The role of Project 3 is to identify and translate the neurobiological mechanisms underlying the ‘dark side of addiction’ studied across Projects 1 and 2. Women have higher stress reactivity and higher rates of depressive symptoms than men that may underlie their increased likelihood of chronic drinking. Since women are particularly sensitive to effects of stress on negative reinforcement drinking (NRD, see Overall Section), a primary aim of Project 3 is to identify mechanisms related to NRD and related treatments. The amygdala is a sexually-dimorphic structure essential for stress reactivity, and both norepinephrine (NE) and GABA signaling are critical for stress-induced behaviors. Based on the established role of GABA and NE in response to both stress and alcohol use, we hypothesize that the increased susceptibility to chronic alcohol use and relapse in women is partly due to sex differences in GABA-NE balance in subregions of the amygdala. We have shown that guanfacine, a NE agonist at α2A receptors, decreases activity of amygdala neurons and induces anxiolytic and antidepressant-like effects, with sex-dependent patterns of neuronal activation. We therefore hypothesize that targeting pre- and postsynaptic NE receptors or activating GABA neurons will counteract NRD, and could have synergistic effects on amygdala neuronal activity and behaviors induced by stress. We further hypothesize that these neuronal mechanisms interact with neuroinflammatory pathways, such as microglial activation, to modify synaptic structure in the brain area, and that targeting these neuroadaptations could alter NRD in a sex-dependent manner. Finally, we know that alcohol metabolism differs in men and women, and have identified a greater effect of decreased brain metabolism of ethanol in female compared to male mice. In Project 3, we will 1) determine whether targeting noradrenergic receptors decrease overall ethanol intake, as well as NRD in female and male mice, 2) determine whether NE manipulations and GABA neuron activity in the amygdala have synergistic effects on NRD in female and male mice using molecular genetics to target specific GABAergic circuit mechanisms, 3) determine the effects of noradrenergic receptors and GABA neuron activity on ethanol-induced microglia alteration and synaptic density in female and male mice, and 4) identify interactions between brain alcohol metabolism and these signaling pathways in NRD. These studies will provide mechanistic data relevant for studies in Projects 1 and 2 to identify brain mechanisms that may modulate stress-related alcohol use in a sex-dependent manner, and to determine how this contributes to sex differences in alcohol-related behaviors.