Advertisement

Combined Fluorometric and Electrophysiological Recordings

  • Hartmut Schmidt
  • Jens Eilers
Part of the Neuromethods book series (NM, volume 35)

Abstract

Combined electrophysiological and fluorometric recordings have proven to be a powerful tool, especially in the field of neuro-biology. With this combination it became possible to overcome three major limitations of pure electrophysiological measurements. First, high-resolution recordings from cellular compartments distant to the recording electrode became feasible, allowing, for example, the quantification of the occupancy of postsynaptic receptors (Mainen et al., 1999) and the density of voltage-gated Ca2+channels (Sabatini and Svoboda, 2000) at the level of single dendritic spines. Second, the analysis of cellular responses not directly associated with electrical signals became possible. Examples include synaptically evoked Ca2+release from intracellular stores (Takechi et al., 1998; Finch and Augustine, 1998) and Ca2+buffering by endogenous Ca2+-binding proteins (Zhou and Neher, 1993). Third, the spatio-temporal extent of second messenger signals, e.g., during subthreshold synaptic activity (Eilers et al., 1995a) and during the induction of synaptic plasticity (Eilers et al., 1997a) can be monitored by means of fluorescence imaging.

Keywords

Excitation Intensity Optical Resolution Intracellular Solution Baseline Recording Electrophysiological Signal 
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.

References

  1. Adams, S. R., Harootunian, A. T., Buechler, Y. J., Taylor, S. S., and Tsien, R. Y. (1991) Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697.PubMedCrossRefGoogle Scholar
  2. Carrington, W. A., Lynch, R. M., Moore, E. D., Isenberg, G., Fogarty, K. E., and Fay, F. S. (1995) Superresolution three-dimensional images of fluorescence in cells with minimal light exposure. Science 268, 1483–1487.PubMedCrossRefGoogle Scholar
  3. Chalfie, M., Tu Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.PubMedCrossRefGoogle Scholar
  4. Denk, W., Strickler, J. H., and Webb, W. W. (1990) Two-photon laser scanning fluorescence microscopy. Science 248, 73–76.PubMedCrossRefGoogle Scholar
  5. Dodt, H. U. and Zieglgänsberger, W. (1990) Visualizing unstained neurons in living brain slices by infrared DIC-videomicroscopy. Brain Res. 537, 333–336.PubMedCrossRefGoogle Scholar
  6. Eilers, J., Augustine, G. J., and Konnerth, A. (1995a) Subthreshold synaptic Ca2+signalling in fine dendrites and spines of cerebellar Purkinje neurons. Nature 373, 155–158.PubMedCrossRefGoogle Scholar
  7. Eilers, J., Callewaert, G., Armstrong, C., and Konnerth, A. (1995b) Calcium signaling in a narrow somatic submembrane shell during synaptic activity in cerebellar Purkinje neurons. Proc. Natl. Acad. Sci. USA 92, 10,272–10,276.PubMedCrossRefGoogle Scholar
  8. Eilers, J., Takechi, H., Finch, E. A., Augustine, G. J., and Konnerth, A. (1997a) Local dendritic Ca2+signaling induces cerebellar LTD. Learn. Mem. 4, 159–168.PubMedCrossRefGoogle Scholar
  9. Eilers, J., Hof, D, and Konnerth, A. (1997b) The Flaginserter: a reliable event marker for video recordings. J. Neurosci. Methods 78, 151–156.PubMedCrossRefGoogle Scholar
  10. Eilers, J. and Konnerth, A. (1999) Dye loading with patch-pipettes, in Imaging Neurons: A Laboratory Manual (Yuste, R., Lenny, F., and Konnerth, A., eds.), Cold Spring Harbor Laboratory Press, New York, pp. 35.1–35.10.Google Scholar
  11. Fan, G. Y., Fujisaki, H., Miyawaki, A., Tsay, R. K., Tsien, R. Y., and Ellisman, M. H. (1999) Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. 76, 2412–2420.PubMedCrossRefGoogle Scholar
  12. Finch, E. A. and Augustine, G. J. (1998) Local calcium signalling by inositol-1,4,5-trisphosphate in Purkinje cell dendrites. Nature 396, 753–756.PubMedCrossRefGoogle Scholar
  13. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440–3450.PubMedGoogle Scholar
  14. Helmchen, F. (1999) Calibration of fluorescent calcium indicators, in Imaging Neurons: A Laboratory Manual (Yuste, R., Lenny, F., and Konnerth, A., eds.), Cold Spring Harbor Laboratory Press, New York, pp. 32.31–32.11.Google Scholar
  15. Hernández-Cruz, A., Sala, F., and Adams, P. (1990) Subcellular calcium transients visualized by confocal microscopy in a voltage-clamped vertebrate neuron. Science 247, 858–862.PubMedCrossRefGoogle Scholar
  16. Inoué, S. and Spring, K. R. (1997) Video Microscopy-The Fundamentals, 2nd ed. Plenum Press, plNew York.CrossRefGoogle Scholar
  17. Lanni, F. and Keller, H. E. (1999) Microscopy and microscope systems, in Imaging Neurons: A Laboratory Manual (Yuste, R., Lenny, F., and Konnerth, A., eds.), Cold Spring Harbor Laboratory Press, New York, pp. 1.1–1.72.Google Scholar
  18. Mainen, Z. F., Malinow, R., and Svoboda, K. (1999) Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151–155.PubMedCrossRefGoogle Scholar
  19. Morris, S. A., Correa, V., Cardy, T. J., O’Beirne, G., and Taylor, C. W. (1999) Interactions between inositol trisphosphate receptors and fluorescent Ca2+indicators. Cell Calcium 25, 137–142.PubMedCrossRefGoogle Scholar
  20. Müller, W. and Connor, J. A. (1991) Dendritic spines as individual neuronal compartments for synaptic Ca2+responses. Nature 354, 73–76.PubMedCrossRefGoogle Scholar
  21. Neher, E. (1999) Some quantitative aspects of calcium fluorimetry, in Imaging Neurons: A Laboratory Manual (Yuste, R., Lenny, F., and Konnerth, A., eds.), Cold Spring Harbor Laboratory Press, New York, pp. 31.31–31.11.Google Scholar
  22. Pusch, M. and Neher, E. (1988) Rates of diffusional exchange between small cells and a measuring patch pipette. Pflügers Arch. 411, 204–211.PubMedCrossRefGoogle Scholar
  23. Rose, C. R., Kovalchuk, Y., Eilers, J., and Konnerth, A. (1999) Two-photon Na+imaging in spines and fine dendrites of central neurons. Pflüg. Arch. 439, 201–207.CrossRefGoogle Scholar
  24. Rexhausen, U. (1992) Bestimmung der Diffusionseigenschaften von Fluoreszenz-farbstoffen in verzweigten Nervenzellen unter Verwendung eines rechner-gesteuerten Bildverarbeitungssystems. University of Göttingen, Diploma thesis.Google Scholar
  25. Sabatini, B. L. and Svoboda, K. (2000) Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589–593.PubMedCrossRefGoogle Scholar
  26. Schiefer, J., Kampe, K., Dodt, H. U., Zieglgänsberger, W., and Kreutzberg G. W. (1999) Microglial motility in the rat facial nucleus following peripheral axotomy. J. Neurocytol. 28, 439–453.PubMedCrossRefGoogle Scholar
  27. Takechi, H., Eilers, J., and Konnerth, A. (1998) A new class of synaptic responses involving calcium release in dendritic spines. Nature 396, 757–760.PubMedCrossRefGoogle Scholar
  28. Uhl, R. (1999) Arc lamps and monochromators for fluorescence microscopy, in Imaging Neurons: A Laboratory Manual (Yuste, R., Lenny, F., and Konnerth, A., eds.), Cold Spring Harbor Laboratory Press, New York, pp. 2.1–2.8.Google Scholar
  29. Xu, C., Zipfel W., Shear, J. B., Williams, R. M., and Webb, W. W. (1996) Mul-tiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10,763–10,7658.PubMedCrossRefGoogle Scholar
  30. Zhou, Z. and Neher, E. (1993) Mobile and immobile calcium buffers in bovins chromaffin cells. J. Physiol. 469, 245–273.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Hartmut Schmidt
    • 1
  • Jens Eilers
    • 1
  1. 1.Department of NeurophysiologyMax-Planck-Institute for Brain ResearchFrankfurtGermany

Personalised recommendations