Cellular and molecular bases for rhythmic GnRH release

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Reproductive health is a window to overall health. Fifteen to 20% of couples have difficulty conceiving; failures of the reproductive system thus affect a substantial population. Beyond fertility, sex steroids alter development and function of many systems, for example, bone, the central nervous system and the cardiovascular system. Episodic release of gonadotropin-releasing hormone (GnRH) is required for fertility in vertebrates of both sexes, and shifts in frequency of these pulses are essential for female reproductive cycles. The goal of this proposal is to increase our fundamental understanding of the generation of episodic release of GnRH as a critical link to understanding and modulating fertility. The proposed work will define intrinsic properties of GnRH neurons, local network interactions, and roles of glia and postulated driver neurons located in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B and dynorphin (KNDy neurons). Our working hypothesis is that intrinsic and network mechanisms interact in local circuits to produce the final episodic output of the GnRH neurosecretory system. We will study this system using state-of-the-art electrophysiological, imaging and chemogenetic approaches combined with local measures of GnRH release in transgenic mice in which specific neurons are identified by expression of fluorescent reporter proteins. We will also extend our studies with mathematical modeling to generate hypotheses and return these hypotheses to the lab for testing. We have two specific aims. In Aim 1, we will examine how autocrine and paracrine factors from GnRH neurons and glia alter the relationship among action potentials, intracellular calcium and GnRH release, as well as how steroid feedback in males and females modifies these relationships. We will apply this knowledge to study how GnRH neurons are coordinated to produce pulses. We hypothesize factors from these circuit elements (GnRH neurons, afferent neurons, and glia) are needed for pulse generation. We will use pharmacologic and chemogenetic approaches to study the specific roles of these elements in organizing the GnRH network. In Aim 2, we will characterize intrinsic properties and synaptic input to GnRH neurons in intact vs. castrate mice of both sexes. We will use these data to move beyond independent characterization of isolated parameters and directly study how synaptic transmission interacts with intrinsic properties of GnRH neurons to alter action potential generation. Dynamic clamp is a method that allows these interactions to be studied. This is accomplished by careful modeling of these conductances, which can then be added to and/or subtracted from cells during recording. This occurs in real time through iterative interaction with the cell’s membrane potential, so that voltage-dependent changes in conductance are precisely controlled, and effects of the conductances on the membrane potential and firing of the cell are recorded. By examining pulse generation from these two angles, an integrated picture will emerge that adds to our understanding of this phenomenon. All necessary animal models and methods are in place to complete these studies.