Project Summary Current NIAAA strategic plan calls for efforts to identify novel molecular actions of alcohol and new remedies to mitigate alcohol-impacted brain regions. Ever-changing research tools have exponentially accelerated our understanding of brain regions and circuits affected by alcohol use and comorbid affective disorders. However, efforts towards the understanding of molecular mechanisms for altered neural activity in these impacted regions have lagged behind. To fill in this gap, we will explore a previously unknown molecular mechanism whereby ethanol modulation of brain cholesterol in the prefrontal cortex (PFC) leads to aberrant mGluR2 function and glutamate transmission in a rodent model of chronic intermittent ethanol (CIE) exposure. This modulation may thus contribute to alcohol drinking and anxiety-like behavior, commonly observed in patients with alcohol use disorder (AUD). Using a CIE rodent model, we show that CIE increases glutamate release in the PFC along with increased alcohol drinking and anxiety-like behavior. Further, we and others report that ablation of PFC neuron projection to subcortical regions (e.g. BLA or NAc) abolishes these behaviors. Thus, the PFC is a critical hub for regulating dependence-related neural and behavioral activity. However, the molecular mechanisms underlying CIE-induced disruption of glutamate transmission in the PFC remain elusive. Our preliminary data strongly suggest mGluR2 involvement in this region: a) CIE spatially segregates mGluR2 from Gαo subunit and reduces mGluR2 stimulation of Gαi/o; b) CIE abolishes mGluR2 inhibition of Gβγ-mediated presynaptic glutamate release and postsynaptic intrinsic excitability of PFC neurons; and c) CIE impairs mGluR2 inhibition of anxiety-like behavior. Such a deficit in mGluR2 function may be governed by membrane cholesterol. We found that CIE drastically increases cholesterol content in the PFC by enhancing the activity of HMG-CoA-reductase (HMGCR), a cholesterol synthesis enzyme. Importantly, reduced, CIE- dependent mGluR2 function can be reversed by ex vivo cholesterol removal and mimicked by ex vivo cholesterol addition to naïve tissue. We hypothesize that blockade of cholesterol increase will attenuate CIE- dependent disruption of mGluR2-mediated G-protein activation, neurophysiology and dependence-related behaviors. We will use CRISPR/Cas9, cellular, electrophysiological and behavioral approaches to test our hypothesis. The proposal is conceptually and technically innovative and significant, investigating a largely overlooked and potentially impactful effect of CIE exposure on cholesterol modulation of mGluR2 function, glutamate transmission and anxiety-like behavior.