Table Of ContentMethods in
Molecular Biology 1018
Renping Zhou
Lin Mei Editors
Neural
Development
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
Neural Development
Methods and Protocols
Edited by
Renping Zhou
Department of Chemical Biology, Susan Lehman Cullman Laboratory for Cancer Research,
Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA
Lin Mei
Institute of Molecular Medicine and Genetics, Georgia Regents University, Augusta, GA, USA
Editors
Renping Zhou Lin Mei
Department of Chemical Biology Institute of Molecular Medicine and Genetics
Susan Lehman Cullman Laboratory Georgia Regents University
for Cancer Research Augusta, GA, USA
Ernest Mario School of Pharmacy
Rutgers University
Piscataway, NJ, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-443-2 ISBN 978-1-62703-444-9 (eBook)
DOI 10.1007/978-1-62703-444-9
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013937290
© Springer Science+Business Media, LLC 2 013
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfi l ms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this
legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for
the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work.
Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the
Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions
for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution
under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of publication, neither
the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be
made. The publisher makes no warranty, express or implied, with respect to the material contained herein.
Printed on acid-free paper
Humana Press is a brand of Springer
Springer is part of Springer Science+Business Media ( w ww.springer.com )
Preface
The brain is a remarkable organ that coordinates all the activities necessary for survival. The
human brain consists of many different types of cells. There is an astronomically large num-
ber of neurons, 14 times (!) more than that of human beings on Earth. They are located in
strategic regions in the brain including different layers in the cortex and nuclei deep in the
brain. Each neuron could make 1,000 or more synapses with other neurons in the brain or
target cells in tissues or organs outside the brain. The contacts of a given neuron are esti-
mated to be more than the number of friends an average Facebook user has. Neurons per-
ceive signals from the environment, integrate them, and govern the body’s responses. In
between neurons are glial cells that are thought to represent 80 % of cells in the brain and
outnumber neurons by 10–50 times. They support neurons, insulate nerve tracks, and
regulate neural development and synaptic transmission.
How the myriad types of neurons and glia are generated and wired to form a functional
nervous system is the central question of developmental neurobiology. Neural inductive
activity was fi rst discovered by Spemann and colleagues in the 1920s, and the growth cone
and its chemotactic ability were proposed by Cajal in late 1900. Since these landmark dis-
coveries, research in developmental neuroscience has made tremendous progress. A family
of inhibitors for the bone morphogenic protein (BMP) pathway has been identifi ed as the
Spemann Organizer activity for neural induction. Transcription factor combinations that
determine different neuronal fate are being defi ned. With these advances, we are now on
the verge of generating neurons of specifi c types from many nonneural tissues, providing
hope that one day degenerated dopaminergic neurons in Parkinson’s disease or damaged
spinal cords can be repaired with the specifi c neurons generated from the skin fi broblasts of
the patients. Research in the last 20 years also identifi ed numerous proteins that regulate
directional migration of growth cones, and the guidance functions of these factors have
been con fi rmed in vivo in many cases. Current efforts are being directed toward elucidating
the intricate interactions among the guidance signals in the specifi cation of neural circuits
that control particular behavior. Understanding the molecular and cellular mechanisms
underlying the development of specifi c neural circuits is not just an intellectual curiosity but
also central to our ability to develop therapeutic approaches to repair damaged pathways in
the future.
Scientifi c progress depends in a large degree on the invention of new assays and the
deployment of new technology. Techniques and methods for current developmental studies
are likely to be used in the future. However, many have only been published in research
papers and often short of details because of the limited space of journals. We collect here
commonly used protocols to facilitate future research in developmental neuroscience.
Loosely re fl ecting the developmental sequence of the nervous system, this book consists of
4 parts. The fi rst part is on techniques of culturing neurons and glia as well as their growth
and differentiation. Modern neurobiological research depends critically on the ability to
manipulate expression of specifi c genes in neurons. Methods of gene delivery and down-
regulation are presented in the second part. The third part focuses on protocols for analyzing
v
vi Preface
axon growth and guidance and synapse formation. The fi nal fourth part includes basic
methods to analyze brain morphology and axon pathways in developing animals. The pro-
tocols presented here are written in enough detail to provide useful guidance for students
and postdoctoral fellows new to developmental neurobiology.
We are deeply indebted to the authors of the chapters in this book. We would also like
to thank the series editor, Dr. John M. Walker, and the Humana Press staff, especially David
Casey, for valuable suggestions and patience, and Ms. Erica DiPaola for editorial assistance.
Piscataway, NJ, USA Renping Zhou
Augusta, GA, USA Lin Mei
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I NEURON AND GLIAL CELL CULTURE
1 Isolation and Maintenance of Cortical Neural Progenitor Cells In Vitro . . . . . 3
Mi-Yeon Kim, Byoung-San Moon, and Kang-Yell Choi
2 Neural Induction with a Dopaminergic Phenotype
from Human Pluripotent Stem Cells Through a Feeder-Free Floating
Aggregation Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Asuka Morizane, Daisuke Doi, and Jun Takahashi
3 Nucleoside Analog Labeling of Neural Stem Cells and Their Progeny . . . . . . . 21
Erik Michael DeBoer and Mladen-Roko Rasin
4 Culture of Dissociated Hippocampal Neurons. . . . . . . . . . . . . . . . . . . . . . . . . 39
Yun Peng, Wen-Cheng Xiong, and Lin Mei
5 Culturing Mouse Cerebellar Granule Neurons . . . . . . . . . . . . . . . . . . . . . . . . 49
Tharakeswari Selvakumar and Daniel L. Kilpatrick
6 Preparation of Primary Cultured Dopaminergic Neurons
from Mouse Brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Won-Seok Choi, Hyung-Wook Kim, and Zhengui Xia
7 Culturing Astrocytes from Postnatal Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Yangyang Huang and Cheryl F. Dreyfus
8 Culturing Oligodendrocyte Lineage Cells from Neonatal Rats. . . . . . . . . . . . . 81
Clifton G. Fulmer and Cheryl F. Dreyfus
9 Isolation and Culture of Schwann Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Yanmei Tao
PART II GENE EXPRESSION AND ANALYSIS
10 DNA Transfection: Calcium Phosphate Method . . . . . . . . . . . . . . . . . . . . . . . 107
Munjin Kwon and Bonnie L. Firestein
11 The Gene-Gun Approach for Transfection and Labeling
of Cells in Brain Slices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Anna Dunaevsky
12 Lentiviral Vector Production, Titration, and Transduction
of Primary Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Baojin Ding and Daniel L. Kilpatrick
vii
viii Contents
13 In Ovo Electroporation in Embryonic Chick Spinal Cords . . . . . . . . . . . . . . . 133
Hui Wang and Michael P. Matise
14 Gene Transfer in Developing Chick Embryos: In Ovo Electroporation . . . . . . 141
Emma K. Farley
15 In Utero Electroporation in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Chunlei Wang and Lin Mei
16 General Introduction to In Situ Hybridization Protocol Using
Nonradioactively Labeled Probes to Detect mRNAs on Tissue Sections. . . . . . 165
Daehoon Lee, Shan Xiong, and Wen-Cheng Xiong
17 Immunofluorescence Staining with Frozen Mouse
or Chick Embryonic Tissue Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Hui Wang and Michael P. Matise
18 b-Galactosidase Staining of LacZ Fusion Proteins
in Whole Tissue Preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Margaret A. Cooper and Renping Zhou
19 Chromatin Immunoprecipitation Assay of Brain Tissues
Using Percoll Gradient-Purified Nuclei. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Baojin Ding and Daniel L. Kilpatrick
20 In Vivo Dual Luciferase Reporter Assay with Chick Neural Tube
In Ovo Electroporation System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Hui Wang and Michael P. Matise
PART III AXON PATHWAYS AND SYNAPSES
21 Growth Cone Collapse Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Xin Yue, Alexander I. Son, and Renping Zhou
22 The Stripe Assay: Studying Growth Preference and Axon Guidance
on Binary Choice Substrates In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Markus Weschenfelder, Franco Weth, Bernd Knöll, and Martin Bastmeyer
23 Microcontact Printing of Substrate-Bound Protein Patterns
for Cell and Tissue Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Martin Fritz and Martin Bastmeyer
24 Semiautomated Analysis of Dendrite Morphology in Cell Culture. . . . . . . . . . 261
Eric S. Sweet, Chris L. Langhammer, Melinda K. Kutzing,
and Bonnie L. Firestein
25 Monitoring Synaptic Plasticity by Imaging AMPA Receptor Content
and Dynamics on Dendritic Spines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Hiroshi Makino and Bo Li
26 Morphological Analysis of Neuromuscular Junctions
by Immunofluorescent Staining of Whole-Mount Mouse Diaphragms. . . . . . . 277
Haitao Wu and Lin Mei
Contents ix
PART IV MORPHOLOGICAL ANALYSES OF THE DEVELOPING NERVOUS SYSTEM
27 Routine Histology Techniques for the Developing
and Adult Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
W. Geoffrey McAuliffe
28 Cryosectioning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Alexander I. Son, Katie Sokolowski, and Renping Zhou
29 The Golgi–Cox Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Gitanjali Das, Kenneth Reuhl, and Renping Zhou
30 Neuroanatomical Tract-Tracing Using Biotinylated Dextran Amine. . . . . . . . . 323
Nikolai E. Lazarov
31 Retrograde Tracing Technique for Neonatal Animals. . . . . . . . . . . . . . . . . . . . 335
Kengo Funakoshi, Akira Yoshikawa, and Yoshitoshi Atobe
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
