Patch-Clamp Applications and Protocols

, by Boulton, Alan A.; Baker, Glen A.; Walz, Wolfgang

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ISBN: 9780896033115 | 0896033112
Cover: Hardcover
Copyright: 8/1/1995

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Patch-Clamp Applications and Protocols presents the broadest coverage of techniques for measuring and recording directly activity within a cell. The first part of the book offers modern developments associated with the technology of path-clamp electrodes, of cell-free ion-channel recording, and of the whole-cell patch clamp technique. Chapters on such recent offspring of the technique as the concentration clamp technique, the pressure clap method, and the perfusion of patch clamp electrodes are contributed by authors intimately involved with their development. Also covered is the loose patch-clamp technique, single-channel recording, the perforated patch-clamp technique, molecular biological aspects of the technique, and the use of the technique in brain slices. Every scientist working with path-clamp methods will find this protocols manual invaluable to their continued use and development of this powerful technique.

Preface to the Series

(2)

Preface

vii

(10)

List of Contributors

xvii

Technology of Patch-Clamp Electrodes

(36)

Richard A. Levis

James L. Rae

1. Introduction

(2)

2. General Properties of Pipet Glass

(2)

3. Whole-Cell Pipet Properties: Practical Aspects

(5)

3.1. Choice of Glass

(2)

3.2. Pulling Whole-Cell Electrodes

(1)

3.3. Elastomer Coating Whole-Cell Electrodes

(1)

3.4. Firepolishing Whole-Cell Electrodes

(2)

4. Patch Electrode Fabrication for Single-Channel Recording

(7)

4.1. Choice of Glass

(1)

4.2. Pulling Single-Channel Electrodes

(1)

4.3. Coating Single-Channel Pipets with Elastomers

(2)

4.4. Firepolishing Single-Channel Pipets

(1)

4.5. Fabrication Methods Specific to Quartz

(2)

4.6. Low-Noise Recording

(2)

5. Noise Properties of Patch Pipets

(20)

5.1. Noise Contribution of the Pipet

(2)

5.2. Thin-Film Noise

(2)

5.3. Distributed RC Noise

(3)

5.4. Dielectric Noise

(4)

5.5. R(e)-C(p) Noise

(1)

5.6. Seal Noise

(1)

5.7. Summary of Pipet Noise Sources

(2)

5.8. Noise Sources for Whole-Cell Voltage Clamping

(4)

References

(1)

Whole-Cell Patch-Clamp Recordings

(38)

Harald Sontheimer

1. Introduction

(1)

2. Principles (Why Voltage Clamp?)

(1)

3. Procedure and Techniques

(7)

3.1. Pipets

(1)

3.2. Electronic Components of a Setup

(2)

3.3. Recording Configuration

(2)

3.4. Experimental Procedure

(2)

4. Data Evaluation and Analysis

(19)

4.1. Data Filtering/Conditioning, Acquisition, and Storage

(3)

4.2. Leak Subtraction

(3)

4.3. Determination of Cell Capacitance

(1)

4.4. Dissecting Current Components

(5)

4.5. I-V Curves

(6)

4.6. Fitting of Time-Constants

(1)

4.7. Data Presentation

(1)

5. Limitations, Pitfalls, and Errors

(5)

5.1 Series Resistance and Its Consequences

(3)

5.2. Voltage Clamp Errors

(2)

6. Special Applications

(2)

7. Conclusions

(3)

Acknowledgments

(1)

Recommended Readings

(1)

References

(2)

Pressure/Patch-Clamp Methods

(14)

Owen P. Hamill

Don W. McBride, Jr.

1. Introduction

(1)

2. General Cell-Attached Patch Recording Procedures

(1)

3. Methods of Applying Suction

(2)

3.1. Steady-State Methods

(1)

3.2. Perturbation Methods

(1)

4. Properties of the Pressure Clamp

(4)

4.1. Stimulation Protocols

(2)

4.2. Speed of the Pressure Clamp

(1)

4.3. Sensitivity and Noise of the Pressure Clamp

(1)

4.4. Range of the Pressure Clamp

(1)

5. Applications of Pressure/Patch-Clamp Methods

(2)

5.1. Sealing Protocols and Determination of Functional Membrane-Cytoskeleton Interactions

(1)

5.2. Membrane Viscoelastic and Mechanical Properties

(1)

5.3. Characterization of Mechano-Gated Channels

(1)

6. Conclusion

(4)

Acknowledgments

(1)

References

(4)

Cell-Free Ion-Channel Recording

(34)

C. G. Nichols

M. B. Cannell

A. N. Lopatin

1. Introduction

(1)

2. Making an Inside-Out Membrane Patch: The Problem of Vesicle Formation

(5)

2.1. How to Tell When You Have a Vesicle

(4)

2.2. How to Deal with the Problem

(1)

3. Analysis of Ion Channels in Cell-Free Patches: Dealing with the Problem of Channel "Rundown"

(6)

3.1. Mechanisms of Rundown

(1)

3.2. Accounting for Rundown: Statistical Approaches for Analyzing Current Records

(3)

4. Methods for Rapid Change of the Solution Bathing Cell-Free Membrane Patches

101

(6)

4.1. Methods for Rapid Bulk Application of Solution

102

(1)

4.2. Laminar Flow Methods of Separating Parallel Solutions

103

(1)

4.3. Separation of Solutions Using an "Oil-Gate"

103

(3)

4.4. Separation of Solutions Using an "Air Gate"

106

(1)

5. Analysis of Responses to Rapid Concentration Changes

107

(7)

5.1. Modeling the Pipet Geometry

107

(1)

5.2. Time Course of Solution Change: The Effects of Pipet Geometry

108

(1)

5.3. Experimental Measurement of Diffusion Delays

109

(3)

5.4. Correcting for Diffusion Delays in Analysis of Concentration Jump Experiments

112

(1)

5.5. Advantages and Disadvantages of the Analysis

113

(1)

6. Twenty Eight Hints and Tips for Successful Cell-Free Ion-Channel Recording!

114

(4)

7. Concluding Remarks

118

(5)

References

119

(4)

Perfusion of Patch Pipets

123

(18)

John M. Tang

F.N. Quandt

R. S. Eisenberg

1. Introduction

123

(1)

2. Methods

124

(5)

2.1. Patch-Clamp of Neuroblastoma Cells

124

(1)

2.2. Internal Perfusion Technology

125

(4)

3. Results

129

(6)

3.1. Time Course of Exchange of Internal Solution

129

(1)

3.2. Efficiency of Exchange of Internal Solution

129

(2)

3.3. Selectivity of K Channels Measured by Reversal Potentials

131

(2)

3.4. Pharmacology of 4-Aminopyridine

133

(1)

3.5. Parameters Controlling the Rate of Exchange of Solution

134

(1)

4. Discussion

135

(6)

4.1. Applicability of the Technique

135

(2)

4.2. Possible Problems

137

(1)

4.3. Improvements

138

(1)

References

139

(2)

Concentration Clamp Techniques

141

(14)

Norio Akaike

1. Introduction

141

(1)

2. Setup of Rapid Solution Change

142

(2)

3. Preparations

144

(2)

3.1. Whole-Cell Recording Mode

144

(2)

3.2. Excised Cell Membrances

146

(1)

4. Kinetic Studies Using Concentration Clamp Technique

146

(5)

4.1. Receptor-Mediated Ionic Currents

146

(1)

4.2. Voltage-Dependent Ionic Currents

147

(1)

4.3. Rapid Change of Physical Conditions

148

(2)

4.4. G-Protein Mediated Response

150

(1)

4.5. Measurement of Ca(2+) Release from Intracellular Ca(2+) Store Sites

151

(1)

5. Limitations

151

(4)

References

151

(4)

Perforated Patch-Clamp Techniques

155

(18)

Wolfgang Walz

1. Introduction

155

(1)

2. Dialysis of Cytoplasm by the Patch Micropipet Filling Solution

155

(3)

3. Strategies Used to Prevent Dialysis

158

(2)

3.1. Increase of Pipet Resistance

158

(1)

3.2. Addition of a Cytosolic Extract to the Pipet Solution

159

(1)

3.3. Use of ATP

160

(1)

3.4. Use of Polyene Antibiotics

160

(1)

4. Use of Nystatin

160

(7)

4.1. Properties of Nystatin Pores

160

(2)

4.2. Perforating the Patch Membrane with Nystatin

162

(1)

4.3. Intrapipet Dialysis of Nystatin

163

(1)

4.4. Composition of Pipet Solutions

164

(1)

4.5. Detailed Protocol for Use of Nystatin

165

(1)

4.6. Use of a Nystatin-Fluoroscein Mixture

166

(1)

5. Use of Amphotericin B

167

(1)

6. Special Application: The Perforated Vesicle

168

(1)

7. Conclusions

169

(4)

Acknowledgment

170

(1)

References

170

(3)

The Loose Patch Voltage Clamp Technique

173

(20)

J. H. Caldwell

R. L. Milton

1. Introduction

173

(1)

2. Techniques

174

(12)

2.1. Amplifier

174

(3)

2.2. Pipets

177

(2)

2.3. Equipment

179

(1)

2.4. Procedure for Performing an Experiment

180

(2)

2.5. Possible Sources of Error

182

(4)

3. Variations of the Method

186

(4)

3.1. Concentric Electrodes

186

(1)

3.2. Current Collector

187

(1)

3.3. Ionophoresis with Loose Patch

188

(2)

4. Conclusion

190

(3)

Acknowledgments

190

(1)

References

190

(3)

Patch-Clamp Recording and RT-PCR on Single Cells

193

(40)

Bertrand Lambolez

Etienne Audinat

Pascal Bochet

Jean Rossier

1. Introduction

193

(2)

2. Materials and Methods

195

(11)

2.1. Labware, Reagents

195

(1)

2.2. Solutions

196

(2)

2.3. Design of the Oligos

198

(1)

2.4. Thermocycle PCR Programs

199

(1)

2.5. Test of the Sensitivity of the PCR

200

(1)

2.6. Contamination

201

(1)

2.7. Electrophysiology and Cellular RNA Harvesting

202

(2)

2.8. RT-PCR on Single-Cell Step by Step

204

(2)

3. AMPA Receptor Subunits in Purkinje Cells: GFAP in Glial Cells

206

(12)

3.1. Experimental Procedures

206

(9)

3.2. Specificity of the PCR and Quantification

215

(1)

3.3. Proportional Amplification of the Fragments

216

(1)

3.4. Discussion

217

(1)

4. AMPA Receptor Subunits and GAD in Hippocampal Cells

218

(15)

4.1. Experimental Procedures

219

(3)

4.2. Results

222

(7)

References

229

(4)

Patch-Clamp Technique in Brain Slices

233

(26)

T. D. Plant

J. Eilers

A. Konnerth

1. Introduction

233

(1)

2. Methods

234

(25)

2.1. Brain Slices for Patch-Clamp Studies

234

(2)

2.2. Patch-Clamp Recording in Brain Slices

236

(14)

2.3. Combinations of the Patch-Clamp Techniques in Slices with Other Methods

250

(6)

Acknowledgments

256

(1)

References

256

(3)

Xenopus Oocyte Microinjection and Ion-Channel Expression

259

(48)

T. G. Smart

B. J. Krishek

1. Introduction

259

(2)

1.1. History of the Xenopus laevis Oocyte as an Expression System

259

(1)

1.2. Application to Ion Channel Expression Studies

260

(1)

2. Husbandry of Xenopus laevis

261

(6)

2.1. Source and Identification of Xenopus laevis

261

(2)

2.2. Housing and Environment

263

(1)

2.3. Feeding

264

(1)

2.4. Diseases and Parasites

265

(2)

3. Removal of Ovary Tissue

267

(8)

3.1. Anesthesia of Xenopus laevis

267

(1)

3.2. Removal of Oocytes

268

(4)

3.3. Preparation of Oocytes for Injection

272

(2)

3.4. Removal of the Vitelline Membrane

274

(1)

4. Selection of Oocytes

275

(2)

4.1. Stages of Oocyte Development

276

(1)

4.2. Separation of Stage V/VI Oocytes

276

(1)

5. Microinjection of Xenopus Oocytes

277

(8)

5.1. Injection Equipment

277

(1)

5.2. Preparation of Glassware and RNA/DNA Solutions

278

(1)

5.3. Fabrication of Injection Micropipets

279

(2)

5.4. Cytoplasmic RNA Injection

281

(1)

5.5. Nuclear DNA Injection

282

(1)

5.6. Optimization of Receptor/Ion-Channel Expression

284

(1)

6. Culture/Incubation of Injected Oocytes

285

(2)

6.1 Optimal Culture Conditions for Protein Expression

286

(1)

7. Electrophysiological Recording from Xenopus Oocytes

287

(9)

7.1. Two-Electrode Voltage Clamp

287

(5)

7.2. Patch-Clamp Recording

292

(4)

8. Comparison of Xenopus Oocytes with Alternative Expression Systems

296

(11)

Appendix 1: Composition of Physiological Solutions

297

(3)

Acknowledgments

300

(1)

References

301

(6)

Index

307

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