Effect of GABRA2 expression in the central nucleus of the amygdala on anxiety and alcohol's anxiolytic capacity in C57BL/6J mice

Abstract

The GABRA2 gene, which encodes the α2 subunit of GABAA receptors, is one of the genes most frequently associated with alcohol-related behavior in human studies (Demers, Bogdan, & Agrawal, 2014). Polymorphisms in GABRA2 have been found to be associated with alcohol dependence, changes in drinking frequency, and alcohol’s stimulating and euphoric effects (Arias et al., 2014; Dick et al., 2014; Edenberg et al., 2004). However, the GABRA2-alcohol relationship may not be direct, as anxiety and impulsiveness have been found to be mediating factors (Enoch, Schwartz, Albaugh, Virkkunen, & Goldman, 2006; Villafuerte, Strumba, Stoltenberg, Zucker, & Burmeister, 2013). Comorbidity of anxiety and alcohol use disorders is both prevalent and clinically relevant (J. P. Smith & Randall, 2012), and GABAA receptors play a significant role in each. Benzodiazepines, primary pharmacologic treatments for anxiety disorders and alcohol withdrawal, facilitate signaling at GABAA receptors, and their anxiolytic effects appear to depend on the presence of α2 subunits in these receptors (Low et al., 2000). The amygdala is widely implicated in both anxiety disorders as well as addiction (Janak & Tye, 2015), and its central nucleus is an important mediator of responses to both alcohol- and stress-related stimuli (Roberto, Gilpin, & Siggins, 2012), some of which may be related to GABRA2 expression within this region (Jin et al., 2014). The aim of the current study was to explore the role of Gabra2 (mouse ortholog of GABRA2) expression within the central nucleus of the amygdala (CeA) in anxiety-related behavior and alcohol’s anxiolytic effects in mice. C57BL/6J (B6) mice underwent surgery for bilateral infusion of GFP-tagged lentivirus targeting Gabra2 or a scramble control lentivirus into the CeA. Following 12-13 days of recovery, mice were assessed for anxiety-like behavior in the elevated plus maze (EPM) naïve or following IP injection of 0, 0.75, or 1.5 g/kg ethanol. After assessment, brains were extracted and sectioned through the CeA. Finally, GFP was quantified, the CeA was collected via laser microdissection, and α2 protein was quantified via ELISA. In mice expressing GFP in the CeA, α2 protein concentrations were lower for Virus mice relative to Control mice. The EPM was anxiogenic, and alcohol was found to be anxiolytic. In naïve mice, while there was no difference between Control mice and Virus mice on any behavioral measure, there were significant correlations between CeA α2 protein concentration and time spent in closed arms as well as both total and average time spent in open arms. In mice receiving injection of 0, 0.75, or 1.5 g/kg ethanol, there was a main effect of dose on several behavioral measures, but no interaction between viral condition and dose, and only a main effect of viral condition on average time spent in closed arms. There were no significant correlations between CeA α2 protein concentration and behavioral measures within any injected dose. These results are consistent with GABRA2-anxiety associations and effects of Gabra2 manipulation on anxiety-like behavior. Furthermore, they suggest that CeA α2 protein concentration is positively related to basal anxiety, which could affect alcohol use through various routes. However, these results also suggest that CeA α2 protein concentration is not related to alcohol’s anxiolytic capacity, at least when acutely administered in alcohol-naïve animals.