Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Analysis of calcium channels in single spines using optical fluctuation analysis

Nature volume 408, pages 589–593 (2000)Cite this article

Abstract

Most synapses form on small, specialized postsynaptic structures known as dendritic spines1. The influx of Ca2+ ions into such spines—through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)—triggers diverse processes that underlie synaptic plasticity2. Using two-photon laser scanning microscopy3, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices4. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1–20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (∼0.5) following dendritic action potentials. Activation of GABAB receptors reduced this probability in apical spines to ∼0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Action-potential-evoked Ca2+ influx in spines and dendrites.
Figure 2: Variance analysis.
Figure 3: Failures of action-potential-evoked Ca2+ influx in spines.
Figure 4: GABAB receptor activation reduces Ca2+ influx in apical spines.
Figure 5: Number of VSCCs opened by an action potential in apical (squares) and basal (circles) spines plotted as a function of spine volume.

Similar content being viewed by others

References

  1. Harris, K. M. & Kater, S. B. Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Annu. Rev. Neurosci. 17, 341–371 (1994).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  5. Jaffe, D. B. et al. The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons. Nature 357, 244–246 ( 1992).

    Article  ADS  CAS  Google Scholar 

  6. Svoboda, K., Tank, D. W. & Denk, W. Direct measurement of coupling between dendritic spines and shafts. Science 272, 716– 719 (1996).

    Article  ADS  CAS  Google Scholar 

  7. Hillyard, D. R. et al. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 9, 69– 77 (1992).

    Article  CAS  Google Scholar 

  8. Triggle, D. J. & Rampe, D. 1,4-Dihydropyridine activators and antagonists: structural and functional distinctions. Trends Pharmacol. Sci. 10, 507–511 ( 1989).

    Article  CAS  Google Scholar 

  9. Kavalali, E. T., Zhuo, M., Bito, H. & Tsien, R. W. Dendritic Ca2+ channels characterized by recordings from isolated hippocampal dendritic segments. Neuron 18, 651– 663 (1997).

    Article  CAS  Google Scholar 

  10. Barak, L. S. & Webb, W. W. Diffusion of low density lipoprotein-receptor complex on human fibroblasts. J. Cell Biol. 95, 846–852 (1982).

    Article  CAS  Google Scholar 

  11. Katz, B. & Miledi, R. Membrane noise produced by acetylcholine. Nature 226, 962–963 (1970).

    Article  ADS  CAS  Google Scholar 

  12. Koch, C. & Zador, A. The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization. J. Neurosci. 13, 413–422 (1993).

    Article  CAS  Google Scholar 

  13. Christie, B. R., Eliot, L. S., Ito, K. I., Miyakawa, H. & Johnston, D. Different Ca2+ channels in soma and dendrites of hippocampal pyramidal neurons mediate spike-induced Ca2+ influx. J. Neurophysiol. 73, 2553– 2557 (1995).

    Article  CAS  Google Scholar 

  14. Magee, J. C. & Johnston, D. Characterization of single voltage-gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. J. Physiol. 487, 67–90 (1995).

    Article  CAS  Google Scholar 

  15. Schiller, J., Schiller, Y. & Clapham, D. E. Amplification of calcium influx into dendritic spines during associative pre- and postsynaptic activation: the role of direct calcium influx through the NMDA receptor. Nature Neurosci. 1, 114–118 (1998).

    Article  CAS  Google Scholar 

  16. Wu, L. G., Borst, J. G. & Sakmann, B. R-type Ca2+ currents evoke transmitter release at a rat central synapse. Proc. Natl Acad. Sci. USA 95, 4720–4725 (1998).

    Article  ADS  CAS  Google Scholar 

  17. Bean, B. P. Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence. Nature 340, 153– 156 (1989).

    Article  ADS  CAS  Google Scholar 

  18. Kuo, C. C. & Bean, B. P. G-protein modulation of ion permeation through N-type calcium channels. Nature 365, 258–262 (1993).

    Article  ADS  CAS  Google Scholar 

  19. Zamponi, G. W. & Snutch, T. P. Modulation of voltage-dependent calcium channels by G proteins. Curr. Opin. Neurobiol. 8, 351–356 ( 1998).

    Article  CAS  Google Scholar 

  20. Boland, L. M. & Bean, B. P. Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence. J. Neurosci. 13, 516–533 (1993).

    Article  CAS  Google Scholar 

  21. Page, K. M., Canti, C., Stephens, G. J., Berrow, N. S. & Dolphin, A. C. Identification of the amino terminus of neuronal Ca2+ channel α1 subunits α1B and α1E as an essential determinant of G-protein modulation. J Neurosci. 18, 4815–4824 ( 1998).

    Article  CAS  Google Scholar 

  22. Isaacson, J. S., Solis, J. M. & Nicoll, R. A. Local and diffuse synaptic actions of GABA in the hippocampus. Neuron 10, 165– 175 (1993).

    Article  CAS  Google Scholar 

  23. Petersen, C. C., Malenka, R. C., Nicoll, R. A. & Hopfield, J. J. All-or-none potentiation at CA3-CA1 synapses. Proc. Natl Acad. Sci. USA 95, 4732–4737 ( 1998).

    Article  ADS  CAS  Google Scholar 

  24. Tottene, A., Moretti, A. & Pietrobon, D. Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons. J. Neurosci. 16, 6353–6363 (1996).

    Article  CAS  Google Scholar 

  25. Maravall, M., Mainen, Z. M., Sabatini, B. & Svoboda, K. Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys. J. 78, 2655– 2667 (2000).

    Article  CAS  Google Scholar 

  26. Mainen, Z. F., Malinow, R. & Svoboda, K. Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151–155 (1999).

    Article  ADS  CAS  Google Scholar 

  27. Markram, H., Helm, P. J. & Sakmann, B. Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J. Physiol. 485, 1–20 ( 1995).

    Article  CAS  Google Scholar 

  28. Emptage, N., Bliss, T. V. P. & Fine, A. Single synaptic events evoke NMDA receptor-mediated release of calcium from internal stores in hippocampal dendritic spines. Neuron 22, 115–124 ( 1999).

    Article  CAS  Google Scholar 

  29. Rice, S. O. in Noise and Stochastic Processes (ed. Wax, N.) 133– 294 (Dover, New York, 1954).

    Google Scholar 

  30. Jones, R., OIiver, C. J. & Pike, E. R. Experimental and theoretical comparison of photon-counting and current measurements of light intensity. Appl. Opt. 10, 1673–1680 (1970).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

We thank B. J. Burbach and P. O'Brien for help with experiments, and Z. Mainen, R. Malinow, M. Maravall, W. Regehr, R. Weinberg and R. Yasuda for comments on the manuscript. This work was supported by the Pew, Klingenstein and Mathers Foundations, the Howard Hughes Medical Institute, NIH and a Helen Hay Whitney Fellowship.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, 11724, New York, USA

    Bernardo L. Sabatini & Karel Svoboda

Authors
  1. Bernardo L. Sabatini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karel Svoboda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karel Svoboda.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sabatini, B., Svoboda, K. Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589–593 (2000). https://doi.org/10.1038/35046076

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/35046076

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing