Psychiatry Research, 12, 277-285 Else&r

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Loxapine and Clozapine Decrease Serotonin (S,) But Do Not Elevate Dopamine (D2) Receptor Numbers in the Rat Brain

Tyrone Lee and Siu Wa Tang

Received September 28. 1983; revised version received January 25, 1984; accepted June 12, 1984.

Abstract. Chronic administration of loxapine or clozapine in rats for 4 weeks or 10 weeks did not produce enhancement of striatal dopamine receptor density. However, there was a marked reduction (50-60s) of cortical serotonin receptor density associated with clozapine or loxapine administration. Acute doses of clozapine or loxapine produced the same potent effect. The possibility that these two antipsychotic drugs act via the serotonin system in the brain is proposed.

Key Words. Atypical neuroleptics, dopamine, serotonin, receptor density.

The blockade of central dopamine receptors has been proposed to be the major action of antipsychotic drugs such as haloperidol or chlorpromazine (Carlsson and Lindqvist, 1963) in controlling schizophrenic symptoms. This observation led to the proposal of the dopamine hypothesis of schizophrenia by Van Rossum (1966); the hypothesis states that schizophrenia is associated with hyperdopaminergic neuro- transmission in the brain.

Chronic administration of some antipsychotic drugs in animals is known to induce dopamine receptor supersensitivity in the central nervous system (Fjalland and Moller-Nielsen, 1974; Gianutsos et al., 1974; Tarsy and Baldessarini, 1974; Smith and Davis, 1975; Yarbrough, 1975; Clow et al., 1979). In the human, this induction of dopaminergic supersensitivity by neuroleptics is thought to be the probable cause of tardive dyskinesia in chronic schizophrenics (Klawans and Rubovits, 1972; Tarsy and Baldessarini, 1977). On the other hand, some antipsychotic drugs, like clozapine, seem less likely to induce tardive dyskinesia (Wyatt and Delisi, 1984). Tardive dyskinesia may also be caused by an imbalance of the dopaminergic-cholinergic systems in the basal ganglia (Gerlach et al., 1974; Casey and Denney, 1977) an imbalance between indolamines and catecholamines (Prange et al., 1973) or a y-aminobutyric acid (GABA) deficiency (Ananth, 1979; Singh et al., 1982).

Tyrone Lee, Ph.D., is Assistant Director, Psychopharmacology Unit, Clarke Institute of Psychiatry, and Assistant Professor of Pharmacology and Psychiatry, University of Toronto. Siu Wa Tang, M.B., B.S., Ph.D., is Director, Psychopharmacology Unit, Clarke Institute of Psychiatry, and Assistant Professor of Psychiatry and Pharmacology, University of Toronto. (Reprint requests to Dr. T. Lee, Psychopharma- cology Unit, Clarke Institute of Psychiatry, 250 College St., Toronto, Ont.. Canada M5T I R8.)

0165-1781/84/$03.00 @ 1984 Elsevier Science Publishers B.V

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Commonly used antipsychotic drugs that potently block dopamine receptors (such as the butyrophenones and phenothiazines) are more effective in treating the positive symptoms manifested in chronic schizophrenic patients (Angrist et al., 1980), whereas the negative symptoms such as flattening of affect and poverty of speech are less responsive. Central nervous system (CNS) dopamine receptor pathology may not be the cause of certain symptoms seen in schizophrenia, namely, the negative symptoms. Haase et al. (1974) reported that chronic schizophrenic patients given sulpiride, a weak dopamine receptor blocker, at doses of about 200 mg/day showed an elation of mood and clearing of negative signs. Using sulpiride as a trial drug in the treatment of hospitalized schizophrenic patients, Elizur and Davidson (1975) also found it is effective in improving the autistic state of these patients.

We have studied the effects of three antipsychotic drugs-loxapine, clozapine, and sulpiride-on rat brain neurotransmitter receptors. These three antipsychotic drugs have previously been reported either to show minimal extrapyramidal and dyskinetic effects or to have beneficial effects on some of the negative symptoms of schizophrenia (Shopsin et al., 1979; Edwards et al., 1980). Haloperidol, as a typical neuroleptic drug, was also included in this study.

Methods

Chronic Drug Treatment. Adult male Wistar rats (150-I 75 g) were used. They were housed in groups of four with water and food ad lib and were allowed to adapt to the environment for a period of 7 days before drug administration began. Animals were divided into two treatment groups: chronic daily administration for a duration of either 28 days or 10 weeks. Drugs administered and the corresponding doses were: loxapine (5 mg/ kg), clozapine (30 mg/ kg), sulpiride (100 mg/kg), haloperidol (5 mg/ kg), and vehicle (0.1 M tartaric acid, 1 ml per injection). All drugs were dissolved in vehicle and given intraperitoneally, 1 ml per day.

The acute effect of clozapine and loxapine was studied by injecting a separate group of rats with a single dose of the drugs and then sacrificing the animals 24 hours later.

Brain Tissue Preparation. Two days after the last injection, animals were sacrificed by decapitation (around 9 a.m.) and brains were immediately removed. Tissues from the frontal cortex and corpus striatum were dissected out on ice. These brain regions from each rat were separately homogenized with a Teflon-glass homogenizer (clearance: 0.13-0.18 mm) in 20 volumes of ice cold TEAN buffer containing 15 mM tris-HCI, 5 mM Na2EDTA, 1.1 mM ascorbic acid, and 12.5 pM nialamide with pH adjusted to 7.4 using 0.1 N HCI. The crude homogenates, obtained after homogenizing with 20 up-and-down strokes, were centrifuged at 39,000g for 15 minutes at 4°C. The supernatant was then discarded and the pellet was resuspended in fresh TEAN buffer. At this step, tissue homogenate from the frontal cortex was then frozen and stored at -2O’C for radioreceptor assay at a later date. Tissue suspension from corpus striatum was further centrifuged and resuspended in buffer, and the same process was repeated one more time (total three times). At the end of the third centrifugation, the pellet was resuspended in buffer at a tissue concentration of about 20 mg wet tissue per ml of buffer. They were then frozen and stored at -20°C for future radioreceptor assays.

Dopamine (Dz) Receptor Assay. The method is essentially the same as described previously (Lee and Tang, 1982). The frozen homogenate from corpus striatum was thawed and centrifuged at 39,000g for I5 minutes at 4’C. They were then resuspended in TEAN buffer such that the final membrane protein concentration was about 0.2 mg/ ml. The tissue suspension was further homogenized with a Brinkmann Polytron (PT-IO, full scale of IO) for 20 seconds at a setting of 6. Then, 0.2 ml aliquots of such membrane suspension were added to test tubes already

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containing 0.2 ml of 3H-spiperone (New England Nuclear Corp., speci$ic activity = 25-30 Ci/mmole) at various concentrations and 0.2 ml of either buffer or IO- M sulpiride. The ingredients were thoroughly mixed with a vortex and allowed to incubate at room temperature for 30 minutes. At the end of the incubation period, 0.5 ml of the incubate was rapidly filtered through Whatman GF/ B glass fiber filters and immediately followed by a wash with 10 ml of buffer. The filters were then transferred to scintillation vials and 9 ml of Aquasol was added to each vial. The radioactivity was measured by a liquid scintillation counter after the filters were equilibrated in Aquasol overnight.

Serotonin (SJ Receptor Assay. Essentially the method is the same as described above. Crude homogenate from frontal cortex was thawed and then polytroned for 20 seconds before use. 3H-Spiperone was used to label the receptors and 50 nM mianserin was used to displace the radioligand from the binding sites.

The radioreceptor binding assays were carried out using different concentrations of radioligand (0.05-I nM for dopamine receptors and 0.05-3 nM for serotonin receptors), thus allowing determination of receptor densities (B,,,) and receptor affinities (Kd) by Scatchard analysis. Statistical significance was tested using the two-tailed Student’s t test.

Results

As shown in Table 1, haloperidol given to rats for a duration of either 4 or 10 weeks produced a marked enhancement of dopamine (D2) receptor density of 47% @ < 0.01) and 52% (p < O.OOl), respectively. Loxapine and clozapine did not produce any significant increase in dopamine receptor density during this period up to 10 weeks. Sulpiride induced an increase of 22% (p < 0.05) in receptor density after 4 weeks of treatment, but such elevation was not detectable at 10 weeks of treatment.

Table 1. Dopamine (Dz) receptor densities after chronic neuroleptic treatment in rat brain

4 Weeks 10 Weeks

H % B MX Control K, n L Control K, n

Control 45Ok 871 100 0.110 f 0.0382 5 411f29 100 0.094 Ik 0.029 6

Haloperidol 66Ok 65 1473 0.120 f 0.054 5 624f81 1524 0.113 f 0.029 5

Loxapine 484f105 108 0.106 fO.029 6 462269 112 0.086 f 0.016 7

Clozapine 477+ 65 106 0.130 f 0.072 5 426+56 104 0.087 fO.009 5

Sulpiride 547$ 39 1225 0.100 + 0.025 6 431 f47 105 0.075 f 0.012 6

1. h&,x= Mean + SD, maximum binding of sH-spiperone to rat striatum in femtomoles/mg protein 2. Kd = Mean t SD, dissociation constants in nanomoles/iiter. 3. p c 0.01, by Student’s t test (two-tailed), compared to control group. 4. p < 0.001, by Student’s t test (two-tailed), compared to control group. 5. p < 0.05, by Student’s t test (two-tailed), compared to control group.

Table 2 shows loxapine and clozapine ind&ed a very significant reduction (more than 50%) of serotonin (S,) receptor density after 4 weeks or 10 weeks of daily injection in the rat. Sulpiride did not show any significant effect on serotonin receptor density. Haloperidol, on the other hand, produced an increase (200/o) of serotonin receptor density after 4 weeks of treatment. This effect appeared to be weaker at IO-week exposure to haloperidol and the increase became nonsignificant.

Table 2. Serotonin (S,) receptor densities after chronic neuroleptic treatment in rat brain

4 Weeks 10 Weeks

% % t3 m.r Control K, n t3 max Control K, n

Control 403 + 431 100 0.370$0.1012 5 358 f 69 100 0.34OkO.112 5

Haloperidol 482 2 36 1203 0.460 f 0.134 5 407 + 121 114 0.390 + 0.112 5 Loxapine 167234 414 0.350 + 0.215 5 1422 36 404 0.250 2 0.128 5

Clozapine 190+76 474 0.303 Z?I 0.167 6 164k 72 465 0.230 _+ 0.112 5

Sulpiride 346 + 69 86 0.290 z!z 0.141 5 3722 114 104 0.340 _+ 0.112 5

1. Bmax= mean k SD, maximum binding of sH-spiperone to rat frontal cortex in femtomoles/mg protein 2. Kd = mean k SD, dissociation constants in nanomoles/liter. 3. p < 0.02. by Student’s f test (two-tailed), compared to control group. 4. p c 0.001. by Student’s t test (two-tailed), compared to control group. 5. p c 0.01, by Student’s t test (two-tailed), compared to control group.

Tables 3 and 4 show the acute effect of clozapine and loxapine, respectively, on dopamine and serotonin receptors. A single dose of clozapine did not alter the dopamine receptor density but had a strong effect in lowering the density of serotonin receptors (48% reduction, p < 0.001, Table 3). Similarly, acute loxapine did not change dopamine receptor density but greatly reduced serotonin receptor density (47% reduction, p < 0.0 1, Table 4).

All drugs under investigation did not show any significant change on the affinities of receptors (K,,). The range for dopamine receptors was 0.08-o. 13 nMand the range for serotonin receptors was 0.23-0.46 nM.

Table 3. Acute effects of clozapine on rat dopamine and serotonin receptors

Dopamine receptor Serotonin receptor

% % B m.x Control Kd n a,,, Control K, n

Control 473 + 401 100 0.083 f 0.0252 5 436 2 56 100 0.290 ? 0.058 5

Clozapine 494 + 58 105 0.107 * 0.089 5 226 f 69 523 0.380 k 0.239 5

1. &ax = mean + SD, maximum binding of sH-spiperone in femtomoles/mg protein 2. Kd = mean + SD, dissociation constants in nanomoles/liter. 3. p c 0.001, by Student’s t test (two-tailed).

Table 4. Acute effects of loxapine on rat dopamine and serotonin receptors

Dopamine receptor Serotonin receptor

% 6 “._” Control K, n 8 m.x Cozhol K, n

Control 498 k 491 100 0.055kO.1312 5 4552116 100 0.380 4 0.154 5

Loxaoine 308 f 69 623 0.059 f 0.022 5 239 2 47 533 0.280 + 0.089 5

1. Elmax = mean + SD, maximum binding of sH-spiperone in femtomoles/mg protein 2. Kd = mean + SD, dissociation constants in nanomoles/liter. 3. p < 0.01. by Student’s t test Itwo-tailed).

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Discussion

Antipsychotic drugs like haloperidol or chlorpromazine have been demonstrated to increase the dopamine receptors in the rat after chronic treatment (Burt et al., 1977; Theodorou et al., 1981). Our present data indicated that loxapine and clozapine, however, did not increase dopamine receptor density in the rat brain compared to haloperidol. This finding is compatible with reports by several investigators that clozapine did not cause any change in the dopamine system in animals (Kobayashi et al., 1977; Tye et al., 1979; Rebec et al., 1982; Seeger et al., 1982). The finding that sulpiride caused a 22% increase in dopamine receptor density in the present study is in agreement with some recent reports (Hall et al., 1981; Lau et al., 1983) that chronic sulpiride induced dopaminergic supersensitivity in the rat. Bannet et al. (1980) and Montanaro et al. (1982) however, reported that chronic sulpiride treatment in the rat had no effect on dopamine receptors.

The possibility that residual drug left on the membrane might have affected receptor binding can be ruled out since the affinity constants (K,) were not significantly different among the various drug groups compared to the vehicle-control group.

Clozapine and sulpiride have been used to treat schizophrenic patients because of their minimal extrapyramidal side effects (Matz et al., 1974; Mielke et al., 1977; Gelenberg and Doller, 1979; Lapierre et al., 1980; Rama Rao et al., 1981). Since the chronic administration of Ioxapine and clozapine in this study did not seem to produce any significant increase in dopaminergic receptor numbers, the probability of developing tardive dyskinesia after long-term use may be less likely, although loxapine has not been shown to be devoid of extrapyramidal side effects.

In our present study, loxapine and clozapine were very effective in altering the serotonin neuronal system, causing a significant reduction of serotonin (S,) receptor density. Reynolds et al. (1983) also recently reported the down-regulation of cortical Sz receptors by chronic clozapine treatment, Acute clozapine and loxapine were also effective in reducing S, receptor number. Thus far, the unusual effect on S2 receptors has only been reported for tricyclic (Bergstrom and Kellar, 1979; Peroutka and Snyder, 19806; Barbaccia et al., 1983; Friedman et al., 1983) and some other antidepressant drugs (Peroutka and Snyder, 1980~; Fillion and Fillion, 1981; Wong and Bymaster, 198 1; Blackshear and Sanders-Bush, 1982). Even more intriguing is the potent effect of clozapine and loxapine in reducing the S, receptor after a single dose. Again, this same action has only been so far reported in some atypical antidepressant drugs, e.g., mianserin (Blackshear and Sanders-Bush, 1982). The elevation of ‘& receptor number by haloperidol after 4 weeks of treatment is difficult to interpret but may signify the interaction between the serotonin and dopamine systems. This possibility needs to be studied further.

Some schizophrenic patients who do not respond to the classical neuroleptics such as haloperidol and chlorpromazine have been shown to have enlarged cerebral ventricles (Weinberger et al., 1980; Andreasen et al., 1982; Frangos and Athanassenas, 1982; Nasrallah et al., 1982). These patients have been diagnosed as primarily having negative symptoms such as flattening of affect and poverty of speech in the course of their illness (Crow, 1980, 1981; Mackay, 1980; Andreasen and Olsen, 1982). Post- mortem human brain studies have demonstrated a subgroup of schizophrenic patients

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showing normal numbers of dopamine receptors in the brain (Lee and Seeman, 1980a, 19806, 1982). These brains may have been from patients who had large ventricles and were also resistant to neuroleptic drug treatment. Nasrallah et al. (1980) failed to show the neuroleptic-potentiating effect of a-methyl-p-tyrosine, a dopamine synthesis inhibitor, in schizophrenic patients with large ventricles. Potkin et al. (1983) reported an abnormality of the serotonin system in schizophrenic patients with large ventricles. All these observations suggest that the etiology of a complex disease such as schizophrenia cannot be explained by dopamine abnormality alone.

Wyatt and DeLisi (1984) pointed out that clozapine-like drugs may have characteristics that are not shared with the conventional neuroleptics such as haloperidol. In the present study, we have obtained evidence showing that loxapine and clozapine have possible effects on serotonergic neuronal systems that distinguish them from typical antipsychotic drugs.

In conclusion, it is possible that the effect of some antipsychotic drugs such as loxapine and clozapine on the serotonin system may contribute to their therapeutic action. The absence of dopamine (D2) receptor increase after chronic clozapine or loxapine administration suggests that these drugs may not induce tardive dyskinesia in patients.

Acknowledgments. The authors thank Ms. Lydia Burkitt for her excellent technical assistance. Thanks are due to the following companies for their donation of drugs: McNeil Pharmaceuticals (Canada) Ltd. (haloperidol); Delagrange International (sulpiride); Cyanamid Canada Inc. (loxapine); and Sandoz (Canada) Ltd. (clozapine). This study has been supported by the Research Fund of the Clarke Institute of Psychiatry. Dr. SW. Tang is an Ontario Mental Health Foundation Scholar.

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