Control of rem sleep by ventral medulla gabaergic neurons

ABSTRACT Rapid eye movement (REM) sleep is a distinct brain state characterized by activated electroencephalogram and complete skeletal muscle paralysis, and is associated with vivid

dreams1,2,3. Transection studies by Jouvet first demonstrated that the brainstem is both necessary and sufficient for REM sleep generation2, and the neural circuits in the pons have since

been studied extensively4,5,6,7,8. The medulla also contains neurons that are active during REM sleep9,10,11,12,13, but whether they play a causal role in REM sleep generation remains

unclear. Here we show that a GABAergic (γ-aminobutyric-acid-releasing) pathway originating from the ventral medulla powerfully promotes REM sleep in mice. Optogenetic activation of ventral

medulla GABAergic neurons rapidly and reliably initiated REM sleep episodes and prolonged their durations, whereas inactivating these neurons had the opposite effects. Optrode recordings

from channelrhodopsin-2-tagged ventral medulla GABAergic neurons showed that they were most active during REM sleep (REMmax), and during wakefulness they were preferentially active during

eating and grooming. Furthermore, dual retrograde tracing showed that the rostral projections to the pons and midbrain and caudal projections to the spinal cord originate from separate

ventral medulla neuron populations. Activating the rostral GABAergic projections was sufficient for both the induction and maintenance of REM sleep, which are probably mediated in part by

inhibition of REM-suppressing GABAergic neurons in the ventrolateral periaqueductal grey. These results identify a key component of the pontomedullary network controlling REM sleep. The

capability to induce REM sleep on command may offer a powerful tool for investigating its functions. Access through your institution Buy or subscribe This is a preview of subscription

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ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A MEDULLARY HUB FOR CONTROLLING REM SLEEP

AND PONTINE WAVES Article Open access 03 July 2023 GABAERGIC NEURONS IN THE ROSTROMEDIAL TEGMENTAL NUCLEUS ARE ESSENTIAL FOR RAPID EYE MOVEMENT SLEEP SUPPRESSION Article Open access 07

December 2022 A CLUSTER OF MESOPONTINE GABAERGIC NEURONS SUPPRESSES REM SLEEP AND CURBS CATAPLEXY Article Open access 25 October 2022 REFERENCES * Aserinsky, E. & Kleitman, N. Regularly

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recordings. _Neuroscience_ 131, 1–11 (2005) Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank A. Popescu for the help with _in vivo_ physiology, M. Bikov and S.

Chung for technical assistance, the University of North Carolina Virus Core for supplying AAV, and T. Kilduff and J. Cox for discussions. This work was supported by EMBO and Human Frontier

Science Program postdoctoral fellowships (to F.W.). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Neurobiology, Department of Molecular and Cell Biology, Helen Wills Neuroscience

Institute, Howard Hughes Medical Institute, University of California, Berkeley, 94720, California, USA Franz Weber, Shinjae Chung, Min Xu & Yang Dan * Department of Biology, Howard

Hughes Medical Institute, Stanford University, Stanford, 94305, California, USA Kevin T. Beier & Liqun Luo Authors * Franz Weber View author publications You can also search for this

author inPubMed Google Scholar * Shinjae Chung View author publications You can also search for this author inPubMed Google Scholar * Kevin T. Beier View author publications You can also

search for this author inPubMed Google Scholar * Min Xu View author publications You can also search for this author inPubMed Google Scholar * Liqun Luo View author publications You can also

search for this author inPubMed Google Scholar * Yang Dan View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS F.W. and Y.D. conceived and

designed the experiments. F.W. performed all optogenetic stimulation experiments and optrode recordings. S.C. performed a subset of pharmacogenetic experiments and fluorescence microscopy.

K.T.B. and L.L. provided viral reagents for rabies-mediated trans-synaptic experiments. M.X. designed the optrodes used in this study. F.W. and Y.D. wrote the manuscript, and all authors

participated in the revision of the manuscript. CORRESPONDING AUTHOR Correspondence to Yang Dan. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests.

ADDITIONAL INFORMATION All primary histological, electrophysiological, and behavioural data have been archived in the Department of Molecular and Cell Biology, University of California,

Berkeley. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1 COMPARISON OF SPONTANEOUS AND LASER-INDUCED REM SLEEP. A, Mean EEG spectrogram and EMG amplitude before and after laser

onset (averaged across all trials with laser onset falling on NREM sleep). B, Mean EEG spectrogram and EMG amplitude before and after spontaneous REM onset outside laser stimulation periods

(only REM episodes with duration >70 s were included). C, Comparison of EEG power spectra during spontaneous (grey) and laser-induced (blue) REM sleep. Blue shading, s.e.m. for

laser-induced REM sleep. EXTENDED DATA FIGURE 2 EFFECT OF LASER STIMULATION ON BRAIN STATES IN EYFP CONTROL MICE. A, Brain states in all trials from five mice aligned by time of laser

stimulation (top) and probability of wake, NREM, or REM states before, during, and after laser stimulation (bottom). Shading, 95% CI. Blue bar, period of laser stimulation. Laser stimulation

caused no significant change in the probability of any brain state (_P_ > 0.34, bootstrap). B, Similar to A, for trials in which laser onset fell on NREM sleep. C, Trials in which laser

onset fell on wakefulness. EXTENDED DATA FIGURE 3 OPTOGENETIC ACTIVATION OF VM GLUTAMATERGIC NEURONS INDUCES WAKEFULNESS. A, Probability of wake, NREM, or REM states before, during, and

after laser stimulation (20 Hz, 120 s) in VGLUT2-Cre mice injected with AAV expressing ChR2–eYFP into the vM (_n_ = 3 mice). Shading, 95% CI. Blue bar, period of laser stimulation. Laser

stimulation caused a significant increase in wakefulness (_P_ < 0.001, bootstrap) and decrease in NREM sleep (_P_ < 0.001). B, Mean durations of REM sleep episodes with and without

laser stimulation. Each pair of dots represents data from one mouse. Laser stimulation shortened the duration of REM sleep episodes (_n_ = 3 mice, _P_ = 0.008, paired _t_-test). Error bar,

s.d. C, Probability of wake (left), NREM (middle), and REM (right) states before, during, and after laser stimulation (20 Hz, 120 s) at different laser powers (colour coded). EXTENDED DATA

FIGURE 4 EFFECT OF LASER STIMULATION OF VM GABAERGIC NEURONS ON THE TRANSITION PROBABILITY BETWEEN EACH PAIR OF BRAIN STATES IN CHR2 AND EYFP CONTROL MICE. A–F, ChR2 control mice. A,

Probability of NREM (N) to REM (R) state transition within each 20 s period in ChR2 mice. Blue shading, period of laser stimulation (20 Hz, 120 s). Error bar, s.d. (bootstrap, _n_ = 6 mice).

Probability of baseline transition (red dashed line) was computed after excluding the laser stimulation period. The probability during laser stimulation was significantly higher than the

baseline (_P_ = 0.02, bootstrap). B, Similar to A, for NREM to wake (W) transition. The probability during laser stimulation was not significantly different from baseline (_P_ = 0.24). C,

REM to wake, probability during laser stimulation was significantly lower than baseline (_P_ < 0.001), consistent with the effect of vM GABAergic neurons on prolonging REM duration (Fig.

2). D, REM to NREM, which rarely occurs in rodents. Laser stimulation caused no significant effect (_P_ > 0.99). E, Wake to REM, which rarely occurs in normal mice. Laser stimulation had

no significant effect (_P_ > 0.99). F, Wake to NREM. Laser stimulation caused a significant reduction in the transition probability (_P_ < 0.001), indicating that during wakefulness vM

GABAergic neuron activity has a wake-maintenance effect. G–L, Similar to A–F, for eYFP control mice. Laser stimulation had no significant effect on any transition probability (_P_ >

0.05). EXTENDED DATA FIGURE 5 PHARMACOGENETIC INACTIVATION OF VM GABAERGIC NEURONS REDUCES REM SLEEP. A, Brain states in a control (vehicle injection) and a CNO session from an example

mouse. The recording session started 20 min after vehicle or CNO injection. B, Probability of each brain state during the first 2.5 h of the recording session, after injection of vehicle

(grey) or two dosages of CNO (different shades of blue). Error bar, s.e.m. (_n_ = 6 mice). *_P_ < 0.05; **_P_ < 0.01; one-way analysis of variance with post hoc Dunnett’s test. C,

Similar to B, but during the second half of the recording session (2.5–5 h). There was no significant difference between control and CNO at any dosage (_P_ > 0.12). D, Latency of first

REM sleep episode (from the beginning of each recording session). E, Frequency of REM episodes during the first 2.5 h of the recording session. F, Duration of REM episodes during the first

2.5 h of the session. The reduction of REM sleep caused by pharmacogenetic inactivation of vM GABAergic neurons appears to be due to the reduction of frequency rather than duration of REM

episodes. EXTENDED DATA FIGURE 6 OPTOGENETIC IDENTIFICATION OF VM GABAERGIC NEURONS. A, Left, reliability and temporal jitter of laser-evoked spikes in identified (blue) and unidentified

(grey) units recorded in the vM. Note that the identified and unidentified units form two distinct clusters, with high reliability and low jitter for identified units. Dark/light symbols,

data during 30/15 Hz laser stimulation. Middle, distribution of delays of laser-evoked spiking for 21 identified GABAergic neurons. Delay is defined as timing of the first spike after each

laser pulse. Right, distribution of correlation coefficient between laser-evoked and spontaneous spike waveforms for all 21 identified vM GABAergic neurons. B, Fluorescence image of a

coronal section showing the position of an electrolytic lesion at the end of the optrode tract (arrow). Blue, DAPI staining. C, Positions of the 21 identified vM GABAergic neurons from 5

mice. Each dot indicates one neuron. All 20 neurons with recording periods encompassing all three brain states showed maximal firing rates during REM sleep. The wakeful behaviour during

which the neuron showed the maximal firing rate is colour-coded. Black, neurons for which the recording period did not include all wakeful behaviours. Schemes of brain sections adapted from

Allen Mouse Brain Atlas (Website: © 2015 Allen Institute for Brain Science. Allen Mouse Brain Atlas [Internet]. Available from: http://mouse.brain-map.org). EXTENDED DATA FIGURE 7 ACTIVITY

OF VM GABAERGIC NEURONS AT REM SLEEP ONSET AND OFFSET. A, Mean firing rates of vM GABAergic neurons at REM onset. Shading, s.e.m. Dark blue, period in which firing rate was significantly

higher than baseline (_P_ < 0.05, Wilcoxon signed-rank test); baseline was defined as the average firing rate during the 10 s intervals 60 s before and after REM sleep. B, Mean firing

rates at REM offset. EXTENDED DATA FIGURE 8 EFFECT OF LASER STIMULATION OF VM NEURONS ON SEVERAL WAKEFUL BEHAVIOURS. A, Probability of moving, running, eating, and grooming before, during,

and after laser stimulation (20 Hz, 120 s) in GAD2-Cre mice injected with AAV expressing ChR2–eYFP into the vM (_n_ = 8 mice). Unclassified behaviours are not shown. Blue bar, period of

laser stimulation. Laser stimulation caused a significant increase in eating (_P_ = 0.008, Wilcoxon signed-rank test). B, Similar to A, but in control mice expressing eYFP (_n_ = 4 mice).

EXTENDED DATA FIGURE 9 FIRING RATES OF UNIDENTIFIED VM NEURONS. A, Firing rates of unidentified units in the three brain states. Each line represents data from one neuron. Gray bar

represents average over units (_n_ = 24). B, Left, relative firing rates of the units across brain states. The firing rates of each unit were normalized by its maximum. Right, normalized

firing rates across different wakeful behaviours. C, Mean firing rates of unidentified vM neurons at REM onset. Shading, s.e.m. Dark blue, period in which firing rate was significantly

higher than baseline (_P_ < 0.05, Wilcoxon signed-rank test). D, Mean firing rates of unidentified vM neurons at REM offset. EXTENDED DATA FIGURE 10 CO-EXPRESSION OF GABA AND GLYCINE IN

VM NEURONS. A, Fluorescence image of ChR2–eYFP expressing neurons in the vM of a GAD2-Cre mouse injected with Cre-inducible AAV. B, Immunohistochemical staining for glycine. C, Superposition

of eYFP expression and glycine staining. In total, 94% (273/289) of eYFP-expressing cells were glycine positive (_n_ = 3 mice). SUPPLEMENTARY INFORMATION OPTOGENETIC ACTIVATION OF VM

GABAERGIC NEURONS INDUCES REM SLEEP The video shows 3 laser stimulation trials, including 1 min before and after each laser stimulation period. In the first two trials the animal was in NREM

sleep at laser onset, and he was awake in the third trial. The EEG spectrogram, EMG amplitude and hypnogram are shown on the right. Laser stimulation periods are depicted by the blue

shading on the top right and additionally indicated as a blue square in the upper right corner of the movie frame; 10x speedup. (MOV 2003 kb) FIRING RATES OF AN IDENTIFIED VM GABAERGIC

NEURON DURING SLEEP AND WAKEFUL BEHAVIORS The video shows two recording periods of an identified unit. During the first period the animal was asleep, during the second period he was awake

and engaged in multiple behaviors (ET – eating, GR – grooming, MV – moving, RU – running, QA – quiet awake). EEG spectrogram, EMG amplitude, hypnogram and firing rate are shown on the right.

The time points of single spikes are represented as vertical lines on the bottom left. Laser stimulation periods (15 or 30 Hz) are shown as blue shadings along with the firing rate and are

indicated by a blue square within the movie frame; 10x speedup. (MOV 2400 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT

SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Weber, F., Chung, S., Beier, K. _et al._ Control of REM sleep by ventral medulla

GABAergic neurons. _Nature_ 526, 435–438 (2015). https://doi.org/10.1038/nature14979 Download citation * Received: 14 May 2015 * Accepted: 23 June 2015 * Published: 07 October 2015 * Issue

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