Abstract
Gonadotrophin-releasing hormone (GnRH) is an integral part of the system regulating sexual development and fertility in mammals. GnRH is secreted in a pulsatile manner from neurones located in the hypothalamus under the control of the neural oscillator known as the hypothalamic GnRH pulse generator, thought to be located in the arcuate nucleus of the hypothalamus. Pulses of GnRH control the secretion of luteinising hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. These hormones, in turn, are responsible for the downstream regulation of gonadal activity, completing the pathway known as the hypothalamic-pituitary-gonadal (HPG) axis.It is not well understood how the activity of the GnRH pulse generator is generated and modulated. It is thought that a population of recently discovered kisspeptin neurones, which co-express the neuropeptides Neurokinin B (NKB) and Dynorphin (Dyn) and are therefore called KNDy neurones, are crucial for hypothalamic regulation of the GnRH pulse generator. Arcuate KNDy neurones have also been shown to be under the influence of estrogenic, glutamatergic and GABAergic signalling. Additionally, recent research suggests that a crucial upstream regulator of the GnRH pulse generator may involve GABAergic neurones in a classical GABA-GABA disinhibitory system in the posterodorsal medial amygdala (MePD) that have projections contacting KNDy neurones in the arcuate nucleus.
Using transgenic cre- mouse models and cutting edge optogenetic stimulation techniques, we set out to elucidate KNDy neurone firing dynamics that dictate the activity of the GnRH pulse generator. Through a dual optogenetic and pharmacology approach we hope to gain a better understanding of the external modulation of the GnRH pulse generator. The downstream effects of manipulation of the GnRH pulse generator will be measured by an ultra-sensitive LH ELISA. Serial blood sampling will allow us to record changes in LH pulse frequency as a proxy for GnRH pulses. Optogenetic and pharmacological manipulation of arcuate KNDy neurones was guided and supported by an innovative mathematical model constructed by Professor K. Tsaneva-Atanasova and Dr. M. Voliotis of The University of Exeter. This model predicts the behaviour of the GnRH pulse generator is based on the crucial parameters of basal activity of the KNDy neurones and rate of secretion of Dyn and NKB. The model assumes that the GnRH pulse generator operates as a relaxation oscillator and predicts the response of the system to external manipulation using optogenetics and neuropharmacological techniques. Using the mathematical model to gain a deeper understanding of the system and interpret our results, we aim to solve long-standing inconsistencies in experimental observations and elucidate core mechanisms of the GnRH pulse generator.
We demonstrated that through optogenetic stimulation of KNDy neurones at specific frequencies in intact female mice to drive their basal activity, the system is sensitive to small yet distinct changes in stimulatory frequency, corresponding to observable and statistically significant changes in LH pulse frequency. These results confirm that ARC KNDy neurones are a critical component of the GnRH pulse generator and through varying levels of basal activity are able to fine-tune GnRH pulse generator frequency. Through interpretation of our results alongside the mathematical model, we present deep insights into the inner mechanisms of the GnRH pulse generator, showing how low frequency optogenetic stimulation at 5Hz drives an increase in basal activity and subsequently GnRH pulse frequency. Further increases in stimulatory frequency and basal activity show that the GnRH pulse generator exists in clearly defined states with upper and lower limits, mathematically deafened as bifurcations, that dictate when the GnRH pulse generator is able to produce pulses. These findings clarify and shed light on previously uncertainties about the GnRH pulse generator. Additionally, we show that glutamate signalling acts as a stimulatory influence to GnRH pulse generator activity, primarily affecting KNDy neurone excitability, with both AMPA and NMDA receptors being crucial in this relationship. Oestrogenic signalling was found to affect the basal activity of the GnRH pulse generator. We demonstrate how KNDy neurone signalling dynamics vary across the estrous cycle, and use the mathematical model to provide a mechanistic explanation of the effects of optogenetic stimulation and resulting changes in LH pulsatility, providing a unique window into GnRH pulse generator modulation. Finally, we demonstrate that arcuate KNDy neurones are under the influence of negative upstream GABAergic modulation that originates in the MePD, providing evidence for our hypothesized GABAergic disinhibitory mechanism. This finding implicates the MePD as an important additional external regulator of the GnRH pulse generator. Surprisingly, we also uncovered an unexpected possible link between GABAergic modulation of KNDy neurones and increased feeding behaviour. These data provide future questions that need to be answered about the role of GABAergic signalling in the ARC. In summary, we provide new insight into the GnRH pulse generator using a novel inter-disciplinary approach and answer crucial questions of a structure that is intrinsic to mammalian sexual development and reproduction.
| Date of Award | 1 May 2022 |
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| Original language | English |
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| Supervisor | Lucilla Poston (Supervisor) & Kevin O'Byrne (Supervisor) |