PROJECT SUMMARY Exposure to psychosocial stress has widespread deleterious effects on the body and brain that lead to the development of many physiological and neuropsychiatric disorders. Importantly, conditions such as anxiety and opioid use disorder (OUD) are two of the most common stress comorbidities that not only cause drastic effects on the patient but also represent an extreme public health crisis. Notably, females often suffer from these conditions at twice the rate of men, but research into the potential sex differences that allow for this susceptibility has traditionally been under studied. Further, the underlying neural mechanisms by which stress confers susceptibility to future psychiatric illness has not been fully explored. The overall goal of this project is to determine the neural alterations induced by psychosocial stress in discrete stress-sensitive brain regions in animal models utilizing both male and female rats. Specifically, these experiments aim to investigate neuroimmune signaling within the locus coeruleus (LC) to determine how stress-induced alteration of microglial activation influences noradrenergic output and subsequent anxiety-like and drug seeking behaviors. Increased neuroimmune signaling in this brain region has been associated with increased norepinephrine (NE) output, but the downstream impact of stress-induced microglial alterations of LC-NE activity on projection regions involved in the control of anxiety and drug seeking behaviors has not been assessed. Therefore, these experiments have been designed to test the hypothesis that stress exposure augments microglial signaling within the LC which, in turn, affects noradrenergic tone in downstream regions including the basolateral amygdala (BLA), a region known for its control of anxiety- and drug-related behaviors. This hypothesis will be tested using a variety of techniques including behavioral pharmacology, chemogenetics, and microdialysis to modulate and measure the impact of stress-induced LC microglial activation on anxiety-like and opioid seeking behaviors. Male and female rats will be exposed to our model of vicarious social stress, witness stress, where a rat experiences the sensory and psychological aspects of a social defeat encounter. Aim 1 is designed to determine the effects of LC microglia modulation, through intra-LC DREADD mediated inactivation of microglia, on anxiety-like and opioid reinstatement behaviors using marble burying and opioid conditioned place preference testing. These animals will also be implanted with microdialysis probes within the BLA for Aim 2 which is designed to determine the downstream effects of LC microglial modulation on NE activity in the BLA. These studies have the potential to identify novel mediators of maladaptive stress responses that escalate to opioid use disorder with the ultimate goal of determining preventative treatments for stress-induced comorbidities. These data support the mission of the NIH in the searc...