Analysis of exocytotic events recorded by amperometry

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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.

References

  1. 1

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

  2. 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).

  3. 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).

  4. 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).

  5. 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).

  6. 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).

  7. 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).

  8. 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).

  9. 9

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

  10. 10

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

  11. 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).

  12. 12

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

  13. 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).

  14. 14

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

  15. 15

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

  16. 16

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

  17. 17

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

  18. 18

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

  19. 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).

  20. 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).

  21. 21

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

  22. 22

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

  23. 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).

  24. 24

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

  25. 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).

  26. 26

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

  27. 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).

  28. 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).

  29. 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).

  30. 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).

  31. 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).

  32. 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).

  33. 33

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

  34. 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).

  35. 35

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

  36. 36

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

  37. 37

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

  38. 38

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

  39. 39

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

  40. 40

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

  41. 41

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

  42. 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).

  43. 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).

  44. 44

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

  45. 45

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

  46. 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).

  47. 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).

  48. 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).

  49. 49

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

  50. 50

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

  51. 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).

  52. 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).

  53. 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).

  54. 54

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

  55. 55

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

  56. 56

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

  57. 57

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

  58. 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).

  59. 59

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

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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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The authors declare no competing financial interests.

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) doi:10.1038/nmeth782

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