Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at Single Cells

Michelle L. Mundorf1, R. Mark Wightman1

1 The University of north carolina at Chapel Hill, Chapel Hill, North Carolina
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.14
DOI:  10.1002/0471142301.ns0614s18
Online Posting Date:  May, 2002
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Abstract

Amperometry and cyclic voltammetry are two electrochemical techniques that enable the detection of electroactive neurotransmitters that are released from single cells. These techniques have provided the first chemical view of the events that occur during exocytosis. This unit describes the isolation of several cell types known for their exocytotic properties, the fabrication and calibration of carbon fiber microelectrodes, as well as some of the equipment and software requirements for obtaining electrochemically generated data.

     
 
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Table of Contents

  • Basic Protocol 1: Amperometry
  • Basic Protocol 2: Cyclic Voltammetry
  • Support Protocol 1: Chromaffin Cell Isolation from Bovine Adrenal Glands
  • Support Protocol 2: Chromaffin Cell Isolation from Rat
  • Support Protocol 3: Mast Cell Isolation from Mouse
  • Support Protocol 4: Microelectrode Preparation
  • Support Protocol 5: Electrode Calibration Using a Flow Injection System for Amperometry and Cyclic Voltammetry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amperometry

  Materials
  • 35‐mm culture plate with attached cells (e.g., see protocol 3Support Protocols 1, protocol 42, or protocol 53)
  • Amp/CV buffer(see recipe)
  • Pulled glass pipet (unit 6.3) with 10‐µm opening filled with desired secretagogue connected to a pressure‐ejection device (e.g., Picospritzer; Parker Hannifin Corporation)
  • Calibrated and back‐filled microelectrodes (see protocol 6Support Protocols 4 and protocol 75)
  • Potentiostat—i.e., Axopatch series of patch clamp amplifiers (e.g., 200B); Axon Instruments
  • Reference electrode (e.g., Ag/AgCl)
  • Micromanipulators (Burleigh Instruments)
  • Data acquisition and analysis software (e.g., Axon Instruments)
  • Computer storage device (e.g., A/D VCR adapter, VCR, and software package; Axon Instruments)

Basic Protocol 2: Cyclic Voltammetry

  Materials
  • Culture plate with adherent cells (e.g., see protocol 3Support Protocols 1, protocol 42, or protocol 53)
  • Amp/CV buffer(see recipe)
  • Pulled glass pipet (unit 6.3) with 10‐µm opening filled with desired secretagogue attached to a pressure ejection device (e.g., Picospritzer; Parker Hannifin)
  • Calibrated and back‐filled microelectrodes (see protocol 6Support Protocols 4 and protocol 75)
  • Potentiostat—i.e., Axopatch series (e.g., 200B) of patch clamp amplifiers (Axon Instruments)
  • Reference electrode (Ag/AgCl)
  • Micromanipulators (Burleigh Instruments)
  • Data acquisition and analysis software (see Introduction )
  • 100‐kHz 16‐bit A/D board

Support Protocol 1: Chromaffin Cell Isolation from Bovine Adrenal Glands

  Materials
  • Bovine adrenal glands, fresh and undamaged
  • 1× physiological buffer without nystatin, ice‐cold (see recipe)
  • 1× physiological buffer with nystatin, 37°C (see recipe)
  • Digestion mixture, 37°C (see recipe)
  • 7.5% and 15% Renografin solutions, well‐shaken (see recipe)
  • Elevated calcium and magnesium physiological buffer with nystatin (see recipe)
  • Plating medium(see recipe), fresh
  • Trypan blue
  • Growth medium, fresh (see recipe)
  • 10‐ml syringes
  • Glass dissection dish
  • Submersible stir plate
  • 250‐µm nylon mesh
  • 50‐ml centrifuge tubes
  • 35‐mm culture plates
  • Additional reagents and equipment for determining cell number a hemocytometer ( appendix 3B)
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 2: Chromaffin Cell Isolation from Rat

  Materials
  • 300‐ to 400‐g male Sprague‐Dawley rats
  • Rat adrenal buffer, 4°C (see recipe)
  • Rat adrenal buffer containing 0.3% (w/v) BSA/0.15% (w/v) collagenase, 37°C
  • 7.5% and 15% Renografin solutions(see recipe)
  • Plating medium(see recipe)
  • Growth medium, fresh (see recipe)
  • Stereoscope
  • 250‐µm nylon mesh
  • Culture plates
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 3: Mast Cell Isolation from Mouse

  Materials
  • Mice: 2 months or older
  • Ether
  • Mouse mast cell buffer (see recipe)
  • 70% ethanol
  • Growth medium(see recipe)
  • 5‐ml syringe equipped with 25‐G × 0.625‐in. needle
  • 15‐ml centrifuge tubes
  • 35‐mm plastic culture plates
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.

Support Protocol 4: Microelectrode Preparation

  Materials
  • Shell EPON 828 epoxy resin and MPDA hardener (Miller‐Stephenson Chemical Company)
  • Carbon fibers (various diameters available from Good Fellow Metals)
  • Glass capillaries
  • Aspirating device: 0.25‐in.‐i.d. Tygon tubing with small rubber septum to place capillary in
  • Narishige model PE‐2 micropipet puller (Narishige USA)
  • Wooden applicator stick

Support Protocol 5: Electrode Calibration Using a Flow Injection System for Amperometry and Cyclic Voltammetry

  Materials
  • Isopropyl alcohol
  • 4 M potassium acetate/150 mM potassium chloride
  • Amp/CV buffer(e.g., see recipe)
  • Neurotransmitters of interest (e.g., catecholamines at 10 mM) stored in 0.1 N HClO 4 (perchloric acid). Dilute to desired concentration with Amp/CV buffer
  • Carbon fiber microelectrode (see protocol 6)
  • Diamond dust–embedded beveling wheel (Sutter Instruments)
  • Paint or marker
  • 25‐G needle
  • Ag/AgCl reference electrode
  • Flow Cell (Figure )
  • Potentiostat (Axopatch series of patch clamp amplifiers; Axon Instruments)
  • Six‐port injection valve
  • Data acquisition system: CV6 or LabVIEW system (recommended)
  • Oscilloscope
  • Syringe pump
  • Computer controlled six‐port injection valve
  • Faraday cage (copper mesh will suffice)
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Figures

Videos

Literature Cited

Literature Cited
   Bard, A.J. and Faulkner, L.R. 1980. Electrochemical Methods: Fundamentals and Applications. John Wiley & Sons, New York.
   Baur, J.E., Kristensen, E.W., May, L.J., Wiedemann, D.J., and Wightman, R.M. 1988. Fast‐scan voltammetry of biogenic amines. Anal.Chem. 60: 1268‐ 1272.
   Del Castillo, J. and Katz, B. 1954. Quantal components of the end‐plate potential. J. Physiol. 124: 560‐ 573.
   Fatt, P. and Katz, B. 1952. Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117: 109‐ 128.
   Kawagoe, K.T., Zimmerman, J.B., and Wightman, R.M. 1993. Principles of voltammetry and microelectrode surface states. J. Neurosci.Methods. 48: 225‐ 240.
   Kristensen, E.W., Wilson, R.L., and Wightman, R.M. 1986. Dispersion in flow injection analysis measured with microvoltammetric electrodes. Anal. Chem. 54: 986‐ 988.
   Martin, A.R. 1966. Quantal nature of synaptic transmission. Physiol. Rev. 46: 51‐ 66.
   Neher, E. and Marty, A. 1982. Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal medullary cells. Proc. Natl. Acad. Sci. U.S.A. 79: 6712‐ 6716.
   Pihel, K., Hsieh, S., Jorgenson, J.W., and Wightman, R.M. 1995. Electrochemical detection of histamine and 5‐hydroxytryptamine at isolated mast cells. Anal. Chem. 67: 4514‐ 4521.
   Travis, E.R., Wang, Y.M., Michael, D., Caron, M., and Wightman, R.M. 2000. Differential quantal release of histamine and 5‐hydroxytryptamine from mast cells of vesicular monoamine transporter 2 knockout mice. Proc. Natl. Acad. Sci. U.S.A. 97: 162‐ 167.
   Wehmeyer, K.R. and Wightman, R.M. 1985. Scan rate dependence of the apparent capacitance at microvoltammetric electrodes. J. Electroanal. Chem. 196: 417‐ 421.
   Wightman, R.M., Jankowski, J.A., Kennedy, R.T., Kawagoe, K.T., Schroeder, T.J., Leszczyszyn, D.J., Near, J.A., Diliberto, E.J. Jr., and Viveros, O.H. 1991. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. U.S.A. 88: 10754‐ 10758.
   Wilson, S.P. 1987. Purification of adrenal chromaffin cells on Renografin gradients. J. Neurosci. Methods. 19: 163‐ 171.
   Wilson, S.P. and Viveros, O.H. 1981. Primary culture of adrenal medullary chromaffin cells in a chemically defined medium. Exp. Cell Res. 133: 159‐ 169.
   Zimmerberg, J., Curran, M., Cohen, F.S., and Brodwick, M. 1987. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc. Natl. Acad. Sci. U.S.A. 84: 1585‐ 1589.
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