Abstract The induction of pro-inflammatory signaling is a key pathologic feature of alcohol use disorder (AUD). This includes increased expression of Toll-like receptor (TLR) signaling systems as well as activation of a variety of proinflammatory cytokines. Since many TLRs are involved in AUD pathology, it is important to study downstream mediators that are shared by multiple TLRs, in circuits that regulate AUD-related behaviors. Therefore, it is important that we find we find that agonists to TLR3 and 7 as well as chronic ethanol exposure all result in robust downstream induction of the proinflammatory transcription factor interferon regulator factor-7 (IRF7) in the anterior insular cortex (aIC) and nucleus accumbens core (AcbC). This induction in these brain regions is relevant as our work implicates aIC→AcbC circuitry as critical in the maintenance of alcohol self-administration. Interestingly, IRF7 was primarily localized in neurons along with pro-inflammatory activation of microglia. Microglia are key initiators of inflammatory signaling in brain and are capable of promoting neuronal and circuit adaptations to ethanol. Microglial pro-inflammatory cytokines further alter neuronal excitatory/inhibitory (E/I) balance – an overall theme of the UNC Alcohol Research Center (ARC) – as well as functional connectivity. Therefore, we hypothesize that pro-inflammatory microglia in aIC promote neuronal IRF7 and subsequent excitatory outflow from the aIC to AcbC to promote alcohol self-administration. The overall hypothesis of the present project is that neuronal IRF7 induction and pro-inflammatory microglial activation as a consequence of alcohol vapor exposure together alter E/I balance across aIC→Acb circuitry to promote increases in alcohol self- administration. Experiments in Aim 1 assess IRF7 signaling in aIC projection neurons and determine if IRF7 promotes escalations in alcohol self-administration following alcohol vapor exposure. Experiments in Aim 2 will examine proinflammatory microglia signaling in the aIC including E/I balance using gene expression analyses and determine the functional role of aIC proinflammatory microglia in vapor-induced increases in alcohol self- administration. Lastly, the collaborative studies in Aim 3 will directly determine the effects of the alcohol vapor exposure on the activity of aIC→Acb projection neurons using electrophysiology in collaboration with Research Component 1 and investigate the role of microglia on brain-wide connectivity across binge ethanol models using resting state functional connectivity magnetic resonance imaging (rs-fcMRI) and magnetic resonance spectroscopy (MRS) in collaboration with Research Components 3 and 4 and the Scientific Resource Core. The overall goal of the UNC ARC is to increase understanding of molecular and cellular pathogenesis in alcohol use disorder. To this end, the studies in Research Component 2 will determine the molecular consequences of chronic high-dose alcohol expos...