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  • Review Article
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Analysis of exocytotic events recorded by amperometry

Abstract

Amperometry is widely used to study exocytosis of neurotransmitters and hormones in various cell types. Analysis of the shape of the amperometric spikes that originate from the oxidation of monoamine molecules released during the fusion of individual secretory vesicles provides information about molecular steps involved in stimulation-dependent transmitter release. Here we present an overview of the methodology of amperometric signal processing, including (i) amperometric signal acquisition and filtering, (ii) detection of exocytotic events and determining spike shape characteristics, and (iii) data manipulation and statistical analysis. The purpose of this review is to provide practical guidelines for performing amperometric recordings of exocytotic activity and interpreting the results based on shape characteristics of individual release events.

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Figure 1: An overview of amperometric data acquisition and analysis.
Figure 2: Detection of amperometric events.
Figure 3: Calculation of spike parameters.
Figure 4: Spikes with different falling phase kinetics.

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References

  1. Kissinger, P.T., Hart, J.B. & Adams, R.N. Voltammetry in brain tissue-a new neurophysiological measurement. Brain Res. 55, 209–213 (1973).

    Article  CAS  PubMed  Google Scholar 

  2. Gonon, F. et al. In vivo continuous electrochemical determination of dopamine release in rat neostriatum. C.R. Acad. Sci. Hebd. Seances. Acad. Sci. D 286, 1203–1206 (1978).

    CAS  PubMed  Google Scholar 

  3. Leszczyszyn, D.J. et al. Nicotinic receptor–mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis. J. Biol. Chem. 265, 14736–14737 (1990).

    CAS  PubMed  Google Scholar 

  4. Wightman, R.M. et al. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. USA 88, 10754–10758 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Chen, T.K., Luo, G. & Ewing, A.G. Amperometric monitoring of stimulated catecholamine release from rat pheochromocytoma (PC12) cells at the zeptomole level. Anal. Chem. 66, 3031–3035 (1994).

    Article  CAS  PubMed  Google Scholar 

  6. Pothos, E., Davila, V. & Sulzer, D. Presynaptic recording of quanta from midbrain dopamine neurons and modulation of the quantal size. J. Neurosci. 18, 4106–4118 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhou, Z. & Misler, S. Amperometric detection of stimulus induced quantal release of catecholamines from cultured superior cervical ganglion neurons. Proc. Natl. Acad. Sci. USA 92, 6938–6942 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chen, G. & Ewing, A.G. Multiple classes of catecholamine vesicles observed during exocytosis from the Planorbis cell body. Brain Res. 701, 167–174 (1995).

    Article  CAS  PubMed  Google Scholar 

  9. Alvarez de Toledo, G., Fernandez-Chacon, R. & Fernandez, J.M. Release of secretory products during transient vesicle fusion. Nature 363, 554–558 (1993).

    Article  CAS  PubMed  Google Scholar 

  10. Bruns, D. & Jahn, R. Real-time measurement of transmitter release from single synaptic vesicles. Nature 377, 62–65 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Huang, L., Shen, H., Atkinson, M.A. & Kennedy, R.T. Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode. Proc. Natl. Acad. Sci. USA 92, 9608–9612 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Paras, C.D. & Kennedy, R.T. Electrochemical detection of exocytosis at single rat melanotrophs. Anal. Chem. 67, 3633–3637 (1995).

    Article  CAS  PubMed  Google Scholar 

  13. Chow, R.H. & von Ruden, L. Electrochemical detection of secretion from single cells. in Single-channel recording (eds. Sakmann, B. & Neher, E.) 245–276 (Plenum Press, New York, 1995).

    Chapter  Google Scholar 

  14. Bruns, D. Detection of transmitter release with carbon fiber electrodes. Methods 33, 312–321 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Dernick, G. et al. Patch amperometry: high-resolution measurements of single-vesicle fusion and release Nat. Methods 2, 699–708 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Rettig, J. & Neher, E. Emerging roles of presynaptic proteins in Ca2+-triggered exocytosis. Science 298, 781–785 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Jahn, R., Lang, T. & Sudhof, T.C. Membrane fusion. Cell 112, 519–533 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Burgoyne, R.D. & Morgan, A. Secretory granule exocytosis. Physiol. Rev. 83, 581–632 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Chow, R.H., Von Rueden, L. & Neher, E. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356, 60–63 (1992).

    Article  CAS  PubMed  Google Scholar 

  20. Zhou, Z., Misler, S. & Chow, R.H. Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells. Biophys. J. 70, 1543–1552 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lindau, M. & Alvarez de Toledo, G. The fusion pore. Biochim. Biophys. Acta 1641, 167–173 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Albillos, A. et al. The exocytic event in chromaffin cells revealed by patch amperometry. Nature 389, 509–512 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Wightman, R.M., Troyer, K.P., Mundorf, M.L. & Catahan, R. The association of vesicular contents and its effects on release. Ann. NY Acad. Sci. 971, 620–626 (2002).

    Article  CAS  PubMed  Google Scholar 

  24. Rahamimoff, R. & Fernandez, J.M. Pre- and postfusion regulation of transmitter release. Neuron 18, 17–27 (1997).

    Article  CAS  PubMed  Google Scholar 

  25. Sombers, L.A. et al. The effects of vesicular volume on secretion through the fusion pore in exocytotic release from PC12 cells. J. Neurosci. 24, 303–309 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Amatore, C. et al. Correlation between vesicle quantal size and fusion pore release in chromaffin cell exocytosis. Biophys. J. 88, 4411–4420 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Curran, M.J. & Brodwick, M.S. Ionic control of the size of the vesicle matrix of beige mouse mast cells. J. Gen. Physiol. 98, 771–790 (1991).

    Article  CAS  PubMed  Google Scholar 

  28. Marszalek, P.E., Farrell, B., Verdugo, P. & Fernandez, J.M. Kinetics of release of serotonin from isolated secretory granules. II. Ion exchange determines the diffusivity of serotonin. Biophys. J. 73, 1169–1183 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Borges, R., Travis, E.R., Hochstetler, S.E. & Wightman, R.M. Effects of external osmotic pressure on vesicular secretion from bovine adrenal medullary cells. J. Biol. Chem. 272, 8325–8331 (1997).

    Article  CAS  PubMed  Google Scholar 

  30. Amatore, C., Bouret, Y., Travis, E.R. & Wightman, R.M. Interplay between membrane dynamics, diffusion and swelling pressure governs individual vesicular exocytotic events during release of adrenaline by chromaffin cells. Biochimie 82, 481–496 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Ales, E. et al. High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat. Cell Biol. 1, 40–44 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Henkel, A.W. & Almers, W. Fast steps in exocytosis and endocytosis studied by capacitance measurements in endocrine cells. Curr. Opin. Neurobiol. 6, 350–357 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Palfrey, H.C. & Artalejo, C.R. Vesicle recycling revisited: rapid endocytosis may be the first step. Neuroscience 83, 969–989 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Tabares, L., Lindau, M. & Alvarez de Toledo, G. Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry. Biochem. Soc. Trans. 31, 837–841 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Sulzer, D. & Pothos, E.N. Presynaptic mechanisms that regulate quantal size. Rev. Neurosci. 11, 159–212 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Staal, R.G., Mosharov, E.V. & Sulzer, D. Dopamine neurons release transmitter via a flickering fusion pore. Nat. Neurosci. 7, 341–346 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Berg, H.C. Diffusion: macroscopic theory. in Random Walks in Biology (ed. Berg, H.C.) 17–36 (Princeton University Press, Princeton, NJ, 1983).

    Google Scholar 

  38. Schroeder, T.J. et al. Zones of exocytotic release on bovine adrenal medullary cells in culture. J. Biol. Chem. 269, 17215–17220 (1994).

    CAS  PubMed  Google Scholar 

  39. Travis, E.R. & Wightman, R.M. Spatio-temporal resolution of exocytosis from individual cells. Annu. Rev. Biophys. Biomol. Struct. 27, 77–103 (1998).

    Article  CAS  PubMed  Google Scholar 

  40. Schroeder, T.J. et al. Temporally resolved, independent stages of individual exocytotic secretion events. Biophys. J. 70, 1061–1068 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wang, C.T. et al. Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles. Science 294, 1111–1115 (2001).

    Article  CAS  PubMed  Google Scholar 

  42. Heinemann, S.H. Guide to data acquisition and analysis. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 53–91 (Plenum Press, New York, 1995).

    Chapter  Google Scholar 

  43. Schroeder, T.J. et al. Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis. Anal. Chem. 64, 3077–3083 (1992).

    Article  CAS  PubMed  Google Scholar 

  44. Xu, T., Binz, T., Niemann, H. & Neher, E. Multiple kinetic components of exocytosis distinguished by neurotoxin sensitivity. Nat. Neurosci. 1, 192–200 (1998).

    Article  CAS  PubMed  Google Scholar 

  45. Segura, F. et al. Automatic analysis for amperometrical recordings of exocytosis. J. Neurosci. Methods 103, 151–156 (2000).

    Article  CAS  PubMed  Google Scholar 

  46. Jankowski, J.A., Schroeder, T.J., Ciolkowski, E.L. & Wightman, R.M. Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells. J. Biol. Chem. 268, 14694–14700 (1993).

    CAS  PubMed  Google Scholar 

  47. Jankowski, J.A., Finnegan, J.M. & Wightman, R.M. Extracellular ionic composition alters kinetics of vesicular release of catecholamines and quantal size during exocytosis at adrenal medullary cells. J. Neurochem. 63, 1739–1747 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Sorensen, J.B. et al. Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23. Cell 114, 75–86 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Alvarez de Toledo, G. & Fernandez, J.M. Compound versus multigranular exocytosis in peritoneal mast cells. J. Gen. Physiol. 95, 397–409 (1990).

    Article  CAS  PubMed  Google Scholar 

  50. Hafez, I., Stolpe, A. & Lindau, M. Compound exocytosis and cumulative fusion in eosinophils. J. Biol. Chem. 278, 44921–44928 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Glavinovic, M.I., Vitale, M.L. & Trifaro, J.M. Comparison of vesicular volume and quantal size in bovine chromaffin cells. Neuroscience 85, 957–968 (1998).

    Article  CAS  PubMed  Google Scholar 

  52. Tang, K.S., Tse, A. & Tse, F.W. Differential regulation of multiple populations of granules in rat adrenal chromaffin cells by culture duration and cyclic AMP. J. Neurochem. 92, 1126–1139 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Colliver, T. et al. Quantitative and statistical analysis of the shape of amperometric spikes recorded from two populations of cells. J. Neurochem. 74, 1086–1097 (2000).

    Article  CAS  PubMed  Google Scholar 

  54. Van der Kloot, W. Statistics for studying quanta at synapses: resampling and confidence limits on histograms. J. Neurosci. Methods 65, 151–155 (1996).

    Article  CAS  PubMed  Google Scholar 

  55. Ott, R.L. & Longnecker, M. An Introduction to Statistical Methods and Data Analysis (Duxbury Press, Belmont, California, 2001).

    Google Scholar 

  56. Pothos, E. et al. D2-like dopamine autoreceptor activation reduces quantal size in PC12 cells. J. Neurosci. 18, 5575–5585 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Baur, J.E. et al. Fast-scan voltammetry of biogenic amines. Anal. Chem. 60, 1268–1272 (1988).

    Article  CAS  PubMed  Google Scholar 

  58. Colquhoun, D. & Sigworth, F.J. Fitting and statistical analysis of single-channel records. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 483–587 (Plenum Press, New York, 1995).

    Chapter  Google Scholar 

  59. Gomez, J.F. et al. New approaches for analysis of amperometrical recordings. Ann. NY Acad. Sci. 971, 647–654 (2002).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Lindau, R. Staal and Y. Schmitz for critique of the manuscript and J.B. Sørensen, R. Borges and other participants of 12th International Symposium on Chromaffin Cell Biology for helpful discussion. Supported by Parkinson's Disease Foundation, Picower Foundation and National Institute of Drug Abuse grant 07418.

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Correspondence to Eugene V Mosharov.

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Supplementary information

Supplementary Fig. 1

Finding spike beginning and end. (PDF 106 kb)

Supplementary Fig. 2

Statistical analysis of different foot subpopulations. (PDF 160 kb)

Supplementary Fig. 3

Analysis of overlapping spikes. (PDF 148 kb)

Supplementary Table 1

Statistical analysis of different spike subpopulations. (PDF 192 kb)

Supplementary Methods (PDF 151 kb)

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Mosharov, E., Sulzer, D. Analysis of exocytotic events recorded by amperometry. Nat Methods 2, 651–658 (2005). https://doi.org/10.1038/nmeth782

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