SUMMARY The overarching working model of The Scripps Research Institute Alcohol Research Center (TSRI-ARC) is that the infralimbic subdivision of the medial prefrontal cortex (mPFC) represents a critical hub for the dysregulation of brain network activity by chronic intermittent ethanol (CIE) exposure, driven in part by inputs from the hypothalamus and impairing top-down control over the central nucleus of the amygdala (CeA). Within this framework, the Molecular Component, led by Drs. Contet and Dunning, will test the hypothesis that reduced abundance of metabotropic glutamate receptor 3 (GRM3) in mPFC astrocytes contributes to behavioral phenotypes of alcohol dependence in mice by maintaining a local hyperglutamatergic tone that elevates the inhibitory input of local interneurons onto pyramidal neurons projecting to the CeA. This hypothesis is supported by results generated during the previous funding period, which revealed a long-lasting impact of CIE-induced excessive alcohol drinking on the abundance of astrocytic markers, with GRM3 reaching proteome-wide significance, in both prelimbic and infralimbic mPFC samples. GRM3 is strongly enriched in astrocytes where it controls glutamate uptake, but it can also regulate synaptic transmission and plasticity in neurons. Specific Aim 1 of this project will thus be to determine whether the GRM3 abundance reduction detected in bulk proteomes stems from a reduction in astrocytes vs. neurons. To do this, we will leverage state-of-the-art quantitative mass spectrometry from metabolically labeled brain samples to tease apart protein abundance changes in different cell types. We anticipate that CIE-induced mPFC GRM3 deficiency will be specific to astrocytes and will be associated with reduced abundance of glutamate transporters. We will also probe a potential role of CRF1 receptor signaling in this effect. We will then test the hypotheses that reduced GRM3 abundance is both necessary (Specific Aim 2) and sufficient (Specific Aim 3) to produce the behavioral phenotypes of CIE withdrawal in mice with a history of chronic excessive alcohol drinking, using gene expression manipulations targeted to mPFC astrocytes. Analyzing the consequences of GRM3 overexpression and knockdown on cell type-specific proteomes, extracellular glutamate levels, and synaptic transmission will provide mechanistic insights into how these behavioral outcomes might be generated. Furthermore, Specific Aim 2 will include a translational approach to rescue GRM3 signaling via inhibition of glutamate carboxypeptidase II (GCPII). Altogether, this project is anticipated to establish the relevance of astrocytic GRM3 as an important regulator of glutamate homeostasis in the mPFC that can be targeted to reduce excessive alcohol drinking. It will benefit from the expertise and resources provided by the Neuroproteomics and Animal Models Cores and will interact both conceptually and experimentally with the four other TSRI-ARC Research Components.