Fig 1.
Recombination of Ai35D and ChAT-Cre mice.
(A) Diagram of heterozygous mice with conditional expression of Arch-GFP after recombination of the Stop-loxP gene and ChAT-Cre recombinase. (B) PCR analysis. Ai35D heterozygous mice had one 242-bp and one 246-bp band, while WT mice had only one 242-bp band. The positive Cre band was at 350-bp.
Fig 2.
Specific and efficient expression of Arch-GFP in cholinergic BF neurons of ChAT-Arch mice.
(A–C) Representative photomicrographs of the BF depicting Arch-GFP expression (A, green), ChAT immunoreactivity (B, red), and overlaid images (C) from a ChAT-Arch mouse. Scale bars, 50 μm. (D) Statistics of the total co-expression of GFP and ChAT immunofluorescence from ChAT-Arch mice (n = 5) in 30-μm sections from the rostral-to-caudal ends of the BF (anteroposterior, -0.4 to -0.8). Cell counts are represented as mean ± s.e.m. (E) Yellow light inhibits the activity of Arch-expressing cholinergic BF neurons. Action potentials recorded under current-clamp from a neuron expressing Arch-GFP in a BF slice in response to 20 Hz yellow light pulse trains (20 ms per pulse) or continuous yellow light for 30 s (upper trace). Action potentials recorded under current-clamp from a neuron expressing Arch-GFP in a BF slice in response to 20 Hz yellow light pulse trains (20 ms per pulse) or continuous yellow light for 60 s (lower trace). (F) Representative photomicrographs of Arch-GFP expression in BF from a ChAT-Arch mouse. White arrow indicated a fiberoptic lesion track. 3V, 3rd ventricle; D3V, dorsal 3rd ventricle. Scale bars, 200 μm.
Fig 3.
Photoinhibition of cholinergic BF neurons during wakefulness, SWS, and REM sleep in the inactive period.
(A–D) Left panel, representative EEG and EMG recordings showing that photoinhibition (yellow bars, continuous yellow light for 30 s) applied during waking (B), SWS (C), or REM sleep (D) in a ChAT-Arch mouse and during waking (A) in a wild-type mouse. Right panel, relative cortical EEG power (0.38-Hz binned frequencies, 30-s duration) at baseline (black trace) and after the onset of photostimulation (yellow trace) in ChAT-Arch mice. n = 3 mice, 10 stimulations per mouse in A–D. p >0.05, two-tailed Student’s t test between baseline and ChAT-Arch mice with bilateral inactivation of cholinergic BF neurons.
Fig 4.
Silencing of cholinergic BF neurons prolongs SWS duration but does not affect REM sleep.
(A, C) Duration of individual SWS episodes at baseline and 30-s unilateral or bilateral continuous inactivation during the inactive period (A) and active period (C). (B, D) Duration of individual REM sleep episodes at baseline and 30-s unilateral or bilateral continuous inactivation during the inactive period (B) and active period (D). The experiments were performed from 13:00 to 17:00 during the inactive period and from 22:00 to 02:00 during the active period. The baseline was determined by analyzing the duration of individual SWS or REM sleep episodes about the same time on the day before inactivation. n = 5 mice, *p <0.05, **p <0.01, two-way ANOVA between stimulation condition and animal type followed by Tukey’s post-hoc test.
Fig 5.
Silencing of cholinergic BF neurons reduces transitions from SWS but does not affect transitions from wakefulness and REM sleep.
(A, D) Percentage probability of transition to wakefulness or REM sleep or maintained SWS from SWS within a single 30-s unilateral or bilateral continuous inactivation in the inactive period (A) and the active period (D). (B, E) Percentage probability of transition to SWS or maintained wakefulness from wakefulness within a single 30-s unilateral or bilateral continuous inactivation in the inactive period (B) and the active period (E). (C, F) Percentage probability of transition to wakefulness or maintained REM sleep from REM sleep within a single 30-s unilateral or bilateral continuous inactivation in the inactive period (C) and the active period (F). n = 6 mice, *p <0.05, **p <0.01, ***p <0.001, one-way ANOVA for unilateral or bilateral continuous inactivation compared with control followed by Tukey post-hoc test.
Fig 6.
Six-hour inactivation of cholinergic BF neurons during the inactive or active period.
(A, B) Time the ChAT-Arch mice spent in wakefulness, SWS, and REM sleep during 24 h with 6-h inactivation (continuous, 60 s, once every 2 min) in the inactive period (13:00–19:00) (A) and the active period (19:00–01:00) (B). Each symbol represents the hourly mean time in each stage. The baseline was determined by analyzing the hourly mean time in each stage on the day before inactivation. (C, D) Percentage of total times the ChAT-Arch mice spent in wakefulness, SWS, and REM sleep during 24 h with and without light inactivation. n = 6 mice, *p <0.05, **p <0.01, two-tailed Student’s t test between baseline and with light inactivation.
Fig 7.
SWS episode duration increases during 6-h inactivation of cholinergic BF neurons during the active period.
(A, B) Numbers of episodes of wakefulness, SWS, and REM sleep during 6 h with and without light inactivation. n = 5 ChAT-Arch mice. (C, D) Durations of episodes of wakefulness, SWS, and REM sleep during 6 h with and without light inactivation. n = 5 ChAT-Arch mice. (E, F) Numbers of episodes of wakefulness during 6 h with and without light inactivation. n = 5 ChAT-Arch mice. (G, H) Numbers of episodes of SWS during 6 h with and without light inactivation. n = 5 ChAT-Arch mice. **p <0.01, ***p <0.001, two-tailed Student’s t test between baseline and with light inactivation.