Project Summary/Abstract Excessive ethanol consumption is a major public health issue that can drive a range of serious health problems. There are multiple mechanisms that contribute to the motivation to consume ethanol, and it has been shown that ethanol has both aversive and rewarding properties. Considerable attention has been paid to the reinforcing effects of ethanol and how these effects motivate ethanol intake. However, another factor that contributes to the motivation to consume ethanol is the sensitivity to the aversive effects. It has been hypothesized that tolerance to the aversive properties of ethanol contributes to excessive intake. One pre-clinical behavioral assay for the aversive effects of ethanol is the development of conditioned taste aversion (CTA). When a taste is paired with a treatment which produces aversive internal symptoms, a strong aversion to the taste develops. Studies comparing different rodent strains suggest a link between sensitivity to the aversive effects of ethanol and the propensity to voluntarily ingest ethanol. Importantly, it has been shown that as a dependence-like state emerges in mice, sensitivity to the aversive effects of ethanol also declines as measured by CTA. This would suggest that the tolerance to the aversive effects of ethanol that emerges with dependence may be another factor that drives dependence-induced ethanol drinking. Because the neurocircuitry underlying the aversive effects of ethanol is still poorly understood, we propose to combine cutting-edge tools to characterize the neurocircuit and molecular mechanisms that regulate tolerance to the aversive properties of ethanol. Based on previous studies and compelling pilot data, we will test the hypothesis that aversive properties of ethanol are encoded in the insular cortex (aIC) and that chronic ethanol disrupts excitatory/inhibitory (E/I) balance in the aIC, disrupting the aversive properties of ethanol, contributing to escalated ethanol consumption. Specific Aim 1 will use electrophysiology and chemogenetic approaches to explore the plasticity in aIC to BLA outputs (aICBLA) as well as the necessity of PV interneurons (aICPV) during retrieval of ethanol-induced CTA. Specific Aim 2 will use chemogenetics and electrophysiology to explore plasticity in both aICPV and aICBLA neurons during retrieval of ethanol CTA following long-term ethanol drinking. Specific Aim 3 is a collaborative aim and it will incorporate slice physiology as well as channelrhodopsin-assisted circuit mapping to explore changes in frontal-limbic connectivity between aIC, nucleus accumbens (Acb), and BLA. In addition, using fiber photometry we will measure activity of pyramidal neurons and interneurons simultaneously in the aIC and BLA. Together, all these studies will help us to identify a novel circuit mechanism that drives tolerance to the aversive properties of ethanol, which is a hallmark of alcohol use disorder (AUD).