A Tale of Two “CBs”: Epigenetics and Ethanol in Cerebellar Ataxia and the Forebrain Cannabinoid System

The cerebellum is crucial in mediating the motor impairments induced by ethanol and the endogenous cannabinoid system plays a key role in emotional and motivated behaviors including anxiety and voluntary ethanol drinking. These two “CBs,” the cerebellum and cannabinoid system, were evaluated in several different ethanol exposure paradigms to comprise this thesis: a tale of two “CBs.” Ethanol is a pharmacologically complex drug with complicated neurological, psychological, and behavioral effects. It is also a drug with a huge societal impact due to its widespread use and associated medical, legal, social, and economic harms. We focused on three specific ethanol-related phenotypes to hone in on neurobiological substrates relevant to the behaviors as well as use appropriate ethanol administration models for studying ataxia, anxiety, and voluntary ethanol drinking. These three alcohol-related phenotypes are interestingly implicated in both the acute and chronic effects of ethanol, therefore allowing for investigation of epigenetic effects relevant to the binge/intoxication stage of Alcohol Use Disorder as well as the withdrawal/negative affect and the preoccupation/craving stages. Ataxia is one of the most consistent manifestations of acute ethanol intoxication, and cerebellar neurodegeneration and ataxia are also prominent in individuals with a history of heavy ethanol drinking. Ataxia is also prominent in Fragile X-Associated Tremor/Ataxia Syndrome, an epigenetic disorder featuring dysregulated Fragile X Mental Retardation 1 (FMR1) expression and ethanol has been shown to epigenetically increase FMR1 expression. We hypothesized a role for the epigenetic regulation of cerebellar FMR1 in the ataxia produced by both acute and chronic ethanol. In two different ethanol exposure paradigms in rats, we show that both acute and chronic ethanol produce ataxia as measured in the accelerating rotarod behavioral assay. Biochemically, we also show that FMR1 expression is increased in the cerebellum at the same time-points at which ataxia behavior is occurring. Furthermore, we show ethanol epigenetically induces alterations in FMR1 expression via histone modifications at the FMR1 promoter. Finally, we show that the several glutamatergic RNA binding targets of the FMR1 protein are similarly upregulated in the cerebellum. We propose the epigenetic induction of FMR1 by ethanol and subsequent stabilization of several glutamatergic genes disrupts the excitatory-inhibitory balance underlying the ataxic behavior observed following ethanol exposure. These studies represent an important expansion of ethanol-related FMR1 studies into the cerebellum and make the connection between Fragile X-associated ataxia and ethanol-associated ataxia. Overall, the results increase understanding of mechanisms underlying ethanol-induced ataxia, illuminating gene candidate FMR1 as a potential epigenetic and neuroregulatory target for treating Alcohol Use Disorder-associated ataxia. Anxiety is reduced by acute ethanol intoxication, but tolerance to this effect develops and ultimately anxiety is increased during withdrawal from chronic ethanol. The last phenotype, ethanol drinking itself, is the main mode of alcohol administration in humans. In the second set of experiments, we studied the effects of the neutral cannabinoid 1 (CB1) receptor antagonist AM4113 for its effects on anxiety and voluntary ethanol drinking as well as molecular and epigenetic effects in the amygdala, a brain region crucial in anxiety behavior and the negative affective state within Alcohol Use Disorder. This class of drug shows promise for reducing drug consumption without psychiatric side effects and current Alcohol Use Disorder treatments have modest effect sizes and don’t target the endocannabinoid system. We hypothesized CB1 neutral antagonist AM4113 would reduce ethanol drinking without altering anxiety-like behavior as well as produce epigenetic changes in amygdaloid Neuropeptide Y (NPY), a neuromodulator with overlapping downstream G-protein signaling effectors with the CB1 system. We show that AM4113 treatment reduces short-term continuous access 10% ethanol drinking while producing a subtle anxiolytic effect in the light-dark box anxiety-like behavior test in male mice. Furthermore, we show that these behavioral effects are associated with an epigenetic upregulation of NPY signaling in the amygdala; histone acetyltransferase expression and global and NPY-specific histone acetylation is increased in the amygdala following AM4113 treatment. Overall, we identify an important molecular mechanism by which CB1 receptors epigenetically regulate NPY function in the amygdala and our data further supports the potential use of CB1 receptor neutral antagonists for the treatment of excessive alcohol drinking without psychiatric liability. Finally, we conducted another set of experiments with AM4113 in the context of chronic ethanol drinking where we evaluated the efficacy of neutral CB1 antagonism in reducing ethanol drinking as well as the effects of chronic ethanol drinking itself of endocannabinoid system gene expression in several addiction-related brain regions. Additionally, we tested the ethanol-specificity of AM4113 by assessing effects on sucrose drinking as an alternative caloric reinforcer and studied sex differences in ethanol drinking and AM4113 response. We find AM4113 reduces chronic ethanol drinking in male and female mice without effects on sucrose drinking. Additionally, we show females drank more ethanol in the chronic intermittent paradigm and reach higher blood ethanol concentrations compared to males. We also show upregulations of CB1 receptor expression in the prefrontal cortex and dorsal hippocampus of male but not female mice following chronic ethanol drinking. Finally, we tested anxiety-like behavior following chronic intermittent ethanol access and find no expression of anxiety compared to water-drinking controls during acute withdrawal from chronic drinking. These experiments provide key evidence for the Alcohol Use Disorder pharmacotherapy candidate AM4113 and further the understanding of the endocannabinoid system in the context of ethanol-associated anxiety and ethanol drinking behaviors. We add to the emerging literature that the endocannabinoid system is crucial in ethanol drinking behavior and that CB1 receptors are an intriguing pharmacotherapeutic targets since neutral CB1 antagonists specifically reduce ethanol drinking without metabolic or anhedonic consequences. These tales of cerebellar FMR1 in ethanol-induced ataxia and the forebrain cannabinoid system in anxiety and ethanol drinking show different facets of the diverse ethanol-related phenotypes and the wide epigenetic and molecular landscape altered by ethanol, but they are linked together by Dickens’ “spring of hope” that further elucidation of these biochemical targets and their role in ethanol-related behaviors will lead to improved treatment paradigms for Alcohol Use Disorder patients.