This is a competitive U01 renewal application under the Integrative Neuroscience Initiative on Alcoholism- Neuroimmune (INIA-N) Consortium (Notice# RFA-AA-20-11/12/13). It is designed to integrate multidisciplinary research projects based on the genomic, cellular, and behavioral neuroadaptations related to excessive alcohol consumption. The overall premise is that excessive alcohol consumption produces changes and adaptations in immune-related pathways that promote and sustain drinking. Mounting evidence indicates that immune-related pathways, including cytokines, are critical to regulating neuronal functions in addiction-related brain regions that underly the excessive drinking and behavioral phenotype associated with Alcohol Use Disorder (AUD). Notably, transcriptomic, and functional results point to interleukin-6 (IL-6) signaling as a strong candidate to test our central hypothesis that cytokines are integral to the neuroadaptations occurring in GABAergic and glutamatergic synapses and play a critical role in excessive drinking and anxiety-like behaviors associated with ethanol dependence and withdrawal. Specifically, we hypothesize that chronic ethanol-induced activation of IL-6 signaling is a major mediator of the central amygdala (CeA) and medial prefrontal cortex (mPFC) synaptic changes that contribute to the escalation of drinking and the associated negative affect. We anticipate that chronic ethanol will produce sex-, brain region- and cell type-specific IL-6 functional changes that subsequently contribute to ethanol dependence-associated behavioral phenotypes and persist (or be exacerbated) with ethanol withdrawal. We will study the IL-6 pathway and its positive and negative regulators (including SOCS3 and IL-10) to unveil potential therapeutic targets to alleviate ethanol-induced inflammation and ameliorate alcohol-related behaviors. In Aim 1 and 2, to induce ethanol dependence, we will use the chronic intermittent ethanol–two-bottle choice (CIE-2BC) paradigm in both male and female mice. We will apply behavioral, biochemical, and electrophysiological approaches to characterize dysregulation of the IL-6 signaling pathways in alcohol-drinking animals. Aim 3 will investigate the behavioral and electrophysiological effects of ethanol- induced neuroimmune responses on physiological functions across species (rodents and rhesus macaques) and cellular and molecular mechanisms of the candidate drugs/neuroimmune signaling that are identified by other INIA-N projects. Thus, we anticipate that our studies will identify cellular mechanisms and neuroimmune targets that contribute to excessive alcohol drinking. Furthermore, our INIA collaborations will promote replicability and translational aspects by testing our key targets in multiple species using multiple complementary approaches to bridge alcohol basic research with the human condition. The key personnel have extensive publications within their areas of expertise and ongoing interactions with ...