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.
