Imaging the Spatial Organization of Calcium Channels in Nerve Terminals Using Atomic Force Microscopy

  • Hajime Takano
  • Marc Porter
  • Philip G. Haydon
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


For many years there has been much interest in identifying the spatial relationship between the organization of calcium channels and the sites for the release of chemical neurotransmitter (for a recent review, see ref. 1). During this time, the debate has been fostered by biophysical studies concerning the relation between calcium and transmitter release. Many studies, for example, have asked whether multiple calcium ions are required for the release of neurotransmitter. With the frequent observation of an apparent cooperative relation between calcium influx and stimulated release, and the demonstration of very short latencies between calcium influx and the onset of the evoked synaptic potential/current, there has been much debate about the organization of calcium channels with respect to the secretory apparatus. Questions that frequently surface are how many channels surround a vesicle, is calcium influx through multiple calcium channels necessary for the release of neurotransmitter, and how closely do calcium channels cluster at a release site? Although these questions frequently surface, there have been few successful attempts to define the spatial organization of calcium channels in nerve terminals. This paucity of information is not owing to a lack of effort, but rather because of technical challenges that are associated with working within the limited space of a nerve terminal.


Calcium Channel Gold Particle Nerve Terminal Calcium Influx Presynaptic Terminal 
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.
    Stanley, E. F. (1997) The calcium channel and the organization of the presynaptic transmitter release face. Trends. Neurosci. 20, 404–409.PubMedCrossRefGoogle Scholar
  2. 2.
    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.PubMedGoogle Scholar
  3. 3.
    Llinas, R., Sugimori, M., and Silver, R. B. (1992) Microdomains of high calcium concentration in a presynaptic terminal. Science 256, 677–679.PubMedCrossRefGoogle Scholar
  4. 4.
    Witcher, D. R., DeWaard, M., Sakamoto, J., Franzini-Armstrong, C., Pragnell, M., Kahl, S. D., et al. (1993) Subunit identification and reconstitution of the Ntype Ca2+ channel complex purified from brain. Science 261, 486–489.PubMedCrossRefGoogle Scholar
  5. 5.
    Pumplin, D. W., Reese, T. S., and Llinas, R. (1981) Are the presynaptic membrane particles the calcium channels? Proc. Natl. Acad. Sci. USA 78, 7210–7213.PubMedCrossRefGoogle Scholar
  6. 6.
    Robitaille, R., Adler, E. M., and Charlton, M. P. (1990) Strategic location of calcium channels at transmitter release sites of frog neuromuscular synapses. Neuron 5, 773–779.PubMedCrossRefGoogle Scholar
  7. 7.
    McIntosh, J. M., Olivera, B. M., and Cruz, L. J. (1999) Conus peptides as probes for ion channels. Methods Enzymol. 294, 605–624.PubMedCrossRefGoogle Scholar
  8. 8.
    Olivera, B. M., et al. (1990) Diversity of Conus neuropeptides. Science 249, 257–263.PubMedCrossRefGoogle Scholar
  9. 9.
    Gray, B. W. R., Olivera, B. M., and Cruz, L. J. (1988) Peptide toxins from venomous Conus snails. Annu. Rev. Biochem. 57, 665–700.PubMedCrossRefGoogle Scholar
  10. 10.
    Olivera, B. M., Cruz, I. J., deSnatos, V., LeChemiuant, G. W., Griffin, D., Zeikus, R., et al. (1987) Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom. Biochemistry 26, 2086–2090.PubMedCrossRefGoogle Scholar
  11. 11.
    Olivera, B. M., Cruz, I. J., de Santos, V., Le Cheminant, G. W., Griffin, D. Zeikus, F., et al. (1985) Peptide neurotoxins from fish-hunting cone snails. Science 230, 1338–1343.PubMedCrossRefGoogle Scholar
  12. 12.
    Haydon, P. G., Henderson, E., and Stanley, E. F. (1994) Localization of individual calcium channels at the release face of a presynaptic nerve terminal. Neuron 13, 1275–1280.PubMedCrossRefGoogle Scholar
  13. 13.
    Binnig, G., Rohrer, H., Gerber, C., and Weibel, E. (1981) Phys. Rev. Lett. 49, 57.CrossRefGoogle Scholar
  14. 14.
    Takano, H., Kenseth, J. R., Wong, S., O’Brien, J. C., and Porter, M. D. (1999) Chemical and biochemical analysis using scanning force microscopy. Chem. Rev. 99, 2845–2890.PubMedCrossRefGoogle Scholar
  15. 15.
    Haydon, V. Lartius, R., Parpura, V., and Marchese Ragona, S. P. (1996) Membrane deformation of living glial cells using atomic force microscopy. J. Microsc. 182, 114–120.PubMedCrossRefGoogle Scholar
  16. 16.
    Parpura, V., Haydon, P. G., and Henderson, E. (1993) Three-dimensional imaging of living neurons and glia with the atomic force microscope. J. Cell Sci. 104, 427–432.PubMedGoogle Scholar
  17. 17.
    Parpura, V., Doyle, R. T., Basarsky, T. A., Henderson, E., and Haydon, P. G. (1995) Dynamic imaging of purified individual synaptic vesicles. Neuroimage 2, 3–7.PubMedCrossRefGoogle Scholar
  18. 18.
    Hoh, J. H. and Schoenenberger, C. A. (1994) Surface morphology and mechanical properties of MDCK monolayers by atomic force microscopy. J. Cell Sci. 107, 1105–1114.PubMedGoogle Scholar
  19. 19.
    Hansma, H. G. and Hoh, J. H. (1994) Biomolecular imaging with the atomic force microscope. Annu. Rev. Biophys. Biomol. Struct. 23, 115–139.PubMedCrossRefGoogle Scholar
  20. 20.
    Henderson, E., Haydon, P. G., and Sakaguchi, D. S. (1992) Actin filament dynamics in living glial cells imaged by atomic force microscopy. Science 257, 1944–1946.PubMedCrossRefGoogle Scholar
  21. 21.
    Florin, E. L., Moy, V. T., and Gaub, H. E. (1994) Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417.PubMedCrossRefGoogle Scholar
  22. 22.
    Parpura, V. and Fernandez, J. M. (1996) Atomic force microscopy study of the secretory granule lumen. Biophys. J. 71, 2356–2366.PubMedCrossRefGoogle Scholar
  23. 23.
    Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., and Gaub, H. E. (1997) Reversible unfolding of individual titin immunoglobulin domains by AFM [see comments]. Science 276, 1109–1112.PubMedCrossRefGoogle Scholar
  24. 24.
    Stanley, E. F. (1993) Single calcium channels and acetylcholine release at a presynaptic nerve terminal. Neuron 11, 1007–1011.PubMedCrossRefGoogle Scholar
  25. 25.
    Stanley, E. F. (1993) Presynaptic calcium channels and the transmitter release mechanism. Ann. NY Acad. Sci. 681, 368–372.PubMedCrossRefGoogle Scholar
  26. 26.
    Stanley, E. F. (1992) The calyx-type synapse of the chick ciliary ganglion as a model of fast cholinergic transmission. Can. J. Physiol. Pharmacol. 70(Suppl.), S73–S77.PubMedGoogle Scholar
  27. 27.
    Stanley, E. F. (1991) Single calcium channels on a cholinergic presynaptic nerve terminal. Neuron 7, 585–591.PubMedCrossRefGoogle Scholar
  28. 28.
    Stanley, E. F. and Goping, G. (1991) Characterization of a calcium current in a vertebrate cholinergic presynaptic nerve terminal. J. Neurosci. 11, 985–993.PubMedGoogle Scholar
  29. 29.
    Stanley, E. F. and Cox, C. (1991) Calcium channels in the presynaptic nerve terminal of the chick ciliary ganglion giant synapse. Ann. NY Acad. Sci. 635, 70–79.PubMedCrossRefGoogle Scholar
  30. 30.
    Stanley, E. F. and Atrakchi, A. H. (1990) Calcium currents recorded from a vertebrate presynaptic nerve terminal are resistant to the dihydropyridine nifedipine. Proc. Natl. Acad. Sci. USA 87, 9683–9687.PubMedCrossRefGoogle Scholar
  31. 31.
    Stanley, E. F. (1987) Light microscopic visualisation of the presynaptic nerve terminal calyx in dissociated chick ciliary ganglion neurons. Brain Res. 421, 367–369.PubMedCrossRefGoogle Scholar
  32. 32.
    Vesenka, J., Manne, S., Giberson, R., Marsh, T., and Henderson, E. (1993) Colloidal gold particles as an incompressible atomic force microscope imaging standard for assessing the compressibility of biomolecules. Biophys. J. 65, 992–997.PubMedCrossRefGoogle Scholar
  33. 33.
    Wong, S. S., Harper, J. D., Lansbury, P. T., and Lieber, C. M. (1998) Carbon nanotube tips: high resolution probes forimaging biological systems. J. Am. Chem. Soc. 120, 603,604.CrossRefGoogle Scholar
  34. 34.
    Dai, H., Hafner, J. H., Rinzler, A. G., Colbert, D. T., and Smalley, R. E. (1996) Nanotubes as nanoprobes in scanning probe microscopy. Nature 384, 147–150.CrossRefGoogle Scholar
  35. 35.
    Hwang, J., Tamm, L. K., Bohm, I., Ramalingham, T. S., Betzig, E., Edidin, M. (1995) Nanoscale complexity of phospholipid monolayers investigated by near-field scanning optical microscopy. Science 270, 610–614.PubMedCrossRefGoogle Scholar
  36. 36.
    Ambrose, W. P., Goodwin, P. M., Martin, J. C., and Keller, R. A. (1994) Alterations of single molecule fluorescence lifetimes in near-field optical micorscopy. Science 265, 364–367.PubMedCrossRefGoogle Scholar
  37. 37.
    Betzig, E. and Chichester, R. J. (1993) Single molecules observed by nearfield scanning optical microscopy. Science 262, 1422–1425.PubMedCrossRefGoogle Scholar
  38. 38.
    Betzig, E. and Chichester, R. J. (1992) Near-field optics: microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science 257, 189–195.PubMedCrossRefGoogle Scholar
  39. 39.
    Kopelman, R. and Tan, W. (1993) Near-field optics: imaging single molecules. Science 1382–1384.Google Scholar
  40. 40.
    Trautman, J. K., Macklin, J. J., Brus, L. E., and Betzi, E. (1997). Near-field spectroscopy of single molecules at room temperature. Nature 369, 40–42.CrossRefGoogle Scholar
  41. 41.
    Trautman, J. K., et al. (1971) Image contrast in near-field optics. J. Appl. Physics 4663Google Scholar
  42. 42.
    Marchese-Ragona, S. P. and Haydon, P. G. (1997) Near-field scanning optical microscopy and near-field confocal optical spectroscopy: emerging techniques in biology. Ann. NY Acad. Sci. 820, 196–206.PubMedCrossRefGoogle Scholar
  43. 43.
    Bui, J. D., et al. (1999) Probing intracellular dynamics in living cells with nearfield optics. J. Neurosci. Methods 89, 9–15.PubMedCrossRefGoogle Scholar
  44. 44.
    Nagy, P., Zelles, T., Lou, H. J., Gallion, V. L., Phillips, M. I., Tan, W. (1999) Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy. J. Cell Sci. 112, 1733–1741.PubMedGoogle Scholar
  45. 45.
    Lewis, A., Radko, A., Ben Ami, N., Palanker, D., and Lieberman, K. (1999) Near-field scanning optical microscopy in cell biology. Trends. Cell Biol. 9, 70–73.PubMedCrossRefGoogle Scholar
  46. 46.
    Shiku, H. and Dunn, R. C. (1999) Near-field scanning optical microscopy. Anal. Chem. 71, 23A–29A.PubMedCrossRefGoogle Scholar
  47. 47.
    Subramaniam, V., Kirsch, A. K., and Jovin, T. M. (1998) Cell biological applications of scanning near-field optical microscopy (SNOM). Cell Mol. Biol. (Noisy-le-grand) 44, 689–700.Google Scholar
  48. 48.
    Meixner, A. J. and Kneppe, H. (1998) Scanning near-field optical microscopy in cell biology and microbiology. Cell Mol. Biol. 44, 673–688.PubMedGoogle Scholar
  49. 49.
    Hwang, J., Gheber, L. A., Margolis, L., and Edidin, M. (1998) Domains in cell plasma membranes investigated by near-field scanning optical microscopy. Biophys. J. 74, 2184–2190.PubMedCrossRefGoogle Scholar
  50. 50.
    Ben-Ami, N., et al. (1998) Near-field optical imaging of unstained bacteria: comparison with normal atomic force and far-field optical microscopy in air and aqueous media. Ultramicroscopy 71, 321–325.PubMedCrossRefGoogle Scholar
  51. 51.
    Bui, J. D., et al. (1999) Probing intracellular dynamics in living cells with nearfield optics. J. Neurosci. Methods 89, 9–15.PubMedCrossRefGoogle Scholar
  52. 52.
    Subramaniam, V., Kirsch, A. K., and Jovin, T. M. (1998) Cell biological applications of scanning near-field optical microscopy (SNOM). Cell Mol. Biol. 44, 689–700.PubMedGoogle Scholar
  53. 53.
    Ben-Ami, N., et al. (1998) Near-field optical imaging of unstained bacteria: comparison with normal atomic force and far-field optical microscopy in air and aqueous media. Ultramicroscopy 71, 321–325.PubMedCrossRefGoogle Scholar
  54. 54.
    Haydon, P. G., Marchese Ragona, S., Basarsky, T. A., Szulczewski, M., and McCloskey, M. (1996) Near-field confocal optical spectroscopy (NCOS): subdiffraction optical resolution for biological systems. J. Microsc. 182, 208–216.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Hajime Takano
    • 1
  • Marc Porter
    • 1
  • Philip G. Haydon
    • 1
  1. 1.Department of Zoology and GeneticsIowa State UniversityAmes

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