Hypothalamic supramammillary neurons that project to the medial septum modulate wakefulness in mice

The hypothalamic supramammillary nucleus (SuM) plays a crucial role in controlling wakefulness, but the downstream target regions participating in this control process remain unknown. Here, using circuit-specific fiber photometry and single-neuron electrophysiology together with electroencephalogram, electromyogram and behavioral recordings, we find that approximately half of SuM neurons that project to the medial septum (MS) are wake-active. Optogenetic stimulation of axonal terminals of SuM-MS projection induces a rapid and reliable transition to wakefulness from non-rapid-eye movement or rapid-eye movement sleep, and chemogenetic activation of SuMMS projecting neurons significantly increases wakefulness time and prolongs latency to sleep. Consistently, chemogenetically inhibiting these neurons significantly reduces wakefulness time and latency to sleep. Therefore, these results identify the MS as a functional downstream target of SuM and provide evidence for the modulation of wakefulness by this hypothalamic-septal projection.

1.The supramammillary region (SuM) neurons projecting to the medial septum (MS)(SuM-to-MS neurons) increased activities more during wakefulness and REM sleep than NREM sleep, using fiberphotometry Ca+2-imaging.2. These neurons were classified into two types: wake-active neurons and REM-sleep active neurons.a. Wake-active neurons: Those increased firing rates at the transition from NREM/REM sleep to wakefulness and decreased firing rates at the transition from wakefulness to NREM sleep.b.REM-sleep active neurons: The other increased firing rates at the NREM-to-REM sleep transition and decreased at the REM sleep-to-wakefulness transition.3. Optogenetic activation of both types of SuM-to-MS neurons induced wakefulness transitioned from the NREM or REM state in a frequency dependent manner.4. Similarly, chemogenetic activation of these neurons during the light phase prolonged wakefulness and shortened NREM and REM sleep. 5. Conversely, chemogenetic inactivation of these neurons increased NREM sleep and reduced wakefulness time.
Overall Evaluation: The study provides valuable data for understanding SuM neuron functions, but improvements are needed for more comprehensive understanding: 1. Clarify questionable data analyses (see below); 2. Provide nuanced interpretation (see below).

Specific Comments:
Lines 36-38: The authors concluded "Therefore, these results identify the MS as a functional downstream target of SuM and provide evidence for a causal role for this hypothalamic-septal projection in wakefulness control".This conclusion is too strong given the results.If the inhibition of SuM neurons prevents animals from transitioning from sleep to wakefulness, I support that the conclusion.But the present study did not provide such evidence.Therefore, it is more adequate to characterize that SuM neuron activity modulates wakefulness.Similarly, it should be avoided to conclude that SuM-to-MS neurons play an essential role in promoting wakefulness (lines 235-237), as it is unclear how essential those neurons are.
Lines 79-81: Although the authors state "the specific cell types of SuM neurons projecting to MS . . .have not been well characterized", there is previous work that has investigated the issue as much as the present study did.Kesner et al. (Nat Commun 2021, 12, 2811) showed that SuM glutamatergic neurons projects to the MS and excite MS neurons in a glutamate receptor dependent manner.Therefore, this statement is a mischaracterization of the previous work.
Lines 211-216: Please confirm if CNO was administered at 8 am, the onset of the light phase.
Lines 220-224: Clarify the methodology used for photostimulation to inhibit SuM-to-MS neurons.Describe the duration and timing of stimulation in relation to the light-dark cycle.
Line 316: Clarify the use of Wilcoxon signed-rank tests for Fig. 2i.This test is inappropriate for comparing three groups and doesn't account for multiple comparisons.
Lines 339-342: The use of paired t-tests for Fig. 3j and 3k is inappropriate due to multiple comparisons.Consider using one-way ANOVAs, as there are six groups in each EEG frequency analysis.
Reviewer #2 (Remarks to the Author): Liang et al reported that approximately half of the SuM neurons projecting to the medial septum (MS) exhibit wake-promoting activity.Optogenetic stimulation of the axonal terminals originating from SuM-MS projection elicits a prompt and dependable transition from non-rapid-eye movement (NREM) or rapid eye movement (REM) sleep to wakefulness.Furthermore, chemogenetic activation of SuM-MS projecting neurons significantly augments the overall duration of wakefulness and extends the latency period preceding sleep onset.Correspondingly, chemogenetic inhibition of these neurons leads to a substantial reduction in wakefulness duration and diminishes the latency to sleep onset.The author took several advanced technology to prove the SuM-MS projection play a causal role for wakefulness control.The data are very solid.I just have few minor questions: 1.The author found that there are both wakefulness-related neurons and REMrelated neurons in SuM.In this study, the author mainly focused on the neural correlates of arousal.
Have the authors paid attention to REM-related neurons?How might these two types of neurons interact with each other?No more experiment is required, but the author should discuss it in detail.2. The authors found that the projection of SuM-MS controls arousal, but did not discuss the specific role of this projection in the whole sleep-wake regulation system.Why does the regulation of wake need this projection?
1.The author found that there are both wakefulness-related neurons and REM-related neurons in SuM.In this study, the author mainly focused on the neural correlates of arousal.Have the authors paid attention to REM-related neurons?How might these two types of neurons interact with each other?No more experiment is required, but the author should discuss it in detail.Thanks a lot for this important suggestion.It is possible that these REM-active SuM MS projecting neurons might participate in certain REM sleep-related functions, such as memory consolidation or cortical plasticity.New experiments, such as projection-specific manipulation and electrophysiological recording, are needed to investigate the interaction of wake-active and REM-active SuM MS projecting neurons in future.We have discussed it in the discussion part.
2. The authors found that the projection of SuM-MS controls arousal, but did not discuss the specific role of this projection in the whole sleep-wake regulation system.Why does the regulation of wake need this projection?
We now included the discussion of the possible circuit mechanisms of SuM-MS projection in arousal control.SuM receives inputs from arousal-related brain regions, including lateral hypothalamus, basal forebrain, locus coeruleus, and dorsal raphe.These arousal-related brain regions may active SuM-MS projection, and then active MS Vglut2 neurons by releasing glutamate.And the MS Vglut2 neurons have been reported to control wakefulness by activating lateral hypothalamus glutamatergic neurons (An et al., 2021).Therefore, this SuM-recruited circuit, acts as one part of the whole sleep-wake regulation system, may support locomotion or memory encoding or wakefulness-related behaviors (Farrell et al., 2021; Chen et al., 2020).