Emma Gnatowski^1,2, Jessikah Buys^1,2, Jensen Goulette^2,3, Andrew A. George¹ and Michael F. Miles^1,2

Acute ethanol reduces anxiety in humans and animal models. Anxiety disorders increase risk for Alcohol Use Disorder (AUD) and human subjects report that stress and anxiety increase ethanol consumption. The Miles laboratory previously identified the microtubule binding protein Ninein (Nin) as a candidate gene underlying ethanol’s acute anxiolytic-like properties in BXD recombinant inbred mice. Here we report on behavioral, gene expression and GABAergic function consequences of Nin deletion in central amygdala (CeA). Deletion of Nin in CeA was done using stereotactic injections of AAV8-hSyn-GFP (control) or AAV8-hSyn-CRE-GFP (deletion) virus in Ninfl/fl mice. CeA Nin deletion increased acute ethanol anxiolysis in the light-dark box assay in male and female mice and reduced intermittent access 2-bottle choice ethanol consumption and preference x 5 weeks in female but not male mice. There were no changes in ethanol sedation (loss-of-righting reflex) or pharmacokinetics. Taste preference for quinine or saccharin were also unaffected. Bulk RNAseq analysis of stereotactic injection sites in CeA revealed striking evidence of neuroinflammatory and GABAergic gene expression alterations in Nin deletion mice. Preliminary electrophysiological studies on CeA IPSP activity measured by voltage clamp analysis showed Nin deletion altered IPSC duration, suggesting a post-synaptic site of action. Conclusions: These studies document that Nin function in CeA modulates the acute anxiolytic and consumption properties of ethanol, with the latter showing a striking sex preference. Initial mechanistic studies suggest that disruption of Nin expression in CeA produces changes in post-synaptic GABA receptor function, with coincident gene expression changes consistent with altered GABAergic neuron homeostasis and possible synaptic remodeling.

¹Dept. of Pharmacology and Toxicology, ²VCU Alcohol Research Center, and ³Dept. of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA USA

Funding Support: NIAAA grants F31AA030727, P50AA022537, and R01AA027581.