The preoptic Kisspeptin/nNOS/GnRH (KiNG) neuronal network regulates rhythmic LH release through a dual activation-inhibition mechanism

Gonadotropin-releasing hormone (GnRH) neurons are the final common target of a complex network of cells cooperating for the central control of reproduction. The balance between excitatory and inhibitory transsynaptic and non-synaptic inputs is crucial for the maintenance of the GnRH rhythms: the pulse and the surge. The precise mechanisms behind this remain under debate. In this work, we challenge the hypothesis that excitatory and inhibitory inputs from kisspeptin and neuronal nitric oxide (NO) synthase (nNOS)-expressing neurons orchestrates GnRH release, in a microcircuit that we call the Kisspeptin/nNOS/GnRH (KiNG) neuronal network. Our work specifically focuses on the role of nNOS neurons located in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MePO). nNOS and kisspeptin neurons interact anatomically and functionally, with the kisspeptin receptor (Kiss1r) being differentially regulated in nNOS-expressing neurons across the female estrous cycle. Using a novel viral tool allowing for the measurement of NO/cGMP levels with exquisite sensitivity, we demonstrate that kisspeptin is able to induce NO-dependent cGMP production in the OV/MePO, including in GnRH neurons in vivo. Using electrophysiological, genetic, chemogenetic and pharmacologic approaches, we reveal that NO production from nNOS neurons in the OV/MePO is needed to fine-tune the GnRH/LH response to kisspeptin, and specifically to turn off GnRH release, thus generating pulses. Our findings provide valuable insights into the tripartite KiNG neuronal network governing the regulation of the GnRH/LH pulse and surge.