Mechanisms of Ethanol Anxiolysis: Role of Ninein Deletion and Splicing on Ethanol and Anxiety-related Behaviors

Alcohol Use Disorder (AUD) is a complex neuropsychiatric chronic-relapsing condition characterized by excessive alcohol intake over time. Its etiology is multifactorial, with genetic, environmental, and psychological factors all contributing to its complex presentation. Anxiety disorders often serve as a predictor of developing AUD with human subjects reporting stress and anxiety as drivers of ethanol consumption. Individual responses to anxiolytic properties of alcohol are though to contribute to the overall development of AUD. The Miles laboratory previously identified Ninein (Nin) as a candidate gene underlying ethanol’s acute anxiolytic-like properties in BXD recombinant inbred mice, using the light-dark box (LDB) transition model of anxiety. Here we present the first comprehensive analysis of Ninein expression and function in the context of ethanol and anxiety-related phenotypes. Here, we utilized both global and region-specific Nin knockout models to dissect the contribution of Nin to ethanol-induced anxiolysis and other ethanol-related behaviors. In particular, our investigation focused on Nin expression in the central amygdala (CeA), which has been characterized as the “hub” region for the overlapping neurobiology of AUD and anxiety disorders. Our results indicate that deletion of Nin, globally or within the CeA, significantly alters the anxiolytic response to ethanol. Intriguingly, selective deletion of Nin in the CeA led to a marked reduction in ethanol consumption in female mice, suggesting a potential sex-specific role for Nin in regulating ethanol intake. Further behavioral assessments revealed that in all Nin knockout models, the deletion of Nin did not affect basal anxiety levels or alter sensitivity to ethanol’s sedative-hypnotic effects. This specificity implies that Nin’s role is predominantly tied to the modulation of ethanol-induced anxiolysis rather than to the generalized regulation of anxiety. To gain insights into the underlying molecular mechanisms, we examined the expression and localization of putative downstream targets of Nin in the CeA. Additionally, we investigated alternative splicing as a post-transcriptional mechanism for strain-specific regulation of overall Nin expression. Across all of our models, validation of our Nin knockout indicated that we did not completely knockout Nin expression, but rather, disrupted Nin expression potentially by disrupting translation initiation. Together, the results presented in this dissertations highlight Nin’s role as a novel contributor mediating the relationship between anxiety and ethanol.