Changed accumbal responsiveness to alcohol in rats pre-treated with nicotine or the cannabinoid receptor agonist WIN 55,212-2

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1 Available online at Neuroscience Letters 433 (2008) 1 5 Changed accumbal responsiveness to alcohol in rats pre-treated with nicotine or the cannabinoid receptor agonist WIN 55,212-2 José Antonio López-Moreno a,, María Scherma b, Fernando Rodríguez de Fonseca c, Gustavo González-Cuevas a, Walter Fratta b,1, Miguel Navarro a,1 a Laboratory of Psychobiology, Faculty of Psychology, Campus de Somosaguas, Complutense University of Madrid, 28223, Madrid, Spain b B.B. Brodie Department of Neuroscience, University of Cagliari, Monserrato, Cagliari, Italy c Fundación IMABIS, Avda Carlos Haya 82, 29010, Málaga, Spain Received 14 April 2007; received in revised form 27 October 2007; accepted 29 November 2007 Abstract Alcohol, nicotine, and cannabinoid acutely increase the activity of the mesolimbic dopamine (DA) pathway. Although polysubstance consumption is a common pattern of abuse in humans, little is known about dopamine release following pre-exposure to these drugs. The purpose of this study was to test whether alcohol-induced dopamine release into the nucleus accumbens (NAc) shell is modified by different pre-treatments: water (i.g.), alcohol (1 g/kg, i.g.), nicotine (0.4 mg/kg, s.c.), and WIN 55,212-2 (1 mg/kg, s.c.). Male Wistar rats were treated (i.g.) for 14 days with either water or alcohol. In the following 5 days rats were injected (s.c.) with vehicle, nicotine, or WIN 55, Finally, a cannula was surgically implanted into the NAc shell and alcohol-induced extracellular dopamine release was monitored in freely moving rats. Alcohol (1 g/kg; i.g.) only increased the release of dopamine when animals were previously treated with water. This DA increase was markedly inhibited by (subchronic) treatment (5 days) with nicotine or WIN as well as by previous (chronic) exposure to alcohol (14 days). These data demonstrate that pre-treatment with nicotine and the cannabinoid agonist WIN 55,212-2 is able to change the sensitivity of the NAc shell in response to a moderate dose of alcohol. Therefore, cannabinoid and nicotine exposure may have important implications on the rewarding effects of alcohol, because these drugs lead to long-lasting changes in accumbal dopamine transmission Elsevier Ireland Ltd. All rights reserved. Keywords: Alcohol; Dopamine; Nicotine; Cannabinoid; Addiction; Polysubstance abuse The mesolimbic dopamine (DA) system has an important role in the motivational aspects of rewarding stimuli (for reviews, see [5,8,17,37] and has an important role in the phenomenon of addiction to drugs such as alcohol, nicotine, and cannabinoids, among others [19,20,33]. Usually, the role of dopamine, expressed as an increase in extracellular DA, has been observed in animal models as well as in human subjects [4,40,41]. A common element in the phenomenon of addiction is polysubstance abuse of several different drugs. Both contingent administration (i.e., administration of drugs simultaneously or spaced by a short period of time [9,30,39]) and noncontingent polysubstance abuse (i.e., the use of one drug during abstinence from another [1,7,31]) in humans are frequent. Corresponding author. Tel.: ; fax: address: jalopezm@psi.ucm.es (J.A. López-Moreno). 1 Both authors have participated equally to this work. In light of these observations, we performed a series of experiments using a model of alcohol relapse: Drug During Deprivation. In brief, this model suggests that exposure to a drug challenge during the deprivation period of a chronically taken drug (distinct to the previous one) will cause a higher probability of relapse after reinstatement of the regularly used drug. In our studies, the drug used regularly was alcohol. With this model we demonstrated that exposure to either nicotine or the cannabinoid receptor agonist WIN 55,212-2 (WIN) during alcohol deprivation, caused a long lasting increase in relapse to alcohol [23,24]. Therefore, in order to explore the neurobiological correlates for these observations, we designed the present study (see Fig. 1). We have tried to establish an analogy with the behavioral data previously shown. We are aware that these data referred to self-administration, but it is well known that forced alcohol intake, as well as alcohol self-administration, increases extracellular DA in the nucleus accumbens (NAc) [11,14,16], particularly in the shell subregion. Similarly /$ see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi: /j.neulet

2 2 J.A. López-Moreno et al. / Neuroscience Letters 433 (2008) 1 5 Fig. 1. Schematic representation of the Drug During Deprivation model used. The critical point is the avoidance of exposing animals to both drugs (either alcohol nicotine or alcohol WIN 55,212-2) concomitantly for evaluating longlasting neurochemical changes. occurrences have been observed with nicotine and WIN [6,12,13]. We consider the key point of this model is alcohol relapse during exposure to a drug during the alcohol deprivation period. Therefore, we considered it of great interest to study the regulation of alcohol-induced extracellular DA after exposure to nicotine or WIN during alcohol deprivation. Male Wistar rats (Harlan, Italy) initially weighing between 200 and 250 g were used for these experiments. All animals were housed six per cage with food and water ad libitum except for the 6 h prior to intragastric alcohol (i.g.) treatment, during which they were food deprived. In this way, the stomachs of all animals were normalized in a similar condition previous to alcohol exposure. The animals were kept under a standardized dark/light cycle (light 8:00 a.m. to 8:00 p.m.); 21 ± 1 C room temperature, and 60% relative humidity. All procedures were conducted with strict adherence to the European Community Council Directive (86/609/EEC). The doses and route of administration were chosen based on the results obtained from our previous work [23,24]. The dose of alcohol (1.0 g/kg) corresponds approximately to the highest alcohol intake of a Wistar rat during the 30 min alcohol selfadministration session (approximately, 80 90% of the alcohol intake occurs during the first 10 min). The route of administration of alcohol (i.g.) was chosen because this is the most common route used for alcohol ingestion. Furthermore, intragastric administration by a trained experimenter may produce less stress in rats than other routes of administration (e.g. i.p. injections). The doses and route of administration of nicotine and WIN (0.4 mg/kg and 1 m/kg respectively, s.c.), were chosen according to our previous behavioral data, where their effects were characterized over a range of doses [23,24]. Each animal was exposed to single daily injections for 5 days of either nicotine or WIN during the alcohol deprivation period, in an analogous way to the original behavioral protocol. Alcohol (10% w/v in tap water, 5 ml/kg) was administrated i.g. (1.0 g/kg) for 14 consecutive days. WIN 55,212-2 (Tocris Cookson, Bristol, UK), which was dissolved in sterile physiological saline with 0.1% Tween 80, and ( ) nicotine bitartrate (Sigma, Milan, Italy), which was dissolved in saline solution at a dose of 1 ml/kg of body weight, were administered subcutaneously for 5 consecutive days. All rats were habituated to the chronic gavage procedure. For 5 days they were weighed everyday and intubated intragastrically to receive tap water (5 ml/kg) and then returned to their home cages. After that, half of the rats were assigned, in a counterbalanced way, to either alcohol or water treatment. Alcohol treated rats received a daily dose of alcohol (10% w/v in tap water, 1.0 g/kg; i.g.) for 14 consecutive days. Afterwards, they were divided in three counterbalanced groups and assigned to one of the following treatments: vehicle, nicotine, or WIN. Injections were given subcutaneously between the shoulder blades for 5 consecutive days. Rats receiving water followed the same procedure. On day 6 in vivo samplings using microdialysis were carried out in all rats (see Fig. 1 for details). Rats were anesthetized with Equitesin (5 ml/kg i.p.) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). A concentric microdialysis probe with 2 mm dialyzing surface length (AN 69AF; Hospal-Dasco, Bologna Italy; cut-off at 40,000 Da, in vitro recovery about 30%) was implanted vertically into the shell of the NAc and then fixed to the skull using dental acrylic cement. The coordinates relative to the bregma were: AP + 1.7, L ± 0.7, V 7.8 (according to the Paxinos and Watson atlas [29]). Starting at 24 h after the implantation of the dialysis probe, artificial cerebrospinal fluid (acsf) (147 mm NaCl, 4 mm KCl, 1.5 mm CaCl 2, ph 6 6.5) was pumped through the dialysis membrane at a constant rate of 2.5 l/min with a CMA/100 microinjection pump (Carnegie Medicine, Sweden). Dialysate samples (50 l) were taken every 20 min and directly injected into a high performance liquid chromatography (HPLC) system in order to quantify DA levels. The system consisted of an isocratic pump (ESA model 580), a 7125 Rheodyne injector connected to a Hewlett Packard series 1100 column thermostat with a reverse phase column (LC18 DB Supelco, 5 m, 4.6 mm 150 mm), and an ESA Coulochem II detector. The first electrode of the detector analytical cell was set at +400 mv and the second at 180 mv; the column temperature was set at 30 C. The mobile phase was delivered at 1.2 ml/min, consisting of: 50 mm sodium acetate, mm Na 2 EDTA, 0.35 mm OSA, 12% methanol and ph 4.21 with acetic acid. All experiments were performed between 8:00 a.m. and 4:00 p.m. The average value of DA levels in the last three pre-drug (baseline) samples (varying no more than 10%) was taken as 100%, and all subsequent post treatment values were expressed as mean ± S.E.M. percent variation of basal values. Only significant effects (P-values <0.05) from the two-way ANOVA analysis (time: repeated measures factor/treatment: between groups factor) were subjected to Tukey s honestly significant difference test. These analyses were performed using the SPSS (Chicago, IL, USA) statistical software package (version 11.0) for Windows. Pre-treatment with nicotine or WIN in animals exposed chronically to water (Fig. 2) showed a significant blockade of DA levels induced by alcohol in the dialysate recovered from the shell of the NAc with respect to the group pre-treated merely with vehicle (two way ANOVA: between

3 J.A. López-Moreno et al. / Neuroscience Letters 433 (2008) Fig. 2. Changes in dialysate DA levels from the shell of the NAc after acute alcohol administration (1 g/kg; i.g.) in rats chronically exposed to water (i.g.) and later subchronically treated (s.c.) with either vehicle, nicotine or the agonist cannabinoid WIN 55, Values are expressed as percentage of baseline, * P < 0.05; ** P < 0.01 (Tukey s post hoc test), vehicle vs. nicotine and vehicle vs. WIN 55, treatments F 2,16 = P = 0.001; within time F 8,128 = 2.38 P < 0.05; interaction time treatments F 16,128 = 2.77 P = 0.001). The alcohol-induced increase in extracellular levels of DA in the NAc observed here (Fig. 2) is consistent with other studies [14]. In addition, other works have shown that WIN and nicotine increase the efflux of DA in the NAc [6,12]. Therefore, alcohol, nicotine, and WIN can act directly or indirectly over a common and limited resource: an increase in the extracellular levels of DA in the NAc shell subregions. The DA release elicited by alcohol was slightly lower when animals were treated chronically with alcohol and pre-exposed to vehicle (Fig. 3). The peak DA concentrations in these animals were around 18% above baseline, which was not a significant difference (two way ANOVA: between treatments F 2,16 = 2.45 n.s.; within time F 8,128 = 1.84 n.s.; interaction time treatments F 16,128 = 0.70 n.s.). This suggests that DA release into the shell of the NAc is slightly modified after moderate alcohol exposure. Therefore, DA responsiveness would show a process of tolerance. Nevertheless, it is important to point out that the groups that were exposed to alcohol and pre-treated with nicotine and WIN did not show DA release evoked by alcohol. This could be due to an increased tolerance to alcohol caused by the combination of nicotine or cannabinoid. However, this hypothesis can be discarded because when the animals were never exposed to alcohol, but were pre-treated with nicotine and WIN (see Fig. 2), we observed the same altered extracellular DA responsiveness. Therefore, the decrease in alcohol-induced extracellular levels Fig. 3. Changes in dialysate DA levels in the shell of the NAc after acute alcohol administration (1 g/kg; i.g.) in rats chronically exposed to alcohol (1 g/kg; i.g.) and later subchronically treated (s.c.) with either vehicle, nicotine or the agonist cannabinoid WIN 55, Values are expressed as percentage of baseline. No significant differences were found between vehicle, nicotine and WIN 55,212-2 groups. of DA is due to a phenomenon of cross-tolerance after exposure to nicotine or cannabinoid, and not due to a summation of effects from either alcohol nicotine or alcohol WIN. Finally, the basal extracellular levels (fmol/ l of dialysate, mean ± S.E.M.) of DA in the NAc from different group of rats did not differ significantly (one way ANOVA, P = 0.25 n.s.). These levels were the following (see Fig. 1 for group details): group water-vehicle: 55.6 ± 7.5 fmol/50 l; group water-win: 55.6 ± 8.6 fmol/50 l; water nicotine: 54.3 ± 9.8 fmol/50 l; group alcohol-vehicle: 64.5 ± 6.7 fmol/50 l; group alcohol WIN: 52.5 ± 7.9 fmol/50 l; group alcohol nicotine: 85.8 ± 18.9 fmol/50 l. Only the results derived from rats with correctly positioned dialysis probes were included in the data analysis. The location of the probe was verified histologically at the end of each experiment in coronal brain sections (50 m) stained with cresyl violet. Fig. 4 shows a schematic illustration of the placement of the probes (see electronic supplementary material). The major findings in the present study are the following: First, the administration of a moderate dose of alcohol (1 g/kg) increased DA levels in the NAc shell when animals were exposed to water and pre-treated with vehicle. Second, water exposed rats pre-treated with either nicotine or the cannabinoid receptor agonist WIN showed a significant inhibition of the alcohol-induced DA release. Third, no differences in DA levels were found between rats chronically exposed to alcohol and pre-treated with either nicotine or WIN. And fourth, chronic alcohol expo-

4 4 J.A. López-Moreno et al. / Neuroscience Letters 433 (2008) 1 5 sure produced a reduction in the alcohol-induced DA release (a phenomenon of tolerance). The observed ability of alcohol to significantly increase the extracellular levels of DA in the NAc is modest when compared with cocaine and methamphetamine. This finding is consistent with other studies [3,11,14,16]. Furthermore, nicotine [12,13,35] as well as the cannabinoid receptor agonist WIN are found to increase DA levels in the NAc, but the latter effect may not occur by a direct mechanism [6,38]. However, CB1 receptors seem to be crucial for alcohol-induced dopamine release since it has been demonstrated that mice lacking these receptors do not show alcohol-induced dopamine release [15]. Therefore, alcohol, nicotine, and WIN are thought to act over a common mechanism: the increase in the efflux of DA in the NAc. Thus, the release of DA would be compromised after chronic exposure to alcohol, or subchronic exposure to nicotine or to a cannabinoid receptor agonist. This process would be similar to the stable low changed state during dopamine-mediated neurotransmission described by Bonci et al. [5]. Moreover, in vitro studies have shown that chronic alcohol exposure reduces nicotineinduced dopamine release in PC12 cells [9]. All together, these observations suggest that subchronic activation of nicotinic acetylcholine receptors, CB1 receptors, and other receptors affected by alcohol (essentially GABAergic and glutamatergic receptors) may regulate the release of dopamine in the shell of the NAc. However, it should be taken in consideration that alcohol has other actions in the central nervous system (e.g. on the opioid peptide and serotonin systems [18,21]) that may help explain, at least in part, the modest but significant increase of DA release after alcohol treatment. As commented, a previous exposure to nicotine or cannabinoid receptor agonist seems to be more critical than the addition of the two drugs: alcohol cannabinoid or alcohol nicotine. Consequently, two possible explanations are offered for the reduction of alcohol-induced DA release observed when the animals were chronically exposed to alcohol and pre-treated with nicotine or WIN. On the one hand, this effect may be caused by specific cross-tolerance between nicotine and alcohol. Alcohol exposure would affect nicotinic acetylcholine receptors [9,10], and reciprocally, nicotine exposure would influence alcohol-induced modifications in glutamatergic and GABAergic neurotransmission [22]. For example, chronic nicotine exposure causes the deactivation and subsequent upregulation of nicotinic acetylcholine receptors [10]. On the other hand, this diminished alcohol-induced DA release could be due to a specific cross-tolerance between cannabinoids and alcohol. Indeed, CB1 receptors and their functionality can be downregulated after chronic alcohol exposure [2]. Moreover, this effect may result from a more general process caused by the pre-exposure of any drug of abuse that causes the release of dopamine [12 14,28].It is well known that repeated exposure to common drugs of abuse produces long lasting changes in the circuitry of reinforcement (for review, see [26]). Such changes affect dopaminergic, glutamatergic, GABAergic and nicotinic neurotransmitter systems, among others, and their corresponding intracellular signaling pathways. Within this neural circuitry, the shell of the NAc may play a key role. The neurochemical alterations mentioned above, as well as the lack of alcohol-induced DA release following WIN/nicotine pre-treatment shown here, could lead to an adjustment in addictive behaviors in some individuals. It is interesting to note that the blockade of alcohol-induced dopamine release in the shell nucleus acumbens by the administration of nicotine or WIN could explain, in part, the significant increase in alcohol relapse evaluated in operant self-administration when animals were exposed either to nicotine or WIN during alcohol deprivation [23,24]. Thus, we hypothesize that animals were less sensitive to the effects of alcohol, which led to greater alcohol consumption, as we showed previously. Low sensitivity to alcohol has been characterized as one of the best predictors for alcoholism and a determinant of risk for becoming alcoholic [32]. In this line, it has been widely demonstrated that dopamine release and dopamine function are decreased in animal and human studies after chronic alcohol exposure. By using in vivo microdialysis, it has been revealed that forced intoxication of alcohol as well as voluntary alcohol self-administration leads to a decrease in the release of dopamine in the nucleus accumbens [27]. Furthermore, electrophysiological studies show that previous exposure to alcohol (e.g. prenatal and/or chronic exposure) results in lower activity of the ventral tegmental area, which highly innervates the nucleus accumbens [34]. In humans, neuroimaging findings show that alcohol dependence is associated with a dysfunctional dopamine system [25,36]. Taken together, these results support the view of a tolerance to DA release following repeated alcohol exposure. In conclusion, altered alcohol-induced DA release in the shell NAc could explain the greater alcohol relapse after exposure to nicotine and the cannabinoid agonist WIN, as previously reported. The decrease in the release of alcohol-induced DA after nicotine or WIN treatment could change the sensitivity of the mesolimbic DA system. This could be one of the factors underlying the phenomenon of vulnerability to alcohol, assuming this vulnerability is the predisposition to consume large amounts of alcohol. Acknowledgements This work was supported by The European Fith Framework Programme QLRT , Ministerio de Ciencia y Tecnología Grant BFI-2001-C02-01, Fondo de Investigación Sanitaria (Red Trastornos Adictivos), M.S.C (Plan Nacional Sobre Drogas), M.E.C. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.neulet References [1] H.J. Aubin, C. Laureaux, S. Tilikete, D. Barrucand, Changes in cigarette smoking and coffee drinking after alcohol detoxification in alcoholics, Addiction 94 (1999)

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