Fig. 1.
Activities of the dorsal–intermediate lateral septum (LS) γ-aminobutyric acid–mediated neurons across sleep–awake states. (A) Timeline of immediate early gene expression of proteins (c-Fos) staining during sleep–wake and different anesthesia states. (B) Top: Coronal images showing c-Fos expression in the lateral septum at the sleep stage. Bottom: Coronal images showing c-Fos expression in the lateral septum during different periods of anesthesia. Scale bar, 100 µm. (C) The c-Fos expression in dorsal–intermediate lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA followed by Tukey’s post hoc test: F3,32 = 31.34, P < 0.001; P [8 am to 8 pm] < 0.001, P [8 am to 10 pm] < 0.001, P [10 am to 8 pm] < 0.001, P [10 am to 10 pm] < 0.001), and ventral lateral septum (LSV; n = 9 slices from 3 mice/group, one-way ANOVA: F3,32 = 0.89, P = 0.457). (D) With the deepening of anesthesia, the expression of c-Fos gradually decreased and gradually recovered in dorsal–intermediate lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA followed by Tukey’s post hoc test: F3,32 = 39.21, P < 0.001; P [wake-induction] < 0.001, P [wake-maintenance] < 0.001, P [induction-emergence] = 0.017, P [maintenance-emergence] < 0.001) after the cessation of anesthesia. However, c-Fos expression of ventral lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA: F3,32 = 0.52, P = 0.669) did not change under different anesthesia periods. (E) Schematic of the virus injection, fiber photometry setup, and in vivo electroencephalogram (EEG)/electromyography (EMG) recording configuration. (F) Top left: Unilateral viral targeting of adeno-associated virus (AAV)-DIO-GCaMP6s into the lateral septum. Scale bar, 1,000 μm. Top right: Location of the optical fiber in the dorsal–intermediate lateral septum. Scale bar, 100 μm. Bottom row: Magnified image showing the expression of GCaMP6s-positive (green) and glutamic acid decarboxylase 67 (GAD-67) (red) cells and the co-expression of GAD-67 (red) in GCaMP6s-positive (green) cells. Scale bar, 10 μm. (G) Representative fluorescence traces, relative EEG power spectra, and EEG/EMG traces across spontaneous sleep–wakefulness states. Voltage represents the change in fluorescence for the entire time series. (H) Statistics for the fluorescence signals during the awake state, non–rapid eye movement (non-REM) sleep, and REM sleep (each dot represents the average for each mouse, n = 7 mice, 5 sessions per mouse, one-way ANOVA followed by Tukey’s post hoc test: F2,18 = 43.72, P < 0.001; P [non-REM–awake state] < 0.001, [non-REM–REM] < 0.001). (I) Sample EEG power spectrum, EEG-EMG traces, and raw fluorescence trace in a continuous anesthesia state from the awake state to the anesthesia induction period, deep anesthesia, and anesthesia recovery period. (J) Summary of fluorescence signals during preanesthesia (Pre-anesth.), anesthesia (Anesth.), and postanesthesia (Post. anesth.) (each dot represents the average for each mouse, n = 7 mice, 5 sessions per mouse, one-way ANOVA followed by Tukey’s post hoc test; F2,18 = 23.28, P < 0.001; P [preanesthesia-anesthesia] < 0.001, P [anesthesia-postanesthesia] = 0.046). The data represent means ± SD. *P < 0.05; ***P < 0.001. LSD, dorsal lateral septum; LSI, intermediate lateral septum; LV, lateral ventricle; MS, medial septum.

Activities of the dorsal–intermediate lateral septum (LS) γ-aminobutyric acid–mediated neurons across sleep–awake states. (A) Timeline of immediate early gene expression of proteins (c-Fos) staining during sleep–wake and different anesthesia states. (B) Top: Coronal images showing c-Fos expression in the lateral septum at the sleep stage. Bottom: Coronal images showing c-Fos expression in the lateral septum during different periods of anesthesia. Scale bar, 100 µm. (C) The c-Fos expression in dorsal–intermediate lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA followed by Tukey’s post hoc test: F3,32 = 31.34, P < 0.001; P [8 am to 8 pm] < 0.001, P [8 am to 10 pm] < 0.001, P [10 am to 8 pm] < 0.001, P [10 am to 10 pm] < 0.001), and ventral lateral septum (LSV; n = 9 slices from 3 mice/group, one-way ANOVA: F3,32 = 0.89, P = 0.457). (D) With the deepening of anesthesia, the expression of c-Fos gradually decreased and gradually recovered in dorsal–intermediate lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA followed by Tukey’s post hoc test: F3,32 = 39.21, P < 0.001; P [wake-induction] < 0.001, P [wake-maintenance] < 0.001, P [induction-emergence] = 0.017, P [maintenance-emergence] < 0.001) after the cessation of anesthesia. However, c-Fos expression of ventral lateral septum (n = 9 slices from 3 mice/group, one-way ANOVA: F3,32 = 0.52, P = 0.669) did not change under different anesthesia periods. (E) Schematic of the virus injection, fiber photometry setup, and in vivo electroencephalogram (EEG)/electromyography (EMG) recording configuration. (F) Top left: Unilateral viral targeting of adeno-associated virus (AAV)-DIO-GCaMP6s into the lateral septum. Scale bar, 1,000 μm. Top right: Location of the optical fiber in the dorsal–intermediate lateral septum. Scale bar, 100 μm. Bottom row: Magnified image showing the expression of GCaMP6s-positive (green) and glutamic acid decarboxylase 67 (GAD-67) (red) cells and the co-expression of GAD-67 (red) in GCaMP6s-positive (green) cells. Scale bar, 10 μm. (G) Representative fluorescence traces, relative EEG power spectra, and EEG/EMG traces across spontaneous sleep–wakefulness states. Voltage represents the change in fluorescence for the entire time series. (H) Statistics for the fluorescence signals during the awake state, non–rapid eye movement (non-REM) sleep, and REM sleep (each dot represents the average for each mouse, n = 7 mice, 5 sessions per mouse, one-way ANOVA followed by Tukey’s post hoc test: F2,18 = 43.72, P < 0.001; P [non-REM–awake state] < 0.001, [non-REM–REM] < 0.001). (I) Sample EEG power spectrum, EEG-EMG traces, and raw fluorescence trace in a continuous anesthesia state from the awake state to the anesthesia induction period, deep anesthesia, and anesthesia recovery period. (J) Summary of fluorescence signals during preanesthesia (Pre-anesth.), anesthesia (Anesth.), and postanesthesia (Post. anesth.) (each dot represents the average for each mouse, n = 7 mice, 5 sessions per mouse, one-way ANOVA followed by Tukey’s post hoc test; F2,18 = 23.28, P < 0.001; P [preanesthesia-anesthesia] < 0.001, P [anesthesia-postanesthesia] = 0.046). The data represent means ± SD. *P < 0.05; ***P < 0.001. LSD, dorsal lateral septum; LSI, intermediate lateral septum; LV, lateral ventricle; MS, medial septum.

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