Raclopride lessens the ability of clozapine to suppress alcohol drinking in Syrian golden hamsters

Raclopride lessens the ability of clozapine to suppress alcohol drinking in Syrian golden hamsters

Neuropharmacology 61 (2011) 646e652 Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm...

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Neuropharmacology 61 (2011) 646e652

Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Raclopride lessens the ability of clozapine to suppress alcohol drinking in Syrian golden hamsters David T. Chau a, Jayme Ahmed a, Thomas T. Wang a, Haiyi Xie a, b, Ree Dawson c, Alan I. Green a, * a

Department of Psychiatry, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03576, USA Department of Community and Family Medicine, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756, USA c Frontier Science and Technology Research Foundation, 900 Commonwealth Avenue, Boston, MA 02215, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2010 Received in revised form 9 May 2011 Accepted 10 May 2011

Emerging evidence suggests that the atypical antipsychotic clozapine decreases alcohol consumption in patients with schizophrenia, while typical antipsychotics, all of which are potent dopamine (DA) D2 receptor antagonists, do not. We have proposed that clozapine, through its weak DA D2 receptor blocking action, coupled with its ability to potentiate noradrenergic and serotonergic activity, may ameliorate a dysfunction in the mesocorticolimbic DA reward circuitry that underlies alcohol use disorder in patients with schizophrenia. In prior studies, we have demonstrated that clozapine also decreases alcohol drinking in the Syrian golden hamster, but haloperidol does not. The purposes of the current study were: (1) to further assess the effect of clozapine (2 or 4 mg/kg/day, s.c.) on alcohol consumption in hamsters, using a continuous access, 2-bottle choice paradigm; and (2) to examine whether clozapine’s effect on alcohol drinking is affected by increasing its DA D2 blockade through adjunctive use of the potent DA D2 receptor antagonist raclopride (2, 4, or 6 mg/kg/day, s.c.). The major findings were: (1) clozapine suppressed both initiation and maintenance of alcohol drinking in hamsters; and (2) these effects of clozapine were lessened when raclopride was given adjunctively with clozapine. These data suggest that clozapine may limit alcohol drinking in the golden hamster (and possibly in patients with schizophrenia) in part because of its weak blockade of the DA D2 receptor. Ó 2011 Published by Elsevier Ltd.

Keywords: Clozapine Raclopride Dopamine D2 receptor Alcoholism Schizophrenia

1. Introduction Alcohol use disorder occurs commonly in patients with schizophrenia and dramatically worsens their clinical course (Drake et al., 1989; Regier et al., 1990). Despite the problems presented by their alcohol use, few treatment options are available to control alcohol use in these individuals. Typical antipsychotic medications, such as haloperidol, do not appear to decrease their alcohol consumption (Green et al., 2008). However, emerging data from our group and others suggest that the atypical antipsychotic clozapine may substantially decrease alcohol use in patients with schizophrenia and co-occurring alcoholism (Albanese et al., 1994; Drake et al., 2000; Green et al., 2003, 2008; Lee et al., 1998; Marcus and Snyder, 1995; Yovell and Opler, 1994; Zimmet et al., 2000), although it is unclear what pharmacologic action of clozapine allows it to do so.

* Correspondence author. Tel.: þ1 603 650 7549; fax: þ1 603 650 8415. E-mail addresses: [email protected] (D.T. Chau), jayme.ahmed.05@ alum.dartmouth.org (J. Ahmed), [email protected] (T.T. Wang), [email protected] (H. Xie), [email protected] (R. Dawson), alan.i. [email protected] (A.I. Green). 0028-3908/$ e see front matter Ó 2011 Published by Elsevier Ltd. doi:10.1016/j.neuropharm.2011.05.007

Unlike typical antipsychotics, all of which have potent dopamine (DA) D2 receptor blocking ability, clozapine is a broad-spectrum agent with relatively weak affinity for the DA D2 receptor (Ashby and Wang, 1996). Clozapine’s weak affinity for the DA D2 receptor, coupled with its diverse effects on noradrenergic and serotonergic systems, has been proposed to contribute to its atypicality, i.e., its ability to ameliorate positive and negative symptoms without producing significant extrapyramidal symptoms (Deutch et al., 1991), and that its atypicality may be lost when the potency of its DA D2 receptor antagonism is increased (Green et al., 1999; Kapur and Remington, 1996; Svensson, 2003b). For instance, isoclozapine, which has equivalent affinity to clozapine for multiple receptors (e.g., 5-HT1a, 5-HT2, D1, D4, and M1), but 10-fold higher affinity than clozapine for DA D2 receptors, behaves, not like clozapine, but like typical antipsychotics in behavioral assays in rodents, e.g., in tests of catalepsy and conditioned avoidance response, as well as in molecular assays e isoclozapine increases FOS expression in both nucleus accumbens and striatum, whereas clozapine increases it preferentially in the accumbens (Kapur et al., 2002). Studies (mostly in animals) suggest the possibility that clozapine may normalize a dysfunctional mesocorticolimbic DA reward

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circuitry in patients with schizophrenia through a mechanism involving weak DA D2 receptor blockade, coupled with blockade of noradrenergic and serotonergic receptors (Green et al., 1999). For example, clozapine-like drug combinations consisting of low doses of a D2 receptor antagonist and either a noradrenergic a-2 or a serotonergic 5-HT2 receptor antagonist have been shown to enhance the signal detection capacity of dysfunctional mesocorticolimbic circuitry by increasing DA release in the prefrontal cortex and by partially restoring the balance between burst and tonic firing modes of dopaminergic neurons in the ventral tegmental area (Svensson, 2003a; Svensson et al., 1995). Green and colleagues have further proposed, based on these and other data, that (1) patients with schizophrenia have a reward deficiency syndrome, secondary to a dysfunction in the mesocorticolimbic DA reward circuitry, that underlies alcohol use in this population; (2) an important biologic effect of alcohol in patients with schizophrenia may involve a transient amelioration of this brain reward deficiency; and (3) unlike typical antipsychotics, clozapine, through its varied actions on serotonergic and noradrenergic neurons (especially its antagonistic effects on a-2 adrenergic receptors and its ability to chronically elevate norepinephrine in brain and plasma), coupled with its weak dopamine D2 receptor blocking ability, may tend to have a ‘‘normalizing’’ effect on the signal detection capacity of the dysfunctional mesocorticolimbic DA system in such patients, thereby ameliorating the basis of their use of alcohol (Green et al., 1999). While clozapine has been of considerable interest because of its broad-spectrum pharmacological profile, as well as its unusual clinical efficacy, including its ability to limit alcohol and substance use in patients with schizophrenia, only a few studies have assessed its effects on alcohol drinking in animals. Studies in rodents have shown that a single injection of clozapine does not decrease alcohol drinking in the limited access paradigm (Ingman and Korpi, 2006; June et al., 1991; Thrasher et al., 1999); however, other studies in rodents have shown that repeated clozapine administration decreases alcohol drinking in the continuous access paradigm (Chau et al., 2010; Green et al., 2004; Ufer et al., 1999). To further elucidate the effects of clozapine on alcohol consumption, our laboratory has examined the comparative effects of clozapine versus the typical antipsychotic haloperidol in the Syrian golden hamster (Mesocricetus auratus). Our data using a continuous access paradigm have indicated that clozapine, but not haloperidol, decreases chronic alcohol drinking in this animal, as it does in patients with schizophrenia (Chau et al., 2010, 2004). In the current study, we tested the hypothesis that clozapine’s ability to suppress alcohol drinking in hamsters is related in part to the fact that it is a weak, rather than strong, DA D2 receptor antagonist. Specifically, we assessed whether the effects of clozapine on the initiation and maintenance of alcohol drinking in the golden hamster would be blunted if the potent D2 receptor antagonist raclopride was added to clozapine to increase the DA D2 receptor blockade. 2. Material and methods 2.1. Experiment 1: determining the effects of raclopride and clozapine combinations on initiation of alcohol drinking 2.1.1. Animals and experimental procedures Forty adult, male Syrian Golden Hamsters (M. auratus) (100e130 g) were acquired from Harlan Inc. (Indianapolis, IN) and individually housed on an altered 12 h/12 h light/dark cycle (3 AM light on/3 PM light off) in standard home cages with ad libitum access to food and water. To compare the effects of clozapine, raclopride, combinations of clozapine and raclopride, and vehicle on initiation of alcohol drinking, hamsters were randomly assigned to 1 of 6 treatment groups (n ¼ 6e7 per group) receiving subcutaneous (s.c.) injections of: (1) 2 mg/kg/day clozapine [2CLOZ]; (2) 4 mg/kg/day clozapine [4CLOZ]; (3) 4 mg/kg/day raclopride [4RACL]; (4)

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2 mg/kg/day clozapine þ 4 mg/kg/day raclopride [2CLOZ þ 4RACL]; (5) 4 mg/kg/day clozapine þ 4 mg/kg/day raclopride [4CLOZ þ 4RACL]; or (6) vehicle [VEH]. Animals from each group were injected with the respective drug(s) daily over a period of 18 days. Since studies have shown that an injection of 2 mg/kg raclopride in the rat produces peak DA D2 receptor binding >80% after 1e2 h and approximately 50% after 4e8 h (Wadenberg et al., 2000), to maintain high levels of DA D2 receptor blockade throughout the animals’ most active period (i.e., the initial phase of the dark cycle), 2 mg/kg raclopride was given twice a day; the first injection occurred at 1 PM (2 h before the onset of the dark cycle) and the second injection occurred 6 h later, at 7 PM (4 h following the onset of the dark cycle), for a total of 4 mg/kg/day, as noted above. A similar twice-per-day-injection procedure has been previously used by other investigators to assess the effects of raclopride and other drugs with relatively short half-life on free-access alcohol drinking in the rat (Silvestre et al., 1996). The groups that were not given raclopride received a second injection consisting of vehicle. Since data from our prior studies demonstrated that clozapine injected in hamsters once per day, over successive days, substantially decreases their alcohol drinking (Chau et al., 2010; Green et al., 2004), in this experiment, clozapine was given once per day, at 1 PM (2 h before the onset of the dark cycle). Beginning on the 4th day of treatment, all animals were given continuous access to a 15% (v/v) ethanol solution as a second choice of drinking fluid. The positions of the water and alcohol bottles were rotated on a daily basis to prevent positional preference. A technician, blinded to the experimental conditions, measured alcohol and water intake (every 24 h), food intake (every 48 h), and body weight (every 3 days). Measurements took place at 12 noon (3 h before dark). 2.1.2. Drug preparation The clozapine solutions were prepared for injection by first dissolving clozapine (Sigma Chemical Inc.) in 0.5 N acetic acid, then adjusting the volumes to the desired clozapine concentrations using a vehicle solution (0.5 M sodium acetate, pH 5.5). The pH levels of the clozapine solutions were subsequently adjusted to 5.5 to match that of the vehicle. Raclopride solutions were prepared for injection by dissolving the desired amounts of raclopride hydrochloride (Sigma Chemical Inc.) in vehicle solution. The combinations of clozapine and raclopride were prepared by dissolving the desired amounts of raclopride hydrochloride in the clozapine solution. Injections were given in volumes of 2.0 ml/kg body weight. 2.1.3. Data analysis Alcohol intake (g/kg), water intake (ml), food intake (g), and body weight (g) data were analyzed using two-way repeated measures analysis of variance (ANOVA), using time and drug treatment as independent variables. Because 2-way ANOVA did not detect significant effects of treatment group on alcohol drinking but visual inspection of the data suggested otherwise, additional analyses of the alcohol drinking data were conducted using a mixed effects model for repeated measures, implemented in SAS Proc Mixed procedure (SAS Institute, 2008), an extension of the traditional ANOVA that allows more flexible modeling of time effects and correlated observations due to repeated measurement than ANOVA (Jennrich and Schluchter, 1986; Laird and Ware, 1982). When the mixed model analysis indicated that significant differences existed among treatments, post-hoc pair-wise comparisons between groups were made based on the best-fit model of the data. Significance was determined at p < 0.05. 2.2. Experiment 2: determining the effects of raclopride and clozapine combinations on maintenance of alcohol drinking 2.2.1. Animals and experimental procedures Fifty-six adult, male Syrian golden hamsters (100e130 g, Harlan Inc., Indianapolis, IN) were individually housed in standard home cages with ad libitum access to food and water. The room was maintained on an altered 12 h/12 h light/dark cycle (light on at 3 AM/light off at 3 PM). All animals were given continuous access to a bottle of water and a second bottle containing 15% (v/v) alcohol for 20 days prior to randomization into treatment groups. The positions of the two drinking bottles were rotated on a daily basis to prevent positional preference. A technician, blinded to the experimental conditions, measured fluid intake (every 24 h), and food intake and body weight (every 3 days). Measurements took place at the same time each day, at 12 noon (3 h before the onset of the dark cycle). On the 20th day of alcohol drinking, hamsters were divided into 8 treatment groups (n ¼ 7 per group) with similar baseline alcohol intake values (g/kg/day), calculated using data from the last 4 days. Over a period of 20 days, individual treatment groups received subcutaneously (s.c.) injections with one the following: (1) vehicle [VEH]; (2) 2 mg/kg/day raclopride [2RACL]; (3) 4 mg/kg/day raclopride [4RACL]; (4) 6 mg/kg/day raclopride [6RACL]; (5) 4 mg/kg/day clozapine þ 2 mg/kg/day raclopride [4CLOZ þ 2RACL]; (6) 4 mg/kg/ day clozapine þ 4 mg/kg/day raclopride [4CLOZ þ 4RACL]; (7) 4 mg/kg/day clozapine þ 6 mg/kg/day raclopride [4CLOZ þ 6RACL]; or (8) 4 mg/kg/day clozapine [4CLOZ]. All injections occurred once per day, at 1 PM (2 h before the onset of the dark cycle). 2.2.2. Drug preparation The drug preparation procedures used in this experiment were identical to the procedures used in Experiment 1 (see Section 2.1.2).

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2.2.3. Data analysis Alcohol intake (g/kg), water intake (ml), food intake (g), and body weight (g) data were analyzed using two-way repeated measures analysis of variance (ANOVA), using time (3-day blocks) and drug treatment as independent variables. When the analysis indicated that significant differences existed between treatments, post-hoc pair-wise comparisons between groups were made using the Tukey adjustment. Significance was determined at p < 0.05. Data are expressed as mean (M)  standard error of the mean (SEM). 2.3. Ethical considerations All experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and were approved by Dartmouth Institutional Animal Care and Use Committee. Careful efforts were made to minimize stress and pain to the animals and to reduce the number of animals used.

3. Results 3.1. Experiment 1: effects of raclopride and clozapine combinations on initiation of alcohol drinking 3.1.1. Alcohol intake Two-way repeated measures ANOVA revealed a significant effect of time on alcohol intake (g/kg) (F(13,546) ¼ 69.6, p < 0.0001), but no effect of treatment group nor group by time interaction (Fig. 1A and B). Further analysis of the alcohol drinking data was performed using a mixed effects model. To assess trends in alcohol intake, the daily intake values were fitted using time as a continuous variable. The analysis revealed a linear time trend (F(1,508) ¼ 146.85, p < 0.0001), as well as a quadratic time trend (F(1,508) ¼ 93.18, p < 0.0001), in the pattern of alcohol intake over time across all treatment groups. All groups showed similar curvilinear patterns of the rate of rise and stabilization of alcohol intake over time. Thus, the particular treatment did not affect the rate at which hamsters reached a stable level of alcohol intake. Unlike the 2-way ANOVA, the mixed effects model detected significant differences in alcohol intake among the treatment groups during the 14-day assessment period (F(5,34) ¼ 9.52, p < 0.0001), but no group by time (linear or quadratic) interaction. Subsequent pairwise comparisons between individual groups (based on the best-fit model with group, linear time and quadratic time as factors) indicated that animals receiving 2 mg/kg/day clozapine alone drank less alcohol over this period than those receiving vehicle (2CLOZ vs. VEH, t ¼ 2.14, p ¼ 0.04), while those receiving the adjunctive 4 mg/kg/day raclopride treatment did not (2CLOZ þ 4RACL vs. VEH: t ¼ 1.66, p ¼ 0.106). Raclopride by itself also did not affect alcohol drinking (4RACL vs. VEH: t ¼ 0.87, p ¼ 0.390). Adjunctive raclopride treatment did not appear to reverse the ability of the higher dose (4 mg/kg/day) of clozapine used to suppress initiation of alcohol drinking (4CLOZ vs. VEH, t ¼ 4.24, p ¼ 0.0002; 4CLOZ þ 4RACL vs. VEH: t ¼ 5.48, p < 0.0001). The difference in alcohol intake (over the 14-day period) between the clozapine alone groups and the drug combination groups did not reach statistical significance (2CLOZ vs. 2CLOZ þ 4RACL: t ¼ 0.35; p ¼ 0.727; 4CLOZ vs. 4CLOZ þ 4RACL: t ¼ 0.31, p ¼ 0.757). Finally, we note that while alcohol drinking in all groups increased at similar rates over the first week of the study, during the second week, there appeared to be substantial separations between groups, as alcohol intake in each group stabilized to its own level. Thus, we performed another mixed model analysis on the data, focusing on the second half of the study (days 8e14). The result of this post-hoc analysis indicated a near significant difference between the effects of 2CLOZ versus 2CLOZ þ 4RACL (F(1,12) ¼ 4.56, p ¼ 0.054). 3.1.2. Water and food intake, and body weight Two-way repeated measures ANOVA found no significant group mean differences in body weight prior to treatment, indicating that

Fig. 1. Comparative effects of clozapine (CLOZ), raclopride (RACL), combinations of clozapine and raclopride, and vehicle (VEH) on initiation of alcohol drinking in hamsters. For clarity, data involving 2 and 4 mg/kg clozapine are displayed separately (Fig. 1A and B, respectively). Adding 4 mg/kg/day (2 mg/kg, twice a day) raclopride to 2 mg/kg/day clozapine essentially abolished the ability of this dose of clozapine to suppress initiation of alcohol drinking (see text).

the randomization scheme used to assign individual hamsters to treatment groups was effective. Moreover, there were no differences between groups in water intake, food intake, or body weight during the 14-day assessment period when animals had access to alcohol while receiving treatment. 3.2. Experiment 2: effects of raclopride and clozapine combinations on maintenance of alcohol drinking 3.2.1. Alcohol intake Two-way repeated measures ANOVA with time (3-day blocks) and drug treatment as independent variables indicated significant effects of time (F(7,440) ¼ 36.0, p < 0.0001) and treatment group (F(7,48) ¼ 10.9, p < 0.0001) on alcohol consumption (g/kg), as well as a group by time interaction (F(49,399) ¼ 6.6, p < 0.0001) (Fig. 2AeC). Tukey’s post-hoc tests indicated that there was no significant difference between groups in alcohol consumption during the baseline period, indicating effective randomization into groups. Throughout the treatment period (all seven 3-day blocks), clozapine-treated groups (4CLOZ, 4CLOZ þ 2RACL, 4CLOZ þ 4RACL, 4CLOZ þ 6RACL) drank less alcohol than the vehicle-treated group (VEH) (p < 0.05). Alcohol drinking in the groups treated with raclopride alone (2RACL, 4RACL, 6RACL) during this period was not different from that in the VEH group. Moreover, animals treated with the combination of clozapine (4 mg/kg/day) and raclopride (2 or 6 mg/kg, but not 4 mg/kg/day) drank more alcohol than animals treated with clozapine alone (4CLOZ þ 2RACL vs. 4CLOZ: p < 0.001; 4CLOZ þ 6RACL vs. 4CLOZ: p < 0.001).

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Fig. 3. Comparative effects of clozapine (CLOZ), raclopride (RACL), combinations of CLOZ and RACL, and vehicle (VEH) on water and food intake in hamsters. Mean water intake (ml/day) and mean food intake (grams/3 days) for the individual groups during the 21-day treatment period are shown in Fig. 3A and B, respectively. (A) Water: During the treatment period, all clozapine-treated groups drank more water than the VEH group, as they drank less alcohol. However, animals treated with the clozapine (4 mg/kg/day) plus raclopride (2 or 6 mg/kg/day, but not 4 mg/kg/day) combination drank less water than those treated with clozapine alone, reflecting the fact that former group drank more alcohol than the latter. (B) Food: During the treatment period, all clozapine-treated groups (including the ones that received raclopride [2 or 6, but not 4 mg/kg/day] in combination with clozapine) ate more food than the VEH group. Animals treated with the clozapine (4 mg/kg/day) plus raclopride (2, 4, or 6 mg/ kg/day) combination ate a similar amount of food as those treated with clozapine alone. (*p < 0.01, **p < 0.001, vs. VEH; yp < 0.001, vs. 4 mg/kg CLOZ).

treated with raclopride alone at the highest dose (6 mg/kg) drank slightly more water than those treated with vehicle (p < 0.01). The lower doses of raclopride did not affect water drinking. Animals treated with the combination of clozapine (4 mg/kg/day) and raclopride (2 or 6 mg/kg/day, but not 4 mg/kg/day) drank less water than those treated with clozapine alone (p < 0.001).

Fig. 2. Comparative effects of clozapine (CLOZ), raclopride (RACL), combinations of clozapine and raclopride, and vehicle (VEH) on maintenance of alcohol drinking in hamsters. For clarity, data involving 2, 4, and 6 mg/kg/day raclopride are displayed separately (Fig. 2AeC respectively). Adding 2 or 6 (but not 4) mg/kg/day raclopride to 4 mg/kg/day clozapine lessened the ability of clozapine to decrease alcohol drinking.

3.2.2. Water intake Two-way repeated measures ANOVA with time and treatment group as independent variables indicated a significant effect of time (F(7,440) ¼ 41.3, p < 0.0001) and treatment group (F(7,48) ¼ 3.65, p < 0.005) on water drinking (ml), as well as a group by time interaction (F(49,399) ¼ 1.87, p < 0.01) (Fig. 3A). Tukey’s post-hoc tests indicated that baseline water drinking was not different among groups. During the treatment period, all clozapine-treated groups (4CLOZ, 4CLOZ þ 2RACL, 4CLOZ þ 4RACL, 4CLOZ þ 6RACL) drank more water, relative to the VEH group (p < 0.001). Animals

3.2.3. Food intake Two-way ANOVA indicated significant effects of time (3-day blocks) (F(7,440) ¼ 33.0, p < 0.0001) and treatment group (F(7,48) ¼ 3.49, p < 0.005) on food intake, but no group by time interaction (Fig. 3B). Tukey’s post-hoc tests indicated that mean food intake during the treatment period for all clozapine-treated groups (except the 4CLOZ þ 4RACL group) was higher than that seen in the VEH group (p < 0.001). Food intake in the groups treated with raclopride alone (2RACL, 4RACL, and 6RACL) during this period was not different from that in the VEH group. Moreover, among the animals treated with the combination of clozapine (4 mg/kg/day) and raclopride (2, 4 or 6 mg/kg), only those treated with 4 mg/kg/ day CLOZ and 2 mg/kg/day raclopride ate less food than animals treated with clozapine alone (4CLOZ þ 2RACL vs. 4CLOZ: p < 0.001). 3.2.4. Body weight There were significant effects of time (F(7,440) ¼ 59.9, p < 0.001), as animals in both the drug-treated and vehicle-treated groups gained body weight over time (data not shown). However,

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there was no effect of treatment group on body weight, and no group by time interaction. 4. Discussion The primary purpose of the current study was to determine whether clozapine’s effect on alcohol drinking by the Syrian golden hamsters relates in part to its weak blockade of the DA D2 receptor. The major findings were: (1) clozapine suppressed both initiation and maintenance of alcohol drinking; and (2) suppression of both initiation and maintenance of alcohol drinking by clozapine was lessened when the potent D2 antagonist raclopride was coadministered with clozapine. 4.1. Effects of clozapine on initiation and maintenance of alcohol drinking The 4 mg/kg/day dose of clozapine used in each experiment has a peak D2 occupancy rate (approximately 50% in rats) in PET studies that is comparable to that produced by clinically relevant doses of clozapine used in humans (Kapur et al., 2003). We note that our prior studies indicated that maintenance treatment using 4 mg/kg/ day clozapine chronically suppresses alcohol drinking in hamsters (Chau et al., 2010). A lower dose (2 mg/kg/day) was included here to allow for the assessment of the dose-response effects of clozapine alone and in combination with raclopride. These doses of clozapine were the lowest effective doses used in our prior studies e they produced intermediate effects on maintenance of alcohol drinking; higher doses (e.g., 12 mg/kg/day) tended to create a ceiling effect (Chau et al., 2010; Green et al., 2004). In Experiment 1, clozapine treatment (2 and 4 mg/kg/day) initiated in alcohol-naive hamsters reduced the animals’ acquired baseline alcohol intake levels by about 20e25%, relative to vehicle (VEH) (see Fig. 1, Days 8e14). This reduction of “acquired baseline drinking” was substantially less than the reduction of “ongoing drinking” seen in Experiment 2 or in our previous studies of clozapine on chronic alcohol drinking (Chau et al., 2010). In Experiment 2, clozapine (4 mg/kg/day) decreased “ongoing alcohol drinking” by 45% (see Fig. 2). It appears from these data that alcohol-experienced, actively drinking hamsters are more responsive to clozapine treatment than are alcohol-naïve hamsters. Further studies are necessary to clarify why clozapine impacts alcohol drinking somewhat differently in hamsters under these two treatment conditions. 4.2. Adding raclopride to clozapine The 2e6 mg/kg/day doses of raclopride used in this study have been shown in PET studies to be associated with D2 occupancy rates exceeding 80% (Wadenberg et al., 2000). Such D2 occupancy rates are comparable to the rates associated with clinically effective doses of conventional typical antipsychotics (Kapur et al., 2003; Wadenberg et al., 2000). We predicted that with the addition of the potent DA D2 blockade from raclopride, clozapine would behave more like the typical antipsychotic haloperidol in the hamster. As predicted, in Experiment 1, raclopride (4 mg/kg/day) reversed the effect of 2 mg/kg/day clozapine on initiation of alcohol drinking. We note, however, that the same dose of raclopride only partially reversed the effect of 4 mg/kg/day clozapine. One possible explanation for this observation is that the dose of raclopride used might not have been high enough to completely override the action of 4 mg/kg/day clozapine. In Experiment 2, we kept the dose of clozapine fixed at 4 mg/kg/ day and tested multiple doses of raclopride. Also as predicted, clozapine alone decreased ongoing alcohol drinking more than the

clozapine/raclopride combinations e adding raclopride (either 2 or 6 mg/kg/day) to clozapine lessened the effect (suppression of alcohol drinking) of clozapine by approximately 30%. It is interesting to note that the lowest dose (2 mg/kg/day) and the highest dose (6 mg/kg/ day) of raclopride were equally effective in lessening the effect of clozapine, while the middle dose (4 mg/kg/day) was ineffective. This suggests that the dose effect of raclopride added to clozapine could be bimodal. In this regard, we note that the effect of raclopride on brain function have been shown to depend on the dose used (Tarazi et al., 1993). Alternatively, it is possible that the inability of 4 mg/kg/day raclopride to lessen the suppression of ongoing alcohol drinking by clozapine (Experiment 2) was a false negative finding due to the single-injection-per-day procedure used, given that a similar 4 mg/kg/day dose administered in two 2 mg/kg injections was able to lessen the effect of clozapine on initiation of alcohol drinking (Experiment 1). However, since our data clearly indicated that a once per day injection of either 2 or 6 mg/kg raclopride was able to lessen the effect of clozapine on chronic alcohol drinking in the hamsters (Experiment 2), a once-a-day regimen, which should produce less stress on the animals than a twice-per-day schedule, is most appropriate. Nonetheless, since some, but not all, doses of raclopride used lessened the ability of clozapine to limit alcohol drinking in the hamster, additional studies will be necessary to further clarify the effective dose-range of raclopride that may do this. In regard to the direct action of raclopride on alcohol drinking, we noted that other DA D2 antagonists have been shown in some studies to increase alcohol drinking in animals and to produce relapses to alcohol drinking in humans (Levy et al., 1991; Melendez et al., 2005; Walter et al., 2001). However, in the current study, raclopride by itself did not alter hamsters’ alcohol drinking. This is consistent with a prior study indicating that raclopride does not alter alcohol drinking in SpragueeDawley rats, as assessed using a continuous access, 2-bottle choice test (Silvestre et al., 1996). Thus, in the hamster, the reduction in the suppression of alcohol drinking by clozapine with the addition of raclopride was likely due to raclopride interfering with the pharmacological action of clozapine, rather than raclopride than having an effect on its own. Lastly, although raclopride has been shown to cause catalepsy in rats (Wadenberg et al., 2000), this was not an issue here since: visual inspection indicated that the hamsters were freely moving following the injections of raclopride; and raclopride treatment did not decrease their daily food intake. Moreover, raclopride-induced catalepsy would have been expected to impair drinking, but here raclopride increased alcohol drinking when it was given together with clozapine. These data suggest that increasing the potency of the DA D2 receptor blockade (through the use of raclopride) decreases the ability of clozapine to limit alcohol drinking. As discussed above, we have proposed that clozapine’s effect on alcohol drinking in rodents and in patients with schizophrenia relates in part to its weak DA D2 receptor blockade coupled with its varied actions on serotonergic and noradrenergic neurons (especially its antagonistic effects on the norepinephrine a-2 receptor and its ability to chronically elevate norepinephrine in brain and plasma). The findings from this study in hamsters provide further support for the notion that a weak DA D2 receptor blockade is one essential component underpinning clozapine’s unusual clinical effects (Green et al., 1999; Kapur and Remington, 1996; Svensson, 2003b). Interestingly, another atypical antipsychotic medication, quetiapine, which shares with clozapine a relatively weak DA D2 receptor blocking ability and effects on the noradrenergic system (Kalkman and Loetscher, 2003), has been shown to have a modest, but not dramatic, ability to decrease substance use in patients with severe mental illness (Brown et al., 2003; Brunette et al., 2009; Potvin et al., 2006). Whether the effects of clozapine on alcohol

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drinking also relate in part to other mechanisms, such as its facilitation of N-Methyl-D-aspartate (NMDA) receptor functioning in the prefrontal cortex (Coyle, 2006; Marcus et al., 2005), will be important to assess in future studies. 4.3. Other considerations While the Syrian golden hamster is not an animal model of schizophrenia per se, it appears to have some predictive validity as a screen for potential medications for treatment of patients with schizophrenia and co-occurring alcohol use disorder. First, clozapine decreases alcohol drinking in this animal, while haloperidol does not. (Green et al., 2004). Second, clozapine chronically suppresses alcohol drinking in this animal, as it does in patients with schizophrenia (Chau et al., 2010). Third, its pattern of alcohol consumption resembles regular moderate alcohol drinking in patients with schizophrenia. Like patients with schizophrenia, many of whom consume moderate amounts of alcohol on a regular basis, amounts that tend not to lead to physical dependence, the golden hamster consumes alcohol at a steady pace; maintains a stable and moderate (but physiological relevant) blood alcohol level (Chau et al., 2010; Keung et al., 2000; Kulkosky and Cornell, 1979); and does not exhibit signs of withdrawal following cessation of chronic alcohol drinking (McMillan et al., 1977). 5. Conclusion These current data regarding the differential effects of clozapine and the clozapine/raclopride combinations on alcohol drinking in the golden hamster are generally consistent with our hypothesis that clozapine’s ability to suppress alcohol drinking in patients with schizophrenia relates in part to its weak antagonistic action at the DA D2 receptor (Green et al., 1999). In particular, as predicted by our neurobiological formulation, the current study indicates that increasing DA D2 receptor blockade (with the co-administration of raclopride). lessens the ability of clozapine to suppress initiation and maintenance of alcohol drinking in this animal. Nonetheless, additional studies will be required to fully elucidate the effects of clozapine’s weak DA D2 receptor blockade in this animal (e.g., a full dose-response study of the interaction between raclopride and clozapine) and to clarify which of the other components of clozapine’s unique pharmacology (e.g., its noradrenergic or serotonergic action) contribute to its ability to suppress alcohol drinking. Disclosure statement We have two patent applications related to this work: “Combinations of dopamine D2 receptor blocker with norepinephrine reuptake inhibition and with norepinephrine alpha-2 receptor blockade”, No. 00826643.2; and “Treating alcohol and/or substance abuse by antagonizing alpha-2 adrenergic receptors with weak dopamine receptor blocking, No. 10/531,523. Acknowledgements This work was supported by the National Institute for Alcohol Abuse and Alcoholism (NIAAA, 1R03AA014644). We would like to thank Dr. James C. Leiter for comments on an early draft of the manuscript. References Albanese, M.J., Khantzian, E.J., Murphy, S.L., Green, A.I., 1994. Decreased substance use in chronically psychotic patients treated with clozapine. Am. J. Psychiatry 151, 780e781. Ashby Jr., C.R., Wang, R.Y., 1996. Pharmacological actions of the atypical antipsychotic drug clozapine: a review. Synapse 24, 349e394.

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