Skip to main content
SpringerLink
Log in

Imaging of Localized Neuronal Calcium Influx

  • Protocol
Ion Channel Localization

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

  • 569 Accesses

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

REFERENCES

  1. Ghosh, A. and Greenberg, M. E. (1995) Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268, 239–247.

    Article  PubMed  CAS  Google Scholar 

  2. Zucker, R. S. (1999) Calcium-and activity-dependent synaptic plasticity. Curr. Opin. Neurobiol. 9, 305–313.

    Article  PubMed  CAS  Google Scholar 

  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. 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. Augustine, G. J. and Neher, E. (1992) Neuronal Ca2+ signalling takes the local route. Curr. Opin. Neurobiol. 2, 302–307.

    Article  PubMed  CAS  Google Scholar 

  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. 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.

    Article  PubMed  CAS  Google Scholar 

  8. Sabatini, B. L. and Regehr, W. G. (1998) Optical measurement of presynaptic calcium current. Biophys. J. 74, 1549–1563.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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. 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.

    Article  CAS  Google Scholar 

  14. Pawley, J. B. (ed.) (1995) Handbook of Biological Confocal Microscopy. Plenum, New York.

    Google Scholar 

  15. Denk, W., Strickler, J. H., and Webb, W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science 248, 73–76.

    Article  PubMed  CAS  Google Scholar 

  16. Denk, W., Yuste, R., Svoboda, K., and Tank, D. W. (1996) Imaging calcium dynamics in dendritic spines. Curr. Opin. Neurobiol. 6, 372–378.

    Article  PubMed  CAS  Google Scholar 

  17. Denk, W. and Svoboda, K. (1997) Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351–357.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  20. Neher, E. (1995) The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34, 1423–1442.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  24. Borst, J. G. and Helmchen, F. (1998) Calcium influx during an action potential. Meth. Enzym. 293, 352–371.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  26. Koutalos, Y. and Yau, K.-W. (1996) Regulation of sensitivity in vertebrate rod photoreceptors by calcium. TINS 19, 73–81.

    PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  32. Jaramillo, F. (1995) Signal transduction in hair cells and its regulation by calcium. Neuron 15, 1227–1230.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  36. Hudspeth, A. J. (1997) Mechanical amplification of stimuli by hair cells. Curr. Opin. Neurobiol. 7, 480–486.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  38. Tucker, T. and Fettiplace, R. (1995) Confocal imaging of calcium microdomains and calcium extrusion in turtle hair cells. Neuron 15, 1323–1335.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  40. Menini, A. (1999) Calcium signalling and regulation in olfactory neurons. Curr. Opin. Neurobiol. 9, 419–426.

    Article  PubMed  CAS  Google Scholar 

  41. Smith, S. J. and Augustine, G. J. (1988) Calcium ions, active zones and synaptic transmitter release. TINS 11, 458–464.

    PubMed  CAS  Google Scholar 

  42. Neher, E. (1998) Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release. Neuron 20, 389–399.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  45. Llinas, R., Sugimori, M., and Silver, R. B. (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  48. Regehr, W. and Atluri, P. P. (1995) Calcium transients in cerebellar granule cell presynaptic terminals. Biophys. J. 68, 2156–2170.

    Article  PubMed  CAS  Google Scholar 

  49. Mintz, I., Sabatini, B. L., and Regehr, W. G. (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron 15, 675–688.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  51. Dunlap, K., Luebke, J. I., and Turner, T. J. (1995) Exocytotic Ca2+ channels in mammalian central neurons. TINS 18, 89–98.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  53. Regehr, W. G. and Tank, D. W. (1994) Dendritic calcium dynamics. Curr. Opin. Neurobiol. 4, 373–382.

    Article  PubMed  CAS  Google Scholar 

  54. Yuste, R. and Tank, D. W. (1996) Dendritic integration in mammalian neurons, a century after Cajal. Neuron 16, 701–716.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  62. Eilers, J. and Konnerth, A. (1997) Dendritic signal integration. Curr. Opin. Neurobiol. 7, 385–390.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  65. Yuste, R. and Denk, W. (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  69. Svoboda, K. and Mainen, Z. F. (1999) Synaptic [Ca2+]: intracellular stores spill their guts. Neuron 22, 427–430.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biological Computation Research Department,Bell Laboratories, Lucent Technologies, Murray Hill, NJ

    Fritjof Helmchen

Authors
  1. Fritjof Helmchen
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Michigan, Ann Arbor, MI

    Anatoli N. Lopatin

  2. Washington University Medical School, St. Louis, MO

    Colin G. Nichols

Rights and permissions

Reprints and permissions

Copyright information

© 2001 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Helmchen, F. (2001). Imaging of Localized Neuronal Calcium Influx. In: Lopatin, A.N., Nichols, C.G. (eds) Ion Channel Localization. Methods in Pharmacology and Toxicology. Humana Press. https://doi.org/10.1385/1-59259-118-3:327

Download citation

  • DOI: https://doi.org/10.1385/1-59259-118-3:327

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-833-2

  • Online ISBN: 978-1-59259-118-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation