Rimonabant attenuates sensitization, crosssensitization and cross-reinstatement of place preference induced by nicotine and ethanol

Pharmacological Reports 2010, 62, 797 807 ISSN 1734-1140 Copyright 2010 by Institute of Pharmacology Polish Academy of Sciences attenuates sensitization, crosssensitization and cross-reinstatement of place preference induced by nicotine and ethanol Gra yna Bia³a, Barbara Budzyñska Department of Pharmacology and Pharmacodynamics, Medical University of Lublin, Staszica 4, PL 20-081 Lublin, Poland Correspondence: Gra yna Bia³a, e-mail: grazyna.biala@umlub.pl Abstract: The present study focused on the evaluation of behavioral sensitization, cross-sensitization, and cross-reinstatement processes induced by nicotine and ethanol in rodents. First, we showed that nicotine (0.175 mg/kg, base, intraperitoneally, ip) produced a conditioned place preference in rats. When the nicotine place preference was extinguished, nicotine-experienced animals were challenged with nicotine (0.175 mg/kg, ip) or ethanol (0.5 g/kg, ip), which reinstated a preference for the compartment previously paired with nicotine. In the second series of experiments, we demonstrated that after 9 days of nicotine administration (0.175 mg/kg, subcutaneously, sc) every other day and following its 7-day withdrawal, challenge doses of nicotine (0.175 mg/kg, sc) and ethanol (2 g/kg, ip) induced locomotor sensitization in mice. Finally, when we examined the influence of rimonabant (0.5, 1 and 2 mg/kg, ip), we found that this cannabinoid CB receptor antagonist attenuated reinstatement effect of ethanol priming as well as nicotine sensitization and locomotor cross-sensitization between nicotine and ethanol. Our results indicate that similar endocannabinoid-dependent mechanisms are involved in the locomotor stimulant and reinforcing effects of nicotine and ethanol in rodents, and as such these data may provide further evidence for the use of cannabinoid CB receptor antagonists in treatment of tobacco addiction with or without concomitant ethanol dependence. Key words: ethanol, nicotine, reinstatement, rimonabant, rodents, sensitization Abbreviations: CB cannabinoid receptor 1, CNS central nervous system, CPP conditioned place preference, nachrs nicotinic acetylcholine receptors Introduction Drug addiction is a chronic relapsing brain disease characterized by the compulsive use of addictive substances despite adverse consequences. Polydrug use is becoming increasingly more common, and often involves nicotine and alcohol co-abuse. Individuals who are alcohol-dependent present a higher frequency of nicotine dependence than those who do not drink alcohol [25]. Nicotine dependence is also more frequent in alcohol-dependent drinkers [37]. Despite these epidemiological findings, there have been relatively few animal studies on the neurobiological substrates that may underlie this combined nicotine and ethanol addiction. Functional interactions between nicotine and ethanol within the central nervous system (CNS) have been well documented [23, 41]. Experimental studies Pharmacological Reports, 2010, 62, 797 807 797

provide evidence for the involvement of nicotinic acetylcholine receptors (nachrs), the primary mediators of the effects of nicotine, in the modulation of ethanol consumption by showing that nicotine increases ethanol self-administration. This effect is suppressed by the nicotinic antagonist mecamylamine [33]. In addition, ethanol increases the number of nicotine binding sites in the brain [18] and also augments nicotine s locomotor stimulation [50] or reinforcing activity [29]. It is commonly accepted that repeated exposure to drugs produces long lasting changes in the circuitry of reinforcement [28, 39]. Such changes affect many neurotransmitter systems and their corresponding intracellular signaling pathways. Substantial data now point to a role of the endocannabinoid system in triggering drug seeking behavior [36]. Accordingly, rimonabant (SR141716A), a CB 1 cannabinoid receptor antagonist/partial agonist [43], has been shown to counteract the acquisition or expression of many drug-induced motivational effects [13, 15, 20, 22, 34]. Behavioral responses related to a relapse in drug taking can be measured in various animal models e.g., in drug self-administration or the conditioned place preference (CPP) paradigm [11]. After extinction of the drug-reinforced behavior, an acute exposure to the drug (non-contingent injection, priming dose) or non-drug contextual stimuli reinstates drug-seeking behavior [21]. An alternative characteristic of addiction and relapse is a phenomenon termed sensitization or reverse tolerance [45]. Using this paradigm it has been shown that after intermittent chronic exposure to a drug, animals began to develop addiction-like symptoms including continued drug seeking behavior and an escalation of drug intake, increased motivation to obtain drugs and a greater propensity to relapse after enforced abstinence [for review, see 45]. The present studies were undertaken to investigate the incidence of behavioral crossover rewarding and the locomotor effects of nicotine and ethanol. First, we used the nicotine-cpp procedure in rats evaluated in our previous studies [2 4, 6] to further examine the phenomenon of reinstatement of extinguished nicotine- CPP by a priming dose of ethanol. Second, using an experimental procedure previously established in mice [2], we examined whether nicotine-experienced mice develop sensitization to the locomotor stimulating effect of ethanol. Finally, we investigated the influence of rimonabant [43] on the expression of nicotine- and ethanol-induced effects. The results are discussed in the context of behavioral changes induced by subchronic drug treatment that lead to addiction and relapse, especially in connection with the influence of the endocannabinoid system. Materials and Methods Animals The experiments on the CPP paradigm, including cross-reinstatement procedures, were carried out on naive male Wistar rats weighing 250 300 g and experiments for sensitization and cross-sensitization procedures on naive male Swiss mice (Farm of Laboratory Animals, Warszawa, Poland) weighing 20 25 g at the beginning of the experiments. The animals were kept under standard laboratory conditions (12/12-h light/ dark cycle, temperature 21 ± 1 C, humidity 40 50%) with free access to tap water and lab chow (Bacutil, Motycz, Poland) and adapted to the laboratory conditions for at least one week. For all of the CPP procedures, the rats were handled once a day for 5 days preceding the experiments. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Each experimental group consisted of 8 12 animals. The experiments were performed between 9.00 a.m. and 5.00 p.m. All experiments were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the European Community Council Directive of 24 November 1986 for Care and Use of Laboratory Animals (86/609/EEC) and approved by the local ethics committee at the Medical University of Lublin. Drugs The following compounds were tested: ( )-nicotine hydrogen tartrate (Sigma, St. Louis, MO, USA), ethanol (Polmos, Lublin, Poland) and rimonabant (SR 141716A, 5-(p-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-Npiperidinopyrazole-3-carboxamide hydrochloride, gift of Sanofi-Synthelabo, Montpellier, France). Nicotine was dissolved in (0.9% NaCl). The ph of the nicotine solution was adjusted to 7.0. Ethanol was prepared for injections by diluting 95% ethanol to ob- 798 Pharmacological Reports, 2010, 62, 797 807

and nicotine-induced sensitization and place preference Gra yna Bia³a and Barbara Budzyñska tain a concentration of 10% (v/v). was suspended in one drop of 1% Tween 80 solution (Sigma, St. Louis, MO, USA) and diluted in. Fresh drug solutions were prepared on each day of experimentation. Agents were administered subcutaneously (sc) or intraperitoneally (ip) in a volume of 10 ml/kg (mice) or 5 ml/kg (rats), and, except for nicotine, drug doses refer to the salt form. Control groups received injections at the same volume and by the same route. Apparatus Experimental procedure and treatment CPP The CPP-reinstatement paradigm in rats took place on 9 consecutive days and consisted of the following phases: pre-conditioning (pre-test), conditioning, postconditioning (test), extinction and reinstatement. This method (biased design) was similar to that used in our previous experiments based on data indicating that the rewarding action of nicotine in the CPP paradigm can be observed after restricted doses and under specific biased conditions in rats [3, 4, 10]. CPP The testing apparatus for the CPP paradigm, similar to that used by Spyraki et al. [48], has already been validated in our laboratory. Each of six rectangular boxes (60 35 30 cm) was divided into three compartments, two large compartments (20 35 cm) that were separated by removable guillotine doors from a small central area (10 10 cm). One of them had its walls and floor painted white while the walls of the other were painted black. The central grey area constituted a neutral chamber, which serves as a connection and starting compartment. The testing boxes were kept in a soundproof room with neutral masking noise and dim 40-lx illumination. Horizontal locomotor activity of rats Locomotion was recorded individually using square actometer cages (Opto-Varimex-3, Columbus Instruments, Columbus, USA) that were situated in a soundattenuated experimental room. Each cage (60 60 50 cm) was equipped with two rows of 15 infrared light-sensitive photocells each, located 45 and 100 mm above the floor. Horizontal locomotor activity of mice Locomotion was recorded individually in round actometer cages (Multiserv, Lublin, Poland; 32 cm in diameter) kept in a sound-attenuated experimental room. Two photocell beams located across the long axis measured the animal s movements. Pre-conditioning On the first day, each animal was placed separately in the neutral area with the guillotine doors removed to allow access to the entire apparatus for 15 min. The amount of time that the rats spent in each of the two large compartments was observed on a monitor through a video camera system and measured to provide a baseline preference. Conditioning One day after pre-conditioning, the rats were randomized and subsequently conditioned with paired with the preferred (black) compartment (the morning sessions) and nicotine (0.175 mg/kg, ip) with the other (white) compartment (the afternoon sessions) for 30 min. Sessions were conducted twice each day with an interval of 6 8 h for 3 consecutive days (day 2 4). Injections were administered immediately before confinement in one of the two large compartments. A dose of 0.175 mg/kg (base) nicotine was chosen for conditioning because it is known to produce reliable conditioned place preference in rats, which we also found under our experimental conditions. The control group received in each session, every day. The neutral zone was never used during conditioning and was blocked by guillotine doors. Post-conditioning (test) On the 5 JD day, conducted one day after the last conditioning trial, animals were placed in the neutral area with the guillotine doors removed and allowed free access to all compartments of the apparatus for 15 min. The time spent in the - and drug-paired com- Pharmacological Reports, 2010, 62, 797 807 799

partments was recorded for each animal by an observer blind to the drug treatment. No injections were given on the day of this preference test. Extinction training One day after the preference test, rats were given extinction training daily for 3 days. In each trial, the rat was placed in the neutral area and allowed to explore both chambers for 15 min. No injections were given during this extinction period. The amount of time that rats spent in each chamber was measured on day 6 (Extinction 1), 24 h after the initial preference test, and on day 8 (Extinction 2), 72 h after this preference test. Reinstatement One day after the last extinction trial (day 9), separate groups of rats received, ethanol (0.5 g/kg, ip), or rimonabant (0.5 and 1 mg/kg, ip) 30 min before a priming injection of ethanol (0.5 g/kg, ip), and were immediately tested for reinstatement of CPP. During this reinstatement test, rats were allowed free access to the entire apparatus for 15 min, and the time spent in each chamber was measured. The control group was -primed. Locomotor sensitization in mice During the pairing phase (day 1 9), mice received either (ip) + (sc) or (ip) + nicotine (0.175 mg/kg, sc) every other day for five sessions. This method was similar to that used in our previous experiments based on data indicating that this dose of nicotine produces robust locomotor sensitization in mice under our laboratory conditions [5, 6]. The mice remained drug free for one week, and on day 16 the experimental groups were challenged with nicotine (0.175 mg/kg, sc) or ethanol (2 g/kg, ip). Locomotor activity was recorded for 60 min. Because the experiments revealed that nicotine-experienced mice developed sensitization to nicotine and ethanol challenges, a second experiment was designed to investigate whether pretreatment with rimonabant modified the expression of sensitization and cross-sensitization. To test this, on the challenge day (day 16) the mice were injected with rimonabant (0.5, 1 and 2 mg/kg, ip) 30min before the nicotine or ethanol injection. Locomotor activity of rats Animals naive for any drug treatment were injected with rimonabant (0.5, 1 and 2 mg/kg, ip) or for the control group, and immediately placed in the activity chamber. Horizontal locomotor activity, i.e., total distance travelled (in m), was automatically recorded for 30 and 60 min. Locomotor activity of mice Animals naive for any drug treatment were injected with rimonabant (0.5, 1 and 2 mg/kg, ip) or for the control group, and immediately placed in the activity chamber. Horizontal locomotor activity, i.e., the number of photocell beam breaks was automatically recorded for 30 and 60 min. Statistical analysis The data are expressed as the mean ± SEM. For the CPP paradigm, the data are expressed as the mean ± SEM of scores (i.e., the differences between postconditioning and pre-conditioning time spent in the drug-associated compartment). For the CPP procedures, the statistical analyses were performed using repeated measure analysis of variance (ANOVA) comparing treatments between subjects and sessions within subjects as variables. For locomotor sensitization, data were analyzed using ANOVA with treatment as an independent factor and days as repeated measures. The response to drugs on the challenge day was compared using one-way ANOVA. Post-hoc comparison of means was carried out with the Tukey test for multiple comparisons when appropriate. The confidence limit of p < 0.05 was considered statistically significant. Results CPP The time spent in the initially less preferred (white) and in the initially more preferred (black) side did not significantly differ between groups on the preconditioning day. This preference was not signifi- 800 Pharmacological Reports, 2010, 62, 797 807

and nicotine-induced sensitization and place preference Gra yna Bia³a and Barbara Budzyñska scores (s) 400 300 200 100 0 100 conditioning Test Extinction 1 Extinction 2 Reinstatement ethanol (0.5 g/kg) ^^^ ethanol (0.5 g/kg) scores (s) 200 100 0 100 200 conditioning Reinstatement ethanol (0.5 g/kg) rimonabant (0.5 mg/kg) + ethanol (0.5 g/kg) rimonabant (1 mg/kg) + ethanol (0.5 g/kg) Fig. 1. Reinstatement of nicotine-cpp in rats caused by a priming dose of ethanol. The place preference procedure consisted of preconditioning, three conditioning sessions with nicotine (0.175 mg/kg, ip) and a post-conditioning test followed by an extinction period, i.e., repeated test trials at 24 h (Extinction 1) and 72 h (Extinction 2) after the preference test. One day after the last extinction trial, rats were injected with a priming dose of ethanol (0.5 g/kg, ip) or. Data represent the mean ± SEM and are expressed as scores, i.e., the difference (in s) between post- and pre-conditioning time spent in the drug-associated compartment. n = 8 12 rats per group. p < 0.001 vs. -conditioned control group; p < 0.001 vs. conditioned rats primed with ethanol; ^^^ p < 0.001 vs. nicotineconditioned rats primed with (Tukey test) Fig. 2. Effects of rimonabant (0.5 and 1 mg/kg, ip) on the reinstatement of the nicotine-cpp caused by a priming dose of ethanol. The place preference procedure consisted of pre-conditioning, three conditioning sessions with nicotine (0.175 mg/kg, base, ip) and a post-conditioning test followed by extinction period, i.e., repeated test trials, 24 h (Extinction 1) and 72 h (Extinction 2) after the preference test. One day after the last extinction trial, the extinguished nicotine-cpp was reinstated by a priming dose of ethanol (0.5 g/kg, ip) preceded by an injection of rimonabant or. Data represent the mean ± SEM and are expressed as scores, i.e., the difference (in s) between post- and pre-conditioning time spent in the drugassociated compartment. n = 8 12 rats per group. p < 0.001 vs. nicotine-conditioned and -primed group; p < 0.001 vs. nicotine-conditioned and ethanol-primed group, Tukey test cantly changed when was paired with both compartments during the conditioning sessions. As shown in Figure 1, in - and nicotineconditioned rats given or ethanol injection on the reinstatement test, two-way ANOVA analysis revealed that there was a significant effect of treatment and session [treatment: F(1,80) = 391.4, p < 0.0001; session: F(4,80) = 45.33, p < 0.0001; treatment session: F(4,80) = 55.58, p < 0.0001]. On the test day, post-hoc analysis showed that there were significant differences in scores between the -conditioned and nicotine-conditioned groups (p < 0.001, Tukey test). Figure 1 also shows that the time spent in the nicotine-paired chamber gradually diminished over days of repeated test training. The increase in time spent in the drug-paired compartment on day 6 (first test for extinction, Extinction 1, conducted 24 h after the preference test) was greater for the nicotine-paired animals than for the -paired animals, whereas on day 8 (second test for extinction, Extinction 2, 72 h after the initial preference test) there was no difference in the time spent in the drug-paired compartment between these two groups indicating that the nicotine-cpp had been extinguished by repeated test trials. In Figure 1, it can also be seen that the priming injection of ethanol (0.5 g/kg, ip) reinstated the extinguished nicotine-conditioned place preference (p < 0.001vs. -conditioned group given injection during reinstatement test, Tukey test). Furthermore, the data show differences in the scores between nicotine-conditioned and ethanol-primed rats and -conditioned and ethanol-primed rats (p < 0.001, Tukey test), indicating that a prior CPP is necessary for an ethanol prime to produce an increase in time spent in the drug-paired compartment. Moreover, nicotine-conditioned rats show no reinstatement of the CPP after a injection (p < 0.001 vs. nicotine-conditioned and ethanol-primed group, Tukey test). Pretreatment with rimonabant (0.5 and 1 mg/kg, ip) 30 min before the ethanol priming dose inhibited the priming effect of ethanol in nicotine-conditioned rats [treatment effect on the reinstatement test in nicotineconditioned rats: [one-way ANOVA: F(3,28) = 80.94, Pharmacological Reports, 2010, 62, 797 807 801

p < 0.0001] (Fig. 2). Indeed, post-hoc individual comparisons indicated a significant effect of both doses of rimonabant (p < 0.001 vs. nicotine-reinstated group, Tukey test) which completely abolished the reinstatement of the previously established nicotine conditioned place preference (Fig. 2). Locomotor sensitization Two-way analysis of variance of the locomotor response after administration of nicotine (0.175 mg/kg, sc) or vehicle during the pairing phase (day 1 9) revealed a treatment effect [F (3,84) = 21.23, p < 0.0001], a day effect [F(3,84) = 99.15, p < 0.0001] and an interaction effect [F(6,84) = 21.07, p < 0.0001]. In nicotine-treated mice, locomotor activity increased progressively during daily injections [one-way ANOVA, F(5,42) = 39.96, p < 0.0001]. After the last nicotine injection (day 9), a significant difference between the response was observed when compared to the first injection of nicotine (p < 0.001) or to the response to in animals treated with repeated (p < 0.001, Tukey test) (Fig. 3). The locomotor activity of treated mice did not change significantly over time. Photocell beam breaks/60 min 2000 1500 1000 500 0 day 1 day 9 day 16 ^^^ &&& &&& ^^^ ethanol (2 g/kg) ethanol (2 g/kg) After 9 days of nicotine administration every other day and following its 7-day withdrawal, a challenge dose of induced marked behavioral sensitization observed as an increase in locomotor activity compared to that seen after the first injection of nicotine in the same mice (p < 0.001) or to the response to acute nicotine challenge in animals treated repeatedly with (p < 0.001, Tukey test) (Fig. 3). On day 16, when one group of the nicotinepretreated mice received an ethanol challenge (2 g/kg, ip), a significant difference in the response was observed compared to the first injection of nicotine (p < 0.001) or to the response to acute ethanol challenge in animals pretreated with (p < 0.001, Tukey test) (Fig. 3). These findings indicate that pre-exposure to nicotine results in locomotor sensitization to nicotine itself as well as in cross-sensitization to ethanol challenge. at doses of 0.5 and 1 mg/kg, but not 2 mg/kg, injected before the challenge dose of nico- Photocell beam breaks/60 min 1000 750 500 250 0 day 1 day 16 ^^^ ^^^ + + rimo (2 mg/kg) + nicotine (0.175) rimo (1 mg/kg) + rimo (0.5 mg/kg) + Fig. 3. Effect of a challenge nicotine (0.175 mg/kg, base, sc) or ethanol (2 g/kg, ip) injection on locomotor activity of mice pretreated with or nicotine (0.175 mg/kg, sc). Nicotine (0.175 mg/kg, sc) or was injected daily for 9 days, every other day; on day 16, and nicotine-experienced mice were given a challenge dose of nicotine (0.175 mg/kg, sc) or ethanol (2 g/kg, ip). Data represent the mean ± SEM; n = 8 10 mice per group. p < 0.001 vs. the first pairing day; &&& p < 0.001 vs. -treated mice; ^^^ p < 0.001 vs. -treated mice challenged with nicotine or ethanol, respectively (Tukey test) Fig. 4. Effects of rimonabant (rimo) (0.5, 1 and 2 mg/kg, ip) on the expression of locomotor sensitization to nicotine in mice. Nicotine (0.175 mg/kg, base, sc) or was injected daily for 9 days, every other day; on day 16 (a test for expression of sensitization) mice were given nicotine (0.175 mg/kg, sc) or rimonabant + nicotine. Data represent the mean ± SEM; n = 8 10 mice per group. p < 0.001 vs. the first pairing day; ^^^ p < 0.001 vs. -treated and nicotinechallenged mice; p < 0.001 vs. nicotine-treated and nicotinechallenged mice (Tukey test) 802 Pharmacological Reports, 2010, 62, 797 807

and nicotine-induced sensitization and place preference Gra yna Bia³a and Barbara Budzyñska Photocell beam breaks/60 min 2000 1500 1000 500 0 day 1 day 16 ^^^ Tab. 1. Effect of rimonabant (0.5, 1 and 2 mg/kg, ip) on locomotor activity (the mean ± SEM, in meters) of rats measured 30 and 60 min after injection. n = 12 rats per group. * p < 0.05, one-way ANOVA, post-hoc Tukey test Treatment time Saline 30 min 25.61 ± 3.60 60 min 36.54 ± 4.23 0.5 mg/kg 9.96 ± 4.29* 20.03 ± 5.82 1 mg/kg 30.17 ± 3.36 47.97 ± 7.87 2 mg/kg 27.37 ± 3.63 34.01 ± 4.80 + ethanol (2 g/kg) + ethanol (2 g/kg) rimo (2 mg/kg) + ethanol (2 g/kg) rimo (1 mg/kg) + ethanol (2 g/kg) rimo (0.5 mg/kg) + ethanol (2 g/kg) Tab. 2. Effect of rimonabant (0.5, 1 and 2 mg/kg, ip) on locomotor activity (the mean ± SEM, photocell beam breaks) of mice measured 30 and 60 min after injection. n = 8 10 mice per group Treatment time Saline 30 min 389.10 ± 19.30 60 min 555.50 ± 27.09 0.5 mg/kg 386.50 ± 46.49 541.30 ± 74.32 1 mg/kg 397.70 ± 26.92 518.30 ± 33.29 2 mg/kg 385.20 ± 24.28 530.50 ± 36.23 Fig. 5. Effects of rimonabant (rimo) (0.5, 1 and 2 mg/kg, ip) on the expression of locomotor cross-sensitization between nicotine and ethanol in mice. Nicotine (0.175 mg/kg, base, sc) or was injected daily for 9 days, every other day; on day 16 (a test for expression of sensitization) mice were given ethanol (2 g/kg, ip) or rimonabant + ethanol. Data represent the mean ± SEM; n = 8 10 mice per group. p < 0.001 vs. the first pairing day; ^^^ p < 0.001 vs. treated and ethanol-challenged mice; p < 0.001 vs. nicotinetreated and ethanol-challenged mice (Tukey test) tine was effective in blocking the expression of nicotine sensitization (Fig. 4). One-way ANOVA revealed a significant treatment effect on day 16 [F(4,35) = 9.25, p < 0.0001]. When rimonabant was injected before the ethanol challenge, it prevented the expression of crosssensitization between nicotine and ethanol at all doses (0.5, 1 and 2 mg/kg) (Fig. 5). One-way ANOVA showed a treatment effect on day 16 [F(4,35) = 7.65, p < 0.0001]. Locomotor activity of rats The CB1 receptor antagonist, rimonabant at 1 and 2 mg/kg, ip caused no changes in the locomotor activity measured 30 and 60 min after injection (Tab. 1) compared to the control -injected group. administered at a dose of 0.5 mg/kg caused a decrease in locomotor activity of rats 30 min after injection (p < 0.05), but this effect was not observed after 60 min. Locomotor activity of mice at all doses tested (0.5, 1 and 2 mg/kg, ip) caused no changes in the locomotor activity measured 30 and 60 min after injection (Tab. 2) compared to the control -injected group. Discussion The present study focused on the evaluation of behavioral sensitization, cross-sensitization, and crossreinstatement processes induced by nicotine and ethanol in rodents. Additionally, we investigated the effects of rimonabant, a cannabinoid CB receptor antagonist/partial agonist, on the behavioral influences of both drugs. First, we used the CPP paradigm to study the extinction and reinstatement of the extinguished nicotine place conditioning. In these experiments, rats were initially conditioned to associate an environment with nicotine administration. Once established, nicotine place preference was extinguished by repeated daily testing. Rats were then given one Pharmacological Reports, 2010, 62, 797 807 803

priming injection of ethanol which caused the reappearance of a preference for the compartment previously paired with nicotine. In the present study, we examined the effects of rimonabant on ethanolinduced reinstatement of nicotine place conditioning as our recent studies have already demonstrated that another selective cannabinoid CB receptor antagonist, AM 251, dose-dependently prevented the reinstatement of the nicotine-induced place preference induced by a priming dose of nicotine [9]. In the second set of experiments, our results indicated that repeated daily injections of nicotine produced progressive increases in locomotor activity in mice, especially subsequent to a nicotine challenge. One of the main findings of the present study is that locomotor crosssensitization occurred between nicotine and ethanol. Indeed, nicotine-experienced mice showed an enhanced response to ethanol injection compared to both the first pairing day and the response to acute ethanol challenge in animals pre-exposed to. In the third set of experiments, we investigated the effect of rimonabant on ethanol reinstatement of nicotineinduced place conditioning as well as the expression of cross-sensitization between ethanol and nicotine. Interestingly, we found that this CB receptor antagonist, when administered before a priming injection of ethanol or prior to a challenge dose of nicotine and ethanol, dose-dependently attenuated the ethanolinduced reinstatement of nicotine-cpp as well as the cross-sensitization provoked by these drugs. Our findings support the hypothesis that similar neural endocannabinoid-dependent mechanisms may be involved in the psychomotor and rewarding effects of nicotine and ethanol. Our results are in accordance with other animal experimental studies showing interactions between alcohol and nicotine. Both agents produced similar effects in several behavioral models, i.e., sensitization, place preference or self-administration [2, 26, 44]. In addition, several reports demonstrated cross-tolerance or cross-sensitization between ethanol and nicotine [1, 5, 17]. Other reports revealed behavioral interactions between these drugs in tests of motor activity, learning/memory and anxiety [14, 37, 42]. Moreover, a low dose of nicotine increased voluntary drinking of alcohol [47] and ethanol self-administration by rodents [14]. The mechanism underlying these described interactions is still not well understood but it is suggested that nicotine and ethanol share similar neurochemical mechanisms of action in the brain. The results indicate that the central cholinergic system is a site at which nicotine and ethanol interact. It has been well established that all major pharmacological effects of nicotine are mediated through several different types of nachrs located within the brain [52, 53]. Ethanol alters the function of different ionotropic receptors, including the GABA A /benzodiazepine receptor complex and glutamatergic receptors, but this drug can produce both up regulation and down regulation of nachrs depending on the brain region or the cell line used [8, 19, 31, 46]. Interestingly, ethanol may also exert its effects via enhancement of extracellular acetylcholine levels in the ventral tegmental area that subsequently stimulate dopamine overflow in the nucleus accumbens [30]. An additive or synergistic effect, especially on the release of accumbal dopamine induced by simultaneous administration of ethanol and nicotine, could further contribute to their coabuse. Moreover, in mice, ethanol-induced enhancements of locomotor activity and brain dopamine turnover were partially counteracted by mecamylamine, a blood-brain barrier penetrating antagonist of nicotinic receptors [7, 32]. Altogether, these findings indicate that ethanol may serve as a co-agonist with acetylcholine in some brain areas. A variety of frequently abused drugs, including nicotine and ethanol, appear to exert their rewarding effect via the activation of a common neuronal substrate, especially in the mesolimbic dopamine pathways. One could argue that these drugs can prime responses to one another because they share the property of activating the reward system, which becomes sensitized after repeated drug use. Accordingly, neurochemical studies indicate that both nicotine and ethanol increase the extracellular dopamine level in the nucleus accumbens and this effect seems to be mediated by nachrs [18, 49]. Thus, the crossover effects between nicotine and ethanol observed in our present and previous experiments [3] may also result from the above mentioned mechanism (i.e., release of dopamine in the reward system by activation of the cholinergic system). A growing body of evidence suggests that the endogenous cannabinoid system modulates the addictive properties of drugs of abuse as a component of the brain reward system. This system contributes to the primary rewarding effects of ethanol, opioids, nicotine, psychostimulants or cannabinoids through the release of endocannabinoids that act as retrograde messengers that inhibit the release of classical neuro- 804 Pharmacological Reports, 2010, 62, 797 807

and nicotine-induced sensitization and place preference Gra yna Bia³a and Barbara Budzyñska transmitters [40]. Regarding the dependence-producing action of nicotine, it has been shown that rimonabant decreased nicotine self-administration, including context-induced relapse to nicotine seeking or CPP in rats [15, 21, 34]. In support of this idea, knock-out mice lacking cannabinoid CB 1 receptors failed to display nicotine-induced place preference [12]. Furthermore, following chronic nicotine treatment, endocannabinoid levels are increased in the limbic forebrain region of rats [24]. Altogether, these results suggest that the endocannabinoid system may be tonically involved in modulating the rewarding properties of nicotine through a cannabinoid CB 1 receptor-related mechanism. In the present study, we have shown that rimonabant suppresses the expression of nicotine sensitization. In this respect, given the well-established role of dopamine in many aspects of addiction, it has been already pointed out that rimonabant attenuated nicotine-evoked dopamine release in the nucleus accumbens [16] eliminating the sensitized increase in the accumbal and striatal dopamine level. Thus, rimonabant may target the motivational properties of nicotine associated with drug-taking and drug-seeking behaviors. Moreover, in our studies, we observed that rimonabant suppressed the cross-sensitization and cross-reinstatement (i.e., dopamine-related) effects of ethanol in animals pre-exposed to nicotine. It is worth mentioning that microdialysis studies already demonstrated that rimonabant is able to abolish ethanolstimulated dopamine release in the nucleus accumbens [16] while this effect was absent in cannabinoid CB 1 receptor knock-out mice [27], confirming that these receptors are also crucial for ethanol-induced dopamine release. As the mesolimbic dopamine system is involved in the mediation of ethanol reinforcement and intake [51], one may suppose that a cannabinoid CB 1 receptor antagonist is capable of removing dopamine-mediated appetitive and reinforcing properties of ethanol. A common element in the phenomenon of addiction is polysubstance abuse, in which several different drugs are abused. Relapse is a major characteristic of drug addiction and could be used to study the neuronal mechanisms underlying drug craving. The present findings, which reveal the development of locomotor sensitization and reinstatement of nicotine- CPP, show analogies to a similar phenomenon described in ex-smokers and support the addictive role of nicotine in tobacco smoking. Our results also showed cross-reinstatement between nicotine and ethanol after short-term extinction and crosssensitization to the locomotor stimulant effects of nicotine and ethanol, providing circumstantial evidence for ethanol and nicotine interactions. One of the main findings of the present work is that administration of rimonabant completely prevented the expression of nicotine sensitization or cross-sensitization between nicotine and ethanol as well as the reinstatement effects of ethanol priming. Our data may further indicate similar endocannabinoid-dependent mechanisms involved in the stimulating and reinforcing effects of both drugs. Conclusion It is reasonable to conclude that the endocannabinoid system may play an important role in the nicotineand ethanol-induced neural and behavioral plasticity underlying the development of drug addiction and relapse. Because cannabinoid CB receptor antagonists (e.g., rimonabant or AM 251) [9, present study] have been shown to diminish the reward and stimulant properties of addictive drugs, this class of compounds may offer therapeutic advantages in the treatment of tobacco dependence with or without concomitant alcoholism. Although rimonabant has been withdrawn from clinical use in humans because of its serious side effects [35], this CB cannabinoid receptor antagonist/partial agonist will permit validation of the animal models of addiction to suggest potential cannabinoid-dependent mechanisms underlying the phenomenon of drug and polydrug dependence. Acknowledgments: The authors would like to thank Sanofi-Synthelabo (Montpellier, France) for the generous gift of rimonabant. This work was supported by the Statutory Funds of the Medical University of Lublin (DS 23/08) and received no special grant from any funding agency in the public, commercial or not-for-profit sectors. All authors declare that they have no conflicts of interest to disclose. References: 1. Al-Rejaie S, Dar MS: Possible role of mouse cerebellar nitric oxide in the behavioral interaction between chronic intracerebellar nicotine and acute ethanol administration: Pharmacological Reports, 2010, 62, 797 807 805

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