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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Evaluation Summary:
This manuscript is of considerable interest to neuroendocrinologists and other neuroscientists because it provides important insights into mechanisms controlling the synchronous activity of a specific subpopulation of hypothalamic neurons. Luteinizing hormone is secreted from the pituitary gland in pulses which vary over the estrous cycle. The pulses arise by patterned secretion of a hypothalamic 'releasing factor', the secretion of which is itself governed by a population of hypothalamic neurons that express the neuropeptide kisspeptin. The paper by Voliotis et al. combines novel experimental evidence from transgenic mice with an elegant mathematical model to analyze how the kisspeptin neurons generate the varying pulsatile patterns.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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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.
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Evaluation Summary:
This manuscript is of considerable interest to neuroendocrinologists and other neuroscientists because it provides important insights into mechanisms controlling the synchronous activity of a specific subpopulation of hypothalamic neurons. Luteinizing hormone is secreted from the pituitary gland in pulses which vary over the estrous cycle. The pulses arise by patterned secretion of a hypothalamic 'releasing factor', the secretion of which is itself governed by a population of hypothalamic neurons that express the neuropeptide kisspeptin. The paper by Voliotis et al. combines novel experimental evidence from transgenic mice with an elegant mathematical model to analyze how the kisspeptin neurons generate the varying pulsatile patterns.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
This manuscript is of considerable interest to neuroendocrinologists and other neuroscientists because it provides important insights into mechanisms controlling the synchronous activity of a specific subpopulation of hypothalamic neurons. Luteinizing hormone is secreted from the pituitary gland in pulses which vary over the estrous cycle. The pulses arise by patterned secretion of a hypothalamic 'releasing factor', the secretion of which is itself governed by a population of hypothalamic neurons that express the neuropeptide kisspeptin. The paper by Voliotis et al. combines novel experimental evidence from transgenic mice with an elegant mathematical model to analyze how the kisspeptin neurons generate the varying pulsatile patterns.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In previous work these authors developed a mathematical model for the pulsatile activity of KNDy neurons responsible for episodic GnRH and LH secretion. Here they use this model and experimental manipulations to test the hypothesis that the KNDy neural network responds differently to optogenetic stimulation on different days of the estrous cycle and that these changes reflect changes in the excitability of this population driven by glutamate signaling. In the first experiment, they demonstrate that optogenetic stimulation increases episodic LH secretion on estrous when endogenous activity is low and inhibits LH pulse frequency on diestrus when endogenous activity is elevated. In the second experiment, they show that pharmacological inhibition of glutamate signaling blocks the stimulatory actions of …
Reviewer #1 (Public Review):
In previous work these authors developed a mathematical model for the pulsatile activity of KNDy neurons responsible for episodic GnRH and LH secretion. Here they use this model and experimental manipulations to test the hypothesis that the KNDy neural network responds differently to optogenetic stimulation on different days of the estrous cycle and that these changes reflect changes in the excitability of this population driven by glutamate signaling. In the first experiment, they demonstrate that optogenetic stimulation increases episodic LH secretion on estrous when endogenous activity is low and inhibits LH pulse frequency on diestrus when endogenous activity is elevated. In the second experiment, they show that pharmacological inhibition of glutamate signaling blocks the stimulatory actions of optogenetic stimulation on estrous and inhibits endogenous LH pulses on diestrus. Morever, the latter effect is partially overcome with optogentic stimulation. The modelling portion of the manuscript provides a simple explanation for these observations and supports the hypothesis that glutamate signaling that increases the network excitability of this population plays an important role in the changes between diestrus and estrus.
Overall, this work has been carefully done and the overall conclusions are consistent with the experimental observations and model. I do have three concerns about the interpretation and discussion of these results. First, although the key characteristic of the proposed model (that there is a limited range of excitability levels compatible with the episodic activity of the KNDy network) provides an elegant explanation for previous work describing apparent paradoxical effects of many neurotransmitters on LH pulses, it also complicates the design of experiments to test it. Thus, if a pharmacological manipulation will either decrease or increase episodic LH secretion depending on the endogenous activity of the network, which may be difficult to determine, then any experimental result is consistent with the model. This caveat appears to be inherent to their model, but it may be prudent to soften the conclusions in light of it. For example, any pharmacological manipulation that affects the level of excitability of the network might have similar effects as the blockade of glutamatergic transmission reported here. Thus, the data are consistent with the proposed role of glutamatergic input to KNDy neurons, but they do not establish that this is the only, or even the most important, input. Second, how do the authors reconcile the positive correlation of NKB and dynorphin signaling with most data indicating that these two neuropeptides usually have opposite effects on episodic LH secretion. Further consideration of these potentially conflicting data and the implications of this positive correlation to the functioning of the KNDy network would strengthen the Discussion. Finally, the discussion on changes in the characteristics of KNDy neurons on different days of the estrous cycle focuses exclusively on the effects of estrogen on this neural network. However, the relevance to these estrogen effects to the changes reported between diestrus and estrus is unclear because the KNDy neural network (based on bursts of calcium and LH pulse frequencies) does not change from diestrus though proestus when estradiol concentrations are increasing from a nadir to peak levels. Since, as pointed out in the Introduction, the low LH pulse frequency on estrus is caused by the proestrous increase in progesterone concentrations some consideration of this steroid should be included in the Discussion.
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Reviewer #2 (Public Review):
The paper by Voliotis et al. combines novel experimental evidence with mathematical modelling. The experiments were designed to analyse the effects of activating the kisspeptin population in the arcuate nucleus on the generation of pulsatile LH secretion at different phases of the ovarian cycle, and the involvement in this of glutamate signalling. Experimental data come from transgenic mice engineered to express Cre in kisspeptin-expressing cells that had been injected stereotaxically in the arcuate nucleus (bilaterally) with an adenoviral vector to transduce expression of channelrhodopsin in arcuate Kisspeptin neurones. The authors measured luteinising hormone (LH) in small blood samples taken from the tail tips of conscious mice while these kisspeptin cells were activated optogenetically using an implanted …
Reviewer #2 (Public Review):
The paper by Voliotis et al. combines novel experimental evidence with mathematical modelling. The experiments were designed to analyse the effects of activating the kisspeptin population in the arcuate nucleus on the generation of pulsatile LH secretion at different phases of the ovarian cycle, and the involvement in this of glutamate signalling. Experimental data come from transgenic mice engineered to express Cre in kisspeptin-expressing cells that had been injected stereotaxically in the arcuate nucleus (bilaterally) with an adenoviral vector to transduce expression of channelrhodopsin in arcuate Kisspeptin neurones. The authors measured luteinising hormone (LH) in small blood samples taken from the tail tips of conscious mice while these kisspeptin cells were activated optogenetically using an implanted optic fibre. Experiments were performed during icv administration of glutamate antagonists or aCSF.
The experiments are a methodological tour de force, necessitating stress-free sampling and stimulation procedures and precise targeting of adenoviral injections. The design of the experiments is elegant, inspired by a simple mathematical model that generates a concise simulation of the role of kisspeptin neurones in the generation of pulsatile LH secretion.
Kisspeptin neurones co-express two other neuropeptides, dynorphyn and neurokinin, leading them to be known by the acronym KNDy neurones, and these are thought to influence LH secretion by the actions of these peptides on neurones that release GnRH. This paper reproduces previous work by the same authors in showing that optogenetic stimulation of KNDy neurons stimulates pulsatile LH secretion in estrous mice but inhibits it in diestrous mice.
These results are consistent a mathematical model which proposes that that the transition between estrus and diestrus reflects orchestrated changes in the excitability of the KNDy population. On the premise that the excitability of the KNDy cells is primarily controlled by glutamate neurotransmission either between the KNDy cells or from external inputs, the present study studied the consequences of blocking glutamate signaling. Blocking glutamate signalling in diestrous animals inhibited LH pulses, and this inhibition could be mitigated by optogenetic stimulation of the KNDy cells. In estrous mice, blocking glutamate signalling inhibited the pulsatile LH secretion generated by optogenetic stimulation of the KNDy cells.
The model characterises the KNDy cells as a single element that secretes dynorphin and neurokinin independently, in a manner that varies during the ovarian cycle. These two secreted products decay at different rates, and have different (autocrine) feedback effects on the KNdy cells: neurokinin is excitatory and decays rapidly; dynorphin is inhibitory, is secreted at a lower rate but decays more slowly, so accumulates. These characteristics establish a bistable oscillatory system, that oscillates at a frequency determined mainly by the time constants of degradation.
I like the model, it is very simple yet capable of elegantly explaining apparently complex behaviour. However, it seems to me that the assumptions that underlie the model should be explicitly stated and the relevant evidence supporting them clearly stated.
In L209 the authors state "In the equations above neuronal activity stimulates secretion of both neuropeptides, and Dyn represses NKB secretion."
Thus one key assumption is that dynorphin and neurokinin secretion are independent: this seems to require either that they are packaged in separate neurosecretory vesicles that are subject to independent regulation, or that they are predominantly expressed in mainly separate (and functionally distinct) populations of kisspeptin neurones in the arcuate nucleus. I am unsure how tenable either of these assumptions is. I don't mind if they are bold and speculative, but they should be clearly recognised is such. (Key questions appear to be whether dynorphin, NK and kisspeptin are contained in the same or separate vesicles, whether their synthesis is differentially regulated through the estrus cycle, and if their secretion from different compartments is regulated differently).
2. The model collapses two very different roles of glutamate neurotransmission into a single rather vaguely defined variable. The authors recognise that glutamate is likely to be the major excitatory transmitter arising from external inputs that may perhaps be presumed to establish a constant excitatory 'tone.' Understanding 'excitability' as the strength of a tonic input seems reasonable. However, the authors also recognise that glutamate signaling between KNDy neurones might be important in synchronising neural activity in a way important for pulse generation. This seems to be a very different role, a role that is not incorporated in the model.
It seems to me therefore that the experiments don't really test the model predictions because they have a major effect on something not included in the model that would reasonably be expected to have a substantial effect on the way the system behaves.
Nevertheless, this work is an imprtant step forward. It is highly original; its importance in my view is in establishing a clear framework for understanding the function of a complex neuronal system that plays a critically important role in reproduction, and in providing an exemplar of how simple mathematical models can inspire the generation of experimentally testable hypotheses by proposing simple explanations of apparently complex behaviours. The authors have made the raw data from their experiments fully and openly available, as I have checked for myself.
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