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NEUROPHARMACOLOGY

Long-Term Effects of Olanzapine, Risperidone, and Quetiapine on Ionotropic Glutamate Receptor Types: Implications for Antipsychotic Drug Treatment

Mailman Research Center, McLean Division of Massachusetts General Hospital, Belmont, Massachusetts (F.I.T., R.J.B., N.S.K., K.Z.); and Consolidated Department of Psychiatry and Neuroscience Program, Harvard Medical School, Boston, Massachusetts (F.I.T., R.J.B., N.S.K., K.Z.)

Received April 7, 2003; accepted June 6, 2003.

	Abstract

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Levels of ionotropic glutamate (Glu) N-methyl-D-aspartate (NMDA), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainic acid (KA) receptors in rat forebrain regions were compared by quantitative in vitro receptor autoradiography after continuous treatment for 28 days with the atypical antipsychotics olanzapine, risperidone, and quetiapine, or vehicle controls. All three treatments significantly decreased NMDA binding in caudate-putamen (CPu; by 30, 34, and 26%, respectively) but increased AMPA receptor levels in same region (by 22, 30, and 28%). Olanzapine and risperidone, but not quetiapine, also reduced NMDA receptor labeling in hippocampal CA1 (21 and 19%) and CA3 (23 and 22%) regions. KA receptors were unaltered by any treatment in the brain regions examined. These findings suggest that the antipsychotic effects of olanzapine and risperidone may be mediated in part by NMDA receptors in hippocampus, and perhaps AMPA receptors in CPu. The findings also support the hypothesis that down-regulation of NMDA receptors by atypical antipsychotic agents in CPu contributes to their low risk of extra-pyramidal side effects. Inability of olanzapine, risperidone, and quetiapine to alter KA receptors suggests their minimal role in mediating the central nervous system actions of these drugs.

Ionotropic Glu receptors have complex structures. NMDA receptors are comprised of four or five subunits that are encoded by genes NMDAR-1 and NMDAR-2A to NMDAR-2D (Hollmann and Heinemann, 1994). The NMDAR-1 subunit is essential for expression of functional NMDA receptors and determines the pharmacology of receptor binding site (Hollmann and Heinemann, 1994). It has several critical sites, including a phencyclidine (PCP) binding site located within the ion channel that binds PCP, ketamine, and other related compounds, a strychnine-insensitive glycine binding site, and others for magnesium, zinc, and polyamines (Javitt and Zukin, 1991; Hollman and Heinemann, 1994). AMPA receptors also are assembled from four or five subunits derived from a family of four genes (gluR1-gluR4). The pharmacological profile of the assembled AMPA receptor depends on the composition of each subunit (Hollmann et al., 1991; Hollmann and Heinemann, 1994). KA receptors are composed of different subunits derived from genes for the low-affinity gluR5 to gluR7 and high-affinity KA1 and KA2 subunits (Hollmann and Heinemann, 1994). Functional KA receptors are assembled from five identical or nonidentical subunits into homomeric or heteromeric complexes that differ in their pharmacological properties (Lerma, 1998).

Dysfunction in glutamatergic neurotransmission may contribute to the pathophysiology of psychotic disorders, including schizophrenia (Goff and Coyle, 2001; Tsai and Coyle, 2002). Ionotropic Glu receptors, particularly of the NMDA type, have been implicated as a critical site of action of psychotomimetic agents, including PCP, ketamine, and other anesthetics that can produce behavioral and cognitive deficits that resemble some symptoms of psychotic disorders (Javitt and Zukin, 1991; Tsai and Coyle, 2002). Agonists at the modulatory glycine binding site of the NMDA receptor complex are reported to improve negative (amotivation and cognitive) symptoms of schizophrenia (Goff and Coyle, 2001; Tsai and Coyle, 2002). In addition, pathological abnormalities and alterations in Glu receptor densities have been found in postmortem forebrain tissue from patients diagnosed with schizophrenia compared with healthy controls (Meador-Woodruff and Healy, 2000; Goff and Coyle, 2001). However, it is not clear whether observed changes in Glu receptors in such brain specimens reflect the neuropathology of schizophrenia or adaptation to ante-mortem drug exposure.

Preclinical studies indicate that the three ionotropic Glu receptors are altered by treatment with antipsychotic drugs (APDs), although the direction of reported changes has been inconsistent. Different studies report increases, decreases, or no change in levels of these receptors after long-term treatment with various APDs (Meshul et al., 1996; Tarazi et al., 1996; Giardino et al., 1997; McCoy et al., 1998; Spurney et al., 1999). Moreover, contradictory and often opposite findings have been reported in the expression of subunits composing different Glu receptors after chronic administration of APDs (Fitzgerald et al., 1995; Riva et al., 1997; Healy and Meador-Woodruff, 1997). Typical neuroleptics such as haloperidol, as well as the atypical antipsychotic agent clozapine were commonly used in these studies. However, both types of APDs are associated with specific adverse neurological effects, e.g., extrapyramidal side effects (EPS), particularly parkinsonism, dystonia, and tardive dyskinesia in case of typical neuroleptics, excessive sedation, and dose-dependent risk of epileptic seizures with clozapine (Baldessarini and Tarazi, 2001; Tarsy et al., 2002).

In recent years, several APDs have emerged with low risks of EPS (Waddington and Casey, 2000; Baldessarini and Tarazi, 2001). Among them are the clozapine analogs olanzapine and quetiapine and the benzisoxazole derivative risperidone. These compounds have undergone extensive pharmacological, neurochemical, and behavioral characterization in animals (Arnt and Skarsfeldt, 1998; Waddington and Casey, 2000; Tarazi et al., 2001, 2002), as well as extensive clinical testing and application (Baldessarini and Tarazi, 2001; Tarsy et al., 2002). However, their long-term effects on ionotropic Glu receptors in mammalian forebrain are not well defined nor have they been compared quantitatively with those of other antipsychotics. Accordingly, we applied quantitative in vitro receptor autoradiography to assess regulation of NMDA, AMPA, and KA receptors in selected forebrain regions of interest after long-term infusion of olanzapine, quetiapine, or risperidone in rats. We hypothesized that these test agents would induce regionally selective changes in tissue levels of specific Glu receptors more closely resembling those associated with treatment with clozapine than with haloperidol as a representative typical neuroleptic.

	Materials and Methods

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Materials and Animal Subjects. Radiochemicals from PerkinElmer Life Sciences (Boston, MA) were Glu receptor ligands: [3-³H](+)-5-methyl-10,11-dihydro-[5H]-dibenzo[a,d]cyclohepten-5,10-imine (MK-801; 23.9 Ci/mmol for NMDA receptors), [5-³H]AMPA; 83.4 Ci/mmol for AMPA receptors), and [vinylidene-³H]kainic acid (Ci/mmol for KA receptors). Tritium autoradiography standards were from Amersham Biosciences, Inc. (Piscataway, NJ). Tritium-sensitive Hyperfilm and D-19 photographic developer and fixative were from Eastman Kodak (Rochester, NY).

Donated drugs included olanzapine (Eli Lilly & Co., Indianapolis, IN), risperidone (Janssen Pharmaceuticals, Beerse, Belgium), and quetiapine fumarate (Zeneca, Cheshire, UK). 6-Cyano-7-nitroquinoxaline (CNQX), KA, ketamine hydrochloride, potassium thiocyanate (KSCN), and spermine tetrahydrochloride were obtained from Sigma/RBI (Natick, MA), EDTA from Fisher Scientific Co. (Fairlawn, NJ), as well as L-glutamic acid (Glu), L-glycine hydrochloride, and Tris hydrochloride from Sigma-Aldrich (St. Louis, MO).

Subjects were male Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) initially weighing 200 to 225 g, maintained under artificial daylight (on, 7:00 AM-7:00 PM), in a temperature- and humidity-controlled environment with free access to standard rat chow and tap water in a USDA-inspected, veterinarian-supervised, small animal research facility of the Mailman Research Center of McLean Hospital (Belmont, MA). Animal procedures were approved by the Institutional Animal Care and Use Committee of McLean Hospital, in compliance with pertinent federal and local regulations.

In Vitro Ionotropic Glu Receptor Affinity. The three antipsychotic drugs olanzapine, risperidone, and quetiapine initially were tested for affinity at the NMDA, AMPA, and KA receptors, using a rat brain preparation as detailed previously (Reynolds et al., 1987; Wullner et al., 1994). For binding affinities of three APDs to NMDA receptors, rat brain minus cerebellum was frozen, thawed, and homogenized by Polytron (at 50% maximum power) in 3 volumes of buffer (20 mM HEPES containing 1 mM EDTA, pH 7.4, at 4°C) for 0.5 min and then centrifuged at 48,000g for 10 min, and rehomogenized and recentrifuged five more times. The resulting tissue pellet was suspended in buffer and frozen overnight and then thawed and centrifuged again three times in the same buffer without EDTA. The final pellet was suspended in the EDTA-free buffer at 200 mg/ml and stored at -70°C for use within 3 weeks. Thawed tissue was diluted with the same buffer to provide the equivalent of 15 mg of original wet weight of tissue per assay, and incubated with 1.7 nM final concentration of [³H]MK-801 as the assay radioligand. Glutamate (50 µM), glycine (30 µM), and spermine (50 µM) were added to the HEPES buffer to achieve maximum binding affinity of the ligand (Tarazi et al., 1998). Specificity was determined by 200 µM ketamine. Assay tubes were incubated for 60 min at 23°C, filtered (32 S&S filters; ISC Bioexpress, Kaysville, UT), and counted in minivials containing 4.5 ml of Emulsifier-Safe (PerkinElmer Life Sciences) in a beta scintillation counter (Beckman Coulter, Inc., Fullerton CA) at approximately 50% efficiency.

For binding affinities of three APDs to AMPA and KA receptors, rat cortical tissue was prepared as stated above. Assay buffer for AMPA receptors contained 50 mM Tris-HCl (pH 7.3), 2.5 mM CaCl₂, and 30 mM KSCN (Wullner et al., 1994), whereas the KA receptor assay buffer contained only 50 mM Tris-HCl (pH 7.3). Radioligands were [³H]AMPA (6.4 nM) to label AMPA receptors, and [³H]kainate (4.63 nM) for KA receptors. Nonspecific binding was defined using excess L-Glu (1 mM) for AMPA receptors and excess unlabeled kainate (100 µM) for KA receptors. Assay tubes were incubated for 60 min on ice and then filtered and counted as described above. The three drugs were initially screened for affinity at NMDA, AMPA, and KA receptors at a concentration of 10,000 and 100,000 nM, with lower concentrations added after initial inhibition of radioligand binding by at least 50%, to support estimates of IC₅₀ and K_i values.

Drug Treatment and Tissue Preparation. Four groups of rats (n = 6) received control vehicle, olanzapine (5.0), risperidone (3.0), or quetiapine fumarate (10.0 mg/kg/day) by osmotic minipumps (Alzet, Palo Alto, CA) implanted s.c. on the upper back of each animal to provide continuous infusions for 28 days. Doses are based on those typically reported to be behaviorally and neurochemically active in rats (Moore et al., 1992; Ellenbroek et al., 1996; Tarazi et al., 2001, 2002). After 4 weeks of treatment, residual drug solution in each minipump was <5% of the original volume, as predicted, indicating adequate drug delivery. On day 28, rats were decapitated; brains were removed, quick-frozen in isopentane on dry ice, and stored at -80°C until autoradiographic analysis. Frozen sections (10 µm) were prepared in a cryostat at -20°C, mounted on gelatin-coated, glass microscopic slides, and stored at -80°C until use. Coronal brain sections were taken through caudate-putamen (CPu), nucleus accumbens (NAc) septi, hippocampal regions CA1 and CA3, dorsolateral-frontal (DFC), and mesioprefrontal (MPC) cerebral cortex, and the entorhinal cortical (EC) region. These selected extrapyramidal, limbic, and cortical brain regions of interest mediate cognitive, emotional, and motor behaviors that are typically disturbed in patients with psychotic disorders and believed to be altered by antipsychotic drug treatment (Baldessarini and Tarazi, 2001).

Receptor Autoradiography. Brain sections from all drug-treated rats, and matching controls, were evaluated at the same time in each radioreceptor assay to minimize experimental variability. Sections were first preincubated for 60 min at room temperature (RT) in the appropriate specified buffer before incubating them with the radioligand to remove endogenous Glu and wash out any residual drug that may interfere with binding of the radioligands to Glu receptors.

NMDA Receptors. Sections were preincubated for 60 min at RT in 50 mM Tris-HCl buffer (pH 7.4) and then incubated for 150 min at RT in fresh buffer containing 10 nM [³H]MK-801 and 100 µM L-Glu, 100 µM glycine, 1 mM EDTA, and 75 µM spermine to enhance the binding of [³H]MK-801 to its site within the open cation channels associated with NMDA receptors. Nonspecific binding was determined by including 20 µM ketamine. After incubation, slides were washed in ice-cold 50 mM Tris-HCl buffer, twice for 20 min, and dried (Tarazi et al., 1996, 1998, 2000).

AMPA Receptors. Binding protocol was modified from Wullner et al. (1994). Sections were incubated for 60 min at RT in 50 mM Tris-HCl buffer (pH 7.2) and then incubated in fresh buffer containing 30 nM [³H]AMPA, 2.5 mM CaCl₂ and 30 mM KSCN. Nonspecific binding was determined with 30 µM unlabeled CNQX. After incubation, slides were washed in the ice-cold Tris buffer, three times for 10 s, and dried.

Kainate Receptors. Sections were preincubated for 60 min at 4°C in 50 mM Tris-HCl buffer (pH 7.0) at 4°C and then incubated in this buffer containing 20 nM [³H]KA for 60 min at 4°C. Nonspecific binding was determined with 25 µM unlabeled KA. After incubation, slides were washed in ice-cold 50 mM Tris buffer, three times for 10 s, and air-dried (Tarazi et al., 1996, 1998, 2000).

Autoradiography and Image Analysis. Radiolabeled slides and calibrated [³H]standards (Amersham Biosciences, Inc.) were exposed to Hyperfilm (Eastman Kodak). Radiolabeled slides and calibrated [³H]standards were exposed to Hyperfilm for 21 ([³H]AMPA and [³H]KA), or 30 days ([³H]MK-801) at 4°C. Films were developed in Kodak D-19 developer and fixative. Optical density (O.D.) in brain regions of interest was measured with a computerized densitometric image analyzer (MCID-M4; Imaging Research, St. Catherines, ON, Canada). Brain regions of interest were outlined (Fig. 1) and their O.D. was measured. Left and right sides of two contiguous sections represented total binding, and two other sections represented nonspecific binding; the four determinations were averaged for each subject (n = 6 rats/treatment). O.D. was converted to nanocuries per milligram of tissue with calibrated [³H]standards and, after subtracting nonspecific from total binding, specific binding was expressed as femtomoles per milligram of tissue.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Sites of autoradiographic analyses of rat brain regions sampled in 10-µm coronal sections from A 3.2 to 4.2 (A), A 1.7 to 2.2 (B), A 0.7 to 1.2 (C), and A 0.2 to 0.7 mm anterior to bregma (D), according to Paxinos and Watson (1982). CP-L, caudate-putamen lateral; CP-M, caudate-putamen medial; CA1 and CA3, hippocampal regions.

Statistical Analysis. Two-way analysis of variance (ANOVA) was used to evaluate overall changes across treatments and brain regions for each assay. Given overall significance of effects for treatment, Fisher's post hoc tests were used to test for differences due to each drug treatment in preselected anatomical areas. Unless stated otherwise, data are presented as means ± S.E.M. Comparisons were considered significant at p < 0.05 in two-tailed tests, with degrees of freedom (df) based on n = 6 subjects/treatment group.

	Results

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Experiments with rat brain homogenates indicated that olanzapine, risperidone, and quetiapine all had very low affinity at NMDA, AMPA, and KA receptors. At concentrations of 10 to 100 µM, olanzapine, risperidone, and quetiapine inhibited binding of all three radioligands by only 0 to 6% (all K_i values >10 µM).

The observed distribution of ionotropic Glu receptors accorded closely with our previous findings in rat brain (Tarazi et al., 1996, 1998) that NMDA and AMPA receptors are highly expressed in hippocampal areas (CA1 > CA3), followed by cerebral cortex, CPu, and NAc (Tables 1 and 2). In contrast, KA receptors were expressed selectively in the hippocampal CA3 region, followed by MPC, and NAc (Table 3). Two-way ANOVA measuring overall changes across drug treatments and brain regions for NMDA assay was highly significant (p < 0.001). Four weeks of continuous infusion of olanzapine, risperidone, and quetiapine reduced labeling of NMDA receptors in the medial [by 30, 33, and 27%, respectively; F(2,20 df) = 8.7, p < 0.001] and lateral portions of caudate-putamen [by 31, 35, and 24%, F(2,20 df) = 11.3, p < 0.001]. In addition, olanzapine and risperidone, but not quetiapine, significantly decreased NMDA receptor binding in the CA1 [by 21 and 19%, F(2,20 df) = 5.3, p < 0.01] and CA3 (by 23 and 22%, F = 5.3, p < 0.01) regions of hippocampus (Table 1). There were no significant changes in NMDA receptor levels in cerebral cortical MPC, DFC, and EC regions (Table 1).

Two-way ANOVA for AMPA receptor assay was also significant (p < 0.05). Continuous administration of olanzapine, risperidone, and quetiapine increased binding of AMPA receptors in medial CPu [by 19, 30, and 26%, respectively, F(2,20 df) = 4.4, p < 0.02] and lateral (by 24, 31, and 29%, F = 4.9, p < 0.001) regions, with no significant changes in cortical or limbic brain regions (Table 2). Long-term infusion of all test agents failed to alter tissue concentrations of KA receptors in any brain region (Table 3).

	Discussion

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Long-Term Effects of Newer Antipsychotics on NMDA Receptors. Continuous treatment with olanzapine, risperidone, and quetiapine significantly decreased binding of [³H]MK-801 to NMDA receptors in medial and lateral CPu (Table 1). These effects were similar to previously reported effects of clozapine but not haloperidol (Tarazi et al., 1996). Another study also reported a trend to reduced NMDA receptor binding in striatum after chronic treatment with clozapine but not with haloperidol (Spurney et al., 1999). This effect of clozapine may result from its proposed antagonistic action at NMDA receptors (Lidsky et al., 1993). However, it is unlikely that the effects of olanzapine, risperidone, or quetiapine result from direct NMDA receptor blockade because the three drugs showed very low affinity for MK-801 binding sites (all K_i values >10 µM) based on our in vitro assays. Reductions in NMDA receptor binding induced by olanzapine, risperidone, and quetiapine in the CPu may arise indirectly from neurochemical changes initiated by known interactions of these drugs with other neurotransmission systems, including those for 5-hydroxytryptamine (serotonin) (5-HT) or DA, both of which may modulate glutamatergic neurotransmission (Aghajanian and Marek, 2000; Carlsson et al., 2001). Such mechanisms would seem to implicate post-transcriptional changes at the protein level since chronic treatment with olanzapine and quetiapine, was reported not to alter expression of mRNA levels for NMDA-forming subunits in rat striatum (Tascedda et al., 1999, 2001).

The three APDs tested in this study have potent interactions at serotonin (5-HT) receptors (Baldessarini and Tarazi, 2001), and continuous treatment with the same drugs increased concentrations of 5-HT_1A receptors and decreased 5-HT_2A receptor levels in rat frontal cortex (Tarazi et al., 2002). Drug-induced changes in availability and functional status of these 5-HT receptors in cerebral cortex may suppress Glu neurotransmission in corticostriatal projections innervating CPu, and lead to decreased expression of striatal NMDA receptors. There also is evidence that NMDA and DA D₂ receptors are coexpressed in the same striatal neurons (Ariano et al., 1997; Tarazi et al., 1998), and indications that close and often antagonistic functional, behavioral, and cellular interactions occur between the same receptors (Cepeda et al., 1993; Carlsson et al., 2001). Accordingly, blockade and up-regulation of D₂ receptors in rat CPu after continuous administration of olanzapine and risperidone (Tarazi et al., 2001) may contribute to the observed decreases in NMDA receptor labeling in that brain region.

More importantly, NMDA receptor activation may contribute to induction of the extrapyramidal side effects of typical neuroleptics. Conversely, NMDA receptor antagonism has reduced neuroleptic-induced catalepsy (Schmidt and Bubser, 1989; Yoshida et al., 1991) and blocked neuroleptic-induced expression of immediate early gene c-fos in striatal tissue (Boegman and Vincent, 1996). In contrast, NMDA receptor agonists potentiated haloperidol-induced catalepsy (Yoshida et al., 1991). Suppression of striatal NMDA receptor activity by the three APDs included in the present study may contribute to their relatively benign impact on the extrapyramidal system (Baldessarini and Tarazi, 2001; Tarsy et al., 2002).

Continuous treatment with olanzapine and risperidone decreased NMDA receptor binding in hippocampal CA1 and CA3 regions (Table 1). Functional impairment of Glu neurotransmission within the hippocampal formation might contribute to the pathophysiology of psychosis (Gao et al., 2000; Goff and Coyle, 2001; Tsai and Coyle, 2002). Lower levels of hippocampal NMDA receptors may act in synchrony with higher levels of hippocampal D₂ receptors induced by anti-psychotic treatment (Tarazi et al., 2001), to improve psychotic symptoms by ameliorating hippocampal DA hyperactivity and restoring NMDA-sensitive Glu-mediated outputs from hippocampus to limbic and cortical brain areas (Krieckhaus et al., 1992; Gao et al., 2000). It is tempting to speculate that changes in hippocampal NMDA receptors may contribute uniquely to the beneficial clinical effects of olanzapine and risperidone because other antipsychotic agents, including clozapine as well as quetiapine and haloperidol, did not induce such effects in hippocampus (Table 1; Tarazi et al., 1996).

Long-Term Effects of Newer Antipsychotics on AMPA and KA Receptors. Continuous treatment with olanzapine, risperidone, or quetiapine significantly increased labeling of AMPA receptors in medial and lateral CPu, and not in other forebrain regions (Table 2). This finding, based on labeling with the agonist [³H]AMPA, contrasts to a previously reported lack of effect of long-term administration of haloperidol or clozapine on AMPA receptors labeled with the antagonist [³H]CNQX (Tarazi et al., 1996). Differences in the binding sites or receptor-states labeled by each radioligand may have contributed to this discrepancy. The agonist radioligand [³H]AMPA selectively labels a high-affinity state, whereas the antagonist [³H]CNQX binds to both high- and low-affinity states of AMPA receptors with similar affinity (Nielsen et al., 1990; Hall et al., 1993). With AMPA receptors in CPu, long-term treatment with APDs seems to increase the high-affinity binding state selectively. This effect may be difficult to observe when both binding states of AMPA receptors are radiolabeled with an antagonist. Other studies also found elevations of [³H]AMPA binding, with minimal changes in [³H]CNQX binding, after long-term administration of clozapine, risperidone, or haloperidol (McCoy et al., 1996, 1998). These changes in AMPA receptors probably reflect post-transcriptional modifications, because olanzapine and quetiapine did not alter expression of mRNA encoding different AMPA subunits in striatum (McCoy et al., 1998; Tascedda et al., 1999, 2001).

Our present findings also suggest that AMPA receptors represent a novel common site of action that may contribute to beneficial clinical effects of olanzapine, risperidone, and quetiapine. Antipsychotic-induced up-regulation of AMPA receptors may restore cortico-striato-limbic Glu neurotransmission by normalizing reduced glutamatergic activity suggested as a pathophysiological contribution in schizophrenia (Goff and Coyle, 2001; Tsai and Coyle, 2002). In support of this hypothesis, ampakines, drugs that act as positive modulators of the AMPA receptor complex and enhance Glu neurotransmission via AMPA receptors, have improved cognitive impairments in schizophrenia patients treated with clozapine (Goff et al., 2001).

Similar to NMDA receptors, it is unlikely that effects of olanzapine, risperidone, or quetiapine on AMPA receptors in CPu result from direct receptor blockade because we found all three APDs to have very low affinity for all three ionotropic Glu receptors (all K_i values >10 µM). However, indirect actions arising from the effects of these drugs on the central 5-HT system, again, may contribute to the increased AMPA receptor binding found in CPu (Table 2). These effects include opposite long-term effects of olanzapine, risperidone, and quetiapine on cortical 5-HT_1A (increases) and 5-HT_2A (decreases) receptors (Tarazi et al., 2002). Additional evidence for a direct interaction between 5-HT_1A/2A/AMPA receptors arises from studies finding that 5-HT_2A receptor stimulation increased release of Glu by pyramidal cells in layer-V of prefrontal cortex, which produces corticostriatal and corticotectal projections (Miller, 1988). The mechanism involved depends on stimulation of AMPA receptors (Aghajanian and Marek, 2000). In contrast, stimulation of 5-HT_1A receptors decreased AMPA-evoked electrical stimulation in prefrontal cortex (Cai et al., 2002). The changes in cortical 5HT_1A (increase) and 5HT_2A receptors (decrease) after continuous treatment with the APDs included in the present study may alter corticostriatal AMPA-mediated Glu neurotransmission and lead to an increase in post-transcriptional expression of postsynaptic AMPA receptors in CPu.

Alternatively, the observed increase in AMPA receptors in rat CPu may result from antipsychotic-induced up-regulation of D₂ receptors (Tarazi et al., 2001), because both receptors may be expressed on the same striatal neurons (Ariano et al., 1997). It is noteworthy that antipsychotic-induced changes in 5-HT and DA receptors produced opposite effects on NMDA (decrease) and AMPA (increase) receptors in CPu, suggesting that these ionotropic Glu receptor subtypes respond differently to long-term changes in forebrain 5-HT and DA neurotransmission.

Long-term infusion of olanzapine, risperidone, or quetiapine did not alter the binding of [³H]kainate to KA receptors in any brain region examined (Table 3). Lack of change in tissue levels of KA receptors may result from the very low affinity of three APDs to KA receptors, as well as a lack of indirect effects of such treatment on secondary neural mechanisms that may trigger changes in KA receptor binding. This finding agrees with previous autoradiographic studies that did not find changes in KA receptor levels after chronic administration of the dissimilar antipsychotic agents clozapine, haloperidol, and raclopride (Tarazi et al., 1996; Spurney et al., 1999; Gao et al., 2000). In contrast, long-term treatment of rats with haloperidol or clozapine increased KA2 mRNA levels in the CPu. Clozapine treatment also caused an increase in gluR7 mRNA expression, and a decrease in gluR3 mRNA expression in both cortex and striatum (Healy and Meador-Woodruff, 1997). However, these brain region-specific alterations in mRNA levels of KA receptor subunits was not associated with changes in KA receptor densities after treatment with haloperidol or clozapine, suggesting that post-transcriptional factors may also contribute to maintaining KA receptors at constant levels in brain tissue during exposure to APDs.

Changes in levels of KA receptors have been reported after various experimental manipulations in animals. Lower levels of KA receptors were found in mouse cerebral cortex after chronic barbiturate treatment (Short and Tabakoff, 1993). In contrast, an increase in KA receptors was observed in rat hippocampus 24 h after withdrawal from chronic treatment with PCP or ethanol (Gao and Tamminga, 1994; Carta et al., 2002), and in rat striatum after long-term nigrostriatal DA denervation (Tarazi et al., 2000). In addition, changes in the expression of KA receptor proteins or the mRNAs encoding their different subunits have been observed in postmortem tissue from some patients with schizophrenia compared with healthy controls, although these findings have not been consistently replicated (Meador-Woodruff and Healy, 2000). It is likely that the reported abnormalities in KA receptors in postmortem schizophrenia brain tissue are not the result of ante-mortem drug exposure, because KA receptors have resisted adaptations to long-term treatment with typical, atypical, and newer atypical antipsychotic agents and are less likely to mediate the actions of dissimilar classes of APDs.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Similar to the actions of clozapine, and in contrast to a lack of effect of haloperidol, long-term treatment of rats with olanzapine, risperidone, or quetiapine significantly down-regulated NMDA receptors in medial and lateral CPu (Table 1). These new findings add support to the hypothesis that these receptor decreases of NMDA receptors in the basal ganglia may contribute to the relatively benign profile of clinical EPS with these agents (Baldessarini and Tarazi, 2001; Tarsy et al., 2002). In addition, both olanzapine and risperidone decreased levels of NMDA receptors in hippocampal CA1 and CA3 regions but not other cortical areas (including DFC and EC), suggesting a possible common site contributing to beneficial effects of newer atypical antipsychotics.

At behaviorally and neurochemically effective doses, olanzapine, risperidone, and quetiapine also increased abundance of AMPA receptors in medial and lateral CPu, indicating that AMPA receptors in these brain regions constitute common targets that mediate the actions of newer APDs. Failure of these atypical APDs to alter abundance of KA receptors in any rat brain region examined adds support to the view that this ionotropic Glu receptor type is unlikely to contribute to the clinical actions of various kinds of antipsychotic agents.

	Acknowledgements

 
Drug substances were generous gifts of Eli Lilly & Co., Janssen Pharmaceuticals, and Zeneca.

	Footnotes

 
This study was supported by Stanley Medical Research Institute, National Alliance for Research on Schizophrenia and Depression Young Investigator Award, and research awards from Eli Lilly & Co. and Janssen Pharmaceuticals (to F.I.T.); Adam Corneel Award (to K.Z.); and a grant from the Bruce J. Anderson Foundation and funds of the McLean Hospital Private Donors Neuropharmacology Research Fund (to R.J.B.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.052597.

ABBREVIATIONS: Glu, glutamate; NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; KA, kainic acid; NMDAR, N-methyl-D-aspartate receptor; PCP, phencyclidine; APD, antipsychotic drug; EPS, extrapyramidal side effect; CNQX, 6-cyano-7-nitroquinoxaline; KSCN, potassium thiocyanate; CPu, caudate-putamen; NAc, nucleus accumbens; DFC, dorsolateral-frontal cerebral cortex; MPC, mesioprefrontal cortex; EC, entorhinal cortex; T, room temperature; O.D., optical density; ANOVA, analysis of variance; 5-HT, 5-hydroxytryptamine (serotonin); DA, dopamine.

Address correspondence to: Dr. Frank I. Tarazi, Mailman Research Center, McLean Hospital, Harvard Medical School, 115 Mill St., Belmont, MA 02478. E-mail: ftarazi{at}hms.harvard.edu

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