Genetic Versus Pharmacological Assessment of the Role of Cannabinoid Type 2 Receptors in Alcohol Reward-Related Behaviors.

AN ESTIMATED 8.5 % of Americans suffer from alcohol use disorders (AUDs) in the United States. Estimated costs associated with AUDs exceed $223 billion and approximately 79,000 lives, with approximately 2.3 million years of potential life lost annually (Harwood, 2013). Because the burden of AUDs on society is so great, elucidating the mechanisms involved in the disease progression is critical. More specifically, identifying neurochemical systems involved in alcohol-related behaviors could pave the way for therapeutic targets that will ultimately reduce the burden of AUDs on society. One such system, the endocannabinoid system (ECS), has gained attention in recent years because it appears to play an important role in a variety of neuropsy-chiatric conditions (Abood and Martin, 1992; Hirst et al., 1998; Navarro and Rodriguez de Fonseca, 1998), including AUDs (see reviews by Justinova et al., 2009; Maldonado et al., 2006). The ECS consists of 2 known receptor subtypes (cannabinoid receptor 1 [CB1R] and CB2R), 2 main endogenous ligands that act on these receptors (anandamide [AEA] and 2-arachidonoylglycerol [2-AG]), and the enzymes responsible for endocannabinoid degradation (monoacylglycerol lipase and fatty acid amide hydrolase). CB1R is highly expressed in brain reward circuitry and can regulate the excitatory and inhibitory inputs to the dopaminergic neurons of the mesocorticolimbic reward pathway (see review by Gardner, 2005). CB2Rs are found in a number of tissue and cell types in the periphery, including spleen, tonsils, and immune cells such as microphages and T cells. In the central nervous system, CB2Rs are expressed on neurons and activated microglia (Cabral et al., 2008; Garcia-Gutierrez et al., 2010; Onaivi, 2006). While there is still ongoing debate as to the localization of CB2R in the brain, there is evidence for expression in the caudate putamen, cingulate cortex, amygdala, hippocampus, ventromedial hypothalamic nucleus, arcuate nucleus, thalamus, substantia nigra, dorsal raphe nucleus, medial raphe nucleus, ventral tegmental area (VTA), and the nucleus accumbens (NAc) (Garcia-Gutierrez et al., 2010; Onaivi, 2006). The presence of CB2R in reward-related brain areas (i.e., NAc and VTA) suggests the possible involvement of this receptor in drug reward-related behaviors. Both alcohol and activation of CB1R increase dopamine (DA) levels in the NAc, and cannabinoids (both endogenous and exogenous) can enhance the firing of dopaminergic neurons (Diana et al., 1998; French, 1997; French et al., 1997; Gessa et al., 1998; Hungund et al., 2003). Activation of CB2R has been shown to reduce cocaine-induced extracellular DA release and stimulated locomotor behavior (Xi et al., 2011), as well as reduce VTA DA neuronal firing (Zhang et al., 2014). These data, together with the observed localization of CB2Rs in the VTA, suggest that CB2Rs modulate drug reward-related behaviors. Behavioral evidence further supports a role for CB2R in drug reward-related behaviors. Pharmacological activation of CB2Rs with JWH-133 dose-dependently reduced cocaine self-administration in wild-type (WT) and CB1R knockout (KO) mice, but not in CB2R KO mice (Xi et al., 2011). Similarly, transgenic mice that overexpress CB2Rs self-administered less cocaine than WT controls (Aracil-Fernandez et al., 2012). CB2R KO mice showed reduced nicotine reward-related behaviors (Ignatowska-Jankowska et al., 2013; Navarrete et al., 2013) and pharmacological blockade of CB2Rs with SR144528 reduced nicotine-induced conditioned place preference (CPP) in C57BL/6 mice (Ignatowska-Jankowska et al., 2013). Relevant to the current study with alcohol, Onaivi and colleagues (2008) showed reduced CB2R gene expression in the ventral midbrain region of inbred mice that developed a preference for alcohol compared to inbred mice that did not. In addition, chronic treatment with a CB2R agonist and a CB2R antagonist enhanced and reduced alcohol consumption, respectively, in stressed but not control inbred mice (Ishiguro et al., 2007; Onaivi et al., 2008). CB2R KO mice consumed more alcohol in an unlimited-access 2-bottle choice study and developed more robust alcohol-induced CPP compared to WT littermate controls (Ortega-Alvaro et al., 2015). Also, CB2R activation, via the agonist β caryophyllene, reduced sensitivity to alcohol-induced sedation, alcohol intake, and alcohol-induced CPP in C57BL/6 mice (Al Mansouri et al., 2014). These findings are compelling and suggest that modulation of CB2Rs affects alcohol reward-related behaviors. The purpose of this study was to assess the role of CB2R in modulating alcohol reward-related behaviors in 2 genetic mouse models and using 2 well-established models of alcohol reward: home cage limited-access 2-bottle choice alcohol drinking and alcohol-induced CPP. First, we pharmacologically assessed CB2R involvement in alcohol reward-related behaviors in selectively bred high-alcohol-preferring (HAP2) mice. Evidence suggests that genetic alterations in the ECS, including CB1R, may influence alcohol-related behaviors in rodents (e.g., Cippitelli et al., 2005; Hansson et al., 2007; Hungund and Basavarajappa, 2000). The HAP2 mouse line is a relevant model when studying genetically influenced mechanisms of AUDs in humans. We previously showed that HAP2 mice were more sensitive to pharmacological ECS manipulation compared to their low-alcohol-preferring counterparts (Powers et al., 2010), suggesting that genetic propensity toward high alcohol preference is associated with changes in ECS function. Second, we used mice with genetic deletion of CB2R (CB2R KO) to replicate the findings of Ortega-Alvaro and colleagues (2015) and to compare pharmacological versus genetic approaches in the study of the role of CB2R in alcohol reward-related behaviors. We hypothesized that pharmacological blockade and genetic deletion of CB2Rs would increase alcohol drinking and increase the expression of alcohol-induced CPP based on findings of Ortega-Alvaro and colleagues (2015), who showed that CB2R KO mice developed an alcohol-induced CPP, while WT controls did not.