PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) afflicts millions of individuals and their families each year. AUD is frequently comorbid with anxiety and depression, indicating potential sources for the motivation to consume alcohol. Alcoholics commonly list stressors and negative affective states as leading triggers of cravings and relapse, and severity of the disease state correlates with relapse susceptibility. The health and financial burden associated with AUD highlights the pressing need for more research focused on understanding the interaction between AUD and negative affective disturbances. Identifying key neuroadaptations driving this debilitating disease is essential for developing better diagnostic tools and treatments for affective symptoms in alcohol abstinence. Converging evidence suggests the transition from social use to negative reinforcement-driven alcohol seeking involves a set of brain structures collectively referred to as the extended amygdala. The bed nucleus of the stria terminalis (BNST), a component of the extended amygdala network, is a critical node for stress-related disorders such as anxiety, depression, and addiction. The BNST plays a prominent role in many facets of alcohol addiction including binge drinking and cravings in withdrawal. The BNST interacts with many cortical, subcortical, midbrain, and hindbrain regions to determine general affect. Our recent work identified a functional connection between the insular cortex (insula) and the BNST. The insula is involved in interoceptive awareness, cognitive control, and sensory processing, and mounting evidence suggests a role for the insula in alcoholism and negative affective disturbances. We used a mouse model of chronic drinking followed by forced abstinence (CDFA) to outline a definitive role for the insula-BNST pathway in abstinence-induced negative affect, and this proposal will significantly build on this foundational evidence. We will combine in vivo calcium fiber photometry and ex vivo electrophysiology with genetic mouse lines and a variety of sophisticated viral- genetic techniques to isolate circuit-specific neuronal ensembles. The mentored (K99) phase will provide training in fiber photometry and complex viral-genetic manipulation strategies to determine the functional and physiological state of BNST neurons receiving insular inputs (Aim 1) and insular neurons that project to the BNST (Aim 2) in the protracted abstinence phase of CDFA. The independent (R00) phase will identify 2nd order inputs onto insulaBNST neurons, with the goal of further delineating the complex neurocircuitry regulating abstinence-induced negative affect. The proposed studies and related career development training plan in this Pathway to Independence Award collectively provide the ideal mechanism to transition the applicant to a career as an independent addiction neuroscientist. The results will significantly advance our understanding of the neural adaptations that occur in protracted abstinence following chronic alcohol abuse.