Project Summary/Abstract Binge drinking is a major contributor to the harms of alcohol use disorder (AUD), with high-intensity binge intake being particularly hazardous. However, little is known about the mechanism(s) that drive front-loading (strong initial intake rate) and sustained high drinking. Component 1 human results show that familial history/density of AUD relates to shorter time to reach binge levels, suggestive a heritable risk for excessive intake, but there is critical unmet need to understand mechanism(s) that drive excessive drinking. To discover critical brain circuits that drive excessive intake with heritable risk, we use rat lines selected for high alcohol consumption, P and HAD rats, which may model heritable AUD risk, and moderate-drinking Wistar rats as controls. Importantly, P rats show greater behavioral responses involving stimulus salience and vigor, while HAD rats have higher anxiety. Thus, these two lines may model reward versus relief drinking in AUD. Excessive drinking is likely driven by the strong, salient motivational properties of alcohol, which implicates the brain’s salience network (SN) which identifies and rapidly responds to important events. We focus on anterior insula (aINS), a critical regulator of SN and many aspects of motivation- and emotion-driven behavior. Also, aINS inhibition reduces rodent alcohol drinking, and human studies implicate aINS in both cue- and negative affect-driven drive for alcohol. However, little is known about specific mechanism(s) whereby aINS promotes front-loading and sustained high drinking. We specifically examine aINS projections to Nucleus Accumbens core (NAcb) and Basolateral Amygdala (BLA), since NAcb has widely been linked to vigor and reward-directed action, with BLA implicated in anxiety expression. Thus, we hypothesize that P rat drinking will involve greater aINS-NAcb neuronal firing during drinking and greater reduction of consumption when inhibiting aINS-NAcb (vs HAD and Wistar rats). In contrast, HADs will have higher aINS-BLA encoding of drinking and greater reduction of intake when inhibiting aINS-BLA. Aim 1 looks for rat line differences in the anatomical size of aINS-NAcb and aINS-BLA projections. Aim 2 uses cutting edge in vivo neuronal recording to discover firing patterns during front-loading in aINS-NAcb and aINS-BLA neurons (identified by “opto-tagging,” light-based stimulation to identify particular cells). We predict that greater aINS firing will correlate with higher front-loading, especially aINS-BLA in HAD and aINS-NAcore in P rat. Before alcohol, we also examine aINS activity during anxiety, sedation, and sucrose exposure, to determine whether aINS encoding of these states predicts excessive intake level. Aim 3 uses projection-specific inhibition methods to specifically suppress aINS-NAcore or aINS-BLA and test the hypothesis that excessive alcohol intake is more affected by aINS-NAcore inhibition in P rats and aINS-BLA inhibition in HADs. Together, proposed studies have strong interactions and synergy with other Components, and, if successful, the positive impact of our work will be the discovery of key aINS mechanisms that drive front-loading and sustained high alcohol drinking, which would improve AUD treatment.