Table Of ContentNEVILLE N. OSBORNE
Microchemical Analysis
of Nervous Tissue
PERGAMON PRESS
OXFORD • NEW YORK • TORONTO • SYDNEY
Pergamon Press Ltd., Headington Hill Hall, Oxford
Pergamon Press Inc., Maxwell House, Fairview Park, Elmsford,
New York 10523
Pergamon of Canada Ltd., 207 Queen's Quay West, Toronto 1
Pergamon Press (Aust.) Pty. Ltd., 19a Boundary Street,
Rushcutters Bay, N.S.W. 2011, Australia
Copyright © 1974 Pergamon Press Ltd.
All Rights Reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted, in any
form or by any means, electronic, mechanical, photocopying,
recording or otherwise, without the prior permission of
Pergamon Press Ltd.
First edition 1974
Library of Congress Cataloging in Publication Data
Osborne, Neville N.
Microchemical analysis of nervous tissue.
(Methods in life sciences, v. 1)
1. Brain chemistry. 2. Microchemistry. I Title.
[DNLM: 1. Microchemistry. 2. Neurochemistry. Wl
ME9615S / WL104 081]
QP356.3.082 1974 611'.0188 74-17043
ISBN 0-08-018100-7
TO JANE
Preface
IN the pioneer years of neurochemistry, homogenisation of whole brain and the chemical
analysis of its content was the technique generally employed. Many of the data obtained
in this way are to be found listed in different textbooks. Although this approach still offers
more possibilities, today the trend is towards a more detailed analysis. The availability of
more and more sensitive methods together with the rapid development in molecular
biology in the past years have also changed the neurochemical point of view. One of the
most fascinating aspects in modern science is to use brain-power for the objective analysis
of the molecular aspects of just this very brain-power.
It is generally accepted that a good method is the backbone of any experimental result.
It is often also thought that results obtained with micromethods are inaccurate and that
working with microprocedures is reserved for only a few really artistic experimenters.
This monograph, however, demonstrates clearly that these two prejudices are wrong. On
the contrary, micromethods need only be handled as carefully and skilfully as with normal
macromethods and the results will have the same distribution error. The time necessary
for learning a micromethod is also more or less the same as a normal experienced experi
menter needs to learn the correct handling of any new method. Furthermore, micromethods
often have the added advantage in that the performance of a technique in microscale is
much less time-consuming and often less expensive than the corresponding macro-
procedure.
It will probably be only a question of time till physiologists have mapped out to a great
extent all types of spikes from all the different brain areas and cell types, and yet there will
still be but little information on the biochemistry behind these spikes. Only the teamwork
between physiologists and neurochemists familiar with as many neurochemical micro-
methods as possible can assist in finding the necessary correlation between the physio
logical observations and the neurochemical background. The choice of the optimal
experimental approach is therefore extremely important. Starting with kilograms of brain
tissue and preparing their different subcellular particles is only meaningful when finding
out the common basic principles, but is meaningless for analysing a physiologically defined
function of a brain area or an identified neuron. This will be the domain of the neuro
chemical micromethods alone.
With regard to the molecular aspects of modern neurochemistry, the use of micro-
methods, including the isolation of defined brain areas and defined single cells or parts of
defined cells, and their subsequent biochemical analysis, is the most promising approach.
This is demonstrated in this monograph, which also includes a review of the literature of
neurochemical analysis performed with different micromethods. It is my firm conviction
that micromethods of the type described here or even more sophisticated and more sen
sitive methods which may be developed on requirement can help to bridge the gap between
the different hypotheses for explaining the complex function of the nervous system on a
molecular basis and the neurochemical facts which have been obtained so far.
V. NEUHOFF
xi
Acknowledgements
FIRST and foremost I should like to thank Professor G. A. Kerkut for suggesting I write
this monograph and for reading the manuscript. My sincere thanks are also due to Profes
sor V. Neuhoff, not only for writing the Preface and reading the manuscript, but also
for his support and encouragement throughout. Drs. H. H. Althaus, W. Dames, H.
Haljamáe, R. Rüchel and D. Wolfrum must be gratefully mentioned because of their
many helpful comments, suggestions and advice on various sections of the book, and
thanks also to Dr. T. W. Waehneldt for photographing a number of specimens, and to
Herr H. Ropte for enthusiastically and expertly producing the photographs and diagrams.
Further I wish to acknowledge the valuable assistance of other members of the neuro
chemical group, in particular Fráulein E. Priggemeier, and the secretarial aid of Frau I. von
Bischoffshausen and Frau B. Rosetz. Last, but by no means least, I thank my wife Jane,
not only for inspiring me to write the monograph, but also for spending many hours
typing and proofreading in the different stages of development.
xiii
CHAPTER 1
General Introduction
WHY a monograph on microprocedures in neurochemistry ? This is not difficult to justify
when one considers that the human brain has approximately 1010 nerve cells, while the
tiny brain of the ant (FormimJugubris) has about 100,000. These vast populations of neu
rons present a formidable challenge to the biologist trying to understand how the nervous
system works. From the mass of electrophysiological and electron microscopical data
which has accumulated it can now generally be concluded that nerve cells are independent
units (see e.g. Bullock, 1967; Bullock and Horridge, 1965; Eccles, 1964; Segundo, 1970;
Horridge, 1968). Furthermore, each neuron has several parts : (1) receptive loci specialized
in transducing the dozens of imputs which impinge on them in several ways ; (2) pacemaker
loci which inject spontaneous rhythms; (3) mixing and integrating loci; (4) threshold loci
for initiating all-or-none nerve impulses in bursts and trains from 1 to 1000 per second;
and (5) transmitter loci at each of the far ends of the nerve cell, where they influence up to
several dozen others. Clearly, biochemical information to be gained from classical studies
using relatively large amounts of nervous tissue (and therefore large numbers of cells
which may have very different properties) is of limited value. This problem is complicated
by the existence of vast numbers of glial cells which form a close and integrated association
with the neurons. Obviously the physiology, morphology, functional role and biochemistry
of individual neurons have to be studied and the neurons in the nervous system related to
one another before a real insight is gained into the intricate mechanism of the nervous
system. However, progress has been slow, basically for two main reasons. Firstly, the
majority of neurons are difficult to characterise and study as entities because of their small
size, and, secondly, there is a lack of suitable microprocedures which would permit the
study of different biochemical parameters in individual neurons.
One way of circumventing these difficulties is either to separate disaggregated nervous
tissue, thus obtaining populations of neurons and glial (Rose, 1968), or to fractionate
homogenates of nervous tissue and secure relatively pure fractions of a constituent part of
the different neurons, e.g. the nerve endings (Whittaker, 1973). Studies of this kind have
many advantages, but they, too, suffer from certain drawbacks, such as the possibility
that changes could occur in the constituents, caused by the elaborate separation or frac
tionation procedures employed; moreover, any differences there may be in the properties
of similar structures obtained from the brain cannot be observed. Another approach is to
analyse small defined areas of the nervous system or individual neurons where possible.
This presupposes the presence of suitable microchemical procedures. Perhaps a distinction
should be made here between macro- (normal), micro- and ultra-procedures, though one
might think that such a distinction is meaningless, since they represent a scale continuum.
1
2 Microchemical Analysis of Nervous Tissue
In theory they do ; however, in practice there is a change from macroscale (i.e. brain homo-
genates) to micro (i.e. one very large neuron, or microquantities of nervous tissue), and
over this range many macroprocedures can be modified and scaled down. The next step,
the ultra-microprocedure (parts of a single (20 //) minute nerve cell), is a 'quantum jump'
and often requires elaborate apparatus and new approaches.
The purpose of this monograph is to describe some microprocedures recently developed
in this laboratory. Special attention will be paid to the choice of biological material and
the various procedures used for the isolation by dissection of defined components of the
nervous system. The microbiochemical methods described will be those related especially
to the study of amines, amino acids, phospholipids and proteins. Many other extremely
sensitive microprocedures (plus-ultra-microprocedures) have been developed within the
last thirty years (see Chap. 5) and though their description is beyond the scope of the
monograph, a brief review of some of these methods and their applications is presented.
Perhaps it should be pointed out that emphasis is often laid only on the applicability of
microprocedures for studying small amounts of tissue, e.g. isolated cells, discrete areas of
brain, biopsy material, etc., whereas they also have othçr important virtues. Some micro-
methods, for example, are less time-consuming than normal procedures, and are for this
reason employed even when the material available is unlimited. Moreover, the cost of
analysing material by micromethods can often be very much less than that of similar
normal scale studies.
References
BULLOCK, T. H. (1967) Signals and neuronal coding. In: The Neurosciences: a Study Program (Eds.
G. C. Quarton, T. Melnechuk, and F. O. Schmitt), pp. 347-452. The Rockefeller University Press,
New York.
BULLOCK, T. H. and HORRIDGE, G. A. (1965) Structure and Function in the Nervous Systems of
Invertebrates. W. H. Freeman, San Francisco.
ECCLES, J. C. (1964) The Physiology of Synapses. Academic Press, New York.
HORRIDGE, G. A. (1968) Interneurons. W. H. Freeman, San Francisco.
ROSE, S. P. R. (1968) The biochemistry of neurones and glia. In: Applied Neurochemistry (Eds. A. N.
Davison and J. Dobbing), pp. 332-355.
SEGUNDO, J. P. (1970) Functional possibilities of nerve cells for communication and for coding.
Acta Neurol. Latinoamer. 14, 340-344.
WHITTAKER, V. P. (1973) The biochemistry of synaptic transmission. Naturwissenschaften, 60,281-289.
CHAPTER 2
Choice of Biological Material for Microanalysis
As previously mentioned, the fact that the mammalian brain contains a great number of
neurons presents a problem. The difficulty lies in the choice of appropriate experimental
objects. Ideally one needs a nervous system that produces a reasonably complex repertoire
of behaviour and has only a few cells, each of which can be recognised so that suitable
experiments can be carried out on them. In this respect certain invertebrate nervous sys
tems offer a number of advantages, in that they are organised in an orderly manner, have
fewer nerve cells than the vertebrates, have specialised giant neurons and can be individually
characterised. There are certain vertebrate preparations which do contain populations of
giant neurons, though they are difficult to characterise individually. Another important
advantage of the invertebrate neurons is that they can retain their functional activity after
dissection and survive for several hours or even days (see e.g. Strumwasser, 1967). This
therefore makes it possible to perform in vitro experiments on invertebrate nervous systems,
monitoring the activity of individual neurons by means of intra- or extracellular recording
while the environment of the cell can be controlled or changed by adding or substituting
ions, inhibitors, or drugs, etc. These and the many other advantages in using invertebrate
neurons in the analysis of individual cells are summarised in Table 1.
There is not only an enormous variety of invertebrate cell preparations (see Table 2),
but also of invertebrate preparations of giant synapses and giant axons (see Table 3) which
TABLE 1. ADVANTAGES OF INVERTEBRATE OVER VERTEBRATE CELL PREPARATIONS FOR MICRO-
CHEMICAL ANALYSIS
1. Invertebrate neurons can be easily dissected from the surrounding nervous tissue.
2. The neurons can overcome slight environmental changes which often prove disastrous for vertebrate
cells.
3. The cell bodies are generally larger (diameter of up to 1 mm in Aplysid), which means that more
material is available for biochemical analysis.
4. The same neuron is easy to identify in different preparations, reducing the variability of the system.
5. The neurons retain their functional activity after dissection for several hours or even days.
6. The functional activity of the neuron can be tested and altered by changing the environment of the
cell. The alterations in functional activity can then be related to the cell's biochemistry.
7. The study of individual identifiable neurons can be followed through from very young animals
weighing, for example, a few grams in the case of Aplysia, to adult animals of 700-800 g.
8. Glial material is easy to isolate, making it possible to study individual neuron-glial relationships.
9. Some invertebrates have also large axons and synapses, both of which can be isolated and analysed
for their chemical contents.
10. There are smaller numbers of neurons and simpler organisations of the different pathways in the
invertebrates to simplify the study.
3
4 Microchemical Analysis of Nervous Tissue
are suitable for biochemical analysis. In addition, glial cells can often be dissected from
invertebrates (see Kuffler and Nicholls, 1966) and analysed by biochemical procedures,
thus allowing the study of the relationship between specific glial and nerve cells. Despite
all these advantages, some scientists hesitate to compare the properties of neurons of the
vertebrate central nervous systems with those of the invertebrate nervous system. Physical,
biochemical and pharmacological differences do exist between vertebrate and invertebrate
neurons, though they seem to consist mainly of differences in pharmacological sensitivity
and some chemical characteristics rather than fundamental functional mechanisms and
the mode of response of the cells to transmitters and drugs. Although electrophysiological
properties of all neurons appear to be identical and in general comparable with those of
electrogenetic cells, there are detailed electrophysiological differences between invertebrate
TABLE 2. INVERTEBRATE AND VERTEBRATE CELL BODIES SUITABLE FOR
MlCROANAYLSIS
Cell body
Species Preparation diameter
Crustacea (invertebrate)
Astacus fluviatilis (crayfish) Stretch receptor 50- 80/x
(slow-adapting)
Astacus astacus (crayfish) Stretch receptor 50- 85 /x
(slow-adapting)
Homar us americanus (lobster) Stretch receptor 75- 120/x
(slow-adapting)
Insecta (invertebrate)
Periplaneta americana (cockroach) Thoracic ganglion 50- 120 /x
Mollusca (invertebrate)
Aplysia californica (sea slug) Visceral ganglion 400- 800 /x
Helix aspersa (snail) Visceral ganglion 40- 320 /x
Helix pomatia (snail) Visceral ganglion 60- 360 xi
Cerebral ganglion 40- 180/x
Parietal ganglion 260- 400/x
Tritonia gilberti (sea slug) Pleural ganglia 500- 800/x
Parietal ganglia 500-1000 ix
Loligo (squid) Pedal ganglion 150- 170/x
Octopus (octopus) Cerebral ganglion 20- 80/x
Annelida (invertebrate)
Lumbricus (earthworm) Ventral nerve cord 30- 60 ¡JL
Hirudo medicinalis (leech) Ventral nerve cord 30- 65 ¿i
Fishes (vertebrates)
Goldfish Mauthner's cells 30- 40/x
Puffer fish Supramedullary cells 200- 400 [x
Amphibians (vertebrate)
Frog Spinal ganglion ^- 20^
Sympathetic ganglion 10- 35 /x
Mammals (vertebrate)
Rabbit Deiters'cells 50- 100/x
Cortical cells 20- 40/x
Cells of nucleus supraopticus 30- 45 p
Spinal ganglion cells 60- 150 ¡JL
Anterior horn cells 20- 50/x
Granular cells of cerebellum 10- 20/x
Hippocampus cells 10- 30 ft
Cat a-Motoneurons of lumbar 40- 80 xt
spinal cord region
Choice of Biological Material for Microanalysis 5
and vertebrate nerve cells. Furthermore, the anatomy of invertebrate neurons differs in
many ways from that of the vertebrates (Cohen, 1970). It is for these reasons that the
biochemical analysis of mammalian cell preparations is of particular importance.
TABLE 3. INVERTEBRATE GIANT AXONS AND GIANT SYNAPSES SUITABLE
FOR MICROANALYSIS
Giant axon
of synapse
Species Preparations diameter
Crustacea
Homarus americanus (lobster) Inhibitory and motor axons 30- 50 ju
in leg
Carcinus meanus (crab) Inhibitory axon in leg 20- 30 ft
Procambarus ciarkii (crayfish) Giant axons in ventral nerve 100- 220 ft
cord
Cambarus (crayfish) Giant axons in ventral cord 150- 200 ft
Synapses in septal segment 150-240 ft
Insecta
Periplaneta americana (cockroach) Giant axons in ventral nerve 20- 45 ft
cord
Mollusca
Aplysia californica (sea slug) Axons in peripheral nerves 25- 50 ft
Loligo peatii (squid) Giant nerve fibre axon 500- 700 ft
Stellate ganglion, axon 50- 900 ft
Loligo forbesi (squid) Giant nerve fibre axon 700- 900 ft
Annelida
Lumbricus (earthworm) Giant axon in ventral nerve 75- 100 /x
cord
Synapses in septal segmental 150- 200 ft
axon
Myxicoly (marine worm) Giant axon in ventral nerve 100-1700 ft
cord
The autonomic ganglia, which comprise a few thousand nerve cells organised in physio
logical units, have proved suitable material for identifying neurons with different pharma
cological and biochemical properties. Single neurons isolated by microdissection and
subcellular fractions of the ganglia have been analysed by microprocedures so as to follow
changes involved in the process of synaptic plasticity (e.g. Giacobini, 1970). Populations
of large nerve cell bodies also exist in the following mammalian tissues : cerebellum,
hippocampus, anterior horn, spinal ganglion, nucleus supraopticus, cortex and Deiters'
nucleus (Table 2), and can be hand-dissected in excellent morphological condition (Fig. 1)
from pieces of nervous tissue. However, care must be taken in order not to damage the
chemical integrity of the neurons, since dyes (e.g. methylene blue) often have to be used
to help in the identification process and also because vertebrate neurons are exceedingly
susceptible to minute environmental changes. It may be worthwhile noting that the elegant
experiments of Hydén showing changes in brain protein during learning were carried out
on isolated hippocampus cells (Hydén, 1967; Hydén and Lange, 1970). In addition to
Hydén (1972), Lowry and Passoneau (1972) and Giacobini (1970) have all been pioneers
in the chemical analysis of isolated vertebrate neurons, and their work has contributed a
great deal to our understanding of a number of important aspects in neurobiology. The
