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.
