Project Summary/Abstract Alcohol use disorder (AUD) exacts a major personal, economical, and societal toll. Given its high lifetime prevalence, there is an urgent need to understand neurobiological mechanisms underlying AUD pathophysiology to develop effective therapeutic strategies. Dysfunction of stress-related neuroendocrine and autonomic arousal pathways are highly associated with alcohol-related behaviors. Repeated alcohol use increases arousal. Moreover, higher arousal levels in treatment-seeking AUD patients correlates with higher rates of relapse. These findings suggest reciprocal interactions between arousal and alcohol use, such that higher basal arousal promotes alcohol drinking, which further exacerbates arousal. A challenge in understanding the neurobiological mechanisms mediating interactions between arousal and drinking is the lack of preclinical animal models that allow quantification of drinking together with longitudinal measurements of arousal and neuronal activity. We addressed this by designing a voluntary ethanol consumption paradigm for head-fixed mice combined with two- photon calcium imaging for neuronal activity recordings and pupillometry for measuring arousal. Our preliminary experiments show that the anterior cingulate cortex (ACC) subdivision of the prefrontal cortex potently increases arousal. Basal levels of arousal and arousal-related ACC activity are correlated with the amount of ethanol consumption, suggesting that the ACC contributes to arousal modulation of drinking. Cortical activity is critically shaped by inhibition from local interneurons. The vasoactive intestinal polypeptide (VIP) expressing interneurons are particularly important as they inhibit other interneurons, leading to disinhibitory excitation of the ACC. We hypothesize that ACC VIP neurons are a key node for reciprocal interactions between arousal and ethanol consumption. In Aims 1 and 2, we test the hypothesis that VIP neuron-mediated disinhibition increases arousal and ethanol consumption. In Aim 3, we will determine if repeated ethanol consumption increases VIP-mediated disinhibition, leading to hyperarousal that further promotes drinking. We will test these hypotheses using a combination of advanced approaches including multicolor two-photon calcium imaging, multicolor optogenetics, machine vision-assisted closed-loop optogenetics, and ex-vivo slice electrophysiology. Together, these mechanistic studies will determine how VIP neurons both contribute to and are affected by drinking, establishing them as a key therapeutic target for modulating drinking driven by aberrant arousal.