Research reportEthanol self-administration and ethanol conditioned place preference are reduced in mice lacking cannabinoid CB1 receptors
Introduction
EtOH abuse and addiction is a complex social and psychiatric problem that involves a variety of neurotransmitter systems, such as dopamine (DA) [13], [29], [55], [56], GABA [24], glutamate [22], serotonin [32], acetylcholine [38] and the cannabinoid system [25]. EtOH abuse also involves a variety of receptors, second messenger systems and genes [14]. The cannabinoid system is not only involved in the effects of marijuana [19], but may be involved in the reinforcing properties of major abused drugs, such as EtOH and opiates [18] as well as other psychiatric or neurological diseases as supported by various genetic studies [8], [16], [48]. The cannabinoid CB1 receptor in particular has been widely examined and reported to have an interesting though complex role in addiction.
CB1 receptor activation increases the activity of DA neurons in the ventral tegmental area, thereby increasing DA release in the NAc [20], [51] which may have an effect in modulating the brains reward circuitry. Prior research has reported that the CB1 receptor appears to be the site of reinforcing effects within the cannabinoid system [33]. Previous studies examining the role of CB1 in addiction have shown that CB1 KO mice exhibited conditioned place preference for cocaine but not for morphine [34]; failed to self administer morphine but did self administer cocaine, d-amphetamine and nicotine [10]. In contrast, very recently it was reported that CB1 receptors play a role in the acquisition and maintenance of cocaine self-administration and that CB1 KO mice showed diminished operant responding to self-administer cocaine [50].
The human CB1 receptor gene (CNR1) was recently analyzed in alcoholics versus non-alcoholic controls [48]. The observed frequency of the A allele was 31.2% for controls and 42.1% for alcoholics. These results suggested that that homozygous genotype CNR1 1359A/A confers vulnerability to alcoholism [48]. Chronic alcohol exposure caused a decrease in anandamide in the midbrain [21], while it increased anandamide content in the limbic forebrain [3], [21]. These results suggested the involvement of endocannabinoid transmission in the reward circuit activated by alcohol [21].
Several animal studies have indicated that the cannabinoid system can influence the rewarding effects of EtOH. This is based on several observations: lower alcohol consumption in C57BL/six mice [1]; a reduction in EtOH intake by SR141716A [7], [41] and ethanol sensitivity [39] and withdrawal symptoms completely absent in cannabinoid CB1 receptor-deficient mice [39], [42].
SR141716A has been shown to be a very selective ligand for the CB1 receptor and a potent antagonist [46]. SR141716A has been shown to block the actions of the cannabinoids in several behaviors [40], such as suppressing alcohol intake [28], [41]. Sardinian alcohol-preferring (sP) rats, selectively bred for high EtOH preference and consumption were used to study the efficacy of SR141716A, in reducing voluntary EtOH intake [7]. Rats were given limited access to food and (10%) EtOH (4 h/day) and were treated with acute i.p. administration of 2.5 and 5 mg/kg SR1417116A. Results showed that 2.5 and 5 mg/kg of SR1417161A decreased EtOH intake while 10 mg/kg reduced both food and EtOH intake [7]. In another study, C57BL/6 in a two-bottle choice procedure were given free access to water and (10%) EtOH [1], when treated with SR141716A (0.3–3 mg/kg), significantly reduced EtOH consumption. More recently, Long Evans rats trained on a fixed ratio operant response protocol to self-administer EtOH were treated with SR141716A (0.3–3 mg/kg, i.p.), produced dose-related decreases the number of lever presses and EtOH intake [15].
Based on the previous studies with SR141716A, we hypothesized that wild type homozygous, heterozygous and CB1 receptor deficient mice would display differences in their alcohol-drinking behavior. In the present study, we examined the role of the CB1 receptor using CB1 transgenic mice, in a two-bottle choice paradigm, to measure EtOH preference, and total EtOH consumption. We thus hypothesized that CB1 homozygous, heterozygous and CB1 deficient mice would display differences in their alcohol-drinking behavior, and that treatment with SR141716A would modulate that behavior. Furthermore, the present study examined CB1 transgenic mice in a conditioned place preference (CPP) paradigm for ethanol. In this procedure, the animals tendency to approach or avoid environmental cues previously paired with the drug [2], [4], [27], [52]. Several studies have demonstrated that as with other drugs of abuse, animals display CPP to EtOH [5], [6], [12], [44], [49]. Therefore, another objective of the present study was to determine if the CB1 receptor is important in Pavlovian conditioning to the EtOH-paired environmental cues. We hypothesized that CB1-deficient mice would not show EtOH-paired CPP as compared to their wild type littermates.
Section snippets
Experiment 1: two-bottle choice EtOH drinking
Adult male (N = 29) CB1 transgenic mice (CD1 strain, obtained from C. Ledent in Universite libre de Bruxells, Belgium) [31] were individually housed in a 12/12 h reverse light/dark cycle, as well as a temperature and humidity controlled room. Details on the generation of these animals has been previously described [31]. Briefly, using the 129/Sv mouse genome library, the CB1 gene was cloned and the single coding exon was mapped and sequenced. Using R1 cells and aggregation with CD1 eight-cell
EtOH preference
The CB1 WT mice showed the highest EtOH preference on average when compared to the CB1 HT and the CB1 KO. A one way repeated measures ANOVA on percent ethanol preference across all three strains of mice during baseline, SR141716A and vehicle drinking sessions revealed a significant difference (F = 9.953; d.f. = 93.11; p < 0.001; Fig. 1).
All pairwise multiple comparison procedures (using the Holm–Sidak method) revealed several significant differences (p < 0.05; Fig. 1). Specifically, SR141716A (12.9 +
Discussion
Dopaminergic transmission has been shown to be preferentially increased by natural rewards (food, water, sex) under normal and deprivation conditions, as well as in response to novel stimuli. While no CB1 receptor mRNA or protein has been shown in dopamine neurons in the VTA; increased dopamine release and firing of dopaminergic neurons have been found after systemic administration of CB1 agonists, or blocked by CB1 antagonists [20], [51], [57]. This suggested that the endocannabinoid system
Acknowledgements
This work was supported by the NIAAA (AA 11034 & AA07574, AA07611) and by the U.S. Department of Energy under contract DE-AC02-98CH10886. We also thank the DOE SULI program for funding ESD. Finally, we would like to thank Dr. C. Ledent for providing the CB1 mice for this study.
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2019, Pharmacological ReportsCitation Excerpt :However, after eight days of conditioning training (administration of ethanol 20%v/v, in the dose of 2 g/kg alternating with normal saline), this preference was reversed i.e. mice spent more time in drug-paired chamber (white) than the non-drug paired chamber (black). This clearly showed that mice had significant expression of CPP and that CPP paradigm is a useful model for studying ethanol’s rewarding effects in mice as reported previously in several studies [18–20]. In post-conditioning phase after 24 h of expression phase, test drug i.e. lacosamide was administered and then it was observed that mice spent a significant more time in the non-drug paired chamber (Zone B) than the drug-paired chamber (Zone A).