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Patch Amperometry and Intracellular Patch Electrochemistry

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Chromaffin Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2565))

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Abstract

Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector—carbon fiber electrode (CFE)—is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

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References

  1. Fernandez JM, Neher E, Gomperts BD (1984) Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312:453–455

    Article  CAS  Google Scholar 

  2. Sharma S, Lindau M (2018) The fusion pore, 60 years after the first cartoon. FEBS Lett 592:3542–3562

    Article  CAS  Google Scholar 

  3. Breckenridge LJ, Almers W (1987) Currents through the fusion pore that forms during exocytosis of a secretory vesicle. Nature 328:814–817

    Article  CAS  Google Scholar 

  4. Spruce AE, Breckenridge LJ, Lee AK, Almers W (1990) Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle. Neuron 4:643–654

    Article  CAS  Google Scholar 

  5. Hartmann J, Lindau M (1995) A novel Ca(2+)-dependent step in exocytosis subsequent to vesicle fusion. FEBS Lett 363:217–220

    Article  CAS  Google Scholar 

  6. Lindau M (1991) Time-resolved capacitance measurements: monitoring exocytosis in single cells. Q Rev Biophys 24:75–101

    Article  CAS  Google Scholar 

  7. Wightman RM et al (1991) Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc Natl Acad Sci U S A 88:10754–10758

    Article  CAS  Google Scholar 

  8. Schroeder TJ, Jankowski JA, Kawagoe KT, Wightman RM, Lefrou C, Amatore C (1992) Analysis of diffusional broadening of vesicular packets of catecholamines released from biological cells during exocytosis. Anal Chem 64:3077–3083

    Article  CAS  Google Scholar 

  9. Alvarez de Toledo G, Fernández-Chacón R, Fernandez JM (1993) Release of secretory products during transient vesicle fusion. Nature 363:554–558

    Article  CAS  Google Scholar 

  10. Neher E, Marty A (1982) Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells. Proc Natl Acad Sci U S A 79:6712–6716

    Article  CAS  Google Scholar 

  11. Lollike K, Borregaard N, Lindau M (1995) The exocytotic fusion pore of small granules has a conductance similar to an ion channel. J Cell Biol 129:99–104

    Article  CAS  Google Scholar 

  12. Debus K, Lindau M (2000) Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles. Biophys J 78:2983–2997

    Article  CAS  Google Scholar 

  13. Dernick G, de Toledo GA, Lindau M (2007) The patch amperometry technique: design of a method to study exocytosis of single vesicles. In: Michael AC, Borland LM (eds) Electrochemical methods for neuroscience. CRC Press, Boca Raton (FL), pp 315–336

    Google Scholar 

  14. Dernick G, Gong LW, Tabares L, Alvarez de Toledo G, Lindau M (2005) Patch amperometry: high-resolution measurements of single-vesicle fusion and release. Nat Methods 2:699–708

    Article  CAS  Google Scholar 

  15. Albillos A, Dernick G, Horstmann H, Almers W, Alvarez de Toledo G, Lindau M (1997) The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389:509–512

    Article  CAS  Google Scholar 

  16. Gong LW, Alvarez De Toledo G, Lindau M (2003) Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells. J Neurosci 23:7917–7921

    Article  CAS  Google Scholar 

  17. Chow RH, Rüden L, Neher E (1992) Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356:60–63

    Article  CAS  Google Scholar 

  18. Gong LW, de Toledo GA, Lindau M (2007) Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore. Nat Cell Biol 9:915–922

    Article  CAS  Google Scholar 

  19. Dernick G, Alvarez de Toledo G, Lindau M (2003) Exocytosis of single chromaffin granules in cell-free inside-out membrane patches. Nat Cell Biol 5:358–362

    Article  CAS  Google Scholar 

  20. Mosharov EV, Gong LW, Khanna B, Sulzer D, Lindau M (2003) Intracellular patch electrochemistry: regulation of cytosolic catecholamines in chromaffin cells. J Neurosci 23:5835–5845

    Article  CAS  Google Scholar 

  21. Mosharov EV et al (2006) Alpha-synuclein overexpression increases cytosolic catecholamine concentration. J Neurosci 26:9304–9311

    Article  CAS  Google Scholar 

  22. Mosharov EV et al (2009) Interplay between cytosolic dopamine, calcium, and alpha-synuclein causes selective death of substantia nigra neurons. Neuron 62:218–229

    Article  CAS  Google Scholar 

  23. Choi SJ, Panhelainen A, Schmitz Y, Larsen KE, Kanter E, Wu M, Sulzer D, Mosharov EV (2015) Changes in neuronal dopamine homeostasis following 1-methyl-4-phenylpyridinium (MPP+) exposure. J Biol Chem 290:6799–6809

    Article  CAS  Google Scholar 

  24. Lieberman OJ et al (2017) alpha-synuclein-dependent calcium entry underlies differential sensitivity of cultured SN and VTA dopaminergic neurons to a parkinsonian neurotoxin. eNeuro 4:ENEURO.0167-17.2017. https://doi.org/10.1523/ENEURO.0167-17.2017

    Article  PubMed  PubMed Central  Google Scholar 

  25. Michael DJ, Joseph JD, Kilpatrick MR, Travis ER, Wightman RM (1999) Improving data acquisition for fast-scan cyclic voltammetry. Anal Chem 71:3941–3947

    Article  CAS  Google Scholar 

  26. Bucher ES, Wightman RM (2015) Electrochemical analysis of neurotransmitters. Annu Rev Anal Chem (Palo Alto Calif) 8:239–261

    Article  CAS  Google Scholar 

  27. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch/Eur J Physiol 391:85–100

    Article  CAS  Google Scholar 

  28. Penner R (1995) A practical guide to patch clamping. In: Sakmann B, Neher E (eds) Single channel recording. Plenum Press, New York, pp 3–30

    Chapter  Google Scholar 

  29. Tabares L, Ales E, Lindau M, Alvarez De Toledo G (2001) Exocytosis of catecholamine-containing and catecholamine-free granules in chromaffin cells. J Biol Chem 276:39974–39979

    Article  CAS  Google Scholar 

  30. Alés E, Tabares L, Poyato JM, Valero V, Lindau M, Alvarez de Toledo G (1999) High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism. Nat Cell Biol 1:40–44

    Article  Google Scholar 

  31. Jankowski JA, Schroeder TJ, Ciolkowski EL, Wightman RM (1993) Temporal characteristics of quantal secretion of catecholamines from adrenal medullary cells. JBC 268:14694–14700

    Article  CAS  Google Scholar 

  32. Jankowski JA, Finnegan JM, Wightman RM (1994) Extracellular ionic composition alters kinetics of vesicular release of catecholamines and quantal size during exocytosis at adrenal medullary cells. J Neurochem 63:1739–1747

    Article  CAS  Google Scholar 

  33. Wightman RM, Schroeder TJ, Finnegan JM, Ciolkowski EL, Pihel K (1995) Time course of release of catecholamines from individual vesicles during exocytosis at adrenal medullary cells. Biophys J 68:383–390

    Article  CAS  Google Scholar 

  34. Schroeder TJ, Borges R, Finnegan JM, Pihel K, Amatore C, Wightman RM (1996) Temporally resolved, independent stages of individual exocytotic secretion events. Biophys J 70:1061–1068

    Article  CAS  Google Scholar 

  35. Segura F, Brioso MA, Gomez JF, Machado JD, Borges R (2000) Automatic analysis for amperometrical recordings of exocytosis. J Neurosci Methods 103:151–156

    Article  CAS  Google Scholar 

  36. Sulzer D, Pothos EN (2000) Regulation of quantal size by presynaptic mechanisms. Rev Neurosci 11:159–212

    Article  CAS  Google Scholar 

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Acknowledgments

The preparation of this chapter was supported by NIH grant R35GM139608 to M.L. and a JPB grant (PI: D. Sulzer) for E.V.M.

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Authors and Affiliations

  1. Departments of Psychiatry and Neurology, Columbia University Medical Center, New York State Psychiatric Institute, New York, NY, USA

    Eugene V. Mosharov

  2. Department of Physiology and Biophysics, University of Miami, Miller School of Medicine, Miami, FL, USA

    Manfred Lindau

Authors
  1. Eugene V. Mosharov
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  2. Manfred Lindau
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Editor information

Editors and Affiliations

  1. Department of Physical Medicine and Pharmacology, Universidad de La Laguna, La Laguna, Spain

    Ricardo Borges

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Mosharov, E.V., Lindau, M. (2023). Patch Amperometry and Intracellular Patch Electrochemistry. In: Borges, R. (eds) Chromaffin Cells. Methods in Molecular Biology, vol 2565. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2671-9_17

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  • DOI: https://doi.org/10.1007/978-1-0716-2671-9_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2670-2

  • Online ISBN: 978-1-0716-2671-9

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