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. 2023 Feb 7;9:16. doi: 10.1038/s41421-022-00498-9

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a Schematic diagram depicting Ca²⁺ imaging from hM₃Dq-expressing astrocytes in brain slices. b Left, confocal image showing GCaMP-expressing astrocyte in a brain slice (scale, 50 µm); right, Ca²⁺ signals of the individual region of interest (ROI). c Quantification of CNO-evoked Ca²⁺ signals. AUC, area under the curve. AUC measures the area of Ca²⁺ signals during baseline (the 2-min period prior to CNO infusion) and after bath application of CNO (2 min). hM₃Dq group: n = 99 ROIs; ***P < 0.001. tdTomato group: n = 51 ROIs; P = 0.093. (Paired t-test). d–f Chemogenetic activation of BF astrocytes in live mice. d Schematic diagram depicting fiber photometry recording of chemogenetic activation-induced astrocyte Ca²⁺ signal. AAVs expressing GfaABC₁D-tdTomato and GfaABC₁D-GCaMP6s were injected into the BF of one hemisphere, and AAVs expressing GfaABC₁D-hM3Dq and GfaABC₁D-GCaMP6s were injected into the contralateral BF. e Example traces showing astrocyte Ca²⁺ signal from the BF expressing tdTomato (green) or hM₃Dq (yellow), respectively. Free-behaving mice were injected with CNO (1 mg/kg, i.p.) or saline (i.p.). Scale, 2 normalized z-score. f Quantitative comparison of astrocyte Ca²⁺ signal from the BF expressing tdTomato (green) or hM₃Dq (yellow) in response to CNO or saline injections. The mean ratio between the size of astrocyte Ca²⁺ events from the hM₃Dq- and tdTomato-expressing BF was calculated before and after CNO or saline injections. CNO injection significantly increased the size of GCaMP events in hM₃Dq-expressing BF (186% increase, ***P < 0.001, Paired t-test), whereas saline injection had no detectable effect (P = 0.43, Paired t-test). CNO injections, n = 13 sessions from 4 mice; Saline injections, n = 7 sessions from 4 mice. g Schematic diagram depicting chemogenetic activation of astrocytes in the BF in wide-type (WT) mice. h–n Chemogenetic activation of BF astrocytes in WT mice significantly changed sleep–wake behavior (within the 4 h after CNO injection). hM₃Dq group, n = 9 mice; tdTomato group, n = 10 mice. The statistical method was two-way repeated measures ANOVA, followed by Tukey’s post hoc multiple comparison test. Time percentage (h), bout number (i), and bout duration (j) of the three brain states after CNO or saline injections. In h, hM₃Dq-CNO vs tdTomato-CNO: Wake, P = 0.084; NREM, P = 0.57; REM, ***P < 0.001. hM₃Dq-Saline vs hM₃Dq-CNO: Wake, P = 0.98; NREM, P = 0.92; REM, *P = 0.012. tdTomato-Saline vs tdTomato-CNO: Wake, P = 0.54; NREM, P = 0.49; REM, P = 1.0. In i, hM₃Dq-CNO vs tdTomato-CNO: Wake, **P = 0.0026; NREM, **P = 0.0028; REM, *P = 0.033. hM₃Dq-Saline vs hM₃Dq-CNO: Wake, P = 0.051; NREM, P = 0.054; REM, P = 0.065. tdTomato-Saline vs tdTomato-CNO: Wake, P = 0.87; NREM, P = 0.88; REM, P = 0.96. In j, hM₃Dq-CNO vs tdTomato-CNO: Wake, P = 0.22; NREM, *P = 0.029; REM, P = 0.91. hM₃Dq-Saline vs hM₃Dq-CNO: Wake, P = 0.34; NREM, P = 0.62; REM, P = 0.31. tdTomato-Saline vs tdTomato-CNO: Wake, P = 1.0; NREM, P = 0.67; REM, P = 0.79. k Normalized NREM EEG power after the injection of CNO or saline in hM3Dq- or tdTomato-expressing mice. l Quantification of NREM EEG power in delta, theta, sigma, and beta bands. hM₃Dq-CNO vs tdTomato-CNO: delta, *P = 0.039; theta, *P = 0.014; sigma, P = 0.54; beta, P = 0.60. hM₃Dq-Saline vs hM₃Dq-CNO: delta, P = 0.27; theta, P = 0.083; sigma, P = 1.0; beta, P = 1.0. tdTomato-Saline vs tdTomato-CNO: delta, P = 0.79; theta, P = 0.99; sigma, P = 0.46; beta, P = 0.42. m, n Same as k, l, except that REM EEG was analyzed. In n, hM₃Dq-CNO vs tdTomato-CNO: delta, P = 0.065; theta, **P = 0.0041; sigma, P = 0.16; beta, P = 0.17. hM₃Dq-Saline vs hM₃Dq-CNO: delta, P = 0.091; theta, **P = 0.0012; sigma, P = 0.71; beta, P = 0.86. tdTomato-Saline vs tdTomato-CNO: delta, P = 0.99; theta, P = 1.0; sigma, P = 1.0; beta, P = 1.0. o–q IP₃R₂ knockout (KO) reduced astrocyte Ca²⁺ activity during both wakefulness and REM sleep. o Schematic diagram depicting fiber photometry recording of astrocyte Ca²⁺ signal in the BF to examine the effect of IP₃R₂-KO. p Example astrocyte Ca²⁺ signal from the BF expressing Cre (red, IP₃R₂^−/−) or control virus (light blue, IP₃R₂^+/+), respectively. Scale bar = 200 s and 5 z-score. q Quantitative comparison of astrocyte Ca²⁺ signal from the BF expressing Cre (red, IP₃R₂^−/−) or control virus (light blue, IP₃R₂^+/+) during different brain states. Wake, reduced by 64%, ***P < 0.001, Paired t-test; NREM, P = 0.078, Wilcoxon signed-rank test; REM, reduced by 21%, *P = 0.021, Paired t-test; n = 12 recordings from 4 mice (3 recordings per mouse). r–t IP₃R₂-KO in BF astrocytes reduced wakefulness in the late phase of the night. r Schematic diagram of suppressing astrocyte Ca²⁺ elevation in the BF using the IP₃R₂^flox mice. s Circadian variation of wakefulness in the two groups. n = 6 and 8 mice for Cre group and tdTomato group, respectively. t Percentage of wakefulness in the entire 24 h, during the day, during the night, and the early (ZT13–17) or late phase (ZT18–24) of the night. 24 h, P = 0.21; Day, P = 0.79; Night, *P = 0.049; ZT13–17, P = 0.90; ZT18–24, *P = 0.022; Student’s t-test.