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0022-3565/01/2972-711–717$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics JPET 297:711–717, 2001 Vol. 297, No. 2 3387/901353 Printed in U.S.A. Long-Term Effects of Olanzapine, Risperidone, and Quetiapine on Dopamine Receptor Types in Regions of Rat Brain: Implications for Antipsychotic Drug Treatment FRANK I. TARAZI, KEHONG ZHANG, and ROSS J. BALDESSARINI Mailman Research Center, McLean Division of Massachusetts General Hospital, Belmont, Massachusetts; and Consolidated Department of Psychiatry & Neuroscience Program, Harvard Medical School, Boston, Massachusetts Received September 29, 2000; accepted January 31, 2001 This paper is available online at http://jpet.aspetjournals.org Dopamine (DA) exerts its effects in mammalian brain by interacting with several DA receptor types that include D1like (D1, D5) and D2-like (D2, D3, D4) receptor families (Baldessarini and Tarazi, 1996). DA receptors, and particularly the D2-like family, have been implicated in the pathophysiology of psychotic disorders and, more directly, in the pharmacodynamic basis of beneficial effects of antipsychotic drugs (Baldessarini and Tarazi, 2001). They are also implicated in centrally mediated adverse effects typical of most neuroleptic-antipsychotics, including extrapyramidal neurological reactions (particularly, parkinsonism and tardive dyskinesia) and hyperprolactinemia (Baldessarini and Tarsy, 1980; Baldessarini and Tarazi, 2001). The introduction of the prototype “atypical” antipsychotic agent clozapine was a major step in developing drugs with less risk of the adverse neurological effects that are typical of This work was supported by National Alliance for Research on Schizophrenia and Depression Young Investigator Award, The Grable Foundation, and a research award from Eli Lilly Corporation (F.I.T.), National Institutes of Health Grants MH-34006 and MH-47370, a grant from the Bruce J. Anderson Foundation, and funds of the McLean Private Donors Neuropharmacology Research Fund (R.J.B.). (65%, 32%), and HIP (61%, 37%). D1-like and D3 receptors in all regions were unaltered by any treatment, suggesting their minimal role in mediating actions of these antipsychotics. The findings support the hypothesis that antipsychotic effects of olanzapine and risperidone are partly mediated by D2 receptors in MPC, NAc, or HIP, and perhaps D4 receptors in CPu, NAc, or HIP, but not in cerebral cortex. Selective up-regulation of D2 receptors by olanzapine and risperidone in CPu may reflect their ability to induce some extrapyramidal effects. Inability of quetiapine to alter DA receptors suggests that nondopaminergic mechanisms contribute to its antipsychotic effects. standard antipsychotics but with similar or even superior beneficial effects. Clozapine is usually well tolerated, with very limited adverse extrapyramidal signs (EPS) or hyperprolactinemia and substantial evidence of superior antipsychotic effectiveness (Baldessarini and Frankenburg, 1991; Brunello et al., 1995). The pharmacological basis of the unusual clinical properties of this unique agent remains unclear. Clozapine interacts with high or moderate potency at serotonergic (5-HT2A, 5-HT2C, and others), acetylcholinergic (muscarinic), adrenergic (␣1, ␣2, 2), histaminic (H1), and other neurohumor receptors. In contrast, it has only moderate affinity for both D1 and D2 DA receptors (Baldessarini and Frankenburg, 1991; Brunello et al., 1995; Baldessarini and Tarazi, 1996). Clozapine also displays somewhat greater affinity for D4 than other DA receptors (Van Tol et al., 1991), suggesting that these receptors may represent potential sites of action of clozapine and perhaps other antipsychotic agents. D4 receptors also may be increased in postmortem brain tissue of schizophrenic patients (Seeman et al., 1993; Murray et al., 1995), although these findings remain inconsistent (Lahti et al., 1996). In addition, repeated administration of clozapine, ABBREVIATIONS: DA, dopamine; CPu, caudate-putamen; DFC, dorsolateral-frontal cerebral cortex; EC, entorhinal cortex; HIP, hippocampus; MPC, mesioprefrontal cortex; NAc, nucleus accumbens septi (core and shell subdivisions); EPS, extrapyramidal signs; 7-OH-DPAT, R(⫹)-[2,33 H]7-hydroxy-N,N-di-n-propyl-2-amino-1,2,3,4-tetrahydronaphthalene; DTG, 1,3-ditolylguanidine; RT, room temperature; SCH-23390, R(⫹)-[Nmethyl-3H]7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine. 711 Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017 ABSTRACT Changes in members of the dopamine (DA) D1-like (D1, D5) and D2-like (D2, D3, D4) receptor families in rat forebrain regions were compared by quantitative in vitro receptor autoradiography after prolonged treatment (28 days) with the atypical antipsychotics olanzapine, risperidone, and quetiapine. Olanzapine and risperidone, but not quetiapine, significantly increased D2 binding in medial prefrontal cortex (MPC; 67% and 34%), caudate-putamen (CPu; average 42%, 25%), nucleus accumbens (NAc; 37%, 28%), and hippocampus (HIP; 53%, 30%). Olanzapine and risperidone, but not quetiapine, produced even greater up-regulation of D4 receptors in CPu (61%, 37%), NAc 712 Tarazi et al. Experimental Procedures Materials and Animal Subjects Radioligands were from NEN Life Science Products, Inc. (Boston, MA): R,S(⫾)-[N-methyl- 3 H]nemonapride (86 Ci/mmol), S(⫺)[methoxy-3H]raclopride (74 Ci/mmol), and R(⫹)-[N-methyl-3H]7-chloro8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-23390; 81 Ci/mmol); or from Amersham (Arlington Heights, IL): R(⫹)-[2,3-3H]7-hydroxy-N,N-di-n-propyl-2-amino-1,2,3,4-tetrahydronaphthalene (7-OH-DPAT; 116 Ci/mmol). Tritium-sensitive Hyper- films were from Amersham. D-19 photographic developer and fixative were from Eastman Kodak (Rochester, NY). Donated drugs included olanzapine (Eli Lilly, Indianapolis, IN), risperidone (Janssen, Beerse, Belgium), and quetiapine fumarate (Zeneca, Cheshire, UK). 1,3-Ditolylguanidine (DTG), S(⫺)-eticlopride hydrochloride, cis-flupenthixol dihydrochloride, fluphenazine dihydrochloride, ketanserin tartrate, pindolol, and S(⫺)-sulpiride were purchased from RBI-Sigma (Natick, MA). Cation hydrochlorides, guanosine-5⬘-triphosphate sodium (GTP), and tris-(hydroxymethyl)-aminomethane (Tris) hydrochloride were from Sigma (St. Louis, MO). Animals were male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), initially weighing 200 to 225 g, maintained under artificial daylight (on, 7:00 AM–7:00 PM) in a temperatureand 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. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of McLean Hospital, in compliance with pertinent federal and state regulations. Drug Treatment and Tissue Preparation Four groups (N ⫽ 7) of rats received control vehicle, olanzapine (5.0 mg/kg/day), risperidone (3.0 mg/kg/day), or quetiapine fumarate (10.0 mg/kg/day) by osmotic minipumps (Alzet, Palo Alto, CA) implanted subcutaneously to ensure continuous and steady infusion of drugs and overcome variations in tissue drug levels that would result from daily injections. Doses are based on typical behaviorally active doses in rats (Moore et al., 1992; Ellenbroek et al., 1996). After 4 weeks of treatment, rats were decapitated; brains were removed, quick-frozen in isopentane on dry ice, and stored at ⫺80°C until autoradiographic analysis. Frozen coronal sections (10 m) were cut in a cryostat at ⫺20°C, mounted on gelatin-coated microscopic slides, and stored at ⫺80°C until use. Tissue sections were obtained from CPu, NAc, hippocampus (HIP); areas of cerebral cortex including dorsolateral-frontal (DFC) and mesioprefrontal (MPC), and entorhinal (EC) regions; islands of Calleja including the major island, and the olfactory tubercle. These selected cortical, limbic, and extrapyramidal brain regions mediate cognitive, emotional, and motor behaviors that are typically disturbed in patients with psychotic disorders and altered by antipsychotic drug treatment (Benes, 2000; Baldessarini and Tarazi, 2001). Receptor Autoradiography Brain sections from all drug-treated rats were evaluated at the same time in each radioreceptor assay to minimize experimental variability. Sections were first preincubated for 1 h at room temperature (RT) in 50 mM Tris-HCl buffer (pH 7.4) containing (mM): NaCl (120), KCl (5), CaCl2 (2), and MgCl2 (1), for the D1-like, D2, and D4 assays, or with slight modification for D3 assays (with 0.3 mM GTP, 40 mM NaCl, and no MgCl2 added). D1-Like Receptors. Rat forebrain sections were incubated for 1 h at RT in the incubating buffer containing 1 nM [3H]SCH-23390 with 100 nM ketanserin to block 5-HT2A/2C receptors. Nonspecific binding was determined with excess (1 M) cis-flupenthixol. After incubation, slides were washed twice for 5 min in ice-cold buffer, dipped in ice-cold water, and dried under a stream of air (Florijn et al., 1997; Tarazi et al., 1997a, 1998). D2 Receptors. Sections were incubated for 1 h at RT in the same buffer containing 1.0 nM [3H]nemonapride with 0.5 M DTG and 0.1 M pindolol to mask sigma (1,2) and 5-HT1A sites, respectively. Nonspecific binding was determined with 10 M S(⫺)-sulpiride. After incubation, slides were washed twice for 5 min in ice-cold buffer, dipped in ice-cold water, and air-dried (Tarazi et al., 1997b,c, 1998). Although the resulting radioligand binding may include traces of binding to D3 or D4 sites, most of the signal is believed to represent D2 receptors. Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017 as well as other typical and atypical antipsychotics, increased D4 receptor abundance in rat caudate-putamen (CPu) and nucleus accumbens septi (NAc) (Schoots et al., 1995; Florijn et al., 1997; Tarazi et al., 1997a,c, 1998) and enhanced D4 mRNA expression in monkey striatum (Lidow and GoldmanRakic, 1997). These agents also up-regulated D2 receptors in rat and monkey prefrontal cortex (Lidow and Goldman-Rakic, 1994; Florijn et al., 1997; Tarazi et al., 1997c, 1998). The findings support the view that D4 receptors in CPu and NAc, as well as D2 receptors in prefrontal cerebral cortex, are common sites of action of both typical and atypical antipsychotics, although the physiological consequences of these molecular changes remain incompletely defined. In contrast, typical neuroleptics, but not clozapine, also increased D2 receptor binding and expression in rat and monkey CPu (Lidow and Goldman-Rakic, 1997; Tarazi et al., 1997a,c, 1998). This selective increase in D2 receptor labeling in the extrapyramidal CPu probably reflects higher average occupancy of such receptor sites by typical neuroleptic agents and appears to parallel the risk of acute EPS as well as lateremerging tardive dyskinesia. Despite its favorable characteristics, clinical use of clozapine is complicated by its high risk of potentially fatal bone marrow toxicity, as well as excessive sedation and dosedependent risk of epileptic seizures (Baldessarini and Frankenburg, 1991). There is a keen interest in developing novel drugs with less adverse risk than clozapine but comparable antipsychotic effects. Several newer agents have emerged (Arnt and Skarsfeldt, 1998; Waddington and Casey, 2000; Baldessarini and Tarazi, 2001). Among them are the clozapine analogs olanzapine and quetiapine and the benzisoxazole derivative risperidone. Like clozapine, these compounds have multiple sites of molecular interaction and greater affinity for serotonin 5-HT2A than DA D2 receptors (Bymaster et al., 1996; Schotte et al., 1996; Gunasekara and Spencer, 1998). This receptor-interaction pattern may contribute to low EPS risk (Meltzer et al., 1989). Olanzapine, quetiapine, and risperidone have undergone extensive pharmacological and behavioral characterization in animals (Arnt and Skarsfeldt, 1998; Tarazi et al., 2000; Waddington and Casey, 2000), but their long-term effects on DA receptors in mammalian forebrain are not well defined or quantitatively compared with those of other antipsychotics. Accordingly, we applied quantitative in vitro receptor autoradiography to assess regulation of D1-like, D2, D3, and D4 receptors in different forebrain regions following long-term infusion of olanzapine, quetiapine, or risperidone in rats. Our working hypothesis was that these novel atypical antipsychotics would induce regionally selective changes in tissue levels of specific DA receptors more closely resembling those of clozapine than typical neuroleptics. Effects of Novel Antipsychotics on Dopamine Receptors D3 Receptors. Sections were preincubated for 1 h in Tris buffer modified as stated to minimize labeling of the high-affinity agonist binding state of D2 receptors, then incubated for 1 h in the same buffer containing 3 nM [3H]7-OH-DPAT, with 5 M DTG to mask sigma sites. Nonspecific binding was determined with 1 M S(⫺)eticlopride. After incubation, slides were washed twice for 3 min in ice-cold fresh buffer and dried (Tarazi et al., 1997c, 1998). D4 Receptors. Tissue sections were preincubated for 1 h at RT in the D2 assay buffer, and then for 1 h with 1.0 nM [3H]nemonapride, 300 nM S(⫺)-raclopride to occupy D2/D3 sites, and other masking agents (0.5 M DTG and 0.1 M pindolol) used in the D2 assay. Nonspecific binding was determined with 10 M S(⫺)-sulpiride. The highly D4-selective ligands L-745,870 and RBI-257 displaced approximately 80% of binding remaining in the presence of raclopride in adult CPu and NAc tissue, indicating that most of the racloprideinsensitive binding sites are D4 receptors (Tarazi et al., 1997b,c, 1998). Autoradiography and Image Analysis Given overall significance of effects for drug or brain region, post hoc Dunnett t tests were used to test for significant 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 ⫽ 7 subjects/treatment group. Results Four weeks of continuous infusion of all test agents failed to alter tissue concentrations of D1-like receptors in any brain region (Table 1). In contrast, olanzapine and risperidone significantly increased labeling of D2 receptors in several forebrain areas, including MPC (by 67 and 34%, respectively), NAc (37 and 28%), CPu (by a lateral and medial average of 42 and 25%), and HIP (53 and 30%), but did not alter D2 binding in DFC or EC (Table 2). Similar to D1-like receptors, there were no changes in D3-selective labeling in any brain region analyzed or with any agent (Table 3). Even more than signals representing mainly D2 receptors (uncorrected for overlap with D3 or D4 receptors), D4 labeling was up-regulated in several regions by treatment with olanzapine and risperidone, including NAc (by 65 and 33%, respectively), CPu (average of 61 and 37%), and HIP (61 and 37%), with no significant changes in several regions of cerebral cortex (Table 4). It is particularly noteworthy that quetiapine was anomalous and the only tested antipsychotic that did not alter expression of any DA receptor type in any brain region examined. Discussion Statistical Analysis Long-Term Effects of Newer Antipsychotics on the D1 Receptor Family Two-way analysis of variance (ANOVA) was employed to evaluate overall changes across treatments and brain regions for each assay. Continuous subcutaneous infusion of olanzapine, quetiapine, or risperidone did not alter binding of [3H]SCH-23390 in Fig. 1. Sites of autoradiographic analyses of rat brain regions sampled in 10-m coronal sections from A (A 3.2– 4.2), B (A 1.7–2.2), C (A 0.7–1.2), and D [A 0.2– 0.7 mm anterior to bregma, according to Paxinos and Watson (1982)]. CP, caudate-putamen (L, lateral; M, medial); ICj, islands of Calleja; ICjM, major island of Calleja; Olf Tub, olfactory tubercle. Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017 Radiolabeled slides and calibrated 3H-standards (Amersham) were exposed to Hyperfilm (Eastman Kodak) for 2 to 5 weeks at 4°C. [3H]SCH-23390- and [3H]nemonapride-labeled brain sections were exposed for 2 (CPu and NAc) or 5 weeks (cortex and HIP) and [3H]7-OH-DPAT for 4 weeks. 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. Catharines, 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 ⫽ 7 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. 713 714 Tarazi et al. TABLE 1 D1-like receptor binding after 4 weeks of continuous infusion of antipsychotic drugs Data are mean ⫾ S.E.M. values (N ⫽ 7 rats/group) for binding [fmol/mg of tissue and (percentage of control)], determined by quantitative autoradiography following continuous subcutaneous infusion of vehicle or antipsychotic drugs for 4 weeks, all as described under Experimental Procedures. No statistically significant drug effect was found. Brain Region Cerebral cortex Medial-prefrontal Dorsolateral Nucleus accumbens Caudate-putamen Medial Lateral Hippocampus Entorhinal cortex a Controls Olanzapine Risperidone Quetiapine Clozapinea Fluphenazinea 14.0 ⫾ 0.5 (100) 7.5 ⫾ 0.9 (100) 85.7 ⫾ 4.1 (100) 13.3 ⫾ 0.6 (95) 6.2 ⫾ 0.8 (82) 86.0 ⫾ 4.3 (100) 11.9 ⫾ 1.5 (85) 9.0 ⫾ 0.8 (120) 88.4 ⫾ 7.3 (103) 15.5 ⫾ 1.0 (107) 8.2 ⫾ 0.8 (109) 81.1 ⫾ 6.0 (95) (93) (100) (92) (102) (96) (96) 87.6 ⫾ 7.1 (100) 90.9 ⫾ 7.9 (100) 7.5 ⫾ 1.0 (100) 10.8 ⫾ 0.7 (100) 90.0 ⫾ 10.3 (103) 98.8 ⫾ 11.6 (109) 8.6 ⫾ 0.5 (115) 11.3 ⫾ 1.1 (105) 78.0 ⫾ 4.6 (99) 78.4 ⫾ 4.3 (103) 8.3 ⫾ 0.7 (111) 12.5 ⫾ 0.6 (116) 74.8 ⫾ 7.2 (85) 83.3 ⫾ 6.3 (92) 8.9 ⫾ 0.6 (119) 12.6 ⫾ 0.4 (117) (99) (91) (101) (104) Data (percentage of control) for clozapine (40 mg/kg/day) and fluphenazine (1 mg/kg/day) were determined previously (Tarazi et al., 1998) and are shown for comparison. TABLE 2 D2 receptor binding after 4 weeks of continuous infusion of antipsychotic drugs Brain Region Cerebral cortex Medial-prefrontal Dorsolateral Nucleus accumbens Caudate-putamen Medial Lateral Hippocampus Entorhinal cortex a Controls Olanzapine Risperidone Quetiapine Clozapinea Fluphenazinea 18.4 ⫾ 0.6 (100) 13.0 ⫾ 0.7 (100) 153.3 ⫾ 7.2 (100) 30.8 ⫾ 1.0 (167)* 14.9 ⫾ 0.5 (107) 210.4 ⫾ 14.4 (137)* 24.7 ⫾ 1.3 (134)* 12.6 ⫾ 0.4 (91) 196.8 ⫾ 9.8 (128)* 20.1 ⫾ 0.5 (109) 14.7 ⫾ 0.5 (106) 164.4 ⫾ 6.3 (107) (160)* (111) (103) (146)* (92) (167)* 169.9 ⫾ 8.5 (100) 224.5 ⫾ 12.1 (100) 35.3 ⫾ 1.0 (100) 15.1 ⫾ 1.0 (100) 244.9 ⫾ 21.7 (144)* 311.4 ⫾ 16.3 (139)* 54.4 ⫾ 3.5 (153)* 16.4 ⫾ 1.1 (108) 215.9 ⫾ 12.6 (127)* 276.2 ⫾ 18.0 (123)* 46.0 ⫾ 2.1 (130)* 15.4 ⫾ 1.2 (102) 168.1 ⫾ 11.1 (99) 231.4 ⫾ 17.4 (103) 37.5 ⫾ 2.6 (106) 16.9 ⫾ 0.4 (112) (104) (109) (126)* (117)* Data (percentage of control) for clozapine (40 mg/kg/day) and fluphenazine (1 mg/kg/day) were determined previously (Tarazi et al., 1997c) and are shown for comparison. TABLE 3 D3 receptor binding after 4 weeks of continuous infusion of antipsychotic drugs Data are mean ⫾ S.E.M. values (N ⫽ 7 rats/group) for binding [fmol/mg of tissue and (percentage of control)], determined by quantitative autoradiography following continuous subcutaneous infusion of vehicle or antipsychotic drugs for 4 weeks, all as described under Experimental Procedures. No statistically significant drug effect was found. Risperidone Quetiapine Clozapinea Fluphenazinea 32.8 ⫾ 1.9 (89) 17.9 ⫾ 1.7 (96) 34.5 ⫾ 1.7 (93) 20.0 ⫾ 1.3 (108) 36.5 ⫾ 1.8 (99) 18.8 ⫾ 1.0 (101) (97) (102) (100) (102) 17.9 ⫾ 0.7 (100) 7.9 ⫾ 0.5 (100) 18.7 ⫾ 1.3 (104) 8.5 ⫾ 0.4 (108) 17.4 ⫾ 0.6 (97) 9.0 ⫾ 0.6 (114) 19.7 ⫾ 0.8 (110) 8.8 ⫾ 0.3 (112) (93) (97) (108) 3.4 ⫾ 0.2 (100) 3.7 ⫾ 0.3 (100) 3.3 ⫾ 0.2 (97) 3.2 ⫾ 0.2 (86) 3.3 ⫾ 0.1 (97) 3.3 ⫾ 0.1 (89) 3.4 ⫾ 0.2 (100) 3.6 ⫾ 0.1 (97) (100) (100) (118) (118) Brain Region Controls Islands of Calleja Olfactory tubercle Nucleus accumbens Shell Core Caudate-putamen Medial Lateral 36.9 ⫾ 1.9 (100) 18.6 ⫾ 0.7 (100) a Olanzapine Data (percentage of control) for clozapine (40 mg/kg/day) and fluphenazine (1 mg/kg/day) were determined previously (Tarazi et al., 1997c) and are shown for comparison. any region of rat forebrain considered (Table 1). This radioligand labels both highly abundant D1 receptors and rarer D5 sites (Baldessarini and Tarazi, 1996). Lack of adaptive changes in D1-like binding with the three newer antipsychotic agents tested agrees with similar previous findings following treatment with dissimilar agents including haloperidol, fluphenazine, raclopride, and clozapine (Florijn et al., 1997; Tarazi et al., 1997a, 1998). Interest in D1-like receptors as potential targets for novel antipsychotic drugs was encouraged by findings in positron emission tomography studies that clozapine in clinically relevant doses occupied D1-like sites more than typical neuroleptics did, suggesting that D1-like receptors may contribute to the unique clinical properties of clozapine (Farde et al., 1989). However, clinical trials have yielded no support for the hypothesis that D1 antagonists may exert antipsychotic activity (Debeaurepaire et al., 1995). Lack of adaptive changes in D1-like receptor levels after prolonged administration of olanzapine, risperidone, or quetiapine (Table 1) as well as clozapine and typical neuroleptics (Florijn et al., 1997; Tarazi et al., 1997a, 1998) adds to the impression that these receptors are not likely to play a major role in mediating effects of antipsychotic agents. Nevertheless, open-minded caution is required concerning the pharmacological potential of D1 antagonists since current understanding of the physiology of these most abundant of the cerebral DA receptors remains remarkably limited (Baldessarini and Tarazi, 1996). Long-Term Effects of Olanzapine, Risperidone, and Quetiapine on the D2 Receptor Family D2 Receptors. D2 receptor potency of tested agents ranks risperidone (Ki ⫽ 6.0 nM) ⬎ olanzapine (Ki ⫽ 31 nM) ⬎ quetiapine (Ki ⫽ 380 nM) (Schotte et al., 1996). Infusion of olanzapine and risperidone, but not quetiapine, increased binding of [3H]nemonapride in MPC (Table 2). Such increases mainly reflect up-regulation of D2 receptors since Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017 Data are mean ⫾ S.E.M. values for binding [fmol/mg of tissue and (percentage of control)], determined by quantitative autoradiography following continuous subcutaneous infusion of vehicle or antipsychotic drugs for 4 weeks, with significant differences from controls indicated in bold (* p ⬍ 0.05, N ⫽ 7 rats/group), all as described under Experimental Procedures. 715 Effects of Novel Antipsychotics on Dopamine Receptors TABLE 4 D4 receptor binding after 4 weeks of continuous infusion of antipsychotic drugs Data are mean ⫾ S.E.M. values for binding [fmol/mg of tissue and (percentage of control)], determined by quantitative autoradiography following continuous subcutaneous infusion of vehicle or antipsychotic drugs for 4 weeks, with significant differences from controls indicated in bold (* p ⬍ 0.05, N ⫽ 7 rats/group), all as described under Experimental Procedures. Brain Region Cerebral cortex Medial-prefrontal Dorsolateral Nucleus accumbens Caudate-putamen Medial Lateral Hippocampus Entorhinal cortex a Controls Olanzapine Risperidone Quetiapine Clozapinea Fluphenazinea 10.0 ⫾ 0.6 (100) 6.3 ⫾ 0.3 (100) 29.3 ⫾ 1.1 (100) 11.3 ⫾ 0.8 (113) 6.8 ⫾ 0.4 (108) 48.2 ⫾ 2.8 (165)* 11.1 ⫾ 0.7 (111) 6.4 ⫾ 0.5 (102) 38.9 ⫾ 3.3 (133)* 10.8 ⫾ 0.3 (108) 6.0 ⫾ 0.6 (95) 28.6 ⫾ 3.3 (98) (117) (97) (171)* (106) (75) (224)* 31.4 ⫾ 2.4 (100) 37.8 ⫾ 1.6 (100) 18.0 ⫾ 0.5 (100) 6.9 ⫾ 0.6 (100) 50.2 ⫾ 3.6 (160)* 61.4 ⫾ 1.3 (162)* 29.0 ⫾ 1.6 (161)* 7.4 ⫾ 0.5 (107) 42.7 ⫾ 1.8 (136)* 51.6 ⫾ 2.6 (137)* 24.7 ⫾ 0.9 (137)* 7.3 ⫾ 0.5 (106) 29.1 ⫾ 2.9 (93) 41.9 ⫾ 4.6 (111) 19.1 ⫾ 1.5 (106) 8.2 ⫾ 0.3 (119) (143)* (143)* (160)* (163)* Data (percentage of control) for clozapine (40 mg/kg/day) and fluphenazine (1 mg/kg/day) were determined previously (Tarazi et al., 1997c) and are shown for comparison. well below risperidone and probably also olanzapine (Gunasekara and Spencer, 1998; Leucht et al., 1999; Baldessarini and Tarazi, 2001; Tarsy et al., 2001). The lack of clinical EPS with quetiapine is paralleled by its low D2 affinity (Schotte et al., 1996) and weak antagonism of behavioral effects of DA microinjected directly into rat CPu (Campbell et al., 1991). Risperidone shares many characteristics of typical neuroleptics, including severe hyperprolactinemia and dose-dependent EPS risk (Fleischhacker and Marder, 2000; Baldessarini and Tarazi, 2001; Tarsy et al., 2001). In addition to its ability to occupy striatal D2 receptors, olanzapine has relatively potent antimuscarinic properties compared with risperidone or quetiapine (Bymaster et al., 1996; Schotte et al., 1996) that probably limits its risk of EPS effects. Other studies using low daily doses of olanzapine (0.35–2 mg/kg) or risperidone (0.25– 0.5 mg/kg) did not find D2 receptor up-regulation in rat or monkey striatum (Kuoppamaki et al., 1995; Lidow and Goldman-Rakic, 1997; Kusumi et al., 2000). In addition, long-term treatment with low doses of olanzapine (0.5–2 mg/kg) resulted in lesser oral chewing movements (a proposed animal model of tardive dyskinesia) than did haloperidol (Gao et al., 1998). Nevertheless, the present findings with higher doses of constantly infused olanzapine and risperidone did include significant apparent upregulation of D2 receptors. Moreover, the present and previous results are consistent with clinical evidence that risks of acute EPS and of tardive dyskinesia with risperidone and olanzapine are substantially greater than with either quetiapine or clozapine (Tarsy et al., 2001). D3 Receptors. These low-abundance proteins have a restricted distribution, with notable levels of expression in mammalian basal forebrain, most prominently in the islands of Calleja, followed by olfactory tubercle and NAc shell, with very low levels in CPu (Sokoloff et al., 1990; Levant, 1997; Table 3). Binding of [3H]7-OH-DPAT under D3-selective conditions was unchanged after prolonged exposure to all test agents in all regions examined. Even risperidone, which has relatively high D3 affinity (Schotte et al., 1996), failed to alter expression of D3 receptors, even in the islands of Calleja and NAc (Table 3), as was found with various other antipsychotics (Levésque et al., 1995; Florijn et al., 1997; Tarazi et al., 1997a,c, 1998). Lack of response of D3 receptors to repeated antipsychotic treatment may reflect their unique molecular mechanisms, including absence of well defined interactions with G-proteins and signal transduction cascades (Sokoloff et al., 1990; Downloaded from jpet.aspetjournals.org at ASPET Journals on June 14, 2017 cortical D4 receptors did not increase significantly (Table 4), and D3 receptors are minimally expressed in rat frontal cortex (Sokoloff et al., 1990; Baldessarini and Tarazi, 1996). Similar D2 receptor up-regulation and increased D2 mRNA expression have been found in cerebral cortex of rats and nonhuman primates after repeated treatment with typical and atypical antipsychotics (Damask et al., 1996; Florijn et al., 1997; Lidow and Goldman-Rakic, 1997; Tarazi et al., 1997c, 1998). D2 receptor up-regulation in MPC by olanzapine and risperidone further supports the importance of D2 receptors in this region as a common target for both typical and atypical antipsychotics. Prolonged treatment with olanzapine and risperidone also enhanced binding to D2 receptors in HIP but neither in DFC nor EC (Table 2). Such enhancement of D2 expression in HIP probably reflects an increase in the transcriptional activity of the D2 receptor gene since repeated treatment with clozapine increased D2 mRNA expression in HIP but not EC (Ritter and Meador-Woodruff, 1997). D2 receptors in HIP, where DA innervation is limited, evidently are regulated differently from those in EC, where DA innervation is much denser (Goldsmith and Joyce, 1994). D2 receptors in HIP, but not EC, may act as additional common targets of olanzapine, risperidone, and perhaps other antipsychotics. Blockade and up-regulation of hippocampal D2 receptors by antipsychotics may contribute to improvement of delusions and hallucinations of psychotic patients by ameliorating DA hyperactivity postulated to occur in their HIP (Krieckhaus et al., 1992). Repeated treatment with olanzapine and risperidone, but not quetiapine, also increased D2 receptor labeling in CPu (Table 2). Similar responses have been found after long-term treatment with typical neuroleptics but not clozapine (Florijn et al., 1997; Tarazi et al., 1997a,c, 1998). Up-regulation of D2 receptors in CPu may disturb neurotransmission in circuits involved in regulating movement and posture (Albin et al., 1989) and generally correlates with EPS risk. Similar responses to newer antipsychotics (olanzapine, risperidone) with relatively low EPS risk were, therefore, unexpected. Additional indications that these drugs can occupy D2 receptors in human basal ganglia come from positron emission tomography studies showing that relatively high, but clinically encountered, doses of olanzapine and risperidone lead to occupation of striatal D2 receptors similar to that produced by typical neuroleptics and much more than that produced by clozapine (Farde et al., 1989; Kapur et al., 1999) or quetiapine (Gefvert et al., 1998). Quetiapine has the most benign clinical EPS risk of the three novel agents tested, ranking 716 Tarazi et al. Conclusions Similar to other typical and atypical antipsychotics, longterm treatment of rats with olanzapine and risperidone significantly up-regulated D2 receptors in MPC, as well as D4 receptors in NAc and CPu. These new findings add support to the hypothesis that these receptors and brain regions may be involved in the beneficial clinical effects of antipsychotics. In addition, both olanzapine and risperidone increased levels of D2 as well as D4 receptors in HIP (but not other cortical areas, including DFC and EC), suggesting a third possible site contributing to beneficial effects of atypical antipsychotics. At behaviorally effective doses, olanzapine and risperidone also increased abundance of D2 receptors in CPu, similar to the effects of long-term treatment with typical, but not atypical, antipsychotics. This finding parallels the moderate, dose-dependent risk of clinical EPS with these agents, consistent with their status as quantitatively atypical agents (Baldessarini and Tarazi, 2001; Tarsy et al., 2001). Lack of effects of olanzapine and risperidone on D1-like or D3 receptors in any brain region examined adds support to the view that these receptors are probably not prominently involved in the actions of various kinds of antipsychotics. Failure of quetiapine to alter abundance of any DA receptor type in any brain region examined is consistent with its low affinity for all DA receptors and suggests that nondopaminergic, and perhaps serotonergic or histaminergic, mechanisms may contribute to the clinical actions of this novel agent. Acknowledgments Drug substances were generous gifts of Lilly, Janssen, Lundbeck, and Zeneca Corporations. References Albin RL, Young AB and Penney JB (1989) The functional anatomy of basal ganglia disorders. Trends Neurosci 12:366 –375. 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Alternatively, the high avidity of D3 receptors for DA and their selective protection from alkylation by very low concentrations of DA (Zhang et al., 1999) suggests that occupation by endogenous DA may limit their interaction with potential antagonists to preclude up-regulation. It also follows that D3 receptors are less likely to be critical for the actions of known or novel antipsychotics. Instead, D3 receptors may be involved in stimulant reward and dependence (Pilla et al., 1999). D4 Receptors. As in previous studies (Tarazi et al., 1997b,c, 1998), D4 receptors accounted for relatively high proportions (46 –54%) of total D2-like receptors in MPC, DFC, HIP, and EC, but much less (17–19%) in CPu and NAc (Tables 2 and 4). Prolonged administration of olanzapine and risperidone, but not quetiapine, significantly increased D4 receptors in CPu and NAc (Table 4), presumably reflecting adaptive responses to D4 blockade since both olanzapine and risperidone have much higher D4 affinity than quetiapine (Schotte et al., 1996). D4 receptors were found to increase in rat CPu and NAc after long-term administration of several typical and atypical antipsychotics (Florijn et al., 1997; Tarazi et al., 1997a,c, 1998), as well as olanzapine and risperidone (Table 4), supporting the impression that striatolimbic D4 receptors represent a common target of dissimilar antipsychotics. Lack of D4 up-regulation by quetiapine may, again, reflect its low affinity for all D2-like receptors (Schotte et al., 1996; Baldessarini and Tarazi, 2001). D4 up-regulation is also consistent with the proposal that reported increases in D4-like labeling in postmortem striatum tissue of patients diagnosed with schizophrenia (Seeman et al., 1993; Murray et al., 1995) may reflect responses to antemortem antipsychotic treatment rather than a neuropathology of schizophrenia (Baldessarini and Tarazi, 2001). Long-term treatment with olanzapine and risperidone increased D4 receptor labeling in HIP and not EC (Table 4), similar to responses of D2 receptors (Table 2). This is the first evidence that D4 receptors in HIP can be affected by novel antipsychotics. Moreover, these receptors may act in synchrony with D2 receptors in HIP to mediate beneficial clinical actions of antipsychotics. Increased D4 binding probably reflects post-transcriptional modifications or reduced D4 receptor turnover since D4 mRNA expression was decreased after repeated treatment of rats with haloperidol or clozapine (Ritter and Meador-Woodruff, 1997). Lack of response of D4 receptors in EC to antipsychotic treatment (Table 4) parallels the nonresponse of D2 receptors in that relatively well DAinnervated region (Table 2). In spite of their profound up-regulating effects on D4 receptor proteins in CPu and NAc, in frontal cerebral cortex, olanzapine and risperidone had only minor effects, and quetiapine had none (Table 4). Other antipsychotics also have had little effect on rat cortical D4 receptors (Tarazi et al., 1997a,c). Regional differences in the regulation of D4 mRNA transcription or protein synthesis may account for regional differences in adaptive increases of D4 receptors and in responses to typical versus atypical antipsychotics. Consistent with this interpretation is a lack of increase in D4 mRNA or receptor protein in rat cerebral cortex after repeated treatment with clozapine but significant increases in both with haloperidol (Schoots et al., 1995). 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