"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca(2+) imaging.
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" Ca(2+) imaging in this preparation. We demonstrate that bolus injection of membrane-permeable Ca(2+) 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 Ca(2+) signals simultaneously from a large population of spinal neurons with single-cell resolution. To test the method, Ca(2+) 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 Ca(2+) transients were blocked by the application of strychnine. Sensory stimuli that trigger escape reflexes in mobile zebrafish evoked Ca(2+) transients in distinct neurons of the spinal network. Moreover, long-term recordings revealed spontaneous Ca(2+) 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.
Brustein
E
E
McGill Centre for Research in Neuroscience and Department of Biology, McGill University, H3G 1A4, Montreal, Quebec, Canada.
Marandi
N
N
Kovalchuk
Y
Y
Drapeau
P
P
Konnerth
A
A
D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D004396:Coloring Agents; D003952:Diagnostic Imaging; D018846:Excitatory Amino Acids / Q000037:antagonists & inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D018684:Glycine Agents / Q000494:pharmacology; D066298:In Vitro Techniques; D007814:Larva / Q000502:physiology; D009415:Nerve Net / Q000187:drug effects / Q000254:growth & development / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D013116:Spinal Cord / Q000166:cytology / Q000254:growth & development / Q000502:physiology; D013331:Strychnine / Q000494:pharmacology; D015027:Zebrafish / Q000502:physiology*
D004396:Coloring Agents; D018846:Excitatory Amino Acids; D005456:Fluorescent Dyes; D018684:Glycine Agents; D013331:Strychnine; D002118:Calcium