New Insights in the synchronisation dynamics of the KNDy neural network shaping reproduction

Context Reproduction is essential for human and animal survival yet is exquisitely sensitive to perturbations. Reproductive problems, including stress-related infertility, have enormous socioeconomic impact and understanding underlying mechanisms will facilitate innovative treatments and enhance wellbeing. Challenge Successful reproduction requires normal function of a neural oscillator, the gonadotrophin-releasing-hormone (GnRH) pulse generator, which drives pulsatile release of gonadotrophic-hormones (LH/FSH) to control ovulation and spermatogenesis. This oscillator comprises hypothalamic Kisspeptin neurones co-expressing Neurokinin-B (NKB) and Dynorphin (acronym-KNDy). Despite recent insights into the KNDy neural network, major questions remain unanswered. How do NKB and dynorphin, and intrinsic-glutamatergic neurotransmission collectively orchestrate the intermittent synchronisation of KNDy neurones which underlie GnRH pulsatility? How does stress suppress this synchronisation to cause infertility? Our preliminary work using real-time monitoring of single-KNDy neurones in-situ has revealed for the first-time a complex repertoire of mini-synchronised events (mSEs; synchronisation between 2-or-more neurones) building up to the full-blown SEs characteristic of LH pulses. Through computational modelling we have begun to uncover the precise role of the key neurotransmitters (NKB, dynorphin and glutamate) in orchestrating the emergence of these mSEs that somehow contrive to facilitate synchronisation, as well as understanding how this system collapses under stress. This insight provides a unique opportunity to establish how individual KNDy neurones entrain and synchronise to produce the dynamic SE architecture underlying normal-reproduction and stress-related conditions. To further scrutinise the system, we will integrate our in-silico modelling of the biophysical mechanisms with cutting-edge in-vivo GRadient-INdex-(GRIN)-lens miniendoscopy and electrophysiology, and use the latest in-vivo CRISPR-Cas9-gene-editing methods to selectively knockdown glutamate, NKB, or dynorphin expression in KNDy neurones. Aims and Objectives Hypothesis: Synchronisation in the KNDy neural network is a dynamic phenomenon driven by mini-synchronisation events (mSEs) involving intricate interactions between glutamate, neurokinin-B, and dynorphin signalling, which collectively orchestrate GnRH pulse generation required for successful reproduction. These mSEs vary across the ovarian-cycle influencing the network’s predisposition to synchronisation, while stress-related inputs disrupt synchronisation patterns, dampen pulse generation and impact on fertility. Objective-1: Using in-vivo GRIN-lens and electrophysiological data from single-KNDy neurones across the female ovarian-cycle and in males, to develop a mathematical framework to capture the transient dynamics leading to synchronisation in the KNDy-network, and predict the balance of neuropeptide and neurotransmitter signalling in the synchronisation process. Objective-2: Using CRISPR-Cas9 mutagenesis, and leveraging in-silico insights from Objective-1, to interrogate the integrative role of neurokinin-B, dynorphin and glutamate transmission in synchronising the KNDy-network to initiate and sustain pulsatile dynamics of the GnRH pulse generator. Objective-3: Using in-vivo and in-silico approaches, to develop a quantitative understanding of how stress-activated external inputs to the KNDy-network of female and male mice influence single-neurone and network activity, leading to dysregulation of GnRH pulse generator frequency. Beneficiaries Outcomes from this research will characterise mechanisms fundamental to the control of the GnRH pulse generator. Primarily the livestock industry is a likely beneficiary through improved fertility, quality and productivity. Also, millions of people may benefit from development of novel-therapeutics for infertility, and other reproductive disorders including PCOS and delayed puberty. Academics, clinicians and industry also stand to gain, as our approach aligns with the BBSRC Strategic Delivery Plan objectives “Capacity and Capability in Core-Bioscience Disciplines” by combining in-silico modelling and experimental science, “Understanding the rules-of-life”, relating to human and animal health and wellbeing, and “Enabling world-leading bioscience, and its transition into economic and societal benefits”.