Electrophysiological Analysis of G Protein–Coupled Receptors in Mammalian Neurons

William R. Proctor1, Thomas V. Dunwiddie1

1 Veterans Administration Medical Research Service, Denver, Colorado
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.2
DOI:  10.1002/0471141755.ph1102s07
Online Posting Date:  May, 2001
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Abstract

This unit describes general techniques that are useful for recording electrophysiological responses that are mediated via the activation of G‐protein coupled receptors (GPCRs). It includes a brief description of preparations, but focuses primarily on experiments using hippocampal brain slice preparations. Techniques for the preparation of brain slices, electrodes, filling solutions, and the recording protocols that are suitable for assessing the activity of GPCRs using electrophysiological techniques are summarized, and various protocols for the activation of these receptors are discussed.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Measurement of Responses from Drug‐Activated GPCRs: Bath Application
  • Support Protocol 1: Fabrication of Brain‐Slice Storage Chamber
  • Support Protocol 2: Construction of a Manifold for Addition of Compounds
  • Support Protocol 3: Fabrication and Use of Recording Microelectrodes and Pressure Pipets: Whole‐Cell Recordings Using Whole‐Cell Patch Electrodes
  • Support Protocol 4: Fabrication and Use of Recording Microelectrodes: Intracellular Recordings Using Sharp‐Point Electrodes
  • Support Protocol 5: Preparation of Hippocampal Brain Slices from Young Adult Rats: Tissue Chopper Method
  • Support Protocol 6: Preparation of Hippocampal Brain Slices from Young Adult Rats: Vibratome Method
  • Support Protocol 7: Data Collection and Analysis
  • Alternate Protocol 1: Local Application of Drugs/Transmilters
  • Alternate Protocol 2: Transmitter Release Evoked by Electrical Stimulation
  • Support Protocol 8: Fabrication of Stimulation Apparatus
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Responses from Drug‐Activated GPCRs: Bath Application

  Materials
  • Internal electrode solution (see recipe; Table 11.2.1)
  • aCSF (see recipe)
  • Sample tissue: cultured or isolated cells, or brain slices (see Support Protocols protocol 65 and protocol 76) in a brain‐slice storage chamber (see protocol 2)
  • 95%/5% (v/v) O 2/CO 2
  • 2 mM γ‐amino‐n‐butyric acid (GABA; Sigma) in high‐purity water
  • 3 mM baclofen (Sigma) in high‐purity water
  • 10 mM adenosine (Sigma) in high‐purity water
  • Incubator for maintenance and storage of cell cultures and acutely dissociated cells
  • Electrophysiological setup (see Strategic Planning; unit 11.1; Stuart, 1998)
  • Adapter port (or manifold, Warner Instrument; or see protocol 3) with calibrated syringe pump
  • Recording microelectrodes (see Support Protocols protocol 43 and protocol 54)
  • Recording chamber, e.g.:
  •  small chamber to hold 12‐mm glass cover slips for cultured or isolated cells (Warner Instrument)
  •  chamber appropriate for “visualized slice” cell recording in brain slices (Warner Instrument) using a differential interference contrast (DIC)–equipped microscope with water‐immersion optics
  •  larger recording chamber for use with brain slices utilizing a dissecting microscope (“blind recording” setup for cell recording; H & H Woodworking)
  • Computer with analog‐to‐digital (A/D) board (e.g., Digidata 1320, Axon Instruments; ISC‐16, RC Electronics) and software (see protocol 8)
    Table 1.2.1   MaterialsComposition of Solutions

    Chemical External Internal
    1× aCSF (mM) Potassium gluconate (mM) KCl (mM) Cesium gluconate (mM)
    NaCl 126
    Potassium gluconate 125
    KCl 3 5 120
    CsOH ∼125
    Gluconic acid 125
    CaCl 2 2.4 0.1 1 0.1
    MgCl 2 1.5 2 2 2
    EGTA 1 5 1
    HEPES 10 10 10
    NaH 2PO 4 1.2
    Sodium bicarbonate 25.9
    Glucose 11
    Mg2+‐ATP 2 3 2
    Na+‐GTP 0.3 0.3 0.3
    pH 7.3 7.3 7.4 7.3
    mosM 300‐305 280‐290 280‐290 280‐290

Support Protocol 1: Fabrication of Brain‐Slice Storage Chamber

  Materials
  • aCSF (see recipe)
  • 95%/5% (v/v) O 2/CO 2
  • 400‐ml glass beaker
  • Fluorescent light fixture diffuser, with 0.5‐in. (1.3 cm) square compartments with 0.5‐in.‐high walls (hardware or building supply store)
  • Cyanoacrylate glue
  • Polyethylene mesh (526‐µm mesh opening, 540‐µm thickness; e.g., Spectra/Mesh, Spectrum)
  • 10‐ml plastic centrifuge tube
  • Sintered‐glass gas diffuser (gas dispersion tube with fritted cylinder; Fisher Scientific)
  • Plastic pipet tip
  • Parafilm
  • 21° to 35°C water bath

Support Protocol 2: Construction of a Manifold for Addition of Compounds

  Materials
  • Concentrated stock solution of drug: 100 to 1000× desired final bath concentration dissolved in high‐purity water or freshly oxygenated artificial cerebrospinal fluid (aCSF; see recipe)
  • Gum‐rubber septum (Millipore)
  • 18‐ and 22‐G syringe needles
  • 24‐G wire (single or multi‐stranded copper)
  • PE‐50 and PE‐160 polyethylene tubing (Intramedic/Clay Adams, Becton Dickinson)
  • Recording chamber (see protocol 1Basic Protocol 1, Materials)
  • 1‐liter glass media bottle
  • 10‐ to 12‐ml plastic syringe
  • Calibrated syringe pumps (Razel Scientific Instruments)

Support Protocol 3: Fabrication and Use of Recording Microelectrodes and Pressure Pipets: Whole‐Cell Recordings Using Whole‐Cell Patch Electrodes

  Materials
  • Internal electrode solution (see recipe; Table 11.2.1)
  • aCSF (see recipe)
  • Hippocampal slices (see protocol 6 and protocol 7)
  • Medium‐wall capillary glass: 0.86‐mm i.d., 1.5‐mm o.d., 10‐cm length, internal filament (Sutter Instruments)
  • Two‐ or three‐stage electrode/pipet puller (e.g., P‐87, Sutter Instruments)
  • Microscope with 100× and 600× magnification
  • PE‐10 and PE‐160 polyethylene tubing (Intramedic/Clay Adams, Becton Dickinson)
  • 200‐µl plastic pipet tips
  • Cyanoacrylate glue
  • 1‐ and 10‐ml plastic syringes
  • 0.2‐µm‐pore‐size, 13‐mm‐diameter syringe filters (Nylon Acrodisc 13, Gelman Sciences)
  • Electrode holder with pressure port (specific for particular recording setup; e.g., AxoClamp‐2B)
  • 1‐liter glass bottle
  • H‐size tank of 95% (v/v) O 2/5% CO 2 gas with two‐stage regulator (VWR) and fittings
  • Recording chamber (specific to microscope brand and configuration: for dissecting microscope setup, chamber from H & H Woodworking; for DIC microscope setup, chamber from Warner Instrument)

Support Protocol 4: Fabrication and Use of Recording Microelectrodes: Intracellular Recordings Using Sharp‐Point Electrodes

  • Thin‐wall capillary glass: 0.69‐mm i.d., 1.2‐mm o.d., 10‐cm length, internal filament (Sutter Instruments)
  • 3‐ml plastic syringes
  • 22‐G spinal syringe needle
  • Recording chamber for dissecting microscope setup (H & H Woodworking)

Support Protocol 5: Preparation of Hippocampal Brain Slices from Young Adult Rats: Tissue Chopper Method

  Materials
  • 6‐ to 8‐week‐old rats (Sprague‐Dawley; Charles Rivers)
  • aCSF (see recipe), ice cold
  • 55‐mm filter paper disks (Whatman no. 3; e.g., Fisher)
  • Plastic spoon
  • Small spatulas or plastic knives that have been modified by sharpening the edges
  • Small‐animal guillotine (e.g., Stoelting)
  • Small stainless‐steel surgical scissors, rongeurs, and flat weighing spatula
  • Tissue chopper (McIlwain, Stoelting) equipped with a new razor blade and Whatman filter paper fastened to pedestal (per chopper instructions)
  • Small paintbrush (0 or 00 size; art/craft store)
  • Brain‐slice storage chamber (see protocol 2)

Support Protocol 6: Preparation of Hippocampal Brain Slices from Young Adult Rats: Vibratome Method

  • 2‐ to 4‐week‐old rats (Sprague‐Dawley; Charles River)
  • ∼6 × 15–mm rectangles of filter paper (Whatman)
  • Prep blades (Weck)
  • Tissue vibratome (e.g., Vibratome 1000, Ted Pelco) with plastic chamber and stainless‐steel platform (H & H Woodworking) and blades (injector style; Pelco)
  • Cyanoacrylate glue pen (e.g., Krazy Glue)

Support Protocol 7: Data Collection and Analysis

  Materials
  • Computer with analog‐to‐digital (A/D) board (Digidata 1320, Axon Instruments; ISC‐16, RC Electronics) and for storing sweep data (e.g., pClamp and/or AxoTape, Axon Instruments; NeuroPro, RC Electronics), spread sheet analysis (e.g., Excel, Microsoft), and graphics (e.g., Origin, Microcal Software; pClamp, Axon Instruments) programs
  • Chart recorder (Gould; Cole‐Parmer) for continuous analog measurements of voltage and current changes (instead of the items above)

Alternate Protocol 1: Local Application of Drugs/Transmilters

  • 50 µM baclofen (Sigma) in freshly oxygenated artificial cerebrospinal fluid (aCSF; see recipe)
  • 200 µM adenosine (Sigma) in freshly oxygenated artificial cerebrospinal fluid (aCSF; see recipe)
  • 3 mM bicuculline methiodide (BMI; Sigma) in high‐purity water
  • 10 mM 3‐amino‐2‐(4‐chlorophenyl)‐2‐hydroxypropane‐sulfonic acid (2‐hydroxysaclofen; Sigma) in high‐purity water
  • 100 µM CGP 55845A (Novartis) in high‐purity water
  • 20 mM theophylline (Sigma) in high‐purity water
  • Pressure controller (e.g., Picospritzer II, General Valve)
  • Glass micropipet (see protocol 4, preparation of the electrode)
  • Pressure pipet holder, sealed (Warner Instrument)

Alternate Protocol 2: Transmitter Release Evoked by Electrical Stimulation

  • Receptor agonist:
  •  2 mM 6,7‐dinitroquinoxaline‐2,3(1H,4H)‐dione (DNQX; see recipe)
  •  5 mM 2‐amino‐5‐phosphonovaleric acid (APV or 5‐AP; Sigma) in high‐purity water
  •  3 mM bicuculline methiodide (BMI; Sigma) in high‐purity water
  • Stimulus isolation unit (e.g., AMPI, Digitimer)
  • Bipolar stimulation apparatus (see protocol 11)
  • Stimulator holder (Warner Instrument)

Support Protocol 8: Fabrication of Stimulation Apparatus

  Materials
  • Formvar‐coated nichrome or tungsten wire (0.002‐in. bare, 0.0026‐in. coated diameter; A‐M Systems)
  • ∼7‐ to 8‐cm stainless‐steel spinal syringe needles (22‐G)
  • Cyanoacrylate glue
  • Small, gold double connectors, male and female (e.g., GM‐2 and GF‐2, Microtech)
  • Soldering iron and solder
  • 24‐ to 22‐G multistranded insulated copper wire
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Figures

Videos

Literature Cited

Literature Cited
   Axon Instruments. 1993. The Axon Guide. Axon Instruments, Foster City, Calif.
   Blanton, M.G., LoTurco, J.J., and Kriegstein, A.R. 1989. Whole cell recording from neurons in slices of reptilian and mammalian cerebral cortex. J. Neurosci. Methods 30:203‐210.
   Castillo, P.E., Malenka, R.C., and Nicoll, R.A. 1997. Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons. Nature 388:182‐186.
   Gähwiler, B.H., Capogna, M., Debanne, D., McKinney, R.A., and Thompson, S.M. 1997. Organotypic slice cultures: A technique has come of age. Trends Neurosci. 20:471‐477.
   Gilchrist, A., Mazzoni, M.R., Dineen, B., Dice, A., Linden, J., Proctor, W.R., Lupica, C.R., Dunwiddie, T.V., and Hamm, H.E. 1998. Antagonists of the receptor‐G protein interface block G1‐coupled signal transduction. J. Biol. Chem. 273:14912‐14919.
   Hille, B. 1994. Modulation of ion‐channel function by G‐protein coupled receptors. Trends Neurosci. 17:531‐536.
   Johnson, S.W., Mercuri, N.B., and North, R.A. 1992. 5‐Hydroxytryptamine1B receptors block the GABAB synaptic potential in rat dopamine neurons. J. Neurosci. 12:2000‐2006.
   Kuner, R., Kohr, G., Grunewald, S., Eisenhardt, G., Bach, A., and Kornau, H.C. 1999. Role of heteromer formation in GABAB receptor function. Science 283:74‐77.
   Neher, E. and Sakmann, B. 1992. The patch clamp technique. Sci. Am. 266:28‐35.
   Sakmann, B. and Neher, E. 1984. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46:455‐472.
   Sekiguchi, M., Sakuta, H., Okamoto, K., and Sakai, Y. 1990. GABAB receptors expressed in Xenopus oocytes by guinea pig cerebral mRNA are functionally coupled with Ca2+‐dependent Cl− channels and with K+ channels, through GTP‐binding proteins. Mol. Brain Res. 8:301‐309.
   Sodickson, D.L. and Bean, B.P. 1996. GABAB receptor‐activated inwardly rectifying potassium current in dissociated hippocampal CA3 neurons. J. Neurosci. 16:6374‐6385.
   Stuart, G. 1999. Patch‐pipet recording in brain slices. In Current Protocols in Neuroscience (J.N. Cley, C.R. Gerfen, R. McKay, M.A. Rogawski, D.R. Sibley, P. Skolnick, eds.) pp. 6.7.1‐6.7.10. John Wiley & Sons, New York.
   Trussell, L.O. and Jackson, M.B. 1985. Adenosine‐activated potassium conductance in cultured striatal neurons. Proc. Natl. Acad. Sci. U.S.A. 82:4857‐4861.
   Vignes, M. and Collingridge, G.L. 1997. The synaptic activation of kainate receptors. Nature 388:179‐182.
   Weiner, J.L., Zhang, L., and Carlen, P.L. 1994. Potentiation of GABAA‐mediated synaptic current by ethanol in hippocampal CA1 neurons: possible role of protein kinase C. J. Pharmacol. Exp. Ther. 268:1388‐1395.
Key References
   Axon Instruments. 1993. See above.
  This manual can be obtained from Axon Instruments at no cost. Excellent explanations and descriptions cover almost all aspects of electrophysiological recording.
   Stuart, G. 1999. See above.
  Step‐by‐step procedure for making whole‐cell recordings from rat hippocampal brain slices.
Internet Resources
  http://www.axon.com/MR_Axon_Guide.html
  Axon Instruments Web site for a very complete summary of techniques and considerations for conducting electrophysiological recording studies.
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