PROJECT SUMMARY Alcohol use disorders (AUDs) manifest from a convergence of characteristics of the individual, the environment, and the alcohol itself, and is strongly associated with pain disorders. In addition to the well-known analgesic and anesthetic effects of alcohol intoxication, alcohol withdrawal is associated with hyperalgesic states which contribute to relapse in patients with AUD-pain comorbidities. Here we will test the preclinical efficacy of endocannabinoid (eCB) augmentation for the alleviation of alcohol withdrawal-associated hyperalgesic states and determine the underlying neurobiological mechanisms subserving these effects. eCB augmentation and cannabis products are known to exert analgesic effects. Our preliminary data demonstrate that pharmacological augmentation of eCB signaling, specifically the eCB ligand 2-arachidonoylglycerol (2-AG), exerts reliable anti- hyperalgesic effects in mouse models, while depletion of 2-AG levels worsens and prolongs hyperalgesic states associated with alcohol withdrawal. Based on these data we will rigorously and comprehensively test the global hypothesis that pharmacological augmentation of 2-AG levels will alleviate hyperalgesic states associated with alcohol withdrawal via actions at CB1 and CB2 cannabinoid receptors. We will also test the hypothesis that endogenous 2-AG serves a physiological role to counteract withdrawal-associated hyperalgesic states. We will next test the hypothesis that 2-AG regulates an amygdala-prefrontal cortical (PFC)-periaqueductal gray (PAG) neural circuit. We will use optogenetics, retrograde tracing, and ex vivo electrophysiology to test the hypothesis that 2-AG signaling suppresses amygdala-mediated inhibition of PAG-projecting PFC neurons. We hypothesize that 2-AG signaling exerts analgesic actions in alcohol withdrawal via inhibition of glutamatergic transmission preferentially onto PFC GABA neurons thus increasing the excitation/inhibition ratio onto PAG-projecting PFC neurons, which contribute to descending pain modulation. Lastly, we will use in vivo calcium imaging of PAG- projecting PFC neurons to test the hypothesis that noxious mechanical stimulation-induced activity of these neurons is reduced in alcohol withdrawal and normalized by pharmacological 2-AG augmentation, and that activity of these neurons is required for the analgesic effects of 2-AG augmentation during alcohol withdrawal. Completion of these studies could provide preclinical validation for 2-AG augmentation in the treatment of AUD- pain comorbidity and provide novel mechanistic insight into how 2-AG signaling regulates descending pain circuits under physiological conditions and during alcohol withdrawal.