Abstract
KNDy neurons are named for their co-expression of three neuropeptides, kisspeptin, neurokinin B and dynorphin A (Dyn A). These cells, located in the hypothalamic arcuate nucleus (ARC), are assumed to be the generator of GnRH/LH pulses that control follicular growth, spermatogenesis and steroidogenesis. It is essential to clarify the mechanisms that regulate GnRH pulse generator to understand the reproductive functions in mammals. However, the inhibitory mechanism of GnRH pulse generator by Dyn A is unclear including the location of κ-opioid receptors (KOR; the receptor for Dyn A) in the hypothalamus. To elucidate the mechanism, we analyzed the localization and projections of KOR neurons in rats. We used a lentiviral vector that contains GFP and wheat germ agglutinin (WGA) connected with picornaviral 2A peptide, which are driven by KOR promoter, to visualize KOR neurons. This lentiviral vector was administrated into the region close to the arcuate nucleus (ARC) and ventromedial hypothalamic nucleus (VMH) in female rats. Two weeks after the injection, the rats were sacrificed to analyze the localization and projections of KOR neurons by in situ hybridization (ISH) and immunohistochemistry (IHC). GFP was strongly expressed in cells in the VMH. Double staining of KOR ISH and GFP IHC revealed that all GFP-positive cells were Oprk1 (KOR gene) positive in the VMH, demonstrating that we succeeded to obtain animals that specifically express GFP in KOR neurons. Double staining of GFP and kisspeptin IHC detected the axons of kisspeptin neurons near the cell body of GFP-positive neurons. Likewise, axons of GFP-positive neurons were observed near the Kiss1-positive cells by double staining of GFP IHC and Kiss1 (kisspeptin gene) ISH. In addition, we found axons expressing WGA in the ARC by IHC. Taken together, we found the localization of KOR neurons in the VMH and projections of KOR neurons to KNDy neurons and those of KNDy neurons to KOR neurons as well. These results suggest that KOR neurons in the VMH, which receive Dyn A signal from KNDy neurons, directly inhibit the activity of KNDy neurons. WGA-positive axons in the ARC imply the existence of unknown neurons mediating signals of KOR neurons to KNDy neurons. Our rats fluorescently labeled with KOR neurons will be useful to analyze detailed characteristics of KOR neurons, which will make major progress in elucidating the inhibitory mechanism of reproductive function.