Project Summary Women are twice as likely as men to suffer from stress-related affective disorders, and as a result, are also at a greater risk of developing comorbid cardiovascular disease. This proposal investigates the estrogen (E)- mediated neural mechanisms that we suggest are initiated in the central amygdala (CeA) to promote innate stress vulnerability to both the emergence of negative valence and exaggerated cardiac sympathetic activation. The proposed series of studies relies on the use of a witness stress paradigm whereby a male or female rodent is located in a protected region of a dominant resident's cage and witnesses a social conflict between the resident and a smaller male intruder. Following stress exposure, females with either intact ovarian hormones, or ovariectomized with E replacement (OVX+E) exhibit negative valence (decreased sucrose preference, increased burying) and exaggerated cardiac sympathetic levels (elevated resting blood pressure) while OVX with vehicle replacement (OVX+V) and intact males are resilient, making this model ideal to study the role of estrogen on increased stress susceptibility. Aim 1 expands upon preliminary data that support the hypothesis that ovarian hormones, in particular E, exacerbate stress susceptibility. These studies will identify sex differences and estrogenic effects on behavioral and sympathetic indices of stress-related pathology. Aim 2 utilizes direct intra-CeA administration of an E receptor (ER) agonist (DPN) or antagonist (PHTPP), to activate or block, respectively, the ERs in the CeA. We hypothesize that inhibiting the ER in this brain region will enhance stress resiliency in the innate susceptible (intact-cycling) and induced susceptible (OVX+E) female groups, while activating the receptor will promote vulnerability in the induced resilient group (OVX). One known effect of E is its ability to increase the stress-related neuropeptide corticotropin-releasing factor (CRF), a peptide that is abundant in the CeA and is capable of inducing enhanced behavioral and sympathetic fear responses. Our data indicate that intact females exhibit increased CRF in the CeA, but only if they have a history of stress exposure. Therefore, Aim 2 will also identify if these ER treatments affect CRF expression in the CeA. Finally, Aim 3 will use virus-mediated gene transfer to reduce CRF levels in the CeA to determine whether the susceptibility-enhancing effects of the E are dependent upon CRF. Moreover, using in vivo microdialysis, studies in Aim 3 will identify whether increased CRF in the CeA has consequences on the major stress sensitive target the locus coeruleus (LC). CRF release and neuronal activity will be measured in the LC during stress/control and will identify if intact females exhibit increased CRF release in the LC and whether this translates to elevated activity. The ability of shRNA CRF knockdown in the CeA to affect LC activity and CRF release will also be assessed. Together, these studie...