PROJECT SUMMARY Chronic alcohol exposure is associated with the development of tolerance to alcohol’s aversive properties, which serve to limit drinking. The brain undergoes a host of neuroadaptive changes as a result of chronic alcohol exposure but the neural mechanisms underlying this tolerance are unknown. Impaired signaling in circuits encoding aversion is a likely candidate mechanism. The rostromedial tegmental nucleus (RMTg) is characterized for its involvement in aversion including signaling the aversive properties of ethanol. The aims of the parent grant are designed to test the hypothesis that dependence-induced plasticity in RMTg-projecting neurons in discrete subregions of the medial prefrontal cortex (mPFC) facilitates chronic tolerance to ethanol’s aversive properties. This hypothesis is supported by the presence of dense connectivity and significant functional overlap between the mPFC and RMTg. However, the RMTg also receives input from the anterior insular cortex (AIC). The AIC is critically involved in processing interoceptive information to guide behavioral responding to environmental stimuli including those associated with reward and aversion. In particular, loss of AIC function results in behavioral deficits in response to aversive stimuli. While the function of this neural circuit is not yet known, data from our lab suggests that both the AIC and RMTg play a role in signaling information regarding the aversive properties of ethanol. Like the mPFC, significant neuroadaptive changes are also observed in the AIC following chronic ethanol exposure. The current proposal is designed to investigate whether dependence-induced alterations in AIC-RMTg circuitry are also mechanistically involved in tolerance to ethanol’s aversive properties via a set of experiments that parallel those proposed in the parent grant. Aim 1 will use in vivo fiber photometry to measure changes in calcium signal in RMTg-projecting AIC inputs during the development of tolerance. Aim 2 will use whole-cell patch-clamp slice electrophysiology to identify dependence-induced changes in glutamatergic signaling underlying tolerance to ethanol’s aversive properties. Additional studies will use a virally-mediated intersectional approach to determine whether changes at the physiological level that occur after tolerance are also associated with changes in dendritic spine density and morphology. The results of these studies will augment the findings generated in the parent grant and shed new light on the possible synergistic role played by the AIC and mPFC in the mechanisms underlying chronic tolerance to the aversive properties of ethanol. In addition, this award provides an opportunity for the minority candidate to gain new training in cutting-edge approaches in addiction neuroscience in a rich scientific environment under the mentorship of a highly successful NIAAA-funded PI within the Center for Alcohol Research in Epigenetics. Findings generated with the support of th...