LIST OF TITLES

Already published
A Biochemical Approach to R. A. Freedland. S. Briggs
Nutrition
Biochemical Genetics (second R. A. Woods
edition)
Biological Energy Conservation C. Jones
(second edition)
Biomechanics R. McN. Alexander
Brain Biochemistry (second H. S. Bachelard
edition)
Cellular Degradative Processes R. T. Dean
Cellular Development D. R. Garrod
Cellular Recognition M. F. Greaves
Control of Enzyme Activity P. Cohen
Cytogenetics of Man and other A. McDermott
Animals
Differentiation of Cells M. Bownes
Enzyme Kinetics P. C. Engel
Functions of Biological Membranes M. Davies
Genetic Engineering: Cloning D. Glover
DNA
Hormone Action A. Malkinson
Human Evolution B. A. Wood
Human Genetics J. H. Edwards
Immunochemistry M. W. Steward
Insect Biochemistry H. H. Rees
Isoenzymes C. C. Rider. C. B. Taylor
Metabolic Regulation R. Denton. C. I. Pogson
Metals in Biochemistry P. M. Harrison. R. Hoare
Molecular Virology T. H. Pennington. D. A. Ritchie
Motility of Living Cells P. Cappuccinelli
Plant Cytogenetics D. M. Moore
Polysaccharide Shapes D. A. Rees
Population Genetics L. M. Cook
Protein Biosynthesis A. E. Smith
RNA Biosynthesis R. H. Burdon
The Selectivity of Drugs A. Albert
Transport Phenomena in Plants D. A. Baker

In preparation
Bacterial Taxonomy D. Jones. M. Goodfellow
Biochemical Systematics J. B. Harborne
The Cell Cycle S. Shall
Gene Structure and Function M. Szekely
Invertebrate Nervous Systems G. Lunt
Membrane Assembly J. Haslam
Editor's Foreword

The student of biological science in his final years as an undergraduate

and his first years as a graduate is expected to gain some familiarity
with current research at the frontiers of his discipline. New research
work is published in a perplexing diversity of publications and is
inevitably concerned with the minutiae of the subject. The sheer
number of research journals and papers also causes confusion and
difficulties of assimilation. Review articles usually presuppose a back-
ground knowledge of the field and are inevitably rather restricted in
scope. There is thus a need for short but authoritative introductions
to those areas of modern biological research which are either not dealt
with in standard introductory textbooks or are not dealt with in suffi-
cient detail to enable the student to go on from them to read scholarly
reviews with profit. This series of books is designed to satisfy this need.
The authors have been asked to produce a brief outline of their subject
assuming that their readers will have read and remembered much of
a standard introductory textbook on biology. This outline then sets
out to provide by building on this basis, the conceptual framework
within which modern research work is progressing and aims to give
the reader an indication of the problems, both conceptual and practical,
which must be overcome if progress is to be maintained. We hope that
students will go on to read the more detailed reviews and articles to
which reference is made with a greater insight and understanding of
how they fit into the overall scheme of modern research effort and may
thus be helped to choose where to make their own contribution to this
effort. These books are guidebooks, not textbooks. Modern research
pays scant regard for the academic divisions into which biological
teaching and introductory textbooks must, to a certain extent, be
divided. We have thus concentrated in this series on providing guides
to those areas which fall between, or which involve, several different
academic disciplines. It is here that the gap between the textbook and
the research paper is widest and where the need for guidance is greatest.
In so doing we hope to have extended or supplemented but not sup-
planted main texts, and to have given students assistance in seeing
how modern biological research is progressing, while at the same time
providing a foundation for self help in the achievement of successful
examination results

General Editors:
W.1. Brammar, Professor of Biochemistry,
University of Leicester, UK
M. Edidin, Professor of Biology,
Johns Hopkins University, Baltimore, USA
Brain Biochemistry

H. S. Bachelard
Professor of Biochemistry,
St. Thomas's Hospital Medical School,
London

Second edition

Chapman and Hall

London and New York
First published in 1974
Reprinted in 1976
Second edition published in 1981 by
Chapman and Hall Ltd
11 New Fetter Lane, London EC4P 4EE
Published in the USA by
Chapman and Hall
in association with Methuen, Inc.
733 Third Avenue, New York, NY 10017
© 1974, 1981 H. S. Bachelard

ISBN-13: 978-0-412-23470-5

This paperback edition is sold subject to the condition that it shall not,
by way of trade or otherwise, be lent, re-sold, hired out, or otherwise
circulated without the publisher's prior consent in any form of binding
or cover other than that in which it is published and without a similar
condition including this condition being imposed on the subsequent
purchaser.
All rights reserved. No part of this book may be reprinted, or re-
produced or utilized in any form or by any electronic, mechanical or
other means. now known or hereafter invented, including photo-
copying and recording, or in any information storage and retrieval
system, without permission in writing from the Publisher.

British Library Cataloguing in Publication Data

Bachelard, Herman Stanton

Brain biochemistry. - 2nd ed. - (Outline studies in
biology).
1. Brain chemistry
I. Title II. Series
599 .01 '88 QP376

ISBN-13: 978-0-412-23470-5 e-ISBN-13:978-94-009-5941-5

DOl: 10.1007/978-94-009-5941-5
Contents

I Introduction 7
1.1 Regional cerebral metabolism 8
1.2 Cerebral requirements for glucose and oxygen 9
References 10

2 Appearance of the brain 11

2.1 Gross appearance II
2.2 Fluid compartments 13
2.3 Microscopic appearance 14
2.3.1 Neurones 15
2.3.2 Glial cells 16
2.3.3 The synapse 17
References 21

3 Neurotransmission 21
3.1 The resting potential 21
3.2 The sodium pump 23
3.3 The action potential and nerve conduction 24
3.4 Chemical events at the synapse 26
3.4.1 Identification and occurrence of neurotransmitters 28
3.4.2 The quantum hypothesis 32
3.4.3 Metabolism of acetylcholine 34
3.4.4 Catecholamines: noradrenaline and dopamine 37
3.4.5 5-Hydroxytryptamine 38
3.4.6 Breakdown of the biogenic amines 39
3.4.7 Metabolism of the neuroactive amino acids 40
3.4.8 The neuroactive peptides 40
3.5 Origin of synaptic vesicles 41
3.6 Post-synaptic events 42
3.6.1 Involvement of cyclic nucleotides 44
3.6.2 Receptors 46
3.7 Neurone-axonal transport 48
3.7.1 Mechanism of transport in axoplasmic flow 49
3.7.2 Axonal protein synthesis 50
References 51

4 Adaptive processes in the brain 54

4.1 Inducible enzymes 54
4.1.1 Adaptation to specific substrates 56
4.1.2 Adaptation to the product of an alternate pathway 61
 4.1.3 Adaptation involving coenzyme 62
4.1.4 Adaptation in response to hormones 63
4.2 Adaptation to the environment 65
4.2.1 Light 65
4.2.2 The pineal gland 66
4.3 Drug tolerance and dependence 68
4.3.1 Morphine 69
4.3.2 Amphetamines 71
4.3.3 Ethanol 74
4.4 Learning and memory as adaptive processes? 75
References 76
Index 79
1 Introduction

The brain is the most complex and highly specialised of all mammalian
organs. Understanding the complexity of its function remains man's
greatest challenge. The functional unit is the neurone, or excitable
nerve cell, making anatomical and chemical connections with other
units in the system. Many of the essential biochemical connections of
the nerve cell are dependent upon special morphological features:
synaptic contact is mediated by chemical molecules, 'neuro-trans-
mitters' which ensure the continued propagation of electrical impulses
through sequential units of the system. Also closely related to the
morphology of the nervous system is the chemical energy expended in
maintaining distribution gradients of cations across cellular mem-
branes. Chemical neurotransmission results in an alteration in cation
distribution and while the energy-utilising mechanisms which underly
their redistribution are not peculiar to the nervous system, they are of
particular importance to neural function. The mechanisms of chemical
transmission, in contrast, are peculiar to the nervous system.
Nerve cells are unique in their ability to trigger off and maintain
conduction of electrical impulses over long distances, which may
be measured in metres, without significant loss of strength of the
conducted impulse. Remarkable also is the specificity of their con-
nections, not only with other nerve cells, but also with non-neural
target cells in sites such as the endocrine glands or muscles.
These unique features rest in the possession of semi-permeable
excitable membranes which can be caused, rapidly and transiently, to
undergo changes in permeability to small chemical molecules and to
cations. The highly specialised nature of the constituent cells, with their
unique function and specificity, is closely related to the structure of
the whole tissue. The underlying chemical processes cannot be dis-
cussed or seen in perspective without constant awareness of related
aspects of physiology and morphology. The brain is structurally extra-
ordinarily complex in its distinct anatomical regions, each of which
is heterogeneous in the types and structures of the constituent cells.
One aspect of the biochemical function of the brain can be seen in its
efficient production of the energy required to support the unique
processes referred to above. This energy, essentially stored as ATP,
is produced from the oxidation of glucose by mechanisms common to
all biological cells. The importance in the brain of these processes is
quantitative, rather than qualitative. The brain depends absolutely
for its ability to function normally on a constant supply of glucose
and oxygen from the blood stream. It has virtually no reserves of
7
chemical energy, compared with other tissues and organs. Stored
concentrations of glucose and glycogen (each of the order
of 1-2 Ilmoles/g) and of ATP (3 Ilmoles/g) are sufficient to maintain
function in isolation for minutes only, if permanent damage is not to
ensue and under normal circumstances, the brain cannot utilise
alternative sources for its energy requirements [1]. The importance
of the constant blood supply of essential nutrients can be readily
appreciated if we remember that this organ, only some 3% of the total
adult body weight, consumes some 20% of the glucose required by
the whole body. This supply is in fact supported by the blood: one-fifth
of the output of the heart passes through the brain. The brain is there-
fore the most sensitive part of the body to failure in oxygen or glucose.
In the absence of either of these, fainting occurs within seconds,
and if not corrected, coma and death follow rapidly. It is usually the
first organ to suffer. Its peculiar sensitivity to abnormalities in energy
metabolism can also be seen in the features of vitamin deficiency,
especially of those vitamins such as the B group which function as
coenzymes in intermediary energy metabolism. Although any defi-
ciency affects the same metabolic pathways in the same way through-
out the body, one of the most profound consequences is impaired
mental function and in children, often mental retardation. It must be
stressed that this is due, not to specialised qualitative metabolism
by the brain, but to its very high sensitivity to any impairment in the
normal processes of energy production.
This is of particular importance in the nutrition of the underdeve-
loped 'third world', where deficiency or dietary imbalance may cause
irreparable mental damage to the developing child, and which has
been the concern of special symposia [2, 3]. Not only is an inadequate
environment increasingly suspected of leading to impaired intelligence
in the poorer parts of the world, but evidence is also to hand that this
can be seen in countries normally regarded as rich and developed.
Although current discussions on the relative influences of heredity
and of environment on the development of intelligence are heated
and controversial, studies such as those on Scottish children over a
15 year period indicate that a consistent if small increase in intelligence
can result from progressive improvement in their environment [4].
Further indications of the sensitivity of the brain to general metabolic
impairment arise from the high proportion of inherited metabolic
disorders which result in mental disturbance or retardation so im-
portant in Neurology and Psychiatry [5];

1.1 Regional cerebral metabolism

The great dependence of the brain on its supplies of glucose and oxygen,
noted above, was originally assumed with good reason to be associated
solely with energy production, yet early pioneering studies on the rates
at which the normal adult brain uses these nutrients showed no diffe-
rence with function. That is, the brain seemed to require the same
energy whether the subject was responding to sensory stimuli, was
8
thinking intensively or resting [6]. These studies were based on arterio-
venous differences over the whole brain, and regional variations could
not be assessed. Since then, measurements of regional variations in
rates of cerebral blood flow have shown changes in specific regions in
response to sensory stimuli or mental effort [7]. However this does
not tell us if metabolic rates are changing in a similar manner with
function: in epilepsy or induced fits in animals, overall rates of cerebral
consumption of glucose and oxygen increase to a considerably greater
extent than does the blood flow rate.
Some evidence for regional rates of glucose utilization in experimen-
tal animals is emerging from use of the autoradiographic 2-deoxy-
glucose technique [8], based on knowledge of the transport and
metabolism of deoxyglucose in the brain [9]. While this provides
much information about regional variations in metabolism with
function in animals, it is subject to limitations in the types of function
that can be studied. Furthermore it cannot be applied directly to
studies in man, requiring as it does removal of the brain for autoradio-
graphy. The method has now been adapted for use in man. The 18F_
isotope emits positrons which penetrate the skull, so its localization
within the brain can be monitored externally. The attachment of
18F to the No.2 position of the deoxyglucose molecule is thought
not to affect its metabolism and it is being used, by positron emission
computed tomography. to study regional cerebral glucose metabolism
in conscious normal man [10]. It too has one essential limitation
in that the isotope has a very short half-life, and a cyclotron is required
to produce it in the vicinity of the research or clinical laboratory.
Its expense renders it unlikely to come into general use, but so much
promise in research and diagnosis is offered that one can foresee
extensive use in selected specialist centres.

1.2 Cerebral requirements for glucose and oxygen

A direct relation between function and the requirements of glucose
and oxygen for energy production is also being questioned in view of
recent observations in man and experimental animals that mild
hypoglycaemia and hypoxia show changes in the EEG and in behaviour
without any perceptible energy deficit. This poses two possibilities: 1)
that small regional variations in energy production are escaping
detection; 2) that the brain is responding to changed availability of
these nutrients in a protective homeostatic manner. The first possibility
is regarded as an unlikely answer in itself (though it may contribute)
because of the extraordinary rapidity with which an experimental
animal can be aroused from deep hypoglycaemic coma by giving
glucose: the metabolic machinery is thought not to be able to respond
so fast. The second possibility is receiving much attention. This
puzzling apparent anomaly is seen in a general sense if various aspects
of energy metabolism and synaptic function are compared. We know
that maintenance of synaptic function depends to a great extent on
the cation pumping processes which require energy (Chapter 2).
9
Yet in addition to the conditions of hypoglycaemia and hypoxia
noted above, treatment with various centrally active drugs (anaes-
thetics, depressants and excitants) may also cause changes in the
EEG and in behaviour, and in synaptic function, without detectable
change in the energy state [11, 12]. This has now been confirmed
in vitro: electrical activity evoked from slices of hippocampus in-
cubated in vitro can be affected markedly by slightly lower concen-
trations of glucose (2mM) which seem to have no effect on the inter-
mediary metabolism of the tissue [13]. No clear pointers on the basis for
this have emerged so far. but it may have important clinical significance
in our understanding of such conditions as coma, stroke. epilepsy
and also perhaps. dementia.
To be able to understand what he is trying to achieve, the bio-
chemist who studies brain function must acquaint himself with related
aspects of morphology, physiology and pharmacology; the chemical
function of the brain cannot be separated from the architectural
integrity of the cellular relationships. For a small book of this type
the topics selected concentrate on the chemical events related to excit-
ability and transmission and to the adaptability of the brain to react
to various stimuli both from within and without the body. To this end,
a brief description of the associated morphology and physiology
seems an essential requirement and is treated first. Then follows a
description of membrane permeability phenomena and neurotrans-
mission. The final section of the book is concerned with aspects of
the chemical response of the brain to its immediate environment and
as a result of some of the hormonal signals reaching the brain.

References
[I] McIlwain, H. and Bachelard, H. S. (1971), Biochemistry and the Central
Nervous System (4th ed.), Churchill, London.
[2] CIBA Foundation Symposium (1972), Lipids, Malnutrition and the Develop-
ing Brain, Elsevier, Amsterdam.
[3] DiBenedetta, c., Balazs, R., Gombos, G. and Porcellati, G. (eds) (1980),
Multidisciplinary Approach to Brain Development, Elsevier, Amsterdam.
[4] Scottish Council for Research in Education (1949), The Trend of Scottish
Intelligence,London University Press, London.
[5] Davison. A. N. (ed.) (1976), Biochemistry and Neurological Disease, Black-
wells. London.
[6] Kety. S. S.. Woodford. R. B., Harmel. M. H .. Freyhan. F. A .. Appel. K. E.
and Schmidt, C. F. (1948), 'Cerebral blood flow and metabolism in
schizophrenia: effects of barbiturate semi-narcosis, insulin-coma and
electroshock', Am. J. Psychiat., 104, 765-770.
[7] Lassen, N. A. and Ingvar, D. H. (1972), 'Radio-isotopic assessment of
regional cerebral blood flow', Progress in Nuclear Medicine 1, 376-409.
[8] Sokoloff, L., Reivich, M., Kennedy, c., De Rosiers. M. H., Patlak, C. S.,
Pettigrew, K. D., Sakurada, O. and Shinohara, M. (1977), 'The C4 C)-
deoxyglucose method for the measurement of local cerebral glucose
utilization: theory, procedure, and normal values in the conscious and
anesthetized albino rat', J. Neurochem., 28, 897-916.
10
 [9] Horton, R. W., Meldrum, B. S. and Bachelard, H. S. (1973) 'Enzymic and
cerebral metabolic effects of 2-deoxY-D-glucose', J. Neurochem., 21,
507-520.
[10] Reivich, M., Kuhl, D. and Wolf, A. (1977), 'Measurement of local cerebral
glucose metabolism in man with 18F-2-fluoro-2-deoxY-D-glucose', Acta
Neurol. Scand., 56 (Suppl. 64), 188-190.
[11] Bachelard, H. S. (1981), 'Cerebral Metabolism and Hypoglycaemia', In:
Hypog/ycaemia (ed. Marks, V. and Rose, F.) Blackwells, Oxford.
[12] Siesjo, B. K. (1978), Brain Energy Metabolism, Wiley, Chichester.
[13] Cox, D. W. G. and Bachelard, H. S. (1980), The effect oflowered glucose on
field potentials evoked from the hippocampal slice in vitro', Neuroscience
Letts., Suppl. 5, S448.

2 Appearance of the brain

2.1 Gross appearance

Biochemists tend to study the brains of small mammals and consciously
or subconsciously extrapolate to what might occur in the human brain,
itself subject to obvious limitations in opportunities for chemical
exploration. Yet what do we mean by the 'mammalian' brain, since
the brain of a rat or guinea pig is obviously far different in appearance
and many functions from that of Man? The brain has evolved and
specialised within mammalian species more than any other organ of
the body: Fig. 2.1 shows a comparison of the brains of a selected
group of mammals and it should be remembered that increasing
size is not necessarily associated with increased intelligence or sophisti-
cation of function. The main discernible change that has occurred
during evolution of the mammalian brain is in the size and complexity
of the cerebral cortex. The increase in surface area per unit of volume
of the cortex has been effected by increased folding so that the con-
volutions of the human cerebral cortex are considerably more extensive
than of the rat or rabbit. The function of the cortex has altered also:
the 'primary cortex' concerned with sensorimotor function (Fig. 2.1)
has remained proportionately much the same, but the areas devoted
to 'association', i.e. areas concerned with higher functions of learning
and decision-taking, have increased considerably [1].
Other areas, such as the limbic system (Fig. 2.1), concerned with
more primitive functions of homeostasis, motivation and especially
emotion [2], are phylogenetically older, and have changed little in
relative size, as the shaded areas show [3, 4]. For the non-anatomically
trained, the nomenclature of the regions and specialised parts of an
organ as complex as the brain is daunting. In fact the brain should
11
 Rat (11) Cat (30)

(a) Man (86) Rabbit (22) Monkey (64)

Rat (2 .5) Cat (30)

(b) Man (1350) Rabbit (14)

Fig. 2.1 Comparison of some functional areas of the brains of various mammals.
(a) Proportions of sensorimotor and association (shaded) areas. Values in parenthesis
are percentages of association cortex. (b) Schematic representation of the relative
areas of the limbic system. Values in parenthesis are the weights of the brains in
grams. The drawings are approximately half-size (linear).

really be regarded as a collection of highly specialised organs rather

than as a single organ. Often various areas of the brain are cited
in biochemical articles as experimental material and the untutored
reader may be uncertain of the site and significance of the part named.
The major areas of the human brain which are likely to be referred to
are shown in Fig. 2.2 (see also Table 3.2 of Chapter 3). The first part
of the illustration (a) shows the lateral aspect of the whole brain,
viewed from the left hand side: the next drawing (b) shows some of
the internal parts which are seen if the brain is divided into the two
hemispheres. This is the right hemisphere viewed from the left hand
side. The whole brain has been divided into four main parts for con-
venience: the cerebrum, the cerebellum, the mid-brain and the brain
stem, and it is the latter which contains a large number of specialised
parts indicated in Fig. 2.2. The third illustration (c) shows the interior
of the brain cut horizontally and viewed from above.
12
 , ]

[Movement] Parietal lobe

Frontal
Occipital
lobe

01 factory bulb [ Vision I

[Smell)

Fig. 2.2 Drawings of the human brain, (a) View from the left side, showing major
areas with some indication of function,

2.2 Fluid compartments

One prominent feature of the gross anatomy of the brain is its ex-
tensive blood supply-perhaps not surprisingly, since it uses about
one-fifth of the total blood used in the body, The blood volume of
the brain is only about 3% of the total brain volume and the efficiency
of the supply is ensured by the extensive ramified system of capillaries,
Exchange of solutes between the various fluid compartments (blood,
cerebrospinal fluid and the extracellular tissue space) and the cells
themselves, exhibits features not always found in other parts of the
body. The most studied of these features is the 'blood-brain barrier',
Originally this concept arose from the limited penetration of injected
dyestuffs from the bloodstream to the brain substance, which was
also found to occur with a variety of small highly water-soluble
chemicals of a wide range of classes: sugars such as fructose and
sucrose, and charged molecules such as thiocyanate and most amino
acids. Microscopic examination of the endothelial cells of the walls of
the blood capillaries of the brain indicated that they were packed
more tightly together than in capillaries outside the brain, so there
seemed to be a sound basis for a physical permeability barrier at the
capillary wall, This suffices for relatively large molecules like proteins
but is inadequate to explain the apparent limited permeability of
substances such as glutamate. Indeed if radioactive glutamate is
present in the blood-stream it equilibrates rapidly with the glutamate
within the brain, to judge from the extent of its labelling, but a massive
increase in concentration of the external glutamate does not materially
change its internal concentration. For substances like glutamate,
and many others (including precursors of amines with specific neural
function, Chapter 3), the 'blood-brain barrier' can be regarded as a
homeostatic mechanism whereby the internal concentration is main-
tained by active processes of extrusion [5].
13
 Corpus callosum Forn ix

~\..er,eDra cortex

_wn,,'A maner
Septum lucidum

_ _-.Jf.?:r - Pineal gland

Thalamus ------l~+.4----!

Hypothalamus _ _ -'\B@irtffB%~~J;

Pituitary
4th ventricle

Spinal cord

Fig. 2.2 Drawings of the human brain. (b) Cut-away view as in (a) showing some of
the internal features.

The peculiarities of the 'blood-brain barrier', which is believed

to fulfil a protective role for the highly sensitive brain, can therefore
impede potential treatment of brain disorders by rendering difficult
the internal accumulation of drugs or metabolites. Such a difficulty
might be overcome by a knowledge of the biochemistry of the system
at fault. Thus in Parkinson's disease, a degenerative and progressive
disorder associated with muscular tremor and akinesia, anatomical
observation showed degeneration of certain nerve tracts and histo-
chemical analysis revealed a parallel loss of dopamine (see Chapter
3). Dopamine (3, 4-dihydroxyphenylethylamine) is one of those
substances which are not easily transported into the brain, but its
immediate metabolic precursor, Dopa (dihydroxyphenylalanine),
is readily taken up. Marked improvement in many patients has been
achieved by treatment with Dopa.

2.3 Microscopic appearance

The first appreciation of the morphology and cytology of nervous
tissues, the network of individual cells and their processes, came to-
wards the end of the nineteenth century with the application of the
improved light microscope and the development of new staining
methods. One of these, based on silver salts, causes selective staining
of neuronal cell bodies and their processes in thin sections with such
clarity that the structures seem to stand out in almost a three-dimen-
sional picture. This is the Golgi stain. An alternative method, the
14
 Caudate nucleus

Corpus callosum
Cerebral cortex

White matter Lateral ventricle

(anterior horn)

Septum
lucidum--"""f:!jS , -_ _
L-'-:~-=m-- Fornix

Hippocamp Rt-e~r---4--Thalamus

3rd ventricle
Pineal alarlO- - -

Superior
colliculus

Cerebellum
Fig. 2.2 Drawings of the human brain. (c) Cut-away view from above, with the front
of the brain to the top of the drawing.
Nissl stain, shows the cell bodies of neurones and glia but not their
processes. The observations, made using the light microscope, of
cell bodies, axons, dendrites and dendritic spines (see below), were
sufficient to lead to the 'neuronal hypothesis' with the concept of
synaptic junctions some seventy-five years ago [6]. Further insight
into fine structure, especially of the synapses, and confirmation of
the neuronal hypothesis, had to wait until the advent of the electron
microscope some forty years ago.
Light microscopy had clearly demonstrated the occurrence of a
variety of cell types, classified into two main groups, neurones (the
excitable nerve cells) and glial cells (non-excitable). Within each
group, different types have been discerned.

2.3.1 Neurones
These (Fig. 2.3) may have large or small cell bodies (perikarya) but
all are characterised in possessing a large nucleus containing a promi-
nent nucleolus, a high content of ribosomes in the cytoplasm (either
free or attached to an extensive endoplasmic reticulum) and a high
content of mitochondria. Such features are compatible with active
synthetic and secretory activities and the large capacity for energy
production referred to in Chapter I. Essential characteristics are the
prominent processes which fonn extensions of the outer cell membrane :
15
axons and dendrites. Axons are usually long, relatively thin, and emerge
from a swelling in the cell body - the axon hillock. The axons are
sometimes branched and usually, but not always, covered by an
insulating sheath, the myelin sheath, consisting of a spiral (giving
the impression of concentric rings in cross section) of membranes.
Myelinated axons form the main routes for the efficient rapid con-
duction of the electric impulse from the neurone (efferent) to another
part of the system and the connections are made through synapses
(below). Dendrites are usually thicker, shorter and highly branched,
do not have a myelin sheath and carry the impulse from synapses to
the nerve cell (afferent). These processes contain neurotubules, appa-
rently identical with the microtubules of the mitotic apparatus and
of contractile tissues, and are thought to be associated with axonal
transport of materials from the perikaryon through the axon (Chapter
3).
Three main types of nerve cell can be identified by means of their
processes. 'Unipolar' cells contain only one axon and examples of
these are sensory cells of ganglia. 'Bipolar' cells have two processes,
an axon and a dendrite, and are found as sensory receptor cells con-
cerned with sight, smell, and hearing. The majority of the neurones
are multipolar, having one axon and many dendrites. Multipolar
cells fall into two main classes, named according to their shapes:
the pyramidal cells of Fig. 2.4 and stellate cells.

2.3.2 Glial cells

Glial cells (Fig. 2.5) do not possess the excitable characteristics of the
nerve celL are generally smaller, but also have processes emanating
from their cell bodies. These processes are relatively short and often
highly branched. There are three main types. Astrocytes often occur
close to blood vessels: their processes terminate in 'end-feet' which
make contact with the blood capillary wall. These are thought to
be concerned with nutrition, possibly acting as mediators in the trans-
port of materials from the blood stream to the neurones. Indeed a
highly specific means of causing degeneration of glial cells
without direct and immediate damage to the neurones, is by
promoting hyperammonaemia. This can occur naturally, as
a result of severe liver damage, or experimentally by the porto-
caval shunt technique [7]; the astrocytes become swollen and
vacuolated. The oligodendroglia are also satellite cells and are
intimately concerned in the central nervous system with the myelin
sheath of the axon, which they produce. The third group, the Schwann
cells, perform the same function in myelination of peripheral nerves
outside the brain. Fig. 2.6 is a diagrammatic representation of the
process of myelination. The myelinating cell wraps itself around
the axon so that its plasma membrane forms a spiral. The nucleus of
the cell can be seen lying close to the axon. Each of the myelinating
cells (oligodendroglia or Schwann cells) forms a unit of myelin along
part of the length of the nerve and many such may be required for the
16
 Axon

Myelin sheath Nodes of

Ranvier

II

Nerve ending A
II
Post-synaptic
~Jl ~ cell
Synapse~

Fig. 2.3 Schematic drawing of a neurone.

entire length of that nerve. At the points where the myelin from one
glial cell ends and that from the next begins, is a small gap where the
nerve is not covered by the sheath: the 'node of Ranvier' (Fig. 2.3,
see also Chapter 3.)

2.3.3 The synapse

The junction of one nerve cell with another (or of a nerve with inner-
vated target cells such as in muscle or the endocrine glands) has been
17
Fig. 2.4 Light microscopic picture of neurones. The section of the cerebral cortex
was stained by the Golgi method, magnification x 360. The inset shows a higher
magnification (x2870) of the dendritic spines. The photograph was kindly supplied
by Professor E. G. Gray, University College, London .

Fibrous astrocyte Oligodendroglia

Fig. 2.5 Glial cells.

18
 GlialCell

~ Nucleus

Axon

(a) (b)
Fig. 2.6 Formation of the myelin sheath. (a) An electron micrograph of rat cerebral
cortex showing myelinated axons, containing mitochondria. Magnification x 35000.
(b) During the process of myelination, the glial cell (a Schwann cell in the peripheral
nervous system or an oligodendroglial cell in the central nervous system) wraps
itself around the axon and with a spiral motion, forms the concentric circles of myelin
from its outer plasma membrane.

noted to be at the specialised synapse (Fig. 2.7). As the axon approaches

its point of contact with the subsequent or post-synaptic cell, it en-
larges into a specialised structure, known as the nerve ending. It is
completely surrounded by membrane which, with few exceptions, is
not fused with the membrane of the postsynaptic system, but is separat-
ed from it by a gap, some 200A in width, known as the synaptic cleft.
It is across this gap that chemical mediation of nerve transmission
occurs (Chapter 3). The exceptions, where the pre-synaptic and
post-synaptic membranes are fused, are known as electrical synapses.
These have been positively identified only rarely [8]; so far they seem
to occur in motor synapses in primitive systems such as the earthworm
and crayfish, with spinal electro motor neurones of the electric fish
and with ciliary ganglionic neurones in the chick . For the major
part, especially in mammalian systems, synaptic transmission is
chemically-mediated and the synapses are of the generalised structure
shown in Fig. 2.7. Their internal morphology is characterised by
pre-synaptic mitochondria and synaptic vesicles, believed to store
the chemical transmitter molecules, and by the post-synaptic apparatus
19
 (a )

Mitochondrion
:. ...I---=- Synaptic vesicles

-- - - Nerve ending
Post -synaptic thickening

(b)
Fig. 2.7 The synapse. (a) Electron micrograph of a dendritic synapse in rat cerebral
cortex, magnification x 28175 (Courtesy of Professor E. G. Gray, University College,
London). (b) Schematic drawing of a dendritic synapse, illustrating the constituent
parts.

20
depicted. The synaptic vesicles may be smooth as shown in Fig. 2.7
and are usually about 500A in diameter. Granular vesicles are also
known to occur and may be of similar size or larger, up to loooA.
Synaptic vesicles of different appearance are believed to be associated
with adrenergic transmission, involving catecholamines, rather than
cholinergic transmission, involving acetylcholine (Chapter 3). The
post-synaptic system may be the cell body of another neurone, or
the dendrite of another neurone, where the dendritic post-synaptic
membrane is often swollen to form the spine of Fig. 2.7 (see also
Fig. 2.3).
The molecular events surrounding the process of chemical trans-
mission have stimulated much interest for biochemists, and are now
described.

References
[I] Campbell, H. J. (1965), Correlative physiology ofthe nervous system. Academic
Press, London and New York.
[2] White, L. E. (1965), A morphological concept of the limbic lobe. Int. rev.
Neurobiol.,8,1-34.
[3] Cajal, S. R. (1955), Studies on the cerebral cortex: limbic structures. (trans!.
by Kraft, L. M.), Lloyd-Luke, London.
[4] McLean, P. D. (1954), Studies on limbic system (visceral brain) and their
bearing on psychosomatic problems, in Recent Developments in Psychoso-
matic Medicine (ed. Wittkower, E. D. and Cleghorn, R. A.). Pitman,
London, pp. 101-125.
[5] Bradbury, M. (1979), The Concept ofa Blood-Brain Barrier. Wiley, Chichester.
[6] Sherrington, C. (1906), The integrative action of the nervous system. Cam-
bridge University Press, Cambridge.
[7] Cavanagh. J. B., Lewis, P. D., Blakemore, W. F. and Kyu, M. H. (1972).
Changes in the cerebellar cortex in rats after portocaval anastomosis.
J. neurol. Sci., 15, 13-26.
[8] Phillis, J. W. (1970). Pharmacology of Synapses. Pergamon. London.

3 Neurotransmission

3.1 The resting potential

To understand the principles of neurotransmission, we need some
grasp of the bioelectric properties of the excitable nerve cell membrane.
All cells are surrounded by semi-permeable membranes with a dis-
equilibrium of the charged molecules on each side of that membrane,
to give a charged field. Thus all cells have a difference in electrical
potential across their outer cell membranes, in the range of -10 to
21
-90 millivolts, resulting from the relative distribution of ions between
the intracellular and extracellular regions, and are said to be 'polarised'.
The ions concerned are mainly K + and negatively charged macro-
molecules inside the cell, with Na + outside. The resting potential
(-60 to -70 millivolts in most neurones) has been concluded to be
due to the peculiar permeability properties of cell membranes: they
are considerably more permeable to K + and Cl- than to Na +. This
selective permeability to these ions may be a matter of size since the
hydrated sodium ion is some 50% larger than the hydrated potassium
ion. Nerve cells, in common with most cells, contain high concen-
trations of K + (I 00-120mM) and low concentrations of Na + (20mM)
relative to ·the concentrations existing outside in the extracellular
fluids (SmMK + and l40mMNa +). The cell membrane therefore
separates two compartments with unequal concentrations of NaCl
and KCI in each. From a knowledge of these concentrations, the
potential difference across the membrane can be calculated.
If the concentrations of Na +, K + and CI- inside and outside the
cell are known, an approximation to the resting membrane potential
is given by the Goldman equation [1]:

E = RT In [PKK o + PNaNao +fClCljJ

F PKKj + PNaNaj + Pc1Cl o
where R = gas constant, T = absolute temperature and F = Faraday
(the electric charge/g. equivalent of a monovalent ion). PK' PNa and
PCl are the permeabilities of the ions and Ko, Na o' Cl o and Ki' Nap
Clj are their concentrations outside and inside respectively. Since
Cl has been found to contribute only slightly to the membrane potential,
the equation is simplified to:

E = RT In [PKK o + PNaNaoJ
F PKKj + PNaNa j

or E = -RT In
F
[K 0
Kj+bNaj
P
+ bNa 0 ] ,where b = ~
PK
Since R, T and F are constants, at 37" and converting to loglo' the
relation becomes
..
E(mlliIvolts) = 6210g 1o [K +
0
Kj
+bNaJ
b o.
Na j
If the extracellular and intracellular cerebral concentrations of the
ions (mM) are taken as K o5, Na o 140, Kj 112, Naj 20, and b as 0.04 (2),
E becomes - 60mv which is similar to the membrane potential measur-
ed directly for cerebral neurones.
The Goldman equation is derived from the Nernst equation, as
the sum of the Nernst equations for each ion species. It gives the
equilibrium potential due to the asymmetric distribution of each
ion across the semipermeable membrane:
22
 E=RTln Ao
F Ai
where Ao and Ai are the activity coefficients of the ion species outside
and inside. For practical purposes, since activity coefficients are not
known, concentrations are used instead. However the Goldman
equation is an approximation only: it assumes passive diffusion of the
ions through the membrane, or that active movement of one ion
species is coupled to active movement of another, i.e. it assumes that
movement of ions is 'electroneutral' rather than 'electrogenic'. The
basis for the derivation of these equations is given by Hodgkin and
Katz [la J, see also Woodbury [1 b Jand a previous book in this series
by Davies [3J.
We know that active cation transport does occur and produces a
'steady state' situation in the resting cell, where ion concentrations
and potentials are maintained, so that net ion fluxes (active plus
passive) are zero. Efflux ofNa+ has been shown to take place against
cuncentration gradients of Na + and requires external K +. It is an
energy-consuming process and movement of K + is coupled to move-
ment ofNa+ in the opposite direction, not necessarily on a I : I basis.
The ratio for transport across the erythrocyte membrane is approxi-
mately 2K + transported for 3Na + [4], and while this is likely to vary,
a similar ratio may be the case elsewhere, including the brain. Cer-
tainly part of the influx of K + is linked to the efflux of Na + in the
brain. The tendency for ions to diffuse across the membrane, K +
out and Na + in, is countered by the active transport of the cations
'uphill', i.e. against concentration gradients so that Na+ is pumped
out into the extracellular environment of high Na + ; K + outside
moves to the high K + environment within the cell. So any passive
leakage of ions is compensated by the continued expenditure of
energy of the active transport process. On the discharge of the excitable
cell (Section 3.3) when more rapid efflux of Na + occurs, this cation
redistribution then becomes an integral part of the recovery process
and is effected through participation of a membrane-bound enzyme.
This enzyme, which perhaps uses as much as one-third of the energy
produced metabolically in the brain and stored as ATP [2], hydrolyses
the ATP to produce ADP and inorganic phosphate, and is the meta-
bolic basis for the 'sodium pump' (below).
Thus the development of the resting membrane potential results
essentially from the efflux of N a + and from the differential permeabi-
lity of the membrane to K + and Na +. The necessary unequal distri-
bution of these cations is maintained at the expenditure of energy
by the sodium pump.

3.2 The sodium pump

Membrane-bound 'ATPases' were recognised for some years before
Skou [5] suggested this activity to be associated with active transport
of Na +. He worked with crab nerve and his results were shortly
23
followed by an immense study of the activity in many tissues; the
brain was found to be particularly active [2]. Association of the
Na +, K +-adenosine triphosphatase with active cation transport
rests on convincing if circumstantial evidence.
(1) full enzymic activity requires both Na + (lOOmM) and K +
(6mM) in concentrations comparable to the extracellular Na + and
the extracellular K + (above). The Michaelis constants found for
both cations for the enzyme are similar to those calculated for cation
transport.
(2) the cation specificity of the enzyme is identical to that for mono-
valent cation transport (e.g. NH+ can replace K + but not Na +).
(3) high concentrations of Na + 4are inhibitory to K + in both pro-
cesses.
(4) ouabain (strophanthin g) in similar micromolar concentrations
inhibits both processes to a similar extent.
Perhaps the most convincing evidence arose from studies on re-
constituted erythrocyte 'ghosts', in which the internal contents of the
cell can be partially replaced. This elegant work demonstrated the
'vectorial' properties of the ATPase: the activity of the enzyme was
increased by higher internal Na + [6]. The enzyme reaction involves
intermediate phosphorylation of the enzyme (Fig. 3. I). After some
speculation on its nature (the intermediate was originally suspected to
be a phosphorylated serine residue), experiments using ATP labelled
on the y-position with 32p, with hydroxylamine which removes the
phosphate, and studies on the enzyme activity against synthetic
substrates, together provided strong evidence that it is an acyl phos-
phate. Subsequently, use of tritiated propyl hydroxylamine enabled
the group to be isolated and identified as a y-glutamyl phosphate [7].

3.3 The action potential and nerve conduction

The unique feature of the excitable cell is seen when the 'resting
state' is upset. The earliest technique, still used, was to apply an elec-
trical impulse to the cell by means of an electrode. This pulse caused
'depolarisation'. If a red blood cell or a liver cell were to be stimulated
in this way the depolarisation of the membrane is seen in the slow
passive loss of the potential difference across it. If a nerve cell is
stimulated, a very different series of events ensues. The potential
changes from about -60mV to -70mV but this does not continue
in the same way as it does in the non-excitable cell: there is a rapid
overshoot to the extent that the potential may become positive, to

Enz. + MgATP'- -----1__ E: P + MgADP-

E:P - E+P

[Overall reaction:- ATP - - ADP + P J

Fig. 3.1 Na +. K + -Adenosine Triphosphatase.

24
 >' +30
E
co
'':; \------Action potential
c
ac.
Q)

Q)
c
li'"
E
Q)

,-t------r- Resting potential

-70r---'-...J
\
I
o 2
Time (msec)
Fig. 3.2 An action potential. The arrow shows the time at which the stimulus was
applied,

some + 10 to + 30mV (Fig. 3.2). This is associated with movement

of cations: the membrane becomes more permeable to Na + [1].
Presumably some change has occurred in the porosity of the membrane
so that permeability to Na + becomes less restricted. This could result
from changes in the conformation of macromolecules constituting
the lipo-protein matrix of the membrane. For convenience, although
we do not understand the mechanisms, we think in terms of an increase
in the 'pore-size' of the membrane, i.e. the pores which under the
resting state were too small to permit passage of Na +, have now
increased in size to the extent that rapid passage of Na + can occur.
Sodium flows into the cells and potassium out until near electrochemi-
cal equilibrium is reached. This depolarisation usually lasts about
half a millisecond and is known as the action potential.
The increases in permeability to Na + and K + are not simultaneous:
Na + permeability increases first and initially to a greater extent.
Subsequently Na + permeability decreases and K + permeability
increases; the process becomes self-limiting and the potential difference
returns to its original value. The system remains inexcitable for a few
milliseconds, the 'refractory period', and this transient period is
considered to result from a condition when changes in membrane
permeability are such that it is temporarily non-permeable to Na +
and freely permeable to K +. This knowledge came from use of the
'voltage-clamp' technique which enabled the investigator to prevent
the uncontrolled explosive occurrence of the action potential and to
control changes in the membrane potential, during which current and
ion flux rates could be measured [8]. The process can be summarised
as follows: excitation causes first an increase in permeability to Na + ,
which flows in. This is followed by a decrease in the permeability to
25
Na +, coupled with increased permeability to K +. The cell has the
capacity for many repeated depolarisations before its cation balance
reaches the stage where no further excitation can occur. This does
not occur normally because energy is continually being expended
to return the cations to their original distribution. This re-distribution
is effected through participation of the Na +, K + -ATPase, described
above.
The action potential has certain clear characteristics. There is a
critical size of the stimulus which produces it, smaller stimuli having
no effect and larger stimuli producing no greater effect. The critical
size is the threshold and the process is all-or-none, in that the size
of the action potential is independent of the size of the stimulus,
provided it is at or above the threshold. However this does not mean
that the 'signal' is uncontrolled. Control at the nerve cell which is
depolarised may be exerted by the sequence of the stimuli in frequency
and in size, i.e. the latency, the interval of time between stimulation
and the action potential, may be decreased with repeated stimuli
of increasing size and the frequency of the stimuli can effect the post-
synaptic response (Section 3.6). Other factors also operate: the
properties of the axon which conducts the signal, of the nerve endings
and their chemical transmitter, and of the post-synaptic system.
Once the nerve cell has 'fired' the action potential is conducted along
the axon very rapidly (the rate may be as high as hundreds of metres
per second) until it reaches the nerve ending where chemical trans-
mission usually produces the post-synaptic response. Non-myelinated
axons, which are not 'naked' but surrounded by a membrane of glial
origin, are generally short and some loss or dissipation of the amplitude
of the impulse may occur. Continued renewal of the propagated
impulse is a feature of myelinated axons which may be many centi-
metres in length. The myelin sheath acts as a very efficient insulator
and there is little loss of strength of signal in the axon between the
nodes of Ranvier. It is at these nodes, the gaps in the myelin sheath,
were the signal is renewed. Provided that the action potential reaching
the node is still above threshold, the excitable axonal membrane
is exposed to and surrounded by the extra-cellular fluid; depolarisa-
tion again occurs. The action potential moves through the next mye-
linated section. The action potential can therefore pass down the
entire length of a myelinated axon without decreasing in size, by
'leaping' from node to node and being continually reinforced. The
rapidity of the conduction depends very much on the diameter of
the axon and on the thickness of the myelin sheath.

3.4 Chemical events at the synapse

It should be noted that we know quite well what happens, but we
have very little understanding of the precise mechanisms which
underly chemical neurotransmission. When the action potential
reaches the nerve ending, it causes release of specialised chemicals
almost certainly from their storage sites in the synaptic vesicles. This
26
 Presynaptic membrane
Synaptic cleft

Nerve
Axon
- -./I.r ending Postsynaptic membrane
~ .

Mitochondrion
Synaptic vesicles
Na +
(a)

(b)
Fig. 3.3 Schematic drawing of synaptic transmission. On arrival of the action potential
(Jt.) at the nerve ending (a), transmitter molecules are released and react with receptors
(R) on the post-synaptic membrane. The permeability of the membrane to Na + and
K + changes (b).

release can also be caused by local application of strong solutions of

potassium salts in experiments with isolated preparations. There is
some doubt as to the storage sites of the chemical transmitter mole-
cules, and for acetylcholine there appear to be at least two, and possibly
three, sites available: a 'free' site and two 'bound' sites. One of these,
a tightly-bound site, does not seem to be the source of the active
transmitter which is believed to come from the other, a relatively
'loosely-bound' site. Both bound sites are considered to reside on or
in the synaptic vesicles. The free site may act as a reserve pool outside
the synaptic vesicles in the cholinergic synaptosomal cytoplasm.
Again there is argument and speculation about the way in which
the transmitter is released from the synaptic vesicles into the synaptic
cleft. Three theories have been advanced: (I) that the whole vesicle
passes into the cleft where it disintegrates, discharging its contents
C'exocytosis'), (2) that the vesicle, on coming into contact with the
pre-synaptic membrane, opens up into a pore in the synaptic membrane
through which the transmitter passes, (3) less widely held now, that
the stimulus causes the synaptic vesicle to discharge its contents
into the nerve-ending in the immediate vicinity of the pre-synaptic
membrane and the transmitter diffuses through that membrane.
There is some morphological and histochemical evidence in support
of exocytosis of noradrenaline vesicles in adrenergic systems, espe-
cially from observations of release of stoichiometric amounts of A TP
27
and protein with the catecholamine. Whatever the mechanism in
cholinergic systems (most workers favour exocytosis), it is certain
that the transmitter is released into the 200 A wide synaptic cleft
(Fig. 2.3 and 3. 3) and diffuses across it to react with specific receptor
sites on the post-synaptic membrane. The result is a change in ion
permeability of the post-synaptic membrane to cause either the depo-
larisation or hyperpolarisation described below under 'post-synaptic
events' (Section 3.6).
We do not know how the reaction of the transmitter with its post-
synaptic receptors causes the response; all we can really do is to assume
that, by mechanisms unknown, the reaction causes a change in -mem-
brane permeability to cations, perhaps by modifying the conforma-
tion of the lipoprotein matrix of the membrane, to open an 'ionophore'
(Section 3.6). This is not unreasonable since there are many examples
in biology of changes in protein conformation resulting from specific
interactions with simple small molecules. Much of the recent effort
of biochemists and biophysicists has been devoted to identification
and isolation of these receptors (Section 3.6). It is an incredibly
difficult task because the special function of the receptor could well
reside in its architectural relationship with the membrane in which
it is situated.
3.4.i identification and occurrence of neurotransmitters
The first and best characterised of the chemicals identified is acetyl-
choline (Table 3.1) mainly from elegant studies on the neuromuscular
junction [9]. It fulfils the requirements initially regarded as the essential
criteria for identification of a neurotransmitter, although it must
be noted that these criteria were formulated largely from evidence
which accumulated for cholinergic systems! These were:
(1) The chemical must be stored in the nerve endings from which
it is released.
(2) It must be released upon pre-synaptic stimulation and shown
to be present in the extra-cellular fluid in the vicinity.
(3) When applied post-synaptically it must mimic the action seen
when the pre-synaptic system is stimulated.
(4) Specific antagonists should be recognised which prevent the
action of both the chemical and electrical stimulation. This usually
means a pharmacological agent which blocks the interaction of the
transmitter with its receptor.
(5) Mechanisms for destruction of the transmitter in the post-
synaptic region were thought to be essential to limit its duration of
action (see below).
Historically the first indications of the role of acetylcholine came
from observations that it mimicked the action of stimulation of para-
sympathetic nerves and a few years later that it was released as a
result of stimulation of the vagus nerve. The classical experiments
were on the neuromuscular junction, where Dale and his co-workers
[10] demonstrated both its release on stimulation and that its applica-
28
tion mimicked the action of neurotransmission there. Demonstra-
tion of the blockage of transmission by antagonists such as curare
soon followed. The importance of rapid subsequent destruction
of acetylcholine was recognised from the effects of anticholinesterase
agents in potentiating nerve stimulation and by the lethal effects of
such inhibitors of the enzyme involved, acetylcholinesterase, which
destroys the transmitter by hydrolysis. The remaining criterion (stor-
age) awaited recognition of the nerve ending by electronmicro-
scopy and its separation as a distinct entity which was achieved in the
early 1960's [II].
If the cells of most tissues are disrupted by carefully controlled
homogenisation in aqueous iso-osmotic media, the internal cellular
constituents (nuclei, mitochondria, soluble cytoplasm and fragments
of membranous material from the outer cell membrane and the endo-
plasmic reticulum) can be separated reasonably well by centrifugation
in centrifugal fields of increasing force. If similar techniques are applied
to the brain, the 'mitochondrial' fraction which results consists of many
fragments in addition to mitochondria, including myelin fragments
and nerve ending particles. The latter, named 'synaptosomes', can be
isolated due to a fortuitous circumstance: on gentle disruption of the
tissue the axon breaks near the point where it swells to form the nerve
ending and the broken membrane apparently re-seals to produce an
ending which is usually intact. The majority of these isolated nerve
endings do not seem to be 'leaky', at least as far as soluble enzymes
such as lactate dehydrogenase are concerned [12]. Chemical and enzy-
mic analysis of the synaptosomes revealed that they were enriched in
acetylcholine and also in the enzyme responsible for its synthesis
(Fig. 3.4). If the synaptosomes are disrupted by osmotic shock (by
resuspension in water instead of the iso-osmotic or hyper-osmotic
environment in which they have been prepared) the synaptic vesicles
can be collected and shown to be rich in acetylcholine or other trans-
mitters.
One of the most important of the above criteria to be satisfied has
been thought to be the need for rapid destruction of the transmitter,
due to the dire consequences if this is prevented in cholinergic systems.
However other chemicals, now considered to be neurotransmitters,
fulfil many of the criteria except this one - there is no obvious mech-
anism for their rapid destruction. These are amines; noradrenaline,
dihydroxyphenylethylamine ('dopamine'), 5-hydroxytryptamine
(,serotonin '), amino acids: e.g. glycine and y-aminobutyric acid, or
various peptides (Fig.3.1 0). None of these has a true enzymic equivalent
to the esterase which destroys acetylcholine. All of the degradative
enzymes are slower acting than acetylcholine esterase and some of the
amines and amino acids have been shown to be readily transported
across membranes [13]. It is now felt that diffusion away from the
synaptic cleft is sufficiently rapid to allow cessation of their activity,
particularly since aminergic systems show generally slower responses
than cholinergic systems. The release of all transmitters shows an
30
 10000g 100000 9 ~
~ ~ supernatant

P, p. P,
Homogenate ('Nuclear') (, Mitochondrial') ('Microsomes')

Resuspended in sucrose
Sucrose (M 1 Present after
original centrifugation
M sucrose
p.- 0 '3
W///h A [Myelin)
Cytoplasm

0 ,8 0 ·4 Synaptic vesicles
~ B (Vesicles, debri~
1·0
150000g
3 hr
O'6} Membrane
fragments
~ C (Nerve endings] 0,8
1-2 1·0 Unlysed
~ o [Nerve endings)
Mitochondria
synaptosomes
1·4 1·2 Synaptosomal
E [ Mitochondria] mitochondria
I 1I ill

~
c.. 60
.:
E
ill
eQ. Hexokinase
.'!:
0 (main ly mitochondrial , a
little cytoplasmic)
'"
<:
:~
0
Succinate dehydrogenase
~ (exclusively mitochondrial)
~
:~
ti
'"
'0 D Acetylcholines terase
*-
20
• Acetylcholine

~ Choline acetylase

0~~~~A~~~~8 C
o E
[ Myelin) [ Nerve endings] [ Mitochondria]

Fig. 3.4 Preparation of synaptosomes and synaptic vesicles [II]. The crude
mitochondrial fraction (P 2)' obtained by centrifugation of a sucrose homogenate,
is resuspended in 0.3 M sucrose and layered over a stepwise gradient of sucrose (I).
Centrifugation in swing-out buckets gives the separation shown in (II). Resuspension
of subfraction C in water lyses the constituent synaptosomes to release the contents,
which can be separated by density-gradient centrifugation (III). Chemical and enzymic
analyses of the subfractions of (II) are shown in (IV).

31
absolute requirement for Ca 2 + and is blocked by Mg2 + in vitro, causing
paralysis in nerve-muscle preparations.
The main transmitters that have been identified are shown in Table
3.1 and Fig. 3.10. They vary very considerably in their distribution
throughout the nervous systems; for example, acetylcholine is most
concentrated in parts of the brain stem, the spinal cord and in the
ganglia of the autonomic system (Table 3.2). This transmitter occurs
in lower concentrations in the cerebellum and in minute amounts in
the hippocampus, in contrast to the amines shown in Table 3.2.
Whereas noradrenaline is most highly concentrated in the sympathetic
system and the hypothalamus, dopamine occurs in the greatest amounts
in the caudate nucleus, the corpus striatum and the basal ganglia of
the brain stem. Serotonin (5-hydroxytryptamine) occurs mainly in
the hypothalamus, the caudate nucleus and the pineal gland.
Knowledge of the identity of some ofthe transmitters in various areas
and regions of the nervous system has emerged also from elegant histo-
chemical studies using fluorescent microscopy. The methods are based
primarily on exposure of thin sections of frozen or freeze-dried tissue to
formaldehyde vapour [14]. Dopamine and noradrenaline are converted
to dihydroisoquinolines (Fig. 3.5) which fluoresce with a green or yel-
low-green light of wavelength about 470 nm. Serotonin and other
amines produce a yellow fluorescence with a higher wave-length
(520 nm). By such techniques suspected amine transmitters can be
specifically detected within the limits of resolution of light micro-
scopy, i.e. with precision in different small regions of the brain, but
with less precision within the cells.

3.4.2 The quantum hypothesis

The suggestion [15] that chemical transmitters are released in discrete
packages or 'quanta' resulted from quantitative studies on the minute,
random, spontaneous electrical discharges detected at the neuro-
muscular junction, known as miniature endplate potentials. These occur
at rates of about I per second and have an amplitude of about 0.5
millivolt. The hypothesis has received much support and is believed
to hold, not only for the neuromuscular junction, but also for chemical
synapses everywhere, including those in the central nervous system.
Miniature end plate potentials show the same qualitative properties
as the action potential in cholinergic systems: they are blocked by
curare (which blocks the receptor) and they are enhanced in both
amplitude and duration by inhibitors of acetylcholinesterase (below)
which normally would remove the transmitter by destroying it.
The conclusion is that the miniature end plate potentials arise from
reactions of small amounts of transmitter with post-synaptic receptors.
They are well below threshold so no action potential ensues, or in the
case of muscle, no contraction occurs. They are additive, and analysis
of the distribution of amplitudes of the spontaneous potentials and
those evoked by stimulation showed that they were integral multiples
of a unit component, the 'quantum'. One quantum is the amount of
32
Table 3.2 Regional Distribution of Neurotransmitters

Region Part Acetylcholine Noradrenaline Dopamine Serotonin

Cerebrum Cortex 15-25 1·2 (0·3) 0·3 (0·5) 2 (0,2)

Olfactory bulb 9 0·3 2·5
Cerebellum 1·5 0·6 0·1 0·5
Mid brain Superior colliculi 30 1·2 10
Substantia nigra (I) (7) (3)
Brain stem Hippocampus ~O I 4.5 3
Caudate nucleus 0·6 (0'6) 50 (25) 9 (2)
Corpus striatum 0·1 (25) 0·2
Basal ganglia 50 I 45
Lateral geniculate 25 0·6
Pineal gland 50-100·
Hypothalamus 15 8 (6) 3· 5 (5) 8 (3)
Thalamus 20 I (I) 0·3 (1'5) 4 (I)
Pons 20 1·2 0·7 2.5
Medulla 15 2.5 (0'6) o· 7 (0,1) 3·5 (2)
Spinal Cord Dorsal, ventral
horns 15 1·5 ~o 3
Ven tral roots 100
Dorsal roots I
Peripheral
nerve Sciatic 35
Ganglia Superior Cervical 200 ~o ~o
------ - -_._--- ------~--

Values are approximate only, as nmoles/g fresh weight, from dog and cat, and those
for human brain are given in parenthesis [2, 13, 24]. The richest sources are indicated
by heavy type. There is considerable species variation: small rodents such as rat give
higher values generally. Many of these regions are shown in Fig. 2.2.
* Values in the pineal gland are subject to diurnal variation: high by day, low at
night (Chapter 4).

acetylcholine which produces one miniature end plate potential.

Arrival of the nerve impulse at the nerve ending causes the release of
much greater total amounts of transmitter and is due to a massive
increase in the number of quanta, rather than an increase in the
amount of transmitter released per quantum.
The number of molecules of transmitter per quantum has proved
difficult to assess. By preventing replenishment of acetylcholine (using
hemicholinium, a reagent which prevents choline uptake), repeating
stimulation until an of the acetycholine is depleted, and knowing the
total amount of transmitter available or stored pre-synaptically, the
amount of acetylcholine used per quantum can be roughly calculated
from the number of quanta required to produce the action potential
at a neuromuscular junction. About 200 to 300 quanta are released per
impulse at mammalian neuromuscular junctions. which amount
represents less than 0.5% of the total available transmitter. Each
quantum was calculated to contain about 50 000 molecules of acetyl-
choline [15]. The possibility that each quantum of acetycholine re-
presents the content of one synaptic vesicle seems attractive. though
unlikely: independent measurements [16] of the acetylcholine con-
33
 ~~H
HOOCH2
H.CHO+ I
'<::::: "-
iH2
HO)[))
- HO I
H0JCX)

HO § NH2
§ NH -
HO
I § ...e:: N HO~~
Dopamine 3.4- Dihydroxyisoquinoline Ouinanoid form
(green, 405/470 nm)

H0X)d~ H
°mOH

t2 -
H0J(X)oH H°cOo
H.CHO+ I § I§ NH - I § ...e:: N -
HO
~ ~ NH
HO NH2 HO HO
Noradrenaline

HOWCH~ H0Q:O H0(CC)

H.CHO + I /// .NHI ~:22 - "I
"NH
NH I -
'" NH ~
"I
N

5-Hydroxytryptamine 3.4-Dihydro ~-carboline

(yellow, 390/520 nm)
Fig. 3.5 Conversion of mono-amines to fluorescent derivatives.

tent of isolated vesicles indicate that each can contain only about
1600 molecules, at a concentration of about 0.25M, with a concen-
tration in the whole nerve ending of some 0.03M. For 50000 mole-
cules to be there, a concentration of some 10M would be required.
[If one takes the internal diameter of a cholinergic vesicle to be 500 A,
and assuming it is spherical, the internal volume is 6.6 x 10- 17 ml.
Since 1600 molecules are equivalent to 1.6 x 10- 20 moles, the con-
centration is 0.25M. If, instead of 500 A, one used a diameter of 400 A,
the concentration becomes 0.5M.] The number of 1600 molecules
stored is similar to the number (1000 to 2000) calculated to react with
the receptors to produce one miniature end plate potential [17]. We
therefore seem to be faced with the apparent anomaly that 1000-2000
molecules are stored in each vesicle, that a similar number of molecules
is required to produce a miniature end-plate potential, but that 50 000
molecules are released per quantum. Obviously the contents of more
than one vesicle are released per quantum, but there is a further compli-
cation: there is reason to doubt that all of the acetylcholine contained
within a vesicle is released at the same time. The results from studies on
turnover rates oflabelled acetylcholine indicate that it is only the 'hot',
or recently-formed, acetylcholine that is released on stimulation and
that the newly synthesised molecules represent only a small proportion
of the total associated with the vesicles [18]. Much is still lacking there-
fore in our understanding of the quantitative relationships. However,
if one reflects on the size of the vesicle (0.05jl in diameter) and the
minute number of molecules involved, the progress to date seems truly
remarkable!

3.4.3 Metabolism of acetylcholine

Acetylcholine is synthesised from choline and acetylcoenzyme A by
the reaction catalysed by choline acetylase (Fig. 3.6) in the nerve endings
34
 Glucose
I
I
I (Glycolysis)

Pyruvate

Pyruvate dehydrogenase
(with thiamine pyrophosphate,
lipoic acid, coenzyme A)

+
CH 3 · CO. SCoA + HO. CH 2 · CH 2 · N(CH 3 )3
Acetyl coenzyme A Choline

Choline acetylase

+
CH 3 . CO. OCH 2 . CH 2 · N(CH 3 )3
Acetylcholine (bound)

Nerve impulse
(or high K+)

Acetylcholine (free)

Acetylcholine
esterase

+
CH 3 · CO. 0- + HO. CH 2 · CH 2 · N(CH 3 )3
Acetate Choline
Fig. 3.6 Metabolism of acetylcholine.

of cholinergic systems. Since one of the precursors, acetylcoenzyme A,

requires energy for its formation, the synthesis of the transmitter is an
energy-consuming process. This reaction was studied by using eserine,
an inhibitor of acetylcholinesterase, to prevent breakdown of the
product of the reaction. This is essential because the esterase is widely
occurring and extremely active (below).
The caudate nucleus has the most active choline acetylase in the
central nervous system and produces acetylcholine at rates of some 10
35
Esteratic site' 'Anionic site'

~ ~
+ RO'p/F
Rc( ~o e
(Organophosphate)
..
[Esteratic site blocked]

+
H2 0 CH 3 . CO, OCH 2 · CH 2 · N(CH 3 )3
[Acetylcholine]

e
.. +
+HO. CH, CH, N(CH,),

[Hydrolysis]

Fig. 3.7 Active site of acetylcholinesterase. The hydrolysis of the ester is inhibited by
blockage of the esteratic site by organophosphate poisons,
to IS flmoles per gram of fresh tissue per hour, This is much slower
than the rates at which the transmitter can be destroyed (below).
However such rates of synthesis, or the 2 to 3 flmoles/g/hr. produced
in the cerebral cortex, are sufficient to maintain acetylcholine: as noted
above, only a minute proportion of the stored transmitter is used nor-
mally in vivo, so the rate of required renewal will be relatively small.
Synthesis requires a supply of choline which is transported into nerve
endings more readily than is acetylcholine; Na + is also essential,
probably due to its requirement for choline uptake and for acetyl-
choline storage. Hemicholinium blocks acetylcholine formation
essentially by preventing choline uptake. The acetYlcoenZyme A
required to maintain these rates of acetylcholine synthesis was earlier
thought to depend essentially on supplies of pyruvate from glycolysis,
but realization of the reversibility of the choline acetyl transferase has
thrown doubt on this. The results of studies on the turnover rates and
kinetic properties of the enzyme from the electric organ of the Torpedo
suggest that such formation of acetylcoenzyme A from acetylcholine
(the enzyme requires only acetylcholine and coenzyme A for this) may
be some 30 times more efficient than production of acetylcoenzyme
A by acetylcoenzyme A synthase [19]. Such recycling of acetylcoenzyme
A within the nerve ending cytoplasm is likely to occur when the con-
centration of acetylcholine exceeds its normal level of some 30mM
there, in view of the Km of the transferase for acetylcholine of SOmM.
Breakdown of this transmitter is considered to be the most efficient
of all catabolic processes in the body: it is catalysed by perhaps the
most active enzyme known to occur in nature. Hydrolysis of each
molecule can occur in microseconds. The enzyme responsible, acetyl-
choline esterase, occurs very widely not only within but also without
36
 O
CH'.~HCOOH
I I I _ I
HOOCH,YH.COOH HOCfCH,CH,.NH, HOQYH.CH, NH,
NH, _ NH, _ OH
HO ...9 HO -6 HO ...9 HO ...9
Tyrosine Dopa Dopamine Noradrenaline

Qj
I I
r"
~ NH
H,.CH.COOH
r!m, _ H0(JcjCH'.~H.COOH

~
I
NH
I NH,
-----.:~CH, . CH •. NH.

-~NHV
Tryptophan 5- Hydroxytryptophan 5-Hydroxytryptamine
(serotonin)
Fig. 3.8 Synthesis of amine transmitters.

the nervous system, presumably to ensure that 'leaked' transmitter is

promptly destroyed. In nervous tissues it is most active in membrane
fractions derived from the plasma membrane and the endoplasmic
reticulum (the 'microsomes') and particularly in the membranes of
the nerve endings. The reaction is hydrolysis of the ester to form choline
and acetate (Fig. 3.6). The active centre of the esterase has been the
subject of detailed studies and two parts have been recognised:
an 'anionic' site which binds the quarternary nitrogen and an
'esteratic' site which contains a serine OH group. It is this group
which is blocked by 'nerve gases', the organophosphate poisons, and
is the reason for the toxicity of these agents (Fig. 3.7). Organophos-
phates react with many enzymes but it is cholinergic transmission
which is particularly vulnerable due to the necessity of removing
acetylcholine post-synaptically. Failure of respiration is the usual
consequence arising from neuromuscular blockage in the diaphragm.

3.4.4 Catecholamines: noradrenaline and dopamine

These amines are synthesised from the amino acid, tyrosine, by the
decarboxylation and hydroxylation reactions of Fig. 3.8; dopamine is
an intermediate in the formation of noradrenaline. The first step, the
hydroxylation of tyrosine, is catalysed by tyrosine hydroxylase and is
the rate-limiting regulatory stage, being inhibited by the end-products.
The next stage is removal of CO 2 by Dopa decarboxylase and like all
amino acid decarboxylating enzymes, it requires pyridoxal phosphate
as co-enzyme. Dopamine is converted enzymically to noradrenaline
by oxidation of the side chain. The enzyme is dopamine p-hydroxylase.
These two 'hydroxylating' enzymes are of great interest: both require
molecular 0 . so 'hydroxylase' seems a misnomer. They are really
oxidases. Dopamine P-hydroxylase, a Cu2+ -protein, requires ascorbic
acid as well as 0 and is one of the few cerebral enzymes known to
show a requirem€nt for this chemical which is a vitamin in man and
the guinea pig. Neither of these species can synthesise its own ascorbic
acid, which in the guinea pig brain occurs in concentrations of 1~2
,umoles/g. Tyrosine hydroxylase, which requires O 2 , Fe2+ and a
pteridine cofactor, is found in the brain in the nerve endings, and as
noted above, is the regulatory enzyme for the pathway. Dopamine
P-hydroxylase is the only one of these hydroxylases so far detected
37
(a) Monoamine oxidase (neuronal)

HO~CH,.CH' NH, HO~~:.CH'.NH' HO~CH'.CH'.NH'

HO.J(/ HO~ ~NH)

Dopamine Noradrenaline Serotonin

Aldehyde intermediates

~ ~ COOH
~ COOH
::~CH'.COOH H00JCH,
I
HOOCH
6H
HO #" NH

3.4-Dihydroxy- 3.4- Dihydroxy- 5- Hydroxyindole-

phenylacetic acid mandelic acid acetic acid

(b) Catecholamine-O-methyl transferase (extra-neuronal)

H0X)CH,.CH,. NH, COOH

H0X)~:CH'NH' H~CH' COOH H
I
H°X)9
OH
HO #" HO # HO # HO #"
Dopamine Noradrenaline 3.4- Dihydroxy- 3.4- Dihydroxy-

~
phenylacetic acid mandelic acid

CH3DCH,.CH,.NH, CHryb:·CH,.NH, CH300CH,. COOH COOH

CH3°X)9H
OH I
HO ~ HO # HO # HO #
3-Methoxy- Normetanephrine Homovanillic acid Vanillyl-
tyramine mandelic acid

Fig. 3.9 Catabolism of amines.

within the synaptic vesicles; the others occur in the nerve endings,
apparently in the extravesicular cytoplasm rather than inside the
vesicles.

3.4.5 5-Hydroxytryptamine
This amine, commonly known as 'serotonin', is synthesised from tryp-
tophan by similar series of reactions, hydroxylation and decarboxy-
lation, to those producing the catecholamines. Tryptophan is oxidised
by tryptophan hydroxylase to form 5-hydroxytryptophan (Fig. 3.8)
which is then decarboxylated by a pyridoxal-phosphate requiring
enzyme: 5-hydroxytryptophan decarboxylase, which seems to be
identical to Dopa decarboxylase.
38
 Tryptophan hydroxylase, like the enzyme which oxidises tyrosine,
also requires 02' Fe 2 + and a pteridine cofactor. Its activity in the
brain is very low indeed and so it has proved difficult to detect and
to characterise. It is distinct from tyrosine hydroxylase. and also
from phenylalanine hydroxylase (which produces tyrosine), and is
the rate-limiting enzyme for serotonin synthesis. Other amines (hista-
mine and octopamine) are also now thought to possess centrally-
active properties.

3.4.6. Breakdown of the biogenic amines

Fig. 3.9 gives the pathways for the catabolism of the amines: these
are partly neuronal, catalysed by mono-amine oxidases, and partly
extra-neuronal, catalysed by catechol-O-methyl transferase. The major
excretion products are shown in Fig. 3.9. The mono-amine oxidases
are of particular interest; they are mitochondrial in origin and can
only be efficiently extracted from the mitochondria by treatment
with detergents. Electrophoresis of partially purified extracts seemed
originally to separate up to four forms ('isoenzymes'), but this is now
believed to be due to varying proportions of phospholipid bound to
the enzyme protein. It now appears to be agreed that two forms occur.
Designated MAO A and MAO B, they differ in sensitivity to inhibitors
and in substrate specificity: MAO A acts preferentially on serotonin
and noradrenaline, and is sensitive to inhibition by Clorgyline, whereas
MAOB prefers amines such as tryptamine, benzylamine and j1-phenyl-
ethylamine. It is sensitive to inhibition by Pargyline and the tricyclic
antidepressant drugs, Imipramine and Chlorpromazine. Both forms
of the activity oxidise dopamine, although some species differences
have emerged in that dopamine may be oxidised mainly by MAO B
in human brain and by MAO A in rat brain [20, 21J.
Much attention has been given to the amine transmitters due to
the suspicion that they may be involved in psychiatric disorders.
While there is little solid evidence to support the hypothesis that
defects in amine metabolism may be involved in schizophrenia (the
hypothesis is based largely on the hallucinogenic effects of drugs
such as L.S.D. and mescaline. which bear some chemical resemblance
to the biogenic amines), there is some circumstantial evidence that
they may be of importance in depression. The basis for this is quite
empirical- a group of antidepressive drugs was subsequently found
to have striking inhibitory action on monoamine oxidases, the enzymes
which destroy the amines (Fig. 3.9). Since that realisation, many drugs
have been developed which act to modify endogenous amine concen-
trations, either by increasing or decreasing them. One difficulty is
that most of these drugs are relatively unspecific in that, if they alter
the level of one amine, they tend also to have similar effects on
other amines. However, the results from studies on the effects
of antidepressive drugs on individual forms of the oxidase give
some hope that in the future specific drugs can be tailored to be
effective against a potential enzymic abnormality in each individual
39
case of depression. The amines are certainly involved in many aspects
of mood and behaviour, as modifications in hypothalamic amine
concentrations show.

3.4.7 Metabolism o/the neuroactive amino acids

The two amino acids of Table 3.1 (y-aminobutyrate and glycine)
have been shown to exert inhibitory action on cerebral preparations;
however they may also participate in general reactions of intermediary
metabolism in the brain. y-Aminobutyrate (Gaba) is formed by
decarboxylation of glutamate, a dicarboxylic amino acid. The enzyme
which is specifically responsible, glutamate decarboxylase, occurs
enriched in nerve ending preparations (Fig. 3.4) as does Gaba itself.
Gaba is removed by a mitochondrial transmination reaction; the
succinic semialdehyde which results feeds back into the tricarboxylic
acid cycle. Some 10% of the flux of carbon through this cycle normally
passes through the 'Gaba shunt'. A further product of Gaba meta-
bolism is y-hydroxybutyrate, thought to be important in sleep.
Glycine, like glutamate and Gaba, is rapidly produced from glucose
in the brain; its metabolism is closely linked to that of serine by enzymic
reactions (such as the serine hydroxymethyl transferase) found
throughout the body. Other amino acids which also have inhibitory
action when applied to cerebral preparations include p-alanine and
taurine; some are excitatory and include glutamate, aspartate,
cysteine sulphinate and cysteine sulphonate (cysteate).
All of these amino acids can be regarded as putative neurotrans-
mitters, but their widespread occurrence in relatively high concen-
tration, together with their involvement often in more general aspects
of cerebral metabolism, render difficult the assignment of a truly
specific neurotransmitter function; some may be important as modula-
tors of excitability [22].

3.4.8 The neuroactive peptides

'Substance P' (Fig. 3.10) was originally discovered in 1931 [23] in
spinal cord and subsequently demonstrated in the brain (especially
in the substantia nigra, hippocampus, globus pallidus and hypo-
thalamus); though it is suspected of neurotransmitter function, it
may act to modulate the neurotransmission of other agents [24, 25].
Interest in centrally-active peptides became acute on the discovery
of the enkephalins and endorphins [26, 27] which showed morphine-
like opioid properties. Morphine is the most effective analgesic known,
but the dangers of addiction with the accompanying social conse-
quences have stimulated the search for a non-addictive alternative
analgesic. The discovery of endogenous compounds, which react
with morphine receptor sites in the brain and which produce analgesia,
fired hopes that the alternative had been found. Fig. 3.10 shows the
amino acid sequence (91 residues) of P-lipotropin which contains
one of the two enkephalins, methionine-enkephalin (sequence 61-65).
The other is leucine-enkephalin, where leucine replaces the C-terminal
40
Suhstance P
Arg- Pro- Lys - Pro-Gin-Gin - Phe- Phe-Gly- Leu- Met

Neurotensin
Glu- Leu- Tyr-Glu -Asn- Lys-Pro-Arg-Arg-Pro- Tyr-Ile-Leu

~-Lipotropin (containing enkephalin and endorphin)

IGlu-Leu - Thr-Gly-Glu -Arg-Leu-Glu-Gln-Ala- Arg--Gly -Pro -Glu-Ala 11
16Gln-Ala -Glu-Ser -Ala -Ala-Ala-Arg-Ala -Glu --Leu-Glu-Tyr -Gly-LeuJ~
31Yal_ Ala -Glu -Ala-Glu Ala - Ala -Glu--Lys -Lys -Asp--Ser -Gly- Pro - Tyr4~
46 Lys - Met-Glu-His -Phe--Arg-Trp-Gly-Ser -Pro --Pro - Lys -Asp - Lys -Arg 6Q
6l Tyr -Gly -Gly-Phe- Met-Thr--Ser -Glu- Lys-Ser -Gin - Thr -Pro -Leu- YaF-~
76Thr-Leu -Phe-Lys-Asn-Ala -Ile -Ile- Lys -Asn -Ala -His - Lys - Lys -Gly _Gln91
Fig. 3.10 Some centrally-active peptides.

methionine. Another major opioid peptide is fJ-endorphin (sequence

61-69). The metabolic and functional relationships of the endorphins
to the enkephalins are unclear: though studies on the regional dis-
tribution of their occurrence and of their receptor-binding affinities
indicate that they occur and act independently, the topic remains
controversial. Enkephalins are very labile in the brain in vivo due to
peptidase activity; a stable analogue where the glycine of position 62
is replaced by D-alanine showed longer-lasting analgesic activity
[28].
The list of centrally-active peptides grows almost daily, with N-
terminal acetylaspartyl-, glutamyl-, y-aminobutyryl-, fJ-alanyl- and
histidyl-groups. One of these, fJ-alanyl-L-histidine (carnosine) is of
particular interest in the olfactory system [29, 30]. The detection of
some gastric hormones in the brain stimulated much immunochemical
screening of cerebral preparations for related hormones; to date
angiotensin II (8 amino acid residues), bombesin, bradykinin (9 resi-
dues), cholecystokinin (8 residues), gastrin (17 residues), somatostatin
and VIP (28 residues) have all been detected using immunochemical
techniques, though not all have been isolated and chemically identified.
This is important because the use of radio-immunochemical methods,
with the attendant dangers of cross-reactivity, may not provide
sufficient proof of occurrence. Neurotensin, with 13 amino acid
residues, occurs widely in the brain as well as in the ileum and jejunum
[31, 32]. As noted above with respect to the amino acids, it is not clear
whether many of these peptides act as modulators or transmitters,
but they represent a fascinating group of centrally-active agents
with many more almost certainly awaiting discovery.

3.5 Origin of synaptic vesicles

This is still an open question but two alternatives seem most likely:
either they are formed in the perikaryon and migrate through the axon
to their functional sites in the nerve ending (Section 3.7), or they
are formed locally within the nerve ending. Although much of the
earlier work was carried out on cholinergic vesicles, use of the fluore-
scent histochemical technique (Fig. 3.5) for catecholamines has given
41
us a clearer picture of the occurrence of adrenergic vesicles. Some of
the evidence is as follows:
(1) ligation of adrenergic nerves is followed by accumulation of
noradrenaline above (proximal to) rather than below (distal to) the
ligature;
(2) depletion of noradrenaline by reserpine is followed by re-
appearance of fluorescent material initially in the cell body;
(3) isotopically-labelled noradrenaline is transported through the
axon in a proximodistal direction together with protein. The fastest
rates ofaxoplasmic transport (Section 3.7) have been recorded for
noradrenaline granules. However, whether the vesicles are produced
in the cell body or in the nerve ending, the evidence currently available
points clearly to the n~rve endings as the sites of transmitter synthesis,
though not necessarily within the synaptic vesicles. Most workers
agree that acetylcholine is synthesised within the nerve endings but
not in the synaptic vesicles, jUdging from the distribution of choline
acetylase activity.
The same seems to be the case for Gaba: glutamate decarboxylase
activity is thought to be present in nerve ending cytoplasm rather
than in the vesicles. Like acetylcholine, it is the newly-synthesized
Gaba which seems to be released on stimulation. In contrast, nor-
adrenaline is thought to be synthesized within the synaptic vesicles,
from the presence of dopamine {J-hydroxylase there.

3.6 Post-synaptic events

The post-synaptic structure may be a motor end-plate, the membrane
of a nerve cell body or a dendritic spine (Fig. 2.7). The dendrite is
similar to the axon in one aspect: it has a resting potential, but it is
thought to differ in that it may not be electrically excitable. It is,
however, chemically excitable in that reaction of the transmitter
with the receptor will produce a change in polarisation of the dendritic
membrane. This is then conducted to the neurone which in turn will
change in polarisation. The dendritic post-synaptic response (Fig. 3.11)
has a lower amplitude and is of longer duration than the action poten-
tial. There seems to be no threshold, unlike the action potential,
and it is not an 'all-or-none' process since it is clearly additive, i.e.,
repeated rapid release of transmitter may have an additive effect
on increasing the amplitude of the post-synaptic potential (see the
discussion of miniature end-plate potentials in Section 3.4.2).
The post-synaptic dendritic response may be one of depolarisation
or of hyperpolarisation (Fig. 3.11): depolarisation, as described
above, is when the potential changes in a positive direction from
about - 60mV towards zero or a positive value; hyperpolarisation
is when the potential becomes more negative. In that case, since the
potential of the cell is more negative, it is more resistant to depolarisa-
tion so that there is a tendency to inhibit excitation and the formation
of an action potential. Depolarisation of the post-synaptic system is
therefore excitatory and the resultant potential is known as the EPSP
42
 +5l + 5O i
I

or 01
-50 -50

-100

-150
-100

-150
\J
Fig. 3.11 Post-synaptic potentials. (a) An excitatory post-synaptic potential (EPSP)
becomes from + 20 to + 40mV, from the resting potential of about - 70mV, a change
of some + 100m V in depolarisation. (b) An inhibitory post-synaptic potential (IPSP)
becomes about - 120 to - 140mV, i.e. a change to a more negative potential in
hyperpolarisation.

(excitatory post-synaptic potential). On the other hand, hyper-

polarisation of the post-synaptic system is inhibitory and the potential
is known as the IPSP (inhibitory post-synaptic potential). In excitatory
synapses the transmitter acts to increase the permeability of the
post-synaptic membrane to Na +, causing depolarisation. In inhibitory
synapses the transmitter acts to increase the permeability of the
post-synaptic membrane to chloride and perhaps also to K +', giving
hyperpolarisation.
Whereas it is believed, on good evidence, that only one type of
transmitter is stored and released from each individual nerve ending,
some neurones, e.g. the Renshaw cells of the spinal cord, may have a
multiplicity of types of synapse - in this case there seem to be two
excitatory and one inhibitory. There is evidence that the input (A) to
neurones of the sympathetic ganglia (Fig. 3.12) involves both nicotinic
and muscarinic cholinergic receptors, with intermediation by inter-
neuronal aminergic receptors. Here the responses to the inputs would
be: fast excitatory at the nicotinic cholinergic receptor (i), slow excita-
tory at the muscarinic cholinergic receptor (ii), and slow inhibitory
at the dopaminergic receptor (iii). The signal (B) from the ganglionic
neuron will thus be modulated according to the balance of excitatory
and inhibitory inputs [33]. This generalised, over-simplified picture
shows only cholinergic and catecholaminergic events; all of the
neurotransmitters discussed in this chapter are capable of such inter-
actions. Further, there is recent evidence for pre-synaptic receptors,
which may regulate more directly the release of neurotransmitters
from the nerve ending. Some transmitters (e.g. acetylcholine in the
cerebral cortex) may be excitatory or inhibitory, depending on the
type of receptor. Others, such as the amino acids, glycine and }'-
amino butyrate , seem to be essentially inhibitory at the synapses
that have been studied so far. For obvious reasons of accessibility
43
 Muscarinic

/i- A
-....,~~---
A
1--.....-8

Fig. 3.12 Modulation of excitability in sympathetic neurones.

more work has been done on the spinal cord, the neuromuscular
junction, and on peripheral nerves than on the central nervous system
so far and it is still far from clear which pathways in the brain are
mediated by which of the transmitters yet identified. It seems almost
certain that there are transmitters still awaiting detection and identi-
fication. A diagrammatic representation of the major events occurring
at cholinergic and adrenergic synapses is given in Fig. 3.13.

3.6.1 Involvement of cyclic nucleotides

Recent studies on various aspects of adrenergic transmission have
raised the possibility of involvement of the cyclic adenine nucleo-
tide, 3', Y-cyclic AMP, in subsequent metabolic events. Noradrena-
line has been shown to activate adenyl cyclase (the enzyme which
produces cyclic AMP from ATP) in inhibitory pathways in Purkinje
cells of the cerebellum. Mediation by cyclic AMP of neurotrans-
mission in sympathetic ganglia has also been suggested: in these
ganglia, with preganglionic cholinergic nerve endings and adrenergic
post-synaptic neurones, dopaminergic interneurones have also been
suggested to be present (Fig. 3.12). According to the hypothesis,
these dopaminergic cells receive signals from the cholinergic nerve
endings and their nerve endings form synapses with the post-ganglionic
neurones. The release 9f dopamine from the interneurones is suggested
to activate adenyl cyclase in the post-ganglionic cell membrane,
thus causing a local accumulation of cyclic AMP. This is then sug-
gested to change the permeability of the post-synaptic cell membrane
by causing phosphorylation of membrane proteins, which results
in hyperpolarisation to render the post-ganglionic neurone less
responsive to excitation. This hypothesis therefore invokes modula-
tion of sympathetic transmission by cyclic AMP [33] and evidence
in support is being sought. A direct link between synthesis of cyclic
AMP and post-synaptic events has yet to be demonstrated. More-
over questions on the time sequence remain to be answered: it seems
unlikely that the sequence of events postulated in the hypothesis
(enzyme activation, diffusion of product to another enzyme, which
is then activated to catalyse phosphorylation of membrane-bound
proteins) can occur sufficiently quickly in relation to the rapid post-
synaptic responses known to take place [34]. Certainly cyclic AMP is
44
 Presynaptic terminal Post-synaptic cell
Synaptic Vesicles

Nicotinic receptor
(autonomic ganglia.

t
Glucose
skeletal muscle)
Mitochondrion
Pyruvate

Curare

M uscari nic receptor

(cerebral cortex,
smoot h muscle)

(a) Cholinergic transmission

Tyrosine
Irs

cd
Tyrosine --.:.-- Dopamine ___ 0
.___e o
e

Receptors

Mitochondrion ~ ~ 0
(ATPLcYClic AMP)

, 00

~
If
Products

NA us

Products Cocaine" ....-

amphetamines f
Products
(b) Adrenergic transmission
Fig. 3.13 Schematic representation of neurotransmission. trs., transport, -If--
inhibition. (a) Cholinergic transmission. ACh, acetylcholine; a, choline acetylase: b,
acetylcholine esterase (membrane bound). (b) Adrenergic transmission. NA,
noradrenaline; c, tyrosine hydroxylase; d, Dopa decarboxylase; e, dopamine hydroxy-
lase (vesicular); f, COMT; g, MAO; h, adenyl cyclase.

45
known to accumulate, the catecholamines are known to activate
adenyl cyclase and the metabolic role most clearly eludicated for
cyclic AMP in many systems, including micro-organisms, is activa-
tion of protein kinases. This was the basis of its discovery: its role
in activating the protein kinase involved in glycogen phosphorylase
activation [35], and relevant protein kinases have been described in
neural tissues [36]. Cyclic AMP is also suspected of being involved
with the functions of peptides, such as substance P, and with prosta-
glandins. Cyclic GMP seems intimately concerned with muscarinic
cholinergic receptor function.

3.6.2 Receptors
While chemical analysis of neural membrane fractions has not shown
any clear basis for the difference between excitable and non-excitable
membranes, there is a wealth of pharmacological and physiological
evidence for the presence of sites on the post-synaptic membrane
which react specifically with the known chemical transmitters to
cause rapid, transient and reversible changes in cation permeability.
These receptor sites have mainly resisted attempts at isolation and
purification so the evidence on the mechanisms of interaction with
transmitters which cause conformational changes in the membrane
macromolecules remains indirect. As noted above (Table 3.1), two
types of cholinergic receptor have been detected pharmacologically:
'muscarinic', where transmission is mimicked by muscarine and
blocked by atropine; 'nicotinic', mimicked by nicotine (Fig. 3.14)
and blocked by curare. At present cholinergic receptors in the central
nervous system seem to be essentially muscarinic. Both are distinct
from the active site of cholinesterase. It was previously held by many
workers that they were identical but recent work has eliminated this.
The use of structural analogues of acetylcholine showed differences
in specificity between enzyme and receptor; the two differed markedly
in sensitivity to inhibitors (e.g. dithiothreitol). Also, denervated
muscle showed increased sensitivity to acetylcholine but decreased
enzymic activity. Some information on the nature of the receptor
sites has also come from studies with local anaesthetics. Procaine
(Fig. 3.14) interferes with interaction of acetylcholine at its receptor
sites and so blocks transmission. If a butyl group is substituted on to
the aromatic amino group, as in tetracaine, increased anaesthetic
potency results, possibly due partly to increased penetration of the
molecule to the receptor site.
Some of the approaches to identifying cholinergic receptors involve
'affinity-labelling' techniques whereby a stable, irreversible complex
is formed between the receptor and an easily-identified chemical
or biological label. A chemical approach was used by Karlin and
his co-workers, making use of the disulphide groups known to occur
near or at receptor sites. The free -SH groups, formed by reduction
of the disulphides with dithiothreitol (Fig. 3.14) were complexed
with substituted N-ethyl maleimides. The compound with the greatest
46
9-0
Nicotine
N

+
NH(C2H')2
I
CH 2
r
+

CH 2
CH3 )3
+
i(CH 3)3

CH 2

I
CH 2
I
CH 2
I
CH 2

I I I
°I °I °I

¢ o
C=O C=O C=O

I
CH 3

NH2 NH
I /CH 3
Procaine Acetylcholine CH 2 -CH
"CH 3
Tetracaine

G' < /-c"""",

o
N·(Benzyl trimethylammonium)
maleimide
HO",

HO/
CH

I
/CH 2 SH

CH
'-...CH 2

Dith ioth reitol

SH

Fig. 3.14 Chemical structures.

affinity of those tested was N-benzyl trimethylammonium maleimide

(Fig. 3.14). Using the tritiated compound, a protein complex of about
42 000 molecular weight was separated by gel electrophoresis from
the electroplax organs of Torpedo fish, which contain cholinergic
synapses with nicotinic receptors. The labelling of this protein was
decreased in the presence of snake neuro-toxins [36]. The other main
approach has been to label the membranes with radioactive neuro-
toxins, such as oc-bungarotoxin [37]. A protein of comparable molecu-
lar weight was then isolated. This nicotinic receptor protein has an
apparent molecular weight of around 250 000 and in the electron
microscope appears as a cluster of 6 units, each thought to be of about
40 000 molecular weight. Reaction of transmitter with this receptor
somehow opens a sodium channel or 'ionophore'; the architectural
relationship of an ionophore to the acetylcholine binding site is un-
known [38]. It seems possible that the 'hole' which is seen in the centre of
47
the receptor cluster (above) may represent the ionophore. It is at
least feasible that a change in conformation of cluster units on reaction
with acetylcholine could alter the size of the hole. Some hope of in-
creasing our knowledge comes from indications that a toxin (histrio-
toxin) found in South American tree frogs interacts with the ionophore
without detectable effects on acetylcholine binding. The treatments
inherent in isolating and purifying protein membrane-components
will almost certainly effect the native configuration of the protein so
that it would be surprising indeed if its specialised biological function
were retained. There is no doubt that drug-binding macro-molecules
have been isolated and the lowering of the radioactivity of the isolated
labelled protein in the presence of receptor-blocking agents argues
for some degree of specificity. But the acid test is biological activity.
This is a problem confronting many investigators working with nervous
tissues. So many of the important functions reside in membrane-
bound molecules which may only show their specific biological
activity if the architectural integrity of their immediate environment
is preserved.
The nicotinic cholinergic receptor is the best characterised to date,
due essentially to its rich, homogeneous occurrence in electric organs;
less progress has been made on isolation of cerebral receptors to
other agents, although considerable pharmacological and immuno-
chemical evidence for their occurrence is accumulating. Some caution
should be exercised in assessing receptor identification from binding
studies, either with pharmacological agents or with antisera. Un-
certainties which may be present include pharmacological or immuno-
logical specificity of the agent employed, distinction between binding
to receptor sites or to transport sites, the identity of the membrane
fraction to which the ligand binds; the final emphasis must be on
isolation and characterisation of the suspected receptor.

3.7 Neurone-axonal transport

The mechanism for replenishment of molecules, large and small,
at nerve endings which may be many centimetres from their own cell
bodies (Chapter 2) has been the subject of intense effort for a decade
or more. Two possible processes may be involved: renewal by loca-
lised synthesis, or renewal by transport of pre-formed material synthe-
sised in the nerve cell body. It has been clear for over 30 years that the
material of the axoplasm moves in a proximodistal direction (i.e. in a
direction away from the cell body towards the nerve ending). If a
clamp or restriction is applied to a nerve so as to squash it, a build-up
of material proximal to the 'bottleneck' can be seen under the light
microscope (Fig. 3.15). Mitochondria accumulate here also and are
less obvious on the distal side of the clamp. These facts, together
with observations of higher enzyme concentrations (e.g. the mito-
chondrial enzyme, succinate dehydrogenase) on the proximal side,
gave clear indications of a damming of the natural flow of materials
down the axon [39].
48
Fig. 3.15 Swelling of a nerve. proximal to a constriction. The black block represents
the constriction to the nerve. To the left. on the proximal side (nearer to the cell
body), the nerve becomes swollen within a few weeks and its contents present a
beaded, convoluted appearance suggestive of damming of a stream. flowing from
left to right [39].
Reproduced with the permission of Professor Paul Weiss. Rockefeller University,
and 1. Expt!. Zoo!.

Measurements of the rates of flow of a variety of molecules through

the axon have been made using radio-isotopes. The general technique
has been to inject the labelled compound into the cisterna magna or
the spinal cord of laboratory animals, and subsequently to measure
the rate of flow of the incorporated material through the appropriate
nerve fibre. Estimation of radioactivity has been effected by direct
counting in dissected segments of the nerve, or by autoradiography.
The rate of flow has been found to vary to a certain extent according
to the type of material being transported: two, and possibly three,
types seem to occur. The slowest rates (1 to 2 mm/day) have been
observed for proteins [40] labelled with tritiated leucine and for lipids
labelled with 32p-phosphate. Intermediate rates of 10 to 40mm/day
have also been noted using the same tracers. Really rapid rates of
200 to 500 mm/day [41] have been described for proteins and es-
pecially for catecholamine-containing granules [42]. The enzyme,
choline acetylase, is apparently transported at the intermediate rate.
Many of the putative neurotransmitters, including peptides such
as substance P, are now known to be transported in this way, as are
many of the related synthetic enzymes. However it is not clear what
proportion of the renewal of transmitters in nerve endings is due to
flow and what to local synthesis. Although with rapid flow of enzymes
it seems likely that most of the synthesis of neurotransmitters occurs
at the nerve ending, there is some evidence for local synthesis of enzyme
(below).

3.7.1 Mechanism of transport in axoplasmic flow

While little is known about this mechanism, flow due to constant
pressure being exerted from the neuronal cell body seems unlikely
and the kinetic properties of the process are incompatible with simple
49
diffusion. A peristaltic process may be involved and has been seen in
time-lapse cinematographic studies of nerve fibres in tissue culture
[43]. The internal structures of the axon are certainly concerned
with axoplasmic flow. Colchicine, an inhibitor of cell mitosis, sup-
presses some types of flow: the rapid type seems very sensitive to the
drug and can be fully blocked, whereas the intermediate and especially
the slow types are less affected. Colchicine binds specifically to the
proteins of the neurotubules of the axoplasm. These tubules, about
250A in diameter, run longitudinally down the interior of the axon,
and seem identical in physical and chemical properties to the micro-
tubules of the mitotic apparatus. Nerve tissue is the richest known
source of colchicine-binding tubule proteins. The brain has about
2 to 3 times the binding capacity per unit weight than the mitotic
apparatus of various cells, and isolated axoplasm, about 10 times
the capacity. Colchicine has been shown to inhibit tubule function
by dissociating the tubular protein (,tubulin') into its inactive subunits.
Dissociation is also caused by low temperature (3°C) which also
inhibits axoplasmic flow. Tubulin is a protein of molecular weight
about 120000, consisting of two subunits. It is different from the
globular protein (molecular weight 80 000) of another constituent of
the axoplasm, the neurofilaments. These structures are smaller (80 to
100A) than the neurotubules, do not seem to be associated with axo-
plasmic flow, and do not bind colchicine. There is an apparent change
during development: the immature brain contains a higher proportion
of neurotubules than the mature brain, which contains relatively
more neurofilaments. Relative activities ofaxoplasmic flow correlate
well with this change. In the neurotubules, 1 molecule of tubulin
dimer binds 1 molecule of colchicine and the binding requires Mg2 + .
The nucleotide, GTP, is also required: 2 molecules are associated with
each tubulin molecule. One of these is thought to stabilise the dimer
and occupies a 'stable' site on the molecule. The second is more labile
and its role is not clear. Interest in neurotubules and neurofilaments
has been heightened by observations of 'neurofibrillary tangles' in
the hippocampus and temporal lobe in ageing, and particularly in
senile dementia. The precise relationship of the material of the tangles
to these normally-occurring materials has not been resolved [44]
but it seems possible that the tangles could contribute to cellular
degeneration, perhaps by affecting axonal flow. While there is un-
doubted evidence for these very active and rapid transport mechanisms,
not all material of the axons and nerve endings is renewed in this way.

3.7.2 Axonal protein synthesis

Independent protein synthesis has been shown to occur, not only
in nerve endings, but also all along the axon. This has been most
clearly demonstrated by studies on acetylcholinesterase activity,
which can be irreversibly inhibited by organophosphate poisons.
After such inactivation in peripheral nerves (mammalian sciatic and
hypoglossal nerves, and in the axon of the Mauthner giant neurone of
50
goldfish), the enzymic activity re-appears no more rapidly at sites
near the neuronal cell body than it does at sites remote from the body.
If the enzyme had been synthesised solely in the cell body, a gradation
of renewed activity would have been expected, appearing first at
proximal sites and subsequently in more distal segments of the nerve.
In some experiments with paired mammalian nerves (e.g. the right
and left hypoglossal nerves) both nerves were poisoned with organo-
phosphate but only one was treated also with careful local appli-
cations of puromycin to inhibit any local protein synthesis [45].
Reappearance of enzymic activity was inhibited in the puromycin-
treated nerve, but not in the untreated nerve. The results of the use
of inhibitors are puzzling: the protein synthesis showed inhibition
properties similar to those which occur in ribosomes rather than in the
mitochondria also present within the axons. The RNA present in
the axon, in preparations carefully stripped of surrounding myelin
sheath and their glial cells, has been concluded (from its base-ratio
analysis) to be of ribosomal type. Yet electron microscopists have
failed to detect any ribosomes in the axons. There seems no doubt
that a local axonal site for protein synthesis exists, but the mechanism
of this synthesis is unclear at present.
Local sites in nerve endings for synthesis of enzymes have not been
established but, as noted earlier in this Chapter, many of the enzymes
required for transmitter synthesis have been detected there.
Protein therefore seems to be renewed at the nerve ending by a
combination of local synthesis and transport of pre-formed protein;
at present there is insufficient evidence to provide any basis for an
assessment of the relative contribution or importance of each of the
two processes. If significant amounts of protein do reach the nerve
endings by transport it seems reasonable to expect active localised
mechanisms for protein breakdown, but little knowledge on this is
currently available.
In this Chapter we have been very briefly concerned with neuronal
function and the interaction with endogenous chemicals intimately
and directly involved; we can now turn to consider some aspects of
the regulation of brain function by 'external' factors, either within
the body external to the brain or belonging to the environment ex-
ternal to the body.

References
[I a] Hodgkin, A. L. and Katz, B. (1949). The effect of sodium ions on the
electrical activity of the giant axon of the squid'. J. Physiol. 108.37- 77
[Ib] Woodbury, 1. W. (1965). 'Action potential: properties of excitable mem-
branes'. Neurophysiology (Ruch, T. C, Patton, H. D., Woodbury, J. W.
and Towe. A. L.), Chapter 2, London; Saunders.
[2] McIlwain, H. and Bachelard. H. S. (1971) Biochemistry and the Central
Nervous System (4th. ed.). Churchill, London.
[3] Davies, M. (1973), Functions of biological membranes. Chapman and Hall.
London.

51
 [4] Glynn, I. M. (1968) 'Membrane adenosine triphosphatase and cation
transport'. Brit. Med. Bull. 24, 165-169.
[5] Skou, J. C. (1957), The influence of some cations on an adenosinetriphos-
phatase from peripheral nerves'. Biochim. Biophys. Acta, 23, 394-401.
[6] Dunham, E. T. and Glynn, I. M. (1961) 'Adenosine triphosphatase activity
and the active movement of alkali metal ions'. J. Physiol. 156, 274- 293.
Whittam, R. H. (1962) The asymmetrical stimulation of a membrane
adenosine triphosphatase in relation to active cation transport'. Bio-
chem. J. 84,110-118.
[7] Kahlenberg, A, Galsworthy, P. R. and Hokin, L. E. (1968) 'Studies on the
characterization of the sodium-potassium transport adenosinetriphos-
phatase'. Arch. Biochem. Biophys. 126, 331-342.
[8] Hodgkin, A L. (1958), Tonic movements and electrical activity in giant
nerve fibres'. Proc. Roy. Soc. B. 148, 1-37.
[9] Dale, H. H .. Feldberg, W., and Vogt, M. (1936), 'Release of acetylcholine
at voluntary motor nerve endings', J. Physiol. 86, 353-380.
[10] Brown, G. L.,Dale, H. H.and Feldberg, H. (1936), 'Reactions of the normal
mammalian muscle to acetylcholine and eserine'.
J. Physiol. 87, 394-424.
[lla] Gray, E. G. and Whittaker, V. P. (1962), The isolation of nerve endings
from brain'. J. Anat. Lond. 96, 79-88.
[lib] DeRobertis, E., de Iraldi, A. P., de Lores Arnaiz, G. R. and Salganicof, L.
(1962), 'Cholinergic and non-cholinergic nerve endings in rat brain'.
J. Neurochem. 9, 23-35.
[12] Marchbanks, R. M. (1967). 'The osmotically sensitive potassium and so-
dium compartments of synaptosomes'. Biochem. J. 104, 148-157.
[13] Iversen, L. L. (1967), The uptake and storage ofnoradrenaline in sympathetic
nerves. Cambridge University Press.
[14a] Falck, B. (1962), 'Observations on the possibility of the cellular localisation
of monoamines by a fluorescent method'. Acta Physiol. Scand. 56,
supp!. 197.
[l4b] Dahlstrom A. and Fuxe, K. (1964), 'Evidence for the existence of mono-
amine-containing neurones in the CNS'. Acta Physiol. Scand. 62,
supp!. 232.
[15] Katz. B. (1966), Nerve, muscle and synapse. New York, McGraw-Hili.
[16a] Whittaker, V. P. and Scheridan, M. N. (1965). 'The morphology and
acetylcholine content of isolated cerebral cortical synaptic vesicles'.
J. Neurochem. 12, 363-372.
[16b] Wilson, W. S.. Schultz, R. A and Cooper. J. R. (1973), The isolation of
cholinergic synaptic vesicles from bovine superior cervical ganglion and
estimation of their acetylcholine content'. J. Neurochem. 20, 659-667.
[17a] Katz, B. and Miledi, R. (1972), 'The statistical nature of the acetylcholine
potential and its molecular components'. J. Physiol. 224, 665-669.
[17b] Katz, B. and Miledi, R. (1973), The binding of acetylcholine to receptors
and its removal from the synaptic cleft'. J. Physiol. 231. 549-574.
[18] Marchbanks, R. M. and IsraeL M. (1972), The heterogeneity of bound
acetylcholine and synaptic vesicles'. Biochem. J. 129, 1049-1061.
[l9] Diebler, M.-F. and Morot-Gaudry, Y. (1977). 'Biosynthesis of acetyl-
coenzyme A in the electric organ of Torpedo marmorata in relation to
acetylcholine metabolism'. Biochem. J., 166,447-453.
[20] Achee, F. M., Gabay, S. and Tipton, K. F. (1977), 'Some aspects of mono-
amine oxidase activity in brain', Progr. Neurobiol., 8, 325-348.

52
 [21] Johnston, J. P. (1968), 'Some observations upon a new inhibitor of mono-
amine oxidase in brain tissue', Biochem. Pharmac., 17, 1285-1297.
[22] Bachelard, H. S. (1981), 'Biochemistry of centrally active amino acids',
In Amino Acid Transmitters (ed. Mandel, P. and DeFeudis, F. V.),
Raven Press, Yew York.
[23] Von Euler, U. S. and Gaddum, J. H. (\931), 'An unidentified depressor
substance in certain tissue extracts', J. Physiol., 72, 74-87.
[24] Malthe-~renssen, D., Cheney, D. L. and Costa, E. (1978), 'Modulation
of acetylcholine metabolism in the hippocampal cholinergic pathway by
intraseptally injected substance P', J. Pharmac. Exp. Ther., 206, 21-28.
[25] Henry, J. L., Krnjevic, K. and Morris, M. E. (1975), 'Substance P and
spinal neurons', Can. J. Physiol. Pharmac., 53, 423-432.
[26] Hughes, 1., Smith, T. W., Kosterlitz, H. W., Fothergill, L. A., Morgan,
B. A. and Harris, H. R. (1975), 'Identification of methionine-enkephalin
structure',Nature, 258,577-579.
[27] Bradbury, A. F., Smyth, D. G. and Snell, C. R. (1976), 'Lipotropin:
precursor of two biologically-active peptides', Biochem. Biophys. Res.
Commun., 69, 950-956; Prohormones of beta-melanotropin and corti-
cotropin: structure and activation, CIBA Found. Sympos., 41, 61-75.
[28] Pert, A. (1976), 'Behavioral pharmacology of D-alanine 2-methionine
enkephalin amide and other long-acting opiate peptides', In: Opiates
and Endogenous Opioid Peptides (ed. Kosterlitz, H. W.), p. 87-94,
North-Holland, Amsterdam.
[29] Emson, P. c., Hunt, S. P., Gilbert, R. T. F., Wu, 1. Y., Rehfeld, 1. F. and
Fahrenkrug, J. (1980), 'Current knowledge of the storage and release
of transmitter candidate amino acids and neuropeptides', In: Synaptic
Constituents in Health and Disease (eds. Brzin, M., Sket, D. and Bache-
lard, H.), pp. 57-80, Pergamon, Oxford.
[30] Harding, 1. and Margolis, F. L. (1976), 'Denervation in the primary ol-
factory pathway of mice. III. Effects on enzymes of carnosine metabo-
lism',Brain Res., 110, 351-360.
[31] Carraway, R. and Leeman, S. E. (1976), 'Characterization of radioim-
munoassayable neurotensin in the rat', J. BioI. Chem., 251, 7045-7052.
[32] Polak, J. M., Sullivan, S. N., Bloom, S. R., Buchan, A. M. J., Facer, P.,
Brown, M. R. and Pearse, A. G. E. (1977), 'Specific localization of
neurotensin to the N cell in human intestine by radioimmunoassay
and immunocytochemistry', Nature, 270, 183-184.
[33] Greengard, P. (1976), 'Possible role for cyclic nucleotides and phosphory-
lated membrane proteins in postsynaptic actions of neurotransmitters',
Nature,260,101-108.
[34] Rodnight, R. (1979), 'Cyclic nucleotides as second messengers in synaptic
transmission', In: Physiological and Pharmacological Biochemistry
(ed. Tipton, K. F.), 26, 1-80, Univ. Park Press, Baltimore.
[35] Sutherland, E. W., and Rail, R. W. (1960), 'The relation of adenosine-3',
5'-phosphate and phosphorylase to the actions of catecholamines and
other hormones'. Pharmacol. Revs. 12, 265-299.
[36] Keitler, M. 1., Cowburn, D. A., Prives, J. M. and Karlin, A. (1972),
'Affinity labelling of the acetylcholine receptor in the electroplax'.
Proc. Nat. Acad. Sci. U.S. (I), 1168-1172.
[37a] Miledi, R., Molinoff, P. and Potter, L. T. (1971), 'Isolation of the choliner-
gic receptor protein of Torpedo electric tissue'. Nature, 229, 554-557.
[37b] Meunier, J-C, Olsen, R., Menez, A., Morgat, J-L., Fromageot, P., Ronse-

53
 ray, A. M., Boquet, P. and Changeux, J-P. (1971), 'Quelques proprietes
physiques de la proteine receptrice de l'acetylcholine etudiees a l'acide
neurotoxine radioactive'. CR. Acad. Sci. Paris, 273D, 595-598.
[38] Heidmann, T. and Changeux, J.-P. (1978), 'Structural and functional
properties of the acetylcholine receptor protein in its purified and mem-
brane-bound states', Ann. Rev. Biochem, 47, 317-357.
[39] Weiss, P. and Hiscoe, H. B. (1948), 'Experiments on the mechanism of
nerve growth'. J. Exptl. Zool. 107,315-395.
[40] Taylor, A. C. and Weiss, P. (1965), 'Demonstration of axonal flow by the
movement of tritium-labelled protein in mature optic nerve fibres'. Proc.
nat. Acad, Sci. U.S. 54,1521-1527.
[41] Ochs, S., Sabri, M. I. and Johnson, J. (1969). 'Fast transport system of
materials in mammalian nerve fibres'. Science, 163,686-687.
[42] Dahlstrom A. and Haggendal, J. (1966), 'Studies on the transport and
life-span of amine storage granules in a peripheral adrenergic neuron
system'. Aera Physiol. Scand. 67. 278-288.
[43] Weiss, P. (1958), The concept of perpetual neuronal growth and proximo-
distal substance convection', in Regional Neurochemistry (ed. Kety, S. S.
& Elkes, J.), Pergamon, London, p. 220-242.
[44] Katzman, R., Terry, R. D. and Bick, K. L. (eds), (1978) Alzheimer's
Disease, Aging, Vol. 7, Raven Press, New York.
[45] Koenig, E. (1969), 'Nucleic acid and protein metabolism of the axon'.
Handbook of Neurochemistry, 2,423-434 (ed. Lajtha, A.), Plenum, N. Y.

4 Adaptive processes In the brain

4.1 Inducible enzymes

Although much has been known for a decade or more about enzyme
induction in micro-organisms and in mammalian organs such as the
liver, analogous processes have only recently been detected in neural
tissues. In a review on neural plasticity some years ago, a statement
[I] that nothing was known about enzyme induction in nerve cells,
could not easily be challenged. However, in the interval many examples
have been described, and these mainly concern enzymes involved in
transmitter metabolism or function.
The preceding chapter of this book included a brief description of
tyrosine hydroxylase as the rate limiting stage of catecholamine
synthesis and there now is evidence that this enzyme is inducible.
Release of noradrenaline on stimulation of adrenergic nerves is follow-
ed immediately by a rise in the rate of its synthesis from tyrosine. This
was believed to be due essentially to the decrease in the end-product
inhibition of tyrosine hydroxylase, normally by noradrenaline. How-
ever subsequent work indicated that the activity of tyrosine hydro-
54
xylase in vitro also increased. If the increase in rate of synthesis of the
catecholamines in vivo had been due solely to de-inhibition of the
enzyme, a prolonged increase in its activity in vitro would not be
expected and the possibility therefore arises of increased enzyme
protein formation in addition to activation of the pre-existing enzyme.
Further evidence that the increase in tyrosine hydroxylase activity
of adrenals and superior cervical ganglia could be prevented by
inhibitors of protein synthesis (e.g. cycloheximide) tended to support
the growing view that an induction of enzyme synthesis was involved
[2]. Formation of the enzyme is thought to occur in the neuronal cell
body from where it is transported to its functional site in the nerve
ending, by the neurone-axonal flow mechanisms described in Chapter
3. Induction of tyrosine hydroxylase has been examined closely in
adrenergic neurones of ganglia from the sympathetic nervous system.
Treatment with reserpine, which releases amines from their storage
sites (Chapter 3), is followed by increased tyrosine hydroxylase activity;
lowering the temperature, which is associated with increased adrenergic
activity, also results in increased enzymic activity [3]. Furthermore,
the increase in the number of ganglionic synapses which occurs during
development is paralleled by an increase in hydroxylase activity;
if the pre-ganglionic nerve trunk is cut, the increased enzymic activity
in post-synaptic neurones is prevented [4]. These observations are
suggested to be consistent with trans-synaptic regulation: i.e. that
events at presynaptic nerve terminals (release of neurotransmitters)
regulate enzymic levels in the post-synaptic cells. Further evidence
has come from the use of high K + concentrations to depolarise cultured
sympathetic ganglia: again tyrosine hydroxylase activity increased [5].
Similar results were obtained with dibutyryl cyclic AMP (Chapter 3).
In the experiments with high K +, the effect was enhanced by the
presence of theophylline (a caffeine-like drug which prevents break-
down of Y, Y-cyclic AMP). There is some evidence, from the use of
inhibitors, that induction of synthesis of new enzyme is involved. The
results with cyclic AMP are also compatible with a post-synaptic
process (Chapter 3). Since the ganglia used in such studies are inner-
vated by cholinergic nerves, it seems likely that it is the pre-synaptic
release of acetylcholine that causes the trans-synaptic increase of
post-synaptic enzyme [2].
This therefore provides a good example of metabolic adaptation, i.e.
of enzyme induction, neuronally mediated in response to excitation
and neurotransmission. While the mechanism of this adaptation
remains unclear, it seems unlikely to be due to induction by the sub-
strate, tyrosine, in the absence of any evidence that the concentration
of tyrosine increases. There is a strong possibility, therefore, that the
enzyme is induced directly by neurohumoural agents, where presyn-
aptic release of a neurotransmitter is followed by enzyme synthesis in
postsynaptic cells. This seems clearly to be the case in the adrenal
medulla and sympathetic ganglia, where immunoassay provided
further evidence for the presence of increased amounts of enzyme
55
protein. However in certain brain regions studied (hippocampus,
hypothalamus and locus coeruleus) the evidence is less firm for syn-
thesis of new protein: while tyrosine hydroxylase activity was increas-
ed under the conditions described above (including treatment with
reserpine and cholinergic stimulation), immunoassay failed to show a
significant increase in enzyme protein [6].
Examples are known of induction of cerebral enzymes by their
specific substrates and there is growing knowledge on adaptive
processes also involving hormones, drugs and environmental stimuli.
Some of these are described in the following sections.

4.1.1 Adaptation to specific substrates

The first example of this is the response of f3-hydroxybutyrate de-
hydrogenase to the presence of substrate in the blood stream. It was
originally conceived as a direct induction of the enzyme by its substrate
but the results of more recent work have thrown doubts on this: the
presence of substrate is now believed to result in the maintenance of
existing enzyme, rather than in induction of synthesis of new enzyme.
The detection of this response of the cerebral dehydrogenase came al-
most simultaneously from independent studies on rat and human
brain, based on the use of quite different techniques. Both were acci-
dental findings. Working with rats, Sokoloff and his co-workers had
become interested in the effects of thyroxin on the developing brain,
in particular its effects on the in vitro incorporation of amino acids into
mitochondrial protein. In such studies, oxidisable substrates (such as
succinate) are presented to the incubated mitochondria to ensure
adequate endogenous supplies of ATP. One of these, f3-hydroxybuty-
rate, was found to support the amino acid incorporation in the mito-
chondria from immature rat brain far more effectively than in
mature mitochondria, due to the efficiency of ATP formation
(Fig. 4.1). Further investigation confirmed that the immature
mitochondria were relatively rich in f3-hydroxybutyrate dehydro-
genase activity (Figs. 4.1, 4.2) whereas the cerebral mitochondria
from mature animals contained very little [7]. Comparisons
of the changes in mitochondrial enzyme activities during develop-
ment showed that, whereas enzymes of the tricarboxylic acid cycle or
of the respiratory chain (e.g. cytochrome oxidase, Fig. 4.1.) increased
over the first few weeks of life to reach a stable level, retained in the
adult, the f3-hydroxybutyrate dehydrogenase activity rose in the first
few weeks, but fell immediately after the young rats were weaned.
Prolongation of weaning resulted in a delay in the fall of the dehy-
drogenase activity and analysis of the maternal rat milk revealed that
it was rich in ketone bodies (f3-hydroxybutyrate and acetoacetate).
The conclusion was that the presence of the substrate had induced
enzymic activity which disappeared when the substrate was no longer
present. However, attempts to re-induce the activity in adult rats by
starvation (when the f3-hydroxybutyrate concentration in the blood

56
 ATP formation
c -
~

:J
o
E
<II
III
>
.;::;

1!'"

/3 - Hydroxybutyrate Succinate
(a)

Enzymic activity

Cytochrome
oxidase

~
:~
t)
<II
o
'E
>
N
C
UJ

p- Hydroxybutyrate
dehydrogenase

90 120
Days after birth
(b)

Fig. 4.1 Metabolism of /3-hydroxybutyrate in young and adult rats. (a): ATP
formation in the mitochondria occurs at similar rates in the presence of succinate
or Ot-oxoglutarate. When /3-hydroxy-butyrate is the substrate, mitochondria from
the adult (clear column) produce less ATP than from the young animals (dark
column). (b) : /3-Hydroxybutyrate dehydrogenase falls after weaning at about 4 weeks
of age, whereas cytochrome oxidase activities of the same mitochondrial
preparations remain constant [7].

57
 (i)
CH 3 . CHOH . CH 2 . COOH + NAD ====-
+
CH 3 . CO . CH 2 . COOH + NADH + W

o (-) 13- Hydroxybutyrate Aceto-acetate

I ,- CH 2 · CO . SCoA
(ii) 1/ I
CH 2 . CO. SCoA
Succinyl CoA

( iii)
CH 3 ' CO. SCoA + CH 3 . CO. SCoA~ CH 3 . CO. CH 2 · CO. SCoA + succinate
2 x Acetyl CoA Aceto-acetyl CoA

1
Tricarboxylic Acid Cycle -----ATP

(i) 13 - Hydroxybutyrate dehydrogenase (EC 1.1.1.30)

(ii) 3-0xo acid-coenzyme A transferase (EC 2.8.3.5)
(iii) Acetoacetyl-coenzyme A thiolase (EC 2.3.1.9)
Fig. 4.2 Oxidation of ketone bodies.

stream becomes elevated as a result of mobilisation of depot fats) prov-

ed unsuccessful. The net utilisation of the fJ-hydroxybutyrate by the
brain increased, but the amounts of available dehydrogenase activity
remained unchanged; the enzyme under fed conditions was presumably
far from being saturated with substrate. So it was argued that in the
adult, the enzyme normally present, though much less than in the
young animal, remained constant and that the increased utilisation of
ketone bodies on starvation was due to increased availability of
substrate rather than to induction of enzymic activity [8]. Further
support for this conclusion was given by observations of increased
cerebral utilisation of acetoacetate in adult rats after acute infusion of
the acetoacetate [9].
In the same year as Sokoloffs initial experiments on the rat, a
similar type of adaptation was reported to occur in man. Prolonged
fasting of obese patients had been carried out under clinical conditions
which allowed sampling of arterial and venous blood. After 5 to 6
weeks' starvation on a diet which contained only water, salt, vitamins
and flavouring, their mental ability was unimpaired, yet the arterio-
venous difference was such that insufficient glucose to maintain
consciousness was apparently being used by the brain. The respiratory
quotient (from measurements of O 2 and CO 2 ) was only 0.63 compared
with the normal value of close to one. Oxygen consumption was in the
normal range, so some substrate other than glucose must have been
consumed. Analysis of the arterial and venous blood showed that
fJ-hydroxybutyrate was being consumed at a rate significantly greater
than that of glucose (Fig. 4.3). It is well established that under normal
conditions the mammalian brain cannot replace glucose as its major
energy source. Few substrates are effective in reversing hypoglycaemic
coma and those that are capable of this are thought to be converted to
glucose elsewhere in the body before utilisation in the brain. Yet here
58
 Excreted from the brain Removed by the brain

Amino acids

Glucose

~ - Hydroxybutyrate

Lactate

0·2 0 ·1 0 0·1 0·2 0·3

Blood concentration of metabolite (nett change, mM)
Fig. 4.3 Cerebral arterio-venous differences during starvation [10].

was a condition in which the human brain was clearly capable of

consuming an alternative to glucose, fJ-hydroxybutyrate [l0],
At present it is not clear if similar mechanisms operate in the adapt-
ation to circulating ketone bodies in man and the rat because com-
parable experiments have not been performed on both species. We do
not know the time course, after beginning the fasting of obese humans,
of the rise in blood concentration of fJ-hydroxybutyrate and whether
the cerebral dehydrogenase activity remains constant or rises during
the period of starvation. For the moment it is assumed that the situa-
tion is similar; i.e., that in man. the enzymic activity remains constant
and that the increased consumption of fJ-hydroxybutyrate reflects
its increased concentration in the circulation. We therefore
are forced to think in terms of two processes: the first a form of
adaptation. is the change in enzyme content of cerebral mitochondria
during development in which the amount of enzyme present
does appear to be conditioned by the substrate available. and
which does not seem to occur in the adult brain. The second is the
increased rate of utilisation of the substrate due to its increased avail-
ability, and is therefore not really an adaptive process since it might be
expected to occur generally with any enzyme system whose substrate
changes from sub-saturating to saturating concentrations. An alter-
native form of adaptation which might be present is in the transport
of the ketone body from the blood to the brain, This is not possible
to assess in the human starvation experiments without the appropriate
time courses of change in blood levels and arterio-venous difference in
the fJ-hydroxybutyrate; prolonged starvation of rats has not proved to
result in increased enzymic activity but could result in increased rates
of transport. In rat brain slices incubated in vitro, preparations from
immature animals oxidised acetoacetate more rapidly than those
from mature animals so the possibility of changes in the capacity for
,transport of ketone bodies can now be studied in immature and mature
rat brain in vitro [I I].
59
 Subsequent studies in vivo have indeed indicated that the capacity
of the transport system is increased on starvation; the investigators
observed an increase in V with no change in K m , and suggested an
induced increase of carrier protein had occurred [12]. This remains to
be confirmed, which might prove difficult. While the use of inhibitors
of protein synthesis can provide circumstantial evidence for induction,
firm evidence rests on proof of increased amounts of protein, usually
by isolation or by immunochemical means which depend on isola-
tion and purification. However while evidence for the presence of
'carrier' proteins in cerebral systems is sound, none has been isolated
to date. The changes in P-hydroxybutyrate and acetoacetate concen-
trations, especially in the case of the immature rats, are essentially
dietary changes; the example which follows involves regulation of
the available amounts of a cerebral enzyme with substrate induction
and product repression.
Glutamate decarboxylase catalyses the formation of y-aminobuty-
rate (GABA) from glutamate (Chapter 3). Both GABA and the
decarboxylase are mainly neural in occurrence, little of either being
found elsewhere in the mammalian body. The functional importance of
the decarboxylase in the brain is that it helps to regulate the relative
proportions of glutamate (an excitant amino acid) and GABA (an
inhibitory amino acid, almost certainly an inhibitory neurotransmitter;
Chapter 3). The enzyme has been shown to occur in the nerve endings.
The activity of mouse brain glutamate decarboxylase, measured in vitro,
was almost doubled some 4hr. after an intraperitoneal injection
of its substrate, L-glutamate. During this period the concentration
of glutamate in the brain increased by less than 50% [13]. Other related
amino acids (L-aspartate, OL-glutamine, GABA) or cortisol were
ineffective. If the animals were pretreated with an inhibitor of protein
synthesis, actinomycin 0 (2mg/kg body weight), the effect of glutamate
was diminished. The possibility that the increase in enzymic activity
had been due to substrate-protection of a labile enzyme was considered
unlikely since the slow decrease in enzymic activity of incubated brain
slices was not affected by the presence of glutamate in the incubation
medium [13]. It seems plausible, therefore, to consider that the increase
in enzymic activity was due to substrate-induced synthesis of new
enzyme. These results are slightly surprising in that many workers have
found that glutamate cannot easily be caused to accumulate in the
brain although it exchanges between blood and brain relatively
readily (see Chapter 2).
Synthesis of this enzyme may also be subject to suppression by the
product; in vivo accumulation of GABA is followed by diminished
enzymic activity. GABA also is not readily taken up from the blood-
stream to the brain so in these experiments an inhibitor of GABA
catabolism was used to effect its accumulation. A subcutaneous injec-
tion of amino-oxyacetic acid into young mice caused a 5-fold increase
in brain GABA content within 6hr. which was followed during the
course of one day by a decrease in glutamate decarboxylase activity
60
 HO
O I
./-,>.
CH'·TH.COOH

NH,

Tyrosine '""", "'>

~,?

.,
~tj.6

H O O CH,. TH . COOH '~CH' CO.COOH

I NH,

HO ./- HOV

3.4- Dihydroxyphenylalanine p- Hydroxyphenylpyruvate

(DOPA)

Fig. 4.4 Alternative pathways of tyrosine metabolism.

[14]. The use of an inhibitor to produce accumulation of an endo-

genous chemical presents problems not faced if the accumula-
tion could be achieved more directly by elevating its concentration
in the bloodstream, so various potential interpretations alternative
to that of supression of enzyme synthesis required assessment. Gluta-
mate decarboxylase is a pyridoxal phosphate requiring enzyme [15];
indeed, apart from pyridoxal kinase, it is the cerebral enzyme most
sensitive to reagents which interfere with binding of pyridoxal phos-
phate. Moreover, like all transaminases, the enzyme which removes
GABA and which in these experiments was inhibited to cause its
accumulation, GABA-a-oxoglutarate transaminase, also requires
pyridoxal phosphate. Pretreatment with pyridoxal phosphate was
found to reverse the effect of amino-oxyacetic acid in that the decrease
in decarboxylase activity was prevented. Other pyridoxine antagonists
were tested~those which caused no change in GABA concentra-
tion (thiosemicarbazide and hydroxylamine) had no effect on the
decarboxylase activity, whereas hydrazine, like amino-oxyacetic
acid, caused both the increase in GABA and the decreased enzymic
activity. The diminution of activity is not likely to be due to direct
product inhibition of decarboxylase by GABA because it does not
inhibit the enzyme in vitro; the results seem therefore to be consistent
with suppression of enzyme synthesis by GABA. The adaptive pro-
cesses which affect the enzyme which produces GAB A may thus
provide a potentially effective and sensitive means of regulating
presynaptic GABA levels.

4.1.2 Adaptation to the product of an alternate pathway

A fascinating example of the product of one enzyme causing an
adaptive response in an enzyme of an alternate pathway in the brain is
given by the stimulation of tyrosine transaminase by 3, 4-dihydroxy-
phenylalanine (Dopa), which is produced by tyrosine hydroxylase
(Fig. 4.4; see also Chapter 3). Brain biogenic amine concentrations can
be decreased by administration of reserpine, which depletes catechol-
amines and serotonin (Chapter 3) by preventing their storage; treat-
61
 Table 4.1 Effect of endogenous L-3, 4-dihydroxy-
phenylalanine (Dopa) on cerebral tyrosine trans-
aminase activity [16].

Treatment Enzymic activity

!% of untreated)
rt.-Methyltyrosine 60
rt.-Methyltyrosine + Dopa 110
Reserpine 65
Reserpine + Dopa 105

ment with IX-methyl tyrosine acts more specifically to decrease cate-

cholamine concentrations by inhibiting tyrosine hydroxylase, but not
tryptophan hydroxylase. Treatment of animals for some hours with
either of these reagents caused a marked decrease in the cerebral
tyrosine transaminase activity [16]. This was reversed by pretreat-
ment with Dopa (Table 4.1). Presumably the transaminase is not
directly affected normally (i.e. in the untreated animals) by Dopa
since the enzymic activity measurements were performed on tissue
extracts in vitro, so it seems possible that the presence of Dopa in-
duces formation of the active transaminase enzyme. This is not certain,
however, especially without directly testing the effects of Dopa on
the isolated transaminase and without some assessment of the effects
of inhibitors of protein synthesis. Normally cerebral transaminase
capacity is some 100 times that of the hydroxylase, as measured in
vitro, so that relatively little tyrosine would be available for Dopa
formation. However, if the endogenous concentrations of Dopa
should fall, the decreased transaminase activity should render more
tyrosine available for Dopa formation. It seems possible, therefore,
that Dopa might act normally to suppress synthesis of the trans-
aminase. In that case, inhibitors of protein synthesis would be expected
to prevent the increase in enzymic activity observed as a result of
pretreatment with Dopa. In contrast to regulation of tyrosine trans-
aminase in the liver [17] this aspect of regulation of the brain enzyme
does not seem to involve the coenzyme, pyridoxal phosphate.

4.1.3 Adaptation involving coenzyme

This has been shown to occur in thiamine-deficient rats, and the
adaptation is not restricted to the brain. Rats made deficient in thia-
mine have lowered mitochondrial pyruvate dehydrogenase activity
in heart, liver and brain [18], even though the activity was measured
in the presence of the coenzyme, thiamine pyrophosphate. Treatment
of the animals with thiamine then results in a recovery of the enzymic
activity to the normal value within 10 to 12 hr.; the recovery is prevent-
ed by prior treatment with actinomycin D or cycloheximide. Labelling
of nucleic acids with uridine and of proteins with 14C-leucine was
stimulated by thiamine in the thiamine deficient animals (Table 4.2),
where the response was greatest in the brain.
62
 Table 4.2 Cerebral metabolic response to thiamine in thiamine-
deficient rates [18]

Treatment Pyruvate Uridine

dehydrogenase incorporation
(% of control) (% of control)
Thiamine 160 2500
Thiamine + actinomycin 0 106 100
Thiamine + cycloheximide 114

4.1.4 Adaptation in response to hormones

Removal of the adrenals or the pituitary gland from adult rats is
followed by an exponential decrease in glycerolphosphate dehydro-
genase activity of the cerebral hemispheres and especially of the brain
stem (Fig. 4.5). The activity depleted by adrenalectomy could be
restored by injections of cortisol and that depleted after hypophysec-
tomy was restored by injections either of cortisol or of adrenocorti-
cotrophic hormone (ACTH). The enzymic activity in the livers of the
same animals was not affected. Two types of glycerolphosphate
dehydrogenase activity are present in the mammalian brain, as in other
organs: a soluble cytoplasmic NAD+ -requiring enzyme (EC 1.1.1.8)
and a mitochondrial flavoprotein enzyme (EC 1.1.95.5); only the
cytoplasmic enzyme was found to be involved. The mitochondrial
activity and other mitochondrial enzymes (malate dehydrogenase,
isocitrate dehydrogenase) were not affected. Another cytoplasmic
enzyme, lactate dehydrogenase, was also unchanged throughout. The
results of these studies led the investigators to the conclusion of a
specific regulation of cerebral glycerolphosphate dehydrogenase
activity by corticosteroids [19].
The adult mammalian brain contains relatively little of this dehy-
drogenase activity and the maximum peak of activity is reached only
late in the immature brain during development, at a stage coincident
with later stages of myelination. In the developing rat brain this is
between 5 and 6 weeks of age. The rate of increase in enzymic activity
can be accelerated during the first two weeks of age by treatment with
cortisol, and the normal development of the activity can be retarded by
adrenalectomy or hypophysectomy (Fig. 4.5b) and also by X-ray
irradiation. Myelination in the central nervous system is associated
with glial cells (Chapter 2) and requires active lipid synthesis for which
the glycerol3-phosphate is needed. Cultured glial cells have accordingly
been used to study this aspect of hormonal regulation: cortisol in the
culture medium was then shown to induce glycerol-phosphate de-
hydrogenase activity, which was prevented by the presence of inhibitors
of synthesis of RNA and proteins. The induction occurred with a
variety of corticosteroids. but not with steroid sex hormones, insulin or
cyclic AMP. Other enzymes tested were not affected by cortisol. During
the course of these studies on glial cell cultures, lactate dehydrogenase
activity (not affected by treatment with corticosteroids) was increased
63
eE ~ V
0
(,) (Liver) (Muscle)
80
'0
?f2.

60:-
I
!

J 0
I
10
I
20
I
30
Days after adrenalectomy
(a)
..,>
.s; 50
.~
co
(,)

·E
>
N
40
c:
OJ

'0 Cerebrum (control)

l!l
·c 30
;:)

20

10

30 40 50 60
Days of age (hypophysectomy at 20 days of age)
(b)
Fig. 4.5 Rat brain glycerolphosphate dehydrogenase activity [19].

significantly after the addition to the medium of adrenaline; the results

of these studies have been interpreted in terms of regulation of glial
lactate dehydrogenese activity by adrenaline.
The possible involvements of corticosteroids in cerebral adaptation
are often assessed by following the effects of adrenalectomy or by
treatment with dexamethasone or cortisone. Use of such techniques has
indicated that these hormones contribute to regulation of many
transmitter-synthesizing enzymes, such as tryptophan hydroxylase
[20] and the phenylethanolamine N-methyltransferase which converts
noradrenaline to adrenaline [21].
64
4.2 Adaptation to the environment

4.2.1 Light
The retina can be regarded as the external part of the nervous system
and vision has been the sense most studied in terms of mechanisms of
adaptation to the external environment. Fibres pass from the retinal
ganglia. as the optic nerve. to the lateral geniculate body and thence to
the visual cortex (Chapter 2).
Structural changes in the visual cortex are known to result from
visual deprivation: if the animals are reared in darkness, morophologic-
al development of the visual cortex is impaired [22]. Light stimulation
following visual deprivation also causes changes in the size and number
of synapses in the visual cortex. Less clear have been the biochemical
findings. Visual deprivation causes decreases in the content of RNA
in retinal ganglion cells and in the visual cortex. The number of poly-
ribosomes may also vary according to the environmental conditions.
First exposure to light of rats reared in darkness results in a transient

H 10 MT activity

I I I I I I

Noradrenaline

30

oL-~-'----'----L~_~_~~I~-----"-_ ~
8 12 16 20 24 4 8
Time of day
Fig. 4.6 Diurnal variation of serotonin. noradrenaline and 5-hydroxyindole O-methyl
transferase activity (HIOMT) in rat pineal gland [26, 27). The hatched bars give the
periods of darkness.

65
increase in rates of 3H-lysine incorporation into proteins of the visual
cortex [22], and the brains of rats habituated to darkness have been
reported to contain increased numbers of polysomes after exposure
to light. In these animals, the light stimulation also caused increased
rates of protein synthesis, which result correlates well with the in-
creased number of polysomes [23]. However in a split-brain monkey
preparation, unilateral visual stimulation had no effect on rates
of protein synthesis in the subcellular fractions prepared from various
regions [24]. Presumably the increase due to light stimulation does
not occur in animals habituated to light, but requires prior visual
deprivation. I t should be noted that the proportion of ribosomes
present as polysomes can also be changed by temperature variation
and by convulsions [15].
Other biochemical observations in the visual cortex on adaptation
to light include changes in assayable tubulin (Section 3.7.1) and in
muscarinic receptors (Section 3.6.2) [25].

4.2.2 The pineal gland

The incidence of light causes an adaptive change in the biochemistry
of the pineal gland, and provides an excellent example of enzyme
induction caused by a sensory stimulus. The content of 5-hydroxy-
tryptamine (serotonin) of the pineal gland was noted in Chapter 3
(Table 3.2) to be subject to diurnal variation, being high by day and low
at night. In contrast, the serotonin of the hypothalamus remains at a
relatively constant concentration and the variation in this amine seems
peculiar to the pineal. Noradrenaline concentrations also vary in the
reverse direction. being higher in the dark than in the light (Fig. 4.6).
The mammalian pineal gland has evolved from a visual sensory organ
of more primitive animals: in amphibian brains, for example, the
equivalent is found as a photoreceptive area on the roof of the brain.
The primitive pineal responded directly to light with direct transduction
of the stimulus to nerve impulses. The mammalian pineal responds
indirectly to light; the nerve impulse generated in the photoreceptors of
the retina is carried by fibres of the optic nerves to the optic
chiasma where the nerve tracts branch. One branch, the inferior ac-
cessory optic tract, carries the impulse to the pineal. Little is known of
the nerve fibre connections from this tract to the pineal, but innervation
of the gland is by sympathetic nerves from superior cervical ganglia
[26]. The pineal appears to receive no direct input from the rest of the
nervous system and does not send fibres to other parts of the brain.
The changes from light to darkness are associated with changes in
another indole derivative, 5-methoxy- N -acetyltryptamine (,melatonin ').
This is formed from serotonin by the acetylation and methyl transfer
reactions shown in Fig. 4.7. The adaptive response to darkness involves
release of serotonin and regulation of the enzyme which forms mela-
tonin: 5-hydroxyindole O-methyl transferase (HIOMT; Fig. 4.7).
The formation of serotonin itself is not susceptible in this way to
changes in illumination; it is synthesised in the pineal at a relatively
66
 Serotonin N-acetylserotonin
CH 2 NH2 CH 2 ·NH.CO.CH,
N -acetylase

P
CH 2 . NH . CO . CH,

Melatonin

Fig. 4.7 Synthesis of melatonin (5-methoxy-N-acetyltryptamine). Serotonin is formed

from tryptophan (Chapter 3). Melatonin is excreted after hydroxylation and
conjugation in the liver as the sulphate glucuronide.

constant rate. However more serotonin is released from its storage

sites in darkness than in light and so becomes more accessible to
enzymes concerned with its further metabolism: HIOMT and mono-
amine oxidase. Part of the released serotonin is destroyed by the oxidase
and part is converted to melatonin by HIOMT (Fig. 4.7). The methyl
donor for methylation of N-acetylserotonin is S-adenosylmethionine.
As noted above, the level of noradrenaline is out of phase with the
pineal content of serotonin. During daylight hours, noradrenaline is
low and serotonin is high. HIOMT activity is also lower during the
day which indicates that the illumination indirectly causes inhibition of
the enzymic activity. It is not clear which of two alternative processes
occurs: either the arrival of the nerve impulse results in inhibition of
release of a transmitter which normally stimulates or induces HIOMT,
or it results in increased release ofa transmitter which inhibits HIOMT.
Thus it is not certain if the increase in HIOMT can properly be re-
garded as enzyme induction because the change in activity could equally
be due to suppression of enzyme synthesis in daylight. Some indications
that induction may occur have come from studies on the effects of
noradrenaline in increasing melatonin synthesis in pineal glands
maintained in an organ bath. The effects were blocked by cyclohexi-
mide. The serotonin N-acetyl transferase of Fig. 4.7 also responds
to the dark (it is 30-50 times more active in the dark than in daylight)
and to catecholamines. The activity of the enzyme in the pineal gland
in vivo is activated by catecholamines (Dopa, noradrenaline and
adrenaline) and by amine-oxidase inhibitors which prevent breakdown
of catecholamines. Involvement of cyclic AMP was indicated by the
observation of activation of the N-acetyl transferase by theophylline
(which inhibits breakdown of cyclic AMP by phosphodiesterase). The
increased enzymic activity was prevented by treatment with inhibitors
of protein synthesis and by propranolol (which blocks adrenergic
j3-receptors; Chapter 3). The results of these studies suggest that new
transferase enzyme is synthesised as a consequence of adrenergic
67
stimulation of pineal !3-receptors, and involves activation of forma-
tion of cyclic AMP [28]. Thus the two enzymes involved in melatonin
formation from serotonin (Fig. 4.7) are known to respond to cate-
cholamines: it seems reasonable to conclude from the evidence avail-
able at present, that the light-stimulated nerve impulse releases
noradrenaline from the pineal, thus making less available for enzyme
induction [29].
The role of melatonin in mammals seems to be essentially inhibitory:
it inhibits thyroid function and sexual maturation in both males and
females. It also appears to suppress the secretion of luteinizing hor-
more (LH) and it is possibly involved in regulation of adrenocortical
function although the results of research on this aspect are confusing
and sometimes contradictory [26].

4.3 Drug tolerance and dependence

The phenomenon of drug addiction involves a process whereby a
chemical substance which is foreign to the body becomes an intrinsic
part, essential for continued maintenance of normal function. Nor-
mally, first acquaintance with an addictive drug elicits a positive
response by the body. This may be an unpleasant or toxic response or,
as in the case with analgesics and certain stimulants, the response may
be pleasurable or euphoric. If, on continued exposure to the drug,
these responses are lessened or disappear, tolerance to the drug has
developed. If the individual is only 'well' during continued exposure
to the drug, and becomes 'ill' when it is withdrawn, then a state of
physical dependence on the drug has been set up.
Tolerance is usually assessed by measuring the quantitative response
to standard doses of the drug. The response of a tolerant animal is
expressed as a percentage of its response before exposure to the drug
or of the response of a control group of animals. Physical dependence is
assessed by measuring the degree of illness experienced on withdrawal
of the drug or on administration of a specific antagonist to the drug.
The mechanism of the body's response (tolerance or dependence)
to a foreign drug may be similar in principle to the mechanisms which
underly the effects of many of the body's normal endogenous chemicals
in modifying physiological and biochemical function by affecting
the rate of synthesis of a specific enzyme; the chemical may bear no
obvious structural resemblance to the chemicals (substrates and
products) normally involved with that enzyme. Many workers have
therefore approached the phenomenon of drug tolerance and de-
pendence on the assumption that induction or repression of enzyme
synthesis is involved [30). A molecule which acts as an enzyme inducer
needs to have no structural relationship with the enzyme because it
presumably acts at sites, remote from the enzyme itself, to dissociate
specific repressor protein molecules from their sites on the cellular
DNA in the nucleus. It seems possible also that,just as enzyme synthesis
is modifiable in this way, the synthesis of receptor proteins could also
be affected, so as to render the receptor either more or less sensitive
68
to its transmitter, and we need therefore to think in terms of induction
of synthesis of non-enzyme proteins as well as of enzymes. The major
criterion for assessing de novo synthesis of proteins and enzymes
rests on the use of inhibitors of protein synthesis; while these may be
specific in their sites of interaction within the sequence of RNA and
protein synthesis, they are quite unspecific in that they are likely to
inhibit protein synthesis indiscriminately. A table of some of the side
effects of these inhibitors is given by Shuster [30]. One means of
circumventing the grosser artefacts which might result from this
lack of specificity is to use methods of developing tolerance and de-
pendence in animal models sufficiently rapidly (within a few hours)
so that tests with the inhibitors of protein synthesis are less likely to
have indiscriminate effects. This approach has been used successfully
by Cox and his co-workers studying the morphine tolerance and
dependence [31] which follows.

4.3.1 Morphine
Addiction to morphine, the alkaloid principle prepared from opium,
often results from its legitimate prescribed use as an analgesic and
a great amount of effort has been expended in attempts to find a
pharmacological agent which would prevent the addiction without
impairing analgesic function. Great hope that this may be soon
achieved arose from the discovery of the endogenous opiates, enke-
phalins and endorphins (see Chapter 3). However, though they react
with central morphine receptors and produce analgesia, the evidence
currently available indicates that they too are addictive. While it
may prove feasible to produce analogues which are analgesic and
non-addictive, this may depend on whether the analgesia is mediated
by the same mechanisms as are involved in the euphoria which seems
an integral part of the addiction; only if these are different are our hopes
of separating the responses likely to be realised. It has proved relatively
easy to produce animal models: the rat can be made tolerant to mor-
phine, either by subcutaneous implantation of the drug as a pellet,
or by intravenous infusion, where tolerance can be shown to be
developed within a few hours (Fig. 4.8). The animals are then in-
sensitive to the analgesic effect of some 20 times the normal dose.
Their sensitivity to pain in these studies is usually measured by their
response to a tail-flick. The development of tolerance to the drug is
not due to increased destruction or excretion of the drug, and can
be prevented by inhibitors of protein synthesis. The results, Fig. 4.8b,
show that actinomycin D does not overcome tolerance already esta-
blished; it only affects the rats during the period when tolerance is
developing. Use of a range of inhibitors of RNA and protein synthesis
showed the development of tolerance to depend on synthesis of new
messenger RNA [31]. This knowledge has led to attempts to identify
the enzymes (or proteins) whose synthesis might be induced or sup-
pressed by morphine. The brains of tolerant animals show changes in

69
 Morphine, 7·5 mg/kg/hr
',0

)(
0·8 + Actinomycin D, 10 mg/kg/hr
Q)
"C
.!:
u 0·6
'(jj
Q)
Cl
"iii
c:
III0'4l Morphine, 7·5 mg/kg/hr
c:
III
Q)

::!
0·2

0 2 4

(a)

1·0
rDay 1(0)

0·8

)(
Q)
"C
.!:
u 0·6
'(jj
Q)
Cl
"iii

o·t
c:
III
c:
III
Q)

::!

0·2
V DaY3 (t.)
Day 4(.)

0 4 6 8
Time (hr)
(b)

Fig.4.8 Development of morphine tolerance in rats [31]. (a): Morphine was infused
intravenously into rats and the development of tolerance (when the analgesic effects
of the drug were lessened) was prevented by the presence of the inhibitor, actinomycin
D. (b): The tolerance to morphine increases with subsequent days of infusion.
The presence of actinomycin D in the infusion fluid on the 4th day of infusion (0)
shows that it affects the tolerance only during the period when it is developing,
but does not overcome the tolerance previously established.

70
respiration and in the turnover of phospholipids, especially of tri-
phosphoinositide, a phospholipid component of synaptic membranes.
Various neurotransmitters, implicated by different workers, include
acetylcholine, serotonin, noradrenaline and GABA, from observa-
tions that their antagonists affect the rate of onset and the degree of
tolerance attained. The development of physical dependence to the
drug, based on physical and behavioural manifestations which occur
when the drug is withdrawn, has also been suggested to involve one
or all of these transmitters. The one enzyme found to be increased
during the development of tolerance so far is tryptophan hydroxylase
(involved in formation of serotonin, Chapter 3). Cyclic AMP seems
to be involved in the effects of serotonin and its antagonists on the
development of tolerance to and dependence on morphine [32].
Interactions of all neurotransmitters might be affected: results of a
study on the Ca 2 + -activated ATPase suggest this could be the case.
Ca 2 + is an essential requirement for the release of all neurotransmitters
(Chapter 3): the synaptosomal Ca 2 + -ATPase prepared from normal
rats was inhibited by morphine in vitro in contrast to the enzyme from
morphine-tolerant rats, which remained unaffected by exogenous
morphine [33]. If the response to morphine is on transmitter release,
then the specificity of the transmitter involved may be related to the
more sensitive regions of the brain. Lack of interaction of transmitter
with receptor could, from the mechanisms discussed in previous
sections of this chapter, result in increased synthesis of specific enzymes,
such as the increase in tryptophan hydroxylase activity noted above.
This is not the only likely mechanism by which morphine might act:
blockage of transmitter receptors could also result in changes in
synthesis of enzymes involved in transmitter metabolism. The use of
drugs which interfere with serotonergic transmission may not distinguish
between action on transmitter release and action on receptors. p-
ChI oro phenylalanine (PCPA), which inhibits serotonin synthesis,
5, 6-dihydroxytryptamine (DHT), which destroys serotonergic nerve
endings, and methergoline, which blocks serotonin receptors, all
inhibit the development of tolerance and of dependence [32].
The role of the endogenous opiates in pain perception and analgesia
is essentially still a matter for speculation, as is the possibility that
their function is related to acupuncture. Current research on the
mechanisms of function of the enkephalins and endorphins will
determine whether they behave as transmitters or modulators, and
how this is related to release of other transmitters. Such research
should result in further knowledge on the cerebral responses to
morphine, in terms of receptor sensitivity or enzyme induction, and
provide more insight into the mechanisms involved in addiction.

4.3.2 Amphetamines
The group of amphetamine stimulants have long been recognised
as leading to psychological dependence, but unequivocal evidence
for the development of physical dependence is not so well esta-
71
blished. The amphetamines are known to inhibit mono-amine
oxidase and to interfere with re-uptake of catecholamines (Chapter 3).
Prolonged abuse can lead to the development of psychotic
disturbances which tend to disappear on withdrawal of the drug.
Severe addiction. with a high relapse rate. was noted by Connell
[34] to be similar in some aspects to alcohol addiction. Amphetamine
psychosis is a relatively rare consequence of abuse but in Japan,
encouraged use of amphetamines to increase industrial productivity
after the end of the 1939-1945 war led to dependence and psychosis
of almost epidemic proportions. Animal models for psychological
dependence, monitored by self-administration of the drug, proved
relatively easy to develop; models of physical dependence have not
proved so easy to assess, due to variations in behaviour on with-
drawal. The major emphasis on the possible biochemical interactions
of the amphetamines has been on adrenergic transmission [15].
Hyperactivity and stereotyped behaviour in experimental animals
seems to be associated with noradrenaline and dopamine: the effects
are antagonised by propranolol (Chapter 3) and by e.g. rx-methylty-
rosine, which blocks catecholamine formation by inhibiting tyrosine
hydroxylase. Induction of tyrosine hydroxylase by catecholamines
(above) also occurs on administration of amphetamines, and the
increase in enzyme activity is prevented by pretreatment with cyclo-
heximide [35]. Such effects are associated with relatively short-term
administration of the drug. Long-lasting effects on mono-amine
oxidases were observed when methamphetamine was given to guinea
pigs for 3 to 10 weeks: the 30% decrease in activity was reversed on
withdrawal of the drug, when it reached a level some 30% above
normal. Acute administration of the drug resulted in increased cerebral
respiration, which was decreased on chronic administration [36].
The effects of amphetamine on cerebral oxidative metabolism were
assumed to be secondary to the effects on catecholamines, which were
expected to cause changes in mobilisation of glycogen by phosphory-
lase activation mediated by cyclic AMP. This does not seem to be
confirmed by recent studies. Though catecholamine activation,
mediated by cyclic AMP, of cerebral glycogen phosphorylase is
known to occur, the increased rates of cerebral glycolysis after acute
treatment with amphetamines cannot be explained solely in terms
of increased glycogen breakdown. Some evidence for dependence
emerged from these studies. The increase in glycolytic rates shown
after acute administration disappeared on chronic treatment with the
drug, when the rates reverted to normal. Withdrawal of the drug
from chronically-treated rats resulted in rates of glycolysis signifi-
cantly lower than normaL which were similar to those seen in ani-
mals treated with depressant drugs, such as the barbiturates (Fig. 4.9).
Chronic administration of amphetamine to rats also causes profound
changes in cerebral tyrosine hydroxylase activity [38. 39]. only if the
rats fail to develop tolerance to the anorexic eftects of the drug. The
activity recovered within 1 day after withdrawal of the drug (Fig. 4.10).
72
 e-m
.0
a
Ol

----
Ol
.3
Q)
c
m
E 10
Q)
.r::
Q.
E
«

o 30
Time (min) after injection
(a)

c 900, ...0 9000 c

co
.0'" Acute D'" .0
a 800 8000 a
Ol Ol

----
E 700 7000 ----
E
ci. ci.
2- 2-
c 600 6000
Q) ~
Ol co
0
u tl
~ 500,- 5000 .::!
Ol c
C
u
u 400C-- 4000 ~
~

co
~
~

co
0
30) 3000 0
~
I-

..:..- 200 2000 I

100 1000

, _ _"---_ _"--1_ _---'--I _ _ -.-1

4 6 8 10
Time (min) after injection of 14C-glucose
(b)

Fig.4.9 Some effects of amphetamines. (a): Concentrations of 0- or L-amphetamine

in the brain after a single intraperitoneal injection, of 15 mg./kg. body weight, into
mice [40]. (b): Incorporation of 14C from glucose into glycogen (-) and lactate (- --)
in the brains of rats treated with methamphetamine. 'Acute' : one intraperitoneal
injection (5 mg./kg.) I hr. beforehand; 'chronic' : the drug was administered in the
drinking water in increasing concentrations over a 3 week period. Initial intake was
5 mg./kg./day and final intake was 40 mg./kg./day; 'withdrawn': as for chronic
administration but the drug was absent from the drinking water for the final 24 h.
period. The chronic groups gave results identical to those of the control groups of
rats [37].

73
 *

80

~
~ 60
u
'0
Ql
0>
E 40
•
c:
Ql
u
'-
Ql
Q.

Cerebral Striatum Nucleus Hypothalamus Amygdala Pons/Medulla

cortex accumbens
IIID Chronic administration for 15 days ~ Withdrawn from the drug after 15 days
• Chronic administration for 30 days Em Withdrawn from the drug after 30 days
• Significantly different from controls '" Significant change on withdrawal
Fig. 4.10 Tyrosine hydroxylase activity expressed as percentage of activity in sham-
treated controls, in various regions of rat brain after treatment with methamphetamine
[39].

The actIVIty was unchanged in rats which developed tolerance.

Contents of noradrenaline and dopamine. and of their metabolites.
were also decreased in conditions where tyrosine hydroxylase activity
was decreased. Thus biochemical interactions of the amphetamines
have been shown to involve synthesis, transport and destruction of
catecholamines and also to affect glycolysis in the brain. While the
relationship between the two areas of metabolism remains obscure.
both can be used as criteria of the development of dependence. with
the striking metabolic changes which occur on withdrawal (Figs. 4.9,
4.10).
4.3.3 Ethanol
Adaptation to ethanol involves both increased peripheral metabolism
of the drug and alterations in cerebral function. The major metabolites
which appear in the body fluids as a result of oxidation of ethanol,
predominantly in the liver, are acetaldehyde and acetoacetate. The
development of physical dependence on alcohol in man is shown in
the hallucinations and tremors which occur on its withdrawal. The
most pronounced enzymic adaptation to alcohol which has been
observed is the increase in liver alcohol dehydrogenase, an NAD + -
requiring enzyme which produces acetaldehyde. The increase is
74
thought to be due to substrate induction of enzyme synthesis, because
it can be prevented in rats by cycloheximide or by actinomycin D [15].
While the toxic effects of ethanol can be attributed largely to the
unpleasant effects of acetaldehyde, the hallucinations and other
physical and pharmacological effects of dependence, and particularly
of withdrawal, suggest specific neural interactions. In particular, the
hallucinations have stimulated an interest in the metabolism of bioge-
nic amines; increased urinary excretion of tryptamine pointed to a
possible effect on serotonin metabolism [41]. While increased dopamine
breakdown occurs in the striatum with acute ethanol treatment, this
disappears on chronic administration, when changes in sensitivity of
dopamine receptors have been observed. Also chronic treatment
resulted in increased levels of metenkephalin there [42]. Despite
many studies on the effects of ethanol on the storage, release and
oxidation of serotonin and catecholamines, their involvement in the
adaptive response by the brain to ethanol remains unclear [43, 44].
It seems appropriate to note that oxidation of alcohol in the brain by
alcohol dehydrogenase, an NAD-linked enzyme, will tend to lower the
ratio ofNADH to NAD+ which in turn would affect a wide variety of
intermediary metabolic processes [45].

4.4 Learning and memory as adaptive processes?

The recent increase in our understanding of the genetic code and
transference of biological information has prompted renewed interest
in possible macromolecular mechanisms of learning and memory.
Memory fixation has been observed to occur in 2 phases: 'short-
term' memory which lasts about 30 min., which is then fixed as 'long-
term' memory. Studies based on lesions of various brain regions and
consequent disturbance of memory have indicated areas of the hip-
pocampus and amygdala of the limbic system to be associated with
short-term memory, and the 'association regions' of the cortex (Chapter
2) with long-term memory. Due to the knowledge that transfer of
genetic information involves nucleic acid and protein molecules,
much emphasis has been placed by biochemists on these. The cau-
tions necessary in interpreting the results from the use of inhibitors
of protein synthesis (Section 4.3) should be kept in mind but it seems
to be agreed that inhibitors such as puromycin and cycloheximide,
while having no effect on short term memory, can prevent fixation
as long-term memory [46].
Most current theories on memory fixation involve aspects of synaptic
modification, i.e. an adaptive response of synaptic connections to
a learned situation either by activating hitherto dormant inactive
synapses or by rendering previously active synapses inactive. This
could be by modifications in proteins (or lipids) of the lipoprotein
synaptic membranes so as to change their conformation and permea-
bility properties, or to change the sensitivity of specific receptor sites.
Such thoughts have led to searches for 'memory molecules' which might
cause such changes. so far usually as proteins or peptides. Many ex-
75
periments which are based on changes in the random incorporation of
isotopically labelled amino acids into large and diffuse protein fractions
of the brain seem difficult to accept: it appears unlikely that a change
in rate of synthesis of a proteins or small family of proteins, in res-
ponse to the learning of a relatively simple task, could be easily detected
since all of the proteins in the tissue sample will incorporate the label.
Any change in a small proportion of the proteins (or lipids or nucleic
acids, as the case may be) seems almost certain to be masked by the
lack of change in the majority of the related macromolecules. More
acceptable is the attempt to study the labelling of specific proteins
or other molecules peculiar to the nervous system; some progress has
been made in this direction [47].
One major handicap in deciding which type of experiment is likely
to prove fruitful is the uncertainty of the 'memory sites'. While it is
accepted that certain areas noted above are associated with memory,
it is not at all clear if the sites are specific. Animallesioning experiments
indicate little or no cellular specificity whereas the evidence from
studies on man suggests the opposite [48]. It has been difficult to
demonstrate association of any specified localised part of the brain
with any particular learning or memory situation, so the possibility
of an analogy to the hologram has been raised. This is at least compa-
tible with the idea of the relatively wide distribution of a memory
molecule, which reacts on numerous synapses, rather than activation
of specific synapses. This is discussed in detail by Rose [48] who
proposes a combined hypothesis - the 'redundant network, modifiable
synapse' theory-which includes the ability of the brain to replace
loss of function in one site by the same function elsewhere, thus allow-
ing the whole of the organ to continue to function if one part should
fail, with the basic assumption of synaptic modification noted above.
References
[I] Kandel, E. R. and Spencer, W. A. (1968), 'Cellular neurophysiological
approaches in the study ofleaming'. Physiol. Revs. 48, 66-134.
[2] Thoenen, H. (1972), 'Neuronally mediated enzyme induction in adrenergic
neurones and adrenal chromaffin cells'. Biochem. Soc. Symp. 36, 3-15.
[3a] Mueller, R. A., Thoenen, H. and Axelrod, J. (1969), 'Increase in tyrosine
hydroxylase activity after reserpine administration'. J. Pharm. expo
Ther. 169, 74-79.
[3b] Thoenen, H. Meuller, R. A. and Axelrod, J. (1969), 'Trans-synaptic
induction of adrenal tyrosine hydroxylase'. J. Pharm. expo Ther. 169,
249-254.
[4] Black, I. B., Hendry, I. A. and Iversen, L. L. (1971), 'Trans-synaptic
regulation of growth and development of adrenergic neurones in a
mouse sympathetic ganglion'. Brain Res. 34, 229-240.
[5] Mackay, A. V. P. and Iversen, L. L. (1972), 'Increased tyrosine hydroxylase
activity of sympathetic ganglia cultured in the presence of dibutyryl
cyclic AMP. Brain Res. 48, 424-426. 'Trans-synaptic regulation of
tyrosine hydroxylase activity in adrenergic neurones: effect of potassium
concentration on cultured sympathetic ganglia'. Naunyn-Schmiedeberg's
Arch. Pharmacol. 272, 225-229.
76
 [6] Thoenen, H. (1974, 1975), 'Trans-synaptic enzyme induction', Life Sci., 14,
223~235; Adv. Neurol., 6, 67~71.
[7] Klee, G. B. and Sokoloff, L. (1967), 'Changes in the D(-)-f3-hydroxy-
butyrate dehydrogenase activity during maturation in the rat'. J. BioI.
Chern. 242, 3SS0~3SS3.
[Sa] Pull, I. and McIlwain, H. (1971), '3-Hydroxy-butyrate dehydrogenase
of rat brain on dietary change and during maturation'. J. Neurochem.
18, l163~ 1165.
[Sb] Williamson, D. H., Bates, M. W., Page, M. A. and Krebs, H. A. (1971),
'Activities of enzymes involved in acetoacetate utilisation in adult
mammalian tissues.' Biochem. J. 121, 41 ~47.
[9] Hawkins, R. A. (1971), 'Uptake of ketone bodies by rat brain in vivo,'
Biochem. J. 121, 17P.
[10] Owen, O. E., Morgan, A. 0., Kemp. H. G., Sullivan, 1. M., Herrera,
M. G. and Cahill, G. F. (1967), 'Brain metabolism during fasting'.
J. Clin. invest. 46, 15S9~ 1595.
[II] Hoh, T. and Quastel, 1. H. (1970), 'Acetoacetate metabolism in infant and
adult rat brain in vitro'. Biochem. J. 116, 641 ~655.
[12] Gjedde, A. and Crone, C. (1975), 'Induction processes in blood-brain
transfer of ketone bodies during starvation', Am. J. Physiol., 229,
1165~1169.
[l3] Kraus, P. (1970), 'Substrate induction of mouse brain L-glutamate de-
carboxylase'. Hoppe-Seyler's Z. Physiol. Chern. 349, l425~1427.
[14] Sze, P. Y. (1970), 'Possible repression of L-glutamic acid decarboxylase
by gamma aminobutyric acid in developing mouse brain'. Brain Res.
19, 322~325.
[15] McIlwain, H. and Bachelard, H. S. (1971), Biochemistry and the Central
Nervous System. 4th. ed., London: Churchill.
[16] Gibb, J: W. and Webb, J. G. (1969), 'The effects of reserpine, ()(-methylty-
rosine and L-3, 4-dihydroxyphenylalanine on brain tyrosine transa-
minase'. Proc. Nat. Acad. Sci. U.S. 63, 364~369.
[17] Black, I. B. and Axelrod, 1. (196S), 'Elevation and depression of hepatic
tyrosine transaminase activity by depletion and repletion of nore-
pinephrine'. Proc. Nat. Acad. Sci. u.s., 59, 1231~1234.
[IS] Reinauer, H. and Hollmann, S. (1969), 'The co-enzyme-dependent in-
duction of pyruvate dehydrogenase in thiamine deficiency', Hoppe-
Seyler's Z. physiol. Chern. 350, 4O~50.
[19] De Yellis, 1. and Inglish, D. (196S), 'Hormonal control of glycerol phos-
phate dehydrogenase in rat brain'. J. Neurochem. 15, 1061~1070;
Abstr. Second Intern. Neurochem. Meeting (1969), Milan: Tamburini,
p. 152.
[20] Rastogi, R. B. and Singhal, R. L. (197S), 'Adrenocorticoids control 5-
hydroxytryptamine metabolism in rat brain', J. Neural Transm., 42,
63~ 71.
[21] Turner, B. B., Katz, R. J. and Carroll, B. 1. (1979), 'Neonatal corticosteroid
permanently alters brain activity of epinephrine-synthesizing enzyme
in stressed rats', Brain Res., 166, 426~430.
[22] Rose, S. P. R. (1968), 'Biochemical aspects of memory mechanisms' in
Applied Neurochemistry (ed. Davison, A. N. and Dobbing, J .), BlackwelL
Oxford, pp. 356~376.
[23] Appel, S. H., Davis, W. and Scott, S. (1967), 'Brain polysomes: response
to environmental stimulation'. Science, 157, S36~S3S.

77
 [24] Metzger, H. P., Cuenod, M., Grynbaum, A and Waelsch, H. (1967),
The effect of unilateral visual stimulation on synthesis of cortical
proteins in each hemisphere of the split-brain monkey'. J. Neurochem.
14,183-187.
[25] Rose, S. P. R. (1978), 'Macromolecular mechanisms and long-term changes
in behaviour', Biochem. Soc. Trans., 6,844-848.
[26] Wurtman, R. l, Axelrod, l and Kelly, D. E. (1968), The Pineal, New
York, AP.
[27] Axelrod, l, Shein, H. M. and Wurtman, R. J. (1969), 'Stimulation of
14C-melatonin synthesis from 14C-tryptophan by noradrenaline in rat
pineal organ culture'. Proc. Nat. Acad. Sci. U.S. 62, 544-549.
[28] Deguchi, T. and Axelrod, J. (1972), 'Induction and superinduction of
serotonin N-acetyl transferase by adrenergic drugs and denervation
in rat pineal organ'. Proc. Nat. Acad. Sci. U.S. 69, 2208-2211.
[29] Wolstenholme, G. E. W. and Knight, l (ed.), (1971), The Pineal Gland.
London: Churchill.
[30] Shuster, L. (1971), Tolerance and physical dependence' in Narcotic
Drugs (ed. Clouet, D. H.), New York: Plenum, pp. 408-423.
[3Ia] Cox, B. M., Ginsburg, M. and Osman, O. H. (1968), 'Acute tolerance to
narcotic analgesis drugs in rats'. Br. J. Pharmac. Chemother. 33, 245-256.
[31 b] Cox, B. M. and Osman, O. H. (1970), 'Inhibition of the development of
tolerance to morphine in rats by drugs which inhibit ribonucleic acid
or protein synthesis'. Br. J. Pharmac. 38, 157-170.
[32] Way, E. L., Ho, I. K. and Loh, H. H. (1973), 'Some biochemical aspects of
morphine tolerance and physical dependence'. Internat. Neurochem.
Meeting, Tokyo, 3, 32-33,478.
[33] Kaneto, H., Koku, T. and Koida, M. (1973), 'Inhibitory effect of morphine
on synaptosomal Ca 2+ -activated ATPase and its absence in morphi-
nized mice'. Internat. Neurochem Meeting, Tokyo, 3, 479.
[34] Connell, P. H. (1958), Amphetamine Psychosis. London, Chapman and Hall.
[35] Mandell, A J. and Morgan, M. (1970), 'Amphetamine induced increase
in tyrosine hydroxylase activity'. Nature, 227, 75-76.
[36] Utena, H., Ezoe, T., Kato, N. and Hada, H. (1959), 'Effects of chronic
administration of methamphetamine in enzymic patterns in brain
tissue'. J. Neurochem. 4, 161-169.
[37] Manning, D. H., Strang, R. H. C. and Bachelard, H. S. (1974), 'Changes
in cerebral carbohydrate metabolism in the rat after acute and chronic
treatment with, and withdrawal of, methampetamine'. Biochem.
Pharmacol. 23,1205-1209.
[38] Javoy, F., Agid, Y., Bouvet, D. and Glowinski, J. (1974), 'In vivo estima-
tion of tyrosine hydroxylation in the dopaminergic terminals of the
rat neostriatum', J. Pharm. Pharmac., 26,179-185.
[39] Bardsley, M. E. and Bachelard, H. S. (1981), 'Catecholamine levels and
tyrosine hydroxylase activity in rat brain regions after chronic treat-
ment with, and withdrawal of, methampethamine'. Biochem. Pharmac.,
in press.
[40] Benakis, A. and Thomasset, M. (1969), 'Metabolism of amphetamines
and their interaction with other drugs' in Abuse of central stimulants
(ed. Sjoqvist, A. and Tottie, M.). Almqvist and Wiksell, Stockholm,
pp. 409-434.
[41] Schenker, V. J., Kissin, B., Maynard, L. S. and Schenker, A C. (1967),
The effects of ethanol on amine metabolism in alcoholism' in Bio-

78
Glycerolphosphate dehydrogenase, 63 Nucleic acids, 51, 65, 69, 75, 76
Glycine, 29, 30, 40-43 Nutrition, 8
Glycogen, 8, 46, 72, 73
Glycolysis, 36, 72 Opiates, 40, 69, 71
Goldman equation, 22 Organophosphates, 36, 37, 45,50,51
Oxygen, 7, 37, 39, 58
Hemicholinium, 33, 36, 45
Histamine, 39 Peptides, 30, 40, 46, 69
I3-Hydroxybutyrate dehydrogenase, 56, Potassium, 22-27, 43,55
57 Protein synthesis, 50, 51, 65, 76
Hydro6'1ndole.()-methyl transferase, 66, inhibitors, 55, 60, 62, 63, 67-70, 72,
75
Hyperpolarization, 42 Pyridoxal phosphate, 38, 61, 62
Hypoglycaemia, 10 Pyruvate dehydrogenase, 35, 62
Inhibitory transmission, 40-43, 60
Ionophore, 47, 48 Quantum hypothesis, 32, 33

Ketone bodies, 56-59, 74 Receptors, 27-29, 32, 43-48, 65--69, 71,

75
Lactate dehydrogenase, 30, 63 Resting potential, 24, 25
Learning, 8, 75, 76 Ribosomes, 15, 17, 51, 65

Magnesium, 32, 50 Serotonin, 29-34, 37, 38, 61, 65--68, 71,

Melatonin, 66--69 75
Memory, 75, 76 Sodium, 22-27, 36, 43, 47
Methergoline, 29, 71 Starvation, 58--60
Metabolism Strychnine, 29
acetylcholine, 34-37 Substance P, 40, 46
amines, 37, 39 Succinate dehydrogenase, 31, 48
amino acids, 40 Synapse, 16-20, 26, 44-47
energy, 7 Synaptic
regional, 8, 9 modulation, 43, 75, 76
Mitochondria, 15,20,31,40,45,48,56, vesicles, 19, 27, 33, 41, 42, 45
62,63 Synaptosomes, see Nerve endings
Monoamine oxidase, 38, 39, 45, 67, 72
Morphine, 40, 69, 70
Myelin, 16-19,26,31,51,63 Thiamine, 35, 62
Transamination, 40, 61--63
Nernst equation, 22 Transmitters
Nerve identity, 28, 30, 32,41, 71
conduction, 24 occurrence, 28, 33
endings, 17\ 27, 30-36, 49,51,60 Trans-synaptic regulation, 55
Neuromuscular junction, 28, 33, 46 Tryptophan hydroxylase, 37-39, 64, 71
Neurone-axonal flow, 42, 48-50, 55 Tubulin, 50, 65
Neurones, 7,15,17,50 Tyrosine hydroxylase, 45, 54-56, 61, 62,
Neurotoxins, 47, 48 72, 74
Nodes of Ranvier, 17,26
Noradrenaline, 29-34, 42, 54, 65--67, 71, Vesicle hypothesis, 42
72 Vision, 65