Abstract

In humans and other mammalian species, lesions in the preoptic area of the hypothalamus cause profound sleep impairment1,2,3,4,5, indicating a crucial role of the preoptic area in sleep generation. However, the underlying circuit mechanism remains poorly understood. Electrophysiological recordings and c-Fos immunohistochemistry have shown the existence of sleep-active neurons in the preoptic area, especially in the ventrolateral preoptic area and median preoptic nucleus6,7,8,9. Pharmacogenetic activation of c-Fos-labelled sleep-active neurons has been shown to induce sleep10. However, the sleep-active neurons are spatially intermingled with wake-active neurons6,7, making it difficult to target the sleep neurons specifically for circuit analysis. Here we identify a population of preoptic area sleep neurons on the basis of their projection target and discover their molecular markers. Using a lentivirus expressing channelrhodopsin-2 or a light-activated chloride channel for retrograde labelling, bidirectional optogenetic manipulation, and optrode recording, we show that the preoptic area GABAergic neurons projecting to the tuberomammillary nucleus are both sleep active and sleep promoting. Furthermore, translating ribosome affinity purification and single-cell RNA sequencing identify candidate markers for these neurons, and optogenetic and pharmacogenetic manipulations demonstrate that several peptide markers (cholecystokinin, corticotropin-releasing hormone, and tachykinin 1) label sleep-promoting neurons. Together, these findings provide easy genetic access to sleep-promoting preoptic area neurons and a valuable entry point for dissecting the sleep control circuit.

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Acknowledgements

We thank J. Cox, L. Pinto for help with sleep recordings; A. Popescu for help with optrode recordings; C. Ma for help with FISH; D. Leib, C. Zimmerman, and D. Estandian for discussions on TRAP; K. Kao, G. Daly, M. Bikov, F. Virani, and G. Carrillo for technical assistance; and C. Koch for supporting the collaboration with the Allen Institute for Brain Science. This work was supported by a Davis Postdoctoral Fellowship (S.C.), Tourette Syndrome Association Grant (S.C.), EMBO Long-term Fellowship (F.W.), Human Frontier Science Program Fellowship (F.W.), and Howard Hughes Medical Institute (Y.D., L.L.).

Author information

Affiliations

  1. Division of Neurobiology, Department of Molecular and Cell Biology, Helen Wills Neuroscience Institute, Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA

    • Shinjae Chung
    • , Franz Weber
    • , Peng Zhong
    • , Nikolai Hörmann
    • , Wei-Cheng Chang
    • , Zhe Zhang
    • , Johnny Phong Do
    •  & Yang Dan
  2. Department of Physiology, University of California, San Francisco, San Francisco, California 94158, USA

    • Chan Lek Tan
    •  & Zachary A. Knight
  3. Allen Institute for Brain Science, Seattle, Washington 98103, USA

    • Thuc Nghi Nguyen
    • , Shenqin Yao
    • , Bosiljka Tasic
    • , Ali Cetin
    •  & Hongkui Zeng
  4. Department of Biology, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • Kevin T. Beier
    •  & Liqun Luo
  5. Diabetes, Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Michael J. Krashes
  6. National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland 21224, USA

    • Michael J. Krashes

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Contributions

S.C. and Y.D. conceived and designed the study, and wrote the paper. S.C. performed most of the experiments. F.W. wrote the programs for data analysis and sleep recording, and S.C. and F.W. analysed the data. P.Z. performed slice recordings. C.L.T. and Z.A.K. constructed the herpes simplex virus (HSV virus), and performed TRAP experiments. T.N., B.T., and H.Z. performed single-cell RNA-seq. K.T.B. and L.L. provided cTRIO and axon arborization analysis reagents and performed part of the input tracing experiments. N.H. and Z.Z. performed FISH. W.C.C. and J.P.D. performed part of the sleep recording. M.J.K. generated PDYN-IRES-Cre mice. S.Y. and A.C. constructed the lentivirus. Y.D. supervised all aspects of the work.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yang Dan.

Reviewer Information Nature thanks M. Halassa, T. Kilduff, A. Yamanaka and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended data figures

  1. 1.

    Overlap of c-Fos staining of sleep-active POA neurons with retrograde labelling from several brain regions and with GAD1–GFP or VGLUT2–GFP.

  2. 2.

    Innervation of histamine neurons in the TMN by GABAergic neurons in the POA and overlap of lentivirus labelling of GABAPOA→TMN neurons with GAD expression and with c-Fos labelling after sleep rebound.

  3. 3.

    Effect of optogenetic activation of GABAPOA→TMN neurons at low frequencies and effect of laser stimulation in GABAPOA→TMN–eYFP control mice.

  4. 4.

    Optogenetic manipulation of axon projections of GABAPOA neurons to TMN, dorsomedial hypothalamus, and habenula, and effect of anti-histamine on optogenetic activation of the TMN axon projections.

  5. 5.

    Effect of laser stimulation on transition probability between each pair of brain states in GABAPOA→TMN-ChR2, GABAPOA→TMN-Ctrl, and GABAPOA-ChR2 mice.

  6. 6.

    Optogenetic identification of GABAPOA→TMN neurons, firing rates of unidentified POA neurons, and firing rate dynamics of identified GABAPOA→TMN neurons during NREM sleep.

  7. 7.

    Mapping of monosynaptic inputs and axon projections of GABAPOA→TMN neurons and axon projections of POA CCK, CRH, TAC1 neurons.

  8. 8.

    Identification of genetic markers for GABAPOA→TMN neurons using TRAP and single-cell RNA-seq, and overlap between each identified marker and GAD and between the markers in the POA.

  9. 9.

    Effect of laser activation of CCK, CRH, TAC1, and PDYN neurons on transition probability between each pair of brain states.

  10. 10.

    Pharmacogenetic inactivation of CCK, CRH, and TAC1 neurons, optogenetic inactivation of CCK neurons, optogenetic activation of GAL neurons, and optogenetic activation of PDYN neurons in the POA.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Tables 1-2.

Videos

  1. 1.

    Optogenetic activation of GABAPOA→TMN neurons promotes sleep

    2 laser stimulation trials, including 30 s before and after each laser stimulation period. The EEG spectrogram, EMG trace and color-coded hypnogram are shown on the right. Laser stimulation periods are depicted by the blue bar on the top right and additionally indicated as a blue square in the upper right corner of the movie frame. The video is shown at 8× the original speed.

  2. 2.

    Optogenetic activation of GABAPOA neurons promotes wakefulness

    2 laser stimulation trials, including 30 s before and 60 s after each laser stimulation period. The video is shown at 8× the original speed.

  3. 3.

    Optogenetic activation of CCK neurons in the POA promotes sleep

    2 laser stimulation trials, including 30 s before and after each laser stimulation period. The video is shown at 8× the original speed.

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DOI

https://doi.org/10.1038/nature22350

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