FIGURE 4.

Activation of PB glutamatergic neurons during the inactive period (10:00 or ZT3). (A) Extent of transduced neurons (mCherry-positive somas) is shown for individual Vglut2-IRES-cre mice that received bilateral injections of hM3Dq-mCherry-AAV into the PB (PB^{Vglut2–hM3Dq}). (B1) PB^{Vglut2–hM3Dq} whole-cell recording showing an increase in firing frequency in response to bath application of CNO (0.5 μM). (B2) Average firing frequency (±S.E.M.) during the last 2 min of CNO (0.5 μM) application as compared with the 2 min period preceding CNO application (control; N = 6 PB^{Vglut2–hM3Dq} neurons). ^*p < 0.05 Paired Student’s t-tests. (C) Hourly amount of wakefulness (C1), SWS (C2) and REM (C3) sleep following CNO (0.3 mg/kg, N = 5 mice) as compared with control injection. (D1–D3) Percentage of sleep-wake states (±S.E.M.) during the 5 h post-injection period (10:00–15:00), the following 4 h of the light period (15:00–19:00), the subsequent 12 h dark/wake period (19:00–07:00) and first 3 h of the light period of the next day (07:00–10:00, N = 5 mice). (E1,E2) Number of episodes (±S.E.M.) of wakefulness (W) or SWS in each bout length and (E1’,E2’) time-weighted frequency histograms showing the proportion (±S.E.M.) of W or SWS amounts in each bout length as a percentage of the total amount of W or SWS during the 9 h post-injection period (10:00–19:00; N = 6). (E3) Number of episodes (±S.E.M.) of REM sleep (RS) in each bout length and (E3’) time-weighted frequency histograms showing the proportion (±S.E.M.) of RS amounts in each bout length as a percentage of the total amount of W or SWS during the 5 h post-injection period (10:00–15:00; N = 6). (F1)Wake power spectrum changes over baseline during the 3 h (10:00–13:00) post CNO (0.3 mg/kg, N = 5 mice) injection period as compared with control injection. (F2) Quantitative changes (±S.E.M.) in power for the δ (0.4–5 Hz), θ (5–9 Hz), α (9–15 Hz), β (15–30 Hz), low γ (30–60 Hz) and high γ (60–120 Hz) frequency bands (±S.E.M.) following vehicle or CNO (0.3 mg/kg, N = 5 mice) administrations. Power spectral analysis was not performed for slow-wave-sleep and REM sleep due to the low amount of these vigilance stages. (C–F) Control injection in Black, CNO injection in green; ^*p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^*⁣*⁣**p < 0.0001, two-way ANOVA followed by a post hoc Bonferroni test.