The amygdala, a key upstream regulator of the hypothalamic GnRH pulse generator

This project provides a unique opportunity to unravel the mechanism by which psychogenic stress impacts on the control of reproductive function in mammals. Reproduction is critically dependent upon a neural oscillator that is responsible for the episodic secretion of pulses of the neurohormone gonadotrophin-releasing hormone (GnRH), that drives the pulsatile secretion of the gonadotrophic hormones, luteinising hormone (LH) and follicle stimulating hormone (FSH). This neural oscillator, known as the GnRH pulse generator, comprises Kisspeptin neurones that co-express the neuropeptides Neurokinin B and Dynorphin A (known as KNDy neurones) in the hypothalamic arcuate nucleus (ARC). The KNDy oscillator provides the essential episodic stimulatory kisspeptin signal to the GnRH neurones. With the very recent neuronal tract-tracing studies mapping the diverse afferent inputs to the KNDy neurones, there is now a substantial international effort to elucidate the pathways conveying metabolic, circadian, and other key homeostatic signals regulating the KNDy neural network. The amygdala, a part of the limbic brain typically associated with emotions and anxiety, has strong projections to the KNDy system. We have shown that kisspeptin signalling in the amygdala robustly regulates the most critical parameter of the GnRH pulse generator, namely its frequency, which is critical for normal follicular development and spermatogenesis. This finding has facilitated a recent surge of interest in the amygdala's control of fundamental reproductive processes, including pubertal timing, ovulation and fertility. We have shown in preliminary studies that amygdala kisspeptin operates through GABA signalling and that psychological stress-induced suppression of GnRH pulse generator frequency is mediated via the stress neuropeptide urocortin 3 in the amygdala.
The present study aims to functionally characterise the neurocircuitry in the amygdala and its projections to the hypothalamic KNDy neural network. We will combine the latest state-of-the-art neuroscience technologies, enabling simultaneous in-vivo optogenetics manipulations and GCaMP gradient-index (GRIN) lens microendoscopic imaging of real time neurone calcium dynamics, with mathematically modelling. This will allow us to simulate system interactions and make predictions, which can then be tested through biological experiments and feed back into the model to develop it further. We will determine the mechanisms by which the projections from the amygdala regulate rhythmicity of the KNDY oscillator under normal and stress conditions.
This project will reveal the mechanism by which the psychogenic stress systems in the amygdala regulate the hypothalamic GnRH pulse generator, thus improving our fundamental understanding of stress-related disorders of fertility and facilitating developments of more effective treatments in humans and animals.

The GnRH pulse generator, the central regulator of the reproduction, comprises KNDy neurones in the hypothalamic arcuate nucleus. Stress suppresses this KNDy neural oscillator, but the underlying mechanisms are not well established. This project will focus on the amygdala, a part of the limbic brain typically associated with emotions and anxiety, which has strong projections to the KNDy system. Our discovery that kisspeptin signalling in the amygdala is an upstream regulator of GnRH pulse generator frequency, has facilitated the recent surge of interest in the amygdala's control of reproduction. We have shown in preliminary studies that amygdala kisspeptin operates through GABAergic signalling and that psychological stress-induced suppression of GnRH pulse generator is mediated by urocortin 3 in the amygdala. Recent developments in intersectional genetically encoded tools have enabled independent manipulation of a least two variables (eg. using Cre- or Flp-dependent constructs) in the same mouse. Capitalising these tools, this project will use simultaneous in-vivo optogenetic manipulation and gradient-index (GRIN) lens microendoscopic monitoring of GCaMP-expressing neurones in combination with mathematical modelling to, (i) examine the functional relationship between kisspeptin, GABA and glutamate neurocircuitry within the amygdala that underlies upstream regulation of hypothalamic GnRH pulse generator frequency, (ii) interrogate how the KNDy oscillator integrates the dynamic tone of inhibitory GABAergic and stimulatory glutamatergic output projections from the amygdala to regulate its rhythmic frequency, and (iii) determine how stress activated urocortin 3 neurones regulate the kisspeptin-GABA/glutamate neurocircuits in the amygdala to suppress GnRH pulse generator frequency. These studies will improve our understanding of how the higher-order limbic brain regulates the hypothalamic GnRH pulse generator that contributes to stress-dependent suppression of fertility.