PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) is characterized by problematic alcohol consumption that evolves over an individual’s drinking history. While initial alcohol use is thought to be driven by its positive reinforcing effects, following long-term drinking, negative affective states emerge during withdrawal. These states are associated with increases in alcohol seeking, tolerance, and levels of consumption that follow repeated bouts of abstinence. This has led to the hypothesis that negative reinforcement becomes the primary motivational drive – where individuals consume alcohol to alleviate these negative states. However, the circuit-based mechanisms that control this switch to negative reinforcement are unclear. The goal of this proposal is to outline how alcohol-associated cues recruit circuits that control negative reinforcement before and after chronic alcohol exposure. Our preliminary data show that D2 medium spiny neurons (MSNs) are a critical a negative reinforcement circuit and control the motivational drive to avoid aversive stimuli in an environment. This proposal will test the hypothesis, that alcohol-associated cues recruit D2 MSNs activation in vivo and drive drinking after chronic exposure to alcohol. By combining optical approaches to record from and bidirectionally modulate D2 MSNs in the NAc during alcohol drinking I will define exactly when and how their activity is recruited over a history of drinking and how it controls drinking behavior. In Aim 1& 2, I will optically inhibit or activate D2 MSN activity at the time of an alcohol-predictive cue to determine how this controls operant drinking before and after chronic intermittent ethanol (CIE) exposure. In Aim 3, I will determine if the dynamics of D2 MSNs during operant alcohol drinking are predictive of specific drinking patterns by combining fiber photometry with operant behavior and doing deep-phenotyping analysis. I hypothesize that D2 MSNs – which function as a negative reinforcement signal – are recruited by alcohol associated cues only after a history of CIE exposure, when alcohol use is driven by negative reinforcement. Taken together, the experiments in this proposal will determine the negative reinforcement circuits that are recruited in the later phases of alcohol drinking and whether the dynamics of these neuronal circuits could be predictive of alcohol drinking phenotypes. This proposal encompasses technical and theoretical training that will provide the foundational expertise and conceptual thinking needed to address larger questions regarding how long-term exposure of alcohol changes the brain and drives continued alcohol use. Additionally, these findings can ultimately inform our understanding of underlying reward and learning process and lead to more efficacious treatment interventions for AUD.