Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the longelusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.

Psychological stress is linked to infertility by suppressing the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centres, however, their functional significance has not been determined. We hypothesis that MePD GABAergic signalling mediates psychological stress-induced suppression of pulsatile luteinising hormone (LH) secretion. We selectively inhibited MePD GABA neurones during psychological stress in ovariectomised (OVX) Vgat-cre-tdTomato mice, to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurones were virally infected with inhibitory hM4DGi-DREADDs to selectively inhibit MePD GABA neurones. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurones in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fibre implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurones blocked predator odour and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20 Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress-induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator.

Reproduction critically depends on the pulsatile secretion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. This ultradian rhythm drives the secretion of gonadotrophic hormones (LH and FSH) from the pituitary gland, which are critical for gametogenesis and ovulation, and its frequency is regulated throughout the life course to maintain normal reproductive health. However, the precise mechanisms controlling the pulsatile GnRH dynamics are unknown. Here, we propose and study a novel mathematical model of a population of neurones in the arcuate nucleus (ARC) of the hypothalamus that co-expresses three key modulators of GnRH secretion: kisspeptin; neurokinin B (NKB); and dynorphin (Dyn). The model highlights that positive feedback in the population exerted by NKB and negative feedback mediated by Dyn are the two key components of the pulse generator, which operates as a relaxation oscillator.Furthermore, we use the model to study how external inputs modulate the frequency of 1 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/245548 doi: bioRxiv preprint first posted online Jan. 12, 2018; the pulse generator, a prediction that can be readily tested in-vivo using optogeneticallydriven stimulation. Finally, our model predicts the response of the system to various neuropharmacological perturbations and reconciles inconsistent experimental observations following such interventions in-vivo. We anticipate that our model in combination with cutting-edge, in-vivo techniques, allowing for neuronal stimulation and recording, will set the stage for a quantitative, system-level understanding of the GnRH pulse generator.

Pulsatile GnRH release is essential for normal reproductive function. Kisspeptin secreting neurons found in the arcuate nucleus, known as KNDy neurons for co-expressing neurokinin B, and dynorphin, drive pulsatile GnRH release. Furthermore, gonadal steroids regulate GnRH pulsatile dynamics across the ovarian cycle by altering KNDy neurons' signalling properties. However, the precise mechanism of regulation remains mostly unknown. Here we investigate these mechanisms using a combination of mathematical and in-vivo approaches. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. Our mathematical modelling suggests that this differential effect is due to well-orchestrated changes in neuropeptide signalling and the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling in the arcuate nucleus in diestrous animals inhibits LH pulses, and that optic stimulation of the KNDy population mitigates this inhibition. In estrous mice, disruption of glutamate signalling inhibits pulses generated via sustained low-frequency optic stimulation of the KNDy population, supporting the idea that the level of network excitability is critical for pulse generation. Our results reconcile previous puzzling findings regarding the estradiol-dependent effect that several neuromodulators have on the GnRH pulse generator dynamics. Therefore, we anticipate our model to be a cornerstone for a more quantitative understanding of the pathways via which gonadal steroids regulate GnRH secretion dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

There is compelling evidence that head injury is a significant environmental risk factor for Alzheimer’s disease (AD) and that a history of traumatic brain injury (TBI) accelerates the onset of AD. Amyloid-β plaques and tau aggregates have been observed in the post-mortem brains of TBI patients; however, the mechanisms leading to AD neuropathology in TBI are still unknown. In this study, we hypothesized that focal TBI induces changes in miRNA expression in and around affected areas, resulting in the altered expression of genes involved in neurodegeneration and AD pathology. For this purpose, we performed a miRNA array in extracts from rats subjected to experimental TBI, using the controlled cortical impact (CCI) model. In and around the contusion, we observed alterations of miRNAs associated with dementia/AD, compared to the contralateral side. Specifically, the expression of miR-9 was significantly upregulated, while miR-29b, miR-34a, miR-106b, miR-181a and miR-107 were downregulated. Via qPCR, we confirmed these results in an additional group of injured rats when compared to naïve animals. Interestingly, the changes in those miRNAs were concomitant with alterations in the gene expression of mRNAs involved in amyloid generation and tau pathology, such as β-APP cleaving enzyme (BACE1) and Glycogen synthase-3-β (GSK3β). In addition increased levels of neuroinflammatory markers (TNF-α), glial activation, neuronal loss, and tau phosphorylation were observed in pericontusional areas. Therefore, our results suggest that the secondary injury cascade in TBI affects miRNAs regulating the expression of genes involved in AD dementia.