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Kinase signalling in excitatory neurons regulates sleep quantity and depth

Abstract

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1–SIK3–HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

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Fig. 1: Loss-of-function mutations of Hdac4 and Hdac5 increase NREMS.
Fig. 2: SIK3–HDAC4 interactions on sleep and wakefulness.
Fig. 3: Sleep–wakefulness of neural LKB1-deficient and SIK3 kinase activity-modified mice.
Fig. 4: Loss of Hdac4 or Sik3 in excitatory neurons decreases sleep.
Fig. 5: Sik3Slp in excitatory neurons increases sleep.
Fig. 6: Single-nucleus transcriptome changes and the molecular pathway associated with sleep need.

Data availability

Raw and processed snRNA-seq data have been deposited into the NCBI Gene Expression Omnibus database under accession number GSE214337. All detailed statistics for sleep and behaviour studies are included in the Supplementary Information. All other data supporting this study are available from the corresponding authors without restriction. Source data are provided with this paper.

Code availability

Code used in this study is available from the corresponding authors without restriction.

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Acknowledgements

We thank all members of Yanagisawa/ Funato Lab and the International Institute for Integrative Sleep Medicine, especially K. Ebihara, M. Taniguchi, M. Sato for technical support, K. Iida, A. Kuno, M. Lazarus, E. Hasegawa, K. Roy, S. Wakana, T. Suzuki and I. Miura for technical advice and discussion, D. Kawaguchi for sharing Foxg1cre mice, and E. Olson for sharing Hdac4flox mice. This work was supported by the World Premier International Research Center Initiative from MEXT to M.Y., JSPS KAKENHI (17H06095, 22H04918 to M.Y. and H.F.; 17H04023, 17H05583, 20H00567 to H.F.; 26507003, 18968064 to C.M. and H.F.; 15J00393, 18J21517 to F.A.; 18K14811 to T.F.; and 20J12137 to K.I.), JST CREST (JPMJCR1655 to M.Y.), AMED (JP21zf0127005 to M.Y.), JSPS DC2 (17J07957 to S.J.K.), the University of Tsukuba Basic Research Support Program Type A (to S.J.K.), and the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) from JSPS (to M.Y.).

Author information

Authors and Affiliations

Authors

Contributions

S.J.K., N.H.-H., F.A., T.K., K.I., S.N., H.K. and N.A. contributed equally to this work. S.J.K. carried out experiments for all Hdac-modified mice. N.H.-H. performed sleep analyses of the Sik3Slp-flox mice. F.A. produced and performed sleep analysis of Sik3flox mice. T.K. performed sleep analysis of Lkb1-modified mice. K.I. performed snRNA-seq experiments and analysis. S.N. produced and performed sleep analysis of Sik3T221E mice. H.K. produced and performed sleep analysis of Sik3T221A mice. N.A. produced and performed sleep analysis of Hdac4S245A mice. T.M. performed cell culture experiments. A.I. performed western blotting. M.K. and Y.T. performed histology. D.K. performed memory experiments. S.K., J.C., T.T. and A.E. performed sleep analyses. C.M. performed sleep analyses and pharmacology experiments. S. Miyazaki and Y.H. contributed to C.elegans data generation. T.F., S. Mizuno, F.S. and S.T. contributed to gene-modified mouse generation. M.M. and Q.L. contributed to data interpretation. S.J.K., M.Y. and H.F. wrote the manuscript. M.Y. and H.F. conceived and supervised the entire project.

Corresponding authors

Correspondence to Masashi Yanagisawa or Hiromasa Funato.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Splice mutations of Hdac4 and Hdac5 genes increased NREMS.

a. Haplotype analysis of the Slp2 pedigree for wake time based on C57B6J/C57B6N SNPs (N2, n = 31). b. Exome sequencing identified single nucleotide changes within the LOD score peak (upper) and total wake time (lower) of each mouse. Blue and red bars indicate normal and short wake time, respectively. c. RT-PCR showing short Hdac4 transcripts specific to Hdac4Slp2/+ mice. Hrt, heart; Ctx, cerebral cortex; Hyp, hypothalamus. Representative images from three biological replicates are shown. d. Immunoblot of brain homogenates of wild-type and Hdac4Slp2/+ mice using anti-HDAC4 antibody. Anti-β-actin antibody was used as loading control. Quantitation of HDAC4 band intensity against β-actin (Right). Representative blots from three biological replicates are shown. e. Structures of HDAC4,5,7 and 9 with phosphorylation sites and deacetylase (DAC) domain. Numbers on the DAC domain indicate the region encoded by the skipped exon. f. Genomic DNA and transcripts of class IIa Hdacs. Red triangles indicate the splice acceptor site mutations (SA) of Hdac4, 5, 7, and 9 genes. Black triangles indicate stop codon produced due to the frameshift. g, h. RT-PCR of Hdac4SA (g) and Hdac5SA (h) mouse cortical tissues (3 mice per group). Shorter bands (asterisk) corresponding to exon skipping variants. i–k. Total wake time (i), NREMS episode duration (j), and total time spent in REMS (k) of Hdac4 mice (+/+, n = 26; SA/+, n = 26). l. NREMS delta power under the basal condition and after 6-hour sleep deprivation (SD) of Hdac4SA mice. m. Change in NREMS delta power after sleep deprivation relative to each ZT under the basal condition of Hdac4 mice (+/+, n = 10; SA/+, n = 13). n, o. Relative NREMS delta power to the basal condition (n) and time spent in NREMS (o) for 3 h from ZT13. HDAC4 inhibitor, LMK235, was intracerebroventricularly administered at ZT11.5. n = 12. p–r. Total time spent in wake (p), NREMS episode duration (q), and total time spent in REMS (r) of Hdac5 mice (+/+, n = 17; SA/+, n = 21; SA/SA, n = 7). s, t. NREMS delta power under the basal condition and after 6-hour sleep deprivation (s) and change in NREMS delta power after sleep deprivation relative to each ZT under the basal condition (t) of Hdac5 mice (+/+, n = 14; SA/+, n = 21; SA/SA, n = 7). u, v. Diurnal NREMS delta density (u) and EEG power spectra during NREMS (v) of Hdac4SA; Hdac5SA mice. Hdac4+/+; Hdac5SA/+ mice (n = 16) and Hdac4SA/+; Hdac5SA/+ mice (n = 10). Data shown as mean ± s.e.m.; two-sided P values. Unpaired t-test (f, v); two-way ANOVA with Tukey’s test (ik, pr); mixed-effects model (m, t, u), with Dunnett’s test (n, o); two-way repeated measures ANOVA with Sidak’s (l) or Tukey’s test (s).

Source data

Extended Data Fig. 2 Retina and behavioral characterization of HDAC4SA mice.

a. Representative immunostaining from at least 3 biological replicates using anti-rhodopsin antibody of wild-type and Hdac4SA retinas. Sections were also stained with hematoxylin. Scale bar, 150 μm. b, c. Body temperature of Hdac4SA mice (+/+, n = 8; SA/+, n = 7). Data points are averaged every 10 min. d. Daily food intake over 4 days of chow diet of Hdac4SA mice (+/+, n = 14; SA/+, n = 13). e, f. Total distance (e) and time spent in the center area (f) of Hdac4SA mice (+/+, n = 12; SA/+, n = 11). g. Time spent in the open area on the elevated plus maze of Hdac4SA mice (+/+, n = 11; SA/+, n = 14). h. The time of immobility during the last 4 min of the tail suspension test of Hdac4SA mice (+/+, n = 13; SA/+, n = 13). i. The time of immobility during the last 4 min of the forced swim test of Hdac4SA mice (+/+, n = 13; SA/+, n = 14). j–l. Fear conditioning test. Shock reactivity during conditioning (j) was movements for 2 s immediately before shock (Pre-shock) and for 2 s during the first shock (Shock). Freezing in the tone fear conditioning task (k) and in contextual fear conditioning task (l). Mice were placed in the non-conditioned context, and the tone was played during the last 3 min of the session Hdac4SA mice (+/+, n = 14; SA/+, n = 15). Data shown as mean ± s.e.m.; two-sided P values. Two-way repeated measures ANOVA (b); unpaired t-test (cf, hl). Mann-Whitney U-test (g).

Source data

Extended Data Fig. 3 Sleep/wakefulness of Hdac7SA and Hdac9SA mice.

a. RT-PCR of Hdac7SA and Hdac9SA mouse brains. Shorter bands (asterisk) corresponding to exon skipping variants. Representative images from three biological replicates. b–f. Total wake time (b), total NREMS time (c), NREMS episode duration (d), diurnal NREMS delta density (e), and EEG power spectra during NREMS (f) of Hdac7SA mice (+/+, n = 12; SA/+, n = 12). g–k. Total wake time (g), total NREMS time (h), NREMS episode duration (i), diurnal NREMS delta density (j), and EEG power spectra during NREMS (k) of Hdac9SA mice (+/+, n = 15; SA/+, n = 15; SA/SA, n = 13). l. Nuclear HDAC4 intensity at ZT6 and ZT12.5 of the primary motor cortex in constant darkness (ZT6, n = 10; ZT12.5, n = 10). m, n. Representative result of western immunostaining (m) of nucleo-cytoplasmic fractionated cerebral cortex. Densitometric analysis of HDAC4 over β-actin of the cytoplasmic fractions. Six biological replicates (n). o. In vitro kinase assay of SIK3 using HDAC4 peptide. p. Immunoblots of PC12 cell homogenates using HDAC4 antibodies after the addition of HG9-91-01. Two technical replicates. q, r. NREMS episode duration (q) and total REMS time (r) of Hdac4 mice (+/+, n = 35; S245A/+, n = 20). s. NREMS delta power after 6 hour-sleep deprivation of Hdac4 mice (+/+, n = 29; S245A/+, n = 12). Data shown as mean ± s.e.m.; two-sided P values. Two-way ANOVA (bd, gi, q, r) with Tukey’s test (q); mixed-effects model (e, j); unpaired t-test (f, l, n); one-way ANOVA (k), two-way repeated measures ANOVA with Sidak’s test (s).

Source data

Extended Data Fig. 4 Lethargus of C. elegans had-4 and kin-29 mutants.

a. During lethargus, hda-4(oy59) (n = 15) and kin-29(oy39) hda-4(oy59) (n = 14) mutant worms exhibited increased quiescence compared to wild-type worms (n = 42), whereas kin-29(oy39) mutant worms (n = 12) exhibited decreased quiescence. b. Increased quiescence during lethargus in the hda-4(oy59) mutant was rescued by transfection with genomic hda-4 (n = 9), but not with an injection marker alone (n = 10). c, d. Comparison of the duration of bout of motion (c) and quiescence (d) during lethargus revealed that the quiescent bouts were selectively affected. e, f. The quiescent bouts durations were significantly decreased in genomic hda-4 transfected worms. Data shown as mean ± s.e.m.; two-sided P values. Steel-Dwass multiple comparison tests (a, c, d); Mann-Whitney U-tests (b, e, f).

Source data

Extended Data Fig. 5 Sleep/wakefulness of Lkb1 and Sik3 gene-modified mice.

a. In vitro kinase assay for SIK3, SIK3 (T221A) and SIK3 (T221E). b–d. Total wake time (b), NREMS episode duration (c), total REMS time (d) of Syn1creERT; Lkb1flox mice (+/+, n = 9; flox/flox, n = 14). e. NREMS delta power for 2 h after 6-hour sleep deprivation of Syn1creERT; Lkb1flox mice (+/+, n = 8; flox/flox, n = 10). f–i. Total wake time (f), NREMS episode duration (g), total REMS time (h), and NREMS delta power under the basal condition and after 6-hour sleep deprivation (i) of Sik3 mice (+/+, n = 11; T221A/+, n = 12). j–l. Total wake time (j), NREMS episode duration (k), and total REMS time (l) of Syn1creERT; Lkb1flox; Sik3 mice (+/+, n = 14; T221E/T221E, n = 15). m–o. Total wake time (m), NREMS episode duration (n), and total REMS time (o) of Sik3 mice (Slp/+, n = 7; Slp-T221A/+, n = 10). p. NREMS delta power under the basal condition and after 6-hour sleep deprivation of Sik3 mice (Slp/+, n = 6; Slp-T221A/+, n = 10). Data shown as mean ± s.e.m.; two-sided P values. Two-way ANOVA (c, f, g, h, k, o) with Tukey’s test (b, d, j, ln); two-way repeated measures with Sidak’s test (e, i, p).

Source data

Extended Data Fig. 6 Sleep/wakefulness of neuron-type-specific Hdac4-deficient mice.

a. HDAC4 immunoreactivity was abolished in nestincre; Hdac4flox/flox mice (right), compared to nestincre; Hdac4+/+ mice (left). Scale bars, 1 mm. b. EEG power spectra during NREMS of Vglut2cre; Hdac4flox mice (+/+, n = 21; flox/+, n = 13; flox/flox, n = 12). c. NREMS delta power after 6 hour-sleep deprivation of Vglut2cre; Hdac4flox mice (+/+, n = 13; flox/+, n = 13, flox/flox, n = 11). d–g. Total wake time (d), total NREMS time (e), diurnal NREMS delta density (f), and EEG power spectra during NREMS (g) of Camk2acre; Hdac4flox mice (+/+, n = 13; flox/+, n = 11; flox/flox, n = 11). h–k. Total wake time (h), total NREMS time (i), diurnal NREMS delta density (j) and EEG power spectra during NREMS (k) of Vgatcre; Hdac4flox mice (+/+, n = 12; flox/+, n = 9; flox/flox, n = 11). Data shown as mean ± s.e.m.; two-sided P values. Two-way ANOVA (d, e) with Tukey’s test (h, i); mixed-effects model (f, j); one-way ANOVA (b, g, k); two-way repeated measures ANOVA with Tukey’s test (c).

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Extended Data Fig. 7 Histological analysis of HDAC4, SIK3 and Cre drivers.

a–f. X-gal staining of Vglut2cre; ROSA-LacZ (a), Vglut1cre; ROSA-LacZ (b), Camk2acre; ROSA-LacZ (c), Vgatcre; ROSA-LacZ mice (d), Foxg1cre; ROSA-LacZ (e), and Foxd1cre; ROSA-LacZ (f). Scale bars, 1 mm. g. Visualization of SIK3 of the cerebral cortex (left) and the ventromedial nucleus of the hypothalamus (VMH, right) using anti-HA antibody of HA-Sik3 knock-in mouse brains. Scale bars, 40 μm. h, i. Representative result of Sik3 mRNA expression in the brain of nestin+/+; Sik3flox mouse (h) and nestincre; Sik3flox mouse (i). Three biological replicates. High magnification images of visual cortex. Scale bars, 500 μm, 250 μm. j. Representative result of western blotting using anti-SIK3 antibody showing SIK3 expression in nestin+/+; Sik3flox mouse brain, but not in nestincre; Sik3 flox mouse brain. Three biological replicates. k–n. Representative results of three Sik3 mRNA expression in the brains of Sik3flox (k), nestincre; Sik3flox (l), Vglut2cre; Sik3flox (m), and Vgatcre; Sik3flox mouse (n). Vglut2cre; Sik3flox mice showed reduced SIK3 expression in the thalamus (TH) whereas Vgatcre; Sik3flox mice showed reduced SIK3 expression in the thalamic reticular nucleus (TRH). Nestincre; Sik3flox mice showed reduced SIK3 expression in both TH and TRH. Sections were lightly counterstained with Nissl. Scale bars, 400 μm.

Extended Data Fig. 8 Sleep/wakefulness of neuron-type-specific Sik3-deficient mice.

a–c. NREMS episode duration (a), total wake time (b), total REMS time (c) of Vglut2cre; Sik3 flox mice (+/+, n = 16; flox/flox, n = 17). d,e. NREMS delta power under basal condition and after 6-h sleep deprivation (d) and relative NREMS delta power after sleep deprivation to each ZT under the basal condition (e) of Vglut2cre; Sik3 flox mice (+/+, n = 10; flox/flox, n = 17). f—h. NREMS episode duration (f), total wake time (g), total REMS time (h) of Vglut1cre; Sik3 flox mice (+/+, n = 12; flox/flox, n = 12). i–j. NREMS delta power under basal condition and after 6-h sleep deprivation (i) and relative NREMS delta power after sleep deprivation to each ZT under the basal condition (j) of Vglut1cre; Sik3 flox mice (+/+, n = 10; flox/flox, n = 12). k—p. Total NREMS time (k), total wake time (l), total REMS time (m), NREMS episode duration (n), diurnal NREMS delta density (o), and EEG power during NREMS (p) of Camk2acre; Sik3 flox mice (+/+, n = 11; flox/flox, n = 11). q. NREMS delta power under basal condition and after 6-h sleep deprivation of Camk2acre; Sik3 flox mice (+/+, n = 9; flox/flox, n = 11). r–w. Total NREMS time (r), total wake time (s), total REMS time (t), NREMS episode duration (u), diurnal NREMS delta density (v), and EEG power during NREMS (w) of Vgatcre; Sik3 flox mice (+/+, n = 14; flox/flox, n = 8). x. NREMS delta power under basal condition and after 6-h sleep deprivation of Vgatcre; Sik3 flox mice (+/+, n = 7; flox/flox, n = 6). y–aa. NREMS episode duration (y), total wake time (z), total REMS time (aa) of Foxg1cre; Sik3 flox mice (+/+, n = 8; flox/flox, n = 11). ab. NREMS delta power under basal condition and after 6-h sleep deprivation of Foxg1cre; Sik3 flox mice (+/+, n = 8; flox/flox, n = 11). ac–ae. NREMS episode duration (ac), total wake time (ad), total REMS time (ae) of Foxd1cre; Sik3 flox mice (+/+, n = 10; flox/flox, n = 14). af. NREMS delta power under basal condition and after 6-h sleep deprivation of Foxd1cre; Sik3 flox mice (+/+, n = 10; flox/flox, n = 13). Data shown as mean ± s.e.m.; two-sided P values. Two-way ANOVA with Tukey’s test (ac, fh, k-n, ru, yaa, acae); two-way repeated measures ANOVA with Sidak’s test (d, i, q, x, ab, af); mixed-effects model (e, j, o, v); unpaired t-test (p, w).

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Extended Data Fig. 9 Sik3Slp in excitatory neurons increased NREMS.

a–f. NREMS episode duration of Vglut2cre; Sik3Slp-flox mice (a)(+/+, n = 19; Slp-flox/+, n = 17; Slp-flox/Slp-flox, n = 20), Vglut1cre; Sik3Slp-flox mice (b)(+/+, n = 11; Slp-flox/+ n = 11; Slp-flox/Slp-flox, n = 10), Camk2acre; Sik3Slp-flox mice (c)(+/+, n = 11; Slp-flox/+, n = 13; Slp-flox/Slp-flox, n = 10), Vgatcre; Sik3Slp-flox mice (d)(+/+, n = 10; Slp-flox/+, n = 13; Slp-flox/Slp-flox, n = 12), Foxg1cre; Sik3Slp-flox mice (e)(+/+, n = 17; Slp-flox/+, n = 12; Slp-flox/Slp-flox, n = 14), and Foxd1cre; Sik3Slp-flox mice (f)(+/+, n = 27; Slp-flox/+, n = 16; Slp-flox/Slp-flox, n = 21). g–l. Total wake time of Vglut2cre; Sik3Slp-flox mice (g), Vglut1cre; Sik3Slp-flox mice (h), Camk2acre; Sik3Slp-flox mice (i), Vgatcre; Sik3Slp-flox mice (j), Foxg1cre; Sik3Slp-flox mice (k), and Foxd1cre; Sik3Slp-flox mice (l). Colours are as for af, respectively. m-r. Total REMS time of Vglut2cre; Sik3Slp-flox mice (m), Vglut1cre; Sik3Slp-flox mice (n), Camk2acre; Sik3Slp-flox mice (o), Vgatcre; Sik3Slp-flox mice (p), Foxg1cre; Sik3Slp-flox mice (q), and Foxd1cre; Sik3Slp-flox mice (r). Colours are as for af, respectively. s, t. Relative NREMS delta power changes after sleep deprivation to each ZT under the basal condition of Vglu2cre; Sik3Slp-flox mice (s)(+/+, n = 13; Slp-flox/Slp-flox, n = 16) and Foxd1cre; Sik3Slp-flox mice (t)(+/+, n = 10; Slp-flox/Slp-flox, n = 11). u–z. Process S simulation for sleep need dynamics. Td/Ti ratio of Hdac4SA mice (u)(+/+, n = 10; SA/+, n = 13), Hdac5SA mice (v)(+/+, n = 9; SA/+, n = 13; SA/SA, n = 7), Vglut2cre; Sik3flox mice (w)(+/+, n = 10; flox/flox, n = 17), Vglut1cre; Sik3flox mice (x)(+/+, n = 10; flox/flox, n = 12), Vglut2cre; Sik3Slp-flox mice (y)(+/+, n = 16; Slp-flox/Slp-flox, n = 13), and Foxd1cre; Sik3Slp-flox mice (z)(+/+, n = 11; Slp-flox/Slp-flox, n = 10). Data shown as mean ± s.e.m.; two-sided P values. Two-way ANOVA with Tukey’s test when applicable (ar); mixed-effects model (s, t); unpaired t-test (u, wz); one-way ANOVA with Tukey’s test (v).

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Extended Data Fig. 10 Single-nucleus RNA-seq analysis of the cerebral cortex transduced with AAV-Sik3Slp and of sleep-deprived mice.

a. The number of detected reads in nuclei of WPRE-positive cells of each cluster from the cerebral cortex transduced with AAV-Sik3Slp or AAV-Sik3. b. The number of detected reads in nuclei of each cluster from the cerebral cortex of sleep-deprived or ad libitum sleep mice. c, d. The number of genes detected in nuclei of each cluster from the cerebral cortex transduced with AAV-Sik3Slp or AAV-Sik3 (c), and sleep-deprived or ad libitum sleep mice (d). e. WPRE-positive nuclei were displayed on UMAP of nuclei from the cerebral cortex transduced with AAV-Sik3Slp or AAV-Sik3 (6 mice per group). f. Volcano plot of genes in the GABAergic cluster of nuclei from the cerebral cortex transduced with AAV-Sik3Slp or AAV-Sik3. The vertical axis indicates -log10(adjusted P value) of Wilcoxon rank-sum test with Bonferroni correction. The horizontal axis indicates log2(fold change) calculated from AAV-Sik3Slp compared with AAV-Sik3. Each point represents a gene detected in at least 10% of nuclei in any conditions. Black circles, P ≥ 0.05 or |log2(fold change)|≤0.14. Red circles, P < 0.05 and log2(fold change) > 0.14. Blue circles, P < 0.05 and log2(fold change) < −0.14. g–j. Volcano plots of genes in the GABAergic cluster (g), astrocyte cluster (h), oligodendrocyte cluster (i), and microglia cluster (j) of nuclei from the cerebral cortex of sleep-deprived (n = 4) or ad libitum sleep mice (n = 6). k, l. Expression level of Bdnf in nuclei from the cerebral cortex transduced with AAV-Sik3Slp or AAV-Sik3 (k) and the cerebral cortex of sleep-deprived or ad libitum sleep mice (l).

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Kim, S.J., Hotta-Hirashima, N., Asano, F. et al. Kinase signalling in excitatory neurons regulates sleep quantity and depth. Nature 612, 512–518 (2022). https://doi.org/10.1038/s41586-022-05450-1

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