to amphetamine, similar to that observed in schizophrenia.
The glutamate hypothesis links the effects of PCP to γ-aminobutyric acid (GABA)ergic dysfunction. Olney et al. (1991) proposed that NMDA receptor blockade decreases the excitatory drive on GABAergic neurons. This loss of GABAergic inhibition would disinhibit excitatory neurotransmission, possibly leading to increased drive on dopaminergic neurons (Carlsson et al., 2001). Decreased GABAergic function has been identified in hippocampal and cortical regions of postmortem brains from schizophrenic patients (Costa et al., 2004). For example, Benes et al. (1991) found reductions of interneuron density in the cingulate, prefrontal cortex, and hippocampus of postmortem tissue from schizophrenic brains and have shown decreased GABA neurons in some of these areas (Benes and Berretta, 2001). Similarly, binding studies using [3H]muscimol as a potent and selective label for GABAA receptors, or experiments measuring GABA receptor mRNA have reported increases in receptor density in the prefrontal cortex (Hanada et al., 1987; Dean et al., 1999) and hippocampus (Benes et al., 1996) in post-mortem brains of people with schizophrenia, likely attributable to a compensatory response to attenuated GABAergic neurotransmission. Moreover, putative GABAergic neurons identified by parvalbumin staining in the hippocampus and frontal cortex were decreased in number after subchronic treatment with PCP in rats (Schroeder et al., 2000; Reynolds et al., 2004; Abdul-Monim et al., 2007; but see Abe et al., 2005). Greene (2001) reported that PCP preferentially affects NMDA receptors on inhibitory interneurons, providing electrophysiological evidence that GABAergic dysfunction contributes to the effects of PCP. Recently, Pratt et al. (2008) examined rats 72 h after PCP treatment (2.6 mg/kg) for 5 consecutive days and noted a significant reduction of mRNA expression in the voltage-gated potassium channel (3.1 kV) and the calcium binding protein parvalbumin which are both predominantly present in GABAergic interneurons in the rat prefrontal cortex.
In this study, rats were administered PCP subchronically (4.5 mg/kg twice a day for 7 days) and tested 7 days after the last drug administration. We hypothesized that treated rats would show an elevated response to amphetamine in tests of locomotor activity and an increased number of errors in a maze task. It was further hypothesized that GABAA receptor binding parameters would be altered in the hippocampus and frontal cortex of PCP-treated rats.
Male Sprague–Dawley rats (n=72; Charles River, Quebec, Canada) weighing 200–250 g upon arrival, were housed in transparent acrylic thermoplastic cages (45×25×22 cm) with woodchip bedding in a colony room on a reversed 12-h light/dark schedule; lights off: 07.00 h. Rats had free access to water and free or restricted access to food (LabDiet 5001, Missouri, USA). Rats were handled 2 min/day for 6 consecutive days before testing. Rats were maintained according to the guidelines of the Canadian Council on Animal Care and the Animals for Research Act. Experimental procedures were approved by the Queen's University Animal Care Committee.
Activity was measured in six transparent acrylic thermoplastic chambers (50×40×40 cm high) within wooden, styrofoam-insulated enclosures, outfitted with metal rod floors, a 2.5-W incandescent bulb, and a fan for ventilation and background noise. Seven infrared emitter/detector pairs (height 5 cm) measured beam breaks (Beninger et al., 1985).
The gray painted walls of the maze were 15 cm high and 15 cm apart. The 55-cm long corridor was attached to two arms extended 35 cm at an angle of 120° at each end. Removable plastic barriers could be inserted into slots in the arms and corridor to provide 15-cm long compartments. The floor consisted of steel bars spaced 1 cm apart except at the junctions of the arms where the floor consisted of transparent acrylic thermoplastic. Food containers were placed at the end of each arm and in the center of the corridor. Froot Loops cereal (Kellogg, Michigan, USA) used as reward, were randomly placed below the maze to mask odor cues. Various cues (e.g. light, cabinet, door) were visible to the rats (Mallet and Beninger, 1993).
Experiment 1: locomotor activity
Rats (n=24) were housed in pairs with food continually available. After 1 week in the colony room, rats (now approximately 250–300 g) were administered PCP (4.5 mg/kg, intraperitoneal, n=12) or saline (n=12) twice daily for 7 days at 08.30 and 18.00 h. Testing was conducted 7 days after injections for six rats from each group and 1 day later for the other six rats from each group.
Activity was monitored in 10-min bins within three components in a continuous session: habituation (60 min), saline injection (1.0 ml/kg, intraperitoneal, 60 min) and dextroamphetamine sulfate (AMPH) injection (1.5 mg/kg, intraperitoneal, 90 min).
Experiment 2: maze performance
For the first week, rats (n=24) were housed in pairs with food continually available. For subsequent weeks, rats were housed separately and food restricted. Weights were reduced to 85% of free-fed rats by rationing daily food (5–20 g/day). The day before maze training, rats were fed five Froot Loops in their home cages.
In the first habituation session, rats (approximately 250–300 g) were placed in the maze for 5 min. In the next two sessions, each food container held half a Froot Loop. Barriers were removed/inserted as rats progressed through the maze. Habituation to the double Y-maze took three sessions, one per day. Rats were then assigned randomly into training groups; half were trained in the spatial discrimination task while the other half received spatial alternation training. Rats were trained on the opposite task once training was considered complete.
For spatial discrimination training, rats were given a session with 4 practice trials followed by 18 recorded trials per day, 7 days/week. Each trial began by randomly placing the rat in an end arm. The barrier was removed allowing the rat to run either to the opposite arm or corridor, where a removable barrier blocked the second ‘Y’. In the first ‘Y’, only the path to the corridor led to reward. Rats were considered trained when at least 16 of 18 trials were correct for 2 consecutive sessions (3–5 days).
For spatial alteration training, rats were given a session with four practice trials followed by 18 recorded trials per day, 7 days/week. A barrier was inserted behind the rat preventing reentry into the first ‘Y’. The barrier in front was removed to allow access to the second ‘Y’. Rats entered either arm; the arm containing reward was opposite the arm entered in the second ‘Y’ on the previous trial.
The initial trial in the second ‘Y’ of every session was forced choice, implemented by blocking one arm. After an arm entry was made in the second ‘Y’, a barrier was inserted behind the rat. The next trial was started in less than 10 s. An entry was considered completed when the rat's hind legs crossed completely onto the grid floor of the arm. Rats were considered trained when at least 16 of 18 trials were correct for two consecutive sessions (3–7 days).
Once both the components of the double Y-maze were learned, the two tasks were combined in each trial. Rats received one session starting with four practice trials and followed by 18 recorded trials per day, 7 days per week until a criterion of at least 88% (16 of 18) choice accuracy on both memory components was reached over three consecutive sessions (13–20 days). These three consecutive sessions were used as a rat's baseline training session performance.
The next day, rats (approximately 400–500 g) were matched according to criterion performance and randomly assigned to groups receiving PCP (4.5 mg/kg, n=12) or saline (n=9) at 08.30 and 18.00 h for 7 consecutive days. Three rats were not included in the study because they failed to meet criterion before injections (n=2) or failed to perform any trials during testing (n=1). Testing was conducted over 5 sessions, 7 days after the last injection and was identical to the three previous training sessions. Correct arm entries in both parts of the maze were recorded.
Rats tested for locomotion after subchronic PCP (n=12) or saline (n=12) (brains extracted 10 days after last injection, 2–3 days after testing), and rats tested in the double Y-maze following subchronic PCP (n=6) or saline (n=6) (brains extracted 14 days after last injection, 2 days after testing) were euthanized by decapitation under halothane anesthesia. Brains were rapidly excised, weighed, and dissected. The entire hippocampus was used, frontal cortex was isolated by making a coronal cut rostral to the hypothalamus and cutting away the striatum and olfactory bulbs, and striatal tissue was dissected from a coronal section taken approximately 3 mm caudal to the cut used to isolate the frontal cortex. Tissues were frozen on dry ice and stored at −70°C.
Tissue was thawed and homogenized in 10 volumes of buffer (10 mmol/l Tris–HCl, 300 mmol/l sucrose, 2 mmol/l EDTA, pH 7.4) containing protease inhibitor cocktail (Sigma-Aldrich, Oakville, Ontario, Canada). Homogenate was centrifuged at 1000g for 10 min at 4°C. The supernatant was centrifuged at 16 000g for 15 min at 4°C. The pellet (synaptosomal membrane fraction) was resuspended in homogenization buffer, aliquoted, and stored at −70°C.
For binding to GABAA receptors, the highly specific GABAA analog [3H]muscimol was used; like GABA, muscimol binds at the interface of α and β subunits of the GABAA receptor but it can bind to a number of different GABAA receptor subtypes. Aliquots of the synaptosomal membrane fraction were washed through dilution with 5 volumes of wash buffer (50 mmol/l Tris–HCl, 2 mmol/l EDTA, pH 7.4), vortex-mixed and centrifuged at 16 000g for 15 min at 4°C. After repeating this procedure twice the final pellet was resuspended in binding assay buffer (10 mmol/l Tris–HCl, 150 mmol/l NaCl, pH 7.4). A spectrophotometric protein dye-binding assay (Bradford, 1976) established the protein concentration of each sample, standardized to bovine serum albumin. [3H]muscimol saturation binding was determined in reaction vessels containing 150–200 μg of washed synaptosomal membrane fraction, [3H]muscimol (1–40 nmol/l, 25.5 Ci/mmol; Perkin-Elmer, Massachusetts, USA) and binding assay buffer; total volume was 500 μl. Nonspecific [3H]muscimol binding was determined in the presence of 100 μmol/l GABA. Samples were incubated in the dark for 60 min at 4°C. The reaction was terminated by vacuum filtration using Whatman GF/B glass fiber filters (Piscataway, New Jersey, USA) prewet with 5 ml of ice-cold binding assay buffer. Filters were washed twice with 5 ml of ice-cold binding assay buffer. Remaining radioactivity was quantified by liquid scintillation spectrometry using a Beckman LS 6500 scintillation counter (Bailey et al., 2001).
Western immunoblot analysis of GAT-1 and GAD
Aliquots of the synaptosomal membrane preparation were diluted in Tris–HEPES–SDS buffer (Pierce Biotechnology, Rockford, Illinois, USA) to a final concentration of 5 mg protein/ml and 80 μl aliquots were mixed with an equal volume of Laemmli buffer containing dithiothreitol. The mixture was heated for 5 min at 100°C, and 10 μl aliquots (25 μg protein) were added to the wells of pre-cast 8% polyacrylamide gels (Pierce Biotechnology). Gels were run in a Mini-Protean 3 system (Bio-Rad Laboratories, Mississauga, Ontario, Canada) at 150 V for 45 min. Proteins were transferred onto nitrocellulose membranes, washed three times with Tris-buffered saline containing 0.005% Tween-20 (TBS-T), and blocked by incubation in TBS-T containing 5% (w/v) skim milk powder for 70 min at room temperature. Membranes were washed in TBS-T for 15 min, and then incubated with primary antibody diluted in TBS-T containing 5% (w/v) skim milk powder overnight at 4°C. The primary antibodies were rabbit polyclonal anti-GABA transporter-1 (GAT-1) (Chemicon International, Temecula, California, USA) at a dilution of 1 : 100, mouse monoclonal anti-glutamic acid decarboxylase-67 (GAD-67) (Chemicon International) (1 : 2000), or mouse monoclonal anti-β-actin (Sigma-Aldrich) (1 : 2000). Membranes were washed with TBS-T, and incubated for 60 min at room temperature in TBS-T containing 5% (w/v) skim milk powder with horseradish peroxidase-conjugated anti-rabbit (1 : 4000) or anti-mouse (1 : 4000) secondary antibodies (Bio-Rad Laboratories). Blots were washed with TBS-T and treated with ECL chemiluminescence detection system and visualized using Hyperfilm ECL X-ray film (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). Immunoblot bands were quantified by measuring relative optical density (ROD) using Image-J software (National Institutes of Health, Bethesda, Maryland, USA). ROD measurements for the GAT-1 and GAD proteins were normalized to the ROD of the β-actin protein signal within each lane.
1-(1-phenylcyclohexyl) piperidine (PCP; Sigma-Aldrich) and AMPH (USP, Maryland, USA) were dissolved in saline.
Beam breaks during habituation, saline and amphetamine components of the locomotor activity session were analyzed by three separate two-way (group×time) mixed-design analyses of variance (ANOVA) followed by simple effects analysis where appropriate. Proportion correct arm entries in the first or second ‘Y’ of the double Y-maze maze were analyzed with separate two-way mixed-design (group×day) ANOVA. Total correct arm entries were calculated as the combined proportion of correct arm entries in both components of the maze and were analyzed with a two-way mixed-design (group×day) ANOVA.
Radioligand binding is presented as the mean±SEM of 18 rats from each treatment group. Two-way mixed design (group×region) ANOVA analyzed [3H]muscimol binding (BMAX and KD) and the relative expression of GAT-1 and GAD in three brain regions (striatum, hippocampus and frontal cortex). Newman–Keuls post-hoc analyses were conducted where appropriate. All statistical analyses were conducted using SPSS software (Chicago, Illinois, USA). Alpha was set at 0.05.
Mean (±SEM) number of beam breaks (counts/10 min) was calculated for rats previously administered subchronic PCP or saline. During the 60-min habituation session, both the groups showed an initial burst in activity to the novel environment but then showed a decrease over the 10-min bins (Fig. 1). A group×time ANOVA revealed only a significant main effect of time [F(5,110)=127.69, P<0.05].
During the saline session both groups showed a small increase in the number of beam breaks during the first 10-min bin but this activity declined quickly. ANOVA revealed only a significant main effect of time [F(5,110)=6.32, P<0.05].
Before the last 90-min session, all rats were treated with AMPH (1.5 mg/kg) and placed back into the activity chambers. Both groups showed an increase in the mean number of beam breaks early in this period; however this increased activity level was enhanced over time in the PCP group in comparison with controls. ANOVA revealed a significant main effect of time [F(8,176)=9.92, P<0.05]. There was no significant main effect of group [F(1,22)=3.12, P=0.09], but a significant interaction was found [F(8,176)=3.21, P<0.05], revealing that groups differed in some 5-min bins. Tests of simple effects of groups at each 5-min bin revealed that the PCP group was more active than controls in bins 3 and 4 (P<0.05).
The mean proportion of correct arm entries in the spatial discrimination task during training was similar for both groups and did not differ significantly (Fig. 2a). Subchronic PCP-treated rats made fewer correct responses than controls on each of the 5 test days. The two-way ANOVA revealed only a near-significant main effect of group [F(1,19)=4.09, P=0.057]. Spatial alternation task performance of the two groups did not differ significantly during training (Fig. 2b). The PCP group did not improve as much over test days as the control group but ANOVA revealed no significant effects.
When performance in both the tasks of the maze was combined, the proportion of total correct arm entries did not differ significantly between groups during training (Fig. 2c). Over the 5 test days, correct arm entries gradually increased in both the groups but the subchronic PCP group had a lower proportion of total correct arm entries when compared with controls. This was confirmed by ANOVA which revealed a significant main effect of group [F(1,19)=4.46, P<0.05]. The effect of days was also significant [F(4,76)=2.51, P<0.05] and Newman–Keuls post-hoc analysis revealed that both the groups combined performed significantly better on day 5 of testing than on day 1 (P=0.02).
[3H]muscimol binding to GABAA receptors
Subchronic PCP-treatment increased Bmax for [3H]muscimol binding to the high-affinity GABA binding site on GABAA receptors in the striatum, hippocampus, and frontal cortex (Fig. 3a). ANOVA revealed a nonsignificant difference between groups in Bmax when data were combined across brain regions [F(1,34)=3.25, P=0.08]. The effect of drug was significant in the striatum [t(34)=2.45, P<0.05] but not in the hippocampus [t(34)=0.66, NS], or in the frontal cortex [t(34)=1.69, P=0.10].
The average KD for [3H]muscimol binding on GABAA receptors of rats that were administered subchronic PCP-treatment was increased in the striatum, hippocampus, and frontal cortex in comparison with controls (Fig. 3b). ANOVA revealed a significant difference between groups when data were combined across brain regions [F(1,34)=6.11, P<0.05]. The effect of drug was significant in the frontal cortex [t(34)=2.48, P<0.05] but not in the striatum [t(34)=0.99, NS], or in the hippocampus [t(34)=1.33, NS].
Subchronic PCP treatment did not alter the expression of GAT-1 or GAD-67 proteins (relative to β-actin) in frontal cortex, hippocampus, or striatum (Fig 4, Table 1).
Rats received PCP (4.5 mg/kg) twice a day for 7 days followed by a 7-day drug-free period before behavioral testing. The dose of PCP, as well as the drug administration regimen were within the range of subchronic PCP treatments previously described (e.g. Jentsch et al., 1998; Morris et al., 2005; Rodefer et al., 2005; Abdul-Monim et al., 2006; Dunn and Killcross, 2006). In this study, this subchronic PCP treatment protocol resulted in enhanced locomotor activity in response to amphetamine administration and impaired performance in a memory task. Subchronic PCP treatment also resulted in long term alterations in GABAA receptor binding in the frontal cortex, striatum, and hippocampus.
Our finding of an enhanced locomotor activity response to amphetamine in rats treated subchronically with PCP is in agreement with previous observations. Jentsch et al. (1998) showed that PCP (two injections of 5 mg/kg per day for 7 days, followed by a 7-day washout) produced an increased motor response to amphetamine. A more selective NMDA receptor antagonist, MK-801 (0.5 mg/kg twice daily for 7 days, followed by a 7-day washout) resulted in a similar increase in locomotor response to amphetamine (Beninger et al., 2009). Enhanced locomotor activity in the subchronic PCP group from this study as well as the MK-801 group previously reported was observed only after amphetamine administration and not during the habituation or saline component. Amphetamine acts by reversing the dopamine transporter and increasing synaptic dopamine (Fleckenstein et al., 2007). The finding that subchronic PCP treatment enhanced locomotor activity after amphetamine treatment suggests that dopamine function was enhanced by subchronic NMDA receptor blockade. These results support a link between NMDA receptor blockade and elevated dopamine function (Carlsson et al., 2001).
The ability of patients with schizophrenia to carry out set-shifting tasks that require working memory (e.g. Wisconsin Card Sorting Task) is impaired (Goldberg and Weinberger 1988; Beninger et al., 2003). The double-Y maze task allows the dissection of working memory performance (i.e. alternation task) from possible sensory or motor performance (i.e. spatial task) in well-trained rats (Biggan et al., 1991). PCP-treated rats made more errors in both the spatial discrimination and spatial alternation components of the maze. Therefore it cannot be concluded unequivocally that the greater number of errors was indicative of memory-specific impairment. However, the greater number of errors is unlikely to be attributable to a motor deficit, as rats in both the groups did not differ in the locomotor activity test during habituation or after saline administration. Moreover, Audet et al. (2007) recently found that, similar to control animals, rats treated with PCP (5 mg/kg per day for 7 days followed by 7 day withdrawal) showed decreased time spent in an illuminated area and number of contacts with a cat collar, suggesting that this treatment does not induce significant impairments in visual or olfactory detection.
The finding of impaired performance in the double Y-maze task after subchronic PCP is consistent with previous studies. PCP (10 mg/kg daily for 14 days, followed by a 2-day washout period) impaired delayed alternation in a T-maze task at delays of 40, 60 or 80 s but not 0 or 20 s (Jentsch et al., 1997a, b). Stefani and Moghaddam (2002) found that PCP (5.0 mg/kg two times per day, followed by a 9-day washout) did not significantly affect delayed alternation in a T-maze with 5 and 30-s delays, but they did not test longer delays. Treatment with PCP (5.0 mg/kg daily for 5 days, with a 1-day washout) impaired recent memory in a holeboard task with a delay of 180 s from the sample to test period (Schroeder et al., 2000). This suggests that subchronic PCP affects task performance more consistently at long test delays, possibly because these longer delays place a greater demand on working memory. It is possible that the delays imposed on rats in the double Y-maze were too short, producing a subtle cognitive deficit, whereas a longer delay period might augment the extent of impairment. Studies using other tasks that depend on working memory (e.g. operant reversal learning, novel object recognition, conditional discrimination, and set shifting) have similarly reported deficits after subchronic PCP (Rodefer et al., 2005; Abdul-Monim et al., 2006, 2007; Dunn and Killcross 2006; Fujita et al., 2008; Hagiwara et al., 2008; Hashimoto et al., 2008a, b; Rodefer et al., 2008; McLean et al., 2008, 2009a, b).
Audet et al. (2007) have reported less time spent in an illuminated area and fewer contacts with a cat collar in rats, compared with controls, indicative of increased anxiety, after a 7-day withdrawal from subchronic PCP treatment. It is unlikely that the double Y-maze impairment was anxiety related as unpublished data from our laboratory have shown that rats subchronically treated with MK-801 (0.5 mg/kg twice daily for 7 days, followed by a 7-day washout) did not significantly differ from controls in the elevated plus maze task, a rodent model of anxiety. Perhaps future research will dissociate components of performance impairments that are produced by memory dysfunction as opposed to PCP withdrawal.
PCP affects many neurotransmitter systems and the possibility exists that the dysregulation of neurotransmitters other than GABA contributes to behavioral deficits. However, evidence suggests that GABAergic dysfunction plays a major role in cognitive impairments produced by subchronic PCP. Xi et al. (2009) found downregulation of NMDA receptor subunit mRNAs in GABAergic parvalbumin-containing interneurons in the prefrontal cortex of rats treated with the more selective NMDA receptor antagonist MK-801. We found that subchronic MK-801 treatment (0.5 mg/kg for 7 days) resulted in impaired reversal learning in the watermaze in rats 7 days after the last treatment (Beninger et al., 2009). Thus, GABAergic dysfunction may be involved in the cognitive impairments witnessed after subchronic PCP treatment.
Previous studies have examined changes in GABA function following subchronic PCP. Expression of parvalbumin (a calcium binding protein that is selectively expressed in inhibitory chandelier and basket cells) immunoreactivity was decreased in the CA3 region and dentate gyrus of the hippocampus after subchronic PCP treatment (Schroeder et al., 2000; Reynolds et al., 2004; Abdul-Monim et al., 2007). In humans, evidence from postmortem brains of schizophrenic people have shown defects suggestive of decreased GABA function in hippocampal and cortical GABA systems (Benes et al., 1991). Recently, Wang et al. (2008) observed cortical decreases in parvalbumin-containing GABAergic neurons in adult rats treated with perinatal PCP. Similarly, Du Bois et al. (2009) treated rats with 10 mg/kg of PCP on postnatal days 7, 9 and 11 and found increased GABAA receptor binding levels compared with controls in the prefrontal and anterior cingulate cortex on postnatal day 32 and in the thalamus and hippocampus at all ages from postnatal day 18 onwards. Moreover, alterations in GABAA receptor binding after perinatal PCP treatment were highly correlated with alterations in NMDA receptor binding levels highlighting the close relationship between the two neurotransmitter systems.
The observation in this study that the number of high-affinity binding sites for [3H]muscimol was greater in the striatum, frontal cortex, and hippocampus of PCP-treated rats provides further evidence for an increased density of GABAA binding sites, possibly as a compensatory mechanism for decreased GABAergic neurotransmission in these brain areas, although the effect was only near significance (P=0.08). A significant increase in KD for [3H]muscimol binding in these brain regions was found after PCP treatment and is indicative of a decreased binding affinity of GABAA receptors, possibly as a consequence of altered subunit composition of GABAA receptors (Ebert et al., 1997) expressed after PCP treatment. These novel findings suggest that subchronic PCP treatment decreases GABA neurotransmission in the frontal cortex, striatum, and hippocampus of the rat brain and this dysfunction was associated with behavioral changes produced by PCP.
Our finding of altered GABA function in an animal model of schizophrenic symptoms is in agreement with the neurodevelopmental hypothesis of schizophrenia, whereby an as yet unknown insult occurring during the second trimester is thought to affect hippocampal development. According to the hypothesis, signs of this putative insult are not seen until early adulthood when the hippocampus is heavily involved in the refinement of synaptic connections in the frontal cortex (Weinberger and Lipska, 1995). Recent evidence has shown that GABA neurons are of particular importance to this process (Lewis et al., 2004). The frontal cortex regulates the mesocorticolimbic dopaminergic system and such an insult could lead to hyperfunction of the dopamine system associated with symptoms of schizophrenia.
Olney et al. (1991) further implicated GABA dysfunction in schizophrenia, postulating that a decrease in NMDA receptor stimulation would result in a loss of excitatory drive on GABA neurons and therefore a loss of inhibition. Greene (2001) provided electrophysiological evidence that PCP preferentially affects NMDA receptors on inhibitory interneurons. Further studies have found decreased markers for GABAergic neurons in the hippocampus and decreased GABAergic inhibition after PCP treatment (Schroeder et al., 2000; Reynolds et al., 2004; Abdul-Monim et al., 2007). Although we did not find alterations in markers for GABAergic neurons (GAT-1 and GAD-67 protein expression), this observation does not rule out subtle or selective loss of GABAergic neurons and/or innervation in specific regions of the brain structures examined in this study. We chose GAD-67 as it is the more ubiquitous marker for GABAergic neurons (Hendrickson et al., 1994) and has specifically been reported to be decreased in schizophrenia (Guidotti et al., 2000). GAT-1 is a specific marker for GABAergic nerve terminals.
The ability of PCP to produce behavioral effects like those seen in schizophrenia and to reduce GABA-mediated inhibition further implicates GABA dysfunction in the etiology of schizophrenia. GABA deficits are unlikely to exist in isolation as GABA neurons interact importantly with dopamine and glutamate systems that also have been implicated in schizophrenia (Olney and Farber, 1995). A complete understanding of the etiological mechanisms of schizophrenia will include the complex interactions of these systems. Future experiments should investigate the precise nature of the alterations in GABAA receptors that result in decreased binding as well as the exact nature of impaired mnemonic function after subchronic PCP treatment.
The study was supported by a grant from the Ontario Mental Health Foundation to R.J. Beninger and J.N. Reynolds.
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