NEUROMETHODS 䊏 35 Patch-Clamp Analysis NEUROMETHODS Series Editors: Alan A. Boulton and Glen B. Baker 37. Apoptosis Techniques and Protocols, Second 20. Intracellular Messengers, edited by Alan A. Edition, edited by Andréa C. LeBlanc, 2002 Boulton, Glen B. Baker, and Colin W. Taylor, 36. Neural Transplantation Methods, edited by 1992 Stephen B. Dunnett, Alan A. Boulton, and 19. Animal Models in Psychiatry, II, edited by Alan Glen B. Baker, 2000 A. Boulton, Glen B. Baker, and Mathew T. 35. Patch-Clamp Analysis: Advanced Techniques, Martin-Iverson, 1991 edited by Wolfgang Walz, Alan A. Boulton, 18. Animal Models in Psychiatry, I, edited by Alan and Glen B. Baker, 2002 A. Boulton, Glen B. Baker, and Mathew T. 34. Cell Neurobiology Techniques, edited by Martin-Iverson, 1991 Alan A. Boulton, Glen B. Baker, and Alan N. 17. Neuropsychology, edited by Alan A. Boulton, Bateson, 1999 Glen B. Baker, and Merrill Hiscock, 1990 33. Molecular Neurobiology Techniques, edited 16. Molecular Neurobiological Techniques, edited by by Alan A. Boulton, Glen B. Baker, and Alan Alan A. Boulton, Glen B. Baker, and Anthony T. N. Bateson, 1999 Campagnoni, 1990 32. In Vivo Neuromethods, edited by Alan A. 15. Neurophysiological Techniques: Applications to Boulton, Glen B. Baker, and Alan N. Bateson, Neural Systems, edited by Alan A. Boulton, 1998 Glen B. Baker, and Case H. Vanderwolf, 1990 31. G Protein Methods and Protocols: Role of G 14. Neurophysiological Techniques: Basic Methods Proteins in Psychiatric and Neurological Dis- and Concepts, edited by Alan A. Boulton, Glen orders, edited by Ram K. Mishra, Glen B. B. Baker, and Case H. Vanderwolf, 1990 Baker, and Alan A. Boulton, 1997 30. JRarne.,dgu1 Pl9ra9oto7torcyo Plsr,o etediinte Md obdyi Hficuagthio Cn.: HTeecmhmniiqnugess, 13. GPBsoryeuceltnhosonhp,a hwGa,rl1me9na8 c9Bo.l oBgayk, eerd, itaendd bAyn Adrlaewn AJ. 29. Apoptosis Techniques and Protocols, edited 12. Drugs as Tools in Neurotransmitter Research, by Judes Poirier, 1997 edited by Alan A. Boulton, Glen B. Baker, and 28. Transgenic Xenopus: Microinjection Methods Augusto V. Juorio, 1989 and Developmental Neurobiology, by Shlomo 11. Carbohydrates and Energy Metabolism, edited Seidman and Hermona Soreq, 1997 by Alan A. Boulton, Glen B. Baker, and Roger 27. Voltammetric Methods in Brain Systems, ed- F. Butterworth, 1989 ited by Alan A. Boulton, Glen B. Baker, and 10. Analysis of Psychiatric Drugs, edited by Alan Ralph N. Adams, 1995 A. Boulton, Glen B. Baker, and Ronald T. 26. Patch-Clamp Applications and Protocols, ed- Coutts, 1988 Witeodl fbgyanAgl aWn aAlz. ,B1o9u9lt5on, Glen B. Baker, and 9. AThlaen NAe. uBroounltaol nM, Giclreone Bn.v Biraoknemr,e annt,d eWdoitlefdga bnyg 25. Neurotrophic Factors, edited by Alan A. Walz, 1988 Boulton, Glen B. Baker, and Franz Hefti, 1993 8. Imaging and Correlative Physicochemical 24. A Alnainm aAl. MBodueltlos no,f GDlreung AB.d dBiacktieorn, ,a enddi tePde tbeyr TBe. cBhankieqru, easn, de dDitoenda bldy PA.l aBno iAsv. eBrto,u 1lt9o8n8, Glen Wu, 1992 7. Lipids and Related Compounds, edited by Alan 23. Practical Cell Culture Techniques, edited by A. Boulton, Glen B. Baker, and Lloyd A. Alan A. Boulton, Glen B. Baker, and Horrocks, 1988 Wolfgang Walz, 1992 6. Peptides, edited by Alan A. Boulton, Glen B. 22. Animal Models of Neurological Disease, II: Meta- Baker, and Quentin Pittman, 1987 BbyoultAitcel arEwn coAer.pt hBh,oa1ulo9ltp9oa2nth, iGesle ann Bd .t hBea kEepri,l eapnsdie Rs,o egdeirte Fd. 5. 1NB9oe8u6lrtootnra, nGsmleintt eBr. EBnazkyemre, sa, nedi tPedet beyr AHla. nY Au,. 21. Animal Models of Neurological Disease, I: Neurodegenerative Disease, edited by Alan 4. Receptor Binding Techniques, edited by Alan A. Boulton, Glen B. Baker, and Roger F. A. Boulton, Glen B. Baker, and Pavel D. Butterworth, 1992 Hrdina, 1986 NEUROMETHODS 䊏 35 Patch-Clamp Analysis ADVANCED TECHNIQUES Edited by Wolfgang Walz University of Saskatchewan, Saskatoon, Canada Alan A. Boulton Alviva Biopharmaceuticals Inc., Saskatoon, Canada Glen B. Baker University of Alberta, Edmonton, Canada HUMANA PRESS TOTOWA, NEW JERSEY © 2002 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. This publication is printed on acid-free paper. ' ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Kim Hoather-Potter. Cover design by Patricia F. Cleary. Cover illustration: From Fig. 2B in Chapter 4 “Combined Fluorometric and Electrophysiological Recordings,” by Hartmut Schmidt and Jens Eilers, and from Fig. 5C in Chapter 5 “Patch-Clamp Techniques Applied to Brain Slices,” by James R. Moyer, Jr. and Thomas H. Brown. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: Preface to the Series When the President of Humana Press first suggested that a series on methods in the neurosciences might be useful, one of us (AAB) was quite skeptical; only after discussions with GBB and some searching both of memory and library shelves did it seem that per- haps the publisher was right. Although some excellent methods books had recently appeared, notably in neuroanatomy, it was a fact that there was a dearth in this particular field, a fact attested to by the alacrity and enthusiasm with which most of the contribu- tors to this series accepted our invitations and suggested additional topics and areas. After a somewhat hesitant start, essentially in the neurochemistry section, the series has grown and will encompass neurochemistry, neuropsychiatry, neurology, neuropathology, neurogenetics, neuroethology, molecular neurobiology, animal models of nervous disease, and no doubt many more “neuros.” Although we have tried to include adequate methodological detail and in many cases detailed protocols, we have also tried to include wherever possible a short introductory review of the methods and/ or related substances, comparisons with other methods, and the relationship of the substances being analyzed to neurological and psychiatric disorders. Recognizing our own limitations, we have invited a guest editor to join with us on most volumes in order to ensure complete coverage of the field. These editors will add their specialized knowledge and competencies. We anticipate that this series will fill a gap; we can only hope that it will be filled appropri- ately and with the right amount of expertise with respect to each method, substance or group of substances, and area treated. Alan A. Boulton Glen B. Baker v Preface Neher and Sakmann were the first to monitor the opening and closing of single ion channels in the membranes of cells by con- ductance measurements. In 1976, they used firepolished micropipets with a tip diameter of 3–5 μm to record currents from a small patch of the membrane of skeletal muscles, thereby decreasing background membrane noise. In order to reduce the dominant source of background noise, the leakage shunt under the pipet rim between membrane and glass, the muscle membrane had to be treated enzymatically. Despite these early limitations, a new technique was born—the patch- clamp. The final breakthrough came in 1981 when the same workers, in collaboration with Hamill, Marty, and Sigworth, developed the gigaohm seal. Not only did this improve the quality of recordings, it was now possible to gently pull the membrane patch with the attached pipet off the cell and study its trapped ion channels in isolation. Another offshoot of the gigaohm seal technique was the whole-cell patch-clamp technique, in which the path is ruptured without breaking the seal. This technique is really a sophisticated voltage-clamp technique and also allows for the altering of cyto- plasmic constituents if the experimenter so wishes. The first part of this treatise on Patch-Clamp Analysis: Advanced Techniques presents modern developments associated with the basic patch-clamp techniques outlined above. These chapters are supple- mented with information on the newest developments in fast external solution switching to study fast inactivating responses as well as the switching of the pipet solution during recordings. The application of the patch pipet technique not only to clean mem- brane preparations, but also to brain or other tissue slices, was an important development in the last decade. Other offshoots of the patch pipet technique are the loose patch, the perforated patch, as well as the recording from macropatches and the patch-cram detection technique. These are all introduced and described in detail. Perhaps the recent developments in the patch-clamp field with the biggest impact are the combination of two of the most powerful life science technologies: molecular biology and imag- ing. This led to the intertwining of the patch pipet with RT-PCR and fluorometric techniques. vii viii Preface The methods associated with the patch pipet are certain to become even more refined in the future, as new applications involving genomics, proteomics, and sophisticated imaging tech- niques emerge. Wolfgang Walz Alan A. Boulton Glen B. Baker Contents Preface to the Series .............................................................................. v Preface ................................................................................................... vii Contributors .......................................................................................... xi 1. Technology of Patch-Clamp Electrodes Richard A. Levis and James L. Rae ................................................ 1 2. Whole-Cell Patch-Clamp Recordings Harald Sontheimer and Christopher B. Ransom ......................... 35 3. Single-Channel Recording David J. A. Wyllie ......................................................................... 69 4. Combined Fluorometric and Electrophysiological Recordings Hartmut Schmidt and Jens Eilers .............................................. 111 5. Patch-Clamp Techniques Applied to Brain Slices James R. Moyer, Jr. and Thomas H. Brown .............................. 135 6. Perforated Patch-Clamp Technique Raimondo D'Ambrosio ............................................................... 195 7. Fast-Drug Application Manfred Heckmann and Christian Pawlu ................................ 217 8. Pipet Perfusion Methods: Techniques and Applications Gea-Ny Tseng and Minoru Horie .............................................. 231 9. Patch-Cram Detection of Cyclic GMP in Intact Cells Richard H. Kramer...................................................................... 245 10. Loose-Patch Voltage-Clamp Technique Blake D. Anson and William M. Roberts .................................. 265 11. Recording from Macropatches Frances Moody-Corbett and Mark Fry ...................................... 287 12. Combining Patch-Clamp Techniques with RT-PCR Gerald Seifert, Albert Becker, and Christian Steinhäuser ........ 301 Index .................................................................................................... 331 ix Contributors BLAKE D. ANSON • Department of Medicine/Cardiology, University of Wisconsin, Madison, WI GLEN B. BAKER • Department of Psychiatry, University of Alberta, Edmonton, Canada ALBERT BECKER • Institute of Neuropathology, University of Bonn, Bonn, Germany ALAN A. BOULTON • Alviva Biopharmaceuticals Inc., Saskatoon, Canada THOMAS H. BROWN • Department of Psychology, Yale University, New Haven, CT RAIMONDO D'AMBROSIO • Department of Neurosurgery, University of Washington, Seattle, WA JENS EILERS • Department of Neurophysiology, Max-Planck-Institute for Brain Research, Frankfurt, Germany MARK FRY • Department of Physiology, Dartmouth Medical School, Hanover, NH MANFRED HECKMANN • Institut für Physiologie, Albert-Ludwigs- Universität, Freiburg, Germany MINORU HORIE • Department of Cardiovascular Medicine, Kyoto University, Kyoto, Japan RICHARD H. KRAMER • Department of Molecular and Cell Biology, University of California, Berkeley, CA RICHARD A. LEVIS • Department of Physiology, Rush Medical College, Chicago, IL FRANCES MOODY-CORBETT • Division of Basic Medical Sciences, Memorial University of Newfoundland, St. John's, Canada JAMES R. MOYER, JR. • Department of Psychology, Yale University, New Haven, CT CHRISTIAN PAWLU • Institut für Physiologie, Albert-Ludwigs-Universität, Freiburg, Germany JAMES L. RAE • Department of Physiology, Mayo Foundation, Rochester, MN CHRISTOPHER B. RANSOM • Department of Neurobiology, University of Alabama, Birmingham, AL WILLIAM M. ROBERTS • Institute of Neuroscience, University of Oregon, Eugene, OR xi