Abstract
Vertebrate behaviours are produced by activity in populations of neurons, but the techniques typically used to study activity allow only one or very few nerve cells to be monitored at a time. This limitation has prompted the development of methods of imaging activity in the nervous system. The overall goal of these methods is to image neural activity non-invasively in populations of neurons, ideally with high spatial and temporal resolution. We have moved closer to this goal by using confocal calcium imaging to monitor neural activity in the transparent larvae of zebrafish. Neurons were labelled either by backfilling from injections of the calcium indicator (Calcium Green dextran) into muscle or spinal cord of larvae or by injections into blastomeres early in development. The labelled neurons were bright enough at resting calcium levels to allow the identification of individual neurons in the live, intact fish, based upon their dendritic and axonal morphology. The neurons from the live animal could also be reconstructed in three dimensions for morphometric study. Neurons increased their fluorescence during activity produced by direct electrical stimulation and during escape behaviours elicited by an abrupt touch to the head or tail of the fish. The rise in calcium associated with a single action potential could be detected as an increase in fluorescence of at least 7--10%, but neurons typically showed much larger increases during behaviour. Calcium signals in the dendrites, soma and nucleus could be resolved, especially when using the line-scanning mode, which provides 2-ms temporal resolution. The imaging was used to study activity in populations of motoneurons and hindbrain neurons during the escape behaviour fish use to avoid predators. We found a massive activation of the motoneuron pool and a differential activation of populations of hindbrain neurons during escapes. The latter finding confirms predictions that the activity pattern of hindbrain neurons may help to determine the directionality of the escape. This approach should prove useful for studying the activity of populations of neurons throughout the nervous system in both normal and mutant lines of fish. 1998 © Chapman & Hall
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References
Bacskai, B.J., Wallen, P., Lev-ram, V., Grillner, S. & Tsien, R.Y. (1995) Activity-related calcium dynamics in lamprey motoneurons as revealed by video-rate confocal microscopy. Neuron 14, 19–28.
Belliveau, J.W., Kennedy, D.N., Mckinstry, R.C., Buchbinder, B.R., Weisskoff, R.M., Cohen, M.S., Vevea, J.M. & Rosen, B.B. (1991) Functional mapping of the human visual cortex by magnetic resonance imaging. Science 254, 716–19.
Bernhardt, R.R., Chitnis, A.B., Lindamer, L. & Kuwada, J.Y. (1990) Identification of spinal neurons in the embryonic and larval zebrafish. J. Comp. Neurol. 302, 603–16.
Clarke, J.D.W. & Lumsden, A. (1993) Segmental repetition of neuronal phenotype sets in the chick embryo hindbrain. Development 118, 151–62.
Cox, K.J.A. & Fetcho, J.R. (1996) Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish. J. Neurosci. Methods 68, 185–96.
Driever, W., Solnica-krezel, S., Schier, A.F., Neuhauss, S.C.F., Malicki, J., Stemple, D.L., Stainier, D.Y.R., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J. & Boggs, C. (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37–76.
Eaton, R.C., Nissanov, J. & Wieland, C.M. (1984) Differential activation of Mauthner and non-Mauthner startle circuits in the zebrafish: implications for functional substitution. J. Comp. Physiol. 155, 813–20.
Eaton, R.C. & Emberley, D.S. (1991) How stimulus direction determines the trajectory of the Mauthner initiated escape response. J. Exp. Biol. 161, 469–87.
Fetcho, J.R. (1992) Excitation of motoneurons by the Mauthner axon in goldfish: complexities in a 'simple' reticulospinal pathway. J. Neurophysiol. 67, 1574–86.
Fetcho, J.R. & Faber, D.S. (1988) Identification of motoneurons and interneurons in the spinal network for escapes initiated by the Mauthner cell in goldfish. J. Neurosci. 8, 4192–213.
Fetcho, J.R. & O'Malley, D.M. (1995) Visualization of active neural circuitry in the spinal cord of intact zebrafish. J. Neurophysiol. 73, 399–406.
Foreman, M.B. & Eaton, R.C. (1993) The direction change concept for reticulospinal control of goldfish escape. J. Neurosci. 13, 4101–13.
Fritzsch, B. (1993) Fast axonal diffusion of 3000 molecular weight dextran amines. J. Neurosci. Methods 50, 95–103.
Guthrie, S. (1995) The status of the neural segment. Trends Neurol. Sci. 18, 74–9.
Haffter, P., Granato, M., Brand, M., Mullins, M.C., Hammerschmidt, M., Kane, D.A., Odenthal, J., van Eeden, F.J.M., Jiang, Y.-J., Heidenberg, C.-P., Kelsch, R.N., Furutani-seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C. & NÜsslein-volhard, C. (1996) The identification of genes with unique and essential functions in the development of zebrafish, Danio reria. Development 123, 1–36.
Jayne, B.C. & Lauder, G.V. (1993) Red and white muscle activity and kinematics of the escape response of the bluegill sunfish during swimming. J. Comp. Physiol. A 173, 495–508.
Kao, J.P.Y. (1994) Practical aspects of measuring [Ca++] with fluorescent indicators. In A Practical Guide to the Study of Calcium in Living Cells (edited by Nuccitelli, R. ), pp. 155–181. New York: Academic Press.
Kimmel, C.B. & Law, R.D. (1985) Cell lineage of zebrafish blastomeres. III. Clonal analysis of the blastula and gastrula stages. Dev. Biol. 108, 94–101.
Liu, D.W. & Westerfield, M. (1988) Function of identified motoneurons and coordination of primary and secondary motor systems during zebra fish swimming. J. Physiol. 403, 73–89.
Mcclellan, A.D., Mcpherson, D. & O'Donovan, M. J. (1994) Combined retrograde labeling and calcium imaging in spinal cord and brainstem neurons of the lamprey. Brain Res. 663, 61–8.
Metcalfe, W.K., Mendelson, B. & Kimmel, C.B. (1986) Segmental homologies among reticulospinal neurons in the hindbrain of the zebrafish larva. J. Comp. Neurol. 251, 147–59.
Myers, P.Z., Eisen, J.S. & Westerfield, M. (1986) Development and axonal outgrowth of identified motoneurons in the zebrafish. J. Neurosci. 6, 2278–89.
O'Donovan, M.J., 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'Malley, D.M. (1994) Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion. J. Neurosci. 14, 5741–58.
O'Malley, D.M. & Fetcho, J.R. (1996) Calcium signals in the zebrafish Mauthner cell: large size and potentiation with repetitive sensory stimulation. Soc. Neurosci. Abst. 22, 795.
O'Malley, D.M., Kao, Y.-H. & Fetcho, J.R. (1996) Imaging the functional organization of zebrafish hindbrain segments during escape behaviors. Neuron 17, 1145–55.
Rome, L.C., Funke, R.P., Mcneill Alexander, R., Lutz, G., Aldridge, H., Scott, F. & Freadman, M. (1988) Why animals have different muscle fibre types. Nature 335, 824–7.
Sobierajski, L.M., Avila, R.S., O'Malley, D.M., Wang, S. & Kaufmann, A.E. (1995) Visualization of calcium activity in nerve cells. IEEE Computer Graphics Appl. 15, 55–61.
Svoboda, K.R. & Fetcho, J.R. (1996) Interactions between the neural networks for escape and swimming in goldfish. J. Neurosci. 16, 843–52.
Westerfield, M. (1993) The Zebrafish Book. Eugene, OR: Oregon University Press.
Wilson, M.A. & Mcnaughton, B.L. (1994) Reactivation of hippocampal ensemble memories during sleep. Science 265, 676–9.
Wong, R.O.C., Chernjavsky, A., Smith, S.J. & Shatz, C.J. (1995) Early functional neural networks in the developing retina. Nature 374, 716–18.
Wu, J.-Y, Cohen, L.B. & Falk, C.X. (1994) Neuronal activity during different behaviours in Aplysia: a distributed organization? Science 263, 820–3.
Wu, J.-Y., Lam, Y.-W., Falk, C.X., Cohen, L.B., Fang, J., Loen, L., Prechtl, J.C., Kleinfeld, D. & Tsau, Y. (1998) Voltage-sensitive dyes for monitoring multineuronal activity in the intact central nervous system. Histochem. J. 30, 169–187.
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Fetcho, J.R., Cox, K.J.A. & O'Malley, D.M. Monitoring activity in neuronal populations with single-cell resolution in a behaving vertebrate. Histochem J 30, 153–167 (1998). https://doi.org/10.1023/A:1003243302777
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DOI: https://doi.org/10.1023/A:1003243302777