Kisspeptin Neurones do not Directly Signal to RFRP‐3 Neurones but RFRP‐3 may Directly Modulate a Subset of Hypothalamic Kisspeptin Cells in Mice

Abstract

The neuropeptides kisspeptin (encoded by Kiss1) and RFamide-related peptide-3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP-3 might act directly on each other’s neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP-3 systems, we performed double label in-situ hybridization (ISH) for RFRP-3’s receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurons of adult male and female mice, as well as double-label ISH for kisspeptin’s receptor, Kiss1r, in Rfrp-expressing neurons of the hypothalamic dorsal-medial nucleus (DMN). Only a very small proportion (5–10%) of Kiss1 neurons of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co-expression (~25%) existed in Kiss1 neurons in the arcuate nucleus. Thus, RFRP-3 could signal to a small, primarily arcuate, subset of Kiss1 neurons, a conclusion supported by the finding of ~35% of arcuate kisspeptin cells receiving RFRP-3--immunoreactive fibre contacts. In contrast to the former situation, no Rfrp neurons co-expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co-expressed with arcuate kisspeptin neurons) was found in <10% of Rfrp neurons. Moreover, kisspeptin-immunoreactive fibres did not readily appose RFRP-3 cells in either sex, further excluding the likelihood that kisspeptin neurons directly communicate to RFRP-3 neurons. Lastly, despite abundant NKB in the DMN region where RFRP-3 soma reside, NKB was not co-expressed in the majority of Rfrp neurons. Our results suggest that RFRP-3 may modulate a small proportion of kisspeptin-producing neurons in mice, particularly in the arcuate nucleus, whereas kisspeptin neurons are unlikely to have any direct reciprocal actions on RFRP-3 neurons.

RFamide-related peptide-3 (RFRP-3) suppresses sexual maturation in a eusocial mammal

“…In tandem with an unprecedented level of RFRP-3 control over pubertal activation, we also document the presence of RFRP-3-producing neurons in the Arc, a chief site of kisspeptin synthesis in other mammalian species (65). In contrast to the small proportion of Kiss1/GPR147 coexpressing cells in other areas of the rodent brain, 25% of kisspeptin somata in the mouse Arc express GPR147 or GPR74, whereas 35% receive contact from RFRP fibers (66). Further, RFRP KO mice show higher numbers of Kiss1 positive neurons and increased total Kiss1 gene expression, specifically in the Arc (35).…”

Daily successive changes in reproductive gene expression and neuronal activation in the brains of pubertal female mice

“…Strikingly, we found that both the number of Rfrp neurons and total Rfrp mRNA dropped significantly, by 30% and 40%, respectively, between PND 15 and PND 21, after which they stabilized at adult levels. This large decrease in Rfrp expression over the course of a few days may point to this particular peripubertal stage as a critical period for a reduction of RFRP-3-mediated inhibition of GnRH or kisspeptin neurons (Poling et al, 2013). Notably, this drop in Rfrp expression occurs almost a week prior to VO, suggesting that hypothalamic changes in reproductive circuits occur well before external physical signs of pubertal onset.…”

Examination of the Influence of Leptin and Acute Metabolic Challenge on RFRP-3 Neurons of Mice in Development and Adulthood

“…Double-label ISH was performed as previously described (35,36). Briefly, one set of slide-mounted brain sections encompassing the entire DMN were fixed in 4% paraformaldehyde, pretreated with acetic anhydride, rinsed in 2× SSC (sodium citrate, sodium chloride), delipidated in chloroform, dehydrated in ethanols, and air-dried.…”