Abstract
Rapid-eye movement (REM) sleep correlates with neuronal activity in the brainstem, basal forebrain and lateral hypothalamus. Lateral hypothalamus melanin-concentrating hormone (MCH)-expressing neurons are active during sleep, but their effects on REM sleep remain unclear. Using optogenetic tools in newly generated Tg(Pmch-cre) mice, we found that acute activation of MCH neurons (ChETA, SSFO) at the onset of REM sleep extended the duration of REM, but not non-REM, sleep episodes. In contrast, their acute silencing (eNpHR3.0, archaerhodopsin) reduced the frequency and amplitude of hippocampal theta rhythm without affecting REM sleep duration. In vitro activation of MCH neuron terminals induced GABAA-mediated inhibitory postsynaptic currents in wake-promoting histaminergic neurons of the tuberomammillary nucleus (TMN), and in vivo activation of MCH neuron terminals in TMN or medial septum also prolonged REM sleep episodes. Collectively, these results suggest that activation of MCH neurons maintains REM sleep, possibly through inhibition of arousal circuits in the mammalian brain.
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Acknowledgements
We thank B. Jones, M.E. Carter and L. de Lecea for helpful comments on a previous version of the manuscript. We thank the members of the Adamantidis laboratory for their technical help and comments. Optogenetic plasmids were kindly provided by K. Deisseroth (Stanford University). S.D.G. was supported by the Fonds de la Recherche du Québec - Santé. D.B. and A.R.A. were supported by the Human Frontier Science Program. A.R.A. was supported by the Douglas Foundation, McGill University, Canadian Fund for Innovation, Canadian Research Chair (Tier 2), Canadian Institute for Health Research and the Natural Science and Engineering Council of Canada.
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All of the authors designed the experiments. S.J., S.D.G., C.G.H., M.E. and S.J.R. collected data and performed analysis. M.E and J.F. generated the transgenic mouse model. All of the authors discussed the results and S.J., S.D.G., D.B. and A.R.A. wrote the manuscript.
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Supplementary Figure 1 Photomicrographs of coronal brain sections from a Tg(Pmch-Cre) mouse injected with AAVdj-ChETA-EYFP in the LH-ZI area.
Pictures show dense EYFP-expressing MCH neuron projection terminating within sleep-related structures including the medial septum (a), neocortex (b), raphe nucleus (c), and the locus coeruleus (d). Abbreviations used: LC: Locus coeruleus. Scale bar: 200 μm.
Supplementary Figure 2 MCH neurons transfected with AAVdj-ChETA-EYFP increase their firing rate in response to blue light pulses.
a, Schematic of the optrode recording system. Note that the optic fiber is placed in the glass pipette. b, In vivo extracellular recording of ChETA-expressing MCH neuron in the LH showing tonic firing evoked by 1 Hz light pulse trains. Note the spontaneous firing rate of MCH neuron in the absence of light. c, Extracellular waveforms recorded from putative MCH neurons before (black), during (green) and after (red) optical stimulation of LH area. Note the strong similarity between spontaneous and light-evoked waveforms. d, Quantification of the discharge of MCH neurons before, during and after the light stimulation. MCH neurons firing was significantly increased by the 1Hz light pulse trains (n = 4 cells, 4 animals, 12 optical stimulations). Number of spike per second are represented as mean ± SEM. ***: p < 0.001 compared to the firing evoked by light pulse trains, one-way ANOVA within subject design, followed by Tukey post-hoc test.
Supplementary Figure 3 Spontaneous sleep-wake cycle of Tg(Pmch-cre) and Tg(Pmch-cre); Mchr1–/–.
a. Spontaneous duration of wake, NREM and REM sleep (expressed as a percentage of time) of Tg(Pmch-Cre) transduced with ChETA-EYFP (blue, n = 8), SSFO-EYFP (orange, n = 4), NpHR3.0-EYFP (red, n = 6) virus, and their EYFP controls (black, n = 8) during the light and dark phases, and 24h of the light/dark cycle. No significant differences were found between Tg(Pmch-Cre) animals expressing the opsin and their controls demonstrating that expression of the opsin in MCH neurons does not alter the spontaneous sleep-wake cycle of the mice b. Spontaneous duration of wake, NREM and REM sleep (expressed as a percentage of time) of Tg(Pmch-Cre) animals (n = 8) compared to [Tg(Pmch-Cre) X MCHR-1-/- ] animals (n = 8) showing a hyperactive phenotype during the dark phase for the double transgenic animals associated with a significant decrease of both NREM sleep and REM sleep. No significant differences during the light-dark phases and the 24h were found. Mean durations are represented as mean ± SEM. *: p < 0.05, ***: p < 0.001 one-way ANOVA between subject design for viral transduction, followed by Tukey post-hoc test or unpaired two-tailed t test.
Supplementary Figure 4 Sleep-specific stimulation during NREM and REM sleep.
a, timeline of NREM and REM sleep-specific stimulation experiments (right). B, Representative EEG/EMG recordings showing optical stimulation of ChETA (lower trace) and EYFP (control, upper trace) animals time-locked to NREM sleep episode. Note the transition from NREM to REM sleep occurring in ChETA animals after blue light stimulation. Horizontal blue bar (dashed) represents optical stimulations. C, Representative EEG/EMG recordings showing optical stimulation of ChETA (lower trace) and EYFP (control, upper trace) animals time-locked to REM sleep episode. Note that optical stimulation of EYFP animal starts at the onset of REM sleep (i.e., after the transition from NREM to REM sleep is complete). Wakefulness signals the termination of REM sleep. Horizontal blue bar (dashed) represents optical stimulations.
Supplementary Figure 5 Activation of SSFO in MCH neurons leads to increased excitability in response to a replayed current trace.
a, To assess whether SSFO activation resulted in increased excitability, a series of EPSCs and IPSCs in an identified MCH neuron were recorded. The current trace (black) was then replayed to MCH cells transfected with SSFO before (red) and during (blue) activation with a brief pulse of blue light (50 ms pulse width). b, Spike waveforms from MCH neurons before (red) and during (blue) SSFO activation are similar. c, Average data show that activation of SSFO resulted in a 4.5-fold increase in the number of spikes elicited during replay compared to baseline (control : 2.5±1.3 spikes, SSFO: 11.3±0.9 spikes, p = 0.0008, n = 4, at least 3x30 s sweep per cell). *** p<0.001, paired two-tailed t-test.
Supplementary Figure 6 MCH neurons silencing decreases the stability of theta oscillations during REM sleep
a, ArchT-expressing MCH neurons show a persistent outward current (bottom) and concomitant hyperpolarization (top) upon 30 s constant yellow illumination in voltage and current clamp, respectively. Note that cells were depolarized to above-threshold potentials using constant steady-state current injection to elicit spiking. b, Quantification of membrane hyperpolarization (Mean : 34.0 ±8.1 mV) and outward currents (166.2±83.4 pA) of ArchT-expressing MCH neurons upon optical silencing (n=5 cells in 4 slices across 2 animals).
Supplementary Figure 7 Recorded neurons (represented by biocytin labeling in green) colocalized with histamine decarboxylase–positive cells (in red).
Scale bar: 50 μm.
Supplementary Figure 8 Optical fiber positions across experimental conditions over the LH, the TMN, the MS and the DR for Tg(Pmch-cre) and Tg(Pmch-cre); Mchr1–/– mice.
Drawings were generated according to the mouse brain atlas{Paxinos:2004ts}. Scale bar: 500 μm. Abbreviations used: ZI: Zona Incerta, LH: lateral hypothalamus, 3V: third ventricle, Arc: Arcuate hypothalamic nucleus, f: fornix, ns: nigrostriatal tract, LV: lateral ventricle, MS: medial septal nucleus, aca: anterior commisure, Aq: Aqueduct, DR: doral raphe nucleus, MnR: Median raphe nucleus, VTM: ventral tuberomammillary nucleus.
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Jego, S., Glasgow, S., Herrera, C. et al. Optogenetic identification of a rapid eye movement sleep modulatory circuit in the hypothalamus. Nat Neurosci 16, 1637–1643 (2013). https://doi.org/10.1038/nn.3522
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DOI: https://doi.org/10.1038/nn.3522
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