Imaging of Localized Neuronal Calcium Influx

  • Fritjof Helmchen
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


Intracellular Ca2+ controls such diverse processes as growth, cell division, contraction, secretion, and cell death. In neurons Ca2+ influx triggers neurotransmitter release, causes activation of various enzyme cascades, and regulates gene expression (1). Increases in the intracellular calcium concentration ([Ca2+]) also affect membrane excitability and are involved in synaptic plasticity (2). How does Ca2+ accomplish this multitude of tasks, often within the same cell? A clue to the answer is the spatial segregation of Ca2+ signaling pathways in different cellular compartments (3). This compartmentalization is based on the nonuniform cellular distribution of Ca2+-permeable ion channels, intracellular Ca2+-binding proteins, and Ca2+ pumps. Localized Ca2+ signaling enormously increases the cells’ ability and flexibility to use Ca2+ as an intracellular messenger in many parallel ways.


Hair Cell Dendritic Spine Release Site Transduction Channel Diffusible Indicator 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Ghosh, A. and Greenberg, M. E. (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268, 239–247.PubMedCrossRefGoogle Scholar
  2. 2.
    Zucker, R. S. (1999) Calcium-and activity-dependent synaptic plasticity. Curr. Opin. Neurobiol. 9, 305–313.PubMedCrossRefGoogle Scholar
  3. 3.
    Wang, S. S.-H. and Augustine, G. J. (1999) Calcium signaling in neurons: a case study in cellular compartmentalization, in Calcium as a Cellular Regulator (Carafoli, E. and Klee, C., eds.), Oxford University Press, Oxford, UK, pp. 545–566.Google Scholar
  4. 4.
    Tsien, R. Y. (1999) Monitoring cell calcium, in Calcium as a Cellular Regulator (Carafoli, E. and Klee, C., eds.), Oxford University Press, Oxford, UK, pp. 28–54.Google Scholar
  5. 5.
    Augustine, G. J. and Neher, E. (1992) Neuronal Ca2+ signalling takes the local route. Curr. Opin. Neurobiol. 2, 302–307.PubMedCrossRefGoogle Scholar
  6. 6.
    Helmchen, F. (1999) Dendrites as biochemical compartments, in Dendrites (Stuart, G., Spruston, N., and Hausser, M., eds.), Oxford University Press, Oxford, UK, pp. 161–192.Google Scholar
  7. 7.
    Brini, M., Pinton, P., Pozzan, T., and Rizzuto, R. (1999) Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cell. Microsc. Res. Tech. 46, 380–389.PubMedCrossRefGoogle Scholar
  8. 8.
    Sabatini, B. L. and Regehr, W. G. (1998) Optical measurement of presynaptic calcium current. Biophys. J. 74, 1549–1563.PubMedCrossRefGoogle Scholar
  9. 9.
    Etter, E. F., Kuhn, M. A., and Fay, F. S. (1994) Detection of changes in near membrane Ca2+ using a novel membrane associated Ca2+ indicator. J. Biol. Chem. 269, 10,141–10,149.PubMedGoogle Scholar
  10. 10.
    Vorndran, C., Minta, A., and Poenie, M. (1995) New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes. Biophys. J. 69, 2112–2124.PubMedCrossRefGoogle Scholar
  11. 11.
    Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887.PubMedCrossRefGoogle Scholar
  12. 12.
    Yuste, R., Lanni, F., and Konnerth, A. (eds.) (1999) Imaging Neurons: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  13. 13.
    DiGregorio, D. A. and Vergara, J. L. (1997) Localized detection of action potential-induced presynaptic calcium transients at a Xenopus neuromuscular junction. J. Physiol. (Lond.) 505, 585–592.CrossRefGoogle Scholar
  14. 14.
    Pawley, J. B. (ed.) (1995) Handbook of Biological Confocal Microscopy. Plenum, New York.Google Scholar
  15. 15.
    Denk, W., Strickler, J. H., and Webb, W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science 248, 73–76.PubMedCrossRefGoogle Scholar
  16. 16.
    Denk, W., Yuste, R., Svoboda, K., and Tank, D. W. (1996) Imaging calcium dynamics in dendritic spines. Curr. Opin. Neurobiol. 6, 372–378.PubMedCrossRefGoogle Scholar
  17. 17.
    Denk, W. and Svoboda, K. (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357.PubMedCrossRefGoogle Scholar
  18. 18.
    Schiller, J., Helmchen, F., and Sakmann, B. (1995) Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones. J. Physiol. (Lond.) 487, 583–600.Google Scholar
  19. 19.
    Monck, J. R., Robinson, I. M., Escobar, A. L., Vergara, J. L., and Fernandez, J. M. (1994) Pulsed laser imaging of rapid Ca2+ gradients in excitable cells. Biophys. J. 67, 505–514.PubMedCrossRefGoogle Scholar
  20. 20.
    Neher, E. (1995) The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34, 1423–1442.PubMedCrossRefGoogle Scholar
  21. 21.
    Schneggenburger, R., Zhou, Z., Konnerth, A., and Neher, E. (1993) Fractional contribution of calcium to the cation current through glutamate receptor channels. Neuron 11, 133–43.PubMedCrossRefGoogle Scholar
  22. 22.
    Bollmann, J. H., Helmchen, F., Borst, J. G., and Sakmann, B. (1998) Postsynaptic Ca2+ influx mediated by three different pathways during synaptic transmission at a calyx-type synapse. J. Neurosci. 18, 10,409–10,419.PubMedGoogle Scholar
  23. 23.
    Garaschuk, O., Schneggenburger, R., Schirra, C., Tempia, F., and Konnerth, A. (1996) Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones. J. Physiol. (Lond.) 491, 757–772.Google Scholar
  24. 24.
    Borst, J. G. and Helmchen, F. (1998) Calcium influx during an action potential. Meth. Enzym. 293, 352–371.PubMedCrossRefGoogle Scholar
  25. 25.
    Ratto, G. M., Payne, R., Owen, R. G., and Tsien, R. Y. (1988) The concentration of cytosolic free calcium in vertebrate rod outer segment measured using Fura-2. J. Neurosci. 8, 3240–3246.PubMedGoogle Scholar
  26. 26.
    Koutalos, Y. and Yau, K.-W. (1996) Regulation of sensitivity in vertebrate rod photoreceptors by calcium. TINS 19, 73–81.PubMedGoogle Scholar
  27. 27.
    Rieke, F. and Schwartz, E. A. (1996) Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse. J. Physiol. (Lond.) 493, 1–8.Google Scholar
  28. 28.
    Krizaj, D. and Copenhagen, D. R. (1998) Compartmentalization of calcium extrusion mechanisms in the outer and inner segments of photoreceptors. Neuron 21, 249–256.PubMedCrossRefGoogle Scholar
  29. 29.
    Protti, D. A. and Llano, I. (1998) Calcium currents and calcium signaling in rod bipolar cells of rat retinal slices. J. Neurosci. 18, 3715–3724.PubMedGoogle Scholar
  30. 30.
    Denk, W. and Detwiler, P. B. (1999) Optical recording of light-evoked calcium signals in the functionally intact retina. Proc. Natl. Acad. Sci. USA 96, 7035–7040.PubMedCrossRefGoogle Scholar
  31. 31.
    Lenzi, D. and Roberts, W. M. (1994) Calcium signalling in hair cells: multiple roles in a compact cell. Curr. Opin. Neurobiol. 4, 496–502.PubMedCrossRefGoogle Scholar
  32. 32.
    Jaramillo, F. (1995) Signal transduction in hair cells and its regulation by calcium. Neuron 15, 1227–1230.PubMedCrossRefGoogle Scholar
  33. 33.
    Ohmori, H. (1988) Mechanical stimulation and fura-2 fluorescence in the hair bundle of dissociated hair cells of the chick. J. Physiol. (Lond.) 399, 115–137.Google Scholar
  34. 34.
    Lumpkin, E. A. and Hudspeth, A. J. (1995) Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells. Proc. Natl. Acad. Sci. USA 92, 10,297–10,301.PubMedCrossRefGoogle Scholar
  35. 35.
    Denk, W., Holt, J. R., Shepherd, G. M., and Corey, D. P. (1995) Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links. Neuron 15, 1311–1321.PubMedCrossRefGoogle Scholar
  36. 36.
    Hudspeth, A. J. (1997) Mechanical amplification of stimuli by hair cells. Curr. Opin. Neurobiol. 7, 480–486.PubMedCrossRefGoogle Scholar
  37. 37.
    Issa, N. P. and Hudspeth, A. J. (1996) The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog’s sacculus. Proc. Natl. Acad. Sci. USA 93, 9527–9532.PubMedCrossRefGoogle Scholar
  38. 38.
    Tucker, T. and Fettiplace, R. (1995) Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells. Neuron 15, 1323–1335.PubMedCrossRefGoogle Scholar
  39. 39.
    Leinders-Zufall, T., Greer, C. A., Shepherd, G. M., and Zufall, F. (1998) Imagning odor-induced calcium transients in single olfactory cilia: specificity of activation and role in transduction. J. Neurosci. 18, 5630–5639.PubMedGoogle Scholar
  40. 40.
    Menini, A. (1999) Calcium signalling and regulation in olfactory neurons. Curr. Opin. Neurobiol. 9, 419–426.PubMedCrossRefGoogle Scholar
  41. 41.
    Smith, S. J. and Augustine, G. J. (1988) Calcium ions, active zones and synaptic transmitter release. TINS 11, 458–464.PubMedGoogle Scholar
  42. 42.
    Neher, E. (1998) Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20, 389–399.PubMedCrossRefGoogle Scholar
  43. 43.
    Robitaille, R., Adler, E. M., and Charlton, M. P. (1990) Strategic location of calcium channels at transmitter release sites of frog neuromuscular junction. Neuron 5, 773–779.PubMedCrossRefGoogle Scholar
  44. 44.
    Smith, S. J., Buchanan, J., Osses, L. R., Charlton, M. P., and Augustine, G. J. (1993) The spatial distribution of calcium signals in squid presynaptic terminals. J. Physiol. (Lond.) 472, 573–593.Google Scholar
  45. 45.
    Llinas, R., Sugimori, M., and Silver, R. B. (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679.PubMedCrossRefGoogle Scholar
  46. 46.
    Regehr, W. G. and Tank, D. W. (1991) Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J. Neurosci. Meth. 37, 111–119.CrossRefGoogle Scholar
  47. 47.
    Wu, L. G. and Saggau, P. (1994) Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3-CA1 synapses of the hippocampus. J. Neurosci. 14, 5613–5622.PubMedGoogle Scholar
  48. 48.
    Regehr, W. and Atluri, P. P. (1995) Calcium transients in cerebellar granule cell presynaptic terminals. Biophys. J. 68, 2156–2170.PubMedCrossRefGoogle Scholar
  49. 49.
    Mintz, I., Sabatini, B. L., and Regehr, W. G. (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron 15, 675–688.PubMedCrossRefGoogle Scholar
  50. 50.
    Wu, L. G., Borst, J. G., and Sakmann, B. (1998) R-type Ca2+ currents evoke transmitter release at a rat central synapse. Proc. Natl. Acad. Sci. USA 95, 4720–4725.PubMedCrossRefGoogle Scholar
  51. 51.
    Dunlap, K., Luebke, J. I., and Turner, T. J. (1995) Exocytotic Ca2+ channels in mammalian central neurons. TINS 18, 89–98.PubMedGoogle Scholar
  52. 52.
    Wu, L. G., Westenbroek, R. E., Borst, J. G. G., Catterall, W. A., and Sakmann, B. (1999) Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J. Neurosci. 19, 726–736.PubMedGoogle Scholar
  53. 53.
    Regehr, W. G. and Tank, D. W. (1994) Dendritic calcium dynamics. Curr. Opin. Neurobiol. 4, 373–382.PubMedCrossRefGoogle Scholar
  54. 54.
    Yuste, R. and Tank, D. W. (1996) Dendritic integration in mammalian neurons, a century after Cajal. Neuron 16, 701–716.PubMedCrossRefGoogle Scholar
  55. 55.
    Schiller, J., Schiller, Y., Stuart, G., and Sakmann, B. (1997) Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal neurons. J. Physiol. (Lond.) 505, 605–616.CrossRefGoogle Scholar
  56. 56.
    Helmchen, F., Svoboda, K., Denk, W., and Tank, D. W. (1999) In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nature Neurosci. 2, 989–996.PubMedCrossRefGoogle Scholar
  57. 57.
    Magee, J., Hoffman, D., Colbert, C., and Johnston, D. (1998) Electrical and calcium signaling in dendrites of hippocampal pyramidal neurons. Ann. Rev. Physiol. 60, 327–346.CrossRefGoogle Scholar
  58. 58.
    Christie, B. R., Eliot, L. S., Ito, K., Miyakawa, H., and Johnston, D. (1995) Different Ca2+ channels in soma and dendrites of hippocampal pyramidal neurons mediate spike-induced Ca2+ influx. J. Neurophysiol. 73, 2553–2557.PubMedGoogle Scholar
  59. 59.
    Westenbroek, R. E., Ahlijanian, M. K., and Catterall, W. A. (1990) Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons. Nature 347, 281–284.PubMedCrossRefGoogle Scholar
  60. 60.
    Mills, L. R., Niesen, C. E., So, A. P., Carlen, P. L., Spigelman, I., and Jones, O. T. (1994) N-type Ca2+ channels are located on somata, dendrites, and a sub population of dendritic spines on live hippocampal pyramidal neurons. J. Neurosci. 14, 6815–6824.PubMedGoogle Scholar
  61. 61.
    Westenbroek, R. E., Sakurai, T., Elliott, E. M., Hell, J. W., Starr, T. V. B., Snutch, T. P., and Catterall, W. A. (1995) Immunochemical identification and subcellular distribution of the alpha 1A subunits of brain calcium channels. J. Neurosci. 15, 6403–6418.PubMedGoogle Scholar
  62. 62.
    Eilers, J. and Konnerth, A. (1997) Dendritic signal integration. Curr. Opin. Neurobiol. 7, 385–390.PubMedCrossRefGoogle Scholar
  63. 63.
    Regehr, W. G. and Tank, D. W. (1990) Postsynaptic NMDA receptor-mediated calcium accumulation in hippocampal CA1 pyramidal cell dendrites. Nature 345, 807–810.PubMedCrossRefGoogle Scholar
  64. 64.
    Malinow, R., Otmakhov, N., Blum, K. I., and Lisman, J. (1994) Visualizing hippocampal synaptic function by optical detection of Ca2+ entry through the N-methyl-D-aspartate channel. Proc. Natl. Acad. Sci. USA 91, 8170–8174.PubMedCrossRefGoogle Scholar
  65. 65.
    Yuste, R. and Denk, W. (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684.PubMedCrossRefGoogle Scholar
  66. 66.
    Schiller, J., Schiller, Y., and Clapham, D. E. (1998) NMDA receptors amplify calcium influx into dendritic spines during associative pre-and postsynaptic activation. Nature Neurosci. 1, 114–118.PubMedCrossRefGoogle Scholar
  67. 67.
    Koester, H. J. and Sakmann, B. (1998) Calcium dynamics in single spines during coincident pre-and postsynaptic activity depend on relative timing of backpropagating action potentials and subthreshold excitatory postsynaptic potentials. Proc. Natl. Acad. Sci. USA 95, 9596–9601.PubMedCrossRefGoogle Scholar
  68. 68.
    Yuste, R., Majewska, A., Cash, S. S., and Denk, W. (1999) Mechanisms of calcium influx into hippocampal spines: heterogeneity among spines, coincidence detection by NMDA receptors, and optical quantal analysis. J. Neurosci. 19, 1976–1987.PubMedGoogle Scholar
  69. 69.
    Svoboda, K. and Mainen, Z. F. (1999) Synaptic [Ca2+]: intracellular stores spill their guts. Neuron 22, 427–430.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Fritjof Helmchen
    • 1
  1. 1.Biological Computation Research Department,Bell LaboratoriesLucent TechnologiesMurray Hill

Personalised recommendations