PROJECT SUMMARY Individuals diagnosed with anxiety disorders are 2-4 times more likely to develop AUD and this comorbid diagnosis is associated with a telescoped progression of AUD and a much worse prognosis in recovery. Despite the clinical significance of this problem, we still lack a full understanding of the neurobiological substrates responsible for the frequent co-occurrence of these disorders. One major obstacle has been the lack of well-validated animal models that reliably engender a behavioral profile that may be indicative of a heightened risk of developing these diseases. To that end, we have re-examined a common rodent model of early-life stress, adolescent social isolation (aSI) and in a series of studies, have generated strong data supporting the face, predictive and construct validity of aSI as a model of vulnerability to anxiety disorders and AUD in male rats, and have used this model to identified profound adaptations in mesolimbic circuits that may contribute to the “AUD vulnerable phenotype” engendered by this model, including enduring alterations in catecholamine release dynamics in the nucleus accumbens and increased intrinsic excitability in the basolateral amygdala. Despite the promise of this model, several key gaps in our knowledge remain. First, this model has not been carefully validated in female rats. Our preliminary data suggest that it may promote a vulnerable phenotype in females but that this phenotype may be sexually dimorphic. Experiments in Aim 1 will therefore conduct behavioral and neurobiological studies to further validate the utility of the aSI model in females. Second, although aSI alters many behaviors linked with increased risk of AUD, it is not known if aSI actually increases vulnerability in a rodent model of ethanol dependence. Aim 2 will integrate aSI and a well- established rodent model of ethanol dependence (chronic intermittent ethanol vapor, CIE) to test the hypothesis that aSI increases sensitivity to the behavioral consequences of CIE. Complementary neurobiological studies will determine if CIE exacerbates neurobiological adaptations engendered by aSI and test the causal role of these adaptations using pharmacological and chemogenetic interventions. Collectively, these studies will further validate aSI as a model of vulnerability to comorbid AUD and anxiety disorders in male and female rats and may facilitate the discovery of novel treatment strategies that may be particularly effective for individuals suffering from comorbid AUD and anxiety disorders.