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Olanzapine, quetiapine, and risperidone: long-term effects on monoamine transporters in rat forebrain
Neuroscience Letters 287 (2000) 81±84
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Olanzapine, quetiapine, and risperidone: long-term effects on monoamine transporters in rat forebrain Frank I. Tarazi a,b,*, Kehong Zhang a,b, Ross J. Baldessarini a,b a Mailman Research Center, McLean Division of Massachusetts General Hospital, Belmont, MA 02478, USA Consolidated Department of Psychiatry and Neuroscience Program, Harvard Medical School, Boston, MA 02115, USA
b
Received 7 March 2000; received in revised form 10 April 2000; accepted 20 April 2000
Abstract Long-term effects of novel atypical antipsychotic drugs on monoamine transporters are unknown. We compared labeling of dopamine (DAT) and serotonin (SERT) transporter proteins in subregions of rat corpus striatum by quantitative autoradiography with [ 3H]2-b-carbomethoxy-3-b-[4 0 ±iodophenyl]tropane ([ 3H]b-CIT) and [ 3H]paroxetine after 28 days of continuous subcutaneous infusion of olanzapine, quetiapine, risperidone, or vehicle controls. Drug treatment did not signi®cantly alter the abundance of either transporter type in caudate-putamen or nucleus accumbens, indicating that transporter proteins required to inactivate synaptically released dopamine and serotonin resist adaptations to longterm treatment with novel antipsychotics that affect neurotransmission by these amines. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Accumbens; Autoradiography; Atypical antipsychotic drugs; Caudate nucleus; Dopamine; Serotonin; Transporters
Dopamine (DAT) and serotonin (SERT) transporters are large membrane proteins that terminate DA and 5-HT neurotransmission by uptake of the released monoamines into their presynaptic nerve endings [2]. These transporters are implicated in neuropsychiatric disorders including attention de®cit-hyperactivity disorder [9], Tourette's syndrome [17], and major mood disorders [7], and they are important targets for antidepressant [4] and stimulant drugs [2]. However, their responses to long-term treatment with novel antipsychotic drugs are not well de®ned. Treatment for several weeks with the typical neuroleptics haloperidol and raclopride or the atypical antipsychotic agent clozapine did not alter radioligand binding to DAT in several rat brain regions [1,3]. Several antipsychotics, including chlorpromazine, haloperidol and clozapine, showed moderate ability to block DA uptake by genetically transfected cells selectively expressing DAT [14]. Repeated treatment with haloperidol or clozapine did not alter binding of [ 3H]paroxetine to SERT in hippocampal membranes [15], but similar treatment of rats with both drugs increased [ 3H]citalopram binding to SERT in rat
* Corresponding author. Tel.: 11-617-855-3176; fax: 11-617855-3479. E-mail address: [email protected] (F.I. Tarazi).
ventral neostriatum [3]. In addition, treatment with clozapine but not haloperidol led to decreased [ 3H]citalopram binding to SERT in substantia nigra and increases in nucleus raphe dorsalis [3]. Postmortem studies of DAT and SERT levels in brain tissue from psychiatric patients have also yielded inconsistent ®ndings. Density and af®nity of DAT did not differ in postmortem striatal tissue from patients with schizophrenia and normal controls [16]. However, synaptosomal uptake of [ 3H]DA was increased in fresh nucleus accumbens tissue obtained at death from previously medicated schizophrenic patients [10]. An autoradiographic study of [ 3H]cyanoimipramine binding to SERT revealed increases in striatum and decreases in frontal and cingulate cortices of schizophrenic patients compared to non-neurological controls [11]. Another study found reduced binding of [ 3H]paroxetine to SERT in postmortem frontal cerebral cortex of schizophrenics [13]. Yet another similar study using [ 3H]paroxetine found no change in SERT density in frontal cortex or corpus striatum, but found decreased af®nity (increased Kd) of this radioligand in hippocampus [8]. These inconsistent ®ndings with DAT and SERT levels in brain tissue from schizophrenic patients may re¯ect procedural or technical differences between studies. Alternatively, antemortem treatment of schizophrenic patients with different types of antipsychotics may have contributed to the variable ®ndings reported.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 13 0- 7
82
F.I. Tarazi et al. / Neuroscience Letters 287 (2000) 81±84
Therefore, examining the long-term effects of antipsychotic drugs on monoamine transporters in rat forebrain regions should reveal if the reported abnormalities in DAT and SERT in patients with schizophrenia are due to drug treatment or components of the neuropathology of the disease. We quanti®ed the abundance of DAT and SERT in rat caudate-putamen (CPu) and nucleus accumbens septi (NAc) following four weeks of treatment with representative newer antipsychotics, olanzapine, quetiapine and risperidone. These agents have prominent pharmacological effects on cerebral DA and 5-HT neurons [6]. We hypothesized that DAT and SERT might respond adaptively to long-term effects of these drugs. Radioligands, [ 3H]2-b-carbomethoxy-3-b-[4 0 ±iodophenyl]tropane ([ 3H]b-CIT; 87 Ci/mmol) and [phenyl±6± 3H](3Strans)-3-([1,3±benzodioxol±5±yl±oxy]methyl)-4-(4-¯uorophenyl)-piperidine ([3H]paroxetine; 16.5 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Tritium autoradiography standards were from Amersham (Arlington Heights, IL). Tritium-sensitive Hyper®lm and D-19 photographic developer and ®xative were from Eastman±Kodak (Rochester, NY). Olanzapine and ¯uoxetine were donated by Eli Lilly (Indianapolis, IN). Risperidone was donated by Janssen (Beerse, Belgium), and quetiapine fumarate was a gift from Zeneca (Cheshire, UK). Lundbeck (Copenhagen, Denmark) donated cis-¯upenthixol. GBR-12909 dihydrochloride was obtained from Research Biochemicals International (RBI; Natick, MA). Cation hydrochlorides, and Tris(hydroxymethyl)aminomethane hydrochloride (Tris), were obtained from Sigma Chemicals (St. Louis, MO). Young adult, male Sprague±Dawley rats (Charles River, Wilmington, MA) initially weighing 200±225 g, were maintained in arti®cial daylight (on, 07:00±19:00 h) at controlled temp. and humidity with free access to standard rat chow and tapwater in a USDA-inspected, veterinarian-supervised, small-animal research facility of the Mailman Research Center of McLean Hospital, with approval by its Institutional Animal Care and Use Committee (IACUC), in accord with federal guidelines. Care was taken to minimize distress and numbers of animals used. Four groups of N 7 rats received either vehicle or olanzapine (5.0 mg/kg per day), quetiapine fumarate (10.0 mg/kg per day), or risperidone (3.0 mg/kg per day) by osmotic minipumps (Alzet, Palo Alto, CA) implanted subcutaneously, to ensure continuous drug infusion. This method should overcome differences in drug metabolism with variations in drug plasma levels, as well as stress effects that may result from daily drug injections. After 4 weeks, rats were decapitated and brains were quickly removed, frozen in isopentane on dry ice, and stored at 2808C until autoradiographic analysis. Frozen coronal sections (10 mm) representing medial and lateral CPu and NAc were cut in a cryostat at 2208C, mounted on gelatincoated microscopic slides, and stored at 2808C until use. For DAT autoradiography, sections were preincubated (60 min, room temp.) in 50 mM Tris±HCl buffer (pH 7.7) containing 120 mM NaCl and 4 mM MgCl2, then in fresh
buffer (60 min) containing 2 nM [ 3H]b-CIT. Non-speci®c binding was de®ned with 1 mM GBR-12909 [12]. Slides were washed twice for 5 min in ice-cold fresh buffer, dipped in ice-cold water, and air-dried. For SERT autoradiography, tissue sections were preincubated (60 min, room temperature) in 50 mM Tris±HCl buffer (pH 7.7) containing 120 mM NaCl and 5 mM KCl, then in fresh buffer (60 min) containing 4 nM [ 3H]paroxetine. Non-speci®c binding was determined with 30 mM (^)-¯uoxetine hydrochloride [18]. Slides were washed twice for 20 min in ice-cold buffer, dipped in ice-cold water, and air-dried. Radiolabeled slides and calibrated [ 3H]standards were exposed to Hyper®lm for 10 (DAT) or 30 days (SERT) at 48C, and then developed and ®xed at room temperature. The ®lms were analyzed by computed densitometry, with levels of transporter labeling expressed as the means ^ SEM in fmol/mg tissue. Statistical comparisons were carried out by two-way (by region and treatment) analysis of variance (ANOVA), all as detailed elsewhere [18]. Substantial levels of [ 3H]b-CIT binding to DAT were detected in CPu and NAc of vehicle-treated rats (48±69 fmol/mg tissue; Table 1A). High levels of SERT were also found in CPu and NAc of controls (116±124 fmol/mg tissue; Table 1B), consistent with previous observations [3]. Four weeks of continuous infusion of olanzapine, quetiapine, and risperidone had no signi®cant effect on the binding of [ 3H]b-CIT to DAT (Table 1A) or [ 3H]paroxetine to SERT (Table 1B) in medial or lateral CPu or NAc (all ANOVA yielded P . 0:10). Tissue concentrations of DAT and SERT remained unchanged after long-term administration of all three experimental agents. In contrast, identical treatment with olanzapine and risperidone, but not quetiapine, signi®cantly increased the density of labeling DA D2 and D4 receptors in the same forebrain regions [19]. These receptor changes, as well as the established behavioral effectiveness of all drug doses used, indicate that the treatment regimen used was effective in altering monoamine neurotransmission in rat forebrain. In general, monoamine transporters have poorly or inconsistently responded to drug treatments that alter their function or strongly in¯uence the abundance and function of their monoamine substrates. Such resistance to druginduced changes may re¯ect the complex molecular structure of the large monoamine transporter proteins, in which critical reactive groups may be shielded from some drug interactions by facing intracellularly or by binding endogenous molecules [2]. DA and 5-HT receptors are smaller membrane proteins whose reactive regions may be more accessible to antipsychotic drugs, which readily alter their abundance [5]. Alternatively, lack of changes in levels of DAT and SERT in corpus striatum after prolonged treatment with the three atypical antipsychotics tested may re¯ect their reported low af®nity (IC50 . 1 mM) at both transporters selectively expressed in genetically transfected cell lines [20].
F.I. Tarazi et al. / Neuroscience Letters 287 (2000) 81±84
83
Table 1 Binding to monoamine transporters after four weeks of antipsychotic drug treatment a Tissue
Control
Olanzapine
Quetiapine
Risperidone
48.6 ^ 2.9 58.3 ^ 3.2 69.0 ^ 1.7
51.8 ^ 3.3 63.9 ^ 3.4 72.6 ^ 2.7
47.2 ^ 3.1 60.4 ^ 3.9 67.6 ^ 3.3
53.7 ^ 2.9 62.1 ^ 3.1 71.7 ^ 4.0
124.3 ^ 5.2 116.6 ^ 7.3 125.7 ^ 6.4
123.7 ^ 7.3 109.0 ^ 7.3 116.3 ^ 9.3
122.7 ^ 7.1 114.1 ^ 5.5 133.1 ^ 6.4
116.7 ^ 5.6 112.9 ^ 9.9 122.0 ^ 5.9
3
A. Dopamine Transporter ([ H]b-CIT) Nucleus accumbens Medial caudate-putamen Lateral caudate-putamen B. Serotonin Transporter ([ 3H]paroxetine) Nucleus accumbens Medial caudate-putamen Lateral caudate-putamen
a Data are mean ^ SEM (N 7 rats/group) and expressed in fmol/mg tissue. Doses (mg/kg per day): olanzapine (5.0), quetiapine (10.0), risperidone (3.0). No difference from vehicle controls is signi®cant.
In conclusion, tissue levels of DAT and SERT in subregions of rat striatum were unaltered by long-term exposure to three novel antipsychotic drugs with prominent effects on both DA and 5-HT neurotransmission [6,19]. These ®ndings further support the impression that monoamine transporter proteins are regulated differently from receptors, and suggest that DAT and SERT are unlikely to be involved in long-term adaptations to antipsychotic drug actions on DA and 5-HT neurotransmission. Supported by a research award from Eli Lilly Corporation (FIT), NIH grants MH-34006 and MH-47370, a grant from the Bruce J. Anderson Foundation, and funds of the McLean Private Donors Neuropharmacology Research Fund (RJB). Drugs were generously provided by Eli Lilly, Janssen, Lundbeck and Zeneca pharmaceutical corporations. [1] Allard, P., Eriksson, K., Ross, S.B. and Marcusson, J.O., Unaltered [ 3H]GBR-12935 binding after chronic treatment with dopamine active drugs, Psychopharmacology, 102 (1990) 291±294. [2] Amara, S.G. and Kuhar, M.J., Neurotransmitter transporters: recent progress, Ann. Rev. Neurosci., 16 (1993) 73±93. [3] Ase, A.R., Amdiss, F., Hebert, C., Huang, N., van Gelder, N.M. and Reader, T.A., Effects of antipsychotic drugs on dopamine and serotonin contents and metabolites, dopamine and serotonin transporters, and serotonin1A receptors, J. Neural Transm., 106 (1999) 75±105. [4] Baldessarini, R.J., Drugs and the treatment of psychiatric disorders. Depression and mania, In J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, and A.G. Gilman (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, Chapter 19, McGraw-Hill, New York, 1996, pp. 431±459. [5] Baldessarini, R.J. and Tarazi, F.I., Brain dopamine receptors: a primer on their current status, basic and clinical, Harvard Rev. Psychiatry, 3 (1996) 301±325. [6] Baldessarini, R.J. and Tarazi, F.I., Drugs and the treatment of psychiatric disorders. Antipsychotic agents, In J.G. Hardman, L.E. Limbird, P.B. Molinoff, R.W. Ruddon, and A.G. Gilman (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, Chapter 20, McGraw-Hill, New York, 2000 (in press). [7] Collier, D.A., Arranz, M.J., Sham, P., Battersby, S., Vallada,
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
H., Gill, P., Aitchison, K.J., Sodhi, M., Li, T., Roberts, G.W., Smith, B., Morton, J., Murray, R.M., Smith, D. and Kirov, G., The serotonin transporter is a potential susceptibility for bipolar affective disorder, NeuroReport, 7 (1996) 1675± 1679. Dean, B., Opeskin, K., Pavey, G., Naylor, L., Hill, C., Keks, N. and Copolov, D.L., [ 3H]Paroxetine binding is altered in the hippocampus but not the frontal cortex or caudate nucleus from subjects with schizophrenia, J. Neurochem., 64 (1995) 1197±1202. Dougherty, D.D., Bonab, A.A., Spencer, T.J., Rauch, S.L., Madras, B.K. and Fischman, A.J., Dopamine transporter density in patients with attention de®cit hyperactivity disorder, Lancet, 354 (1999) 2132±2133. Habeland, N. and Hetey, L., Studies in postmortem dopamine uptakeII. Alteration of the synaptosomal catecholamine uptake in postmortem brain region in schizophrenia, J. Neural Transm., 68 (1987) 303±313. Joyce, J., Shane, A., Lexow, N., Winkour, A. and Casanova, M.F., Serotonin uptake sites and serotonin receptors are altered in the limbic system of schizophrenics, Neuropsycho-pharmacology, 8 (1993) 315±336. Kula, N.S., Baldessarini, R.J., Tarazi, F.I., Fisser, R., Wang, S., Trometer, J. and Neumeyer, J.L., [ 3H]b-CIT: a radioligand for dopamine transporters in rat brain tissue, Eur. J. Pharmacol., 385 (1999) 291±294. Laruelle, M., Abi-Dargham, A., Casanova, M.F., Toti, R., Weinberger, D.R. and Kleinman, J.F., Selective abnormalities of prefrontal serotonergic receptors in schizophrenia, Arch. Gen. Psychiatry, 50 (1993) 810±818. Lee, S-H., Oh, D-Y., Jung, S-C., Kim, Y-M., Cho, H-K., Koh, JK. and Lee, Y-S., Neuroleptic drugs alter the dopamine transporter-mediated uptake and release of dopamine: a possible mechanism for drug-induced tardive dyskinesia, Pharmacol. Res., 35 (1997) 447±450. Naylor, L., Dean, B., Opeskin, K., Pavey, G., Hill, C., Neks, N. and Copolov, D., Changes in the serotonin transporter in the hippocampus of subjects with schizophrenia identi®ed using [ 3H]paroxetine, J. Neural Transm., 103 (1996) 749±757. Pearce, R.K.B., Seeman, P., Jellinger, K. and Tourtellotte, W.W., Dopamine uptake sites and dopamine receptors in Parkinson's disease and schizophrenia, Eur. Neurol., 30 (1990) 9±14. Singer, H.S., Hahn, I.-H. and Moran, T.H., Abnormal dopamine uptake sites in postmortem striatum from patients with Tourette's syndrome, Ann. Neurol., 30 (1991) 558± 562. Tarazi, F.I., Tomasini, E.C. and Baldessarini, R.J., Postnatal
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F.I. Tarazi et al. / Neuroscience Letters 287 (2000) 81±84
development of dopamine and serotonin transporters in rat caudate-putamen and nucleus accumbens septi, Neurosci. Lett., 254 (1998) 21±24. [19] Tarazi, F.I., Zhang, K. and Baldessarini, R.J., Long-term effects of olanzapine, risperidone, and quetiapine on corti-
cal and striatolimbic dopamine D2-like and D4 receptors, Soc. Neurosci. Abstr., 25 (1999) 1570. [20] Tatsumi, M., Jansen, K., Blakely, R.D. and Richelson, E., Pharmacological pro®le of neuroleptics at human monoamine transporters, Eur. J. Pharmacol., 368 (1999) 277±283.
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Olanzapine, quetiapine, and risperidone: long-term effects on monoamine transporters in rat forebrain Frank I. Tarazi a,b,*, Kehong Zhang a,b, Ross J. Baldessarini a,b a Mailman Research Center, McLean Division of Massachusetts General Hospital, Belmont, MA 02478, USA Consolidated Department of Psychiatry and Neuroscience Program, Harvard Medical School, Boston, MA 02115, USA
b
Received 7 March 2000; received in revised form 10 April 2000; accepted 20 April 2000
Abstract Long-term effects of novel atypical antipsychotic drugs on monoamine transporters are unknown. We compared labeling of dopamine (DAT) and serotonin (SERT) transporter proteins in subregions of rat corpus striatum by quantitative autoradiography with [ 3H]2-b-carbomethoxy-3-b-[4 0 ±iodophenyl]tropane ([ 3H]b-CIT) and [ 3H]paroxetine after 28 days of continuous subcutaneous infusion of olanzapine, quetiapine, risperidone, or vehicle controls. Drug treatment did not signi®cantly alter the abundance of either transporter type in caudate-putamen or nucleus accumbens, indicating that transporter proteins required to inactivate synaptically released dopamine and serotonin resist adaptations to longterm treatment with novel antipsychotics that affect neurotransmission by these amines. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Accumbens; Autoradiography; Atypical antipsychotic drugs; Caudate nucleus; Dopamine; Serotonin; Transporters
Dopamine (DAT) and serotonin (SERT) transporters are large membrane proteins that terminate DA and 5-HT neurotransmission by uptake of the released monoamines into their presynaptic nerve endings [2]. These transporters are implicated in neuropsychiatric disorders including attention de®cit-hyperactivity disorder [9], Tourette's syndrome [17], and major mood disorders [7], and they are important targets for antidepressant [4] and stimulant drugs [2]. However, their responses to long-term treatment with novel antipsychotic drugs are not well de®ned. Treatment for several weeks with the typical neuroleptics haloperidol and raclopride or the atypical antipsychotic agent clozapine did not alter radioligand binding to DAT in several rat brain regions [1,3]. Several antipsychotics, including chlorpromazine, haloperidol and clozapine, showed moderate ability to block DA uptake by genetically transfected cells selectively expressing DAT [14]. Repeated treatment with haloperidol or clozapine did not alter binding of [ 3H]paroxetine to SERT in hippocampal membranes [15], but similar treatment of rats with both drugs increased [ 3H]citalopram binding to SERT in rat
* Corresponding author. Tel.: 11-617-855-3176; fax: 11-617855-3479. E-mail address: [email protected] (F.I. Tarazi).
ventral neostriatum [3]. In addition, treatment with clozapine but not haloperidol led to decreased [ 3H]citalopram binding to SERT in substantia nigra and increases in nucleus raphe dorsalis [3]. Postmortem studies of DAT and SERT levels in brain tissue from psychiatric patients have also yielded inconsistent ®ndings. Density and af®nity of DAT did not differ in postmortem striatal tissue from patients with schizophrenia and normal controls [16]. However, synaptosomal uptake of [ 3H]DA was increased in fresh nucleus accumbens tissue obtained at death from previously medicated schizophrenic patients [10]. An autoradiographic study of [ 3H]cyanoimipramine binding to SERT revealed increases in striatum and decreases in frontal and cingulate cortices of schizophrenic patients compared to non-neurological controls [11]. Another study found reduced binding of [ 3H]paroxetine to SERT in postmortem frontal cerebral cortex of schizophrenics [13]. Yet another similar study using [ 3H]paroxetine found no change in SERT density in frontal cortex or corpus striatum, but found decreased af®nity (increased Kd) of this radioligand in hippocampus [8]. These inconsistent ®ndings with DAT and SERT levels in brain tissue from schizophrenic patients may re¯ect procedural or technical differences between studies. Alternatively, antemortem treatment of schizophrenic patients with different types of antipsychotics may have contributed to the variable ®ndings reported.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 13 0- 7
82
F.I. Tarazi et al. / Neuroscience Letters 287 (2000) 81±84
Therefore, examining the long-term effects of antipsychotic drugs on monoamine transporters in rat forebrain regions should reveal if the reported abnormalities in DAT and SERT in patients with schizophrenia are due to drug treatment or components of the neuropathology of the disease. We quanti®ed the abundance of DAT and SERT in rat caudate-putamen (CPu) and nucleus accumbens septi (NAc) following four weeks of treatment with representative newer antipsychotics, olanzapine, quetiapine and risperidone. These agents have prominent pharmacological effects on cerebral DA and 5-HT neurons [6]. We hypothesized that DAT and SERT might respond adaptively to long-term effects of these drugs. Radioligands, [ 3H]2-b-carbomethoxy-3-b-[4 0 ±iodophenyl]tropane ([ 3H]b-CIT; 87 Ci/mmol) and [phenyl±6± 3H](3Strans)-3-([1,3±benzodioxol±5±yl±oxy]methyl)-4-(4-¯uorophenyl)-piperidine ([3H]paroxetine; 16.5 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Tritium autoradiography standards were from Amersham (Arlington Heights, IL). Tritium-sensitive Hyper®lm and D-19 photographic developer and ®xative were from Eastman±Kodak (Rochester, NY). Olanzapine and ¯uoxetine were donated by Eli Lilly (Indianapolis, IN). Risperidone was donated by Janssen (Beerse, Belgium), and quetiapine fumarate was a gift from Zeneca (Cheshire, UK). Lundbeck (Copenhagen, Denmark) donated cis-¯upenthixol. GBR-12909 dihydrochloride was obtained from Research Biochemicals International (RBI; Natick, MA). Cation hydrochlorides, and Tris(hydroxymethyl)aminomethane hydrochloride (Tris), were obtained from Sigma Chemicals (St. Louis, MO). Young adult, male Sprague±Dawley rats (Charles River, Wilmington, MA) initially weighing 200±225 g, were maintained in arti®cial daylight (on, 07:00±19:00 h) at controlled temp. and humidity with free access to standard rat chow and tapwater in a USDA-inspected, veterinarian-supervised, small-animal research facility of the Mailman Research Center of McLean Hospital, with approval by its Institutional Animal Care and Use Committee (IACUC), in accord with federal guidelines. Care was taken to minimize distress and numbers of animals used. Four groups of N 7 rats received either vehicle or olanzapine (5.0 mg/kg per day), quetiapine fumarate (10.0 mg/kg per day), or risperidone (3.0 mg/kg per day) by osmotic minipumps (Alzet, Palo Alto, CA) implanted subcutaneously, to ensure continuous drug infusion. This method should overcome differences in drug metabolism with variations in drug plasma levels, as well as stress effects that may result from daily drug injections. After 4 weeks, rats were decapitated and brains were quickly removed, frozen in isopentane on dry ice, and stored at 2808C until autoradiographic analysis. Frozen coronal sections (10 mm) representing medial and lateral CPu and NAc were cut in a cryostat at 2208C, mounted on gelatincoated microscopic slides, and stored at 2808C until use. For DAT autoradiography, sections were preincubated (60 min, room temp.) in 50 mM Tris±HCl buffer (pH 7.7) containing 120 mM NaCl and 4 mM MgCl2, then in fresh
buffer (60 min) containing 2 nM [ 3H]b-CIT. Non-speci®c binding was de®ned with 1 mM GBR-12909 [12]. Slides were washed twice for 5 min in ice-cold fresh buffer, dipped in ice-cold water, and air-dried. For SERT autoradiography, tissue sections were preincubated (60 min, room temperature) in 50 mM Tris±HCl buffer (pH 7.7) containing 120 mM NaCl and 5 mM KCl, then in fresh buffer (60 min) containing 4 nM [ 3H]paroxetine. Non-speci®c binding was determined with 30 mM (^)-¯uoxetine hydrochloride [18]. Slides were washed twice for 20 min in ice-cold buffer, dipped in ice-cold water, and air-dried. Radiolabeled slides and calibrated [ 3H]standards were exposed to Hyper®lm for 10 (DAT) or 30 days (SERT) at 48C, and then developed and ®xed at room temperature. The ®lms were analyzed by computed densitometry, with levels of transporter labeling expressed as the means ^ SEM in fmol/mg tissue. Statistical comparisons were carried out by two-way (by region and treatment) analysis of variance (ANOVA), all as detailed elsewhere [18]. Substantial levels of [ 3H]b-CIT binding to DAT were detected in CPu and NAc of vehicle-treated rats (48±69 fmol/mg tissue; Table 1A). High levels of SERT were also found in CPu and NAc of controls (116±124 fmol/mg tissue; Table 1B), consistent with previous observations [3]. Four weeks of continuous infusion of olanzapine, quetiapine, and risperidone had no signi®cant effect on the binding of [ 3H]b-CIT to DAT (Table 1A) or [ 3H]paroxetine to SERT (Table 1B) in medial or lateral CPu or NAc (all ANOVA yielded P . 0:10). Tissue concentrations of DAT and SERT remained unchanged after long-term administration of all three experimental agents. In contrast, identical treatment with olanzapine and risperidone, but not quetiapine, signi®cantly increased the density of labeling DA D2 and D4 receptors in the same forebrain regions [19]. These receptor changes, as well as the established behavioral effectiveness of all drug doses used, indicate that the treatment regimen used was effective in altering monoamine neurotransmission in rat forebrain. In general, monoamine transporters have poorly or inconsistently responded to drug treatments that alter their function or strongly in¯uence the abundance and function of their monoamine substrates. Such resistance to druginduced changes may re¯ect the complex molecular structure of the large monoamine transporter proteins, in which critical reactive groups may be shielded from some drug interactions by facing intracellularly or by binding endogenous molecules [2]. DA and 5-HT receptors are smaller membrane proteins whose reactive regions may be more accessible to antipsychotic drugs, which readily alter their abundance [5]. Alternatively, lack of changes in levels of DAT and SERT in corpus striatum after prolonged treatment with the three atypical antipsychotics tested may re¯ect their reported low af®nity (IC50 . 1 mM) at both transporters selectively expressed in genetically transfected cell lines [20].
F.I. Tarazi et al. / Neuroscience Letters 287 (2000) 81±84
83
Table 1 Binding to monoamine transporters after four weeks of antipsychotic drug treatment a Tissue
Control
Olanzapine
Quetiapine
Risperidone
48.6 ^ 2.9 58.3 ^ 3.2 69.0 ^ 1.7
51.8 ^ 3.3 63.9 ^ 3.4 72.6 ^ 2.7
47.2 ^ 3.1 60.4 ^ 3.9 67.6 ^ 3.3
53.7 ^ 2.9 62.1 ^ 3.1 71.7 ^ 4.0
124.3 ^ 5.2 116.6 ^ 7.3 125.7 ^ 6.4
123.7 ^ 7.3 109.0 ^ 7.3 116.3 ^ 9.3
122.7 ^ 7.1 114.1 ^ 5.5 133.1 ^ 6.4
116.7 ^ 5.6 112.9 ^ 9.9 122.0 ^ 5.9
3
A. Dopamine Transporter ([ H]b-CIT) Nucleus accumbens Medial caudate-putamen Lateral caudate-putamen B. Serotonin Transporter ([ 3H]paroxetine) Nucleus accumbens Medial caudate-putamen Lateral caudate-putamen
a Data are mean ^ SEM (N 7 rats/group) and expressed in fmol/mg tissue. Doses (mg/kg per day): olanzapine (5.0), quetiapine (10.0), risperidone (3.0). No difference from vehicle controls is signi®cant.
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