PROJECT 2. SUMMARY Major depressive disorder (MDD) is a leading cause of morbidity and disability worldwide with abnormalities in the stress response circuitry and central autonomic network. Many of these regions are sexually dimorphic and related with sex differences in mood and hypothalamic-pituitary-adrenal (HPA) axis modulation, the dysregulation of which is associated with alterations of hormone and immune responses to stress, autonomic dysfunction and increased cardiovascular risk. The overall clinical discovery mission of this SCORE proposal is to identify the sex-dependent impact of stress-immune pathway dysregulation, beginning in fetal development, on mood circuitry, MDD per se, immune physiology, and central and peripheral vascular function in midlife. The primary goal of Project 2 is to use non-invasive neuromodulatory stimulation of the vagus (i.e., autonomic nervous system) to target the circuitry associated with stress-immune function and map its neuroanatomic and physiological effects in MDD by sex. Vagal nerve stimulation (VNS), FDA- approved for MDD, modulates brain circuitry implicated in mood/anxiety and autonomic regulation, however, it is implanted and thus invasive. We propose the use of a physiologically-enhanced transcutaneous VNS (tVNS) as a low risk, non-invasive, and inexpensive alternative. While tVNS has had beneficial effects on depressive symptomatology and autonomic regulation, current stimulation parameters are based on historical iVNS data that included mostly male populations. We propose that tVNS effects on the regulation of specific brainstem-cortical pathways is modulated by sex. Moreover, as the dorsal medullary vagal system operates in tune with respiration, we recently demonstrated that tVNS can be optimized by gating stimulation to respiration. Thus, Project 2 proposes to identify the sex-dependent impact of expiratory-gated tVNS on the modulation of stress response circuitry alterations and physiological dysregulation of recurrent MDD. We will evaluate a sample of 80 adults with recurrent MDD (ages 51-64, equally divided by sex) randomized to receive active tVNS or active sham stimulation (earlobe stimulation) during a functional magnetic resonance imaging (fMRI) session. The fMRI session will include a stress challenge designed to elicit a sympatho- excitatory state, with simultaneous mood and physiological assessments, including hormonal and dynamic cardiovagal heart rate variability (HRV) evaluations. We hypothesize that expiratory-gated tVNS will effectively modulate specific brainstem-cortical pathways of the stress response circuitry and will attenuate physiological deficits of recurrent MDD patients. We further hypothesize that tVNS will impact brain activity and physiology in sex-dependent ways. Although Project 2 is not a clinical trial or intervention, ultimately, translation of results may lead to a novel sex-dependent intervention with beneficial effects prior to later-life health outcomes a...