Fig. 7: Causal evidence for transient arousal from REM sleep by activation of the RMTgGABA-LDT circuit.
a Schematic diagram of the experimental protocol (top). The coronal section shows the superimposed virus-injected area (bottom, left: RMTg; right: LDT) and the tips of optical fibers in the LDT (bottom, right). b Representative photographs of coronal sections containing the LDT from a control (top) and a knockdown (bottom) VGAT-Cre mouse. The images show mCherry-expressing neurons (red), glutamate (Glut)-expressing neurons (green, but not the green ChR2-expressing RMTg GABAergic nerve endings), merged neurons (yellow) pointed to by arrows with Glut and mCherry, and 4′,6-diamidino-2-phenylindole (DAPI, blue) staining. Scale bar, 50 μm. c Sleep–wake state changes (top) and probability of brain state transitions (bottom) after photostimulation of RMTg GABAergic axon endings in the LDT during REM sleep lasting no less than 16 s before laser on in all trials from mice in the shCtrl (left) and shVglut2 (right) groups. Shading indicates 95% confidence intervals. d During 120 s laser stimulation of the RMTgGABA-LDT, the cumulative probability for REM sleep termination and wake initiation. Colorful stairs, shVglut2 group. Black stairs, shCtrl group. *p < 0.05, Kolmogorov–Smirnov test. e Probability of first transitions from REM sleep to wakefulness by ChR2-mediated activation of RMTg GABAergic neurons. ChR2: n = 5, mCherry: n = 4. f, g Comparison between the probability of first transitions from REM sleep to wakefulness (f) and REM to NREM sleep (g) by activation of the RMTgGABA-LH (n = 5), RMTgGABA-LDT (n = 5), and RMTgGABA-LDT with shVglut2 (n = 5) and shCtrl (n = 4) microinjection in the LDT, respectively. A 561 nm yellow laser stimulation was used as the control group. A 473 nm blue laser stimulation was used as the experimental group. *p < 0.05, Wilcoxon signed rank test for the experimental group with the 561 nm control group; #p < 0.05, Mann–Whitney U test for the shVglut2 and shCtrl group. Data represent mean ± SEM. Source data are provided as a Source Data file. LDT laterodorsal tegmentum.
