The circadian clock is a fundamental regulator of many aspects of physiology and behavior, including reproduction. Reproductive success depends on appropriate daily timing of neuroendocrine events that control ovulation. Kisspeptin (Kiss1) neurons in the preoptic area (POA) of the hypothalamus play a critical role in this by driving the activity of downstream gonadotropin-releasing hormone (GnRH) neurons to generate the surge in GnRH and LH secretion that triggers ovulation. The surge in rodents is timed by the central circadian clock in the suprachiasmatic nucleus (SCN) to initiate just before the onset of diurnal activity, ensuring that ovulation, which occurs a few hours later, coincides with sexual behavior. Projections from the SCN provide timing signals to the GnRH neuronal network, including to POA Kiss1 neurons. Indeed, reports indicate that arginine vasopressin (AVP)-expressing SCN neurons may play a key role in daily timing of the surge by activating POA Kiss1 neurons. Our prior published studies provide evidence that SCN projections release AVP to stimulate POA Kiss1 neuron electrical activity, and that this circuit is most effective in driving Kiss1 neuron activity on the day the surge occurs. Recently, we have obtained exciting preliminary data that indicate that a distinct SCN population releases GABA and inhibits Kiss1 neuron activity. These new observations, along with our published work, reveal that SCN neurons may bidirectionally control the electrical activity of Kiss1 neurons, through the release of GABA and AVP. This has led us to hypothesize that a shift in the balance of SCN-derived AVP- mediated excitation and GABA-mediated inhibition contributes to gating the activation of POA Kiss1 neurons for the surge. We will employ a combination of anatomical and functional approaches to address this central hypothesis. Our first aim will be to establish that SCN neurons directly project to and release GABA on POA Kiss1 neurons using brain slice electrophysiology and optogenetics. Further, we will determine the functional impact of GABA release on Kiss1 neuron electrical activity across the estrous cycle. In the second aim, we will use tract-tracing and immunohistochemical approaches to establish that SCN neuron projections target those POA Kiss1 cells that are involved in the surge and determine the identity of the cells that contribute these projections as well as their activation patterns prior to the surge. In our third aim, we will first assess the electrical activity of SCN neuronal populations in the hours that precede the preovulatory surge. Using this information, we will then determine how Kiss1 neurons integrate SCN timing signals, mediated through GABA and AVP release, on the day of the surge. Together, this research will provide new information about the circadian control of reproduction, and specifically the daily timing of the neuroendocrine events that trigger ovulation. A better understanding of the neural mechanisms responsible for circadian regulation of these circuits under physiological conditions may open new avenues for potential future treatments of ovulatory dysfunction.

NARRATIVE Circadian timing of the preovulatory surge in GnRH and LH secretion is critical in regulating ovulation and reproductive success in rodents as well as, possibly, in women. This proposal will provide new knowledge of the neural circuits that govern the circadian activation of kisspeptin neurons – a cell population essential for the preovulatory surge under physiological conditions. This research, when completed, may provide the foundation for future opportunities in treating ovulatory dysfunction.