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.
