Table Of Contents:
Preface to the Series v(2)
Preface vii(10)
List of Contributors xvii
Technology of Patch-Clamp Electrodes 1(36)

Richard A. Levis

James L. Rae

1. Introduction 1(2)

2. General Properties of Pipet Glass 3(2)

3. Whole-Cell Pipet Properties: Practical Aspects 5(5)

3.1. Choice of Glass 5(2)

3.2. Pulling Whole-Cell Electrodes 7(1)

3.3. Elastomer Coating Whole-Cell Electrodes 7(1)

3.4. Firepolishing Whole-Cell Electrodes 8(2)

4. Patch Electrode Fabrication for Single-Channel Recording 10(7)

4.1. Choice of Glass 10(1)

4.2. Pulling Single-Channel Electrodes 11(1)

4.3. Coating Single-Channel Pipets with Elastomers 11(2)

4.4. Firepolishing Single-Channel Pipets 13(1)

4.5. Fabrication Methods Specific to Quartz 13(2)

4.6. Low-Noise Recording 15(2)

5. Noise Properties of Patch Pipets 17(20)

5.1. Noise Contribution of the Pipet 17(2)

5.2. Thin-Film Noise 19(2)

5.3. Distributed RC Noise 21(3)

5.4. Dielectric Noise 24(4)

5.5. R(e)-C(p) Noise 28(1)

5.6. Seal Noise 29(1)

5.7. Summary of Pipet Noise Sources 30(2)

5.8. Noise Sources for Whole-Cell Voltage Clamping 32(4)

References 36(1)
Whole-Cell Patch-Clamp Recordings 37(38)

Harald Sontheimer

1. Introduction 37(1)

2. Principles (Why Voltage Clamp?) 38(1)

3. Procedure and Techniques 39(7)

3.1. Pipets 39(1)

3.2. Electronic Components of a Setup 40(2)

3.3. Recording Configuration 42(2)

3.4. Experimental Procedure 44(2)

4. Data Evaluation and Analysis 46(19)

4.1. Data Filtering/Conditioning, Acquisition, and Storage 46(3)

4.2. Leak Subtraction 49(3)

4.3. Determination of Cell Capacitance 52(1)

4.4. Dissecting Current Components 52(5)

4.5. I-V Curves 57(6)

4.6. Fitting of Time-Constants 63(1)

4.7. Data Presentation 64(1)

5. Limitations, Pitfalls, and Errors 65(5)

5.1 Series Resistance and Its Consequences 65(3)

5.2. Voltage Clamp Errors 68(2)

6. Special Applications 70(2)

7. Conclusions 72(3)

Acknowledgments 72(1)

Recommended Readings 72(1)

References 73(2)
Pressure/Patch-Clamp Methods 75(14)

Owen P. Hamill

Don W. McBride, Jr.

1. Introduction 75(1)

2. General Cell-Attached Patch Recording Procedures 76(1)

3. Methods of Applying Suction 77(2)

3.1. Steady-State Methods 77(1)

3.2. Perturbation Methods 78(1)

4. Properties of the Pressure Clamp 79(4)

4.1. Stimulation Protocols 79(2)

4.2. Speed of the Pressure Clamp 81(1)

4.3. Sensitivity and Noise of the Pressure Clamp 82(1)

4.4. Range of the Pressure Clamp 82(1)

5. Applications of Pressure/Patch-Clamp Methods 83(2)

5.1. Sealing Protocols and Determination of Functional Membrane-Cytoskeleton Interactions 83(1)

5.2. Membrane Viscoelastic and Mechanical Properties 84(1)

5.3. Characterization of Mechano-Gated Channels 84(1)

6. Conclusion 85(4)

Acknowledgments 85(1)

References 85(4)
Cell-Free Ion-Channel Recording 89(34)

C. G. Nichols

M. B. Cannell

A. N. Lopatin

1. Introduction 89(1)

2. Making an Inside-Out Membrane Patch: The Problem of Vesicle Formation 90(5)

2.1. How to Tell When You Have a Vesicle 90(4)

2.2. How to Deal with the Problem 94(1)

3. Analysis of Ion Channels in Cell-Free Patches: Dealing with the Problem of Channel "Rundown" 95(6)

3.1. Mechanisms of Rundown 97(1)

3.2. Accounting for Rundown: Statistical Approaches for Analyzing Current Records 98(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