Regulation of serotonin 5-HT2C receptors in the rat choroid plexus after acute clozapine treatment

ejp European Journal of Pharmacology

molecular pharmacology

Molecular Pharmacology Section 269 (1994) 201-208

ELSEVIER

Regulation of serotonin 5-HT2c receptors in the rat choroid plexus after acute clozapine treatment Mikko Kuoppam~iki

a,,, T i m o Sepp~il~i b, E r k k a Syv~ilahti a, J a r m o H i e t a l a

a

a Department of Pharmacology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland b National Public Health Institute, Helsinki, Finland

Received 23 March 1994; revised MS received 15 June 1994; accepted 5 July 1994

Abstract

We studied the effects of acute clozapine and haloperidol treatments on 5-HT2c receptor binding characteristics and 5-HT2c receptor-mediated phosphoinositide hydrolysis in the rat choroid plexus. Scatchard analysis (with [3H]mesulergine) showed that acute clozapine treatment (10 and 25 mg/kg) decreased the density (Bm~) of 5-HTzc receptors by 20-25% with no marked change in the affinity (Kd). Quantitative autoradiography was in accordance with homogenate binding studies showing that acute clozapine treatment, unlike haioperidol (0.5 mg/kg), decreased the number of both agonist ([125I](+)-l-(2,5-dimethoxy-4iodophenyl)-2-aminopropane, [125I]DOI) and antagonist ([3H]mesulergine) labeled 5-HT2c receptor binding sites. The decrease was more robust with the higher dose of clozapine. For comparison, both doses of clozapine, unlike haloperidol, decreased equally the density of 5-HT2A receptors in the frontal cortex by about 45%, whereas none of the treatments altered dopamine D e receptor characteristics in the striatum. The K d value of 5-HT2A receptors was significantly increased after the dose of 25 mg/kg of clozapine. These clozapine treatments failed to decrease the maximal 5-HTzc receptor-mediated phosphoinositide hydrolysis response. The higher dose of clozapine increased 5-HT-induced phosphoinositide hydrolysis response, but also decreased significantly the basal levels of phosphoinositide hydrolysis. Haloperidol did not significantly affect the 5-HT2c receptor-mediated phosphoinositide hydrolysis. To summarize, the present data show that a single injection of clozapine is able to reduce the density of 5-HTzc receptors but fails to cause functional desensitization of 5-HT2c receptors in the rat choroid plexus. Keywords: Atypical antipsychotic; Autoradiography; Choroid plexus; Clozapine; 5-HTec receptor; Phosphoinositide hydrolysis

I. Introduction

Clozapine is an atypical antipsychotic agent with many clinical advantages c o m p a r e d to typical antipsychotics, such as haloperidol. In most of the schizophrenic patients, therapeutic effects of clozapine are observed after treatment of several days to several months (e.g. Kane et al., 1988; Meltzer et al., 1989; Baldessarini and Frankenburg, 1991). Therefore, a mechanism which is differentially (qualitatively or quantitatively) modulated after acute and repeated administration of clozapine, is more likely to be involved in therapeutic effects of clozapine.

* Corresponding author. Tel.: 358-21-6337580; Fax: 358-216337216. 0922-4106/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0922-4106(94)00114-6

A m o n g many alternatives, the modulatory effects of clozapine on serotonergic neurotransmission have been linked to its atypical properties. The 5-HTzA receptor 1 has been considered to be important in this respect, and previous studies have suggested that 5-HT2A receptors are downregulated to an equal amount after acute and chronic administration of clozapine (Matsubara and Meltzer, 1989; Reynolds et al., 1983; Wilmot and Szczepanik, 1989; O'Dell et al., 1990). Recently, it has been shown that clozapine binds with relatively high affinity to 5-HT2c receptors a (Canton et al., 1990; Roth et al., 1992; Kuoppam~iki et al., 1993b), which resemble 5-HTaA receptors in many respects. In addition, we have previously shown that chronic clozapine

15_HT2Aand 5-HT2c receptors have been previouslycalled 5-HT2 and 5-HTlc receptors, respectively.

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treatment downregulates 5-HT2c receptors in rat choroid plexus (Hietala et al., 1992; Kuoppam~iki et al., 1993a). The purpose of the present study was to find out, whether acute clozapine treatment would equally downregulate 5-HTzc receptors. Haloperidol was included in the treatment schedule as a typical antipsychotic drug. 5-HTzc receptor binding characteristics (Bmax and K d) were determined with [3H]mesulergine in choroid plexus homogenates. In a separate experiment, the number of 5-HTzc receptor agonist and antagonist-labeled binding sites were measured using quantitative [125I]DOI ([ lzs I]( _+)-l-(2,5-dimethoxy-4iodophenyl)-2-aminopropane) and [3H]mesulergine autoradiography, respectively. To further evaluate the regulation of 5-HTzc receptors after a single injection of clozapine, we measured the maximal response of 5-HTzc receptor-mediated phosphoinositide hydrolysis to find out whether 5-HTzc receptors might undergo functional desensitization. For comparison, 5-HTzA and dopamine O 2 receptor characteristics were determined from frontal cortical and striatal homogenates, respectively. To exclude major interference of residual clozapine on receptor binding results, we also measured clozapine concentrations in the rat striatum and cortex after single subcutaneous injections of clozapine.

injection to study [125I]DOI and [3H]mesulergine binding to 5-HT2c receptors.

2.3.2. Experiment 2 Three groups of rats (saline and clozapine groups; pooled n = 24 per group) were treated in two separate experiments identically to experiment 1. These experiments gave identical results, and pooled results are presented. Rats were decapitated 24 h after the drug injection. For the 5-HT2c receptor Scatchard analysis, choroid plexi from three rats were pooled. 5-HTzA and D 2 receptor binding characteristics (n = 6-8 per group) and clozapine concentrations (n = 3 per group) were determined from frontal cortical and striatal samples. 2.3.3. Experiment 3 Four groups of rats (n = 6 per group) were treated identically to experiment 1. Rats were decapitated 24 h after the drug injection to measure 5-HT2c receptormediated phosphoinositide hydrolysis in the rat choroid plexus. This experiment was repeated with saline- and clozapine (25 mg/kg)-treated rats (n = 5 per treatment group).

2. Materials and methods

2.3.4. Experiment 4 For the determination of pharmacokinetics of clozapine (10 and 25 mg/kg s.c.) in the rat cortex and striatum, three rats per time point (15 and 30 min., and 1, 2, 4, 8, and 18 h) were used.

2.1. Animals

2.4. Brain dissection

Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, USA) weighing 200-230 g were used. Rats were housed in groups of three under standard laboratory conditions (temperature, 21°C; humidity, 55 + 5%; lights on from 6.00 a.m. to 6.00 p.m.). Free access to standard pelleted food and tap water was available at all times.

For receptor autoradiography, brains were removed quickly after decapitation, frozen on tissue pedestals using dry ice and tissue-glue (Tissue-Tek O.C.T. Compound, Miles Inc., Elkhart, IN, USA), and stored at -70°C. Coronal sections (16 /.~m) of choroid plexus (coordinates A 6860-A 6670 according to K6nig and Klippel's atlas) were cut with a cryostat microtome at -18°C. Sections were thaw-mounted onto gelatincoated slides, air-dried at room temperature, and stored at -70°C with desiccator until used. For the determination of receptor binding characteristics with Scatchard analysis, frontal cortices, striata and choroid plexi were immediately dissected out after decapitation and frozen on dry ice. Fresh choroid plexi were used for 5-HT-induced phosphoinositide hydrolysis. For this purpose, choroid plexi were rapidly dissected out after decapitation and a single choroid plexus was placed in 5 ml of Krebs-bicarbonate (KRB) buffer.

2.2. Drugs Clozapine (Leponex 25 mg/ml; Sandoz Pharma AG, Basle, Switzerland) and haloperidol (Serenase 5 mg/ml; Orion, Finland) were used as commercially available ampules. Haloperidol ampules were further diluted with distilled water. No other pharmacologically active compounds were present in the ampules.

2.3. Study designs 2.3.1. Experiment 1 Four groups of rats (n = 7 per treatment group) received single subcutaneous (s.c.) injections of clozapine (10 or 25 mg/kg), haloperidol (0.5 mg/kg) or an equal volume (1 ml/kg) of saline. For receptor autoradiography, rats were decapitated 24 h after the drug

2.5. Autoradiographic procedures 2.5.1. 5-HT2c receptor autoradiography with [ 3H]mesulergine The 5-HTzc receptor autoradiography with [3H]mesulergine was performed as previously described

M. Kuoppamiikiet al. / European Journal of Pharmacology - MolecularPharmacologySection 269 (1994) 201-208

(Kuoppam~iki et al., 1994). Briefly, the sections were drop-incubated for 2 h at room temperature with 100 /~1 of 170 mM Tris-HC1 buffer (pH 7.5) containing a saturating concentration (5 nM) of [3H]mesulergine (78 Ci/mmol, Amersham). Nonspecific binding was determined by incubating the adjacent sections in the presence of 5 p~M methysergide. Spiperone 100 nM was used to prevent binding to 5-HT2A sites. The slides were apposed to Kodak XAR-5 x-ray films together with plastic [3H]standards (American Radiolabeled Chemicals, St. Louis, MO, USA) for 32 days at 4°C. The [3H]autoabsorption (quenching) of the choroid plexus is minimal (Geary and Wooten, 1985). To avoid overestimation of the number of 5-HTzc receptors (labeled by [3H]mesulergine) and to compare absolute densities of [125I]DOI and [3H]mesulergine binding sites in the choroid plexus, [3H]choroid plexus paste standards were prepared to convert the values of [3H]plastic standards (/zCi/g plastic) to pmol ligand bound/g tissue. This was done as previously described (Kuoppam~iki et al., 1994). 2.5.2. 5-HT2c receptor autoradiography with [125I]DOI The 5-HTzc receptor autoradiography with [125I]DOI was done as previously described (Kuoppam~iki et al., 1994). Briefly, the sections were drop-incubated for 1 h at room temperature with 100 ~zl of 50 mM Tris-HC1 buffer (pH 7.5) containing 10 mM MgSO4, 0.1% (w/v) bovine serum albumin and 0.4 nM [125I]DOI (2200 Ci/mmol, New England Nuclear). Nonspecific binding was determined by incubating the adjacent sections in the presence of 5 /xM methysergide. 100 nM spiperone was used to prevent binding to 5-HT2A sites. In pig choroid plexus, the 5-HT2c receptor K d value of [125I]DOI has been reported to be 1.6 nM (Leonhardt et al., 1992), but a concentration of 0.4 nM of [125I]DOI was used for practical reasons. It is assumed that there are no changes in K 0 value of [125I]DOI, as previously shown with [3H]mesulergine (Kuoppam~iki et al., 1993a) and that the binding primarily reflects changes in receptor density. The slides were apposed to Kodak XAR-5 X-ray films together with plastic [14C]standards (American Radiolabeled Chemicals, St. Louis, MO, USA) for 1-2 days at 4°C. Films were developed with an automated Fuji RG II X-ray film processor. For a comparison between treatment groups, all sections from all groups were exposed to the same sheet of film. 2.6. Image analysis

The images were initially digitized, using a CCD Video Camera (Hamamatsu C3077, Hamamatsu Photonics K.K., Hamamatsu City, Japan), into an array of 640 x 480 pixels, each with a gray value in the range of 0-255. A shading correction was applied to compen-

203

sate for variations in illumination and light transmission of the optical system. The images were analyzed with a computerized image analysis system (MCID, M4 1.12, Imaging Research Inc., St. Catharines, Ontario, Canada). A standard curve was generated by measuring and plotting the optical densities of the standards versus their radioactivity. Choroid plexi were carefully traced with a mouse-controlled cursor and the optical densities corresponding to these areas were measured and the radioactivity interpolated. The results are expressed as pmol bound ligand/g tissue. 2. 7. Homogenate binding studies

2. 7.1. 5-HT2c receptor binding assay The 5-HT2c receptor binding assay was performed with [3H]mesulergine (81 Ci/mmol, Amersham) as described previously (Kuoppam~iki et al., 1993b). Briefly, a [3H]mesulergine concentration range of 0.4 to 6.3 nM was used. The nonspecific binding was determined using 10 /zM 5-HT, and 50 nM spiperone and 10/.tM pargyline were included to occupy 5-HT2A receptors and inhibit monoamine oxidase activity, respectively. The protein measurement was done by the method of Lowry et al. (1951). 2. 7.2. 5-HT2A receptor binding assay The 5-HT2A receptor binding assay was performed with [3H]ketanserin (64.1 Ci/mmol, New England Nuclear) as described previously (Kuoppam~iki et al., 1993b). Briefly, a [3H]ketanserin concentration range of 0.2 to 5.5 nM was used. The nonspecific binding was defined in the presence of 1 /xM methysergide. The protein measurement was done by the method of Bradford (1976). 2. 7.3. D 2 receptor binding assay The dopamine D 2 receptor binding assay was performed with [3H]spiperone (21.3 Ci/mmol, New England Nuclear), as described previously (Hietala et al., 1990). Briefly, a [3H]spiperone concentration range of 0.1 to 1.6 nM was used. The nonspecific binding was defined in the presence of 1 /xM (+)-butaclamol, and 33 nM ketanserin was included to occupy 5-HTzA receptors. The protein measurement was done by the method of Bradford (1976). Radioactivity in each receptor assay was measured by beta-counting (Wallac LKB 1219 Rackbeta, Finland) using OptiFluor-O (Packard, Zurich, Switzerland) scintillation fluid at 50% efficiency, except in D 2 and 5-HTzA receptor binding assays where OptiPhase 'Hisafe' 3 (LKB Wallac, Loughborough, England) was used as a scintillation fluid. Computer processing of the binding data (EBDA (equilibrium binding data

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Table 1 Effects of single injections of clozapine (10 and 25 mg/kg) on 5-HT2¢ receptor characteristics in the rat choroid plexus determined with [3H]mesulergine Treatment

Kd

Bma x

Control Clozapine (10 mg/kg) Clozapine (25 mg/kg) ANOVA

1.75 _+0.17 1.55 _+0.21 1.79 _+0.22 P = 0.653

1760+ 126 1330 + 100 a 1390 _+106 a P = 0.027

2. 9. Determination o f brain clozapine concentrations

Choroid plexi from three rats were pooled, n = 8. Values represent mean_+ S.E.M. a p < 0.05 compared to control group.

a n a l y s i s ) / L I G A N D ) was p e r f o r m e d as previously described (Hietala et al., 1990).

2.8. The 5-HT2c hydrolysis assay

different inositol phosphates. Inositol m o n o p h o s p h a t e was eluted with 10 ml of 200 m M a m m o n i u m f o r m a t e / 0 . 1 M formic acid. OptiPhase ' H i s a f e ' 3 (LKB Wallac, L o u g h b o r o u g h , England) was used as scintillation fluid.

receptor-mediated phosphoinositide

5-HT2c r e c e p t o r - m e d i a t e d phosphoinositide hydrolysis response in the rat choroid plexus was m e a s u r e d using 1 0 / x M 5-HT, as preliminary findings showed that this c o n c e n t r a t i o n of 5 - H T caused a maximal p h o s p h o inositide hydrolysis response in the rat choroid plexus. 5-HTzc r e c e p t o r - m e d i a t e d phosphoinositide hydrolysis was p e r f o r m e d as previously described (Kuoppam~iki et al., 1993b). Briefly, Krebs-bicarbonate ( K R B ) buffer was used as incubation medium. T h e samples were labeled with 1 /zCi of [3H]myo-inositol (New E n g l a n d Nuclear) for 90 min in the presence of O z / C O 2 (95 : 5) p r e c e d e d by extensive preincubation (1 h) and three changes of K R B buffer to wash out possible residual drug. 10 ~ M pargyline and 10 m M lithium were a d d e d and the incubation was c o n t i n u e d for 15 min. T h e r e after, 10 /.~M 5 - H T was a d d e d and the samples were incubated for 30 min. A column of Dowex-1 anion-exchange resin in the f o r m a t e form was used to separate

Clozapine levels in cortical and striatal samples were d e t e r m i n e d gas chromatographically as a heptafluorobutyric acid anhydride derivative, as previously described (Kuoppam~iki et al., 1993a).

2.10. Statistical analysis Statistical analyses of the r e c e p t o r binding data were carried out by one-way A N O V A followed by StudentN e u m a n - K e u l s or T u k e y ' s test for post-hoc analyses. F o r statistical analysis of the 5 - H T l c receptor-mediated phosphoinositide hydrolysis data, D u n n e t t ' s test for multiple comparisons and Student's t-test were used. Commercially available statistical software (Systat, Evanston, IL, U S A ) was used for these purposes. A P value lower than 0.05 was considered to be statistically significant.

3. Results

3.1. Effects of single injections of clozapine and haloperidol on the 5-HTec receptor binding characteristics in the rat choroid plexus Scatchard analysis with [3H]mesulergine showed that both doses (10 and 25 m g / k g ) of clozapine significantly decreased the density (Bmax) of 5-HT2c receptors by

Table 2 Effects of single injections of clozapine (10 and 25 mg/kg) and haloperidol (0.5 mg/kg) on the maximal (10 /zM) 5-HT-stimulated phosphoinositide hydrolysis response in rat choroid plexus [3H]IP-accumulation 5-HT-stimulation vs. basal Basal 5-HT-stimulation absolute increase, cpm relative increase, n-fold cpm cpm Experiment 1 Control Haloperidol(0.5mg/kg) Clozapine (10 mg/kg) Clozapine (25 mg/kg) Experiment 2 Control Clozapine(25mg/kg)

4050 + 245 3900+312 (-4%) 3 340 + 228 ( - 18%) 2 630 :t: 167 b ( _ 35%)

12400 + 653 14300+ 520 12100 -+ 611 11 700 + 1 520

8 350 + 672 10400-r 653(+25%) 8 760 -+ 765 (+ 5%) 9 070 _+1 510 (+ 9%)

3.12 + 0.24 3.80_+0.36 (+22%) 3.75 -+ 0.35 (+ 20%) 4.37 + 0.36 a ( + 40%)

5 430 -+ 434 3430_+118°(-37%)

15 200 -+ 1 920 12600_+ 956

9 770 -+ 1 450 9170_+ 813(-6%)

2.77 -+0.25 3.67 _+0.24 a (+32%)

Absolute values of [3H]IP-accumulation (basal and 5-HT-stimulation) are expressed as cpm. On the right side of the table, values of 5-HT-stimulation vs. basal values are expressed as cpm and as n-fold (relative) over basal. Compared to control group, there were no statistical differences in the absolute (cpm) values of 5-HT-stimulation vs. basal. Percent changes from control are given in parenthesis. Values represent mean + S.E.M. For experiments 1. and 2., statistical analysis was carried out by Dunnett's test for multiple comparisons and Student's t-test, respectively, a p < 0.05; b p < 0.005 compared to control group.

M. Kuoppami~'ki et al. ~European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 201-208 250

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Fig. 1. Effects of single injections of clozapine and haloperidol on 5-HT2c receptor agonist and antagonist binding sites in the rat choroid plexus as determined by quantitative autoradiography with 0.4 nM [tzSI]DOI (on the right side of the figure) and 5 nM [3H]mesulergine (on the left side), respectively. White bars: saline 1 ml/kg; black bars: haloperidol 0.5 mg/kg; cross-hatched bars: clozapine 10 mg/kg; lined bars: clozapine 25 mg/kg. Bars represent mean + S.E.M. a P < 0.001 compared to control group.

20-25% with no alterations in the affinity (K d) (Table 1). In autoradiographic studies, acute treatment with the dose of 25 mg/kg of clozapine significantly decreased the number of 5-HTzc receptor [3H]mesulergine ( - 2 7 % ) and [lZSI]DOI ( - 5 2 % ) binding sites (Fig. 1). There was also a non-significant trend towards a decreased number of 5-HT2c receptor [3H]mesulergine and [125I]DOI binding sites after single injections of 10 mg/kg of clozapine (Fig. 1). Haloperidol (0.5 mg/kg) did not affect the number of 5-HT2c receptor binding sites.

3.2. Effects of single injections of clozapine and haloperidol on the maximal 5-HT2c receptor-mediated phosphoinositide hydrolysis response in the rat choroid plexus Results of 5-HT2c receptor-mediated phosphoinositide hydrolysis are summarized in Table 2. The basal values of [3H]IP-accumulation were decreased after E

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clozapine treatments, the effect of 25 mg/kg of clozapine reaching statistical significance. 10 /xM 5-HT was used to study the maximal response in 5-HT2c receptor-mediated phosphoinositide hydrolysis after acute drug treatments. In the control group, the maximal 5-HT-stimulated [3H]IP accumulation over the basal values was 3.12 + 0.24-fold (mean + S.E.M.). After a single injection of 25 mg/kg of clozapine, the maximal response in 5-HT-stimulated phosphoinositide hydrolysis was increased by 40%, when expressed as n-fold over basal values. However, when the results are expressed in absolute values (i.e. cpm(stimulated)cpm(basal)), there are no significant alterations in this response after 25 mg/kg of clozapine. The dose of 10 mg/kg of clozapine and haloperidol (0.5 mg/kg) did not statistically significantly affect the maximal 5-HT2c receptor-mediated phosphoinositide hydrolysis response. As the increased (n-fold over basal) 5-HTstimulated phosphoinositide hydrolysis response after 25 mg/kg of clozapine was surprising, the phosphoinositide hydrolysis experiment was repeated with identical results (Table 2.).

3.3. Effects of single injections of clozapine and haloperidol on frontal cortical 5-HTeA and striatal D 2 receptor characteristics The results of 5-HT2A and D 2 receptor binding characteristics are summarized in Table 3. 5-HT2A receptors: Acute clozapine treatment significantly decreased 5-HT 2 receptor density in frontal cortex by 43 and 46% with doses of 10 and 25 mg/kg, respectively. The 5-HT2A receptor K d value was increased after the dose of 25 mg/kg of clozapine, whereas the dose of 10 mg/kg of clozapine was without any effect. A single injection of haloperidol (0.5 mg/kg) did not cause any significant alterations in 5-HT2A receptor binding characteristics in frontal cortex. x

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Fig. 2. Concentrations of clozapine ( ~ g / g tissue wet weight) in the rat striatum and cortex after single subcutaneous injections with doses of 10 (©) and 25 mg/kg (e). Clozapine concentrations were measured 15 and 30 min., and 1, 2, 4, 8, 18, and 24 h after administration. The points represent means of three rats + S.E.M. A: Striatum; B: Cortex.

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Table 3 Effects of single injections of clozapine (10 and 25 m g / k g ) and haloperidol (0.5 m g / k g ) on 5-HT2A and D 2 receptor characteristics in the rat frontal cortex and striatum, respectively 5-HT2A receptors Treatment Control Haloperidol (0.5 m g / k g ) Clozapine (10 m g / k g ) Clozapine (25 m g / k g ) ANOVA D 2 receptors Control Haloperidol (0.5 m g / k g ) Clozapine (10 m g / k g ) Clozapine (25 m g / k g ) ANOVA

Kd 0.74 +_0.16 0.81 _+0.16 0.98 +_0.30 1.31 _+0.62 ~ P = 0.017

Bmax 193 +_31.0 191 _+26.0 109 +- 19.4 b 104+ 19.1 b P < 0.001

0.058+_0.010 0.058 +- 0.007 0.056 +- 0.005 0.062 +- 0.011 P = 0.631

611 +86.0 655 +_77.8 578 _+35.2 678 _+69.3 P = 0.087

Radioligands for determining 5-HT2A and D 2 receptor characteristics were [3H]ketanserin and [3H]spiperone, respectively. Values represent m e a n +- S.E.M., n = 6-8. a p < 0.05; b p < 0,001 compared to control group.

receptors: Single injections of clozapine (10 and 25 mg/kg) or haloperidol (0.5 mg/kg) did not affect D 2 receptor characteristics in the striatum.

D2

3.4. Time course of clozapine concentrations in the rat striatum and cortex after single injections (s.c.) of elozapine with doses of 10 and 25 mg / kg Results are summarized in Fig. 2. Peak concentrations (2.87-4.08 /xg/g tissue depending on dose and brain area) of clozapine were measured at 1 h after subcutaneous administration (10 and 25 mg/kg). Clozapine concentrations in the cortex were generally a little higher than in the striatum. The half-life of clozapine was estimated to be about 4 h in the cortex and striatum. 24 h after administration, there were low levels of clozapine left in the cortex and striatum, concentrations being 0.13-0.23 and 0.12-0.28 /xg/g tissue, respectively.

4. Discussion

We have previously shown that chronic treatment with doses of 10 and 25 m g / k g / d a y of clozapine, in contrast to haloperidol, decreases the density of 5-HT2c receptors in the rat choroid plexus by 50-70% without altering the affinity measured with [3H]mesulergine as a radioligand (Kuoppam~ki et al., 1993a). We have also shown this with quantitative autoradiography as chronic clozapine treatment with the same doses decreased the number of 5-HT2c receptor agonist ([125I]DOI) and antagonist ([3H]mesulergine) labeled binding sites by 20-45% and 50-60%, respectively (Kuoppam~iki et al., 1994). Based on these results, we have suggested that the ability of chronic clozapine treatment to modulate

5-HT2c receptors might contribute to some of the atypical effects of clozapine. Considering the clinical time-delay of the therapeutical actions of clozapine, it is of interest to know how acute clozapine treatment would affect 5-HT2c receptors. Therefore, the effects of a single injection of clozapine on the regulation of 5-HT2c receptors was investigated. Scatchard analysis with [3H]mesulergine showed that the density (Bmax) of 5-HT2c receptors in the choroid plexus was decreased equally after both (10 and 25 mg/kg) clozapine treatments, whereas the affinity (K d) was unchanged. The results obtained with quantitative autoradiography were in accordance with homogenate binding studies showing that both doses of clozapine, unlike haloperidol (0.5 mg/kg), decreased the number of 5-HT2c receptor binding sites labeled by an agonist ([125I]DOI) and an antagonist ([3H]mesulergine) radioligand. However, determined with quantitative autoradiography, the effects were more robust after the higher dose of clozapine. There were low concentrations of clozapine left in the cortex and striatum at the time the receptor parameters were determined. We cannot rule out the possibility that there are low levels of clozapine left also in the choroid plexus after these treatments, as we did not measure clozapine concentrations directly from the rat choroid plexus for practical reasons. Thus, low levels of clozapine may affect, to a minor extent, our 5-HT2c receptor binding results (especially [125I]DOI binding after the higher dose), but they are unlikely to explain the whole decrease in the number of 5-HT2c receptor binding sites after acute treatment with clozapine. Thus, in addition to chronic treatment with clozapine (Hietala et al., 1992; Kuoppam~ki et al., 1993a and 1994), 5-HT2c receptors (labeled by [3H]mesulergine and [125I]DOI) can readily be downregulated by a single injection of clozapine. However, it seems likely that repeated administration of clozapine produces more profound effects on the number of 5-HT2c receptor binding sites, especially those labeled by [3H]mesulergine. Similar kinds of results have also been reported earlier after mianserin (Sanders-Bush and Breeding, 1988) treatments. The major finding of the present study was that the function of 5-HTzc receptors (measured by maximal response of 5-HT-stimulated phosphoinositide hydrolysis) was not desensitized after acute clozapine treatment, although the density of 5-HT2c receptors was decreased. The most likely explanation to this finding is a 5-HT2c receptor reserve in the choroid plexus, which can compensate for acute receptor downregulation. The dose of 25 mg/kg of clozapine increased the 5-HT-stimulated phosphoinositide hydrolysis response, when the results were expressed as n-fold over basal. This unexpected phenomenon was replicated in a separate experiment. However, interpretation of the result

M. Kuoppamdki et al. /European Journal of Pharmacology - Molecular Pharmacology Section 269 (1994) 201-208

is complicated, as basal values of [3H]IP accumulation were also decreased after clozapine treatment, especially after the higher dose. In fact, the increased phosphoinositide hydrolysis response disappears, if the results are expressed in absolute values over basal (i.e. cpm(stimulated) - cpm(basal)). The ability of clozapine to decrease basal values of [3H]IP accumulation is not unique, as also other 5-HT2c receptor antagonists may have this property (Barker and Sanders-Bush, 1993). In any case, the effects of acute clozapine on the function of 5-HTlc receptors are different from the effects of chronic clozapine treatment as functional desensitization of 5-HT2c receptors in the choroid plexus can be observed after the chronic clozapine treatment with the dose of 25 m g / k g / d a y (Kuoppam~iki et al., 1994). Single injections of 10 mg/kg of clozapine or haloperidol (0.5 mg/kg) did not affect the function of 5-HT2c receptors, as was the case after chronic treatments (Kuoppam~iki et al., 1994). Findings that therapeutical effects of clozapine are observed primarily after treatment of several days to several months (e.g. Kane et al., 1988; Meltzer et al., 1989; Baldessarini and Frankenburg, 1991), suggest that mechanisms, which are differentially modulated after acute and repeated administration of clozapine, may more likely contribute to the effects of clozapine. It has also been suggested that receptor sensitivity (i.e. function), rather than receptor binding, better correlates with behavioral effects (Smith et al., 1990). Thus, the above mentioned suggestions and the differential effects of acute and chronic clozapine treatments on the f u n c t i o n of 5-HT2c receptors further support the possibility that the effects of clozapine on 5-HT2c receptors may contribute to some of the atypical effects of clozapine. In addition to choroid plexus, there are 5-HT2c receptors also elsewhere in the brain, e.g. in limbic regions (Pazos and Palacios 1985, Mengod et al. 1990, Hoyer et al. 1986). It should be noted, however, that the regulation of 5-HT2c receptors and the effects of clozapine on 5-HT2c receptors are not known in these brain areas which, compared to choroid plexus, are more likely to mediate the therapeutic effects of clozapine. For comparison and control purposes, we also determined 5-HT2A and D 2 receptor characteristics after these treatments, as previous studies have shown that acute treatment with clozapine downregulates 5-HT2A (Lee and Tang, 1984; Matsubara and Meltzer, 1989), but not D 2 receptors (Lee and Tang, 1984). In agreement with these previous studies, single injections of clozapine with doses of 10 and 25 m g / k g significantly and equally decreased the density of 5-HT2A receptors in the rat frontal cortex by 43 and 46%, respectively. In addition, the dose of 25 mg/kg of clozapine significantly increased the K d of [3H]ketanserin, which is in line with our findings that there are low levels of

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clozapine ( ~ 0.25/xg/g tissue) left in the cortex 24 hr after a single (s.c.) injection of 25 mg/kg of clozapine. However, Matsubara and Meltzer (1989) have reported no changes in K a values for 5-HT2A receptor binding ([3H]ketanserin) in the homogenates of whole cortex after a single i.p. injection of 20 mg/kg of clozapine. Minor differences in the clozapine doses (20 vs. 25 mg/kg), ways of administration (i.p. vs. s.c.) and the cortical homogenates used (frontal vs. whole cortex) may contribute to these differential results. None of the treatments affected D 2 receptor characteristics in the striatum which agrees with the study of Lee and Tang (1984). I n s u m m a r y , clozapine in single doses (10 and 25 mg/kg) is able to reduce the number of 5-HT2c receptors in the rat choroid plexus. In comparison, these doses of clozapine caused a more robust downregulation of 5-HT2A receptors in the frontal cortex. Striatal D 2 receptors were unaffected by these treatments. In turn, acute treatment with haloperidol (0.5 mg/kg) failed to affect any of the receptor parameters. Although low levels of brain clozapine were found 24 hr after single (s.c.) injections, concentration measurements showed that alterations in receptor bindings were unlikely due to residual clozapine concentrations. The major finding of the present study was that in contrast to chronic treatment (Kuoppam~iki et al., 1994), a single injection of clozapine did not cause a functional desensitization of 5-HT2c receptors in the rat choroid plexus. This and our previous study suggest that acute and chronic clozapine treatments differentially modulate the function of 5-HT2c receptors in the rat choroid plexus. This further supports the possibility that 5-HT2c receptors may contribute to some of the atypical effects of clozapine, as in a clinical setting the therapeutical effects of clozapine generally appear with a time delay.

Acknowledgements The excellent assistance of Eija Lehtovirta is gratefully acknowledged. This study was financially supported by Pharmacal Ltd, Turku University Society, the Finnish Medical Society Duodecim, the Cultural Foundation of Finland (Satakunta), The Research Foundation of Leiras Ltd, and the Foundation of Vihtori and Tutta Pentti.

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