The important role of kisspeptin neurons in the regulation of GnRH neuron activity is now well accepted. However, the ways in which kisspeptin neurons located in the arcuate nucleus (ARN) and rostral periventricular area of the third ventricle (RP3V) control GnRH neurons are poorly understood. The present study used anterograde and retrograde tracing techniques to establish the neuronal projection patterns of kisspeptin cell populations in the female mouse brain. Anterograde tracing studies revealed that kisspeptin neurons in the ARN innervated a wide number of hypothalamic and associated limbic region nuclei, whereas RP3V kisspeptin neurons projected to a smaller number of mostly medially located hypothalamic nuclei. Retrograde tracing confirmed a major projection of RP3V kisspeptin neurons to the ARN and showed that kisspeptin neurons located in the rostral half of the ARN projected to the rostral preoptic area. Peripheral administration of Fluorogold was found to label the majority of GnRH neurons but no kisspeptin neurons. Together, these studies highlight the complexity of the brain kisspeptin neuronal system and indicate that both ARN and RP3V kisspeptin neurons participate in a variety of limbic functions. In relation to the GnRH neuronal network, these investigations demonstrate that, alongside the RP3V kisspeptin cells, rostral ARN kisspeptin neurons may also project to GnRH neuron cell bodies. However, no kisspeptin neurons innervate GnRH nerve terminals in the external layer of the median eminence. These studies provide a neuroanatomical framework for the further elucidation of the functions of the ARN and RP3V kisspeptin neuron populations.

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The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic ‘KNDy’ neurons co-express kisspeptin, neurokinin B (NKB), and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close, non-synaptic appositions with the GnRH neuron dendron. Electrophysiology and confocal GCaMP6 imaging demonstrated that, despite all three neuropeptides being released from KNDy terminals, only kisspeptin was able to activate the GnRH neuron dendron. Mice with a selective deletion of kisspeptin from KNDy neurons failed to exhibit pulsatile hormone secretion but maintained synchronized episodic KNDy neuron behavior that is thought to depend on recurrent NKB and dynorphin transmission. This indicates that KNDy neurons drive episodic hormone secretion through highly redundant neuropeptide co-transmission orchestrated by differential post-synaptic neuropeptide receptor expression at the GnRH neuron dendron and KNDy neuron.

Kisspeptins are a family of overlapping neuropeptides encoded by the Kiss1 gene that regulate the mammalian reproductive axis by a central action in the hypothalamus to stimulate GnRH release. Kisspeptins and their receptor (GPR54 also called KISS1R) are also expressed in the testes but a functional role in this tissue has not been confirmed. We examined which cell types in the testes expressed kisspeptin and its receptor by staining for β-galactosidase activity using tissue from transgenic mice with LacZ targeted to either the Kiss1 or the Gpr54 genes. Expression of both genes appeared to be restricted to haploid spermatids and this was confirmed by a temporal expression analysis, which showed expression appearing with the first wave of haploid spermatid cells at puberty. We could not detect any kisspeptin protein in spermatids however, suggesting that the Kiss1 mRNA may be translationally repressed. We tested whether kisspeptin could act on Leydig cells by examining the effects of kisspeptin on the immortalized Leydig cell line MA-10. Although MA-10 cells were shown to express Gpr54 by RT-PCR, they did not respond to kisspeptin stimulation. We also tested whether kisspeptin could stimulate testosterone release by a direct action on the testes using explants of seminiferous tubules. The explants did not show any response to kisspeptin. The functional integrity of the MA-10 cells and the seminiferous tubule explants was confirmed by showing appropriate responses to the LH analog, human chorionic gonadotropin. These data suggest that kisspeptin signaling does not have a significant role in testes function in the mouse.