Differential Role of Pontomedullary Glutamatergic Neuronal Populations in Sleep-Wake Control.

Parafacial zone (PZ) GABAergic neurons play a major role in slow-wave-sleep (SWS), also called non-rapid eye movement (NREM) sleep. The PZ also contains glutamatergic neurons expressing the vesicular transporter for glutamate, isoform 2 (Vglut2). We hypothesized that PZ Vglut2-expressing (PZVglut2) neurons are also involved in sleep control, playing a synergistic role with PZ GABAergic neurons. To test this hypothesis, we specifically activated PZVglut2 neurons using the excitatory chemogenetic receptor hM3Dq. Anatomical inspection of the injection sites revealed hM3Dq transfection in PZ, parabrachial nucleus (PB), sublaterodorsal nucleus (SLD) or various combinations of these three brain areas. Consistent with the known wake- and REM sleep-promoting role of PB and SLD, respectively, chemogenetic activation of PBVglut2 or SLDVglut2 resulted in wake or REM sleep enhancement. Chemogenetic activation of PZVglut2 neurons did not affect sleep-wake phenotype during the mouse active period but increased wakefulness and REM sleep, similar to PBVglut2 and SLDVglut2 activation, during the rest period. To definitively confirm the role of PZVglut2 neurons, we used a specific marker for PZVglut2 neurons, Phox2B. Chemogenetic activation of PZPhox2B neurons did not affect sleep-wake phenotype, indicating that PZ glutamatergic neurons are not sufficient to affect sleep-wake cycle. These results indicate that PZ glutamatergic neurons are not involved in sleep-wake control.


INTRODUCTION
Over the past few years, the medullary parafacial zone (PZ) has been identified as a strong sleeppromoting brain area (Anaclet and Fuller, 2017). Both disruption of PZ GABAergic transmission (Anaclet et al., 2012) and chemogenetic inhibition of PZ GABAergic (PZ GABA ) neurons (Anaclet et al., 2014) result in insomnia. More importantly, chemogenetic activation of PZ GABA neurons strongly increases SWS amount and consolidation and enhances cortical EEG slow-wave activity (SWA), a marker of SWS depth (Anaclet et al., 2014). Finally, chemogenetic activation of PZ GABA neurons counteracts the wake-promoting action of psychostimulants (Anaclet et al., 2018). The PZ is generally located dorsal and lateral from the facial nerve but its exact boundaries are not precisely defined. A recent study has shown that, in mouse, the parvicellular reticular nucleus part alpha (PCRtA), ventral from PZ, does not contain sleep-active neurons (Sakai, 2017), indicating that the PZ sleep promoting neuronal population does not include the PCRtA.
In rats, about 40% of PZ neurons are sleep-active (Alam et al., 2018) and cell body specific PZ lesions result in insomnia (Anaclet et al., 2012). More specifically, PZ GABA are involved in slow-wave-sleep (SWS) control. However, cFos expression, a marker of neuronal activity, showed that about half of sleep-active neurons are GABAergic (Anaclet et al., 2012), indicating that in the PZ, non-GABAergic neurons are also involved in sleep control. Within the PZ, the only other known neuronal population is glutamatergic, expressing the vesicular glutamate transporter isoform 2 (Vglut2; in situ hybridization data are available in Allen Mouse Brain Atlas, Allen Institute for Brain Science 1 ). We hypothesized that PZ glutamatergic neurons (PZ Vglut2 ) are also involved in sleep control and act synergistically with sleep-active PZ GABA neurons to promote SWS. To start testing this hypothesis, we chemogenetically activated PZ Vglut2 neurons. Specific targeting of PZ was challenging and sleep phenotypes were difficult to interpret due to the possible transfection of the neighboring parabrachial (PB) and sublaterodorsal (SLD) nuclei that are involved in wakefulness and rapid eye movement (REM) sleep, respectively (Clement et al., 2011;Fuller et al., 2011). To get around these obstacles and specifically test the involvement of PZ glutamatergic neurons in sleep-wake control, we used Phox2B, a transcription factor expressed in PZ but not PB or SLD glutamatergic neurons. Data indicate that PZ glutamatergic neurons are not involved in sleepwake control. Additionally, we found that excitation of PB or SLD glutamatergic neurons promotes wakefulness or REM sleep, respectively, results that are complimentary to the reduction in wakefulness or REM sleep previously observed following lesion of PB or SLD, respectively (Clement et al., 2011;Fuller et al., 2011).

Animals
In order to visualize Vglut2-, Phox2B-and Vgat-expressing neurons, Vglut2-IRES-cre [Jackson Laboratory #016963 ], Phox2B-IRES-cre [Jackson Laboratory #016223 (Rossi et al., 2011)] and Vgat-IRES-cre [Jackson Laboratory #016962 ] mice were crossed with a credependent reporter mouse Flox-L10-GFP [Jackson Laboratory #24750 (Liu et al., 2014)], producing Vglut2-GFP, Phox2B-GFP and Vgat-GFP mouse lines. Thirty two adult male Vglut2-GFP mice, seven adult male Phox2B-GFP and one adult male Vgat-GFP (8-12 weeks, 20-25 g) mice were used in this study. Mice were bred at our animal facility and underwent genotyping both before and after experiments. All procedures were approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center and of University of Massachusetts Medical School.

Sleep-Wake Monitoring
Three weeks after surgery, the mice were housed individually in transparent barrels in an insulated sound-proofed recording chamber maintained at an ambient temperature of 22 ± 1 • C and on a 12 h light/dark cycle (lights-on at 7 A.M., Zeitgeber time: ZT0) with food and water available ad libitum. Mice were habituated to the recording cable for 5-7 days before starting polygraphic recording. Cortical EEG (ipsilateral frontoparietal leads) and EMG signals were amplified (A-M System 3500, United States) and digitalized with a resolution of 500 Hz using Vital Recorder (Kissei, Japan). Mice were recorded for a 24 h baseline period followed by IP injections of saline (control injection) or Clozapine-N-oxide (CNO, NIMH Chemical Synthesis and Drug Supply Program; 0.3 mg/kg in saline). Injections were performed at 10 A.M. (10:00, ZT3, light period, time of high sleep-drive) and 7 P.M. (19:00, ZT12, beginning of the dark period, time of high wake-drive), in a randomized cross-over design, with each injection separated by a 2-3 day washout period. In each experiment, recordings were simultaneously made from an equal number (batches of 2-4) of PZ Vglut2−hM3Dq and PZ Vglut2−wt mice.

Sleep Scoring and Analysis
Using SleepSign for Animal (Kissei, Japan) assisted by spectral analysis using fast Fourier transform, polygraphic records were visually scored in 10 s epochs for wakefulness (W), SWS, and REM sleep. The percentage of time spent in wake, SWS and REM sleep were summarized for each group and each condition. The SWS to REM sleep latency is defined as the time between the onset of the first SWS episode, lasting >20 s, after injection and the onset of the first REM sleep episode, lasting >10 s.
Sleep-wake fragmentation was assessed by analyzing the distribution of each vigilance stage in different bout lengths. Vigilance stages were separated into eight bout lengths (<30, 40-70, 80-150, 160-310, 320-630, 640-1270, 1280-2550, and >2550 s) (Mochizuki et al., 2004;Kantor et al., 2013). For each vigilance stage, the number of episodes and the percentage of the vigilance stages occurring in each bout length were used to produce a time-weighted frequency histogram.
Recordings were scored again in 5 s epochs to allow for performance of an EEG power spectrum analysis. On the basis of visual and spectral analysis, epochs containing artifacts occurring during active wake (with large movements) or containing two vigilance states were visually identified and omitted from the spectral analysis. Recordings containing wake artifacts during more than 20% of the time were removed from the spectral analysis. EEG power spectra were computed for consecutive 5 s epochs within the frequency range of 0.5-120 Hz using a fast Fourier transform routine (FFT). The data were collapsed into 0.5 Hz bins. To determine the effect of injection on sleepwake power spectra, EEG power spectra were analyzed during the 3 h period of time post-injection, starting 10 min after injection as a previous study had shown that CNO injection significantly affected SWS amount during 3 h post-injection and SWS latency was no more than 10 min (Anaclet et al., 2014). The data were standardized by expressing each frequency bin as a percentage relative to the same bin under baseline conditions from the same mouse and from the same time of the day (same Zeitgeber time). To analyze the EEG frequency bands, power bins were summed in δ 0.5-5 Hz, θ 5-9 Hz, α 9-15 Hz, β 15-30 Hz, low γ 30-60 Hz and high γ 60-120 Hz, and expressed in percentage of baseline power band, from the same circadian time.
Statistical analysis was performed using Prism v6 (GraphPad Software, San Diego, CA, United States). Following confirmation that the data met the assumptions of the ANOVA model, twoway ANOVA followed by a post hoc Bonferroni test were used to compare the effects of the drug injection and time period on sleep-wake parameters, the effect of the drug injection and the distribution of vigilance episodes, or the effect of drug injection and power band on cortical EEG power density. Paired Student's t-test was used to compare the effects of the drug injection on SWS to REM sleep latency. Sample size and power calculations were performed post hoc at http://www. biomath.info, using means and standard deviations derived from our analysis. The present study was sufficiently powered to detect effect sizes.

Immunostaining and RNAscope
At the end of the behavioral experiments, mice were deeply anesthetized with ketamine/xylazine (200 and 20 mg/kg, respectively) and perfused transcardially with 20 ml of saline, followed by 100 ml of neutral phosphate-buffered formalin (4%; Thermo Fisher Scientific). Brains were removed from the skull and incubated in neutral phosphate-buffered formalin (4%; Thermo Fisher Scientific) for 2 h, followed by 20% sucrose until they sank.
For RNAscope, using a cryostat (Thermo Scientific, Cryostar NX70), brains were sectioned at 10 µm and mounted onto Surgipath (Leica) adhesive microscope slides, 3 slices per slide. Slides were kept at −80 • C until shortly before in situ hybridization. Slides were first warmed to room temperature and then we performed the RNAscope hybridization using a RNAscope Multiplex Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., Newark, CA, United States). Briefly, according to the manufacturer's instructions, target retrieval was performed at 99 • C after which slices were dehydrated in 100% ethanol and air-dried. Next, sections were treated with protease inhibitor (Protease III, RNAscope) for 30 min at 40 • C. After rinsing in RNAscope wash buffer, we incubated the sections in the RNAscope probes for Vglut2 (Mm-Slc17a6, catalog # 319171) and Phox2B (Mm-Phox2b-C2, catalog # 407861-C2). Additional sections were incubated in the manufacturer-supplied 2-plex positive control (catalog # 320761) and negative control (catalog # 320751) probes. Following the first 3 signal amplification steps, the fourth amplification was performed using Amp 4 Alt C-FL, such that channel 1 (Vglut2) was fluorescently labeled with Alto 550 and channel 2 (Phox2B) was labeled with Alto 647. Fluorescent images were collected with a confocal microscope (Zeiss LSM 700; Figures 6D-P).
Recordings were guided using a combination of fluorescence and infrared differential interference contrast (IR-DIC) video microscopy using a fixed stage upright microscope (Axio Examiner.D1, Zeiss) equipped with a Nomarski immersion lens (40×/1.0) and an infrared-sensitive camera (Orca flash 4.0, Hamamatsu). Images were displayed in real time using Zen2 software (Carl Zeiss). Recordings were conducted in wholecell mode using an EPC-10 USB amplifier and Patchmaster software (Heka).
Recordings were performed in current clamp mode using a K-gluconate based pipette solution containing (in mM): Kgluconate 120, KCl 10, MgCl 2 3, HEPES 10, K-ATP, Na-GTP 0.5. After at least 10 min of stable recording, ACSF containing CNO (500 nM) was perfused into the chamber for 3-5 min before washout. Recordings were analyzed, using Patchmaster software, by comparing the last 2 min before the application of CNO to the last 2 min of the CNO application. Paired Student's t-tests were used to calculate statistical significance.

PZ Vglut2 Neurons Are Not Sleep-Promoting
To test whether activation of PZ Vglut2 neurons affects sleepwake phenotype, Vglut2-IRES-Cre mice were injected into the PZ with a virus vector containing the excitatory hM3Dq receptor (AAV-mCherry-hM3Dq) to specifically express hM3Dq receptors in PZ glutamatergic neurons (PZ Vglut2−hM3Dq mice). First, responses of PZ Vglut2−hM3Dq neurons to the hM3Dq ligand, clozapine-N-oxide (CNO), were tested using whole-cell in vitro recordings ( Figure 1A1). Bath application of CNO (500 nM) significantly increased firing rates in PZ Vglut2−hM3Dq neurons (1.2 ± 0.5 vs. 0.3 ± 0.2 Hz in control condition, p = 0.042; Figure 1A2), confirming that CNO activates PZ glutamatergic neurons. We then tested, in vivo, the sleep-wake phenotypes upon activation of PZ glutamatergic neurons. At the end of the behavioral studies, the injection sites were mapped using mCherry immunostaining. Of the sixteen Vglut2-IRES-Cre mice injected with AAV-mCherry-hM3Dq, six mice displayed more specific expression of mCherry bilaterally in the PZ ( Figure 1B) and were used for the following sleep-wake analysis.
To test the effect of PZ Vglut2 neurons in sleep-wake control, mice were injected in a randomized cross-over design with saline or CNO (0.3 mg/kg) at the beginning of the dark/active period (19:00, ZT12) or during the light/rest period (10:00, ZT3). When injected at 19:00, CNO treatment did not significantly affect the hourly amount of wakefulness (two-way ANOVA, F(23,115) = 1.06, p = 0.40; Figure 1C1), SWS (two-way ANOVA, F(23,115) = 1.02, p = 0.45; Figure 1C2), or REM sleep (two-way ANOVA, F(23,115) = 1.38, p = 0.14; Figure 1C3). Because in previous studies (Anaclet et al., 2014(Anaclet et al., , 2015(Anaclet et al., , 2018 the effect of CNO-mediated neuronal activation or inhibition on sleep-wake cycles was most pronounced during the 3 h post-injection period, we performed a more refined analysis of this period. Neither wakefulness nor REM sleep amount and consolidation were affected by CNO injection during the 3 h post CNO injection as compared with control injection (Figures 1D1,D3,E1,E1' ,E3,E3'). However, SWS amounts were significantly decreased during the 3 h post CNO injection period as compared with control injections (15.0 ± 5.0 vs. 33.5 ± 11.9% of SWS in control condition, p < 0.01; Figure 1D2) but without change in bout length distribution (Figure 1E2'). This SWS decrease is associated with a significant increase in the number of very short SWS bouts (7.0 ± 3.1 vs. 1.8 ± 0.6 bouts lasting between 10 and 30 s, p = 0.0006; Figure 1E2). Wakefulness bout duration was also increased with a significant increase of the proportion of wakefulness from long bout lengths (>40 min long bouts: 72.9 ± 10.1 vs. 33.3 ± 21.1% of total wakefulness after control injection, p = 0.003; Figure 1E1'). These results indicate more labile switching between the two vigilance stages. Cortical EEG power spectral distribution was affected by the treatment in wakefulness (two-way ANOVA, F(243,729) = 1.48, p < 0.0001; Figure 1F1) and SWS (two-way ANOVA, F(243,729) = 3.88, p < 0.0001; Figure 1F2) but not in REM sleep (two-way ANOVA, F(243,729) = 0.82, p = 0.97; Figure 1F3). However, none of the frequency bands displayed any significant difference between CNO and control injection, in any vigilance stage (Figures 1F1-F3). Altogether, activation of PZ glutamatergic neurons at a time when the wake-promoting systems are active, during the active phase, did not induce SWS and showed only minimal effects on sleep-wake phenotype, indicating that PZ Vglut2 neurons are not sleep-promoting.

CNO Does Not Affect Sleep-Wake Cycle in Control Mice
To control for non-specific actions of CNO, non-cre expressing littermate mice were used. No hM3Dq receptor transfection was seen in these control mice. Treatment did not affect the hourly distribution of wakefulness (two-way ANOVA, F(23,92) = 1.53, p = 0.082; Figure 5A1), SWS (two-way ANOVA, F(23,92) = 1.56,  Figure 5A3). Moreover, CNO treatment did not affect the number of episodes or the episode length distribution as compared with control injection (Figures 5B1-B3') in any vigilance state. Finally, the cortical EEG power distribution during wakefulness, SWS and REM sleep was similar after CNO injection, as compared with both control injection and baseline recording (Figures 5C1-C3). These results confirm that the sleep-wake effects seen in PZ Vglut2−hM3Dq , PB Vglut2−hM3Dq and SLD Vglut2−hM3Dq mice is due to the specific activation of glutamatergic neurons.

Phox2B Is a Specific Marker for PZ Glutamatergic Neurons
Because chemogenetic activation of PZ Vglut2 neurons resulted in phenotypes resembling chemogenetic activation of PB Vglut2 and SLD Vglut2 neurons, i.e., wakefulness and REM sleep increase, respectively, we hypothesized that in the PZ Vglut2−hM3Dq mouse group some PB Vglut2 and SLD Vglut2 neurons were transfected and therefore responsible for the phenotypes. To specifically target PZ glutamatergic neurons we took advantage of a specific marker for PZ glutamatergic neurons, Phox2B. In the adult rat medullary and pontine regions in proximity to the PZ, Phox2B expression is restricted to the PZ, with a notable lack of expression in either the PB or the SLD (Kang et al., 2007). Moreover, Phox2B is co-localized with Vglut2 but not with Vgat or GAD67 [Figures 6D-G; (Stornetta et al., 2006)], suggesting that Phox2B is a specific marker for PZ glutamatergic neurons. We first confirmed the presence of Phox2B expression in PZ (Figure 6A) of mouse using Phox2B-GFP mice. No GFP positive neurons were seen in either the PB or the SLD (Figures 6B,C), indicating that Phox2B is specific for PZ glutamatergic neurons. Neurons of the locus coeruleus were GFP positive (LC; Figures 6A,B), which is consistent with previous studies showing that Phox2B is necessary for the differentiation of central noradrenergic and adrenergic neurons (Pattyn et al., 2000;Huber et al., 2005). We then assessed the extent of co-localization between Vglut2 and Phox2B in PZ. In each of the slices containing the PZ (n = 12 from 4 mice), Vglut2 co-localized exclusively with Phox2B and Phox2B was found primarily lateral to the facial nerve, in the entire PZ area (Figures 6H-K). Higher magnification photomicrographs show the cellular details of Vglut2/Phox2B co-localization  Figures 6L-O). Therefore, Phox2B is a specific marker for PZ glutamatergic neurons and Phox2B-IRES-cre mice can be used to specifically activate PZ glutamatergic neurons and study their role in sleep-wake control.

Activation of PZ Phox2B Neurons Does Not Affect Sleep-Wake Cycle
To assess the involvement of PZ glutamatergic neurons, five Phox2B-IRES-Cre mice were injected into the PZ with AAV-hM3Dq-mCherry ( Figure 7A). Three of the five cases also showed partial expression in the LC. Whole-cell in vitro recording ( Figure 7B1) confirmed that bath application of CNO (500 nM) significantly increased firing rate in PZ Phox2B−hM3Dq neurons (3.9 ± 1.8 vs. 1.4 ± 1.2 Hz in control condition, p = 0.028; Figure 7B2). CNO injection was successful in four of the five PZ Phox2B−hM3Dq mice (one mouse displayed an atypical adverse reaction to the injection). CNO treatment during the light period (10:00) did not affect the hourly amounts of wakefulness (twoway ANOVA, F(23,69) = 1.26, p = 0.23; Figure 7C1), SWS (two-way ANOVA, F(23,69) = 1.21, p = 0.27; Figure 7C2) or REM sleep (two-way ANOVA, F(23,69) = 0.92, p = 0.57; Figure 7C3). In order to study the qualitative aspects of the sleep-wake cycle following activation of PZ Phox2B neurons, we studied fragmentation (Figures 7D1-D3') and cortical EEG power distribution (Figures 7E1-E3) of the three vigilance stages during the 3 h post-injection time period. The number of sleep-wake episodes and episode length distribution were similar between CNO and control injections (Figures 7D1-D3). Treatment did not affect the cortical EEG power distribution during wakefulness and SWS. However, during REM sleep, the theta frequency band was significantly increased (118.4 ± 6.8 vs. 96.5 ± 2.5% of baseline power in control condition, p < 0.01; Figure 7D3) while the sigma frequency band was significantly decreased (78.4 ± 3.8 vs. 101.6 2.5% of baseline power in control condition, p < 0.01; Figure 7D3). These results indicate that activation of PZ Phox2B−hM3Dq during the light, inactive, period does not affect the sleep-wake cycle but could be involved in cortical EEG activation during REM sleep. Similar results were obtained when CNO was administrated at the beginning of the active period (19:00; Figure 8).

DISCUSSION
To test the contribution of PZ glutamatergic neurons in sleepwake control, we chemogenetically activated Vglut2-expressing neurons. Based on the sleep-wake phenotype and anatomical confirmation of the injection sites, the mice were separated in three groups: (1) one group, with targeted neuronal transfection mainly in the PZ, displayed increased wakefulness followed by a trend to increased REM sleep during the rest period but not during the active period; (2) a second group, with transfections that included the SLD, displayed a shorter wake increase followed by a significant increase in REM sleep amount; and (3) the third group, which had significant PB transfection, displayed a prominent and long lasting increase in wakefulness, independent of the time of day the injections were performed. Thus, due to the difficulty in targeting only glutamatergic PZ neurons while avoiding glutamatergic neurons in surrounding areas, the role of PZ Vglut2 neurons in sleep-wake control was still unclear. CNO did not affect the sleep-wake cycle in control mice not expressing the hM3Dq receptor, confirming that the phenotypes seen in Vglut2 transfected mice were specifically due to the activation of glutamatergic neurons. To test the specific role of PZ glutamatergic neurons in sleep-wake control, we took advantage of Phox2B, a transcription factor expressed by a subset of brainstem glutamatergic neurons. We first confirmed that Phox2B is a specific marker for PZ glutamatergic neurons in mice. Because chemogenetic activation of PZ Phox2B neurons did not affect sleep-wake phenotypes, we can conclude that PZ glutamatergic neurons are not sufficient to influence the sleepwake cycle.
The absence of a sleep-wake phenotype in control and PZ Phox2B−hM3Dq mice after CNO injection provides additional evidence that CNO, at the dose used in our studies, does not affect the baseline sleep-wake cycle. A recent study had shown that clozapine, a metabolite of CNO, but not CNO, crosses the blood brain barrier and binds chemogenetic receptors with high affinity in rats (Gomez et al., 2017). This finding was subsequently challenged by the observation that both clozapine and CNO cross the blood brain barrier in mice, and that unbound CNO is present in the brain at concentrations sufficient to activate DREADDs, albeit at a higher initial dose than we typically use (Jendryka et al., 2019). We had previously shown that, at a dose of 0.3 mg/kg, CNO does not affect sleep-wake quantity and quality in Vgat-IRES-cre mice (Anaclet et al., 2014), nor does it interfere with the wake-promoting actions of armodafinil and caffeine (Anaclet et al., 2018). In the present study, we confirm the absence of non-specific actions of CNO on the sleep-wake cycle, using two different mouse strains, Vglut2-IRES-cre and Phox2B-IRES-cre mice. Additionally, we confirmed that CNO is able to directly activate PZ Vglut2−hM3Dq , PB Vglut2−hM3Dq , and SLD Vglut2−hM3Dq neurons in vitro, where the short application duration (few minutes) and the absence of hepatic metabolism make back-conversion to clozapine highly unlikely. In summary, CNO was able to activate glutamatergic neurons expressing hM3Dq chemogenetic receptor and did not result in non-specific sleep-wake phenotypes.

Phox2B Is a Specific Marker for PZ Glutamatergic Neurons
The transcription factor Phox2B has been studied for its involvement in the control of breathing and autonomic regulation. Phox2B mutations have been implicated in congenital central hypoventilation syndrome (Moreira et al., 2016). Phox2Bexpressing neurons located in the medullary retrotrapezoid nucleus (RTN), ventral from the facial nucleus, are sensitive to hypoxia (Onimaru et al., 2008), hypercapnic acidosis and serotonin (Wu et al., 2019). Phox2B is necessary for the differentiation of central noradrenergic and adrenergic neurons (Pattyn et al., 2000;Huber et al., 2005). Phox2B is expressed in PZ in adult rats (Kang et al., 2007) but these neurons have no known physiological function. In the present study, we showed that, in PZ, Phox2B is highly co-localized with Vglut2 and therefore, is a specific marker for PZ glutamatergic neurons. Phox2B is also highly co-localized with LC noradrenergic neurons, known to be wake-promoting. However, in this study, the three mice showing partial expression of hM3Dq receptors in LC, did not display an increase in wake amount following CNO injection. It is possible that either the partial coverage of LC was not enough to promote wakefulness or LC noradrenergic neurons were not activated by the chemogenetic ligand. In vitro recordings of LC Phox2B−hM3Dq neurons would be necessary to answer this question.

The Role of PZ Glutamatergic Neurons in Sleep-Wake Control
A previous study has suggested that some PZ non-GABAergic neurons are sleep-active (Anaclet et al., 2012). Because glutamatergic neurons are the only other neuronal population identified in PZ thus far, we tested if chemogenetic activation of PZ glutamatergic neurons affects sleep-wake phenotypes. Specific targeting of PZ glutamatergic neurons using Vglut2-cre mice was challenging. Of the over 29 injected mice, only six displayed hM3Dq expression mainly in PZ (PZ Vglut2−hM3Dq ). Five mice displayed hM3Dq expression mainly in PB (PB Vglut2−hM3Dq ) and six in SLD (SLD Vglut2−hM3Dq ). The remaining mice included seven showing expression of hM3Dq at multiple sites, and five died after surgery or during the sleep recordings. These last two mouse groups were excluded from the study.
Chemogenetic activation of PZ Vglut2−hM3Dq neurons at the beginning of the mouse active phase (19:00) had limited impact on the sleep-wake cycle. On the other hand, chemogenetic activation of PZ Vglut2−hM3Dq neurons during the mouse rest phase (10:00) resulted in an early wake enhancement followed by an increase in REM sleep amount. Because these phenotypes are reminiscent of the phenotypes observed in PB Vglut2−hM3Dq and SLD Vglut2−hM3Dq mice, we hypothesized that they were due to the inadvertent transfection of PB and SLD neurons. In other words, in the PZ Vglut2−hM3Dq group, transfection would not be restricted to PZ. To test this hypothesis and definitively confirm the role of PZ Vglut2 neurons in sleep-wake control, we took advantage of Phox2B, a specific marker for PZ Vglut2 neurons. Using Phox2B-cre mice to specifically target PZ Vglut2 neurons and not neighboring PB and SLD, we showed that PZ Vglut2 neurons are not sufficient to affect the sleep-wake cycle at any time of the day. These results indicate that PZ glutamatergic neurons have no role in sleep or wake induction and/or maintenance. It remains, however, to be tested whether PZ Vglut2 neurons are necessary for normal sleep-wake cycle control, using inhibitory chemogenetic receptors and/or cell body specific lesion.

A New Mouse Model for REM Sleep Enhancement
Rostral to the PZ and PB, the SLD contains Vglut2-expressing neurons that are specifically active during REM sleep recovery (Clement et al., 2011). The SLD contains a large proportion of neurons with tonic discharge patterns immediately prior to and during REM sleep (Sakai, 2015). Cell body specific SLD lesions, knockout of glutamatergic transmission and genetic inactivation significantly reduce REM sleep amount and result in REM sleep without muscle atonia (Lu et al., 2006;Krenzer et al., 2011;Valencia Garcia et al., 2017). In the present study, we show for the first time that chemogenetic activation of SLD Vglut2 neurons results in increased REM sleep amount and reduced SWS to REM sleep latency. Moreover, cortical EEG theta power is significantly enhanced during REM sleep. These data provide a new and unique model of REM sleep enhancement. Such a model will permit probing of the specific role of REM sleep in other neurophysiological functions, such as memory consolidation. However, specific targeting of SLD glutamatergic neurons is challenging due to the close proximity of PB wake-promoting glutamatergic neurons (Fuller et al., 2011). A specific marker for SLD glutamatergic neurons would be very useful.

Additional Evidence for the Importance of PB in Wakefulness
In close proximity to PZ, just dorsal, lateral and rostral, the PB is a critical brainstem wake-promoting system. Following lesions of both PB and precoeruleus (PC), rats can no longer sustain cortical activation and become comatose (Fuller et al., 2011). Since this seminal study, the role of PB glutamatergic neurons in wakefulness has been refined. Specific lesions of medial PB result in hypersomnolence (Kaur et al., 2013). Glutamatergic neurons located in the external lateral PB are activated by hypoxia and are a key component of the vitally important circuitry regulating arousal from sleep apnea episodes (Kaur et al., 2017). In the present study we show that chemogenetic activation of medial PB results in long lasting wake enhancement. Moreover, CNO induced wakefulness was characterized by a decreased delta frequency band power. Because the delta band is considered a marker of EEG synchronization and is more prominent during quiet wakefulness, this result indicates a more active wake state induced by activation of PB Vglut2 neurons. Finally, no sleep rebound was seen after the wake enhancement. This is in accordance with previous studies using chemogenetics to specifically activate wake-promoting neuronal populations (Anaclet et al., 2015;Venner et al., 2016;Pedersen et al., 2017) and indicates that chemogenetic activation of wake-promoting neuronal populations does not enhance the homeostatic drive for sleep. All together, these results confirm the strong wake-promoting action of PB glutamatergic neurons.

CONCLUSION
This study shows, for the first time, that PZ glutamatergic neurons are not sufficient to affect the sleep-wake cycle in mouse. However, chemogenetic activation of PB or SLD glutamatergic neurons results in wake or REM sleep enhancement, respectively. Finally, Phox2B is a specific marker for PZ glutamatergic neurons. All together, these results provide a better understanding on how the brain regulates sleep-wake cycles, forming a framework for future studies characterizing the sleep-promoting subpopulation of the PZ.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.