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"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging

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Abstract

The zebrafish larva is a powerful model for the analysis of behaviour and the underlying neuronal network activity during early stages of development. Here we employ a new approach of "in vivo" Ca2+ imaging in this preparation. We demonstrate that bolus injection of membrane-permeable Ca2+ indicator dyes into the spinal cord of zebrafish larvae results in rapid staining of essentially the entire spinal cord. Using two-photon imaging, we could monitor Ca2+ signals simultaneously from a large population of spinal neurons with single-cell resolution. To test the method, Ca2+ transients were produced by iontophoretic application of glutamate and, as observed for the first time in a living preparation, of GABA or glycine. Glycine-evoked Ca2+ transients were blocked by the application of strychnine. Sensory stimuli that trigger escape reflexes in mobile zebrafish evoked Ca2+ transients in distinct neurons of the spinal network. Moreover, long-term recordings revealed spontaneous Ca2+ transients in individual spinal neurons. Frequently, this activity occurred synchronously among many neurons in the network. In conclusion, the new approach permits a reliable analysis with single-cell resolution of the functional organisation of developing neuronal networks.

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Acknowledgements

We thank O. Garaschuk and J. Davis for comments on the manuscript, R. Maul, S. Schickle, I. Schneider, for technical assistance, C. Gauthier for support on graphical design, L. Bailly-Cuif for providing zebrafish eggs and G. Laliberté and L. Brent for excellent animal care. This work was supported by an HFSP short-term fellowship to E.B. and by grants from the CIHR and NSERC of Canada to P.D.

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Authors and Affiliations

  1. Institut für Physiologie, Ludwig-Maximilians Universität München, Pettenkoferstrasse12, 80336, Munich, Germany

    N. Marandi, Y. Kovalchuk & A. Konnerth

  2. McGill Centre for Research in Neuroscience and Department of Biology, McGill University, Montreal, Quebec, H3G 1A4, Canada

    E. Brustein & P. Drapeau

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  1. E. Brustein
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  2. N. Marandi
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  3. Y. Kovalchuk
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  4. P. Drapeau
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  5. A. Konnerth
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Correspondence to A. Konnerth.

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Brustein, E., Marandi, N., Kovalchuk, Y. et al. "In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging. Pflugers Arch - Eur J Physiol 446, 766–773 (2003). https://doi.org/10.1007/s00424-003-1138-4

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  • DOI: https://doi.org/10.1007/s00424-003-1138-4

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