Fig. 3. Optogenetic inactivation of RMTg GABAergic neurons induced REM sleep generation and inhibited REM sleep transition.

a Schematic illustration of virus injection. Adeno-associated virus (AAV) encoding archaerhodopsin-3 (Arch) fused to an enhanced green fluorescent protein (eGFP) reporter was introduced into the bilateral RMTg. b The coronal section shows the superimposed virus-injected area in five mice. c Yellow laser stimulation induced hyperpolarization in current–clamp mode (top) and outward current in voltage–clamp mode with voltage holding at −60 mV (bottom) in an Arch-eGFP expressing RMTg GABAergic neuron. d The average membrane potential (T₈ = 11.32, p < 0.0001) of RMTg Arch-expressing neurons (N = 9 cells from 3 mice) was significantly decreased by yellow laser illumination. The results from each cell are shown on the scatter plot. e Representative EEG and EMG traces and the corresponding heat map of the EEG power spectrum in different sleep–wake states from a mouse in the eGFP (top) and Arch (bottom) group, respectively. Yellow line, laser stimulation (constant 60 s). f, i Sleep–wake state changes (top) and probability of brain state transitions (bottom) after photostimulation was applied during NREM sleep lasting no less than 60 s before laser on (f) and REM sleep lasting no less than 16 s before laser on (i) in all trials from mice in the eGFP (left) and Arch (right) groups, respectively. Shading indicates 95% confidence intervals. g, h During 60 s laser stimulation illuminated during NREM sleep, the cumulative probability of REM sleep initiation and NREM sleep termination (g) and mean probability of NREM to REM sleep transition in the first eight trials per mouse (h). Colorful stairs, Arch group. Black stairs, eGFP group. *p < 0.05 vs. eGFP group, Kolmogorov–Smirnov test (g). j Mean latency of REM sleep transitions to wakefulness/NREM sleep after yellow laser stimulation during REM sleep in the first eight trials per mouse. p = 0.0159 vs. eGFP, Mann–Whitney U test (h, j). Data represent mean ± SEM. Arch: n = 5, eGFP: n = 4. Source data are provided as a Source Data file.