Estrogen-regulated lateral septal kisspeptin neurons abundantly project to GnRH neurons and the hypothalamic supramammillary nucleus.

While hypothalamic kisspeptin (KP) neurons play well-established roles in the estrogen-dependent regulation of reproduction, little is known about extrahypothalamic KP-producing (KPLS) neurons of the lateral septum. As established previously, Kiss1 expression in this region is low and regulated by estrogen receptor- and GABAB receptor-dependent mechanisms. Our present experiments on Kiss1-Cre/ZsGreen knock-in mice revealed that transgene expression in the LS begins at P33-36 in females and P40-45 in males and is stimulated by estrogen receptor signaling. Fluorescent cell numbers continue to increase in adulthood and, regardless of age, remain higher in females. Viral tracing uncovered that the bulk of KPLS fibers joins the medial forebrain bundle and terminates in the hypothalamic supramammillary nucleus. Smaller subsets of the axons innervate the medial amygdala or project to other limbic structures. One-quarter of gonadotropin-releasing hormone-immunoreactive perikarya in the preoptic area and their dendrites receive appositions from KPLS axons. Ovariectomized adult Kiss1-Cre/ZsGreen mice treated for 4 days with 17β-estradiol or vehicle were used for RNA-Seq studies of laser-microdissected KPLS neurons. The transcriptome included markers of GABAergic and neuropeptidergic (Penk, Cartpt, Vgf) cotransmission and 571 estrogen-regulated transcripts. Estrogen treatment upregulated the acetylcholine receptor transcript Chrm2 and, in slice electrophysiology experiments, caused enhanced muscarinic inhibition of KPLS neurons. Finally, we provided immunohistochemical evidence for homologous neurons in the post mortem human brain, suggesting that KPLS neurons may contribute to evolutionarily conserved regulatory mechanisms. Future studies will need to investigate the putative roles of KPLS neurons in the estrogen-dependent control of gonadotropin-releasing hormone neurons and/or various hypothalamic/limbic functions.Significance Statement Gonadotropin-releasing hormone (GnRH) neurons of the hypothalamus stimulate pituitary gonadotropin secretion to promote gonadal functions. Sex steroids of the gonads serve as feedback signals to control the hypothalamic output of GnRH. According to current consensus, steroid feedback is mainly sensed and communicated to the GnRH neuronal system by sex steroid-sensitive hypothalamic cell groups through the neuropeptide kisspeptin.Anatomical, developmental, spatial transcriptomic and electrophysiological characterization of an extrahypothalamic kisspeptin system in this study unveils the previously unexplored contribution of lateral septal kisspeptin cells to the sex-steroid dependent afferent control of GnRH neurons. Identification of analogous kisspeptin neurons in the post mortem human brain suggests that this estrogen dependent system may play an evolutionarily conserved role in the central regulation of reproduction.