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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References
Ali DW, Drapeau P, Legendre P (2000) Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo. J Neurophysiol 84:1726–1736
Ashworth R, Bolsover SR (2002) Spontaneous activity-independent intracellular calcium signals in the developing spinal cord of the zebrafish embryo. Brain Res Dev Brain Res 139:131–137
Ashworth R, Zimprich F, Bolsover SR (2001) Buffering intracellular calcium disrupts motoneuron development in intact zebrafish embryos. Brain Res Dev Brain Res 129:169–179
Ben-Ari Y (2002) Excitatory actions of GABA during development: the nature of the nurture. Nat Rev Neurosci 3:728–739
Budick SA, O'Malley DM (2000) Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J Exp Biol 203:2565–2579
Buss RR, Drapeau P (2001) Synaptic drive to motoneurons during fictive swimming in the developing zebrafish. J Neurophysiol 86:197–210
Buss R, Ali D, Drapeau P (1999) Properties of synaptic currents and fictive motor behaviors in neurons of locomotor regions of the developing zebrafish (abstract). Soc Neurosci Abstr 25:467.3
Cox KJ, Fetcho JR (1996) Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish. J Neurosci Methods 68:185–191
Creton R, Speksnijder JE, Jaffe LF (1998) Patterns of free calcium in zebrafish embryos. J Cell Sci 111:1613–1622
Drapeau P, Ali DW, Buss RR, Saint-Amant L (1999) In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish. J Neurosci Methods 88:1–13
Feller MB (1999) Spontaneous correlated activity in developing neural circuits. Neuron 22:653–656
Fetcho JR, O'Malley DM (1995) Visualization of active neural circuitry in the spinal cord of intact zebrafish. J Neurophysiol 73:399–406
Gahtan E, Sankrithi N, Campos JB, O'Malley DM (2002) Evidence for a widespread brain stem escape network in larval zebrafish. J Neurophysiol 87:608–614
Garaschuk O, Hanse E, Konnerth A (1998) Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus. J Physiol (Lond) 507:219–236
Garaschuk O, Linn J, Eilers J, Konnerth A (2000) Large-scale oscillatory calcium waves in the immature cortex. Nat Neurosci 3:452–459
Ghosh A, Greenberg ME (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268:239–247
Gleason MR, Higashijima S, Dallman J, Liu K, Mandel G, Fetcho JR (2003) Translocation of CaM kinase II to synaptic sites in vivo. Nat Neurosci 6:217–218
Gomez TM, Spitzer NC (1999) In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397:350–355
Hatta K, Ankri N, Faber DS, Korn H (2001) Slow inhibitory potentials in the teleost Mauthner cell. Neuroscience 103:561–579
Helmchen F, Waters J (2002) Ca2+ imaging in the mammalian brain in vivo. Eur J Pharmacol 447:119–129
O'Donovan MJ, Ho S, Sholomenko G, Yee W (1993) Real-time imaging of neurons retrogradely and anterogradely labelled with calcium-sensitive dyes. J Neurosci Methods 46:91–106
O'Donovan M, Ho S, Yee W (1994) Calcium imaging of rhythmic network activity in the developing spinal cord of the chick embryo. J Neurosci 14:6354–6369
O'Malley DM, Kao YH, Fetcho JR (1996) Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17:1145–1155
Regehr WG, Tank DW (1991) Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J Neurosci Methods 37:111–119
Ritter DA, Bhatt DH, Fetcho JR (2001) In vivo imaging of zebrafish reveals differences in the spinal networks for escape and swimming movements. J Neurosci 21:8956–8965
Saint-Amant L, Drapeau P (2000) Motoneuron activity patterns related to the earliest behavior of the zebrafish embryo. J Neurosci 20:3964–3972
Schoenwolf GC (2001) Cutting, pasting and painting: experimental embryology and neural development. Nat Rev Neurosci 2:763–771
Segal M (2001) Rapid plasticity of dendritic spine: hints to possible functions? Prog Neurobiol 63:61–70
Smetters D, Majewska A, Yuste R (1999) Detecting action potentials in neuronal populations with calcium imaging. Methods 18:215–221
Spitzer NC, Lautermilch NJ, Smith RD, Gomez TM (2000) Coding of neuronal differentiation by calcium transients. Bioessays 22:811–817
Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci USA 100:7319–7324
Takahashi M, Narushima M, Oda Y (2002) In vivo imaging of functional inhibitory networks on the Mauthner cell of larval zebrafish. J Neurosci 22:3929–3938
Westerfield M (1995) The zebrafish book: a guide for laboratory use of zebrafish (Brachydanio rerio). University of Oregon Press, Eugene
Wong RO, Chernjavsky A, Smith SJ, Shatz CJ (1995) Early functional neural networks in the developing retina. Nature 374:716–718
Wu LG, Saggau P (1994) Adenosine inhibits evoked synaptic transmission primarily by reducing presynaptic calcium influx in area CA1 of hippocampus. Neuron 12:1139–1148
Zhang LI, Poo MM (2001) Electrical activity and development of neural circuits. Nat Neurosci 4 (Suppl):1207–1214
Zimprich F, Ashworth R, Bolsover S (1998) Real-time measurements of calcium dynamics in neurons developing in situ within zebrafish embryos. Pflugers Arch 436:489–493
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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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