Dysfunctions of the paraventricular hypothalamic nucleus induce hypersomnia in mice

Essential revisions:

1) The authors need to delineate the potential role of changes in endocrine and autonomic function underlie the change in wakefulness.

As requested, we did more experiments and added a supplement figure. Please see Figure 3—figure supplement 2.

In order to delineate the potential changes in autonomic function, we measured the heart rate and temperature of PVH^vglut2-M3 mice after treatment with vehicle or CNO by implantable telemetry devices. We found that chemogenetic activation of PVH^vglut2 neurons induced an increase in heart rate and temperature for 3 h, which both peaked at about 2 h after injection and returned to baseline level at about 3 h following administration of CNO. As for the endocrine function, we detected serum corticotropin release factor (CRF) and corticosterone (CORT) levels in PVH^vglut2-M3 mice after treatment with vehicle or CNO. As shown in Figure supplement 3C, chemogenetic activation of PVH^vglut2 neurons significantly increased CRF level at 2 h, 3 h and 4 h timepoints after injection of CNO, while the CORT level was only higher than that of vehicle group at 3 h timepoint following administration.

Glucocorticoid hormones (CORT for mouse) are the effector hormones of the hypothalamic-pituitary-adrenal (HPA) axis neuroendocrine system, which are regulated by CRF (secreted in the medial parvocellular portion of the PVH) and produce direct negative feedback inhibition of CRF neurons in the PVH [1] (Robert LSpencer, et al., 2017, Figure 1A). Our results showed that although chemogenetic activation of PVH^vglut2 neurons induced higher CRH levels, it did not cause a continuous high level of CORT. These results suggested that changes in glucocorticoid levels indeed occurred but did not last for 9 h, so the long-lasting effects of wakefulness induced by PVH glutaminergic neurons’ activation could only partly be explained by endocrine and autonomic function changes; the wake-promoting neural circuits of PVH^vglut2 neurons played a more important role in the 9 h of wakefulness.

2) The inclusion of the human data needs to be further developed or justified. It may improve the manuscript by removing it.

We greatly appreciate your suggestion. We deleted the human data in the revised manuscript.

3) More evidence for selective ablation of vglut2-positive neurons is needed.

As requested, we supplemented evidence for selective ablation of vglut2-positive neurons by in situ hybridization. Please refer to the answer for Reviewer #3, Question #1.

4) More detail regarding the statistical analyses and power calculations are required.

We confirmed the number of animals and statistics in each experiment and revised figure legends according to the eLife’s format requirements.

5) The photomicrographs of the anterograde tracing are of poor quality and need to be improved.

As requested, we replaced high-resolution images of anterograde tracing in the revised manuscript. Please see Figure 1 in the revised manuscript.

Reviewer #1:

The authors should address/discuss why single injection of CNO into mice expressing Dq DREADD in the PVH (Figure 4C and Fig5I) can affect sleep up to ~9 hours.

Thank you for your suggestion. First, given that PVH glutaminergic neurons are largely co-expressed with corticotropin-releasing hormone (CRH), prodynorphin (PDYN) and oxytocin (OT) neurons, which are all wake-promoting neurons, the 9-h of wakefulness induced by activation of PVH^vglut2 neurons partly results from activation of these types of neurons. Furthermore, the PVH sends direct projections to the PB, and it was reported that chemogenetic activation of the PB produced ~11 h of continuous wakefulness in rats [2, 3]. Furthermore, as we mentioned in response to Essential Revisions 1, the change of endocrine and autonomic function may also contribute to the long-time wakefulness. All these have been added to the discussion in revised manuscript. Please see pages 11-12, lines 286-302.

In the mouse studies, the methods appear to be solid. The used small volumes but it is unclear how injection sites were assessed in each case. Also, it is not clear when unilateral and bilateral injections were performed. More detail is required.

Thank you for your suggestion. According to our previous studies [4-7], after finishing all related experiments, each mouse was anesthetized with chloral hydrate (360 mg/kg) and then perfused intracardially with 30 mL phosphate-buffered saline (PBS) followed by 30 mL 4% paraformaldehyde (PFA). Their brains were removed and postfixed in 4% PFA overnight and then incubated in 30% sucrose phosphate buffer at 4°C until they sank. Coronal sections (30 μm) were cut on a freezing microtome (CM1950, Leica, Germany), and the fluorescence of injection sites was checked with the location of the PVH according to the histology atlas of Paxinos and Franklin (2001, The Mouse Brain in Stereotaxic Coordinates 2nd edn [San Diego, CA: Academic]) using a microscope (Fluoview 1200, Olympus, Japan). Method details were added in the revised manuscript. Please see page 19, lines 485-493.

Unilateral injections were performed in in vivo fiber photometry (AAV-DIO-GCamp6f), anterograde tracing (AAV-DIO-eGFP), while bilateral injections were used in chemogenetic and optogenetic manipulation (AAV-DIO-ChR2, AAV-DIO-hM3Dq, AAV-DIO-hM4Di) and ablation experiments (AAV-DIO-Caspase3). We added these in the revised manuscript. Please see page 14, lines 350-353.

Reviewer #2:

First a general comment, I'm missing in the Results section the number of animals and the statistics in the text. As it is, it is very descriptive and difficult to figure out the power of the results.

Thank you for your comments. We added the number of animals and statistics in each experiment and revised figure legends according to the eLife’s format requirements.

The neuroanatomical retrograde tracing analysis is very limited and in fact does not bring much to the demonstration. They conclude line 128-129 that the fact that the neurons receive inputs from the PVT, PB, ZI and VLPAG suggest that the PVH might act as key central node for sleep-wake regulation. I found this statement rather weak since so many structures project to the PVH that it is not that surprising that some could be interesting for waking. Further, the involvement in Wake of the mentioned structures is not that well demonstrated. I would prefer a more detailed description of the afferents and to couple it with cFos to determine whether they are wake active. It can be left in the paper as it is but it does not bring crucial information.

Thank you for your suggestions. We found that PVH^vglut2 neurons received direct inputs from many brain regions (Figure 1E and Figure supplement 1). Among them, the PVT expressed a higher level of c-fos during the active period (24:00) than the inactive period (12:00), and activation of PVT^vglut2 neurons induced rapid transitions from sleep to wakefulness [8]. Similarly, chemogenetic activation of PB neurons induced increased expression of c-fos and a potent wakefulness lasting about 9 hours [2]. In contrast, ablation of PB^vglut2 neurons induced hypersomnia-like behaviors [9].

More importantly, we observed higher c-fos expression in the PVH during the active period (23:00) than during the inactive period (11:00) (Figure 1A, B). Therefore, we speculated that PVH^vglut2 neurons might act as a central node for sleep–wake regulation.

I wonder why they choose to study only two efferent structures and not others? Do they believe that only the projections to the given structures is inducing Waking?

Thank you for your insightful questions. The reason for choosing these two efferent structures is that PVH^vglut2 neurons mainly projected to four neuroanatomical sites related to sleep–wake regulation: the PB, LSv, PVT and NTS (Figure 5—figure supplement 1C and Figure 5—table supplement 1). The PVT, PB and NTS have been demonstrated to be essential in controlling wakefulness [2, 3, 8] and the PB innervates PVH^vglut2 neurons to form bidirectional connections. In addition, the PVH^vglut2→LSv pathway is involved in the regulation of feeding, which is a wakefulness-required behavior[10]. Therefore, we explored these four pathways to identify the neuronal circuits mediating the wake-promoting effect of PVH^vglut2 neurons and found that optical stimulation of PVH^vglut2→PB and PVH^vglut2→LSv promoted wakefulness (Figure 5) while activation of PVH^vglut2→NTS or PVH^vglut2→PVT pathways did not alter sleep–wake states (Figure 5—figure supplementary 2).

I'm missing information on whether the three neuropeptides populations of neurons colocalize or not? This is not clear to me? It would be great to know whether each of them are subtypes of glutamatergic PVH neurons.

Thank you for your essential suggestions. The distributions of neuropeptides populations in the PVH have been reported in previous research [11], in which Xu et al., observed marker gene enrichment and pairwise gene co-expression in the PVH regions (Figure S5, Xu et al., 2020). According to the results, almost 100% of neurons expressing CRH, PDYN and OXT co-expressed with Vglut2 in the middle PVH (mPVH) and posterior PVH (pPVH), which were also the main distribution regions of these three neuropeptides populations in the PVH (Figure 2B, Xu et al., 2020).

What is missing a bit in the unit recordings is a more detailed analysis on the discharge pattern during Waking. Indeed, it would be helpful to know whether the PVH Glu neurons change their activity during different behaviors to more deeply understand their function.

Thank you for your insightful questions. In the near future, we would like to analyze them to explore other function of PVH^vglut2 neurons.

The photomicrographs shown for the anterograde tracing are of poor quality and it is difficult to figure out where we are in the brains. The drawings help but are not sufficient to show the location of the fibers. I would favor photomicrographs in which landmarks are visible. There is also no quantification there and it is missing. I wonder also whether there is a strong projection to the median eminence since these neurons are mainly known for their neuroendocrine function? I would like the authors to comment on such role of these neurons in the Discussion.

Thank you for your insightful questions. We have supplemented high-resolution images of anterograde tracing in the revised manuscript. As requested, we provided complementary quantification (“+” represents the fluorescence intensity of axon terminal from PVH^vglut2 neurons). Additionally, we found that PVH^vglut2 neurons project to the median eminence, please see Figure 5—figure supplement 1C and Figure 5—table supplement 1. Related discussion has been added in the revised manuscript (pages 12, lines 303-306).

I never heard of the dorsal terminal nucleus. This does not seem the common name of this region. Please explain.

Sorry for the mistake, it should be “nucleus of the solitary tract”. We corrected all related statements in the revised manuscript.

Reviewer #3:

I have one major comment:

Evidence for selective ablation of vglut2-positive neurons (e.g. vglut2 in-situ hybridization) in figure 6B should be shown. NeuN staining is not enough.

We greatly appreciate your suggestion. As requested, we supplemented evidence for selective ablation of vglut2-positive neurons by in-situ hybridization. Please refer to the Figure 7E.

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