Between 15 and 20% of couples have difficulty conceiving; failures of the reproductive system thus affect many individuals. In females, understanding the control of ovulation is critical for helping infertility patients conceive single vs multiple births while minimizing side effects. Psychosocial stress interferes with homeostasis and disrupts many physiologic systems including reproduction. Completing this research will help us understand how the brain responds to ovarian estradiol to generate the central neural signal that ultimately leads to ovulation and how stress disrupts this. The signal for ovulation is provided by a change in the output of gonadotropin-releasing hormone (GnRH) neurons from strictly episodic, producing on/off GnRH pulses that drive pituitary hormone release, to a surge mode in which GnRH release is continuously elevated for hours. To induce the GnRH surge, estradiol action switches from negative to positive feedback. Ovariectomized (OVX) mice treated with constant physiologic levels of estradiol (OVX+E) undergo daily shifts from negative to positive feedback that are timed to the light-dark cycle, allowing mechanistic studies of estradiol action in a reduced variable model. In cycling ovary-intact mice, this switch in estradiol feedback mode occurs on proestrus. Our previous work indicates cyclical changes in estradiol induce cycle-dependent changes in the properties of the hypothalamic neurons involved in generating the GnRH surge. This work established several mechanisms engaged by estradiol that would lead to suppression of GnRH neurons during negative feedback and activation of these cells during positive feedback. In the proposed work, we will expand upon this base in experiments that range from continued investigation of neurobiological mechanisms to whole animal studies, all aimed at elucidating estradiol feedback and GnRH surge generation, and the effects of stress on this system. In Aim 1, we will study estradiol modulation of three important circuits: from kisspeptin neurons in the anteroventral periventricular (AVPV) nucleus to GnRH neurons, from arcuate kisspeptin to AVPV kisspeptin neurons, and among AVPV kisspeptin neurons. These experiments will move us towards understanding a more complete reproductive neuroendocrine network by studying how synaptic properties are modulated by estradiol. In Aim 2, we will study the mechanisms by which acute stress exposure in adulthood perturbs the shift from estradiol negative to positive feedback, disrupting the LH surge. We will test involvement of the adrenal glands and of neurons producing corticotropin-releasing hormone (CRH). We will examine how acute stress mechanistically changes estradiol positive feedback effects on GnRH neurons. Finally, we will test how the stress neuroendocrine axis interacts with the reproductive neuroendocrine axis by examining connectivity between CRH and GnRH neurons and CRH and kisspeptin neurons and their modulation by estradiol. These...