Project Summary/Abstract Research Component 1. Alcohol use disorder (AUD) is a multiphasic neural and behavioral pathology that is characterized, in part, by dependence-induced escalated alcohol use. Robust prior work shows that synaptic activity of calcium permeable AMPA receptors (CP-AMPARs) is a target of alcohol that mediates the positive reinforcing effects of the drug via activity within the brain reward pathway. Despite this progress in understanding the molecular mechanisms of AUD, the role of CP-AMPAR activity in dependence-induced escalated alcohol self-administration remains to be fully elucidated. Strong preliminary data show that alcohol dependence both escalates the reinforcing effects of alcohol, as measured by operant self-administration, and upregulates AMPAR expression in the basolateral amygdala (BLA) and other brain regions that send glutamatergic projections to the nucleus accumbens (Acb), a central component of the reward pathway. This convergence of molecular, physiological, and behavioral data supports the overall hypothesis that: CP-AMPAR expression and activity in the BLAAcb pathway is a target of alcohol dependence that, in turn, drives escalated alcohol self- administration. We propose to test this hypothesis in three separate but integrated aims using male and female C57Bl/6J mice. First, an integrated set of molecular and electrophysiology studies will evaluate the impact of alcohol dependence, induced by the well-characterized chronic intermittent ethanol vapor (CIE-v) method, on AMPAR subunit expression and CP-AMPAR synaptic activity within the BLAAcb pathway. Second, loss of function behavioral studies will evaluate mechanistic regulation of dependence-induced escalated operant alcohol self-administration by CP-AMPARs in the BLA, and by GluA1-containing AMPARs in BLA neurons that project to the Acb using an innovative CRISPR/Cas9 method for specifically deleting the GluA1 subunit in this pathway. Third, based on strong preliminary data, we will conduct multi-channel fiber photometry to assess the impact of alcohol dependence on calcium signaling in genetically tagged excitatory and inhibitory cells during escalated operant alcohol self-administration. The mechanistic role of upregulated calcium signaling in escalated operant alcohol self-administration will be evaluated by inhibiting calcium/calmodulin-dependent protein kinase II (CaMKII) in the BLA, which is required for CP-AMPAR activity. These groundbreaking studies will move the field forward in understanding how calcium-dependent glutamatergic mechanisms in the BLAAcb circuit regulate the escalated reinforcing effects of alcohol associated with AUD.