SUMMARY – Molecular Component The TSRI-ARC as an integrated whole will focus on the neurobiology of protracted abstinence and its role in relapse and recovery. The Molecular Component will test the hypothesis that alterations in the composition and/or dynamics of the microtubule (MT) cytoskeleton contribute to the motivational and emotional symptomatology of abstinence by driving the structural remodeling of neurons in key brain regions. The first Specific Aim will be to determine alterations in MT composition and dynamics, using state-of-the-art proteomics methodology to quantify the abundance of α- and β-tubulin isotypes, MT-associated proteins known to regulate MT dynamics, and tubulin posttranslational modifications indicative of MT stability. These analyses will be performed in the medial prefrontal cortex (mPFC), anterior insula, and amygdala. Mice will be exposed to chronic intermittent ethanol inhalation (CIE) and brains will be collected 1 and 4 weeks into withdrawal to explore the development and persistence of molecular adaptations in early and late abstinence, respectively. The second Specific Aim will be to test the functional implication of MT alterations in ethanol drinking escalation, negative affect and dendritic remodeling during abstinence. A first approach will involve local knockdown of tubulin isotypes and MT-associated proteins to mimic or reverse abundance changes identified in Specific Aim 1. Another approach will entail systemic treatment with the synthetic pregnenolone derivative MAP4343 to stimulate MT dynamics and accelerate recovery from withdrawal. The third Specific Aim will be to complement research conducted by other TSRI-ARC components. A first experiment will probe the involvement of glucocorticoid receptor signaling in the effect of CIE on MT dynamics. A second experiment will test the role of serotonergic projections to the central amygdala and mPFC in the anxiety-like behavior associated with abstinence from CIE. Altogether, this project is anticipated to identify a molecular mechanism for the homeostatic failure—or allostasis—that characterizes alcohol use disorders and underlies relapse vulnerability during protracted abstinence. The Molecular Component will have strong interactions with the Animal Models Core, will involve collaborations with the Neurophysiology and Neurocircuitry Components, and will provide a mechanistic basis for the testing of compounds by the Clinical Component.