Highly redundant neuropeptide volume co-transmission underlying episodic activation of the GnRH neuron dendron
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Abstract
The necessity and functional significance of neurotransmitter co-transmission remains unclear. The glutamatergic “KNDy” neurons co-express kisspeptin, neurokinin B (NKB) and dynorphin and exhibit a highly stereotyped synchronized behavior that reads out to the gonadotropin-releasing hormone (GnRH) neuron dendrons to drive episodic hormone secretion. Using expansion microscopy, we show that KNDy neurons make abundant close but non-synaptic appositions with the GnRH neuron dendron. Confocal GCaMP6 calcium imaging demonstrated that, of the neurotransmitters co-expressed by KNDy neurons, only kisspeptin was able to activate the GnRH neuron dendron. The selective deletion of kisspeptin from KNDy neurons resulted in mice in which the synchronized behavior of the KNDy neurons was maintained but their ability to drive episodic hormone secretion was abolished. This indicates that KNDy neurons drive episodic hormone secretion through converse modes of highly redundant neuropeptide co-transmission orchestrated by differential postsynaptic neuropeptide receptor expression at their two target sites.
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###Reviewer #3:
Authors aim to test the presence and functional significance of KNDy co-transmission at the GnRH distal dendrites in the ventrolateral ARN. The authors use expansion microscopy to score synaptic connections between KNDy and GnRH distal dendrites. Next, they use ex-vivo slice imaging to report the Ca2+ transients of GnRH distal dendrons during pipette application of candidate neurotransmitters. The authors go on to investigate the functional role of kisspeptin on the pulsatile firing of KNDy neurons and the subsequent release of LH using a combination of fiber photometry and repeated blood sampling. This manuscript is a continuation of a large body of work from this laboratory. Most of the techniques used here have been previously published by this group and are at the cutting edge of this research field. As a reviewer I …
###Reviewer #3:
Authors aim to test the presence and functional significance of KNDy co-transmission at the GnRH distal dendrites in the ventrolateral ARN. The authors use expansion microscopy to score synaptic connections between KNDy and GnRH distal dendrites. Next, they use ex-vivo slice imaging to report the Ca2+ transients of GnRH distal dendrons during pipette application of candidate neurotransmitters. The authors go on to investigate the functional role of kisspeptin on the pulsatile firing of KNDy neurons and the subsequent release of LH using a combination of fiber photometry and repeated blood sampling. This manuscript is a continuation of a large body of work from this laboratory. Most of the techniques used here have been previously published by this group and are at the cutting edge of this research field. As a reviewer I have two points for the authors to consider:
In 2016 Qi, Nestor et al. evaluated the mechanistic properties of synchronous firing of KNDy neurons. Along with this, they demonstrated that the influence of NKB on GnRH neurons was indirect and mediated by kisspeptin from KNDy neurons. Given this, I think it is important for the authors to more specifically compare and contrast the work from Qui, Nestor et al. 2016. While the authors do cite the manuscript, the findings are not thoroughly compared.
The authors show that NKB was sufficient to induce [Ca2+] in KNDy neurons, but not in GnRH dendrons. Given this, I found it curious that a delayed, indirect, spike was not observed in (Fig 2 A,B) from KNDy induction. Can the authors clarify this?
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###Reviewer #2:
In this manuscript Liu and co-workers use in vitro and in vivo experiments to explore KNDy neuronal input onto GnRH nerve-fibers (called dendrons) in the arcuate nucleus median eminence area. The main strength of this work is the in vivo photometry experiment to activate ARN Kiss1 neurons combined with tail blood sampling for measurements of plasma LH as a substitute for GnRH secretion. It is well known that Kiss1 deletion causes infertility. In addition, it is known that in some Kiss1Cre mouse models homozygous animals are designed to be infertile, including the mouse model used in the current study.
Using the infertile homozygous Kiss1Cre mouse, the authors showed that the lack of kisspeptin eliminates LH pulses following photometry stimulation in vivo of KNDy neurons, indicating that kisspeptin is responsible for LH …
###Reviewer #2:
In this manuscript Liu and co-workers use in vitro and in vivo experiments to explore KNDy neuronal input onto GnRH nerve-fibers (called dendrons) in the arcuate nucleus median eminence area. The main strength of this work is the in vivo photometry experiment to activate ARN Kiss1 neurons combined with tail blood sampling for measurements of plasma LH as a substitute for GnRH secretion. It is well known that Kiss1 deletion causes infertility. In addition, it is known that in some Kiss1Cre mouse models homozygous animals are designed to be infertile, including the mouse model used in the current study.
Using the infertile homozygous Kiss1Cre mouse, the authors showed that the lack of kisspeptin eliminates LH pulses following photometry stimulation in vivo of KNDy neurons, indicating that kisspeptin is responsible for LH pulses and is the main output signal from KNDy neurons onto GnRH terminals in the ME area. They also used this animal model to show that the absence of kisspeptin did not affect the synchronous firing of KNDy neurons, illustrating that kisspeptin is not involved in synchronous firing and that synchronous firing alone does not maintain fertility. However, previous studies both in vivo (Wakabayashi et al., 2010) and in vitro (Navarro et al., 2009, Qiu et al., 2016) had already provided substantial evidence for kisspeptin being the main output signal onto GnRH neurons and that NKB and dynorphin are responsible for synchronous firing.
It is interesting that although KNDy neurons release the peptides kisspeptin, NKB and dynorphin as well as the classical neurotransmitter glutamate, only kisspeptin was able to activate GnRH dendrons in the ME area. This is surprising since this group has shown previously (Herde et al 2013) that both GABA and glutamate can depolarize GnRH distal dendrons. Specifically, they showed that puff application of glutamate (500 µM) on distal dendrons in vitro elicited bursts of action potentials. Currently, the authors used a similar concentration of glutamate applied in vitro and found no effect on Dendron calcium activity. Clearly further experiments are needed to sort out these differences. Overall, although this manuscript reports some compelling in vivo studies to ascertain the specific role of kisspeptin in the GnRH distal Dendron and confirm the role of NKB and dynorphin on synchronous firing, it is of limited scope and new information.
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###Reviewer #1:
The authors of this high-quality paper use contemporary viral/genetic technologies to show that KNDy neurons in the ARN regulate GnRH release in median eminence (ME) via kisspeptin signaling only, even though they release all their transmitters there. They monitor GCaMP fluorescence in GnRH dendrons to establish that kisspeptin signals there, but NKB, Dyn and GLU do not, whereas these 3 transmitters signal onto Kiss1-neuron cell bodies, while kisspeptin does not. They also show that loss of kisspeptin signaling in ME prevents LH release.
Fig. 6A Authors should compare dF/F trace of Kiss1-Cre -/- with +/- mice, rather than referring to unpublished results.
Line 337, Authors say, "As such, it is interesting to consider whether the episodic release of NKB and dynorphin from KNDy varicosities in the region of the …
###Reviewer #1:
The authors of this high-quality paper use contemporary viral/genetic technologies to show that KNDy neurons in the ARN regulate GnRH release in median eminence (ME) via kisspeptin signaling only, even though they release all their transmitters there. They monitor GCaMP fluorescence in GnRH dendrons to establish that kisspeptin signals there, but NKB, Dyn and GLU do not, whereas these 3 transmitters signal onto Kiss1-neuron cell bodies, while kisspeptin does not. They also show that loss of kisspeptin signaling in ME prevents LH release.
Fig. 6A Authors should compare dF/F trace of Kiss1-Cre -/- with +/- mice, rather than referring to unpublished results.
Line 337, Authors say, "As such, it is interesting to consider whether the episodic release of NKB and dynorphin from KNDy varicosities in the region of the ventrolateral ARN may impact on other ARN neuronal cell types." It is equally interesting to consider the possibility that KNDy neurons release all their neurotransmitters in the ME and NKB, Dyn and Glu may signal to non-GnRH neurons. It would be useful to include references documenting that NKB, Dyn and GLU are released in ME, even if kisspeptin is the only molecule that can signal to GnRH dendrons. If references do not exist, would it be possible to express GCcMP6 non-specifically ME and express ChR2 in Kiss1-Cre-/- KNDy neurons to show that cells in ME can respond to the other transmitters released by KNDy-neuron activation. Antagonists could then be used to establish which transmitters are released there.
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##Preprint Review
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###Summary:
The importance of kisspeptin signaling from arcuate KNDy neurons (expressing kisspeptin, neurokinin B, dynorphin and glutamate) for fertility is well established. KNDy neurons are thought to be critical for the episodic release of LH by acting on GnRH-neuron terminals in the median eminence. A question posed here is whether kisspeptin is the only transmitter signaling onto GnRH terminals (referred to here as dendrons) in the median eminence. Some evidence suggests that the KNDy neuropeptides can be packaged …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary:
The importance of kisspeptin signaling from arcuate KNDy neurons (expressing kisspeptin, neurokinin B, dynorphin and glutamate) for fertility is well established. KNDy neurons are thought to be critical for the episodic release of LH by acting on GnRH-neuron terminals in the median eminence. A question posed here is whether kisspeptin is the only transmitter signaling onto GnRH terminals (referred to here as dendrons) in the median eminence. Some evidence suggests that the KNDy neuropeptides can be packaged into individual vesicles; thus, it is possible that only those vesicles containing kisspeptin travel to the median eminence. Alternatively, it is possible that all peptides and glutamate are released in the median eminence, but only receptors for kisspeptin are present there. To address this issue, the authors express a calcium indicator in GnRH dendrons and determine which transmitters can generate a calcium signal. They show that only kisspeptin can do so and go on to demonstrate that in the absence of kisspeptin (using KO mice), no signal is generated. This is an important result but does not completely distinguish between the two hypotheses.
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