Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Optical stimulation of specific spinal neurons leads to distinct locomotor behaviours.
Figure 2: The Gal4 s1020t line drives expression in motor neurons and Kolmer–Agduhr neurons.
Figure 3: Optical stimulation of Kolmer–Agduhr cells of Gal4 s1003t line induces a forward swim.
Figure 4: Dissection of the light-evoked responses in Gal4 s1020t and Gal4 s1102t by unilateral stimulation and lesion studies.

Similar content being viewed by others

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 

Download references

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:LiGluR10 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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Herwig 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 Gal4s1020t/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 Gal4s1102t/UAS:LiGluR - see file s1 for full Legend. (MOV 619 kb)

Supplementary Movie 5

This movie file shows light induced response in Gal4s1003t/UAS:LiGluR - see file s1 for full Legend. (MOV 348 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08323

This article is cited by

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.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing