Activation of the LHb–RMTg pathway is required for the effects of chronic stress on wakefulness.

(A) Left: time course changes of REM sleep and wakefulness of mice in different experimental groups. All animals received exposure to chronic stress stimuli (CS), RMTg injection of rAAV2/2–Retro–Cre and i.p injection of CNO (1 mg/kg). CS+EGFP–CNO (n = 6 animals): mice that received LHb injection of AAV2/9–DIO–EGFP. CS+hM4Di–CNO (n = 7 animals): mice that received LHb injection of AAV2/9–DIO–hM4Di–EGFP. Middle: total REM sleep and wakefulness amounts during the whole day (24 h) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. Right: REM sleep and wakefulness amounts during the whole day (24 h), light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. (B) Number of transitions between different pair of brain states during the light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO (n = 6 animals) and CS+hM4Di–CNO (n = 7 animals) groups. W–N: Wake to NREM; N–W: NREM to Wake; N–R: NREM to REM. (C) Left: time course changes of REM sleep and wakefulness of mice in different experimental groups (n = 7 animals/group). All animals received exposure to CS, LHb injection of AAV2/1–Cre and i.p. injection of CNO (1 mg/kg). CS+EGFP–CNO: mice that received RMTg injection of AAV2/9–DIO–EGFP. CS+hM4Di–CNO: mice that received RMTg injection of AAV2/9–DIO–hM4Di–EGFP. Middle: total REM sleep and wakefulness amounts during the whole day (24 h) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). Right: REM sleep and wakefulness amounts during the whole day (24 h), light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). (D) Number of transitions between different pair of brain states during the light phase (ZT 0 –ZT 12) and dark phase (ZT 12 –ZT 24) of mice in CS+EGFP–CNO and CS+hM4Di–CNO groups (n = 7 animals/group). For all figures: one–way ANOVA with Sidak’s multiple comparisons test, *, P P P (TIF)