Fig. 4: DRNGAD2 neurons constrain wakefulness in acute stress condition.
From: A midbrain GABAergic circuit constrains wakefulness in a mouse model of stress
a Schematic of a 10 min restraint stress session following c-Fos staining in DRNGAD2-mCherry mice. b Representative images showing c-Fos expression in DRNGAD2-mCherry mice from control (left) or restraint group (right). Zoomed images are enlarged views of the dashed areas. Arrowheads indicate mCherry and c-Fos co-labeled neurons. Scale bars in the right panels are 25 μm. Aq, aqueduct. c Number of mCherry and c-Fos co-labeled neurons in control and restraint group. n = 5 mice for each group, two tailed unpaired t test, t8 = 11.056, P = 3.99 × 10−6. d Schematic of a 10 min restraint stress session and the following EEG/EMG recordings in DRNGAD2-mCherry or DRNGAD2-DTA mice. e NREM sleep onset latency following a 10 min restraint session. n = 6 for mCherry mice and n = 7 for DTA mice, two tailed unpaired t test, t11 = 3.638, P = 0.0039. f Time spent in wake, NREM sleep and REM sleep. n = 6 for mCherry mice and n = 7 for DTA mice, two tailed unpaired t test, wake: t11 = 3.554, P = 0.00452, NREM: t11 = 3.561, P = 0.00446; REM: t11 = 2.374, P = 0.0369. g Schematic of experimental protocol for c-Fos-based activity tagging method to label restraint stress-activated DRNGAD2 neurons with TRE-hM3Dq-mCherry. h, i Representative images (h) and quantification (i) of the specificity of TRE-hM3Dq-mCherry to label restraint stress-activated DRNGAD2 neurons. Arrowheads indicate TRE-hM3Dq-mCherry and c-Fos co-labeled neurons. n = 7 mice in (i). j NREM sleep onset latency following a 10 min restraint session. n = 9 mice, Wilcoxon signed rank test, W = 45, P = 0.004. k Time spent in wake, NREM sleep and REM sleep post saline or CNO injections in restraint stress conditions. n = 9 mice, two tailed paired t test, wake: t8 = 6.708, P = 1.51 × 10−4; NREM: t8 = 6.618, P = 1.66×10−4. *P < 0.05, **P < 0.01, ***P < 0.001. Data (c, e, f, i–k) are presented as mean ± SEM.
