Optogenetic dissection of a behavioural module in the vertebrate spinal cord (original) (raw)

Nature volume 461, pages 407–410 (2009)Cite this article

Abstract

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements1. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord2,3. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics4 in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer–Agduhr cell5, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments6. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer–Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

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References

  1. Grillner, S. Biological pattern generation: the cellular and computational logic of networks in motion. Neuron 52, 751–766 (2006)
    Article CAS Google Scholar
  2. Fenaux, F. et al. Effects of an NMDA-receptor antagonist, MK-801, on central locomotor programming in the rabbit. Exp. Brain Res. 86, 393–401 (1991)
    Article CAS Google Scholar
  3. Kiehn, O. et al. Excitatory components of the mammalian locomotor CPG. Brain Res. Rev. 57, 56–63 (2008)
    Article ADS Google Scholar
  4. Luo, L., Callaway, E. M. & Svoboda, K. Genetic dissection of neural circuits. Neuron 57, 634–660 (2008)
    Article CAS Google Scholar
  5. Agduhr, E. in Cytology and Cellular Pathology of the Nervous System (ed. Penfield, W.) Vol. 2 535–573 (Hoeber, 1932)
    Google Scholar
  6. Higashijima, S., Mandel, G. & Fetcho, J. R. Distribution of prospective glutamatergic, glycinergic, and GABAergic neurons in embryonic and larval zebrafish. J. Comp. Neurol. 480, 1–19 (2004)
    Article CAS Google Scholar
  7. Douglas, J. R. et al. The effects of intrathecal administration of excitatory amino acid agonists and antagonists on the initiation of locomotion in the adult cat. J. Neurosci. 13, 990–1000 (1993)
    Article CAS Google Scholar
  8. Volgraf, M. et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nature Chem. Biol. 2, 47–52 (2006)
    Article CAS Google Scholar
  9. Gorostiza, P. et al. Mechanisms of photoswitch conjugation and light activation of an ionotropic glutamate receptor. Proc. Natl Acad. Sci. USA 104, 10865–10870 (2007)
    Article ADS CAS Google Scholar
  10. Szobota, S. et al. Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54, 535–545 (2007)
    Article CAS Google Scholar
  11. Scott, E. K. et al. Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat. Methods 4, 323–326 (2007)
    Article CAS Google Scholar
  12. Budick, S. A. & O’Malley, D. M. Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J. Exp. Biol. 203, 2565–2579 (2000)
    CAS PubMed Google Scholar
  13. Liu, K. S. & Fetcho, J. R. Laser ablations reveal functional relationships of segmental hindbrain neurons in zebrafish. Neuron 23, 325–335 (1999)
    Article CAS Google Scholar
  14. Shin, J., Park, H. C., Topczewska, J. M., Mawdsley, D. J. & Appel, B. Neural cell fate analysis in zebrafish using olig2 BAC transgenics. Methods Cell Sci. 25, 7–14 (2003)
    Article CAS Google Scholar
  15. Dale, N. et al. The morphology and distribution of ‘Kolmer–Agduhr cells’, a class of cerebrospinal-fluid-contacting neurons revealed in the frog embryo spinal cord by GABA immunocytochemistry. Proc. R. Soc. Lond. B 232, 193–203 (1987)
    Article ADS CAS Google Scholar
  16. Liao, J. C. & Fetcho, J. R. Shared versus specialized glycinergic spinal interneurons in axial motor circuits of larval zebrafish. J. Neurosci. 28, 12982–12992 (2008)
    Article CAS Google Scholar
  17. Drapeau, P. et al. In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish. J. Neurosci. Methods 88, 1–13 (1999)
    Article MathSciNet CAS Google Scholar
  18. Asakawa, K. et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc. Natl Acad. Sci. USA 105, 1255–1260 (2008)
    Article ADS CAS Google Scholar
  19. Gosgnach, S. et al. V1 spinal neurons regulate the speed of vertebrate locomotor outputs. Nature 440, 215–219 (2006)
    Article ADS CAS Google Scholar
  20. McLean, D. L. et al. Continuous shifts in the active set of spinal interneurons during changes in locomotor speed. Nature Neurosci. 11, 1419–1429 (2008)
    Article CAS Google Scholar
  21. Ritter, D. A., Bhatt, D. H. & Fetcho, J. R. In vivo imaging of zebrafish reveals differences in the spinal networks for escape and swimming movements. J. Neurosci. 21, 8956–8965 (2001)
    Article CAS Google Scholar
  22. Alford, S., Sigvardt, K. A. & Williams, T. L. GABAergic control of rhythmic activity in the presence of strychnine in the lamprey spinal cord. Brain Res. 506, 303–306 (1990)
    Article CAS Google Scholar
  23. Brustein, E. & Drapeau, P. Serotoninergic modulation of chloride homeostasis during maturation of the locomotor network in zebrafish. J. Neurosci. 25, 10607–10616 (2005)
    Article CAS Google Scholar
  24. Vigh, B. & Vigh-Teichmann, I. Actual problems of the cerebrospinal fluid-contacting neurons. Microsc. Res. Tech. 41, 57–83 (1998)
    Article CAS Google Scholar
  25. Stoeckel, M. E. et al. Cerebrospinal fluid-contacting neurons in the rat spinal cord, a gamma-aminobutyric acidergic system expressing the P2X2 subunit of purinergic receptors, PSA-NCAM, and GAP-43 immunoreactivities: light and electron microscopic study. J. Comp. Neurol. 457, 159–174 (2003)
    Article Google Scholar
  26. Furusho, M. et al. Involvement of the Olig2 transcription factor in cholinergic neuron development of the basal forebrain. Dev. Biol. 293, 348–357 (2006)
    Article CAS Google Scholar
  27. Huang, A. L. et al. The cells and logic for mammalian sour taste detection. Nature 442, 934–938 (2006)
    Article ADS CAS Google Scholar
  28. Xiao, T. & Baier, H. Lamina-specific axonal projections in the zebrafish tectum require the type IV collagen Dragnet. Nature Neurosci. 10, 1529–1537 (2007)
    Article CAS Google Scholar
  29. Flanagan-Steet, H., Fox, M. A., Meyer, D. & Sanes, J. Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations. Development 132, 4471–4481 (2005)
    Article CAS Google Scholar
  30. Koster, R. W. & Fraser, S. E. Tracing transgene expression in living zebrafish embryos. Dev. Biol. 233, 329–346 (2001)
    Article CAS Google Scholar
  31. Masahira, N. et al. Olig2-positive progenitors in the embryonic spinal cord give rise not only to motoneurons and oligodendrocytes, but also to a subset of astrocytes and ependymal cells. Dev. Biol. 293, 358–369 (2006)
    Article CAS Google Scholar

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Acknowledgements

We thank M. Volgraf for MAG-1 synthesis, K. Kawakami for the UAS:TeTxLC-CFP line, B. Appel for the Olig2-DsRed line, W. Staub for animal care, D. Li for help with screening BGUG larvae, B. Vigh, C. Girit, E. Brustein, P. Drapeau and S. Hugel for discussions, P.G. de Gennes and Noam Sobel for support and O. Wyart for aesthetic input. We are grateful to K. Best, P. Tavormina, H. Aaron, R. Ayer, B. Nowak and M. Ulbrich for advice on the design of the photostimulation setup. Support for the work was from the Marie Curie Outgoing International Fellowship (with the CNRS – UMR5020 ‘Neurosciences Sensorielles, Comportement Cognition’ laboratory, Lyon, France) (C.W.), the Human Frontier Science Program Long-term Postdoctoral Fellowship (F.D.B.), the National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (5PN2EY018241) (E.Y.I., D.T. and H.B.), the Human Frontiers Science Program (RGP23-2005) (E.Y.I. and D.T.), the Lawrence Berkeley National Laboratory Directed Research and Development Program (E.Y.I. and D.T.), R01 NS053358 (H.B.) and a Sandler Opportunity Award (H.B.).

Author Contributions C.W., F.D.B, H.B. and E.Y.I. made critical primary contributions to this study. C.W. built the photostimulation setup, performed behavioural experiments, lesions, pharmacology, calcium imaging, imaging of the immunolabelled larvae, anatomical analysis based on BGUG imaging and wrote the Matlab scripts for analysing behaviour and imaging. F.D.B. generated the transgenic lines _UAS:LiGluR_10 and Hb9:Gal4, as well as performing the immunochemistry experiments. E.W. participated in the anatomical analysis of BGUG. E.K.S. and H.B. generated the enhancer trap Gal4 screen, which made the ‘intersectional optogenetic’ approach possible11. E.Y.I. and D.T. developed chemical optogenetics with LiGluR8. C.W. and E.Y.I. wrote the manuscript with feedback from H.B. and F.D.B. H.B. and E.Y.I. supervised C.W. and F.D.B. and contributed to the planning of all aspects of this project.

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Author notes

  1. Ethan K. Scott
    Present address: Present address: School of Biomedical Sciences, University of Queensland, Queensland 4072, Australia.,
  2. Claire Wyart and Filippo Del Bene: These authors contributed equally to this work.

Authors and Affiliations

  1. Helen Wills Neuroscience Institute and Department of Molecular and Cell Biology, University of California in Berkeley, Berkeley, California 94720, USA,
    Claire Wyart, Erica Warp & Ehud Y. Isacoff
  2. Department of Physiology, Program in Neuroscience, University of California in San Francisco, San Francisco, California 94158-2324, USA,
    Filippo Del Bene, Ethan K. Scott & Herwig Baier
  3. Department of Chemistry, Ludwig Maximilians-Universität, Munich, Germany
    Dirk Trauner
  4. Physical Bioscience Division and Material Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA,
    Ehud Y. Isacoff

Authors

  1. Claire Wyart
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  2. Filippo Del Bene
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  3. Erica Warp
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  4. Ethan K. Scott
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  5. Dirk Trauner
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  6. Herwig Baier
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  7. Ehud Y. Isacoff
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Corresponding authors

Correspondence toHerwig Baier or Ehud Y. Isacoff.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with Legends and Legends for Supplementary Movies 1-5. (PDF 1791 kb)

Supplementary Movie 1

This movie file shows spontaneous slow swim of wt larva at 5dpf - see file s1 for full Legend. (MOV 74 kb)

Supplementary Movie 2

This movie file shows light induced response in Gal4 s1020t /UAS:LiGluR - see file s1 for full Legend. (MOV 2307 kb)

Supplementary Movie 3

This movie file shows Water puff escape response in a wt larva - see file s1 for full Legend. (MOV 206 kb)

Supplementary Movie 4

This movie file shows light induced response in Gal4 s1102t /UAS:LiGluR - see file s1 for full Legend. (MOV 619 kb)

Supplementary Movie 5

This movie file shows light induced response in Gal4 s1003t /UAS:LiGluR - see file s1 for full Legend. (MOV 348 kb)

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Wyart, C., Bene, F., Warp, E. et al. Optogenetic dissection of a behavioural module in the vertebrate spinal cord.Nature 461, 407–410 (2009). https://doi.org/10.1038/nature08323

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Editorial Summary

Kolmer–Agduhr cells in spinal cord development

In the brief period during which we have known of their existence, light-gated ion channels have been used to assess the function of known cell types to which they are genetically targeted. Here Wyart et al. search for unknown cell types that drive the central pattern generator of locomotion. GAL4 lines of zebrafish in which light-gated glutamate receptors were sparsely expressed in diverse, partially overlapping sets of neurons were screened. Common behavioural effects of light could thus be attributed to activity in a specific cell type when it is the only cell shared between the different lines. The photo-stimulation of one specific cell type, the Kolmer–Agduhr cell, was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. Genetically silencing Kolmer–Agduhr cells reduced the frequency of spontaneous free swimming, indicating that Kolmer–Agduhr cell activity provides necessary tone for spontaneous forward swimming. Kolmer–Agduhr cells have been known for over 75 years, but their function has been mysterious. This work shows that during early development in low vertebrates these cells provide a positive drive to the spinal central pattern generator for spontaneous locomotion.