Journal
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
Volume 446, Issue 6, Pages 766-773Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00424-003-1138-4
Keywords
Ca2+ imaging; in vivo; neuronal network activity; two-photon excitation; zebrafish
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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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