Differential regulation of dopamine receptors after chronic typical and atypical antipsychotic drug treatment

Pergamon

PII:

Neuroscience Vol. 78, No. 4, pp. 985–996, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(96)00631-8

DIFFERENTIAL REGULATION OF DOPAMINE RECEPTORS AFTER CHRONIC TYPICAL AND ATYPICAL ANTIPSYCHOTIC DRUG TREATMENT F. I. TARAZI,*† W. J. FLORIJN‡ and I. CREESE Center for Molecular and Behavioral Neuroscience, Rutgers University, 197 University Avenue, Newark, NJ 07102, U.S.A. Abstract––Changes in dopamine receptor subtype binding in different brain regions were examined after 28 days treatment of rats with haloperidol, raclopride, clozapine or SCH23390 using in vitro receptor autoradiography. [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin binding to dopamine D3 receptors was not changed in any brain region by any of the drug treatments. [3H]SCH23390 was only increased by chronic SCH23390 treatment. Haloperidol significantly increased [3H]nemonapride and [3H]spiperone binding to dopamine D2-like receptors in the caudate–putamen. In contrast, haloperidol caused a small, significant increase in [3H]raclopride binding in the lateral caudate–putamen only. Raclopride also elevated, but to a lesser extent [3H]nemonapride and [3H]spiperone binding in caudate–putamen, whereas it did not affect [3H]raclopride binding. Clozapine did not significantly change D2-like striatal binding of [3H]nemonapride, [3H]spiperone or [3H]raclopride. The differences in radioligand binding suggest that [3H]nemonapride and [3H]spiperone may be binding to additional subsets of dopamine D2-like receptors (including D4-like receptors) that are not recognized by [3H]raclopride, which has high affinity for D2 and D3 receptors only. Quantification of [3H]nemonapride or [3H]spiperone binding in the presence of 300 nM raclopride (to block D2 and D3 receptors) revealed that haloperidol, raclopride and clozapine up-regulated D4-like receptors in the caudate–putamen using either radioligand. These results suggest that D4-like receptors may be a common site of action of both typical and atypical antipsychotics. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: atypical antipsychotics, autoradiography, chronic treatment, dopamine receptors, schizophrenia, typical antipsychotics.

Dopamine receptors can be structurally and pharmacologically divided into two families: the D1-like receptor family, which includes the D1 and D5 receptors, and the D2-like receptor family, which includes the D2, D3 and D4 receptors.5,10,56 Non-subtype selective radioligand binding to D2-like receptors is inhibited by antipsychotic drugs in proportion to their clinical efficacies as antipsychotics, suggesting that D2-like rather than D1-like receptors play a central role in mediating antipsychotic drug action.13,53 Antipsychotic drugs can be classified, based on their liabilities to induce extrapyramidal motor side effects (EPS), into two main categories: typical antipsychotics, which are often associated with EPS of both acute and chronic nature and atypical antipsychotics, which cause a significantly lower incidence of EPS. Animal studies have reported significant upregulation of striatal D2-like receptors following different periods of typical antipsychotic treat*To whom correspondence should be addressed. †Present address: Mailman Research Center, McLean Hospital, Harvard Medical School, 115 Mill Street, Belmont, MA 02178, U.S.A. ‡Present address: Central Chemical Lab, Academic Hospital, Free University, Amsterdam, The Netherlands

ment.7,49,50 Since dopamine depletion with reserpine or dopaminergic denervation following 6hydroxydopamine lesion also increase D2-like receptor binding,14 these results suggest that typical antipsychotics produce an effective in vivo receptor blockade leading to subsequent receptor upregulation. D1-like receptors are also up-regulated by chronic D1-like receptor blockade.15 However, dopamine receptor subtype up-regulation has not been demonstrated following chronic treatment with the atypical antipsychotic clozapine at behaviourallyactive doses.46 It is not clear which brain regions, pathways or dopamine receptor subtypes specifically mediate the therapeutic effects of both typical and atypical antipsychotic drugs or those which are potentially associated with the development of EPS, including tardive dyskinesia. In this study we propose to use changes in receptor levels as a functional index of in vivo antipsychotic drug action, mediated by receptor blockade. Changes in a specific receptor level, whether up- or down-regulation, within a specific brain region during chronic treatment with both typical and atypical antipsychotics may indicate it as a common locus for antipsychotic drug actions. Differences in receptor binding induced by typical but not the atypical

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antipsychotics within a brain region may indicate it as a locus for EPS development. We have conducted an in vitro receptor autoradiographic study to examine the effects of chronic (28 days) treatment with a typical antipsychotic (haloperidol), an atypical antipsychotic (clozapine), the dopamine D2/D3 receptor antagonist (raclopride) which exhibits antipsychotic activity,12,21 and the D1-like receptor antagonist (SCH23390) on the different dopamine receptor subtypes (D1-like, D2-like, D3 and D4-like receptors) in various brain regions. EXPERIMENTAL PROCEDURES

Materials Radioligands were obtained from New England Nuclear– Dupont (Wilmington, DE). Ketanserin hydrochloride was purchased from Research Biochemical Inc. (Natick, MA). Clozapine was a gift from Sandoz (East Hanover, NJ), SCH23390 was a gift from Schering–Plough Research (Kenilworth, NJ) and raclopride was a gift from Astra La¨kemedel AB (So¨derta¨lje, Sweden). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO). Drug treatment Different groups of male Sprague–Dawley rats (Charles Rivers, VA,) weighing 200–220 g on delivery, were maintained under controlled light and temperature conditions but given free access to food and water. These rats were housed in the AAALAC-approved animal care facility of Maryland Psychiatric Research Center (MPRC; Baltimore, MD) and treated for 28 days with four different drugs in the following doses: haloperidol (1.5 mg/kg/day), raclopride (10 mg/kg/day), clozapine (25 mg/kg/day) and SCH23390 (0.5 mg/kg/day). The first three drugs were given in drinking water and the control consisted of tap water adjusted to pH 6.0. The solutions were made based on the average weekly weight of the rats and their daily solution consumption. SCH23390 was given by subcutaneous injections and control rats received injections of vehicle only. The protocols utilized were approved by and veterinary care provided by the staff of the animal care facility of MPRC. Care was taken to minimize animal suffering and to limit the number of animals used. Tissue preparation Immediately after drug treatment, rats were decapitated, their brains quickly removed, frozen in chilled isopentane and stored in liquid nitrogen until use. Coronal sections of 16 µm thickness were cut in a cryostat at "20)C, mounted on cold acid-cleaned, gelatin-coated microscopic slides, and stored at "80)C until use. On the day of the experiment, slides were thawed on a slide warmer and then air dried at room temperature. Receptor binding A number of ligands were used to quantify the different dopamine receptor subtypes. Three ligands were used to quantify the D2-like receptors, [3H]nemonapride (previously called [3H]YM-09151-2), [3H]spiperone and [3H]raclopride. These ligands were selected based on their high affinities for the D2-like receptors. Saturation and competition experiments showed that the binding of the three ligands to striatal sections was saturable and was inhibited by D2-like receptor antagonists.33,42,68 Importantly, these three ligands have different specificities for the different D2-like receptors. [3H]nemonapride and [3H]spiperone bind with high affinity to the three D2-like receptors (D2, D3 and D4) in expression

systems, while [3H]raclopride has high affinity for the D2 and D3 receptors and a much lower affinity for the D4 receptor.65 The D3 receptor was quantified using [3H]7-hydroxyN,N-di-n-propyl-2-aminotetralin ([3H]7-OH-DPAT), the first selective (agonist) radioligand which binds to the D3 receptor with subnanomolar affinity compared to nanomolar affinities for D2 and D4 receptors in D2-like receptor transfected cell lines.36 D1-like receptors were quantified using [3H]SCH23390 according to the method of Dawson et al.18 This radioligand labels both the D1 and D5 receptors.61 D1-like receptor binding Sections were preincubated for 1 h in 50 mM Tris–HCl buffer, pH 7.4, containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2. Sections were then incubated for 1 h at room temperature in buffer containing 1 nM [3H]SCH23390 (specific activity 73.24 Ci/mmol) and 40 nM ketanserin to block 5-hydroxytryptamine2 (5-HT2)-like receptors. Non-specific binding was determined in the presence of 1 µM flupenthixol. D2-like receptor binding Sections were preincubated for 1 h at room temperature in the same buffer as above. Sections were then incubated in buffer containing either 1.0 nM [3H]nemonapride (specific activity 81.4 Ci/mmol), 1.2 nM [3H]spiperone (specific activity 116 Ci/mmol) in the presence of 40 nM ketanserin or 5 nM [3H]raclopride (specific activity 86.5 Ci/mmol) for 1 h at room temperature. Non-specific binding was determined in the presence of 10 µM sulpiride ([3H]nemonapride) or 1 µM flupenthixol ([3H]spiperone and [3H]raclopride). D4-like receptor binding In order to determine the optimal concentration of raclopride to completely block D2/D3 receptors, competition experiments were carried out using unlabelled raclopride versus [3H]nemonapride on striatal sections. Computer fitted curves showed that the raclopride curve is best fitted by assuming a two site model (P<0.05).25 At a concentration of 300 nM raclopride, which completely displaced the high affinity binding site (D2/D3 receptors), approximately 15– 22% residual specific binding (which may represent the D4-like receptors) was detected. Non-specific binding was determined using different concentrations of butaclamol, sulpiride, flupenthixol or eticlopride. The absence of two binding components in two other studies utilizing similar competition experiments,44,54 may have resulted from differences in tissue utilized (rats versus human tissue or cell lines), including differences in the definition of non-specific binding, or even the presence of undefined binding sites labeled by [3H]nemonapride. D4-like receptor binding was carried out by preincubating the sections for 1 h at room temperature in the buffer as above. Sections were then incubated for 1 h at room temperature in buffer containing either 1.0 nM [3H]nemonapride or 1.2 nM [3H]spiperone and 40 nM ketanserin in the presence of 300 nM raclopride for both radioligands. Non-specific binding was determined in the presence of 10 µM sulpiride ([3H]nemonapride) or 1 µM flupenthixol ([3H]spiperone). After each radioligand assay, slides were washed (2#5 min) in ice-cold buffer, followed by a quick dip in ice-cold distilled water then dried under a stream of cold dry air. D3 receptor binding Sections were preincubated for 1 h in 20 mM MOPS buffer, pH 7.2, containing 1 mM EDTA, 10 µM pargyline and 0.1% ascorbic acid. Sections were then incubated in

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Fig. 1. Diagramatic presentation of brain regions of interest used to quantify dopamine receptor subtypes. CP-L, lateral caudate–putamen; CP-M, medial caudate–putamen; DFC, dorsolateral frontal cortex; HIPP, hippocampus; MPC, medial prefrontal cortex; NA, nucleus accumbens; Olf Tub, olfactory tubercle; SNpc, substantia nigra pars compacta; SNpr, substantia nigra pars reticulata; VTA, ventral tegmental area.

buffer containing 3 nM [3H]7-OH-DPAT (specific activity 116 Ci/mmol) for 1 h at room temperature. Non-specific binding was determined in the presence of 1 µM eticlopride. After incubation, slides were washed (2#3 min) in ice-cold buffer then dried under a stream of cold air. Magnesium ions were excluded from the incubating buffer to prevent [3H]7-OH-DPAT from labelling the high affinity agonist binding state of the other D2-like receptors.27 Autoradiography and image analysis The slides, together with calibrated tritium standards (Amersham, IL), were exposed to tritium sensitive films for two to four weeks at 4)C. Films were then developed and fixed in D-19 (Eastman Kodak, NY). Optical densities (ODs) of brain regions were measured using a computerbased densitometer, image analyser (MCID-M1, Imaging Research Inc., Canada). Brain regions of interest were outlined (Fig. 1) and the ODs of these regions were measured on two images representing total binding and two images representing non-specific binding. The left and right sides of each region were measured separately and then averaged. The ODs of the sampled regions were converted to nCi/mg using the calibrated standards. The values of

non-specific binding were subtracted from total binding to yield specific binding values, which were expressed in fmol/mg tissue. Statistical analysis An overall two-way analysis of variance (ANOVA) using the four drinking-treated groups and different brain regions was first conducted. A significant two-way ANOVA (P<0.05) was followed by a one-way ANOVA and post hoc Dunnett t-test to identify statistically significant differences between the four drinking-treated groups across brain regions. The data obtained from the two injection-treated groups were analysed using the Student’s t-test. RESULTS

D1-like receptor autoradiography As previously reported,18 D1-like receptors are highly localized in the caudate–putamen (CP), nucleus accumbens (NA), olfactory tubercle (OT) and substantia nigra, pars reticulata (SNpr) of rat brain with comparable levels of binding. Chronic

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F. I. Tarazi et al. Table 1. Specific [3H]SCH23390 binding following chronic SCH23390 treatment

Caudate–putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

SCH23390 (0.5 mg/kg/day)

181.8&14.2 186.8&13.9

233.8&10.3*(+29%) 239.2&9.2* (+28%)

9.0&1.3 13.1&1.2 11.3&1.1 170.4&12.7 167.7&12.8

9.8&1.1 15.7&0.5 12.8&0.6 248.2&12.5*(+46%) 242.0&8.8* (+45%)

14.4&0.8 168.2&13.0 13.1&0.8

15.2&0.8 225.3&6.6* (+34%) 14.5&2.4

Data (mean&S.E.M.)expressed in fmol/mg tissue, n=6/group. *Values significantly different from control (P<0.05, Student’s t-test). Table 2. Specific [3H]SCH23390 binding following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

Haloperidol (1.5 mg/kg/day)

Raclopride (10 mg/kg/day)

Clozapine (25 mg/kg/day)

196.8&6.4 209.7&6.6

175.5&3.7 191.5&4.3

173.8&3.8 188.6&4.2

176.6&14.1 191.5&13.7

8.8&1.1 11.7&0.9 11.6&1.8 189.4&5.6 207.4&5.6

10.8&0.9 13.6&0.9 9.3&1.2 174.6&5.8 209.1&9.3

9.2&0.6 13.7&1.3 11.4&1.3 192.0&4.9 206.0&8.3

6.3&0.8 11.6&1.8 11.9&1.8 182.6&15.6 197.3&27.4

19.2&1.9 185.2&9.9 11.1&0.9

11.5&0.9 147.5&28.5 9.3&2.4

14.9&0.6 195.2&10.0 12.9&2.2

13.7&1.5 202.5&21.8 10.8&2.1

Data (mean&S.E.M.) expressed in fmol/mg tissue, n=7/group.

SCH23390 treatment significantly elevated [3H]SCH23390 binding in all of these regions and to a comparable extent [NA (+46%), CP (+29%), OT (+45%) and SNpr (+34%)] (Table 1). Much lower (<10%) amounts of specific [3H]SCH23390 binding were detected in the cortex, hippocampus, substantia nigra, pars compacta (SNpc) and ventral tegmental area. Chronic SCH23390 treatment did not elevate binding in any of these brain regions. Chronic haloperidol, raclopride or clozapine treatment did not affect [3H]SCH23390 binding in any of the brain regions analysed (Table 2). D2-like receptor autoradiography High levels of binding of the three D2-like receptor radioligands were detected in CP, NA and OT, followed by the ventral tegmental area and SNpc. This is in agreement with previous studies which examined the autoradiographic localization of D2like receptors using the same radioligands.33,42,68 Chronic haloperidol treatment significantly increased [3H]nemonapride and [3H]spiperone binding in CP (+50% and +39%, respectively), NA [+89%

and +45%, respectively; the effect on [3H]nemonapride binding was significantly greater than on [3H]spiperone binding (P<0.05 Dunnett t-test)] and OT [+53% and +32%, respectively] (Tables 3, 4). In contrast, haloperidol treatment caused a significant increase in [3H]raclopride binding only in the lateral CP [+17%] which was significantly lower (P<0.05, Dunnett t-test) than the increases in [3H]nemonopride binding (+47%) or [3H]spiperone binding (+32%). (Table 5). Chronic raclopride treatment elevated, but to a lesser extent, although not significantly, [3H]nemonapride and [3H]spiperone binding in medial CP [+25% and +27%, respectively], and in NA [+28% and +29%, respectively] (Tables 3, 4), whereas [3H]raclopride binding was not changed in either CP, NA or in other brain regions examined. Chronic clozapine treatment did not significantly change the binding of the three radioligands in any of the brain regions analysed (Tables 3, 4, 5). Similarly, chronic SCH23390 treatment did not affect the binding of these radioligands in any of the brain regions examined (data not shown). Chronic treatment with haloperidol, raclopride or clozapine significantly reduced [3H]spiperone binding

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Table 3. Specific [3H]nemonapride binding following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

Haloperidol (1.5 mg/kg/day)

Raclopride (10 mg/kg/day)

Clozapine (25 mg/kg/day)

88.3&6.5 121.8&8.0

135.2&6.2*(+53%) 179.2&8.9*(+47%)

110.7&5.1*(+25%) 154.2&7.8*(+26%)

93.1&5.0 140.4&8.3

6.1&0.9 6.9&0.7 5.2&0.8 61.4&5.5 70.3&6.8

6.4&1.6 7.2&1.3 3.1&1.9 115.7&8.8*(+88%) 101.5&9.3*(+44%)

10.9&2.6 3.9&0.9 7.7&2.1

11.0&2.4 4.1&1.2 7.4&1.8

7.4&1.2 6.9&1.3 3.1&0.4 79.3&6.5 86.8&9.7

5.6&1.3 6.8&1.8 5.7&1.9 68.4&5.4 76.1&9.4

10.6&3.2 2.8&0.5 9.5&2.4

11.2&1.5 3.7&1.3 7.1&1.0

Data (mean&S.E.M.) expressed in fmol/mg tissue, n=7/group. *Values significantly different from control (P<0.05, Dunnett t-test).

Table 4. Specific [3H]spiperone binding following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

Haloperidol (1.5 mg/kg/day)

Raclopride (10 mg/kg/day)

Clozapine (25 mg/kg/day)

63.7&1.2 86.7&1.9

93.3&4.5* (+46%) 114.4&6.0* (+32%)

80.8&5.0* (+27%) 110.9&7.3* (+28%)

10.1&0.1 11.8&0.3 4.2&0.2 55.5&0.8 54.9&0.8

6.7&0.4* ("34%) 6.1&0.3* ("48%) 4.0&0.3 78.8&5.9* (+42%) 60.2&1.9

6.4&0.5*("37%) 5.4&0.6*("54%) 4.9&0.4 71.0&4.1* (+28%) 50.2&3.5

5.4&0.5*("46%) 7.8&0.7*("34%) 4.3&0.4 50.8&3.0 53.3&3.6

9.6&0.3 4.3&0.2 9.4&0.2

15.9&2.0* (+66%) 9.7&0.9*(+125%) 10.8&0.7

16.6&2.0* (+73%) 7.5&1.6* (+74%) 10.5&0.8

10.1&1.1 6.3&0.8 8.9&1.1

59.7&5.1 85.0&6.3

Data (mean&S.E.M.)expressed in fmol/mg tissue, n=6/group. *Values significantly different from control (P<0.05, Dunnett t-test).

in the medial and dorsolateral regions of the frontal cortex (Table 4). This effect was limited to [3H]spiperone as the three drug treatments did not affect [3H]nemonapride or [3H]raclopride binding in the same brain regions (Tables 3, 5).

[3H]nemonapride binding (+24% and +26%, respectively) and [3H]spiperone binding (+35% and +30%, respectively) in the CP.

D3 receptor autoradiography D4-like receptor autoradiography Quantification of brain sections labelled with 1 nM [3H]nemonapride in the presence of 300 nM raclopride (a saturating concentration for D2 and D3 receptors only) revealed the relative distribution of D4-like receptors in different regions of the rat brain (Table 6). These puative receptors are highly expressed in the frontal cortex followed by CP and NA. Chronic haloperidol treatment induced a significant 70% increase in [3H]nemonapride binding in the CP (Table 7), an effect that was also observed when [3H]spiperone was used as a ligand in the presence of the raclopride mask (Table 8). Chronic raclopride or clozapine treatment also significantly increased

[3H]7-OH-DPAT binding showed that D3 receptors are restricted to only a few brain regions in contrast to the other D2-like receptors. High binding of [3H]7-OH-DPAT was observed in the Islands of Calleja and lobules 9 and 10 of the cerebellum, followed by NA and OT. This unique distribution of [3H]7-OH-DPAT binding to rat brain is identical to what was previously reported by Levesque et al.36 Chronic treatment with haloperidol, raclopride or clozapine did not significantly change [3H]7-OHDPAT binding in any of the brain regions examined (Table 9). In addition, chronic SCH23390 treatment did not significantly affect [3H]7-OH-DPAT binding in any of the brain regions (data not shown).

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F. I. Tarazi et al. Table 5. Specific [3H]raclopride binding following chronic typical and atypical antipsychotic treatment

Caudate-putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

Haloperidol (1.5 mg/kg/day)

Raclopride (10 mg/kg/day)

Clozapine (25 mg/kg/day)

47.8&2.2 59.2&1.7

52.5&3.0 69.6&1.6*(+18%)

49.5&1.3 64.3&1.3

43.1&2.4 60.0&2.8

6.8&1.9 6.8&1.0 6.0&1.8 37.3&2.8 33.4&5.0

5.7&1.6 6.0&1.3 4.1&0.6 43.5&2.9 33.8&5.1

6.1&0.5 6.2&0.5 4.9&2.3 41.3&2.2 24.8&2.4

6.2&2.4 4.4&1.5 3.7&0.8 34.2&3.2 27.7&3.5

7.1&1.6 4.4&1.3 6.2&1.2

5.6&0.9 3.1&0.5 5.1&0.7

6.1&1.4 3.4&0.5 5.1&0.5

6.7&0.9 4.8&0.6 6.6&1.0

Data (mean&S.E.M.) expressed in fmol/mg tissue, n=7/group. *Values significantly different from control (P<0.05, Dunnett t-test).

Table 6. Relative distibution of putative D4 receptors in different brain regions [3H]nemonapride (D2+D3+D4 receptors) fmol/mg tissue (n=7)

[3H]nemonapride+300 nM raclopride (putative D4 receptors) fmol/mg tissue (n=6)

% D4/total

6.9&0.7 6.1&0.9 61.4&5.5

4.9&2.0 3.7&1.4 17.3&1.8

70% 60% 28%

88.3&6.5 121.8&8.0

19.4&1.2 30.3&2.2

22% 25%

10.9&2.6 3.9&0.9 7.7&2.1

0.7&0.2 0.4&0.1 0.5&0.2

6% 10% 6%

Cortex Dorsolateral Medial prefrontal Nucleus accumbens Caudate–putamen Medial part Lateral part Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

DISCUSSION

Effects of typical and atypical antipsychotics on D1-like receptors Up-regulation of striatal D1-like receptors following chronic SCH23390 treatment has been previously reported.15,43 This effect is accompanied by an increase in D1 receptor gene transcription.16 In the present study, the effects of chronic SCH23390 treatment were extended to include up-regulation of D1like receptors in other brain regions where the receptors are highly enriched including the NA, OT, and SNpr. The lack of change in [3H]SCH23390 binding in brain areas with very low ‘‘specific’’ [3H]SCH23390 binding could be the result of a poor signal/noise ratio or the lack of a true D1-like receptor signal. Alternatively, the lack of D1-like receptor up-regulation may indicate the involvement of different regulatory mechanisms in these various brain regions, or possibly a higher ratio of D5 to D1 receptors. It is not known if D5 receptors are regulated in a similar manner to D1 receptors in response to chronic receptor blockade nor what the relative

proportions of these two receptor subtypes are in each brain region, although D5 receptor mRNA expression is lower than that of D1 receptors and is restricted to extrastriatal areas.39 Chronic haloperidol, raclopride or clozapine treatment did not significantly change [3H]SCH23390 binding to the D1-like receptors in any of the brain regions examined. Similarly, in a parallel study where we examined the effects of eight months treatment with the same antipsychotic drugs on D1-like receptors, no significant change in [3H]SCH23390 binding was observed.24 Since this and previous studies have shown that chronic blockade of D1-like receptors lead to their up-regulation, it can be concluded that with the drug regimens used in the current study, a D1-like receptor blockade by the three drugs of the degree required to initiate an increase in D1 gene transcription was not obtained. Parametric studies have never been conducted to determine what degree of receptor blockade is required to initiate dopamine receptor up-regulation. The present results are in contrast to two other studies which reported that chronic clozapine

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Table 7. Specific [3H]nemonapride binding in the presence of 300 nM raclopride (putative D4 receptors) following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part Cortex Dorsolateral Medial prefrontal Hippocampus Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars reticulata Ventral tegmental area

Control

Haloperidol (1.5 mg/kg/day)

Raclopride (10 mg/kg/day)

Clozapine (25 mg/kg/day)

19.4&1.2 30.3&2.2

32.7&1.3*(+67%) 50.4&1.8*(+66%)

23.8&0.9*(+23%) 37.9&2.1*(+25%)

24.5&1.1*(+26%) 38.6+1.8*(+27%)

3.7&1.4 4.9&2.0 1.1&0.2 17.3&1.8 20.1&0.8

4.5&0.9 5.5&1.5 1.2&0.1 27.8&2.6*(+61%) 28.5&3.2

4.4&1.9 4.7&2.0 1.3&0.4 20.8&1.9 23.5&2.0

2.6&1.2 3.2&2.1 1.4&0.4 17.1&1.0 21.2&1.7

0.8&0.2 0.4&0.1 0.5&0.1

1.1&0.2 0.6&0.1 0.8&0.1

0.7&0.2 0.4&0.1 0.5&0.2

0.9&0.3 0.5&0.1 0.6&0.1

Data (mean&S.E.M.) expressed in fmol/mg tissue, n=6/group. *Values significantly different from control (P<0.05, Dunnett t-test).

Table 8. Specific [3H]spiperone binding in the presence of 300 nM raclopride (putative D4 receptors) following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part

Control

Haloperidol(1.5 mg/kg/day)

Raclopride(10 mg/kg/day)

Clozapine(25 mg/kg/day)

13.2&0.6 19.4&0.9

23.8&1.3* (+80%) 31.0&1.0* (+60%)

18.9&1.0* (+43%) 24.8&1.3* (+28%)

17.2&0.8* (+30%) 25.5&1.3* (+31%)

Data (mean&S.E.M.) expressed in fmol/mg tissue, n=6/group. *Values significantly different from control (P<0.05, Dunnett t-test).

Table 9. Specific [3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin binding following chronic typical and atypical antipsychotic treatment

Caudate–putamen Medial part Lateral part Cerebellum Islands of Calleja Nucleus accumbens Olfactory tubercle Substantia nigra Pars compacta Pars Reticulata

Control

Haloperidol(1.5 mg/kg/day)

Raclopride(10 mg/kg/day)

Clozapine(25 mg/kg/day)

4.7&1.0 3.6&0.3 14.0&1.4 45.4&3.6 17.2&0.7 16.8&1.4

3.7&0.5 3.4&0.4 11.1&0.9 44.7&5.6 17.0&0.7 16.0&1.2

3.7&0.5 3.2&0.4 12.5&0.9 45.6&7.0 15.5&0.3 13.3&1.1

4.0&0.4 3.9&0.3 12.9&0.6 42.8&2.9 15.6&0.6 13.6&1.2

6.2&0.4 4.3&0.5

5.2&0.3 3.6&0.3

5.8&0.4 3.9&0.3

5.7&0.5 4.0&0.4

Data (mean&S.E.M.) expressed in fmol/mg tissue (n=6/group).

treatment up-regulated D1-like receptors in the CP and NA in rats.41,48 One major difference between the current study and these studies was the route of drug administration, dissolved in drinking water versus intraperitonial injections or continuous subcutaneous infusion. Pharmacokinetic or metabolic factors may have contributed to a greater D1-like receptor blockade and subsequent receptor upregulation. Another study reported significant reduction in [3H]SCH23390 binding in SNpr of rats that developed severe, but not mild, vacuous chewing

movements (VCMs) following six months treatment with haloperidol.55 We did not, however, subdivide our drug-treated animals according to the frequency of VCMs development, and this may have contributed to the discrepancy in [3H]SCH23390 binding in SNpr between the two studies. A third study reported down-regulation of D1-like receptors in the medial prefrontal and temporal cortices of monkeys treated for six months with clozapine, haloperidol or remoxipride.38 This effect, however, was not duplicated in our study, although there was a trend for

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clozapine, but not haloperidol or raclopride, to reduce [3H]SCH23390 binding in the dorsolateral cortex (Table 2). The increased complexity of the primate cerebral cortex and the diversity of its dopaminergic innervations may have accounted for the differences in D1-like receptor binding regulation between primates and rodents.6 The interest in the D1-like receptors as potential targets for atypical antipsychotics was sparked by positron emission tomography (PET) studies which reported that clinically effective doses of clozapine occupy striatal D1-like receptors to a greater extent than any other antipsychotic drug.22 Several other behavioural and neurochemical findings in animals have suggested that clozapine may mediate some of its therapeutic effects via D1-like receptors19 and have led to the suggestion that selective D1-like receptor antagonists may be potential atypical antipsychotics.3,11,26 Our data do not support a role for D1-like receptors in mediating the effects of antipsychotic drugs. This is in agreement with behavioural, biochemical and recent PET reports which strongly argued against an atypical antipsychotic activity of selective D1-like receptor antagonists.8,20,28,29 Furthermore, there are many effective antipsychotics which lack significant D1-like receptor affinity. Thus, a primary role for D1-like receptors in mediating the effects of antipsychotic drugs can probably be ruled out. However, the greater D1-like receptor occupancy in vivo shown by clozapine compared to typical antipsychotics may contribute to its advantageous clinical profile in some non-obvious manner. Effects of typical and atypical antipsychotics on D2-like receptors The D3 receptor subtype has been suggested to be a target for antipsychotic drugs because its highest levels of expression occur in limbic areas, while it has low expression in extrapyramidal areas.58 Chronic treatment with haloperidol, raclopride, clozapine or SCH23390 failed to alter [3H]7-OH-DPAT binding to D3 receptors in any of the brain regions examined. Similarly, Levesque et al.37 did not detect any significant changes in D3 receptor binding or D3 receptor mRNA levels following two weeks of haloperidol treatment. The D3 receptor may have a different regulatory mechanism from other dopamine receptor subtypes. For example, it has been hard to demonstrate a D3/G-protein coupling or a well-defined D3-linked signal transduction mechanism in expression systems57,58 in contrast to D2 or D4 receptors.9,63 Such basic difference in receptor/effector coupling mechanisms could result in a lack of D3 receptor up-regulation in response to ‘‘adequate’’ and chronic receptor blockade. It is also possible that endogenous dopamine, with its higher affinity for the D3 receptor compared to the other D2-like receptors,58,65 may (semi) permanently occupy the D3

receptors and prevent their occupancy by antipsychotic drugs to the level that is sufficient to trigger in vivo receptor up-regulation. Since none of the drug treatments altered [3H]7-OH-DPAT binding it is not possible to draw firm conclusions as to the role of D3 receptor blockade in antipsychotic drug action. However, the lack of any differential changes in D3 receptor binding following chronic typical or atypical antipsychotic treatment could suggest that this receptor subtype is less likely to be involved in mediating the clinical effects of antipsychotic drugs or in the induction of EPS. Receptor autoradiography revealed differential and significant increases in [3H]nemonapride and [3H]spiperone binding compared to a small and nonsignificant increase in [3H]raclopride binding in CP following chronic haloperidol and raclopride treatment. [3H]raclopride has high affinity (1.8 nM and 3.5 nM) for the expressed cloned D2 and D3 receptors, respectively,58 while it has much lower affinity for the expressed cloned D4 receptor (2000 nM).51 Therefore, in the current study, this radioligand should label predominately the D2 and D3 receptors. [3H]raclopride binding was increased to a small extent by only one drug, haloperidol, and in only one region, lateral CP (+18%). The increase in [3H]raclopride binding in lateral CP probably represents D2 receptors, since [3H]7-OH-DPAT binding to D3 receptors remained unchanged (Table 9). This result suggests that D2 receptors were not significantly upregulated following chronic receptor blockade with our particular drug regimens. Previous studies7,49,50 and the current one have consistently shown that other non-selective D2-like receptor radioligand binding in the CP is increased following chronic haloperidol treatment. These observations suggest that D4-like receptors may be the major D2-like receptor subtype subject to antipsychotic-induced up-regulation. In this study, we used an indirect approach to localize and quantify D4-like receptors by masking D2 and D3 sites with unlabelled raclopride and labelling with a non-selective D2-like receptor radioligand. In the presence of 300 nM raclopride, a saturating concentration for D2 and D3 receptors only,24,25 the remaining binding of [3H]nemonapride and [3H]spiperone should be predominately to the D4-like receptors. Based on this method, D4-like receptors, in contrast to D2 and D3 receptors, are highly expressed in the frontal cortex, the brain area with the highest levels of D4 receptor mRNA expression,65 followed by lower levels in NA and CP. The levels of D4-like receptors in the CP are higher and disproportionate to the low levels of D4 receptor mRNA expressed in the same brain region,65 suggesting that a portion of striatal D4-like receptors may arise on corticostriatal projections as well as on intrinsic intrastriatal neurons.62 The presence of D4, or D4-like receptors in human striata remains uncertain; whereas three independent studies detected

Dopamine receptors and antipsychotic drugs

measurable levels of striatal D4-like receptors,40,51,59 two other studies failed to provide evidence for the presence of the same subset of receptors in the striata of undiseased human tissue.35,44 It is also probable that the abundance of striatal D4-like receptors or its mRNA might vary across species.60,65 Chronic haloperidol treatment significantly upregulated these D4-like receptors in the CP and NA. This haloperidol-induced increase in striatal D4-like receptor levels is consistent with the reported elevation in D4 mRNA levels following chronic haloperidol treatment.47 It is also consistent with the lack of a D2 receptor mRNA increase in the face of a significant increase in D2-like receptor binding; a paradoxical observation which was previously interpreted as suggesting that the increase in D2-like receptor binding was the result of a slowed D2 receptor degradation rate.64 Interestingly, chronic raclopride and clozapine also induced a significant increase in D4-like receptors in CP. The biochemical mechanism behind the up-regulation of D4-like receptors in the CP by raclopride is unclear. A functional interaction may exist between D4-like receptors and D2 receptors in the CP such that blockade of D2 receptors by raclopride may increase D4-like receptor levels. An indirect effect could explain why an increase in [3H]nemonapride (+24%) and [3H]spiperone (+36%) binding to D4like receptors (Tables 7, 8) is not as profound as the effects of haloperidol on D4-like receptors (approximately 70% up-regulation by both radioligands) which would result from direct receptor blockade, although the similar magnitude of effect by clozapine, which has higher affinity for D4 receptors,65 would argue against this hypothesis. It is also possible that both drugs in vivo at these administration protocols produce a similar but lower D4like receptor occupancy than haloperidol. It is still to be determined if chronic raclopride or clozapine treatment also up-regulate D4 receptor mRNA. Cortical D4-like receptors were not up-regulated by any drug treatment, a finding consonant with that of Kusumi et al.34 who used nemonapridedisplaceable [3H]clozapine binding to label these sites. We therefore suggest that the significant increase in D2-like receptor ligand binding in the CP, following our particular drug administration protocol, is mainly due to a significant up-regulation of D4-like receptors in conjunction with a small, generally nonsignificant, increase in D2 receptor levels. It thus implicates D4-like receptors as ‘‘common’’ receptors that may initiate and mediate the therapeutic effects of antipsychotic drugs, as both typical and atypical antipsychotics up-regulated D4-like receptors more profoundly than up-regulating D2 receptors. Post mortem studies have reproducibly shown a significant increase in striatal D2-like receptor binding in brain tissues from patients with schizophrenia,49,50 but it is not clear if this is part of the disease

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process or the result of drug-induced up-regulation caused by prior antipsychotic treatment. PET studies which examined striatal D2-like receptor levels in antipsychotic-naive schizophrenics have not resolved this issue, as one study reported a significant elevation of striatal D2-like receptors67 while two other studies failed to detect significant changes in striatal D2-like receptors in drug-naive patients diagnosed with schizophrenia.23,30 Furthermore, interpretation of past post mortem binding studies is complicated because the majority of studies have used [3H]spiperone; a non-selective D2-like receptor radioligand.65 Recently, three post mortem studies have reported significant elevations in striatal D4-like receptor levels in brain tissue from patients with schizophrenia,40,51,59 although these findings are still debated.44,45 Our present study would suggest that the reported up-regulation of D4-like receptors, or D4like binding sites,52 in patients with schizophrenia may be the result in part of their treatment with antipsychotics. It is difficult to speculate on the exact nature of these D4-like binding sites. However, the common high affinity of both [3H]nemonapride and [3H]spiperone to D2-like receptors,65 and the similar and consistent increase in both radioligands binding to these D4-like binding sites induced by chronic treatment with typical and atypical antipsychotics would suggest an up-regulation of dopamine D4-like receptors, rather than some other neurotransmitter receptor. Nevertheless, the pharmacological characterization of D4-like receptors or binding sites and their actual role in the expression of schizophrenia and/or EPS, as well as their regulation following chronic antipsychotic treatment should be better resolved when selective D4 receptor radioligands become available. Chronic haloperidol treatment resulted in an increase in striatal D2 receptors in the lateral CP (as quantified by [3H]raclopride), an effect not caused by chronic raclopride or clozapine treatment. This may provide an important distinction in the mechanism of action of typical versus atypical antipsychotics, at least in reference to their EPS liabilities. Relative differences in the blockade and up-regulation of D2 receptors in the CP by typical and atypical antipsychotics may alter the interaction between different neurotransmitters in the CP and may disrupt neurotransmission in the basal ganglia, a major brain structure involved in programming and initiation of movement.2 Abnormalities in the basal ganglia– thalamocortical circuitry may be the locus of movement disorders that accompanies chronic typical antipsychotic treatment.1 Chronic haloperidol alone also increased D4-like receptors in the NA (Table 7) suggesting a potential unexpected role for these receptors in generating EPS. Another interesting finding was the significant and similar decrease in [3H]spiperone, but not [3H]nemonapride or [3H]raclopride, binding in the prefrontal and dorsolateral cortex by haloperidol,

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raclopride and clozapine. The nature of these down-regulated binding sites is unclear. Spiperone is known to bind not only to D2-like receptors, but also to 5-HT2 receptors17 and á1-adrenergic receptors.31 Chronic clozapine treatment was reported to significantly down-regulate cortical 5-HT2-like receptors.24,41,66 In contrast, these studies found that haloperidol did not affect cortical 5-HT2-like receptor levels. In addition, raclopride has very low affinity for 5-HT2 receptors.32 It is thus possible that [3H]spiperone is labelling an additional uncharacterized site that is down-regulated by both typical and atypical antipsychotic long-term treatment. This uncharacterized site may represent a new and common locus of antipsychotic drug action. Interestingly, this uncharacterized [3H]spiperone site may also be present in humans, as one study reported a significant reduction in [3H]spiperone binding in cortical areas 8 and 9 in post mortem schizophrenic brain tissue.4 In addition, [3H]spiperone, but not [3H]nemonapride or [3H]raclopride, recognized binding sites in the SNpc and SNpr that were significantly up-regulated following chronic haloperidol and raclopride but not clozapine treatment. This binding site is probably different from the cortical [3H]spiperone-sensitive binding site, as evidenced by its opposite response to chronic antipsychotic treatment. Overall, [3H]spiperone seems to recognize more binding sites than previously reported.

CONCLUSIONS

As determined by in vitro radioligand binding, the different dopamine receptor subtypes displayed different responses to chronic antipsychotic treatment. Up-regulation of D4-like receptors by both typical and atypical antipsychotics highlights the potential importance of these receptors as common mediators of antipsychotic drug activity, and suggests that D4-like receptor affinity may be a critical component of the pharmacological profile used to develop novel atypical antipsychotic drugs. The differential effects of typical versus atypical antipsychotics on D2 receptors in the basal ganglia suggest that these receptors, with their potential contribution to EPS development, should be excluded from the pharmacological profile of atypical antipsychotics. Lack of significant changes in D1-like and D3 receptors by both typical and atypical antipsychotics may indicate that both receptor subtypes play a more neutral role in the mechanisms of chronic antipsychotic drug action. Acknowledgements—This work was supported by MH44211 from the National Institute of Mental Health Center for Schizophrenia Research (PI Dr W. Carpenter), Maryland Psychiatric Research Center; an NIMH RSA award MH00316 (I.C.) and a Sigma Xi research grant (F.I.T.). We thank Dr Robert Schwarcz for supervising the drug treatments of the animals.

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