Amongst invertebrates, few bioactive peptides have been sequenced and, of these, most are from molluscs or arthropods and there are, as yet, no sequenced neuropeptides from the annelids. In spite of this paucity of information, we expect that a range of model systems will be developed within this phylum. The failure to sequence any annelid peptide is therefore unfortunate in view of the experimental opportunities provided by the nervous system (NS) of various species (leeches and polychaetes). From an evolutionary point of view, annelids are basic to the phylogeny of numerous animal groups. Knowledge of the structure of their neuropeptides is therefore of interest for comparison with those of invertebrates either more primitive (Hydra, Schaller et al 1984, see also Chapter 10 this volume) or more advanced (molluscs and arthropods, see Chapters 5-9 and 13-15 this volume).

Studies on neuropeptides are approached in two ways: the first involves a neurophysiological and neurobiological analysis directed towards an understanding of neurone physiology and more particularly the role of peptides in neurotransmission and neuromodulation (O'Shea & Schaffer 1985; see also Chapters 8 and 14 this volume). The pioneering work of Stent has established the leech as an important annelid model for the study of the cellular basis of behaviour (Stent & Kristan 1981) and as a model for neuronal development (Stent et al. 1982). The second approach is neuroendocrinological and is based upon a study of the production of peptides by neurosecretory centres in the central nervous system (CNS). Annelids have no discrete endocrine glands except for the infracerebral region (ICR), a neurohaemal area (fig. 5) found in several polychaetes (Dhainaut-Courtois 1970; Golding & Whittle 1977; Olive 1980; Durchon 1984).

The name ‘femerfamide’ (FMRFamide) has been given (Greenberg 1982) to the molluscan neuropeptide Phenylalanyl–methionyl–arginyl–phenylalanyl–amide (Price & Greenberg 1977). It is a mnemonic for the contained amino acids and was suggested to avoid prejudicing views about its natural role(s). (During the period of its discovery it was called Cardioexcitatory Neuropeptide because this was one notable action of the pep tide. See Price & Greenberg 1977.)

Price and Greenberg and their associates have shown that in molluscs there are several peptides chemically related to FMRFamide (see eg Price 1986): these are FLRFamide, pQDPFLRFamide, GDPFLRFamide and SDPFLRFamide. It is convenient to refer to these peptides with one name, as for instance are the opioid peptides, because like the opioid peptides, they appear to require a particular sequence of amino acids for activity and show a range of effects suggesting that they operate through several receptor types. Consequently we shall refer to the molluscan molecules as the femerfamide peptides.

With the opioid peptides the sequence Tyr–Gly–Gly–Phe (YGGF–) is required for full opioid activity, as with for example Met– and Leu–enkephalin, endorphin and dynorphin. This C–terminal sequence of amino acids has been called the message sequence by Charkin and Goldstein (1981). By contrast, in the femerfamide peptides the C–terminal sequence appears essential for activity, i.e. serves as the ‘message-sequence’: –Phe (or Tyr)–Met (or Leu)–Arg–Phe–NH2 (Price 1986).

Introduction

In insects a variety of functions (e.g. intermediary metabolism, ion and osmoregulation, developmental and neuronal processes) are regulated by peptides from different parts of the nervous system. This diversity necessitates the restriction of the present chapter to a particular group of peptides. We have been interested in the structure and biological functions of peptide hormones regulating intermediary metabolism and fluid secretion in insects and these studies are used to highlight the problems encountered in characterizing these peptides.

Lipid and carbohydrate metabolism in insects can be regulated by adipokinetic (AKH) and so-called hyperglycaemic (HGH) hormones present in the corpora cardiaca (Mayer & Candy 1969; Steele 1961). These peptides increase concentrations of haemolymph lipids or carbohydrates. The physiological functions and modes of action of these hormones are reviewed in Chapter 7. Diuretic hormones (DH) present in the corpora cardiaca but also in other parts of the nervous system, e.g. brain, suboesophageal and thoracic ganglia (Proux et al. 1982; Morgan & Mordue 1984a; Aston & Hughes 1980) regulate fluid secretion.

In recent years investigations have been conducted to isolate and characterize prothoracicotropic hormones (PTTH) which stimulate the release of ecdysone from the prothoracic glands (Williams 1947). In Bombyx mori, the PTTHs have been found to exhibit significant homologies in their N-terminal sequences with insulin and insulin-like growth factors (Nagasawa et al. 1984). These peptides are discussed in Chapter 6 which is devoted to the presence of Vertebrate* peptides in insects. The involvement of peptides in the functioning of neurones as neurotransmitters and/or neuromodulators is covered in Chapter 8.

Ultrastructure of invertebrate nervous systems

Neuroecretory and other neurons

Examination of invertebrate nervous systems reveals that many are richly endowed with neurones resembling classical neurosecretory cells in cytology and ultrastructure. Such cells are clearly specialized for peptide secretion. They contain an abundance of rough endoplasmic reticulum (RER), and secretory granules (variously known as elementary granules, large dense-cored vesicles, etc.) generated by Golgi bodies, accumulate in large numbers within the perikarya. Although many are doubtless endocrine cells, others (Figs. 1-3) have axons which extend not to blood cavities, but into the central neuropile where the secretory material is discharged.

Furthermore, some secretory granules are evident in virtually all neurones (Golding & Whittle 1977), and this is consistent with the finding that many and perhaps all neurones, including those with conventional transmitters, also secrete peptides (review by Hokfelt, Johansson & Goldstein 1984).

Nerve terminals: vesicles and granules

In most nerve terminals, whether in the central or peripheral nervous systems, large numbers of synaptic vesicles are encountered (Figs. 4 & 5). Measuring 20-50nm in diameter, the vesicles in all but a small minority of terminals (Fig. 6) have lucent contents, except following exposure to a mixture of Zinc iodide and Osmium tetroxide (the ZIO reagent) (Fig. 7) which deposits extremely electrondense material within them (May & Golding 1982). The vesicles cluster densely adjacent to sites of specialized contact with other cells. Pre- and postsynaptic thickenings are present and the synaptic clefts are wider and often more regular in form than other intercellular spaces, and contain moderately dense material.

Introduction

The discovery that many central neurones utilize peptides as extracellular chemical messengers has revolutionized our understanding of neuronal signalling. Studies to characterize the structure and functions of neuropeptides have taken various approaches, including purification and biochemical analysis of the peptide products and molecular genetic studies of the genes encoding precursor proteins which give rise to peptide products. These investigations have been greatly aided by the use of non-neuronal tissues, such as epithelial tissue or digestive organs, which are often rich sources of bioactive peptides. Many peptides initially identified in peripheral tissues have been found subsequently in the central nervous system. One preparation, frog skin, has been particularly useful in this regard, and has facilitated the discovery of mammalian peptides related to frog bombesin (Orloff et al. 1984).

Invertebrate nervous systems offer unique advantages in the study of neurotransmitter function. Our understanding of the molecular mechanisms underlying neurotransmitter actions have been greatly facilitated by the use of invertebrate systems due to the smaller number of neurons, their simpler organization, and the often large size of their cell soma (see also Chapter 8). In terms of neuropeptide biology and chemistry a number of questions arise: Can neuropeptides related to vertebrate neuropeptides be found in invertebrates? Can neuropeptides characterized in invertebrate systems be used to identify homologous peptides in mammalian systems? Can invertebrate systems be used to gain further insight into the function, regulation, and evolution of neuroendocrine systems?

Introduction

The collective title protochordates is essentially a convenient ‘umbrella’ term for a diverse assembly of animals sharing relatively few, although important, common features. The protochordate group is usually thought to comprise three subphyla, the hemichordata, urochordata (tunicata) and cephalochordata, although some authorities consider the hemichordates a separate phylum. There are, however, no published reports on the occurrence of peptides or amines in hemichordates and they will not be considered further here.

The tunicates and cephalochordates are highly specialized marine organisms whose close relationship is perhaps rather superficial and, in reality, based more upon similar, ciliary powered, particle feeding mechanisms which have developed around the possession of a perforated pharynx, rather than any genuine and common phylogenetic background. Notwithstanding such reservations the protochordates are probably the only available extant representatives of ancient groups which “bridged the gap” between invertebrates and vertebrates, and as such could provide useful clues to the origins of certain vertebrate features.

The features which have attracted most attention and which have provided the most useful information are the central nervous system (CNS) and the gastrointestinal tract. The tunicate CNS has a relatively simple organisation which is in many respects rather more comparable with simple invertebrate neurosecretory centres than any part of the vertebrate CNS. Similarly the cephalochordate CNS is a simple anterior elaboration of the dorsal nerve cord (characteristic of these animals), and its structure and function reflects the highly specialized nature of the adult organism as well as its unusual and asymmetric development.

Introduction: Hormones and development

Since Kopec's demonstration of a humoral role of the brain in insect development, peptide hormones have had a central historic importance in the study of insect endocrinology: the brain produces prothoracicotropic hormones (PTTH's; see Bollenbacher & Granger 1985), thus stimulating moulting by initiating ecdysone synthesis and release by the prothoracic glands. A second neuropeptide, eclosion hormone (see Reynolds & Truman 1983), initiates the necessary behaviour patterns associated with ecdysis and its timing. Subsequently, a third neuropeptide, bursicon (see Reynolds 1983; 1985), controls the tanning of the new cuticle and stimulates endocuticle deposition. Neuropeptides are also implicated in the control of corpus allatum activity; both allatohibins and allatotropins being identified in various insects (see Tobe & Stay 1985). Unfortunately, chemical characterisation of these peptides has progressed slowly. The physiological actions of most of these peptides concerned with development have been the subject of numerous and extensive reviews (see for example Downer & Laufer 1983; Kerkut & Gilbert 1985) and will not be dealt with here.

Recently considerable progress has been made in the characterisation of PTTH's from Bombyx mori (Nagasawa et al. 1984, 1986). Characterisation of these polypeptides has been hampered by their heterogeneity (in several species high and low molecular weight forms exist) but also, no doubt, due to the impurity of the starting material used. The sequence of 4K-PTTH-II one of the small Bombyx hormones, has now been determined (Nagasawa et al. 1986).

This volume arose from an International Congress held in Bordeaux during 1986 and organised jointly by the Comparative Endocrinology Group of the Society for Experimental Biology, Laboratoire de Neurobiologie, Universite de Bordeaux I and Centre National de la Recherche Scientifique (CNRS).

The chapters which follow have been prepared by the invited seminar series speakers attending that meeting, and are designed as broad overviews of their particular specialities.

For the original meeting in Bordeaux we particularly extend warm and grateful thanks to our friend and colleague Professor Adrien Giradie and his collaborators in the Neurobiology Laboratory, Bordeaux, without whom the symposium could not have taken place, and this volume would not have been produced.

The symposium also benefited from the support of the following organisations: Society for Experimental Biology, UK; Centre National de la Recherche Scientifique, France; Direction de la Cooperation et des Relations Internationales du Minitere de l'Education Nationale, France; Universite de Bordeaux 1; Beckman; Bioblock Scientific; Bordeaux Chimie-Cofralab; Etablissements Laurent; Imperial Chemical Industries pic. (Plant Protection, Jealot's Hill, UK); Laboratory Data Control; Mairie de Bordeaux; Mairie de Gradignan; Office du Tourisme de Bordeaux; Peninsula Laboratories Europe; Pfizer Research Ltd.; Poly-Labo; Rohm Haas Chemical Co; Shell Research Ltd.; Sofranie-Mettler; Wild Leitz France.

Introduction

Study of model-systems has played a major role in the developments which have taken place in neurobiology during the past decades. Studies of opisthobranch and pulmonate molluscs have made important contributions in this area. These animals make excellent models, because they possess a relatively small central nervous system (CNS), which contains a limited number of large (polyploid, e.g. Boer et al., 1977) and readily accessible neurons. Two species have been studied in particular; the opisthobranch Aplysia californica (e.g. Strumwasser et al. 1980) and the pulmonate freshwater snail Lymnaea stagnalis (e.g. Joosse & Geraerts 1983; Roubos 1984).

Immunocytochemistry has become an important tool in neurobiology. In our laboratory the CNS of L. stagnalis has been investigated extensively using immunocytochemical methods (Boer et al., 1979, 1980, 1984a,b, 1986; Schot & Boer 1982; Schot et al 1981, 1983, 1984, see also Chapter 13, this volume). Recently monoclonal antibodies were raised to homogenates of whole CNS from L. stagnalis (Boer & Van Minnen 1985). These investigations, in conjunction with functional and electrophysiological studies can illustrate the central position of the peptidergic neuron in neurotransmission and in neuro-endocrine control processes (Joosse 1986).

Neural and hormonal communication

Animals possess two major systems for the regulation and coordination of body functions: the nervous system and the endocrine system. In the classic view these systems differ in a number of aspects. The chemical messengers of the nervous system (neurotransmitters) are small molecules (acetylcholine, biogenic amines, amino acids), which are released at sites of direct contact (synapse) between neurons and their targets.

Introduction: identifiable insect skeletal mononeurones and why they are important

Insect skeletal muscle is innervated by monopolar motoneurones in the segmental ganglia of the CNS which send motor axons to specific muscle targets. The specificity of muscle innervation allows us to identify motoneurones uniquely by their target muscles. Motoneurones innervating particular muscles have a characteristic position and morphology in the central nervous system, and these characteristics can also be used to identify them. An example is provided by the slow coxal depressor or Ds motoneurone of the cockroach Periplaneta americana. This neurone, as its name suggests, innvervates coxal depressor muscles and it has an invariant position and morphology. It is one of perhaps 5 motoneurones involved in the innervation of the coxal depressors. In addition to being identifiable, skeletal motoneurones innervating particular muscles in insects are few in number (in contrast to vertebrate muscle innervation for example). Thus the large extensor muscle of the locust tibia, the extensor tibialis muscle, is innervated by only 4 motor cells, each of which has been identified (see below). The identifiability of insect motorneurones and their small number have combined to make insect neuromuscular systems important model preparations.

The simplicity of insect neuromuscular systems has obvious advantages for the study of muscle physiology in these organisms. Insect motor neurones differ from the classical vertebrate model by using multiple transmitters. This feature has allowed us to study fundamental aspects of synaptic physiology, pharmacology and neuronal function which perhaps could not be studied as conveniently in other organisms.

It has proved difficult through the study of human amnesics to elucidate several matters critical to understanding the disorder. First, human cases are not usually appropriate for identifying the critical lesions that cause the core memory problems because their lesions often extend into brain regions where damage causes unrelated deficits. Second and relatedly, it is hard to determine from human amnesics the extent to which their memory deficits result from damage to neurons that release specific transmitters, such as acetylcholine or noradrenalin. Third, despite developments with electrophysiological recording techniques and the emergence of the PET scan, study of human amnesics and healthy people is not an effective means of exploring the anatomical connections and physiology of the brain regions lesioned in amnesics in order to gain a clearer idea of precisely what functions are disrupted in patients. These three issues have been more effectively examined through physiological studies with animals and pharmacological studies with animals and humans. The next section discusses animal models of the amnesic state. The third section briefly reviews what light pharmacological studies have thrown on amnesia, and the last section considers animal work involving lesions, electrophysiological recordings, and manipulations of long-term potentiation (LTP). As indicated in chapter 1, LTP is an increase in neural responsiveness, particularly striking in hippocampal neurons, that occurs when brief bursts of high-frequency stimulation are given to the inputs of the relevant neurons. It may persist for weeks and is believed by many to be based on a memory-like change. LTP is of interest here because it has been used to examine the functions of the structures whose damage is believed to be critical in amnesia.

People may do badly at different kinds of memory tasks, and they may do so for different reasons. It is the purpose of memory assessment to ascertain the ways in which a subject's memory is poor and the causes of this poor performance. Clinicians need such assessments to help them see how patients will cope in everyday life, what their prognoses are, whether there are any therapies to which they are likely to respond, and whether there is any response to treatment. Theoreticians are most concerned with identifying the range of memory disorders and their specific causes. Both groups need valid, reliable tests of particular kinds of memory for which normative data exist so that the severity of the deficits can be determined. But whereas clinicians need tests that tap everyday memory performance, which exist in several equivalent forms, theoreticians need standardized tests with normative data so that results from different laboratories can be compared. Furthermore, as theoreticians seek to identify new kinds of memory disorder, they need to develop their own special-purpose tests to compare the performance of a patient on these tests with that of a group of matched control subjects. The theoretical aim is to identify elementary memory deficits that cannot be subdivided into further simpler disorders and to see what, if any, kinds of brain damage cause them.

The problem that the theoretician faces is that most memory disorders are messy, are poorly understood, and involve several kinds of elementary memory breakdown that arise for many reasons.

In this book, five possible groups of elementary organic memory disorders have been discussed. It is still controversial whether the disorders considered are truly elementary or whether at least some of them are composed of two or more independent disorders that could be separately compromised by more selective lesions. For example, the organic amnesia caused by medial temporal lobe lesions may differ from that caused by diencephalic lesions, and lesions of the hippo-campal and amygdalar circuits may disrupt recognition for somewhat different reasons. This kind of issue is hard to resolve, partly because lesions in humans are adventitious and tend not to honour boundaries between functionally distinct brain regions, so that it is often difficult to distinguish between cognitive deficits that are essential to a memory deficit and those that are incidental to it.

Despite the problems, five groups of organic memory deficits can be identified. The first group of memory deficits is caused by lesions to PTO association neocortex. This neocortical region includes parts of the parietal, temporal, and occipital lobes and is not specialized for obvious motor or sensory functions. Instead, as its neurons lie several relays away from the sensory input, it is probably concerned with the later stages of analysis and interpretation of sensory information. Lesions to it can cause breakdowns that comprise several kinds of fairly selective short-term memory deficits specific to certain types of information. These disorders, which are discussed in chapter 3, probably arise for a number of reasons, although it still needs to be shown convincingly that they are ever caused by isolated disruption of short-term storage, rather than of specific encoding and retrieval processes.

Section One: The aims of this book

What were you doing immediately before you picked up this book? This question should cause you little difficulty, but there are people who would find it very hard to answer. For these people, known as organic amnesics, life must be experienced as if they were continually waking from a dream. Brain damage has made them very poor at remembering recently experienced events and at learning new information. It also makes them poor at remembering things that were learnt up to many years prior to the brain trauma. Despite such memory impairments, organic amnesics may have normal or superior intelligence. Not all memory deficits caused by brain damage are like organic amnesia, however. Other patients with lesions different from those responsible for organic amnesia show a very rapid loss of spoken information whilst possessing good longer term remembering of most things. For example, such a person might be unable to repeat back more than two spoken digits even with no delay but be able to give the gist of a newspaper article recounted by someone else on the previous day (something well beyond the powers of an organic amnesic). This kind of short-term memory failure is associated with the language disorder known as conduction aphasia and is clearly distinct from the memory deficits seen in organic amnesia, although both are caused by brain damage.