Fig. 3.

Activity recordings of orexin neurons across vigilance states in prepro-orexin knockout [orexin-Flp (KI/KI)] mice as determined by fiber photometry. (A) Schematics illustrating the injection of AAV-CMV-dFRT-GCaMP6s into homozygous orexin-Flp (KI/KI) mice to induce expression of GCaMP6s expression in orexin neurons (Left) and fiber photometry with EEG/EMG recordings (Middle). Histochemical confirmation of GCaMP6 expression in orexin neurons (Right). The dashed line at the top of the panel indicates the edge of the optic fiber. (B) Schematic of the experimental procedures and data analysis. (C) Representative orexin neural activity across vigilance states measured by fiber photometry. (D) Summary of orexin neuron activity across vigilance states, expressed as Z-scores (all stages from N = 5 mice). (E) Activity of orexin neurons from all 121 NREM to REM transitions recorded (each stage > 1 min) during 24-h recordings in N = 5 mice. (F, Top) Mean Z-score of all the transitions in each individual mouse (gray); mean Z-score of all the N = 5 mice (red). (F, Bottom) Summary of orexin neuron activity of all the mice during NREM–REM state transitions shown as Z-scores. Values are the mean ± SEM. *P < 0.05, **P < 0.01. Statistical analyses are shown in SI Appendix, Table S1. Vigilance state parameters of orexin-Flp (KI/KI) mice (n = 5) in the fiber photometry experiments (24 h) and the 10-min episodes for Z-score (all stages) are described in SI Appendix, Tables S2 and S3, respectively. tNR, the last 30 s during the transition from NREM to REM sleep; PMT, photomultiplier tube; W, wakefulness; NR, NREM; R, REM.