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Prefrontal cortical regulation of REM sleep

Nature Neuroscience volume 26, pages 1820–1832 (2023)Cite this article

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Abstract

Rapid eye movement (REM) sleep is accompanied by intense cortical activity, underlying its wake-like electroencephalogram. The neural activity inducing REM sleep is thought to originate from subcortical circuits in brainstem and hypothalamus. However, whether cortical neurons can also trigger REM sleep has remained unknown. Here we show in mice that the medial prefrontal cortex (mPFC) strongly promotes REM sleep. Bidirectional optogenetic manipulations demonstrate that excitatory mPFC neurons promote REM sleep through their projections to the lateral hypothalamus and regulate phasic events, reflected in accelerated electroencephalogram theta oscillations and increased eye movement density during REM sleep. Calcium imaging reveals that the majority of lateral hypothalamus-projecting mPFC neurons are maximally activated during REM sleep and a subpopulation is recruited during phasic theta accelerations. Our results delineate a cortico-hypothalamic circuit for the top-down control of REM sleep and identify a critical role of the mPFC in regulating phasic events during REM sleep.

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Fig. 1: Optogenetic activation of mPFC Pyr neurons triggers REM sleep.
Fig. 2: Activation of mPFC Pyr neurons maintains REM sleep and promotes phasic θ events.
Fig. 3: Optogenetic activation of mPFC inhibitory neurons suppresses REM sleep.
Fig. 4: The effects on REM sleep and phasic θ events are mediated by LH-projecting mPFC neurons.
Fig. 5: Activity of LH-projecting mPFC neurons during sleep.
Fig. 6: Presynaptic inputs to LH-projecting mPFC neurons.

Data availability

Large raw data files, including electrophysiological, video or imaging data, are available upon request from the corresponding author. Source data are provided with this paper.

Code availability

The code used to analyze data and generate figures is available at https://zenodo.org/record/8035420/. The software for EEG/EMG recordings is available at https://github.com/justin0bk/sleepRecording_v9/. The software to control lasers using Raspberry Pi is available at https://github.com/justin0bk/socketrecv/.

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Acknowledgements

This work was supported by a NARSAD Young Investigator grant by the Brain & Behavior Research Foundation (27799 to F.W.), by a grant from the Margaret Q. Landenberger Foundation (to F.W.) and by the NIH/National Institute of Neurological Disorders and Stroke (NINDS; R01NS110865 to S.J.). We thank J. Baik for help with setting up the sleep recording system, S. Acquaye, Z. Spalding, X. Li, C. Harrison and F. Stauffer for help with histology and sleep annotation, and D. Raizen for critical comments on the paper.

Author information

Authors and Affiliations

  1. Department of Neuroscience, Perelman School of Medicine, Chronobiology and Sleep Institute, University of Pennsylvania, Philadelphia, PA, USA

    Jiso Hong, David E. Lozano, Shinjae Chung & Franz Weber

  2. Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, Irvine, CA, USA

    Kevin T. Beier

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  1. Jiso Hong
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  2. David E. Lozano
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  4. Shinjae Chung
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Contributions

Conceptualization: J.H. and F.W.; funding acquisition: F.W.; experiments—optogenetic and chemogenetic sleep experiments, calcium imaging, pupil tracking and viral tracing: J.H.; project administration: F.W. and S.C.; resources—viruses for rabies tracing: K.T.B.; methodology—setup for pupil tracking: D.E.L., development of software for sleep, calcium imaging analysis and pupil tracking: F.W.; data analysis—analysis of sleep recordings, calcium imaging, pupil tracking and viral tracing: J.H.; writing: J.H. and F.W.

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Correspondence to Franz Weber.

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Extended data

Extended Data Fig. 1 Population activity of mPFC pyramidal neurons during sleep.

(a) Left, schematic of calcium imaging of mPFC pyramidal (Pyr) neurons using fiber photometry. AAV-CaMKII-Cre and AAV-FLEX-GCaMP6s were injected into the mPFC. Right, fluorescence image showing expression of GCaMP6s (green) in the mPFC. Scale bar, 500 μm. Brain atlas images adapted with permission from ref. 61. (b) Position of optic fibers within the mPFC used for imaging of mPFC Pyr neurons. Each diagram depicts the section where the lesion caused by the optic fiber (colored bar) was largest along the rostrocaudal axis (n = 7 mice). PL, prelimbic cortex; IL, infralimbic cortex. (c) Example fiber photometry recording. Shown are EEG spectrogram, EMG amplitude, brain states, and ΔF/F signal. PSD, power spectral density. (d) Average ΔF/F activity during REM, wake, and NREM sleep. One-way repeated-measures (RM) ANOVA, P = 0.0000; t-tests with Holm-Bonferroni correction; REM versus wake, P = 0.0001; wake versus NREM, P = 0.0003; REM versus NREM, P = 0.0000. n = 7 mice. Bars, averages across mice; lines, individual mice; error bars, 95% confidence intervals (CIs). (e) Average EEG spectrogram (normalized by the mean power in each frequency band) and mean calcium activity (ΔF/F, z-scored) at brain state transitions. Horizontal lines indicate the time points for which the ΔF/F activity significantly differed from baseline (−60 to −50 s; Methods). One-way RM ANOVA with 10 s time bins as within-subjects factor; P = 0.0000; paired t-tests with Holm-Bonferroni correction; P < 0.05, n = 7 mice. Black lines, averages across mice; gray lines, individual mice. See Supplementary Table 1 for detailed statistical information. ***P < 0.001.

Source data

Extended Data Fig. 2 Expression of ChR2-eYFP throughout mPFC and effects of laser stimulation in mice expressing ChR2-eYFP or eYFP.

(a) Left, expression of ChR2-eYFP in the mPFC. For each mouse (n = 11), we determined the spread of ChR2-eYFP in four consecutive brain sections along the rostrocaudal axis. The color code indicates in how many mice the virus expression overlapped at the corresponding location. Right, each colored bar represents the location of an optic fiber used for optogenetic stimulation of mPFC Pyr neurons. Brain atlas images adapted with permission from ref. 61. (b) Open-loop stimulation in control mice expressing eYFP in mPFC Pyr neurons. All laser stimulation trials from n = 8 mice were aligned with the laser onset (t = 0 s). Trials were sorted depending on the brain state at laser onset (arrows). (c) Effect of laser stimulation in eYFP control mice on brain states. Two-way RM ANOVA comparing the mean percentage of each brain state between the laser and preceding 120 s baseline interval (interaction, P = 0.4675). n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (d) Cumulative transition probabilities for ChR2 mice to remain in REM sleep (R → R), wake (W → W), or NREM sleep (N → N) during the laser and preceding 120 s baseline interval. Bootstrap; R → R, P = 0.0001; W → W, P = 0.0042; N → N, P = 0.0001; n = 11 mice. Shadings, 95% CIs. (e) Differences of laser-induced changes in the cumulative transition probabilities between ChR2 (n = 11) and eYFP mice (n = 8). Each violin plot represents the sampling distribution of the mean difference in the laser-induced changes between ChR2 and eYFP mice (Methods). Bootstrap; N → R, P = 0.0001; R → W, P = 0.0012; N → W, P = 0.3610; W → N, P = 0.3684; R → R, P = 0.0012; W → W, P = 0.3684; N → N, P = 0.0001. Blue lines, 95% CIs; white dots, distribution means. (f) Top, PSD of the EEG during REM sleep with (ON) and without laser stimulation (OFF) for open-loop activation of mPFC Pyr neurons in ChR2 mice. The colored patches indicate the frequency ranges for the δ (0.5–4.5 Hz), θ (6.0–9.5 Hz), and σ power (10–15 Hz). For each frequency band, we tested whether the power significantly differs between REM sleep with and without laser. Two-way RM ANOVA with laser (ON or OFF, P = 0.0001) and frequency band as within-subjects factors (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.0813; θ, P = 0.0000; σ, P = 0.0000. n = 11 ChR2 mice. Shadings, 95% CIs. Bottom, laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA with frequency band as within-subjects factor and virus (eYFP or ChR2) as between-subjects factor (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0068; θ, P = 0.0001; σ, P = 0.0001. ChR2, n = 11; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (g) Top, PSD during wake with and without laser stimulation. Two-way RM ANOVA (laser, P = 0.0147; interaction, P = 0.0007); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.0046; θ, P = 0.0106; σ, P = 0.0106. n = 11 ChR2 mice. Shadings, 95% CIs. Bottom, laser-induced power changes between wake with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0162; θ, P = 0.0911; σ, P = 0.0911. ChR2, n = 11; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (h) Top, PSD during NREM sleep with and without laser stimulation. Two-way RM ANOVA; (laser, P = 0.0129; interaction, P = 0.0017); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.0020; θ, P = 0.3486; σ, P = 0.0348. n = 11 ChR2 mice. Shadings, 95% CIs. Bottom, laser-induced power changes between NREM sleep with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA (interaction, P = 0.0021); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0078; θ, P = 0.4394; σ, P = 0.2171. ChR2, n = 11; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (i) Top, EMG amplitude during each brain state with and without laser stimulation. Two-way RM ANOVA (laser, P = 0.0290; interaction, P = 0.0421); t-tests with Holm-Bonferroni correction; OFF versus ON: REM, P = 0.3573; wake, P = 0.1089; NREM, P = 0.5518. n = 11 ChR2 mice. Box plots (see Methods for definition); lines, individual mice. Bottom, laser-induced changes in the EMG amplitude during each brain state with and without laser stimulation. Mixed ANOVA (interaction, P = 0.0529). ChR2, n = 11; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

Source data

Extended Data Fig. 3 Effects of closed-loop activation and inhibition of mPFC Pyr neurons on EEG and REM sleep duration.

(a) PSD of the EEG during REM sleep with (ON) and without laser stimulation (OFF) of mPFC Pyr neurons expressing ChR2. Two-way RM ANOVA with laser (P = 0.0057) and frequency band (δ, θ, or σ) as within-subjects factors (interaction, P = 0.0001); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.0094; θ, P = 0.0011; σ, P = 0.0005. n = 11 mice. Shadings, 95% CIs. (b) Laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA with frequency band as within- and virus (eYFP or ChR2) as between-subjects factor (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0041; θ, P = 0.0012; σ, P = 0.0330. ChR2, n = 11; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (c) Duration of REM sleep episodes with (ON) or without laser (OFF) and REM sleep duration in baseline recordings without laser stimulation (Bsl) in ChR2 mice. One-way RM ANOVA (laser, P = 0.0001); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0253; Bsl versus ON: P = 0.0054; OFF versus ON: P = 0.0054. n = 6 mice. Box plots; lines, individual mice. (d) Left, heatmaps depicting expression of iC++-eYFP in the mPFC in four consecutive brain sections (n = 8 mice). Right, location of fiber tracts. Each pair of colored bars represents the location of two optic fibers for bilateral inhibition. Brain atlas images adapted with permission from ref. 61. (e) PSD of the EEG during REM sleep with and without laser stimulation of mPFC Pyr neurons expressing iC++. Two-way RM ANOVA (laser, P = 0.0001; interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.1792; θ, P = 0.0001; σ, P = 0.0281. n = 8 mice. Shadings, 95% CIs. (f) Laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and iC++ mice. Mixed ANOVA (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus iC++: δ, P = 0.8948; θ, P = 0.0000; σ, P = 0.0029. iC++, n = 8; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (g) Duration of laser-on or laser-off REM sleep episodes and REM sleep duration in baseline recordings without laser stimulation in iC++ mice. One-way RM ANOVA (laser, P = 0.0000); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0120; Bsl versus ON: P = 0.0120; OFF versus ON: P = 0.0003. n = 8 mice. Box plots; lines, individual mice. (h) PSD of the EEG during phasic θ events and remaining REM sleep (tonic θ) with (ON) and without laser (OFF) in iC++ mice. Peak frequency, two-way RM ANOVA with type of REM (tonic or phasic, P = 0.0000) and laser (ON or OFF) as within-subjects factors (interaction, 0.2849); t-tests with Holm-Bonferroni correction, tonic versus phasic: OFF, P = 0.0000; ON, P = 0.0000. EEG θ power, two-way RM ANOVA (type, P = 0.0001; interaction, P = 0.0953); t-tests with Holm-Bonferroni correction; tonic versus phasic: OFF, P = 0.0003; ON, P = 0.0003. n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (i) Heart rate during phasic θ events and remaining REM sleep (tonic θ) with (ON) and without laser (OFF) in iC++ mice. Two-way RM ANOVA (type, P = 0.0001; interaction, P = 0.4153); t-tests with Holm-Bonferroni correction, tonic versus phasic: OFF, P = 0.0182; ON, P = 0.0123; n = 5 mice. Box plots; lines, individual mice; bpm, beats per minute. (j) Frequency of phasic θ events as function of time in REM sleep (since REM sleep onset) in baseline recordings from ChR2 and iC++ mice without laser stimulation. For each mouse, we averaged the frequency of phasic θ events across all REM sleep episodes for each 25 s time bin on the x-axis. Each dot corresponds to the mean frequency of one animal. Robust regression; P = 0.0000; n = 14 mice. The line and shading indicate the robust regression fit and 95% CI. (k) Mean duration of all REM sleep episodes (averaged across laser-on and laser-off episodes) in closed-loop experiments for ChR2, iC++ mice, and eYFP controls for activation (eYFP1) and inhibition experiments (eYFP2). One-way ANOVA (P = 0.8359); eYFP1, n = 8; ChR2, n = 11; eYFP2, n = 8; iC++, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 4 mPFC Pyr neuron activity promotes rapid EMs during REM sleep.

(a) Schematic illustrating pupil recordings using an infrared (IR) camera in head-fixed animals. (b) Example of REM sleep with phasic θ events in the EEG and rapid EMs. Two phasic θ events are shown in red in the raw EEG (top) and indicated by dashed lines in the EEG spectrogram (middle). Rapid EMs (blue ticks) are identified as sudden accelerations in the pupil speed (bottom). Inset, consecutive movie frames including a rapid EM. Red circles, current pupil position; blue circles, pupil position in previous video frame; scale bars; 200 µV, 1 s; px/s, pixels per second. (c) Inter-EM distribution calculated for all REM sleep periods in recordings without laser stimulation (n = 12 mice). Red line, threshold for defining EM bursts (250 ms). (d) Frequency of all EMs and EM bursts during phasic θ events and randomly selected epochs of equal duration within REM sleep in baseline recordings without laser stimulation. All EMs, paired t-test, P = 0.0000; EM bursts, paired t-test, P = 0.0000. n = 12 mice. Box plots; dots, individual mice. (e) Duration of REM sleep episodes with (ON) or without (OFF) laser activation of ChR2-expressing mPFC Pyr neurons and REM sleep duration in baseline recordings without laser stimulation (Bsl) in the same animals. One-way RM ANOVA with laser as within-subjects factor (P = 0.0000); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.2029; Bsl versus ON: P = 0.0016; OFF versus ON: P = 0.0005. n = 6 mice. Box plots; lines, individual mice. (f) Frequency of phasic θ events during laser-on and laser-off REM sleep episodes and episodes in baseline recordings without laser in ChR2 mice. One-way RM ANOVA (P = 0.0012); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.1183; Bsl versus ON: P = 0.0427; OFF versus ON: P = 0.0173. n = 6 mice. Box plots; lines, individual mice. (g) Frequency of EMs and EM bursts during laser-on, laser-off and baseline REM sleep episodes in ChR2 mice. Note that REM sleep episodes with duration < 20 s were excluded from this analysis, as EMs only occur with a delay of about 20 s after the onset of spontaneous REM sleep24. All EMs, one-way RM ANOVA (P = 0.0140); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0483; Bsl versus ON: P = 0.0483; OFF versus ON: P = 0.0483. EM bursts, one-way rm ANOVA (P = 0.0030); t-tests; Bsl versus OFF: P = 0.0493; Bsl versus ON: P = 0.0493; OFF versus ON: P = 0.0493. n = 6 mice. Box plots; lines, individual mice. (h) Latency between the onset of REM sleep and the first EM for laser-on and laser-off episodes and episodes in baseline recordings without laser in ChR2 mice. One-way RM ANOVA with laser as within-subjects factor (P = 0.0682). n = 6 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (i) Duration of laser-off or laser-on REM sleep episodes and baseline duration in recordings without laser in mice expressing iC++ in mPFC Pyr neurons. One-way RM ANOVA (P = 0.0051); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.1219; Bsl versus ON: P = 0.0395; OFF versus ON: P = 0.0198. n = 6 mice. Box plots; lines, individual mice. (j) Frequency of phasic θ events during laser-on, laser-off, and baseline REM sleep episodes in iC++ mice. One-way RM ANOVA (P = 0.0031); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.6853; Bsl versus ON: P = 0.0272; OFF versus ON: P = 0.0495. n = 6 mice. Box plots; lines, individual mice. (k) Frequency of EMs and EM bursts during laser-on, laser-off, and baseline REM sleep episodes in iC++ mice. Note that REM sleep episodes with duration < 20 s were excluded from this analysis. All EMs, one-way RM ANOVA (P = 0.0000); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0356; Bsl versus ON: P = 0.0042; OFF versus ON: P = 0.0005. EM bursts, one-way rm ANOVA (P = 0.0000); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0353; Bsl versus ON: P = 0.0048; OFF versus ON: P = 0.0003. n = 6 mice. Box plots; lines, individual mice. (l) Latency between the onset of REM sleep and the first EM for laser-on, laser-off, and baseline episodes in iC++ mice. One-way RM ANOVA (P = 0.8167). n = 6 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (m) Effect of mPFC Pyr neuron activation on the frequency of all EMs and EM bursts for different durations of baseline and laser-on episodes. All EMs, two-way ANOVA with laser (Bsl or ON, P = 0.0000) and REM bout duration as between-subjects factors (interaction, P = 0.0036); t-tests with Holm-Bonferroni correction; 0–40 s, P = 0.0023; 40–80 s, P = 0.0012; 80–120 s, P = 0.3496; > 120 s, P = 0.9968. EM bursts, two-way ANOVA (laser, P = 0.0001; interaction, P = 0.0130); t-tests; 0–40 s, P = 0.0212; 40–80 s, P = 0.0197; 80–120 s, P = 0.8791; > 120 s, P = 0.8791. Bsl, n = 434; ON, n = 240 episodes. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (n) Effect of mPFC Pyr neuron inhibition on the frequency of all EMs and EM bursts for different durations of baseline and laser-on episodes. All EMs, two-way ANOVA (laser, P = 0.0003; interaction, P = 0.0052); t-tests with Holm-Bonferroni correction; 0–40 s, P = 0.1656; 40–80 s, P = 0.0724; 80–120 s, P = 0.0362; > 120 s, P = 0.0088. EM bursts, two-way ANOVA (P = 0.0004; interaction, P = 0.0017); t-tests; 0–40 s, P = 0.2412; 40–80 s, P = 0.1671; 80–120 s, P = 0.0096; > 120 s, P = 0.0096. Bsl, n = 501; ON, n = 216 episodes. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 5 Effects of optogenetic activation of mPFC inhibitory neurons on brain state and EEG.

(a) Placement of optic fibers in the mPFC for ChR2-mediated activation of mPFC Vgat neurons. Each colored bar represents the location of an optic fiber. Brain atlas images adapted with permission from ref. 61. (b) Laser stimulation trials from all n = 7 control mice expressing eYFP in mPFC Vgat neurons, sorted depending on the brain state at laser onset (t = 0 s, arrows). (c) Percentages of all brain states before, during, and after open-loop stimulation in eYFP control mice. Blue patch, laser stimulation interval (120 s, 20 Hz). Two-way RM ANOVA comparing the mean percentage of each brain state between the laser and preceding 120 s baseline interval (interaction, P = 0.1228). n = 7 mice. Lines, averages across mice; shadings, 95% CIs. (d) Cumulative transition probabilities in ChR2 mice to remain in REM sleep (R → R), wake (W → W), or NREM sleep (N → N) during the laser and preceding 120 s baseline interval. Bootstrap, R → R, P = 0.0001; W → W, P = 0.0234; N → N, P = 0.0004. n = 8 mice. Shadings, 95% CIs. (e) Differences of laser-induced changes in the cumulative transition probabilities between ChR2 and eYFP mice (Methods). Bootstrap; N → R, P < 0.0001; R → W, P = 0.0318; N → W, P = 0.6890; W → N, P = 0.0300; R → R, P = 0.0318; W → W, P = 0.0272; N → N, P = 0.0014. ChR2, n = 8; eYFP, n = 7 mice. Blue lines, 95% CIs; white dots, distribution means. (f) PSD of the EEG during REM sleep with (ON) and without laser stimulation (OFF). Two-way RM ANOVA with laser (P = 0.0135) and frequency band (δ, θ, or σ) as within-subjects factors (interaction, P = 0.0002); t-tests with Holm-Bonferroni correction; OFF versus ON: δ, P = 0.0068; θ, P = 0.0059; σ, P = 0.0068. n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (g) Laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA with frequency band and virus as within- and between-subjects factors (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0073; θ, P = 0.0022; σ, P = 0.0073. ChR2, n = 8; eYFP, n = 7 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 6 Axonal projections of mPFC Pyr neurons and molecular identity of mPFC→LH neurons.

(a) Schematic of anterograde tracing experiment. AAV-CaMKII-Cre and AAV-DIO-tdTomato were injected into the mPFC of C57BL/6J mice to label axonal projections of mPFC Pyr neurons (n = 3 mice). (b) Injection site in the mPFC. Scale bar, 500 μm. Histology pictures in (b-g) are from the same animal. (c) tdTomato-labeled axon fibers in the thalamus. MD, mediodorsal thalamic nucleus; VA, ventral anterior thalamic nucleus; AM, anteromedial thalamic nucleus; Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bar, 500 μm. (d) tdTomato-labeled axon fibers in the basolateral amygdala (BLA). Scale bar, 500 μm. (e) Axon fibers in the lateral hypothalamus (LH). Scale bar, 500 μm. (f) Axon fibers in the ventrolateral, lateral and dorsolateral periaqueductal gray (VLPAG, LPAG and DLPAG). Scale bar, 500 μm. (g) Axon fibers in the dorsal pons. LDTg, laterodorsal tegmental nucleus. Scale bar, 500 μm. (h) Top, schematic depicting injection of AAVrg-Cre into the LH of a C57BL/6J mouse. Bottom, fluorescence in situ hybridization (FISH) for Cre in the mPFC. Scale bar, 500 μm. (i) Double FISH was performed for Cre and Slc17A7 (gene encoding vesicular glutamate transporter 1), Slc32a1 (gene encoding vesicular GABA transporter), or Npr3. Top, fluorescence images showing Cre, Slc17A7 probe, and overlay of both channels. Middle, double FISH for Cre and Slc32a1. Bottom, double FISH for Cre and Npr3. Arrowheads indicate co-labeled cells. Scale bars, 25 μm. (j) Percentages of Cre-expressing neurons in the mPFC that also express Slc17A7, Slc32a1, or Npr3. Dots, data from single mice (Slc17A7, n = 4; Slc32a1, n = 4; Npr3, n = 2 mice). Mouse brain figures in (a,h) adapted with permission from ref. 61.

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Extended Data Fig. 7 Effects of optogenetic stimulation of axonal fibers of mPFC Pyr neurons in LH, LDTg, MD, and PAG.

(a) Top, schematic depicting ChR2-mediated activation of axons of mPFC Pyr neurons in the LH. Bottom, fluorescence images showing expression of ChR2-eYFP in the mPFC (left) and optic fiber tract in the LH (right). Scale bars, 500 μm. (b) Placement of optic fibers in the LH for laser-activation of mPFC projections. Each colored bar represents the location of an optic fiber. (c) Percentages of brain states before, during, and after laser stimulation. Blue bar, laser stimulation interval (120 s, 5 Hz). Two-way RM ANOVA comparing the mean percentage of each brain state between the laser and preceding 120 s baseline interval (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; REM, P = 0.0005; Wake, P = 0.0184; NREM, P = 0.0001. n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (d) Graph summarizing changes in the cumulative transition probabilities induced by activation of the mPFC-LH projections. Solid and dashed lines indicate significant and nonsignificant changes in the transition probabilities, respectively. (e) Top, laser-trial-averaged normalized EEG spectrogram (Methods). Bottom, time course of δ, θ, and γ power before, during, and after laser stimulation. Two-way RM ANOVA comparing the mean power of each frequency band between the laser and preceding 120 s baseline interval (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; baseline versus laser: δ, P = 0.0000; θ, P = 0.0013; γ, P = 0.0001. n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (f) Duration of REM sleep episodes with and without closed-loop stimulation of mPFC projections. Paired t-test; P = 0.0003. n = 8 mice. Box plots; lines, individual mice. (g) Frequency of phasic θ events during REM sleep with and without closed-loop stimulation. Paired t-test; P = 0.0005. n = 8 mice. Box plots; lines, individual mice. (h) Top, schematic depicting ChR2-mediated activation of mPFC axons in the laterodorsal tegmental nucleus (LDTg). Bottom, fluorescence images showing expression of ChR2-eYFP in the mPFC (left) and fiber tract in the LDTg (right). Scale bars, 500 μm. (i) Placement of optic fibers in the LDTg. (j) Percentages of brain states before, during, and after laser stimulation. Blue bar, laser stimulation interval. Two-way RM ANOVA (interaction, P = 0.1145). n = 7 mice. Lines, averages across mice; shadings, 95% CIs. (k) Duration of REM sleep episodes with and without closed-loop stimulation. Paired t-test; P = 0.4112. n = 7 mice. Box plots; lines, individual mice. (l) Frequency of phasic θ events during REM sleep with and without closed-loop stimulation. Paired t-test;P = 0.8095. n = 7 mice. Box plots, lines, individual mice. (m) Top, schematic depicting ChR2-mediated activation of mPFC axons in the mediodorsal thalamic nucleus (MD). Bottom, fluorescence images showing expression of ChR2-eYFP in the mPFC (left) and fiber tract in the MD (right). Scale bars, 500 μm. (n) Placement of optic fibers in the MD. (o) Percentages of brain states before, during, and after laser stimulation. Blue bar, laser stimulation interval. Two-way RM ANOVA (interaction, P = 0.7918). n = 7 mice. Lines, averages across mice; shadings, 95% CIs. (p) Duration of REM sleep episodes with and without closed-loop stimulation. Paired t-test; P = 0.8255. n = 7 mice. Box plots; lines, individual mice. (q) Frequency of phasic θ events during REM sleep with and without closed-loop stimulation. Paired t-test; P = 0.0824. n = 7 mice. Box plots; lines, individual mice. (r) Top, schematic depicting ChR2-mediated activation of mPFC axons in the periaqueductal gray (PAG). Bottom, fluorescence images showing expression of ChR2-eYFP in the mPFC (left) and fiber tract in the PAG (right). Scale bars, 500 µm. (s) Placement of optic fibers in the PAG. (t) Percentages of brain states before, during, and after laser stimulation. Blue bar, laser stimulation interval. Two-way RM ANOVA (interaction, P = 0.1030). n = 6 mice. Lines, averages across mice; shadings, 95% CIs. (u) Duration of REM sleep episodes with and without closed-loop stimulation. Paired t-test; P = 0.4870. n = 6 mice. Box plots; lines, individual mice. (v) Frequency of phasic θ events during REM sleep with and without closed-loop stimulation. Paired t-test; P = 0.1491. n = 6 mice. Box plots; lines, individual mice. Brain atlas images in (a,b,h,i,m,n,r,s) adapted with permission from ref. 61. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 8 Effects of laser stimulation in mice expressing ChR2-eYFP or eYFP in mPFC→LH neurons.

(a) Left, heatmaps depicting expression of ChR2-eYFP in the mPFC of mice injected with AAVrg-Cre-mCherry into the LH and AAV-DIO-ChR2-eYFP into the mPFC and location of optic fiber tracts (n = 10 mice). Right, expression of mCherry in the hypothalamus. Brain atlas images adapted with permission from ref. 61. (b) Top, laser-trial-averaged normalized EEG spectrogram in ChR2 mice. Bottom, time course of δ, θ, and γ power before, during, and after laser stimulation. Two-way RM ANOVA comparing the mean power in each frequency band between the laser and preceding 120 s baseline interval (interaction, P = 0.0001); t-tests with Holm-Bonferroni correction; baseline versus laser: δ, P = 0.0005; θ, P = 0.0005; γ, P = 0.0005. n = 10 mice. Lines, averages across mice; shadings, 95% CIs. (c) Laser stimulation trials from all control mice (n = 9) expressing eYFP in mPFC→LH neurons, sorted depending on the brain state at laser onset (t = 0 s, arrows). (d) Effect of open-loop stimulation on brain states before, during, and after laser stimulation in eYFP control mice. Two-way RM ANOVA comparing the mean percentage of each brain state between the laser and preceding 120 s baseline interval (interaction, P = 0.4312). n = 9 mice. Lines, averages across mice; shadings, 95% CIs. (e) Changes in the percentage of each brain state (difference between the 120 s baseline and laser interval) induced by laser stimulation in mice expressing ChR2 or eYFP in mPFC→LH neurons. Mixed ANOVA with brain state as within- and virus as between-subjects factor (interaction, P = 0.0000); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: REM, P = 0.0004; wake, P = 0.2857; NREM, P = 0.0005. ChR2, n = 10 mice; eYFP, n = 9 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (f) Cumulative transition probabilities in ChR2 mice to remain in REM sleep (R → R), wake (W → W), or NREM sleep (N → N) during the laser and preceding 120 s baseline interval. Bootstrap; R → R, P = 0.0001; W → W, P = 0.8396; N → N, P = 0.0001; n = 10 mice. Shadings, 95% CIs. (g) Differences of laser-induced changes in the cumulative transition probabilities between ChR2 and eYFP mice (Methods). Bootstrap; N → R, P = 0.0001; R → W, P = 0.0001; N → W, P = 0.0100; W → N, P = 0.8448; R → R, P = 0.0001; W → W, P = 0.8364; N → N, P = 0.0001. n = 10 mice. Blue lines, 95% CIs; white dots, distribution means. (h) Duration of REM sleep episodes with (ON) or without (OFF) laser activation of ChR2-expressing mPFC→LH neurons and REM sleep duration in baseline recordings without laser stimulation (Bsl) in the same animals. One-way RM ANOVA (P = 0.0000); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.0024; Bsl versus ON: P = 0.001; OFF versus ON: P = 0.0005. n = 10 mice. Box plots; lines, individual mice. (i) Laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and ChR2 mice. Mixed ANOVA with frequency band as within-subjects factor and virus (eYFP or ChR2) as between-subjects factor (interaction, P = 0.0001); t-tests with Holm-Bonferroni correction; eYFP versus ChR2: δ, P = 0.0021; θ, P = 0.0524; σ, P = 0.1380. ChR2, n = 10; eYFP, n = 9 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 9 Effects of opto- and chemogenetic inhibition of mPFC→LH neurons on EEG and brain state.

(a) Left, heatmaps depicting expression of iC++-eYFP in the mPFC of mice injected with AAVrg-Cre-mCherry into the LH and AAV-DIO-iC++-eYFP into the mPFC and location of bilateral optic fiber tracts (n = 10 mice). Right, expression of mCherry in the hypothalamus. (b) Top, laser-trial-averaged normalized EEG spectrogram in iC++ mice. Bottom, time course of δ, θ, and γ power before, during, and after laser stimulation. Two-way RM ANOVA comparing the mean power in each frequency band between the laser and preceding 120 s baseline interval (interaction, P = 0.0004); t-tests with Holm-Bonferroni correction; baseline versus laser: δ, P = 0.0117; θ, P = 0.0023; γ, P = 0.0023. n = 10 mice. Lines, averages across mice; shadings, 95% CIs. (c) Laser stimulation trials from all n = 8 control mice expressing eYFP in mPFC→LH neurons, sorted depending on the brain state at laser onset (t = 0 s, arrows). (d) Effect of open-loop stimulation on brain states before, during, and after laser stimulation in eYFP control mice. Two-way RM ANOVA comparing the mean percentage of each brain state between the laser and preceding 120 s baseline interval (interaction, P = 0.2183). n = 8 mice. Lines, averages across mice; shadings, 95% CIs. (e) Changes in the percentage of each brain state (difference between the 120 s baseline and laser interval) induced by laser stimulation in mice expressing iC++ or eYFP in mPFC→LH neurons. Mixed ANOVA with brain state as within- and virus as between-subjects factor (interaction, P = 0.0077); t-tests with Holm-Bonferroni correction; eYFP versus iC++: REM, P = 0.0012; wake, P = 0.0456; NREM, P = 0.3301. iC++, n = 10; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (f) Cumulative transition probabilities in iC++ mice to remain in REM sleep (R → R), wake (W → W), or NREM sleep (N → N) during the laser and preceding 120 s baseline interval. Bootstrap; R → R, P = 0.0622; W → W, P = 0.3630; N → N, P = 0.4758; n = 10 mice. Shadings, 95% CIs. (g) Differences of laser-induced changes in the cumulative transition probabilities between iC++ and eYFP mice (Methods). Bootstrap; N → R, P = 0.0032; R → W, P = 0.6490; N → W, P = 0.0388; W → N, P = 0.7432; R → R, P = 0.6490; W → W, P = 0.7432; N → N, P = 0.4762. iC++, n = 10; eYFP, n = 8 mice. Blue lines, 95% CIs; white dots, distribution means. (h) Duration of REM sleep episodes with (ON) or without (OFF) inhibition of iC++-expressing mPFC→LH neurons and REM sleep duration in baseline recordings without laser stimulation (Bsl) in the same animals. One-way RM ANOVA (P = 0.0165); t-tests with Holm-Bonferroni correction; Bsl versus OFF: P = 0.3334; Bsl versus ON: P = 0.0107; OFF versus ON: P = 0.0620. n = 5 mice. Box plots; lines, individual mice. (i) Laser-induced changes in the δ, θ, and σ power (Δ power) between REM sleep with and without laser stimulation in eYFP and iC++ mice. Mixed ANOVA with frequency band as within-subjects factor and virus (eYFP or iC++) as between-subjects factor (interaction, P = 0.0503). iC++, n = 10; eYFP, n = 8 mice. Bars, averages across mice; dots, individual mice; error bars, 95% CIs. (j) Chemogenetic inhibition of mPFC→LH neurons. Left, schematic depicting viral strategy to express AAV-DIO-hM4D(Gi) or AAV-DIO-mCherry in mPFC→LH neurons. Right, fluorescence image showing expression of mCherry in the mPFC. Scale bar, 500 µm. (k) Left, heatmaps depicting expression of mCherry in the mPFC of mice injected with AAVrg-Cre-mCherry into the LH and AAV-DIO-hM4D(Gi)-mCherry into the mPFC (n = 9 mice). Right, expression of mCherry in the hypothalamus. (l) Percentage of time spent in REM, NREM sleep, and wake and ratio of REM to total sleep (REM + NREM) during 4 h recordings in hM4D(Gi) and mCherry mice following CNO (5 mg/kg) injection. T-tests; REM, P = 0.0029; wake, P = 0.1213; NREM, P = 0.0254; ratio, P = 0.0007. hM4D(Gi), n = 9; mCherry, n = 7 mice. Box plots; dots, individual mice. (m) Effects of chemogenetic inhibition on the duration and frequency of REM sleep. T-tests; duration, P = 0.0285; frequency, P = 0.3380. hM4D(Gi), n = 9; mCherry, n = 7 mice. Box plots; dots, individual mice. (n) Frequency of phasic θ events during REM sleep. T-test; P = 0.0052. hM4D(Gi), n = 9; mCherry, n = 7 mice. Box plots; dots, individual mice. Brain atlas images in (a,j,k) adapted with permission from ref. 61. See Supplementary Table 1 for detailed statistical information. *P < 0.05, **P < 0.01, ***P < 0.001.

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Extended Data Fig. 10 Calcium imaging of mPFC→LH neurons.

(a) Position of GRIN lenses within the mPFC used for imaging of mPFC→LH neurons. Each diagram depicts the section where the lesion caused by the GRIN lens (colored bar) was largest (n = 8 mice). Brain atlas images adapted with permission from ref. 61. (b) Normalized cross-correlation between EEG θ (6.0–9.5 Hz) or σ power (10–15 Hz) and ΔF/F activity of different mPFC→LH neuron subclasses during NREM sleep (Methods). One-sample t-test; θ: R>W>N, P = 0.0059; R>N>W, P = 0.0000; Wake-max, P = 0.0000; σ: R>W>N, P = 0.0002; R>N>W, P = 0.0000; Wake-max, P = 0.0000; R>W>N, n = 36; R>N>W, n = 43; Wake-max, n = 34. Lines, mean for the different cell subclasses. Shadings, ± standard error of the mean (s.e.m). (c) Correlation between REM sleep duration and calcium activity of R>W>N and R>N>W neurons. Each dot represents the mean activity of one cell during a single REM sleep episode (R>W>N, 36 cells, n = 212 episodes; R>N>W, 43 cells, n = 240 episodes). Line, linear fit using robust regression. Robust regression; R>W>N, P = 0.0002, r = 0.1860; R>N>W, P = 0.2837, r = 0.0947. Shadings, 95% CIs. (d) Activity of Wake-max cells during phasic θ events (onset at t = 0 s). Paired t-test; P = 0.5761; n = 34 cells. Line, average across mice; shadings, ± s.e.m. See Supplementary Table 1 for detailed statistical information.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Supplementary Tables 3 and 4, and legends for Supplementary Tables 1 and 2 and Supplementary Videos 1–3.

Reporting Summary

Supplementary Table 1

Statistical results for figures and extended data figures.

Supplementary Table 2

Statistical information on imaged mPFC → LH cells (related to Fig. 5).

Supplementary Video 1

Induction of REM sleep by optogenetic activation of mPFC Pyr neurons.

Supplementary Video 2

Rapid EMs during REM sleep.

Supplementary Video 3

In vivo calcium imaging of mPFC → LH neurons.

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Hong, J., Lozano, D.E., Beier, K.T. et al. Prefrontal cortical regulation of REM sleep. Nat Neurosci 26, 1820–1832 (2023). https://doi.org/10.1038/s41593-023-01398-1

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