Selective optogenetic activation of arcuate kisspeptin neurons generates pulsatile luteinizing hormone secretion

Abstract

Normal reproductive functioning in mammals depends upon gonadotropin-releasing hormone (GnRH) neurons generating a pulsatile pattern of gonadotropin secretion. The neural mechanism underlying the episodic release of GnRH is not known, although recent studies have suggested that the kisspeptin neurons located in the arcuate nucleus (ARN) may be involved. In the present experiments we expressed channelrhodopsin (ChR2) in the ARN kisspeptin population to test directly whether synchronous activation of these neurons would generate pulsatile luteinizing hormone (LH) secretion in vivo. Characterization studies showed that this strategy targeted ChR2 to 70% of all ARN kisspeptin neurons and that, in vitro, these neurons were activated by 473-nm blue light with high fidelity up to 30 Hz. In vivo, the optogenetic activation of ARN kisspeptin neurons at 10 and 20 Hz evoked high amplitude, pulse-like increments in LH secretion in anesthetized male mice. Stimulation at 10 Hz for 2 min was sufficient to generate repetitive LH pulses. In diestrous female mice, only 20-Hz activation generated significant increments in LH secretion. In ovariectomized mice, 5-, 10-, and 20-Hz activation of ARN kisspeptin neurons were all found to evoke LH pulses. Part of the sex difference, but not the gonadal steroid dependence, resulted from differential pituitary sensitivity to GnRH. Experiments in kisspeptin receptor-null mice, showed that kisspeptin was the critical neuropeptide underlying the ability of ARN kisspeptin neurons to generate LH pulses. Together these data demonstrate that synchronized activation of the ARN kisspeptin neuronal population generates pulses of LH.GnRH | kisspeptin | optogenetics | arcuate nucleus | gonadal steroids

Defining Subpopulations of Arcuate Nucleus GABA Neurons in Male, Female, and Prenatally Androgenized Female Mice

“…Kisspeptin is a potent and critical regulator of GnRH neurons [41] , and there is an ongoing hypothesis that the ARN kisspeptin population is involved in mediating gonadal steroid hormone negative feedback to GnRH neurons [42] . While there is no question that ARN kisspeptin neurons are involved in driving GnRH/LH release [43] , the present data suggest that ARN kisspeptin neurons are almost entirely distinct from the previously reported ARN GABA neurons that send dense, plastic projections to the GnRH neuron cell bodies [15] . This is in line with evidence demonstrating that ARN kisspeptin neurons do not project to GnRH neuron cell bodies in the rostral forebrain, and are more likely to regulate GnRH neurons at dendronic or terminal levels [44] .…”

AgRP to Kiss1 neuron signaling links nutritional state and fertility

“…In females, low or depleted estrogen levels evoke transcriptional activation of Kiss1 ARH neurons (7)(8)(9), and stimulation of genetically defined Kiss1 ARH neurons in vivo is sufficient to evoke gonadotropin release (10). During starvation, gonadal steroid levels plummet, yet Kiss1 ARH neurons are unable to drive gonadotropin release.…”

Definition of the hypothalamic GnRH pulse generator in mice

“…Immunofluorescence was undertaken as reported previously (24). Coronal brain sections (30 μm thick) covering the full length of the ARN were used to determine the locations of optic fibers and/or processed for dual-label analysis.…”

“…We have previously demonstrated that optogenetic activation of ARN KISS neurons for >2 min at 10 Hz generates pulse-like increments in LH secretion in anesthetized mice (24). However, the fiber photometry studies above clearly demonstrate that ARN KISS neuron calcium events last for only ∼1 min.…”

“…Channelrhodopsin was targeted to ARN KISS neurons by unilateral injection of AAV9-EF1-dflox-hChR2-mCherry into the ARN of intact male and female KISS1-Cre mice (24). Intact mice exhibited an LH pulse every 90 to 180 min (12,23), thereby providing a long window of basal LH secretion for optogenetic stimulation experiments.…”