Abstract Protracted withdrawal is characterized by negative emotional symptoms and their alleviation is a driving force for continued alcohol consumption. Our TSRI-ARC focuses on identifying the enduring neuroadaptations in the brain stress systems that drive relapse. In rats with ethanol dependence, an overactivation of the central nucleus of the amygdala (CeA) is driven by recruitment of the corticotropin-releasing factor (CRF) and CRF1 receptors (CRF1). Nociceptin/orphanin FQ (N/OFQ) peptide and its receptor (NOP) exert anti-stress effects by counteracting the function of endogenous CRF in CeA. The infralimbic (IL) subdivision of the mPFC exerts “top- down” control over the amygdala to regulate emotional aspects of goal-directed behaviors. Our main hypothesis is that dysfunction of mPFC contributes to the negative affect that drives withdrawal-induced drinking. We predict that IL pyramidal neurons that project to CeA (ILàCeA) may normally inhibit ethanol seeking and drinking under stress, a function that is compromised (decreased) in AUD, rendering subjects vulnerable to stress-induced negative urgency for ethanol. Preliminary chemogenetic data from Zorrilla’s component point to a causal role of rat ILàCeA inhibition in negative affect. Moreover, Martin-Fardon’s component showed that intra-IL CRF1 or hypocretin/orexin (Hcrt) antagonist infusion reduced stress-induced reinstatement of ethanol seeking in rats. These peptides are highly expressed in IL, but their interplay is understudied. The Neurophysiology Project (Lead: Roberto; Co-Lead: Bajo) will elucidate the mechanisms and neurotransmitters that disrupt IL functions and identify the ILàCeA role in these behavioral phenotypes. We will test the hypothesis that ethanol dependence [induced by chronic intermittent ethanol (CIE)] and abstinence produce an imbalance between brain stress (e.g., CRF) and anti-stress (e.g., N/OFQ) systems within the ILàCeA circuitry. Here we show that 1) CRF decreases spontaneous GABA release but increases glutamate release onto layer V IL pyramidal neurons of naïve male rats, 2) the CRF-associated reduction of GABA release onto ILàCeA neurons is stronger than onto other IL neurons. Our preliminary data support that the ILàCeA neurons represent a subset of neurons with unique electrophysiological properties and responsiveness to dependence/withdrawal that account for a decrease in ILàCeA connectivity. Moreover, CRF-induced facilitation of glutamate release and firing are opposed by N/OFQ in the IL. Thus, this proposal will a) investigate the mechanisms mediating N/OFQ and CRF modulation of overall IL function (Specific Aim 1) and b) will unveil the unique electrophysiological properties of IL pyramidal neurons that project to CeA and their role in negative behavioral symptomatology of ethanol withdrawal (Specific Aim 2). This project will use ex vivo electrophysiology and proteomics of circuit-defined neurons, protein assays, in situ hybridization, neuronal tracing...