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.
