Fig. 6: The balanced roles of LS- and PVT-TMN circuits in sleep-wake regulation.

a Experimental scheme for labeling input neurons in the LS. rAAV-VGAT1-CRE-EGFP-WPRE-hGH polyA mixed with AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-pA or rAAV-CAG-DIO-mCherry-WPRE-hGH polyA were injected into the bilateral LS of WT mice. Bilateral cannula was implanted into mouse brains above the TMN for CNO infusion. b Representative images of hM3Dq expression in LS (left) and TMN (right). c Experimental scheme for labeling input neurons in the PVT. rAAV-VGLUT2-CRE-WPRE-hGH polyA mixed with AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry-WPRE-pA or rAAV-CAG-DIO-mCherry-WPRE-hGH polyA were injected into the PVT of WT mice. Bilateral cannula was implanted into mouse brains above the TMN for CNO infusion. d Representative images of hM3Dq expression in PVT (left) and TMN (right). e–g Representative continuous EMG, EEG, wake, NREM sleep, and REM sleep scoring data recorded from group of mcherry (e), LS-hM3Dq (f), and PVT-hM3Dq (g) for 24 h (n = 4 for each group). h, i Ratio quantification of wake, NREM sleep, and REM sleep from different groups in 24 h (n = 4 for each group). The exact p-values were as follows: 0.0342 (mCherry-wake vs LS-wake), 0.0039 (mCherry-wake vs PVT-wake), 0.0060 (mCherry-NREM vs LS-NREM), 0.0091 (mCherry-NREM vs PVT-NREM), 0.0011 (mCherry-REM vs LS-REM), and 0.0009 (mCherry-REM vs PVT-REM). j Ratio quantification of NREM and REM sleep from different groups in sleep time. (n = 4 for each group). The exact p-values were as follows: 0.0012 (mCherry-REM vs LS-REM), and 0.0037 (mCherry-REM vs PVT-REM). k Transition number of NREM-REM from different groups in 24 h (n = 4 for each group). The exact p-values were as follows: 0.0011 (mCherry vs LS), 0.0009 (mCherry vs PVT), and <0.0001 (LS vs PVT). l, m Ratio quantification of wake, NREM sleep, and REM sleep from different groups during 12 h (light) of the daytime. The exact p-values were as follows: 0.1455 (mCherry-wake vs LS-wake), 0.0240 (mCherry-wake vs PVT-wake), 0.0476 (mCherry-NREM vs LS-NREM), 0.0384 (mCherry-NREM vs PVT-NREM), 0.0249 (mCherry-REM vs LS-REM), and 0.0090 (mCherry-REM vs PVT-REM). n Ratio quantification of NREM and REM sleep from different groups in daytime sleep. (n = 4 for each group). The exact p-values were as follows: 0.0149 (mCherry-REM vs LS-REM), and 0.0162 (mCherry-REM vs PVT-REM). o Transition number of NREM-REM from different groups during 12 h (light) of the daytime (n = 4 for each group). The exact p-values were as follows: 0.0096 (mCherry vs LS), 0.0298 (mCherry vs PVT), and 0.0006 (LS vs PVT). p, q Ratio quantification of wake, NREM sleep, and REM sleep from different groups during 12 h (dark) of the nighttime. The exact p-values were as follows: 0.0422 (mCherry-NREM vs LS-NREM), 0.3022 (mCherry-NREM vs PVT-NREM), 0.0003 (mCherry-REM vs LS-REM), and 0.0608 (mCherry-REM vs PVT-REM). r Ratio quantification of NREM and REM sleep from different groups in nighttime sleep. (n = 4 for each group). The exact p-values were as follows: <0.0001 (mCherry-REM vs LS-REM), and 0.0398 (mCherry-REM vs PVT-REM). s Transition number of NREM-REM from different groups during 12 h (light) of the nighttime (n = 4 for each group). The exact p-values were as follows: 0.0031 (mCherry vs LS), and 0.0004 (LS vs PVT). All data are presented as mean ± SEM. Statistical analyses were conducted using two-tailed Student’s t-tests without adjustment for multiple comparisons. Significance levels were presented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001. LS Lateral septal nucleus, NREM non-rapid eye movement, PVT Paraventricular nucleus of the thalamus, REM rapid eye movement, TMN Tuberomammillary nucleus.