Homeostatic regulation of REM sleep by the preoptic area of the hypothalamus

Reviewer #1 (Public Review):

Summary:
This paper identifies GABA cells in the preoptic hypothalamus which are involved in REM sleep rebound (the increase in REM sleep) after selective REM sleep deprivation. By calcium photometry, these cells are most active during REM, and show more claim signals during REM deprivation, suggesting they respond to "REM pressure". Inhibiting these cells ontogenetically diminishes REM sleep. The optogenetic and photometry work is carried out to a high standard, the paper is well-written, and the findings are interesting.

Points that could be addressed or discussed:
1. The circuit mechanism for REM rebound is not defined. How do the authors see REM rebound as working from the POAGAD2 cells? Although the POAGAD2 does project to the TMN, the actual REM rebound could be mediated by a projection of these cells elsewhere. This could be discussed.

2. The "POAGAD2 to TMN" name for these cells is somewhat confusing. The authors chose this name because they approach the POAGAD2 cells via retrograde AAV labelling (rAAV injected into the TMN). However, the name also seems to imply that neurons (perhaps histamine neurons) in the TMN are involved in the REM rebound, but there is no evidence in the paper that this is the case. Although it is nice to see from the photometry studies that the histamine cells are selectively more active (as expected) in NREM sleep (Fig. S2), I could not logically see how this was a relevant finding to REM rebound or the subject of the paper. There are many other types of cells in the TMN area, not just histamine cells, so are the authors suggesting that these non-histamine cells in the TMN could be involved?

3. It is a puzzle why most of the neurons in the POA seem to have their highest activity in REM, as also found by Miracca et al 2022, yet presumably some of these cells are going to be involved in NREM sleep as well. Could the same POAGAD2-TMN cells identified by the authors also be involved in inducing NREM sleep-inhibiting histamine neurons (Chung et al). And some of these POA cells will also be involved in NREM sleep homeostasis (e.g. Ma et al Curr Biol)? Is NREM sleep rebound necessary before getting REM sleep rebound? Indeed, can these two things (NREM and REM sleep rebound) be separated?

4. Is it possible to narrow down the POA area where the GAD2 cells are located more precisely?

5. It would be ideal to further characterize these particular GAD2 cells by RT-PCR or RNA seq. Which other markers do they express?

Reviewer #2 (Public Review):

Maurer et al investigated the contribution of GAD2+ neurons in the preoptic area (POA), projecting to the tuberomammillary nucleus (TMN), to REM sleep regulation. They applied an elegant design to monitor and manipulate the activity of this specific group of neurons: a GAD2-Cre mouse, injected with retrograde AAV constructs in the TMN, thereby presumably only targeting GAD2+ cells projecting to the TMN. Using this set-up in combination with technically challenging techniques including EEG with photometry and REM sleep deprivation, the authors found that this cell-type studied becomes active shortly (≈40sec) prior to entering REM sleep and remains active during REM sleep. Moreover, optogenetic inhibition of GAD2+ cells inhibits REM sleep by a third and also impairs the rebound in REM sleep in the following hour. Despite a few reservations or details that would benefit from further clarification (outlined below), the data makes a convincing case for the role of GAD2+ neurons in the POA projecting to the TMN in REM sleep regulation.

The authors found that optogenetic inhibition of GAD2+ cells suppressed REM sleep in the hour following the inhibition (e.g. Fig2 and Fig4). If the authors have the data available, it would be important to include the subsequent hours in the rebound time (e.g. from ZT8.5 to ZT24) to test whether REM sleep rebound remains impaired, or recovers, albeit with a delay.

REM sleep is under tight circadian control (e.g. Wurts et al., 2000 in rats; Dijk, Czeisler 1995 in humans). To contextualize the results, it would be important to mention that it is not clear if the role of the manipulated neurons in REM sleep regulation hold at other circadian times of the day.

The effect size of the REM sleep deprivation using the vibrating motor method is unclear. In FigS4-D, the experimental mice reduce their REM sleep to 3% whereas the control mice spend 6% in REM sleep. In Fig4, mice are either subjected to REM sleep deprivation with the vibrating motor (controls), or REM sleep deprivations + optogenetics (experimental mice). The control mice (vibrating motor) in Fig4 spend 6% of their time in REM sleep, which is double the amount of REM sleep compared to the mice receiving the same treatment in FigS4-D. Can the authors clarify the origin of this difference in the text?