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Abel Lajtha (Ed.)
Handbook of
Neurochemistry and
Molecular Neurobiology
Practical Neurochemistry Methods
Volume Editors: Glen Baker, Susan Dunn, Andrew Holt
With 104 Figures and 28 Tables
651210150.001.png
Editor
Abel Lajtha
Director
Center for Neurochemistry
Nathan S. Kline Institute for Psychiatric Research
140 Old Orangeburg Road
Orangeburg
New York, 10962
USA
Volume Editors
Glenn Baker, Susan Dunn, Andrew Holt
Department of Pharmacology
University of Alberta
Edmonton, Alberta, T6G 2H7
Canada
Library of Congress Control Number: 2006922553
ISBN ‐ 13: 978 ‐ 0 ‐ 387 ‐ 30359 ‐ 8
Additionally, the whole set will be available upon completion under ISBN ‐ 13: 978 ‐ 0 ‐ 387 ‐ 35443 ‐ 9
The electronic version of the whole set will be available under ISBN ‐ 13: 978 ‐ 0 ‐ 387 ‐ 30426 ‐ 7
The print and electronic bundle of the whole set will be available under ISBN ‐ 13: 978 ‐ 0 ‐ 387 ‐ 35478 ‐ 1
2007 Springer Science þ Business Media, LLC.
All rights reserved. This work may not be translated or copied in whole or in part without the written permission
of the publisher (Springer Science þ Business Media, LLC., 233 Spring Street, New York, NY 10013, USA),
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
springer.com
Printed on acid ‐ free paper
SPIN: 11417699 2109 ‐ 543210
Preface
When preparing this volume we were well aware that it is not possible to cover all relevant neurochemical
techniques in a single book of this length, but we have done our best to gather a group of techniques that
will have wide general interest and are applicable to many analytical problems currently faced by neuro-
chemists and other neuroscientists. There are also some techniques, e.g., neuroimaging methods such as
magnetic resonance imaging/spectroscopy (MRI/MRS) and positron emission tomography (PET), which
are used extensively in the neurosciences but could take up an entire volume in their own right. These are
better covered in volumes of this handbook focusing on applications of such techniques. However, we feel
that the current volume covers some very interesting techniques and that the chapters, written as they are by
active investigators, provide useful details about theory, instrumentation, potential problems, applications,
and relevant references that will be of considerable interest to both novice and seasoned investigators.
The first chapter deals with gas chromatography. Although this technique has been replaced by high
performance liquid chromatography (HPLC) in many applications, it still represents a useful, relatively
inexpensive, and widely accessible technique, which can be applied to numerous neurochemicals, drugs,
and their metabolites. The topic of the second chapter is HPLC, which is now among the most frequently
used techniques in neurochemistry. In these first two chapters, there is information on basic principles,
columns, and detectors, and examples of applications to several neurochemicals and psychotropic drugs are
then discussed. Cytochrome P450 (CYP) enzymes are involved in metabolism of a wide variety of
endogenous neurochemicals and important drugs, and a comprehensive overview of these enzymes is
provided in Chapter 3. Although there are many review papers in the literature on CYP enzymes, most of
these do not focus on these enzymes in brain; this chapter should be a particularly valuable resource for
neuroscientists interested in CYP enzymes in the central nervous system. Many neurochemists carry out
studies on enzymes, but often their knowledge of enzymology is less than desired. Chapter 4 deals with
practical aspects of enzymology and enzyme kinetics. Chapter 5 provides an interesting overview of the
theory and applications of fluorescence techniques used to provide information about receptor–ligand
interactions. Mass spectrometry is a widely used technique, primarily because of its selectivity and large
number of neurochemical applications. Chapter 6 in this volume deals with basic principles and applica-
tions of mass spectrometry, while the following chapter discusses applications of mass spectrometric
techniques to neuroactive steroids, compounds that interact with several important receptors and seem
to be important in the etiology and pharmacotherapy of a number of psychiatric and neurologic disorders.
Immunochemistry has done much over many years to enhance our knowledge in the neurosciences, and
Chapter 8 focuses on immunohistochemistry, Western blotting, and ELISA assays, with many practical
suggestions provided.
In vivo microdialysis, discussed in Chapter 9, permits neurochemical sampling of extracellular fluid
from brain regions in living, freely moving organisms and has proved to be a popular technique in the
neurosciences. The chapter on this topic discusses many practical aspects of this technique and gives
examples of several diverse applications, including clinical ones. Radiolabeled ligand
‐
binding techniques
have been used for many years to investigate receptors and the drugs that interact with them. Chapter 10
provides information on theory, practical considerations, and limitations and gives some examples of
protocols. Receptor autoradiography, the topic of Chapter 11, permits anatomical localization and quanti-
fication of receptor density in discrete regions of the brain and other tissues. Both in vitro and in vivo
receptor autoradiography are discussed, and the applications section focuses on receptors for insulin ‐ like
growth factors. In silico molecular modeling, the topic of Chapter 12, is useful for studying proteins whose
 
vi
Preface
gated ion
channels. This chapter provides a comprehensive discussion of practical aspects of using the oocyte
expression system.
Polymerase chain reaction (PCR) amplification is a widely used technique for obtaining detectable
amounts of DNA or RNA that can be manipulated. Chapter 15 discusses different approaches to PCR and
gives examples of protocols and practical aspects to consider. The next chapter deals with in situ hybridiza-
tion histochemistry, a technique used widely in studies on genes to examine the spatial and temporal
distribution of their mRNA. Basic methodology and examples of standard applications and some more
novel applications are discussed. Differential display, the topic of Chapter 17, is a technique that employs
PCR and DNA sequencing to amplify, visualize, and identify differences in mRNA levels among cell types or
tissues. This chapter deals with advantages and limitations of the technique, experimental design, protocols,
and verification of differential display products. Chapter 18 deals with gene arrays, which permit simulta-
neous analysis of gene expression of multiple genes in a single sample. Although the chapter focuses on
applications to stroke studies, a great deal of practical information about general experimental design and
analysis and interpretation of data is provided. The yeast two ‐ hybrid system, a genetic ‐ based high through-
put technique for the study of protein–protein interactions, is covered in Chapter 19. Theory, practical
factors to be considered in its use and advantages and disadvantages of the technique are discussed. Two
protein engineering techniques, namely chimeragenesis (formation of chimera between proteins encoded
by homologous cDNAs) and site ‐ directed mutagenesis, are discussed in detail in Chapter 20. These
techniques have been used extensively, often in concert, to identify specific domains or amino acids that
confer unique properties to the protein under examination, and this chapter provides a great deal of useful
information about their applications. The use of cysteine ‐ scanning mutagenesis for mapping binding sites
of ligand ‐ gated ion channels is the topic of Chapter 21. In this technique, cysteines are introduced, one at a
time, into a protein region, and thiol ‐ specific reagents are subsequently applied to the mutant proteins to
determine if these cysteines are accessible to modification. The volume concludes with a chapter on the
theory and applications of protein X
‐
ray crystallography. As indicated by the author, this chapter is
intended to provide the readers with a basic understanding of what is required to conduct crystallographic
experiments and to permit them to determine if collaboration with a crystallographer might enhance their
research.
We hope that this volume will be of use to not only neurochemists but also to many neuroscientists
interested in the functioning of the nervous system and the effects of drugs on that functioning. We realize
that some relevant techniques may well have been omitted in the volume, and would appreciate feedback
from readers about techniques that should be included on the website and in future hard copies of this
volume in the Handbook of Neurochemistry and Molecular Neurobiology.
‐
Glen Baker, Susan Dunn, and Andy Holt
structures are not fully characterized. With this technique, homology modeling, i.e., using a related protein
whose structure is known as a template for model building, is employed. The chapter introduces basic
strategies used with homology models and in investigating the docking of ligands in the binding sites of
protein structures generated in silico; applications discussed in the chapter focus on G protein–coupled
receptors. Chapter 13 deals with flow cytometry, a technique, which uses fluorescence and light scatter to
examine biochemical and biophysical properties of cells in fluid suspension. The technique is used routinely
in many disciplines, including neuroscience, and this chapter has sections on principles and instrumenta-
tion, practical considerations, data acquisition and analysis, applications, and protocols. Oocytes from
Xenopus laevis , the topic of Chapter 14, represent a powerful system for studying the transient heterologous
expression of proteins and have been used widely for studying receptors associated with ligand
 
Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
K. A. Rittenbach . G. B. Baker
Performance Liquid Chromatographic Analysis of Psychotropic and
Endogenous Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
J. Odontiadis . G. Rauw
‐
3 Cytochrome P450 Reactions in the Human Brain . . . . . . . . . . . . . . . . . . . . . 43
R. L. Haining
4 Practical Enzymology
Quantifying Enzyme Activity and the Effects of Drugs Thereupon ....93
A. Holt
5 Fluorescence Measurements of Receptor–Ligand Interactions . . . . . . . . . . 133
S. M. J. Dunn
6 The Analyses of Neurotransmitters, Other Neuroactive Substances, and
Their Metabolites Using Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . 149
B. D. Sloley . G. Rauw . R. T. Coutts
7 The Analysis of Neuroactive Steroids by Mass Spectrometry . . . . . . . . . . . 177
R. H. Purdy . R. L. Fitzgerald . E. T. Everhart . S. H. Mellon . A. A. Alomary .
L. H. Parsons
8 Methods in Immunochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
L. L. Jantzie, V. A
‐
M. I. Tanay . K. G. Todd
9 In Vivo Microdialysis: A Method for Sampling Extracellular Fluid in
Discrete Brain Regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
D. L. Krebs-Kraft . K. J. Frantz . M. B. Parent
# Springer-Verlag Berlin Heidelberg 2007
2 High
 

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