Article | Published:

Circadian neuron feedback controls the Drosophila sleep–activity profile

Nature volume 536, pages 292297 (18 August 2016) | Download Citation

Abstract

Little is known about the ability of Drosophila circadian neurons to promote sleep. Here we show, using optogenetic manipulation and video recording, that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep. To investigate more plastic aspects of the sleep program, we used a calcium assay to monitor and compare the real-time activity of DN1 neurons in freely behaving males and females. Our results revealed that DN1 neurons were more active in males than in females, consistent with the finding that male flies sleep more during the day. DN1 activity is also enhanced by elevated temperature, consistent with the ability of higher temperatures to increase sleep. These new approaches indicate that DN1s have a major effect on the fly sleep–wake profile and integrate environmental information with the circadian molecular program.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & Coordination of circadian timing in mammals. Nature 418, 935–941 (2002)

  2. 2.

    Organization and function of a central nervous system circadian oscillator: the suprachiasmatic hypothalamic nucleus. Fed. Proc. 42, 2783–2789 (1983)

  3. 3.

    , , & Functional analysis of circadian pacemaker neurons in Drosophila melanogaster. J. Neurosci. 26, 2531–2543 (2006)

  4. 4.

    , & Light-arousal and circadian photoreception circuits intersect at the large PDF cells of the Drosophila brain. Proc. Natl Acad. Sci. USA 105, 19587–19594 (2008)

  5. 5.

    et al. Identification of a circadian output circuit for rest:activity rhythms in Drosophila. Cell 157, 689–701 (2014)

  6. 6.

    et al. Modulation of GABAA receptor desensitization uncouples sleep onset and maintenance in Drosophila. Nat. Neurosci. 11, 354–359 (2008)

  7. 7.

    & Studying circadian rhythms in Drosophila melanogaster. Methods 68, 140–150 (2014)

  8. 8.

    , , , & pdf neuropeptide gene mutation and ablation of PDF neurons each cause severe abnormalities of behavioral circadian rhythms in Drosophila. Cell 99, 791–802 (1999)

  9. 9.

    , , & Morning and evening peaks of activity rely on different clock neurons of the Drosophila brain. Nature 431, 869–873 (2004)

  10. 10.

    , , & PDF neuron firing phase-shifts key circadian activity neurons in Drosophila. eLife 3, e02780 (2014)

  11. 11.

    & The Drosophila circadian clock is a variably coupled network of multiple peptidergic units. Science 343, 1516–1520 (2014)

  12. 12.

    , , & Coupled oscillators control morning and evening locomotor behaviour of Drosophila. Nature 431, 862–868 (2004)

  13. 13.

    et al. Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila. Curr. Biol. 24, 2652–2664 (2014)

  14. 14.

    & pySolo: a complete suite for sleep analysis in Drosophila. Bioinformatics 25, 1466–1467 (2009)

  15. 15.

    et al. High-resolution positional tracking for long-term analysis of Drosophila sleep and locomotion using the “tracker” program. PLoS One 7, e37250 (2012)

  16. 16.

    et al. Context-specific comparison of sleep acquisition systems in Drosophila. Biol. Open 4, 1558–1568 (2015)

  17. 17.

    et al. Tools for neuroanatomy and neurogenetics in Drosophila. Proc. Natl Acad. Sci. USA 105, 9715–9720 (2008)

  18. 18.

    et al. DN1(p) circadian neurons coordinate acute light and PDF inputs to produce robust daily behavior in Drosophila. Curr. Biol. 20, 591–599 (2010)

  19. 19.

    , , , & Light and temperature control the contribution of specific DN1 neurons to Drosophila circadian behavior. Curr. Biol. 20, 600–605 (2010)

  20. 20.

    et al. A GAL4-driver line resource for Drosophila neurobiology. Cell Reports 2, 991–1001 (2012)

  21. 21.

    et al. A conserved bicycle model for circadian clock control of membrane excitability. Cell 162, 836–848 (2015)

  22. 22.

    et al. Dual PDF signaling pathways reset clocks via TIMELESS and acutely excite target neurons to control circadian behavior. PLoS Biol. 12, e1001810 (2014)

  23. 23.

    et al. Independent optical excitation of distinct neural populations. Nat. Methods 11, 338–346 (2014)

  24. 24.

    et al. Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila. eLife 3, e04580 (2014)

  25. 25.

    et al. Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship. Nat. Methods 11, 325–332 (2014)

  26. 26.

    et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010)

  27. 27.

    et al. The fruit fly Drosophila melanogaster favors dim light and times its activity peaks to early dawn and late dusk. J. Biol. Rhythms 22, 387–399 (2007)

  28. 28.

    et al. GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008)

  29. 29.

    , , , & Analysis of functional neuronal connectivity in the Drosophila brain. J. Neurophysiol. 108, 684–696 (2012)

  30. 30.

    et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013)

  31. 31.

    et al. Glutamate and its metabotropic receptor in Drosophila clock neuron circuits. J. Comp. Neurol. 505, 32–45 (2007)

  32. 32.

    et al. Plug-and-play genetic access to Drosophila cell types using exchangeable exon cassettes. Cell Reports 10, 1410–1421 (2015)

  33. 33.

    & Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system. Proc. Natl Acad. Sci. USA 110, 10294–10299 (2013)

  34. 34.

    , , , & RNA-seq profiling of small numbers of Drosophila neurons. Methods Enzymol. 551, 369–386 (2015)

  35. 35.

    et al. Differentially timed extracellular signals synchronize pacemaker neuron clocks. PLoS Biol. 12, e1001959 (2014)

  36. 36.

    , , , & Balance of activity between LNvs and glutamatergic dorsal clock neurons promotes robust circadian rhythms in Drosophila. Neuron 74, 706–718 (2012)

  37. 37.

    Differential control of morning and evening components in the activity rhythm of Drosophila melanogaster—sex-specific differences suggest a different quality of activity. J. Biol. Rhythms 15, 135–154 (2000)

  38. 38.

    , , & Natural variation in the splice site strength of a clock gene and species-specific thermal adaptation. Neuron 60, 1054–1067 (2008)

  39. 39.

    & A novel pathway for sensory-mediated arousal involves splicing of an intron in the period clock gene. Sleep 38, 41–51 (2015)

  40. 40.

    , , & How a circadian clock adapts to seasonal decreases in temperature and day length. Neuron 24, 219–230 (1999)

  41. 41.

    , , & Mapping neural circuits with activity-dependent nuclear import of a transcription factor. J. Neurogenet. 26, 89–102 (2012)

  42. 42.

    et al. The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95, 681–692 (1998)

  43. 43.

    , & Circadian oscillations in period gene mRNA levels are transcriptionally regulated. Proc. Natl Acad. Sci. USA 89, 11711–11715 (1992)

  44. 44.

    , , & Transcriptional feedback loop regulation, function, and ontogeny in Drosophila. Cold Spring Harb. Symp. Quant. Biol. 72, 437–444 (2007)

  45. 45.

    , , , & A role for blind DN2 clock neurons in temperature entrainment of the Drosophila larval brain. J. Neurosci. 29, 8312–8320 (2009)

  46. 46.

    , , , & Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J. Neurosci. 26, 13400–13410 (2006)

  47. 47.

    , & Synchronous Drosophila circadian pacemakers display nonsynchronous Ca2+ rhythms in vivo. Science 351, 976–981 (2016)

  48. 48.

    et al. A transcriptional reporter of intracellular Ca2+ in Drosophila. Nat. Neurosci. 18, 917–925 (2015)

  49. 49.

    Analysis of rhythmic gene expression in adult Drosophila using the firefly luciferase reporter gene. Methods Mol. Biol. 362, 131–142 (2007)

  50. 50.

    , , & Light-mediated TIM degradation within Drosophila pacemaker neurons (s-LNvs) is neither necessary nor sufficient for delay zone phase shifts. Neuron 66, 378–385 (2010)

  51. 51.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

  52. 52.

    , & A single pair of neurons links sleep to memory consolidation in Drosophila melanogaster. eLife 4, e03868 (2015)

Download references

Acknowledgements

We thank M. Diaz, N. Nguyen, R. Spann and K. Kerr for generous help, and X. Gao and L. Luo for sending us LexAop-LUC flies, and O. Shafer and A. Sehgal for helpful discussion and comments on early versions of this manuscript. This work was supported in part by NIH R01 MH067284 (LCG).

Author information

Affiliations

  1. Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, Massachusetts 02454, USA

    • Fang Guo
    • , Hyung Jae Jung
    • , Katharine C. Abruzzi
    • , Weifei Luo
    •  & Michael Rosbash
  2. Department of Biology, National Center for Behavioral Genomics and Volen National Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02454, USA

    • Fang Guo
    • , Junwei Yu
    • , Hyung Jae Jung
    • , Katharine C. Abruzzi
    • , Weifei Luo
    • , Leslie C. Griffith
    •  & Michael Rosbash

Authors

  1. Search for Fang Guo in:

  2. Search for Junwei Yu in:

  3. Search for Hyung Jae Jung in:

  4. Search for Katharine C. Abruzzi in:

  5. Search for Weifei Luo in:

  6. Search for Leslie C. Griffith in:

  7. Search for Michael Rosbash in:

Contributions

F.G. and M.R. conceived and designed the experiments. F.G. performed behavioural experiments. F.G. and J.Y. performed immunocytochemistry and imaging experiments. F.G. and H.J.J. setup the recording system. K.C.A. and W.L. performed and quantified the mRNA profiling data. F.G. and J.Y. analysed data. F.G., L.G. and M.R. prepared the figures and wrote the paper, with feedback from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Rosbash.

Reviewer Information

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

Extended data

Supplementary information

Videos

  1. 1.

    Live calcium imaging of circadian neuron responses to DN1 activation

    The calcium responses of clock neurons expressing GCaMP6f to ATP/P2X2-activation of DN1s. The genotype of flies used in the experiment is Clk856-GAL4/UAS-GCaMP6f; Clk4.1M-LexA/LexAop-P2X2. This video is accelerated 15X. ATP was added from 30 S (2S in the video).

  2. 2.

    R18H11-GAL4-labeled DN1 arbors co-localize with VGLUT

    Confocal stack of antibody staining of GFP and VGLUT (red) in the dorsal brain of R18H11-GAL4>UAS-mCD8::GFP flies. Most R18H11-GAL4-labeled DN1s are glutamatergic as VGLUT is highly enriched in their projections.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature19097

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.