Analyzing the endogenous calcium oscilllation of kisspeptin neurons and its effect on GnRH neurons

Abstract

Kisspeptin in the hypothalamus acts as an important regulator of mammalian reproduction by synchronizing gonadotropin-releasing hormone (GnRH) pulse generator. Mainly located in the arcuate nucleus (ARC) and the anteroventral periventricular nucleus in the hypothalamus, kisspeptin neurons are poised to generate GnRH pulse and surge. Our group previously reported that intermittent administration of kisspeptin elicits synchronization of GnRH promoter activity and pulsatile secretion of GnRH. However, the driving force which produces pulsatile kisspeptin output still remains unknown. We hypothesized that kisspeptin neurons would sustain their own rhythm, with information received from neurotransmitter or hormonal inputs integrated. To address the hypothesis, we analyzed endogenous intracellular calcium dynamics from ARC kisspeptin neurons ex vivo. Subsequently, we imaged fluorescent and luminescent signals from organotypic slice culture of Kiss1-IRES-Cre;GnRHp-dsLuc mouse transduced with adenoassociated virus encoding RGECO, a genetically encoded calcium indicator. Calcium oscillation in ARC kisspeptin neurons was highly synchronized and appeared to have 240~300s period, which was abolished by IP3 receptor blocker and induced by neurokinin B agonist. Analysis of kisspeptin neurons’ endogenous calcium oscillation and their regulatory factors would elucidate the mechanism underlying pulsatile kisspeptin secretion. Further, monitoring their effects on GnRH neurons would help understand the neural network regulating GnRH pulse generator.