Abstract

Sleep is conserved from invertebrates to vertebrates, and is tightly regulated in a homeostatic manner. The molecular and cellular mechanisms that determine the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remain unknown. Here we identify two dominant mutations that affect sleep and wakefulness by using an electroencephalogram/electromyogram-based screen of randomly mutagenized mice. A splicing mutation in the Sik3 protein kinase gene causes a profound decrease in total wake time, owing to an increase in inherent sleep need. Sleep deprivation affects phosphorylation of regulatory sites on the kinase, suggesting a role for SIK3 in the homeostatic regulation of sleep amount. Sik3 orthologues also regulate sleep in fruitflies and roundworms. A missense, gain-of-function mutation in the sodium leak channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the use of a forward-genetics approach for studying sleep behaviours in mice, and demonstrate the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

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Acknowledgements

We thank all Yanagisawa/Funato laboratory and WPI-IIIS members, especially T. Motoike, A. Matsui, Y. Goto, M. Takahashi and K. Taniguchi for technical assistance and their participation in the early stage of this project, M. Lazarus, R. W. Greene and K. E. Vogt for discussion and comments on this manuscript. J.S.T. is an investigator and M.Y. is a former investigator of the Howard Hughes Medical Institute. This work was supported by the World Premier International Research Center Initiative from MEXT to M.Y., JSPS KAKENHI (grant number 26220207 to M.Y., H.F., T.K.; 16K15187 to H.F.; 26507003 to C.M., H.F.; 15K18966, 00635089 to T.F.; 16K18358 to T.K.; 15J06369 to T.H.; 16K18583 to M.S.), MEXT KAKENHI (grant number; 15H05935 to M.Y., H.F.), Welch Foundation (grant number; I-1608 to Q.L.), NIH (grant number; GM111367 to Q.L.), Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST program) from JSPS to M.Y., research grants from Uehara Memorial Foundation to M.Y., and from Takeda Science Foundation to M.Y. Nematode strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank A. Hart and H. Huang for technical advices on nematode quiescence measurement, Y. Iino for providing plasmids, M. Ikawa for providing EGxxFP plasmid, and M. Montminy and J. B. Thomas for fly stocks.

Author information

Author notes

    • Chika Miyoshi
    • , Tomoyuki Fujiyama
    • , Takeshi Kanda
    •  & Makito Sato

    These authors contributed equally to this work.

Affiliations

  1. International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Hiromasa Funato
    • , Chika Miyoshi
    • , Tomoyuki Fujiyama
    • , Takeshi Kanda
    • , Makito Sato
    • , Zhiqiang Wang
    • , Jing Ma
    • , Aya Ikkyu
    • , Miyo Kakizaki
    • , Noriko Hotta-Hirashima
    • , Satomi Kanno
    • , Haruna Komiya
    • , Fuyuki Asano
    • , Takato Honda
    • , Staci J. Kim
    • , Kanako Harano
    • , Hiroki Muramoto
    • , Toshiya Yonezawa
    • , Shinichi Miyazaki
    • , Linzi Connor
    • , Yu Hayashi
    • , Qinghua Liu
    • , Joseph S. Takahashi
    •  & Masashi Yanagisawa
  2. Department of Anatomy, Faculty of Medicine, Toho University, Ota-ku, Tokyo 143-8540, Japan

    • Hiromasa Funato
  3. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Makito Sato
    •  & Masashi Yanagisawa
  4. Department of Neuropharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Aichi 467-8603, Japan

    • Shin Nakane
    • , Jun Tomita
    •  & Kazuhiko Kume
  5. Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Seiya Mizuno
    • , Fumihiro Sugiyama
    •  & Satoru Takahashi
  6. Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Vivek Kumar
    •  & Joseph S. Takahashi
  7. The Jackson Laboratory, Bar Harbor, Maine 04609, USA

    • Vivek Kumar
  8. Technology and Development Team for Mouse Phenotype Analysis, RIKEN Bioresource Center, Tsukuba, Ibaraki 305-0074, Japan

    • Ikuo Miura
    • , Tomohiro Suzuki
    •  & Shigeharu Wakana
  9. Laboratory of Research Advancement, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan

    • Atsushi Watanabe
  10. Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan

    • Manabu Abe
    •  & Kenji Sakimura
  11. PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

    • Yu Hayashi
  12. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Qinghua Liu
  13. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA

    • Joseph S. Takahashi
    •  & Masashi Yanagisawa
  14. Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

    • Masashi Yanagisawa

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Contributions

H.F. and M.Y. were responsible for the overall experimental design, based on strategies conceived by M.Y. and J.S.T. M.S. constructed EEG analysis and database systems. C.M., S.K., N.H.-H., A.I., H.K., F.A., T.H., S.J.K. and K.H. conducted EEG recording and analysis. M.K. performed in situ hybridization. T.F., Se.M., F.S. and S.T. produced CRISPR-based gene-modified mice. M.A. and K.S. produced gene-modified mice. Sh.M., L.C. and Y.H. conducted roundworm experiments. T.K., H.M. and T.Y. conducted electrophysiological experiments. Z.W., J.M., A.W. and Q.L. conducted proteomics experiments. S.N., J.T. and K.K. conducted fruitfly experiments. ENU mice production and linkage analysis were conducted by I.M., T.S. and S.W. V.K. and J.S.T. designed B6 substrain-based screening. H.F. and M.Y. wrote the paper, which was reviewed by all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Hiromasa Funato or Joseph S. Takahashi or Masashi Yanagisawa.

Reviewer Information

Nature thanks D.-J. Dijk, A. Sehgal and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    The file contains the raw data for Figure 1,f,h,i, and Extended Data Figures 2e, 3b, 6b,c.

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