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Optogenetic dissection of a behavioural module in the vertebrate spinal cord


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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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.


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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.

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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)

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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).

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