Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2000 May;78(5):2655–2667. doi: 10.1016/S0006-3495(00)76809-3

Estimating intracellular calcium concentrations and buffering without wavelength ratioing.

M Maravall 1, Z F Mainen 1, B L Sabatini 1, K Svoboda 1
PMCID: PMC1300854  PMID: 10777761

Abstract

We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca(2+)] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid). Resting [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.

Full Text

The Full Text of this article is available as a PDF (180.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bers D. M., Patton C. W., Nuccitelli R. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 1994;40:3–29. doi: 10.1016/s0091-679x(08)61108-5. [DOI] [PubMed] [Google Scholar]
  2. Busa W. B. Spectral characterization of the effect of viscosity on Fura-2 fluorescence: excitation wavelength optimization abolishes the viscosity artifact. Cell Calcium. 1992 May;13(5):313–319. doi: 10.1016/0143-4160(92)90066-2. [DOI] [PubMed] [Google Scholar]
  3. Denk W., Strickler J. H., Webb W. W. Two-photon laser scanning fluorescence microscopy. Science. 1990 Apr 6;248(4951):73–76. doi: 10.1126/science.2321027. [DOI] [PubMed] [Google Scholar]
  4. Denk W., Sugimori M., Llinás R. Two types of calcium response limited to single spines in cerebellar Purkinje cells. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8279–8282. doi: 10.1073/pnas.92.18.8279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Denk W., Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron. 1997 Mar;18(3):351–357. doi: 10.1016/s0896-6273(00)81237-4. [DOI] [PubMed] [Google Scholar]
  6. Feller M. B., Delaney K. R., Tank D. W. Presynaptic calcium dynamics at the frog retinotectal synapse. J Neurophysiol. 1996 Jul;76(1):381–400. doi: 10.1152/jn.1996.76.1.381. [DOI] [PubMed] [Google Scholar]
  7. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  8. Harkins A. B., Kurebayashi N., Baylor S. M. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys J. 1993 Aug;65(2):865–881. doi: 10.1016/S0006-3495(93)81112-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Helmchen F., Imoto K., Sakmann B. Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys J. 1996 Feb;70(2):1069–1081. doi: 10.1016/S0006-3495(96)79653-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jaffe D. B., Johnston D., Lasser-Ross N., Lisman J. E., Miyakawa H., Ross W. N. The spread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons. Nature. 1992 May 21;357(6375):244–246. doi: 10.1038/357244a0. [DOI] [PubMed] [Google Scholar]
  11. Kao J. P., Harootunian A. T., Tsien R. Y. Photochemically generated cytosolic calcium pulses and their detection by fluo-3. J Biol Chem. 1989 May 15;264(14):8179–8184. [PubMed] [Google Scholar]
  12. Kennedy H. J., Thomas R. C. Effects of injecting calcium-buffer solution on [Ca2+]i in voltage-clamped snail neurons. Biophys J. 1996 May;70(5):2120–2130. doi: 10.1016/S0006-3495(96)79778-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koester H. J., Baur D., Uhl R., Hell S. W. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys J. 1999 Oct;77(4):2226–2236. doi: 10.1016/S0006-3495(99)77063-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koester H. J., Sakmann B. Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9596–9601. doi: 10.1073/pnas.95.16.9596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kurebayashi N., Harkins A. B., Baylor S. M. Use of fura red as an intracellular calcium indicator in frog skeletal muscle fibers. Biophys J. 1993 Jun;64(6):1934–1960. doi: 10.1016/S0006-3495(93)81564-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lau P. M., Zucker R. S., Bentley D. Induction of filopodia by direct local elevation of intracellular calcium ion concentration. J Cell Biol. 1999 Jun 14;145(6):1265–1275. doi: 10.1083/jcb.145.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lev-Ram V., Miyakawa H., Lasser-Ross N., Ross W. N. Calcium transients in cerebellar Purkinje neurons evoked by intracellular stimulation. J Neurophysiol. 1992 Oct;68(4):1167–1177. doi: 10.1152/jn.1992.68.4.1167. [DOI] [PubMed] [Google Scholar]
  18. Magee J. C., Johnston D. A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons. Science. 1997 Jan 10;275(5297):209–213. doi: 10.1126/science.275.5297.209. [DOI] [PubMed] [Google Scholar]
  19. Mainen Z. F., Maletic-Savatic M., Shi S. H., Hayashi Y., Malinow R., Svoboda K. Two-photon imaging in living brain slices. Methods. 1999 Jun;18(2):231-9, 181. doi: 10.1006/meth.1999.0776. [DOI] [PubMed] [Google Scholar]
  20. Mainen Z. F., Malinow R., Svoboda K. Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature. 1999 May 13;399(6732):151–155. doi: 10.1038/20187. [DOI] [PubMed] [Google Scholar]
  21. Markram H., Helm P. J., Sakmann B. Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J Physiol. 1995 May 15;485(Pt 1):1–20. doi: 10.1113/jphysiol.1995.sp020708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Minta A., Kao J. P., Tsien R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem. 1989 May 15;264(14):8171–8178. [PubMed] [Google Scholar]
  23. Murphy T. H., Baraban J. M., Wier W. G., Blatter L. A. Visualization of quantal synaptic transmission by dendritic calcium imaging. Science. 1994 Jan 28;263(5146):529–532. doi: 10.1126/science.7904774. [DOI] [PubMed] [Google Scholar]
  24. Müller W., Connor J. A. Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses. Nature. 1991 Nov 7;354(6348):73–76. doi: 10.1038/354073a0. [DOI] [PubMed] [Google Scholar]
  25. Nakajima K., Harada K., Ebina Y., Yoshimura T., Ito H., Ban T., Shingai R. Relationship between resting cytosolic Ca2+ and responses induced by N-methyl-D-aspartate in hippocampal neurons. Brain Res. 1993 Feb 19;603(2):321–323. doi: 10.1016/0006-8993(93)91255-q. [DOI] [PubMed] [Google Scholar]
  26. Neher E., Augustine G. J. Calcium gradients and buffers in bovine chromaffin cells. J Physiol. 1992 May;450:273–301. doi: 10.1113/jphysiol.1992.sp019127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O'Malley D. M. Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion. J Neurosci. 1994 Oct;14(10):5741–5758. doi: 10.1523/JNEUROSCI.14-10-05741.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Regehr W. G., Atluri P. P. Calcium transients in cerebellar granule cell presynaptic terminals. Biophys J. 1995 May;68(5):2156–2170. doi: 10.1016/S0006-3495(95)80398-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Regehr W. G., Connor J. A., Tank D. W. Optical imaging of calcium accumulation in hippocampal pyramidal cells during synaptic activation. Nature. 1989 Oct 12;341(6242):533–536. doi: 10.1038/341533a0. [DOI] [PubMed] [Google Scholar]
  30. Regehr W. G., Delaney K. R., Tank D. W. The role of presynaptic calcium in short-term enhancement at the hippocampal mossy fiber synapse. J Neurosci. 1994 Feb;14(2):523–537. doi: 10.1523/JNEUROSCI.14-02-00523.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sabatini B. L., Regehr W. G. Optical measurement of presynaptic calcium currents. Biophys J. 1998 Mar;74(3):1549–1563. doi: 10.1016/S0006-3495(98)77867-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schiller J., Helmchen F., Sakmann B. Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones. J Physiol. 1995 Sep 15;487(Pt 3):583–600. doi: 10.1113/jphysiol.1995.sp020902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schiller J., Schiller Y., Clapham D. E. NMDA receptors amplify calcium influx into dendritic spines during associative pre- and postsynaptic activation. Nat Neurosci. 1998 Jun;1(2):114–118. doi: 10.1038/363. [DOI] [PubMed] [Google Scholar]
  34. Schiller J., Schiller Y., Stuart G., Sakmann B. Calcium action potentials restricted to distal apical dendrites of rat neocortical pyramidal neurons. J Physiol. 1997 Dec 15;505(Pt 3):605–616. doi: 10.1111/j.1469-7793.1997.605ba.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Svoboda K., Denk W., Kleinfeld D., Tank D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. 1997 Jan 9;385(6612):161–165. doi: 10.1038/385161a0. [DOI] [PubMed] [Google Scholar]
  36. Svoboda K., Helmchen F., Denk W., Tank D. W. Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo. Nat Neurosci. 1999 Jan;2(1):65–73. doi: 10.1038/4569. [DOI] [PubMed] [Google Scholar]
  37. Tsien R. Y. Fluorescent probes of cell signaling. Annu Rev Neurosci. 1989;12:227–253. doi: 10.1146/annurev.ne.12.030189.001303. [DOI] [PubMed] [Google Scholar]
  38. Tsien R., Pozzan T. Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol. 1989;172:230–262. doi: 10.1016/s0076-6879(89)72017-6. [DOI] [PubMed] [Google Scholar]
  39. Yuste R., Denk W. Dendritic spines as basic functional units of neuronal integration. Nature. 1995 Jun 22;375(6533):682–684. doi: 10.1038/375682a0. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES