The conditioning of ethanol’s (EtOH) reinforcing effects with environmental stimuli is a major factor in the abuse potential of this drug. EtOH-related stimuli elicit strong EtOH seeking in animal models of relapse and these models are widely employed to study the neurobiological basis of EtOH craving and relapse. However, behavioral and neurobiological information on conditioning factors in EtOH seeking derived from animal models is limited to that from EtOH nondependent animals or, in animals with a dependence history, use of stimuli conditioned to the reinforcing effects of EtOH before dependence induction without consideration of conditioning factors related specifically to EtOH consumption in the dependent state. In alcoholics, a significant positive correlation exists between the history and degree of dependence and the severity of drinking urges induced by alcohol-related environmental stimuli. One process to explain this observation is that repeated consumption of EtOH during withdrawal states allows for learning of the negative contingency between EtOH consumption and adverse withdrawal symptoms, modifying an individual’s reinforcement history to include learning about amelioration/avoidance of adverse states as a novel and essential aspect of EtOH’s reinforcing actions. This proposal is designed to address the implications of this process with regard to EtOH craving and relapse, with the major hypothesis that a consequence of withdrawal-related learning (WDL) the conditioned effects of EtOH- associated environmental stimuli come to exert more powerful control over EtOH-directed behavior than stimuli conditioned to the positive reinforcing effects alone, and thereby play a dominant role in eliciting and maintaining compulsive EtOH seeking. In support of this hypothesis, preliminary data that provide the basis for this proposal show that stimuli conditioned to EtOH availability during withdrawal (WDL) elicit significant reinstatement in a manner that is punishment- and effort- resistant, whereas EtOH seeking induced by stimuli conditioned to EtOH in the nondependent state in rats without a withdrawal-related learning (N-WDL) history is not. The purpose of the proposed project is to solidify and extend the preliminary findings across three Specific Aims: (1) to establish the significance of environmental conditioning to withdrawal relief by EtOH (WDL) in initiating and maintaining compulsive EtOH seeking, (2) to identify neuronal ensembles mediating compulsive ethanol seeking linked to WDL and to confirm their role in this behavior via activity-dependent pharmacogenetic neural inactivation, and (3) to establish the neurochemical/phenotypic profile of behaviorally critical neuronal ensembles in WDL- vs. N- WDL-motivated EtOH seeking utilizing RNAscope®. Successful completion of this project is expected to establish WDL as a major previously not well recognized factor in relapse vulnerability and provide the necessary foundations for future ...