Neurotrophin Protocols

272 Pages·2001·1.725 MB·English

by  James M. Conner (auth.), Robert A. Rush (eds.)

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Neurotrophin
Protocols
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M E T H O D S I N M O L E C U L A R B I O L O G YTM
N e u r o t r o p h i n
Protocols
Edited by
Robert A. Rush
Department of Human Physiology, Flinders University,
Adelaide, Australia
Humana Press Totowa, New Jersey
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iv
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Main entry under title: Methods in molecular biology™.
Neurotrophin protocols / edited by Robert A. Rush.
p. ;cm. —(Methods in molecular biology; v. 169)
Includes bibliogrpahical references and index.
ISBN 0-89603-699-5 (alk. paper)
1. Nerve growth factor—Laboratory manuals. I. Rush, Robert A. II. Series. III.
Methods in molecular biology (Totowa, N.J.); v. 169.
QP552.N36N4855 2000
573.8'4—dc21 00-044824
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Preface
The past decade has seen an extraordinary growth in research interest
in neurotrophic factors, and the study of the neurotrophin family has led this
activity. Nevertheless, this area of research has often struggled as a result of
techniques that were either inadequate or just emerging from other research
fields and disciplines. Neurotrophin Protocols has brought together many
leaders in the neurotrophin field who detail their special expertise in a wide
variety of techniques. Though most procedures are valid across many differ-
ent fields of research, some of those described here have been developed to
address particular issues within the neurotrophic factor field. The protocols
cover a broad range of biochemical, histological, and biological techniques
that are often required by the modern laboratory. However, all have been
written with sufficient detail to allow any laboratory to achieve proficiency
without need of reference to other texts.
Neurotrophin Protocols is divided into four sections dealing with pro-
tein, RNA, recombinant, and in vivo techniques. Protein techniques have in
general been less successfully employed than those dealing with RNA or DNA.
However, procedures that achieve localization and quantification of the
neurotrophins are now being used more extensively. Their inclusion here
should assist further studies at the protein level. Transgenic cell lines and
animals are commonplace in the scientific research literature, but their inclu-
sion in several chapters in this book provide some novel uses that are not
readily available elsewhere. Quantitative and histological methods for the
analysis of neurotrophin mRNA are also included. Although radiotracing tech-
niques have become less common, two useful but distinctly different proce-
dures have been included for specialist investigation of neurotrophin transport
in both the retrograde and anterograde directions. Other procedures include
the increasingly popular use of immunotoxins to study the effects of elimina-
tion of a single class of neurons and the essential stereological method for
estimation of neuronal numbers.
Because of this wide range of protocols and the extensive contributions
of the authors over many years, Neurotrophin Protocols should be of high
interest and utility to both postgraduate students and established investigators
alike.
Robert A. Rush
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Contents
Preface .............................................................................................................v
Contributors......................................................................................................x
PART 1 NEUROTROPHIN QUANTIFICATION AND LOCALIZATION
1 Localization of Neurotrophin Proteins Within the Central Nervous
System by Immmunohistochemistry
James M. Conner...................................................................................3
2 Neurotrophin Immunohistochemistry in Peripheral Tissues
Robert A. Rush and Xin-Fu Zhou.......................................................21
3 Extraction and Quantification of the Neurotrophins
Shu-Hua Zhang, Xin-Fu Zhou, and Robert A. Rush........................31
4 Identifying Novel Proteins in Nervous Tissue Using Microsequencing
Techniques
Selena E. Bartlett.................................................................................43
PART 2 RNA QUANTIFICATION AND LOCALIZATION
5 Quantification of mRNA Levels Using Northern Blotting
Paul Fernyhough.................................................................................53
6 Quantification of mRNA Levels Using Ribonuclease Protection Assay
Pirkko Henttu........................................................................................65
7 Quantification of Neurotrophin mRNA by RT-PCR
Edward Chie, Dennis Liu, Xin-Fu Zhou, and Robert A. Rush........81
8 Sensitive and Nonradioactive In Situ Detection of Neurotrophin
mRNAs in the Nervous System
Cory J. Xian and Xin-Fu Zhou............................................................91
PART 3 CONSTRUCTIONOF RECOMBINANT PROTEINS,AND TRANSGENIC CELLSAND ANIMALS
9 The Expression of Neurotrophic Factors such as GDNF in the
E. coli System
Bing-Ren Huang and Xue-Mei Ma....................................................101
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viii Contents
10 Construction of Cells Expressing Neurotrophins
Jasodhara Ray and Fred H. Gage....................................................115
11 Efficient Generation of Stable Pheochromocytoma (PC12) Cell
Lines Using a Recombinant Retrovirus (LNC)
Matthew E. Cunningham, Jan K. Kitajewski, and
Lloyd A. Greene.............................................................................135
12 Construction and Analysis of Transgenic Animals Overexpressing
Neurotrophins
Kathryn M. Albers and Brian M. Davis............................................149
13 Construction of Transgenic Animals Overproducing Neurotrophins
and Their Receptors
Carrie G. Causing and Freda D. Miller............................................167
PART 4 IN VIVO ANALYSIS
14 Tracing with Radiolabeled Neurotrophins
Christopher S. von Bartheld............................................................195
15 Toxin-Induced Death of Neurotrophin-Sensitive Neurons
Ronald G. Wiley.................................................................................217
16 Oligonucleotides as Inhibitors of Protein Synthesis
W. Ruwan Epa, Graham L. Barrett, and Perry F. Bartlett.............223
17 The IODO-GEN Method for Labeling and the Use of Retrograde
Axonal Transport to Assay Neurotrophins
Ian A. Hendry and Anna J. Reynolds..............................................243
18 Stereological Estimation of Neuronal Number:
The Optical Disector/Cavalieri Combination
Victor Nurcombe, Nigel G. Wreford, and John F. Bertram...........251
Index............................................................................................................269
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Contributors
KATHRYN M. ALBERS • Department of Pathology and Laboratory Medicine,
University of Kentucky College of Medicine, Lexington, KY
GRAHAM L. BARRETT • Department of Physiology and Walter and Eliza Hall
Institute of Medical Research, Royal Melbourne Hospital,
Melbourne, Australia
PERRY F. BARTLETT • Walter and Eliza Hall Institute of Medical Research,
Royal Melbourne Hospital, Melbourne, Australia
SELENA E. BARTLETT • Developmental Biology, Division of Neuroscience,
John Curtin School of Medical Research, Australian National University,
Canberra, Australia
JOHN F. BERTRAM • Department of Anatomy and Cell Biology, University of
Melbourne, Melbourne, Australia
CARRIE G. CAUSING • Center for Neuronal Survival, Montreal Neurological
Institute, McGill University, Montreal, Canada
EDWARD CHIE • Department of Physiology, Flinders University,
Adelaide, Australia
JAMES M. CONNER • Department of Neurosciences, University of California
San Diego, La Jolla, CA
MATTHEW E. CUNNINGHAM • Department of Pathology, Columbia University,
New York, NY
BRIAN M. DAVIS • Department of Anatomy and Neurobiology, University of
Kentucky College of Medicine, Lexington, KY
W. RUWAN EPA • Walter and Eliza Hall Institute of Medical Research, Royal
Melbourne Hospital, Melbourne, Australia
PAUL FERNYHOUGH • Department of Pharmacology, St. Bartholomew’s and
the Royal London School of Medicine and Dentistry, Queen Mary and
Westfield College, University of London, London, UK
FRED H. GAGE • Laboratory of Genetics, Salk Institute for Biological
Studies, La Jolla, CA
LLOYD A. GREENE • Department of Pathology, Columbia University,
New York, NY
IAN A. HENDRY • Developmental Neurobiology Group, Division of
Neuroscience, John Curtin School of Medical Research, Australian
National University, Canberra, Australia
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x Contributors
PIRKKO HENTTU • Department of Pharmacology, St. Bartholomew’s and the
Royal London School of Medicine and Dentistry, Queen Mary and
Westfield College, University of London, London, UK
BING-REN HUANG • Department of Biochemistry and Cell Biology, Institute
of Basic Medical Sciences, Beijing, China
JAN K. KITAJEWSKI • Department of Pathology, Columbia University,
New York, NY
DENNIS LIU • Department of Human Physiology, Flinders University,
Adelaide, Australia
XUE-MEI MA • Department of Biochemistry and Cell Biology, Institute of
Basic Medical Sciences, Beijing, China
FREDA D. MILLER • Center for Neuronal Survival, Montreal Neurological
Institute, McGill University, Montreal, Canada
VICTOR NURCOMBE • Department of Anatomical Sciences, University of
Queensland, St. Lucia, Australia
JASODHARA RAY • Laboratory of Genetics, Salk Institute for Biological
Studies, La Jolla, CA
ANNA J. REYNOLDS • Developmental Neurobiology Group, Division of
Neuroscience, John Curtin School of Medical Research,
Australian National University, Canberra, Australia
ROBERT A. RUSH • Department of Human Physiology and Centre of Neuroscience,
Flinders University, Adelaide, Australia
CHRISTOPHER S. VON BARTHELD • Department of Physiology and Cell Biology,
University of Nevada School of Medicine, Reno, NV
RONALD G. WILEY • Department of Neurology and Pharmacology,
Vanderbilt University and Neurology Service, Nashville, TN
NIGEL G. WREFORD • Department of Anatomy, Monash University,
Melbourne, Australia
CORY J. XIAN • Child Health Research Institute, Women’s and Children’s
Hospital, Adelaide, Australia
SHU-HUA ZHANG • Department of Human Physiology and Centre of Neuroscience,
Flinders University, Adelaide, Australia
XIN-FU ZHOU • Department of Human Physiology and Centre of Neuroscience,
Flinders University, Adelaide, Australia