PROJECT SUMMARY Chronic alcohol exposure is associated with the development of tolerance to alcohol’s aversive properties, which serve to limit drinking. In contrast, the response to alcohol’s rewarding properties, which promote drinking, remain unchanged. 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 prelimbic (PL) medial prefrontal cortex (mPFC) shares important functional similarities with the RMTg including facilitation of aversion learning and regulating the behavioral response to aversive stimuli. The neighboring infralimbic (IL) subregion of the mPFC exerts opposing effects to PL mPFC by facilitating extinction of aversive responding. Our work has uncovered a dense projection from the mPFC to the RMTg that spans both PL and IL subregions. Our findings from experiments investigating the function mPFC inputs to the RMTg support a role for these circuits in regulating aversive responding. These data lead us to hypothesize that subregion- and circuit-specific dependence-induced plasticity in RMTg-projecting mPFC neurons facilitates chronic tolerance to ethanol’s aversive properties. The aims described in the current proposal will use innovative, circuit-specific strategies to test this hypothesis. Aim 1 will use in vivo fiber photometry to measure changes in calcium signal in RMTg-projecting PL and IL mPFC inputs during the development of tolerance. Aim 2 will use closed-loop in vivo optogenetics to dissect the effects of bidirectional manipulation of PL and IL mPFC inputs to the RMTg on measures of chronic tolerance to ethanol’s aversive properties. Aim 3 will use whole-cell patch-clamp slice electrophysiology and a virally- mediated intersectional approach to identify dependence-induced changes in synaptic and structural plasticity underlying tolerance to ethanol’s aversive properties. The results of these studies will provide crucial insight into the circuit-specific neural mechanisms that contribute to uncontrolled alcohol drinking. In doing so, our findings have the potential to uncover new therapeutic targets for the treatment of alcohol use disorder.