Effects of acute or long-term treatment with chlorpromazine, haloperidol or sulpiride on neuropeptide Y-like immunoreactivity concentrations in the nucleus accumbens of rat

European Neuropsychopharmacology 9 (1999) 51–59

Effects of acute or long-term treatment with chlorpromazine, haloperidol or sulpiride on neuropeptide Y-like immunoreactivity concentrations in the nucleus accumbens of rat a, b E. Obuchowicz *, J. Turchan a

b

´ Street, 40 -752 Katowice, Poland Department of Clinical Pharmacology, Silesian University School of Medicine, 18 Medykow Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31 -343 Cracow, Poland Received 23 January 1997; received in revised form 28 November 1997; accepted 8 January 1998

Abstract The effects of acute, subchronic (14 days) or chronic (28 days) intraperitoneal (i.p.) administration of chlorpromazine (2 or 10 mg / kg), haloperidol (0.5 or 2 mg / kg) or sulpiride (50 or 100 mg / kg) on the neuropeptide Y (NPY) system in the rat nucleus accumbens were studied. NPY-like immunoreactivity (NPY-LI) decreased in a dose- and time-dependent manner, and was the lowest after haloperidol. NPY-LI levels increased 8 days after withdrawal of chronic drugs treatment. Acute administration of haloperidol reduced NPY mRNA, while subchronic treatment did not change it. Subchronic i.p. administration of the dopamine D 1 -like antagonist SCH 23390 (1 mg / kg) reduced NPY-LI levels but the a 1 -adrenergic antagonist prazosin (0.2 mg / kg) had no effect. The effect of sulpiride coadministered with SCH 23390 was greater than that of SCH 23390 alone, while prazosin coadministered with sulpiride insignificantly reduced the effect of sulpiride. The dopamine D 2 / D 3 agonist quinpirole given as a single injection (3 mg / kg) did not alter NPY-LI content by itself but antagonized the chlorpromazine-induced decrease and attenuated the haloperidol-induced decrease. Our findings indicate that the accumbens NPY system is markedly affected by the antipsychotics studied, and suggest that their effects may be in part mediated by blockade of D 2 -like (D 2 , D 3 ) and D 1 dopaminergic receptors.  1999 Elsevier Science B.V. / ECNP. All rights reserved. Keywords: Antipsychotics; Neuropeptide Y-like immunoreactivity; NPY mRNA; Nucleus accumbens; Rat

1. Introduction The results of many different studies using a large variety of techniques indicate that classical and atypical antipsychotic drugs have strong effects on the mesolimbic area where dopamine plays a predominant role (Ellenbroek, 1993). Besides the generally accepted theory that the mechanism of antipsychotic action of these drugs is based on their well documented antagonism towards dopamine receptors (Sunahara et al., 1993) several pieces of evidence indicate that, in addition to dopamine, other neurotransmitters and neuromodulators such as serotonin (Iqbal and van Praag, 1995), norepinephrine (Joyce, 1993), glutamate (Ishimaru and Torn, 1997) or sigma ligands (Debonnel and de Montigny, 1996) may be involved in *Corresponding author. Fax: 148 32 2523902.

antipsychotic mechanisms. Biologically active neuropeptides are also considered as potential candidates which may, at least partially, mediate this action. Antipsychotics have been shown to affect metabolism of some neuropeptides (Waters et al., 1995) and the levels and mRNA expression of neurotensin (Myers et al., 1992), enkephalins (Herman et al., 1991), substance P (Humpel et al., 1990) and cholecystokinin (Frey, 1983). Neuropeptide Y (NPY) is abundantly distributed through the central nervous system, where it exhibits remarkable ¨ biological activity (Heilig and Widerlow, 1995). High levels of NPY are found in the nucleus accumbens (De Quidt and Emson, 1986), a major part of the mesolimbic system, with a low density of specific NPY receptors, especially of Y 2 subtype (Dumont et al., 1996). In situ hybridization has shown an abundance of NPY mRNAcontaining neurons in the rat ventrolateral neostriatum

0924-977X / 99 / $ – see front matter  1999 Elsevier Science B.V. / ECNP. All rights reserved. PII: S0924-977X( 98 )00007-8

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

52

(Houdeau and Boyer, 1994). In the nucleus accumbens NPY has been found in aspiny interneurons that form symmetrical (inhibitory) junctions with other neurons (Massari et al., 1988). Synapses between NPY-containing neurons and g-aminobutyric acid-ergic (Massari et al., 1988) or tyrosine hydroxylase-positive terminals (Aoki and Pickel, 1988) have been visualized. Although NPY does not coexist with dopamine, biochemical and immunohistochemical reports suggest that NPY and dopaminergic systems are interrelated. Salin et al. (1990) have found that a-methyl-para-tyrosine treatment as well as 6-hydroxydopamine (6-OHDA) lesion of the nigral dopaminergic pathway significantly decrease the density of accumbens NPY neurons. Apomorphine treatment completely reversed the effect of 6-OHDA at the ipsilateral side and partially at the contralateral side. Multiple administration of selective dopamine receptor antagonists or agonists have been shown to alter accumbens NPY content (Midgley et al., 1994). Little is known about the function of NPY in the nucleus accumbens. Josselyn and Beninger (1993), using the place preference condition paradigm, have shown rewarding properties of NPY injected into this structure. Since the effect of NPY was blocked by cis-flupenthixol pretreatment, the authors suggest that intact dopaminergic neurotransmission is required for manifestation of some NPY behavioural effects. All findings indicating that NPY and dopaminergic systems are interrelated and reciprocally regulated suggest that the NPY system may be affected in conditions of disturbed dopaminergic transmission such as schizophrenia, and may be involved in the mechanism of action of antipsychotic drugs. Furthermore, it has been shown that acute and multiple administration of phencyclidine, a drug that can induce schizophrenia-like psychotic symptoms, have profound effects on NPY systems in the limbic structures of rat (Midgley et al., 1993; Sakai et al., 1996). The main purpose of this study was to evaluate the effects of acute or long-term (14 or 28 days) treatment with two typical antipsychotics, chlorpromazine (CPZ) and haloperidol (HAL) or atypical antipsychotic, sulpiride (SULP) on the NPY-like immunoreactivity (NPY-LI) in the rat nucleus accumbens. NPY mRNA levels after acute or subchronic HAL administration were determined by in situ hybridization. The effects of drug withdrawal after chronic treatment on NPY-LI were also investigated. Additionally, we attempted to partly explain the possible mechanism of the effects observed. The obtained results were partly presented at the First European Congress of Pharmacology, Milan, 1995 and published as an abstract (Obuchowicz et al., 1995)

2. Experimental procedure

2.1. Animals Male Wistar rats (the Animal Farm of the Silesian

University School of Medicine) weighing 280–320 g before decapitation were used in the experiments. The rats were housed seven to eight per cage (55332318 cm) under standard conditions (artificial light from 7 a.m. to 7 p.m.; 22628C ambient temperature) with tap water and standard pellet diet (24% crude protein) ad libitum. The rats adapted to their new environment for 5 days before the experiments. All experiments were performed between 9 a.m. and 3 p.m. This study was approved by the Bioethical Committee of the Silesian University School of Medicine.

2.2. Drug treatments Antipsychotics were administered intraperitoneally (i.p.) in a volume of 2 ml / kg once daily at the following doses (mg / kg): CPZ (Sigma) 2 or 10; HAL (Sigma) 0.5 or 2; (6)SULP (Sigma) 50 or 100. The doses of CPZ are expressed as free base. CPZ hydrochloride was dissolved in 0.9% saline. HAL was dissolved in a minimal quantity of glacial acetic acid (1% v / v) (1 mg of HAL per 50 ml of 1% acetic acid). SULP were initially dissolved in deionized water acidified with glacial acetic acid to pH 5.7. After dilution to an appropriate volume with deionized water the pH of HAL and SULP was adjusted with 1 M sodium hydroxide to 6.2. Drug solutions were daily prepared. The following experiments were carried out.

2.2.1. Experiment 1 Antipsychotics were administered i.p. once, for 14 or 28 consecutive days. Controls were injected with the corresponding volume of neutral saline. Each group consisted of 6–8 rats. Rats were sacrificed approximately 24 h after a single or the last dose of drugs or on the eighth day after drug withdrawal. 2.2.2. Experiment 2 Rats were injected i.p. with the dopamine D 1 -like antagonist SCH 23390 (Research Biochemicals) (1 mg / kg) or the a 1 -adrenergic antagonist prazosin (Sigma) (0.2 mg / kg) alone or in combination with the dopamine D 2 -like antagonist SULP (100 mg / kg) for 14 days. The rats were sacrificed approximately 24 h after the final dose. Before injection, SCH 23390 was dissolved in 25 ml of 50% ethanol and further diluted with deionized water; prazosin was suspended in 1% Tween 80. 2.2.3. Experiment 3 In some groups treated with the higher dose of CPZ, HAL, or SULP for 14 days, the dopamine D 2 / D 3 agonist quinpirole (Research Biochemicals) was administered i.p. (3 mg / kg) approximately 24 h after the final dose of drug and 3 h before killing the rats. Before administration, quinpirole was dissolved in physiological saline. In all experiments, control groups were simultaneously given i.p. injections of saline or 1% Tween 80, respectively (2 ml / kg).

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

2.3. Extraction of brain tissue After decapitation brains were rapidly removed and placed on a Petri dish filled with ice. The nucleus accumbens was isolated according to the method of Horn et al. (1974), frozen on dry ice and weighed. The dissected structures were homogenized in 20 vol of 0.5 M acetic acid at 48C, boiled for 10 min in a water bath and then quickly chilled and centrifuged (19 000 g, 30 min, 48C). The supernatants were lyophilized and stored at 2208C until assayed for NPY-LI content.

2.4. NPY radioimmunoassay Before radioimmunoassay the lyophilized samples were reconstituted with 1 ml of assay buffer and centrifuged (8500 g, 15 min, 48C). The supernatants were diluted (1:5) with buffer. The assay buffer (pH 7.4) consisted of 19 mM Na 2 H 2 PO 4 and 81 mM Na 2 HPO 4 , 10 mM Na 2 –EDTA, 0.1% Triton X-100, 0.01% NaN 3 and 0.5% bovine serum albumin. Synthetic porcine NPY (Peninsula Lab.) was applied as a standard in serial dilutions of 60–1000 pg per tube. The rabbit antiporcine NPY antiserum (RAS 7172, Peninsula Lab.) exhibited 100% cross-reactivity with: NPY (human, porcine, rat), NPY 13 – 36 (porcine), NPY 18 – 36 (porcine), peptide YY (porcine) and 1.6% with pancreatic polypeptide (human) but did not cross-react with VIP, vasoactive intestinal peptide, (human, porcine, rat), amylin, insulin (human) and somatostatin. Duplicate aliquots (100 ml) or standard (100 ml) were mixed with diluted NPY antiserum (100 ml) and incubated at 48C for 48 h. Then 125 I-labelled NPY (IM 170, Amersham) was added to each tube (15 000 cpm / 100 ml) and the reaction mixture was incubated at 48C for 24 h. Free and antibodybound NPY were separated by adding 500 ml of a charcoal suspension containing 0.8% charcoal untreated powder, 100–400 mesh, and 0.08% dextran 70 000. After vortexing tubes for 1 min, the suspension was centrifuged (1500 g, 20 min, 48C). The radioactivity of supernatant was counted in a LKB gamma counter. The assay detection limit was 14 pg per tube. The intra- and interassay coefficients of variation were 5 and 11%, respectively.

2.5. The in situ hybridization histochemistry Twenty-four hours after a single or fourteenth HAL injection, rats (3–4 in each group) were decapitated, and their brains were dissected and frozen on dry ice. Ten consecutive series of coronal sections (12 mm thick), starting at 2.20 mm from bregma according to the stereotaxic atlas of Paxinos and Watson (1986) were made across the brain at 250 mm intervals on a Shandon cryostat (UK). The brain sections were thaw-mounted onto chrome–alum-pretreated slides, postfixed in 4% formaldehyde for 10 min and processed for in situ hybridization according to Young III et al. (1986). A mixture of synthetic deoxynucleotides (New England Nuclear), com-

53

plementary to bases 837–866 of rat NPY mRNA were used. Specific patterns of hybridization, found in brain sections, fully agreed with the well-known distribution of mRNA coding for NPY in those structures, and provided support for the specificity of probes under the present experimental conditions. The probes were labelled using 35 S-dATP, 35 S-deoxyadenosine 59-(a thio)triphosphate (1200 Ci / mmol, New England Nuclear) to obtain a specific activity of about 2310 6 Ci / mol. The prehybridization treatment consisted of acetylation and dehydration and was performed according to Young III et al. (1986). The sections were then hybridized with the oligonucleotide probes (2310 6 cpm / 25 ml) for 20 h at 378C in a humidified incubator. After washing at 408C, as described previously, the sections were exposed to Hyperfilm MP (Amersham) at 108C for 14 days. Hybridization of the [ 35 S]oligonucleotide probes was assessed by optical density measurements, taken from autoradiograms corresponding to the coronal sections of the brain, using an image processing and an analysis system. Quantification of the signals on Hyperfilm MP (Amersham) was performed using the Microcomputer Imaging Device (Imaging Research, Brock University, Canada) software. Quantitative densitometry was performed by calibrating to a set of standards before reading density values. We used isotope 35 S standards coexposed with each film as reference. Quantitative changes were recorded as the relative optical density. The optical density values were calculated after subtraction of the film background density. The optical density was measured in the nucleus accumbens bilaterally with no less than four levels (coronal sections). The mean optical density values were obtained by averaging measurements from the autoradiograms of the brain sections, obtained from 3–4 animals.

2.6. Statistical analysis The results were analysed with Student’s t-test or onefactor analysis of variance followed by Duncan’s test.

3. Results

3.1. Effects of acute treatment with antipsychotics on NPY-LI concentrations in the nucleus accumbens NPY-LI levels decreased insignificantly after single injections of both doses of CPZ (by about 20%) and the higher dose of HAL (26%). Only the lower dose of HAL markedly reduced NPY-LI content (by 30%). SULP did not affect NPY-LI concentrations (Fig. 1).

3.2. Effects of subchronic treatment with antipsychotics on NPY-LI concentrations in the nucleus accumbens NPY-LI concentrations decreased in a dose dependent manner after 14 day administration of the higher dose of

54

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

Fig. 1. The effect of acute administration of antipsychotics: chlorpromazine (CPZ), haloperidol (HAL) or sulpiride (SULP) on accumbens NPY-LI concentrations. Doses of drugs (mg / kg) administered as described in Section 2.2 are given under abcissa of the graph. The horizontal line means an average value for the control NPY-LI levels. Bars represent the mean6S.E.M.; n56–8 rats in each group. * P,0.05 vs. control, Student’s t-test.

CPZ (by 27%), both doses of HAL (by 24 or 35%, respectively); the higher dose of SULP (by 20%) (Fig. 2).

3.3. Effects of chronic treatment with antipsychotics on NPY-LI concentrations in the nucleus accumbens

Fig. 3. The effect of 28 day treatment with antipsychotics: chlorpromazine (CPZ), haloperidol (HAL) or sulpiride (SULP) on accumbens NPY-LI concentrations. Details as in Fig. 1. ** P,0.01 vs. control, Student’s t-test.

of CPZ and HAL and both doses of SULP. Considerable increases in NPY-LI concentrations were seen after withdrawal of HAL (by 69%), CPZ (by 20%) and SULP (by 24 and 19% respectively) in comparison with the values observed 24 h after chronic treatment. Moreover, NPY-LI levels after HAL withdrawal were higher than in the control group (by 24%) (Fig. 4).

NPY-LI content was still markedly decreased after 28 day treatment with the higher dose of HAL (by 28%) and the lower and higher doses of SULP (by 30 and 20% respectively). CPZ had no effect on accumbens NPY-LI (Fig. 3).

3.5. Effects of acute and subchronic treatment with the higher dose of HAL on NPY mRNA in the nucleus accumbens

3.4. Effects of 8 day withdrawal of chronic treatment with antipsychotics on NPY-LI concentrations in the nucleus accumbens

In situ hybridization shows presence of mRNA coding for NPY in the nucleus accumbens (Fig. 5). The levels of NPY mRNA were diminished after acute HAL treatment (by 34%) but remained unchanged after subchronic administration (Fig. 6).

Withdrawal of neuroleptics reversed the effects of chronic treatment in the rats injected with the higher doses

Fig. 2. The effect of 14 day treatment with antpsychotics: chlorpromazine (CPZ), haloperidol (HAL) or sulpiride (SULP) on accumbens NPY-LI concentrations. Details as in Fig. 1. ** P,0.01 vs. control, Student’s t-test.

Fig. 4. The effect of 8 day withdrawal after chronic treatment with antipsychotics: chlorpromazine (CPZ), haloperidol (HAL) or sulpiride (SULP) on accumbens NPY-LI concentrations. (a) 28 day treatment; (b) withdrawal. Details as in Fig. 1. * P,0.05, ** P,0.01 vs. control; 11 P,0.01 drugs vs. withdrawal, Student’s t-test.

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

55

Fig. 7. The effect of 14 day i.p. coadministration of SCH 23390 (1 mg / kg) or prazosin (PRAZ) (0.2 mg / kg) and sulpiride (SULP) (100 mg / kg) on accumbens NPY-LI concentrations. Rats were sacrificed approximately 24 h after the final dose. Results are expressed as percent of respective control6S.E.M.; n57 rats per group. ** P,0.01 vs. control, Student’s t-test; [ P,0.05 vs. SCH 23390, Duncan’s test. Fig. 5. Distribution of NPY mRNA at the level of the striatum and nucleus accumbens of the rat.

concentration but slightly attenuated SULP-induced decrease (Fig. 7).

3.6. Effects of coadministration of the dopamine D2 -like receptor antagonist SULP with the dopamine D1 -like receptor antagonist SCH 23390 or the a1 -adrenergic antagonist prazosin on NPY-LI concentrations in the nucleus accumbens Both SCH 23390 and SULP administered alone for 14 days similarly decreased accumbens NPY-LI content (by 13 or 20% respectively). Coadministration of SCH 23390 and SULP increased NPY-LI content in comparison to SCH 23390 only. Prazosin alone had no effect on NPY-LI

Fig. 6. The effect of acute or 14 day i.p. treatment with haloperidol (HAL) on NPY mRNA in the nucleus accumbens. Rats were sacrificed 24 h after the single or last injection. Results are expressed as percent of control6S.E.M.; n53–4 rats per group. * P,0.05 vs. control, Duncan’s test.

3.7. Effects of the dopamine D2 /D3 receptor agonist quinpirole on NPY-LI concentrations in the nucleus accumbens of rats pretreated with the higher dose of CPZ, HAL or SULP A single injection of quinpirole had no effect on NPY-LI concentrations (Fig. 8). Acute quinpirole administration after 14 day neuroleptic treatment completely blocked the decrease in accumbens NPY-LI content induced by CPZ (from 73% to 109% of control), attenuated the effect of HAL (from 63% to 85% of control), but did not change the effect of SULP (Fig. 8).

Fig. 8. The effect of quinpirole (QUIN) acute administration after 14 day i.p. treatment with antipsychotics: chlorpromazine (CPZ) (10 mg / kg), haloperidol (HAL) (2 mg / kg), sulpiride (SULP) (100 mg / kg) on the NPY-LI concentrations in the nucleus accumbens. QUIN (3 mg / kg i.p.) was administered as described in Section 2.2. Results are expressed as percent of respective control6S.E.M.; n56–7 rats per group. * P,0.05, ** P,0.01 vs. control, Student’s t-test; [ P,0.05 CPZ1QUIN vs. CPZ, Duncan’s test.

56

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

4. Discussion The present findings demonstrate that the typical antipsychotics CPZ and HAL and the atypical antipsychotic SULP decrease NPY-LI concentrations in the nucleus accumbens of naive rats. The NPY-LI levels decreased both after single and multiple administration of drugs; yet multiple administration produced more profound effects, which were dose- and time-dependent (Figs. 1–3). The antipsychotics used in this study are characterized by different pharmacological profiles (Leysen et al., 1993) which show the following main features. CPZ and HAL interact with several brain receptors. They antagonize D 2 like (D 2 , D 3 ) and D 1 dopamine receptors, but more strongly D 2 -like receptors. Among the drugs studied HAL is the most effective mixed D 2 -like / D 1 antagonist. CPZ displays high affinity for a 1 -adrenergic receptors and also acts on 5-HT 2 serotoninergic, H 1 histaminergic receptors. In this regard, HAL is less active than CPZ. Unlike CPZ and SULP, HAL exhibits high affinity for the s-receptor sites, which are postulated to be involved in the pathogenesis of schizophrenia (Debonnel and de Montigny, 1996). SULP preferentially binds to D 2 -like (D 2 , D 3 ) receptors (Leysen et al., 1993). The drugs studied were administered in two different doses, the higher one has been shown to alter the activity of other neuropeptide systems in experiments that were similar to ours (Millington et al., 1985; Angulo et al., 1990; Humpel et al., 1990; Marksteiner et al., 1992; Jaber et al., 1994; Waters et al., 1995). The effect of HAL on NPY-LI levels was most pronounced. Taking into consideration the pharmacological mechanisms of the drugs studied and the fact that accumbens NPY is modulated by the dopaminergic system (Salin et al., 1990; Midgley et al., 1994), this effect may be explained by a distinct affinity of HAL to D 2 -like and D 1 dopamine receptors. An altered function of s-receptor sites in response to HAL treatment may be of significance for the regulation of NPY system activity when one considers that NPY interacts with these binding sites (Roman et al., 1993). HAL was reported to give a similar effect on accumbens NPY-LI content by Miyake et al. (1990). They injected HAL (5 mg / kg i.p.) for 6 or 21 days and found an insignificant 20% decrease in the NPY-LI level accompanied by a marked decrease in the density of NPY immunoreactive fibers and terminals in the rostral part of the nucleus accumbens. A significant decrease in accumbens NPY-LI levels was also found in rats which were given five doses of SULP (80 mg / kg i.p.) (Midgley et al., 1994). Interestingly, a decrease in somatostatin concentrations following a 3 week treatment with CPZ, fluphenazine and HAL has been demonstrated in the nucleus accumbens (Beal and Martin, 1984). The similar response of these two neuropeptides to neuroleptics is in accordance with their identical distribution in this structure (Beal et al., 1986).

Additionally, this study has revealed that the effect of chronic antipsychotic treatment on NPY-LI concentrations is reversible. On the eighth day after CPZ, HAL or SULP withdrawal a significant increase in the NPY-LI levels was detected (Fig. 4). This change was probably due to enhanced neurotransmission, mainly dopaminergic transmission, produced by the late withdrawal after long-term treatment. This well-known alteration termed ‘‘receptor supersensitivity’’ is accompanied by an increased number of dopaminergic receptors. In our study, the highest increase in NPY-LI concentrations was shown after withdrawal of HAL administered at a dose of 2 mg / kg. This increase may be explained by the report of Laruelle et al. (1992) who observed that withdrawal of this HAL dose resulted in a 40% increase in the density of mesolimbic D 2 receptors. Since alterations in peptide content may reflect various changes in peptide metabolism, the accumbens NPY mRNA level after HAL treatment was estimated to identify the mechanism probably responsible for the effects observed. HAL was chosen because it induced a more pronounced effect on NPY system activity. Since a single injection of HAL reduced NPY mRNA, we can conclude that observed lower NPY-LI content is due to, partly at least, the diminished NPY biosynthesis. The reduced NPYLI concentrations observed after 14 day HAL treatment was not accompanied by detectable changes in mRNA level so it may mean that subchronic administration probably causes an excessive NPY release and / or its degradation. Because the antagonism of D 2 -like (D 2 , D 3 ), D 1 dopaminergic and a 1 -adrenergic receptors, although different for each drug studied, plays an important role in the mechanism of their action, we decided to investigate the effect of their blockade on NPY-LI content. In this study, SCH 23390 known as the D 1 dopamine receptor antagonist (Meller et al., 1985) and the D 2 -like dopamine receptor antagonist SULP administered for 14 days produced similar, significant decreases in NPY-LI content, which indicates that the dopaminergic system through these receptors is involved in maintaining the basal NPY levels (Fig. 7). Although it has been show that apart from complete blockade of D 1 receptors, SCH 23390 used in a high dose (3 mg / kg i.p.) has also affinity for serotonin receptors (Meller et al., 1985) and may act as a 5-HT 1 agonist (Skarsfeldt and Larsen, 1988), in our study, the SCH 23390-induced decrease in NPY-LI is rather unlikely to result from its effect on serotoninergic transmission because, as Midgley et al. (1993) showed, the accumbens NPY system is slightly regulated by the serotoninergic mechanism (about 80% depletion of serotonin with DL-pchloroamphetamine produced only a 15% decrease in NPY-LI). Moreover, our data indicate that blockade of a 1 -adrenergic receptors is not significant for the activity of the NPY system. Taking into account the fact that CPZ and HAL simultaneously antagonized more receptor types, we

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

investigated the effect of coadministration of SULP and prazosin or SULP and SCH 23390. Coadministration of SULP and prazosin insignificantly attenuated the SULPinduced decrease but the effect of SCH 23390 and SULP treatment was significantly stronger in comparison with the SCH 23390-induced decrease. The results of this experiment may indicate that: (1) blockade of D 2 -like (D 2 , D 3 ) receptors plays a more important role in the regulation of the accumbens NPY system than blockade of D 1 receptors; (2) D 2 -like (D 2 , D 3 ) and D 1 dopaminergic but not a 1 adrenergic receptors may partly mediate CPZ- or HALinduced decreases in NPY-LI content. In contrast to our data, Midgley et al. (1994) did not observe any changes in accumbens NPY-LI concentrations after five doses of SCH 233390 (0.5 mg / kg i.p.). This suggests that the response of accumbens NPY system to D 1 receptor blockade is dosedependent. Similar to our results, they also observed that coadministration of D 1 and D 2 antagonists (in five doses) decreased accumbens NPY-LI in the manner of SULP alone. To elucidate whether the antipsychotic-induced alterations in NPY-LI levels are reversed by stimulation of D 2 -like (D 2 , D 3 ) dopamine receptors, we injected antipsychotic-pretreated rats with quinpirole, which displays high affinity for D 2 and D 3 receptors (Sokoloff et al., 1990). We have found that the high dose of quinpirole reversed the effect of subchronic CPZ administration, attenuated the effect of HAL, and unexpectedly failed to change the effect of SULP on NPY-LI levels (Fig. 8). It is noteworthy that the effect of quinpirole on rat behaviour (data not shown) was also most pronounced in CPZpretreated rats, while it was moderate in HAL-pretreated rats. In contrast, in SULP-pretreated rats quinpirole gave only mild behavioural alterations. Moreover, prazosin administered 1 h before quinpirole injection antagonized its effect on both NPY-LI concentrations and the behaviour of CPZ-pretreated rats. In this group we observed only minor behavioural alterations similar to the quinpiroleinduced changes in SULP-pretreated rats (data not shown). The results of our study suggest that the varied effect of quinpirole on different antipsychotic-pretreated groups of rats may be explained, apart from the upregulation of D 2 dopamine receptors, by the upregulation of other receptors including a 1 -adrenergic, which allowed quinpirole to affect NPY-LI levels. Our observation and the results reported by Midgley et al. (1994) indicate that a single injection of quinpirole has no effect on accumbens NPY-LI content in naive rats. The proposed explanation is in accordance with data providing evidence of a cooperative / synergistic interaction between D 2 / a 1 and D 1 / D 2 receptors (Jackson and Westlind-Danielsson, 1994). The results of the third experiment seem to support our previous suggestion that CPZ- and HAL-induced alterations in NPY-LI content may be mediated by D 2 -like (D 2 , D 3 ) dopamine receptors. The stimulation of D 3 receptors by quinpirole seems to be less likely to contribute to its

57

effect on NPY-LI levels in the nucleus accumbens, though it cannot be excluded because of the following reasons: (1) nucleus accumbens is characterized by a high density of [ 3 H]quinpirole-binding sites, which represent mainly dopamine D 2 receptors (Gehlert et al., 1992); (2) long term HAL treatment has no effect on the level of D 3 mRNA and D 3 receptor density but increases the level of D 2 mRNA and D 2 receptor density in the rat nucleus accumbens (Hurley et al., 1996). These results confirm our previous assumption that the reduced accumbens NPY-LI concentrations observed after subchronic HAL treatment result from increased NPY release because the short-lasting (3 h) action of quinpirole attenuating HAL effect is probably a consequence of its influence on NPY release. The significance of the NPY-LI decrease observed in naive rats in response to antipsychotics is presently unknown. A few clinical studies on the hypothetical role of NPY in schizophrenia have provided conflicting results. Peters et al. (1990) reported an increase in NPY-LI content in the cerebrospinal fluid (CSF) of drug-free chronic schizophrenic patients. They also observed that withdrawal of HAL maintenance treatment was associated with a significant increase in NPY-LI. Frederiksen et al. (1991) found reduced NPY concentrations in the temporal cortex ¨ et al. (1988); of schizophrenics. In contrast, Widerlov Berrettini et al. (1987) found normal NPY levels in the CSF of schizophrenic patients. Therefore, in the light of clinical results the significance of NPY system alterations remains unclear and further investigations are required. Nevertheless our investigation has provided new information on the complexity of changes due to the antipsychotic action. In summary, the results of this study indicate that the NPY system in the nucleus accumbens is markedly and reversibly affected by treatment with antipsychotics. Acute administration of HAL diminished accumbens NPY synthesis. Although the exact receptor mechanism by which antipsychotics affect accumbens NPY-LI concentrations has not been exactly described, it has been shown that blockade of D 2 -like (D 2 , D 3 ) and D 1 dopamine receptors may partly mediate these alterations.

Acknowledgements The skilful technical assistance of Mr. Z. Sprada is gratefully acknowledged. This work was financially supported by the grants for statutory activity of Silesian University School of Medicine and Institute of Pharmacology, Polish Academy of Sciences.

References Angulo, J.A., Cadet, J.L., McEwen, B.S., 1990. Effect of typical and atypical neuroleptic treatment on protachykinin mRNA levels in the striatum of the rat. Neurosci. Lett. 113, 217–221.

58

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59

Aoki, C., Pickel, V.M., 1988. Neuropeptide Y-containing neurons in the rat striatum: ultrastructure and cellular relations with tyrosine hydroxylase containing terminals and with astrocytes. Brain Res. 459, 205– 225. Beal, M.F., Chattha, G.K., Martin, J.B., 1986. A comparison of regional somatostatin and neuropeptide Y distribution in rat striatum and brain. Brain Res. 377, 240–245. Beal, M.F., Martin, J.B., 1984. Effects of neuroleptic drugs on somatostatin-like immunoreactivity. Neurosci. Lett. 47, 125–130. Berrettini, W.H., Doran, A.R., Kelsoe, J., Roy, A., Pickar, D., 1987. Cerebrospinal fluid neuropeptide Y in depression and schizophrenia. Neuropsychopharmacology 1, 81–83. Debonnel, G., de Montigny, C., 1996. Modulation of NMDA and dopaminergic neurotransmissions by sigma ligands: possible implications for the treatment of psychiatric disorders. Life Sci. 58, 721–734. De Quidt, M.E., Emson, P.C., 1986. Distribution of neuropeptide Y-like imunoreactivity in the rat central nervous system. I. Radioimmunoassay and chromatographic characterisation. Neuroscience 18, 545–616. Dumont, Y., Fournier, A., St-Pierre, S., Quirion, R., 1996. Autoradiographic distribution of [ 125 I]Leu 31 , Pro 34 ]PYY and [ 125 I]PYY 3 – 36 binding sites in the rat brain evaluated with two newly developed Y 1 and Y 2 receptor radioligands. Synapse 22, 139–158. Ellenbroek, B.A., 1993. Treatment of schizophrenia: a clinical and preclinical evaluation of neuroleptic drugs. Pharmacol. Ther. 57, 1–78. ¨ E., Jonsson, ¨ Frederiksen, S.O., Ekman, R., Gottfries, C.-G., Widerlov, S., 1991. Reduced concentrations of galanin, arginine, vasopressin, neuropeptide Y and peptide YY in the temporal cortex but not in the hypothalamus of brains from schizophrenics. Acta Psychiatr. Scand. 83, 273–277. Frey, P., 1983. Cholecystokinin octapeptide levels in rat brain are changed after subchronic neuroleptic treatment. Eur. J. Pharmacol. 95, 87–92. Gehlert, D.R., Gackenheimer, S.L., Seeman, Ph., Schaus, J., 1992. Autoradiographic localization of [ 3 H]quinpirole binding to dopamine D 2 and D 3 receptors in rat brain. Eur. J. Pharmacol. 211, 189–194. ¨ Heilig, M., Widerlow, E., 1995. Neurobiology and clinical aspects of neuropeptide Y. Crit. Rev. Neurobiol. 9, 115–136. Herman, Z.S., Huzarska, M., Kmieciak-Kołada, K., Kowalski, J., 1991. Chronic treatment with chlorpromazine, thioridazine or haloperidol increases striatal enkephalins and their release from rat brain. Psychopharmacology 104, 106–112. Horn, A.S., Cuello, A.C., Miller, R.J., 1974. Dopamine in the mesolimbic system of the rat brain: endogenous levels and the effects of drugs on the uptake mechanism and stimulation of adenylate cyclase activity. J. Neurochem. 22, 265–270. Houdeau, E., Boyer, P.-A., 1994. In situ hybridization study of neuropeptide Y neurons in the rat brain and pelvic paracervical ganglion. Cell Tissue Res. 277, 579–586. Humpel, C., Knaus, G.A., Auer, B., Knaus, H.G., Haring, C., Theodorsson, E., Saria, A., 1990. Effects of haloperidol and clozapine on preprotachykinin-A messenger RNA, tachykinin tissue levels, release and neurokinin-1 receptors in the striato–nigral system. Synapse 6, 1–9. Hurley, M.J., Stubbs, C.M., Jenner, P., Marsden, C.D., 1996. Effect of chronic treatment with typical and atypical neuroleptics on the expression of dopamine D 2 and D 3 receptors in rat brain. Psychopharmacology 128, 362–370. Iqbal, N., van Praag, H.M., 1995. The role of serotonin in schizophrenia. Eur. Neuropsychopharmacol. Suppl. 5, 11–23. Ishimaru, M.J., Torn, M., 1997. The glutamate hypothesis of schizophrenia. CNS Drugs 1, 47–67. Jaber, M., Tison, F., Fournier, M.C., Bloch, B., 1994. Differential influence of haloperidol and sulpiride on dopamine receptors and peptide mRNA levels in the rat striatum and pituitary. Mol. Brain Res. 23, 14–20. Jackson, D.M., Westlind-Danielsson, A., 1994. Dopamine receptors: molecular biology, biochemistry and behavioural aspects. Pharmacol. Ther. 64, 291–369.

Josselyn, S.A., Beninger, R.J., 1993. Neuropeptide Y: intraaccumbens injections produce a place preference that is blocked by cis-flupenthixol. Pharmacol. Biochem. Behav. 46, 543–552. Joyce, J.N., 1993. The dopamine hypothesis of schizophrenia limbic interactions with serotonin and norepinephrine. Psychopharmacology 112, S16–S34. Laruelle, M., Jaskiw, G.E., Lipska, B.K., Kolachana, B., Casanova, M.F., Kleinman, J.E., Weinberger, D.R., 1992. D 1 and D 2 receptor modulation in rat striatum and nucleus accumbens after subchronic and chronic haloperidol treatment. Brain Res. 575, 47–56. Leysen, E.J., Janssen, P.M.F., Schotte, A., Luyten, W.M.H.L., Megens, A.A.H.P., 1993. Interaction of antipsychotic drugs with neurotransmitter receptor sites in vitro and in vivo in relation to pharmacological and clinical effects: role of 5 HT 2 receptors. Psychopharmacology 112, S40–S54. Marksteiner, J., Saria, A., Miller, C.H., Krause, J.E., 1992. Differential increases of neurokinin B- and enkephalin-like immunoreactivities and their mRNAs after chronic haloperidol treatment in the rat. Neurosci. Lett. 148, 55–59. Massari, V.J., Chan, J., Chromwell, B.M., O’Donohue, T.L., Oertel, W.H., Pickel, V.M., 1988. Neuropeptide Y in the rat nucleus accumbens: ultrastructural localization in aspiny neurons receiving synaptic input from GABAergic terminals. J. Neurosci. Res. 19, 171–186. Meller, E., Bohmaker, K., Goldstein, M., Friedhoff, A.J., 1985. Inactivation of D 1 and D 2 dopamine receptors by N-ethoxycarbonyl-2-ethoxy1,2-dihydroquinoline in vivo: selective protection by neuroleptics. J. Pharmacol. Exp. Ther. 233, 656–662. Midgley, L.P., Bush, L.G., Gibb, J.W., Hanson, G.R., 1993. Differential regulation on neuropeptide Y systems in limbic structures of the rat. J. Pharmacol. Exp. Ther. 267, 707–713. Midgley, L.P., Wagstaff, J.D., Singh, N.A., Bush, L.G., Gibb, J.W., Hanson, G.R., 1994. Dynamic dopaminergic regulation of neuropeptide Y systems in discrete striatal and accumbens regions. Eur. J. Pharmacol. 251, 191–199. Millington, W.R., Maiewski, S., O’Donohue, Th.L., Mueller, G.P., 1985. Long-term haloperidol treatment elevates b-endorphin levels in the intermediate pituitary but not in rat brain. Neuropeptides 6, 365–372. Miyake, M., Iguchi, K., Okamura, H., Fukui, K., Nakajima, T., Chihara, K., Ibata, Y., Yanaihara, N., 1990. Effect of haloperidol on immunoreactive neuropeptide Y in rat cerebral cortex and basal ganglia. Brain Res. Bull. 25, 263–269. Myers, B., Levant, B., Bissette, G., Nemeroff, C.B., 1992. Specificity of the increase in neurotensin concentrations after antipsychotic drug treatment. Brain Res. 575, 325–328. ˜ W., 1995. Effects of acute or Obuchowicz, E., Herman, Z.S., Lason, multiple administrations of neuroleptics on neuropeptide Y system in the rat nucleus accumbens. Pharmacol. Res. 31 (Suppl.), 280. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, second ed. Academic Press, Sydney. Peters, J., Van Kammen, D.P., Gelernter, J., Yao, J., Shaw, D., 1990. Neuropeptide Y-like immunoreactivity in schizophrenia. Schizophr. Res. 3, 287–294. Roman, F.J., Martin, B., Junien, J.L., 1993. In vivo interaction of neuropeptide Y and peptide YY with s receptor sites in the mouse brain. Eur. J. Pharmacol. 242, 305–307. Sakai, K., Maeda, K., Okuyama, S., Tanaka, Ch., 1996. Phencyclidine alters regional concentrations of neuropeptide Y and peptide YY in rat brain. Biol. Psychiatry 40, 1246–1254. Salin, P., Kerkerian, L., Nieoullon, A., 1990. Expression of neuropeptide Y immunoreactivity in the rat nucleus accumbens is under the influence of the dopaminergic mesencephalic pathway. Exp. Brain Res. 81, 363–371. Skarsfeldt, T., Larsen, J.J., 1988. SCH 23390—a selective dopamine D-1 receptor antagonist with putative 5HT-1 receptor agonistic activity. Eur. J. Pharmacol. 148, 389–395. Sokoloff, P., Giros, B., Martres, M.-P., Bouthenet, M.-L., Schwartz, J.-C., 1990. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146–151.

E. Obuchowicz, J. Turchan / European Neuropsychopharmacology 9 (1999) 51 – 59 Sunahara, R.K., Seeman, Ph., Van Tol, H.M., Niznik, B.H., 1993. Dopamine receptors and antipsychotic drug response. Br. J. Psychiatry 163 (Suppl. 22), 31–38. Waters, S.M., Konkoy, Ch.S., Davis, T.P., 1995. Neuropeptide metabolism on intact, regional brain slices: effect of dopaminergic agents on substance P, cholecystokinin and met-enkephalin degradation. J. Pharmacol. Exp. Ther. 274, 783–789.

59

¨ E., Lindstrom, ¨ L.H., Wahlestedt, C., Ekman, R., 1988. NeuroWiderlov, peptide Y and peptide YY as possible cerebrospinal markers for major depression and schizophrenia, respectively. J. Psychiatr. Res. 22, 69–79. Young III, W.S., Bonner, T.I., Brann, M.R., 1986. Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc. Natl. Acad. Sci. USA 83, 9827–9831.