Table Of ContentCOMPARATIVE INVERTEBRATE NEUROCHEMISTRY
Edited by G.G. Lunt, Department of Biochemistry, University of Bath, and R.W.
Olsen, Department of Pharmacology, University of California, Los Angeles
Neurochemistry is a rapidly advancing field of research and workers on mammalian
and other vertebrate systems, as well as those interested in insects and other inverte
brates, are turning their attention to invertebrates to show how neural transmission
occurs in 'simple' systems. Hence the subject is attracting interest from many neuro
physiologists and neuropharmacologists as well as from zoologists. It is also of rele
vance in applied fields such as the pesticide industry, since it is possible to produce
pesticides which interfere with nervous transmitter systems of major insect pests, in a
similar way to how drugs act in other systems.
This book presents a review of invertebrate neurochemistry at a level suitable for
the advanced student or researcher in zoology, neuroscience or pharmacology. Most
of the book is concerned with insects, but other invertebrate systems are also
covered and comparisons are made. with mammalian neurochemistry. The book is
organised around the major neurotransmitter classes and in each case both 'pure'
and applied aspects are covered.
Comparative
Invertebrate
Neurochemistry
Edited by G.G. LUNT and R.W. OLSEN
CROOM HELM
London & Sydney
© 1988 G.G. Lunt and R.W. Olsen
Softcover reprint of the hardcover 1s t edition 1988
Croom Helm Ltd, Provident House,
Burrell Row, Beckenham, Kent BR3 lAT
Croom Helm Australia, 44-50 Waterloo Road,
North Ryde, 2113, New South Wales
British Library Cataloguing in Publication Data
Comparative invertebrate neurochemistry.
1. Nervous system - Invertebrates
2. Neurons
I. Lunt, G.G. II. Olsen, R.W.
592'.0188 QL364
ISBN-13: 978-1-4615-9806-0 e-ISBN-13: 978-1-4615-9804-6
DOl: 10.1007/978-1-4615-9804-6
Typeset in Times Roman by Leaper & Gard Ltd, Bristol
Contents
List of Contributors vi
Preface vii
1. Acetylcholine Amira T. Eldefrawi and Mohyee E. Eldefrawi 1
2. Glutamate Ian R. Duce 42
3. GABA Timothy N. Robinson and Richard W. Olsen 90
4. Amine Transmitters and their Associated Second Messenger Systems
Peter F. T. Vaughan 124
5. Invertebrate Neuropeptides Nicholas Platt and Stuart E. Reynolds 175
6. Neuronal Cultures as Experimental Systems David J. Beadle 227
7. Neurotoxins EliahuZlotkin 256
Index 325
List of Contributors
David J. Beadle, School of Biological Sciences and Environmental Health,
Thames Polytechnic, London SE18 6PF, England.
Ian R. Duce, Department of Zoology, University of Nottingham, University
Park, Nottingham NG7 2RD, England.
Amira T. Eldefrawi and Mohyee E. Eldefrawi, Department of Pharmacology
and Experimental Therapeutics, University of Maryland School of Medicine,
Baltimore, Maryland 21201, USA.
George C. Lunt, Department of Biochemistry, University of Bath, Claverton
Down, Bath BA2 7 A Y, England.
Richard W. Olsen, Department of Pharmacology, UCLA School of
Medicine, Los Angeles, California 90024, USA.
Nicholas Platt, Department of Anatomy and Neurobiology, Washington
University School of Medicine, 660 South Euclid Avenue, St Louis, Missouri
63110, USA.
Timothy N. Robinson, Astra Neuroscience Research Unit, 1 Wakefield
Street, London WC11PJ, England
Stuart E. Reynolds, School of Biological Sciences, University of Bath,
Claverton Down, Bath BA2 7 A Y, England.
Peter F. T. Vaughan, Department of Biochemistry, University of Glasgow,
Glasgow G12 8QQ, Scotland.
Eliahu Zlotkin, Institue of Life Sciences, The Hebrew University of
Jerusalem, 91904 Jerusalem, Israel.
vi
Preface
The attractions of invertebrate nervous systems have long been appreciated
by neurophysiologists. Indeed some of the milestones in our understanding
of nervous systems have their foundations in experiments done on
invertebrate preparations, typified by the role of the squid axon in dissect
ing the events that constitute the action potential. More recently we have
seen how the relatively simple nervous system of Aplysia has permitted
new insights into the molecular mechanisms of memory and learning.
Neurochemists, however, have not been enthusiastic about invertebrate
tissues as their experimental material. Much of the biochemical information
on invertebrate nervous systems that has accrued has been incidental,
almost as a by-product of what were primarily physiological investigations.
Fortunately the field is changing, and research groups are making a positive
choice to turn to invertebrate tissues.
Two important factors have contributed to this. First, the study of
analogous systems in invertebrates and vertebrates can tell us much about
the evolution of nervous systems. The application of the techniques of
molecular genetics to the study of such molecules as receptors and ion
channels can provide detailed information about their composition that, in
turn, allows us to better understand their function. By extending such
studies to the invertebrates we should be able to understand how such
systems have developed. Secondly, invertehrate pests are responsible for
enormous losses of agricultural crops and are major vectors of disease in
man. The pesticide industry has in the past adopted a predominantly
empirical approach to the control of pests. However, it is increasingly
apparent that such an approach is no longer tenable. The industry needs
new targets at which to direct more specific, safer, control agents. It is
generally agreed that the nervous system is probably the most vulnerable
target at which to aim sach agents, and consequently there has been a
major effort to promote research aimed at identifying potential pest
specific sites.
It is against this background of increasing emphasis on comparative
studies that we have assembled this volume. We have sought to review our
current understanding of the major neurotransmitter systems in inverte
brates and to highlight their relationship to the generally much better
characterised vertebrate systems. Our authors have all worked with
invertebrate systems for many years and have been in part responsible for
the current upsurge of interest. In addition to considering the major trans
mitter systems we have included two chapters that cover slightly different
areas. Cell culture, and more particularly neuronal cell culture, is a rela
tively new discipline that has had a great impact on biology. Cultured cells
vii
offer possibilities that the natural tissue cannot. Invertebrate neuronal
culture is just beginning to realise its potential and is being enthusiastically
taken up by a number of groups. Neurotoxins have provided neuro
scientists with some of the most specific and potent research tools that we
have; how could we study nicotinic acetylcholine receptors without a
bungarotoxin or the Na+ channel without tetrodotoxin? There are toxins
that differentiate between vertebrate and invertebrate targets: not only
could such toxins provide new probes for the comparative neurochemist,
but they may also give valuable leads to the pesticide chemist. The chapter
on invertebrate neurotoxins constitutes one of the most comprehensive
accounts of these fascinating agents.
A book of this kind depends entirely on the efforts of colleagues, most
of whom would rather be doing neurochemistry than writing a chapter for a
book! We are most grateful to each of them for contributing to our text.
Our thanks must also go to our publisher, who has waited so patiently for
the final manuscript.
G.G. Lunt
Bath
R. W. Olsen
Los Angeles
viii
1
Acetylcholine
Amira T. Eldefrawi and Mohyee E. Eldefrawi
INTRODUCfION
It is estimated that 97 per cent of animal species (1200000) are inverte
brates and 78 per cent are insects. Yet most biochemical research on
cholinergic transmission has been conducted on vertebrates. There are
obvious advantages to studying the invertebrate nervous system. It has
simpler cellular organisation and clearly identifiable neurones, which allow
for the investigation and understanding of the basic principles of neuro
transmission and neuronal integration of signals. Also, the availability of
anatomical, physiological and pharmacological data on certain neurones,
whose regulation of certain behaviours is known (such as learning in the
marine snail Aplysia (Kandel, 1979) and in the American cockroach
Periplaneta americana (Sattelle et aL, 1983), and food aversion learning in
the terrestrial mollusc Limax maxim us (Kelly, 1981)) provides inform
ation on the basic mechanisms that underlie these behaviours. Further
more, the genetics of an insect, Drosophila, are the best studied of all
animal species, which makes possible the systematic and direct generation
of single gene mutants, selection of the relevant ones and identification and
isolation of the altered gene and its product. The mutation can be mapped
accurately in the chromosome and cloned using recombinant DNA tech
nology. The availability of behavioural mutants in Drosophila may help
elucidate the nature of the macromolecular components involved in neuro
chemical mechanisms and their role in normal behaviour.
Acetylcholine (ACh) acts as a neurotransmitter throughout the Animal
Kingdom. It is also present in single-cell animals and even in plants (Jaffe,
1970) with cholinesterase (Fluck and Jaffe, 1975), though its role there is
still unknown. In arthropods, ACh is the transmitter of messages from
sensory neurones to the central nervous system (CNS) and within the CNS,
but not from motor neurones to skeletal muscles where the transmitter is
mostly glutamate, as shown in insects (see Sattelle, 1980), lobster (Barker
et al., 1972) and crabs (Florey, 1973). On the other hand, ACh is a motor
1
ACETYLCHOLINE
excitatory transmitter on to body wall muscles in annelids (leeches and
earthworms) and possibly in velvet worms (see Gardner and Walker,
1982), as it is in vertebrate motor neurones. ACh is localised in certain
neurones in the CNS, as shown in locust brain using anti-ACh antibodies
(Geffard et a/., 1985).
The simultaneous measurement of 17 putative transmitters and meta
bolites in insect tissues revealed a concentration of ACh ranging from
7.6 nmoIlmg protein in housefly brain to 25 and 50 nmoIlmg protein in
locust brain and thoracic ganglion, respectively (Clarke and Donnellan,
1982). By comparison, the fleshfly brain and cotton leafworm nerve cord
had 17.7 and 12.4nmoIlmg protein, respectively. However, ACh is also
widely distributed in tissues that are not cholinergic (Sastry and Sadavong
vivad, 1979), such as locust muscle (54 nmoIlmg protein (Oarke and
Donnellan, 1982», where it may be involved in metabolic processes.
Compared with other putative neurotransmitters in the CNS, the concen
tration of ACh is generally lower than those of glutamate, aspartate,
proline, glycine and alanine (e.g. 476, 135,399, 133,94 nmoIlmg protein,
respectively, in locust brain). However, some of the high concentrations of
these amino acids may be due to their significant involvement in amino
acid metabolism.
ACh may coexist in the same neurone with other transmitters such as
serotonin and octopamine, as shown in Ap/ysia ganglia (Brownstein et aL,
1974), or with peptides such as neurotensin, vasoactive intestinal poly
peptide (VIP) (Said, 1984) or enkephalins in vertebrate peripheral nervous
system (see O'Donohue et a/., 1985). Such cotransmitters may influence
the same or different receptors in the same or different neurones. An excel
lent example is the effect of VIP on the affinity of muscarinic receptors for
ACh (Lundberg et aL, 1982).
The arrival of an action potential from the axon induces ACh release by
exocytosis of vesicles in which it is stored. ACh diffuses to the postsynaptic
membrane in a few microseconds, and binds at the postsynaptic membrane
to ACh receptor (AChR) proteins, inducing changes in their conform
ations. There are two major types of AChR. The nicotinic receptor is
activated by ACh or nicotine and inhibited by d-tubocurarine. It is a single
protein that includes a cationic channel and traverses the membrane. The
muscarinic receptor is activated by ACh and muscarine and is inhibited by
atropine and quinuclidinyl benzilate (QNB). It is located in the extra
cellular portion of the membrane bilayer and is conformationally coupled
to enzymes, such as adenylate cyclase and phosphodiesterase, and regula
tory proteins, such as nucleotide binding protein. Activation of the
nicotinic AChR results in a rapid change in membrane conductance, and
the reaction is usually complete in a few milliseconds. On the other hand,
activation of the cuscarinic AChR results in a much slower reaction, taking
hundreds of milliseconds to seconds, because several proteins and second
2
