Fig. 3
Optogenetic activation of NAc D1R neurons induces a rapid transition from NREM sleep to wakefulness. a Diagram of experiment for in vivo optical stimulation. b Brain section was stained against mCherry and c-Fos to confirm that the ChR2 protein was expressed in the NAc, and arrows indicate the tip of the optical fiber above the NAc. Scale bar: 1 mm. c Higher magnification image of white box in (b) indicates abundant c-Fos immunoreactivity in ChR2-mCherry neurons following photostimulation. Scale bar: 50 μm. d, g Representative EEG/EMG traces, heat map of EEG power spectra show that acute photostimulation (20 Hz/5 ms) applied during NREM sleep induced a transition to wake in a ChR2-mCherry mouse. Scale bar: 8 s. e, h EEG power (mean) before (red trace) and after (blue trace) onset of photostimulation in ChR2-mCherry (e) or mCherry (h) mice. Gray lines indicate statistical differences (n = 5 per group; P < 0.05, paired t test). f, i Heat maps showing the mean probability of NREM sleep-to-wake transitions induced by photostimulation in ChR2-mCherry (f) or mCherry (i) mice, n = 5 per group. Quantification based on an average of 6–12 stimulations per condition per mouse. Stim stimulation. j Latencies of transitions from NREM sleep to wakefulness after photostimulation at different frequencies (n = 6 per group, unpaired t test; Base, t10 = 0.4, P = 0.7; 5 Hz, t10 = 1.3, P = 0.2; 10 Hz, t10 = 5.5, P = 3 × 10−4; 20 Hz, t10 = 18.8, P = 4 × 10−9; 30 Hz, t10 = 19.5, P = 3 × 10−9; 50 Hz, t10 = 26.4, P = 1 × 10−10). k Time course of wakefulness during semi-chronic optogenetic experiment (20 Hz/5 ms, 10 s on/20 s off). The blue column indicates the photostimulation period of the stimulation group (n = 5, repeated-measures ANOVA; F1,8 = 10.5, P = 0.01). l Total amounts of each stage in control and photostimulation groups (n = 5, paired t test; t4 = 3.7 (wake), 3.5 (NREM), 5.0 (REM); P = 0.02 (wake), 0.03 (NREM), 0.007 (REM)). Data represent mean ± s.e.m. *P < 0.05, **P < 0.01
