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Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

ABSTRACT: 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. To better understand these mechanisms, we start by perturbing the KNDy system at different stages of the estrous cycle using optogenetics. We find that optogenetic stimulation of KNDy neurons stimulates pulsatile GnRH/LH secretion in estrous mice but inhibits it in diestrous mice. These in vivo results in combination with mathematical modelling suggest that the transition between estrus and diestrus is underpinned by well-orchestrated changes in neuropeptide signalling and in the excitability of the KNDy population controlled via glutamate signalling. Guided by model predictions, we show that blocking glutamate signalling 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 pulse generator dynamics. Finally, our results could inform useful repurposing of drugs targeting the glutamate system in reproductive therapy.

SUBMITTER: Voliotis M

PROVIDER: S-EPMC8651288 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.

Project description:KEY POINTS:Neurons in the hypothalamus of the brain which secrete the peptide kisspeptin are important regulators of reproduction, and normal reproductive development. Electrical activity, in the form of action potentials, or spikes, leads to secretion of peptides and neurotransmitters, influencing the activity of downstream neurons; in kisspeptin neurons, this activity is highly irregular, but the mechanism of this is not known. In this study, we show that irregularity depends on the presence of a particular type of potassium ion channel in the membrane, which opens transiently in response to electrical excitation. The results contribute to understanding how kisspeptin neurons generate and time their membrane potential spikes, and how reliable this process is. Improved understanding of the activity of kisspeptin neurons, and how it shapes their secretion of peptides, is expected to lead to better treatment for reproductive dysfunction and disorders of reproductive development. ABSTRACT:Kisspeptin neurons in the hypothalamus are critically involved in reproductive function, via their effect on GnRH neuron activity and consequent gonadotropin release. Kisspeptin neurons show an intrinsic irregularity of firing, but the mechanism of this remains unclear. To address this, we carried out targeted whole-cell patch-clamp recordings of kisspeptin neurons in the arcuate nucleus (Kiss1Arc ), in brain slices isolated from adult male Kiss-Cre:tdTomato mice. Cells fired irregularly in response to constant current stimuli, with a wide range of spike time variability, and prominent subthreshold voltage fluctuations. In voltage clamp, both a persistent sodium (NaP) current and a fast transient (A-type) potassium current were apparent, activating at potentials just below the threshold for spiking. These currents have also previously been described in irregular-spiking cortical interneurons, in which the A-type current, mediated by Kv4 channels, interacts with NaP current to generate complex dynamics of the membrane potential, and irregular firing. In Kiss1Arc neurons, A-type current was blocked by phrixotoxin, a specific blocker of Kv4.2/4.3 channels, and consistent expression of Kv4.2 transcripts was detected by single-cell RT-PCR. In addition, firing irregularity was correlated to the density of A-type current in the membrane. Using conductance injection, we demonstrated that adding Kv4-like potassium conductance (gKv4 ) to a cell produces a striking increase in firing irregularity, and excitability is reduced, while subtracting gKv4 has the opposite effects. Thus, we propose that Kv4 interacting dynamically with NaP is a key determinant of the irregular firing behaviour of Kiss1Arc neurons, shaping their physiological function in gonadotropin release.

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Modulation of pulsatile GnRH dynamics across the ovarian cycle via changes in the network excitability and basal activity of the arcuate kisspeptin network.

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