Project Summary In 2021, there was an estimated 29.5 million people that were diagnosed with alcohol use disorder (AUD), resulting in an estimated annual cost of $249 billion to the US economy. AUD is an ongoing, severe health crisis that requires further research to identify therapeutic targets. Stress can drive initial and sustained drug use, as well as trigger relapse during abstinence, however the mechanisms that drive this effect are under active research. The insula is a highly interconnected brain region that has been implicated in multiple disorders, including AUD. Interestingly, fMRI studies have shown that alcohol-dependent individuals have reduced insular cortex volumes, which was correlated with self-reports of impulsivity. PTSD patients have increased connectivity between the insula and the bed nucleus of the stria terminalis (BNST), suggesting this pathway is recruited by salient negative affective states. We have shown that the insula-BNST pathway drives negative affect-like behavior during abstinence, however the molecular mechanisms involved in this pathway are not understood. Nicotinic acetylcholine receptors (nAChRs) have been repeatedly shown to play an important role in reward and stress involved with drug misuse, however further research is needed to understand circuit specific nAChRs in these behaviors. Ethanol (EtOH) acts on nAChRs in a subunit specific manner. Multiple nAChR subunits have been implicated in AUD, one of the most prominent being α4β2, which are highly expressed throughout the brain. Moreover, multiple compounds acting on α4β2 nAChRs reduced binge-like EtOH consumption. Chronic alcohol exposure in rhesus monkeys resulted in decreased α4β2 nAChRs in the insular cortex, suggesting a role for these receptors in driving AUD associated behaviors. The overarching goal of this proposal is to characterize the role of α4β2 nAChRs expressed in mid-insulaBNST neurons in modulating compulsive-like EtOH consumption and abstinence-induced negative affect-like behavior. We will use whole-cell electrophysiology and calcium imaging in Aim 1 to determine the changes in sensitivity to α4β2 nAChRs expressed in insula-BNST neurons following binge-like EtOH drinking, aversion- resistant drinking and abstinence from alcohol. In aim 2, we will use bidirectional genetic manipulation of β2 nAChRs in this specific cell population to determine whether these receptors are sufficient and necessary for binge-like drinking and abstinence-induced negative affect. These studies will highlight the potential for nAChRs expressed in the insula-BNST pathway as a potential therapeutic target for AUD.