THE GABAB RECEPTOR AGONISTS BACLOFEN AND CGP 44532 PREVENT ACQUISITION OF ALCOHOL DRINKING BEHAVIOUR IN ALCOHOL-PREFERRING RATS

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(1)Alcohol & Alcoholism Vol. 37, No. 5, pp. 499–503, 2002. THE GABAB RECEPTOR AGONISTS BACLOFEN AND CGP 44532 PREVENT ACQUISITION OF ALCOHOL DRINKING BEHAVIOUR IN ALCOHOL-PREFERRING RATS GIANCARLO COLOMBO1*, SALVATORE SERRA1,2, GIULIANA BRUNETTI1,2, GIULIANA ATZORI2, MARIALAURA PANI2, GIOVANNI VACCA1,2, GIOVANNI ADDOLORATO3, WOLFGANG FROESTL4, MAURO A. M. CARAI2 and GIAN LUIGI GESSA1,2,5 1. C.N.R. Institute of Neurogenetics and Neuropharmacology, Cagliari, 2Neuroscienze S.c.a r.l., Cagliari, 3Institute of Internal Medicine, Catholic University of Rome, Rome, Italy, 4Research Department, Novartis Pharma AG, Basel, Switzerland and 5‘Bernard B. Brodie’ Department of Neuroscience, University of Cagliari, Cagliari, Italy (Received 23 January 2002; in revised form 26 March 2002; accepted 27 March 2002) Abstract — Aims: The present study investigated the effect of the γ-aminobutyric acid (GABA)B receptor agonists, baclofen and CGP 44532, on the acquisition of alcohol drinking behaviour in selectively bred Sardinian alcohol-preferring (sP) rats. Methods: Baclofen [0, 1 and 3 mg/kg, intraperitoneally (i.p.)] and CGP 44532 (0, 0.1, 0.3 and 1 mg/kg, i.p.) were administered immediately before alcohol presentation to alcohol-naive sP rats. Alcohol was offered under the two-bottle free-choice regimen with unlimited access for 24 h/day. Drug treatment was repeated once daily for 10 consecutive days. Results: Baclofen and CGP 44532, dosedependently and with comparable efficacy, suppressed alcohol intake; compensatory increases in water intake left total fluid intakes virtually unchanged. Conclusions: These results demonstrate that baclofen and CGP 44532 prevent the acquisition of alcohol drinking behaviour in sP rats, and suggest the involvement of the GABAB receptor in the mechanisms underlying the disclosure and experience of the reinforcing properties of alcohol in this rat line.. INTRODUCTION. In close agreement with the above preclinical data, baclofen has recently been reported to be effective in decreasing cocaine craving and use in cocaine addicts in an open-label study (Ling et al., 1998) and preventing limbic activation, as measured by positron emission tomography, during cue-induced craving for cocaine in cocaine-dependent patients (Brebner et al., 2002a). Further, baclofen has been found to suppress the intensity of signs and symptoms of the opiate withdrawal syndrome in opiate addicts in double-blind studies (Akhondzadeh et al., 2000; Ahmadi-Abhari et al., 2001). With regard to alcohol, this laboratory has recently described the ability of non-sedative doses of baclofen to specifically reduce voluntary alcohol intake in selectively bred Sardinian alcohol-preferring (sP) rats offered the choice between an alcohol solution and water, as well as to suppress the signs of alcohol withdrawal syndrome in rats made physically alcohol-dependent by the repeated administration of intoxicating doses of alcohol (Colombo et al., 2000). Although these results found a non-unanimous consensus in the preclinical scientific literature (Tarika and Winger, 1980; Daoust et al., 1987; File et al., 1991; Smith et al., 1992; Humeniuk et al., 1994; Tomkins and Fletcher, 1996; Petry, 1997; Smith et al., 1999), they have been confirmed by studies which investigated the therapeutic effectiveness of baclofen in human alcoholics. Indeed, a recent double-blind survey indicated that baclofen markedly reduces alcohol consumption, craving for alcohol and obsessional thinking about alcohol in alcoholics (Addolorato et al., 2002a). A further study showed that acute administration of baclofen resulted in the rapid disappearance of alcohol withdrawal symptomatology in alcoholic patients (Addolorato et al., 2002b). The above study with alcohol-preferring sP rats (Colombo et al., 2000) evaluated the reducing effect of baclofen on alcohol intake in alcohol-experienced subjects, that is rats in which the consumption of pharmacologically relevant doses of alcohol was already established before baclofen administration. The latter protocol models the ‘maintenance’ phase. In recent years, the γ-aminobutyric acid (GABA)B receptor agonist, baclofen, has attracted some interest in the drug addiction field, because of its ability to reduce the self-administration and severity of withdrawal syndrome of some drugs of abuse in rodents. Specifically, acute administration of baclofen has been reported to decrease the intravenous self-administration of cocaine (Brebner et al., 2002a), heroin (Xi and Stein, 2002; Brebner et al., 2002b), nicotine (Corrigall et al., 2000, 2001; Fattore et al., 2002), and γ-hydroxybutyric acid (Fattore et al., 2001) in rats and mice, as well as to block the development of conditioned place preference to morphine (Tsuji et al., 1996) and methamphetamine (Li et al., 2001) in rats. Further, baclofen has been reported to suppress locomotor stimulation [a possible animal model of the euphorigenic properties of a psychoactive drug (Wise and Bozarth, 1987)] induced by cocaine (Kalivas et al., 1990), amphetamine (Kalivas et al., 1990; Phillis et al., 2001), morphine (Woo et al., 2001; Leite-Morris et al., 2002), the opioid µ-receptor agonist DAMGO (Kalivas et al., 1990), and phencyclidine (Menon et al., 1980) in rats and mice. Recent investigations indicated the ability of the newly synthesized GABAB receptor agonist CGP 44532 [(3-amino2(S)-hydroxypropyl)methylphosphinic acid] to decrease the self-administration of cocaine (Brebner et al., 1999) and nicotine (Paterson et al., 2001) in rats. Finally, baclofen administration reduces the severity of the withdrawal syndrome from morphine (Zarrindast and Mausa-Ahmadi, 1999; Bexis et al., 2001; Diaz et al., 2001; Kemmling et al., 2002), barbital (Benedito and Liete, 1981; Sandoval and Palermo-Neto, 1985) and diazepam (Andrews and File, 1993) in rodents.. *Author to whom correspondence should be addressed at: C.N.R. Institute of Neurogenetics and Neuropharmacology, c/o ‘Bernard B. Brodie’ Department of Neuroscience, University of Cagliari, S.S. 554, km. 4.5, I-09042 Monserrato (CA), Italy. 499. © 2002 Medical Council on Alcohol.

(2) 500. G. COLOMBO et al.. of human alcoholism. To further characterize the anti-alcohol effect of stimulation of the GABAB receptor, the present investigation was designed to assess the effect of baclofen and CGP 44532 on the acquisition of alcohol drinking behaviour in sP rats, that is a model of development of the predisposition toward excessive alcohol consumption in individuals at genetic risk of alcoholism. To this end, the sP rats used in the present study were alcohol-naive at the time of the start of baclofen administration. MATERIALS AND METHODS. RESULTS The present study evaluated, in two separate experiments, the effect of the repeated administration of baclofen and CGP 44532 on the acquisition of alcohol drinking behaviour in sP rats. In both experiments, mean daily alcohol intake in salinetreated rats rose to 5–6 g/kg/day [i.e. the amount of alcohol usually consumed daily by sP rats (Agabio et al., 2000)] within 4–7 days. Treatment with baclofen significantly [Ftreatment(2,216) = 22.597, P < 0.000005] and dose-dependently reduced the acquisition of alcohol drinking (Fig. 1A); in the rat group treated. Animals Male sP rats, from the 51st generation and 75 days old at the start of the study, were used. Rats were derived from a population of sP rats which underwent Caesarean delivery at Charles River (Lyon, France) for production of Specific Pathogen Free individuals. Rats were individually housed in standard plastic cages [size: 425 × 266 × 150 (height) mm] with wood chip bedding. The animal facility was under an inverted 12-h light: 12-h dark cycle (lights on at 23:00), at a constant temperature of 22 ± 2°C and relative humidity of ~60%. Rats were extensively habituated to handling and intraperitoneal (i.p.) injection. Rats were alcohol-naive before the start of the study. Experimental procedure Throughout the duration of the experiment, alcohol was offered in a free-choice regimen between the alcohol solution (10% v/v in tap water) and tap water with unlimited access for 24 h/day. Bottles were refilled every day with fresh solution and their left–right positions interchanged at random to avoid development of position preference. Food pellets (MIL Morini, San Polo d’Enza, RE, Italy) were always available. Rats were divided into three groups (n = 9 each) in the baclofen experiment and into four groups (n = 7 each) in the CGP 44532 experiment. In each experiment, rat groups were matched for body weight. In both experiments, alcohol presentation was initiated at the start of the dark phase of day 1. Rats received their first treatment, baclofen (0, 1 and 3 mg/kg; Sigma Chemical Co., St Louis, MO, USA) or CGP 44532 (0, 0.1, 0.3 and 1 mg/kg; Novartis, Basel, Switzerland), 20–30 min before alcohol presentation. Each drug was dissolved in saline (injection volume: 2 ml/kg) and injected i.p. Drug administration was repeated once a day (20–30 min before lights off) for 10 consecutive days. Alcohol, water and food intakes were monitored by weighing the bottles and food pellets (0.1 g accuracy) once daily immediately before the start of the dark phase. Recording of daily alcohol, water and food intake was performed throughout the 10 days of treatment as well as for an additional 14 (baclofen experiment) and 11 (CGP 44532 experiment) days after termination of treatment. Statistical analysis Data concerning daily alcohol, water, total fluid (i.e. the sum of alcohol solution and water consumed) and food intake during the 10-day treatment period and the post-treatment period of each experiment were expressed in g/kg, ml/kg, ml/kg and g/kg, respectively, and were evaluated separately by two-way (drug treatment; day) analysis of variance with repeated measures on the factor day.. Fig. 1. Effect of the repeated administration of the γ-aminobutyric acid (GABA)B receptor agonist baclofen on the acquisition of alcohol drinking behaviour in Sardinian alcohol-preferring (sP) rats. Baclofen (0, 1 and 3 mg/kg, i.p.) was injected once daily (20–30 min before lights off) for 10 consecutive days. Alcohol (10%, v/v) and water were offered under the two-bottle, free-choice regimen with unlimited access for 24 h/day starting from the first day of treatment with baclofen. Food pellets were always available. Alcohol, water and food intakes were monitored once a day immediately before lights off. The dashed line indicates the completion of the 10-day treatment period and the start of the post-treatment period. Each point is the mean ± SEM for n = 9 subjects..

(3) GABAB RECEPTORS AND ALCOHOL DRINKING BEHAVIOUR. with 3 mg/kg baclofen, daily alcohol intake was virtually suppressed throughout the 10-day treatment period. Reduction in alcohol intake was associated with a compensatory increase in daily water intake [Ftreatment(2,216) = 27.544, P < 0.000001] (Fig. 1B), so that daily total fluid intake remained unchanged [Ftreatment(2,216) = 2.357, P > 0.05] (Fig. 1C). Daily food intake was significantly higher in the rat group treated with 3 mg/kg baclofen than in the saline-dosed control group [Ftreatment(2,216) = 7.854, P < 0.005] (Fig. 1D), probably because in the control group part of the total caloric intake was provided by alcohol. After treatment completion, daily alcohol intake in the 3 mg/kg baclofen-treated group increased progressively, reaching control values after ~2 weeks [Ftreatment(2,312) = 4.481, P < 0.05] (Fig. 1A). Consistently, water intake progressively diminished [Ftreatment(2,312) = 5.206, P < 0.05] (Fig. 1B). Daily total fluid intake [Ftreatment(2,312) = 1.188, P > 0.05] (Fig. 1C) and food intake [Ftreatment(2,312) = 2.651, P > 0.05] (Fig. 1D) did not differ among rat groups during the post-treatment period. Similarly, treatment with CGP 44532 resulted in a marked suppression of the acquisition of alcohol drinking behaviour [Ftreatment(3,216) = 13.992, P < 0.00005] (Fig. 2A); in the rat groups treated with 0.3 and 1 mg/kg CGP 44532, mean alcohol intake was steadily <2.5 and <1.5 g/kg/day, respectively, throughout the 10-day treatment period. Daily water intake was significantly [Ftreatment(3,216) = 13.156, P < 0.00005] increased in the rat groups treated with 0.3 and 1 mg/kg CGP 44532 (Fig. 2B). Total fluid intake [Ftreatment(3,216) = 4.113, P < 0.05] (Fig. 2C) as well as food intake [Ftreatment(3,216) = 3.301, P < 0.05] (Fig. 2D) were minimally affected by treatment with CGP 44532. After treatment completion, 11 days elapsed before alcohol intake in the 0.3 and 1 mg/kg CGP 44532-dosed groups approached the intake recorded in vehicle-treated control rats [Ftreatment(3,240) = 3.049, P < 0.05] (Fig. 2A). During the post-treatment period, water intake progressively decreased [Ftreatment(3,240) = 2.712, P > 0.05] (Fig. 2B), while total fluid intake did not differ among the rat groups [Ftreatment(3,240) = 2.076, P > 0.05] (Fig. 2C). Food intake was higher in the 0.3 and 1 mg/kg CGP 44532-treated groups [Ftreatment(3,240) = 5.961, P < 0.01] (Fig. 2D), presumably as a compensation of the lower caloric intake deriving from alcohol. DISCUSSION The results of the present study demonstrated that repeated daily administration of the GABAB receptor agonists, baclofen and CGP 44532, prevented the acquisition of alcohol drinking behaviour in selectively bred alcohol-preferring sP rats. Specifically, the daily average alcohol intake in the rat groups treated with 3 mg/kg baclofen and 1 mg/kg CGP 44532 was steadily <1 and <1.5 g/kg, respectively, throughout the 10-day treatment period. These results suggest that stimulation of the GABAB receptor results in a virtually complete blockade of the disclosure and experience of alcohol reinforcing properties, which is otherwise a rather rapid phenomenon in sP rats, as indicated by intakes of pharmacologically relevant amounts of alcohol from the very beginning of alcohol exposure in salinetreated rat groups. The doses of 3 mg/kg baclofen (corresponding to 14 µmol/kg) and 1 mg/kg CGP 44532 (corresponding. 501. Fig. 2. Effect of the repeated administration of the γ-aminobutyric acid (GABA)B receptor agonist CGP 44532 on the acquisition of alcohol drinking behaviour in Sardinian alcohol-preferring (sP) rats. CGP 44532 (0, 0.1, 0.3 and 1 mg/kg, i.p.) was injected once daily (20–30 min before lights off) for 10 consecutive days. Alcohol (10%, v/v) and water were offered under the two-bottle, free-choice regimen with unlimited access for 24 h/day starting from the first day of treatment with CGP 44532. Food pellets were always available. Alcohol, water and food intakes were monitored once a day immediately before lights off. The dashed line indicates the completion of the 10-day treatment period and the start of the post-treatment period. Each point is the mean ± SEM for n = 7 subjects.. to 6.5 µmol/kg) displayed comparable efficacy; the relatively higher potency of CGP 44532, compared to that of baclofen, in blocking the acquisition of alcohol drinking in sP rats is consistent with its higher binding affinity for the GABAB receptor (Froestl et al., 1995). The results of the present study extend to the acquisition phase the ability of baclofen to reduce alcohol intake in the so-called ‘maintenance’ model, i.e. in rats that were alcoholexperienced before the start of the baclofen treatment (Colombo et al., 2000). It should be noted, however, that the effect of baclofen on voluntary alcohol intake in rats is a.

(4) 502. G. COLOMBO et al.. matter of debate, as studies conducted so far have yielded equivocal results; indeed, baclofen has been found to reduce (Daoust et al., 1987; Colombo et al., 2000), produce no change on (Tomkins and Fletcher, 1996), or even to stimulate (Smith et al., 1992, 1999) alcohol intake in rats given a choice between an alcohol solution and water. Differences in the rat strain (unselected or selectively bred alcohol-preferring rats), baclofen dose-range, route of baclofen administration, and procedure of alcohol presentation may explain, at least in part, these discrepancies. With regard to the acquisition phase, Smith et al. (1992) reported that baclofen, administered at the single dose of 10 mg/kg, stimulated — although not specifically — the acquisition of alcohol intake in Long–Evans rats offered a choice between water and an alcohol solution in an ascending series of concentrations (2–10%) every other day. Again, several differences in the experimental procedures, including particularly the use of unselected Long Evans rats (presumably not possessing any particular innate predisposition to consume alcohol) or of genetically selected alcohol-preferring sP rats, might be responsible for the opposite outcomes of the study by Smith et al. (1992) and the present study. The cellular mechanism by which GABAB receptor agonists exert their reducing effect on alcohol intake has yet to be defined. Different lines of experimental evidence suggest a role for the mesolimbic dopamine system in the regulation of the positive reinforcing properties of alcohol. Accordingly, administration of low to moderate doses of alcohol have been reported to activate the firing rate of mesolimbic dopamine neurons (Gessa et al., 1985) and stimulate dopamine release in the nucleus accumbens (e.g. Imperato and Di Chiara, 1986; Weiss et al., 1993) in rats. Further, pharmacological manipulation of dopamine D1 and D2 receptor subtypes has been found to influence alcohol drinking behaviour in rodents and humans (e.g. Pfeffer and Samson, 1988; Enggasser and de Wit, 2001). GABAB receptors located in the ventral tegmental area have been suggested to contribute to the control of mesolimbic dopamine neurons, exerting — when stimulated — an inhibitory action (Kalivas, 1993; Yoshida et al., 1994; Westerink et al., 1996). A recent, preliminary microdialysis study from this laboratory demonstrated that acute administration of doses of baclofen, within the dose-range that reduces alcohol intake in rats, inhibits the alcohol-induced stimulation of dopamine release in the rat nucleus accumbens (Carta et al., 2001). Accordingly, baclofen has repeatedly been reported to suppress the stimulation of motor activity [a phenomenon mediated by activation of the mesolimbic dopamine system (see Wise and Bozarth, 1987)] induced by alcohol in mice (Cott et al., 1976; Humeniuk et al., 1993; Shen et al., 1998; Broadbent and Harless, 1999; Chester and Cunningham, 1999). Thus, the reducing effect of GABAB receptor agonists on voluntary alcohol intake might be the consequence of their ability to diminish the reinforcing properties of alcohol by inhibiting alcohol-stimulated dopamine release in the mesolimbic system. With regard to the results of the present study, it might be hypothesized that baclofen and CGP 44532 blocked the acquisition of alcohol drinking behaviour preventing the disclosure of the dopamine-mediated, positive reinforcing properties of alcohol, rendering the alcohol solution modestly attractive to the rat. 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