Stimulation of Hcrt neurons is sufficient to increase sleep-to-wake transitions across the inactive period. A, B, Latencies of wake transitions after stimulation during SWS (A) or REM sleep (B) of Hcrt::ChR2–mCherry transduced animals (n = 6) or Hcrt::mCherry control animals (n = 6). Photostimulation consisted of 15 ms light pulses at either 1 or 20 Hz for 10 s. Photostimulation periods took place at 9:00 A.M. to 1:00 P.M., 1:00 P.M. to 5:00 P.M., or 5:00 P.M. to 9:00 P.M., with the 12 h light inactive period starting at 9:00 A.M. Data analysis is based on an average of 30 or 10 stimulations per frequency and per mouse during SWS and REM sleep, respectively. Latencies represent mean ± SEM. Two-way ANOVA between stimulation parameter and time of day revealed no significant difference between latencies in Hcrt::ChR2–mCherry animals across the inactive period (p > 0.05). However, we found significant differences in the latencies between ChR2–mCherry and mCherry control animals at 20 Hz stimulation at all times tested (*p < 0.05, ***p < 0001 using a two-tailed Student's t test). C, D, Cumulative probability distribution of latencies from SWS (C) or REM sleep (D) after light stimulation (ChR2–mCherry animals, blue curves; mCherry control animals, red curves). The 20 Hz stimulation resulted in a significant shift in the probability curve for both SWS (p < 0.001, KS test) and REM (p < 0.001, KS test). E, Chronic stimulation of Hcrt neurons is sufficient to increase transitions from SWS to wakefulness across the circadian period. We used a chronic stimulation protocol (15 ms light pulses for 10 s every minute for 1 h, 20 Hz stimulation) to stimulate Hcrt::ChR2–mCherry mice (n = 4) or Hcrt::mCherry control mice (n = 4) at 4 h intervals throughout the entire circadian period. y-Axis represents the mean ± SEM number of transitions from SWS to wakefulness divided by the baseline number of transitions during that time interval. Analysis is based on duplicated stimulation sessions for each animal. *p < 0.05 using a two-tailed Student's t test between transduced animals. F, Chronic stimulation of Hcrt neurons is insufficient to increase transitions from REM to wakefulness throughout the circadian period. Same experimental conditions as E.
