Cellular Dynamics in Invertebrate Neurosecretory Systems

Cellular Dynamics in Invertebrate Neurosecretory Systems ALLAN BERLIND Biology Department, Wesleyan Uniuersity, Middletown, Connectictit

I. Introduction . . . . . . , . . . 172 11. Definitions of Neurosecretory Cells . . . . 173 111. Chemical Nature ofSecretory Products . . . . 175 A. Histology and Histochemistry ofthe Secretory Product . 175 B. Isolation and Characterization of Invertebrate Neurohormones . . , . . . . . 181 C. Carrier Proteins and Hormone Precursors . . . . 187 . . . . . . . 195 IV. Neurosecretory Granules . A. Granule Isolation . . . , . . . . 195 B. Formation ofsecretory Granules . . . . 197 C. Active and Inactive Systems (Electron Microscope Evidence) . . , . . . . . . 199 D. Summary . . . . . . . . . . 206 . 206 V. Control of Synthesis and Transport-Radiotracer Studies A. Molluscs . . . . . . , . . . 207 B. Insects . . , . . . . . . . 207 C. Annelids , . . . . . . . 211 D. Summary , . . . . . , . . 212 VI . Transport of Neurosecretory Material . , . 212 VII. Release of Neurosecretory Material-Microscopic Studies . 214 A. Specialization of the Neurohemal Area . . 214 B. Nonterminal Release Sites . . . . . . . 215 C. Release Mechanisms . . , . , . . . 216 VIII. Electrical Activity of Invertebrate Neurosecretory Cells and the Release of Neurosecretory Material , . . 222 A. Introduction . . . . . . . 222 B. Electrical Activity and the Release of Neurosecretory . . . . . . . 223 Material C. Details of .Electrical Activity of Selected Neurosecretory Cells in Molluscs and Insects . . . . 229 IX. Modes of Control of Neurosecretory Cell Activity . . 233 A. Spontaneously Active System . . . . 233 B. MononeuronalNeuroendocrineReflexes . . . 234 C. Synaptic Control in Neuroendocrine Reflexes . . . 236 D. Control of Release a t Neurosecretory Terminals 238 E. Feedback Control of Neurosecretory Systems . . 239 . . , . . 242 X. General SummaryandPerspectives. References , . . . . . . . , . . 244

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I. Introduction Invertebrate neurosecretory cells, classically defined as neurons specialized for the synthesis, storage, and secretion of hormones, are quantitatively more important, and may play more varied roles, than corresponding cells in vertebrates. Much of the current research effort in evaluating neurosecretory systems is aimed at understanding the control mechanisms for the synthesis, storage, and release of active factors by these cells. Classic methods of analysis, especially those involving the use of histological techniques, have often yielded results that are difficult to interpret in terms of dynamic aspects of secretory cell function. Even in studies involving correlation of temporal changes in secretory cell histology with physiological or morphological changes in the animal, the assessment of secretory activity may be ambiguous. Highnam (1965) has stated most clearly the problems of interpretation. Accumulation of stain in the perikaryon or terminals of a secretory neuron may, for example, result from a steady rate of synthesis of neurosecretory material (NSM) but a decrease in release rate, from an increase in synthesis with no change in release rate, or from any relationship in which the rate of synthesis exceeds the rate of release. Similarly, lack of change in histological appearance of a neurosecretory cell during the course of a physiological or morphological change does not necessarily indicate that the cell in question is not involved in the control of that change. If synthesis, transport, and release of a secretory product remain balanced, regardless of rate changes, no alteration in staining would be expected. In principle, the same problems of interpretation exist with all static observations of a secretory cell, whether they involve the content of neurosecretory granules (Wendelaar Bonga, 1971b) or hormone content. It is also clear that observing changes in only one part of a secretory neuron may give misleading information about function. A histologically detected loss of material from the cell body (even if it correlates with a loss of hormone) might imply that hormone release is occurring from cell terminals, but might also indicate that material is simply being moved from synthesizing to storage areas. Without a detailed functional analysis of all parts of the cell a complete understanding of its dynamics cannot be obtained. This article therefore focuses on recent studies which have attempted to analyze, by direct and independent measures, synthesis, transport, and release phenomena. Several reviews on invertebrates dealing with functional aspects of neurosecretory systems as a whole have appeared recently [Maddrell, 1974 (insects); Goldsworthy and Mordue, 1974b (insects); Golding,

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1974 (nonarthropod invertebrates); Fingerman, 1974 (marine invertebrates)]. Since the critical review of arthropod cellular dynamics by Highnam (1965),numerous reports have appeared which should be of value to both vertebrate and invertebrate neuroendocrinologists in the continuing revision of general concepts of neurosecretion and in the formulation of specific experimental procedures. Early concepts of neurosecretion (reviewed by Scharrer and Scharrer, 1963; Gabe, 1966) were broadly based on extensive morphological and physiological studies of invertebrate and vertebrate groups, and valuable generalizations derived support from both sources. With more intensive investigation many researchers, particularly those working on vertebrates or on insects, have tended to restrict their outlook and to focus more or less exclusively on a single phylogenetic group. As a result vertebrate neuroendocrinologists have often ignored valuable ultrastructural, electrophysiological, and biochemical results derived from studies on invertebrates. Investigators of invertebrate systems have tended to draw extensively, and in some cases uncritically, on details of the hypothalamoneurohypophysial system of vertebrates, especially with regard to biochemical aspects of neurosecretory cell function. While many generalizations are undoubtedly applicable to all groups, it is necessary to recognize that significant differences in cellular detail may exist. This article emphasizes studies in which invertebrate preparations have yielded particularly valuable information on cellular dynamics: quantitative electron microscope studies of the secretory cycle, biochemical processing of hormones and hormoneassociated molecules, and aspects of the control of secretory cell activity. 11. Definitions of Neurosecretory Cells

N o single histological or ultrastructural criterion is sufficient to distinguish neurons with secretory function (Highnam, 1965; Bern, 1966; Bern and Knowles, 1966). The functional criteria for neurosecretory cells suggested by Knowles and Bern (1966) include the release of hormone by such cells into the circulatory system or involvement in the control of other endocrine glands. Included under 'this definition are not only neurosecretory cells that terminate in well-defined neurohemal organs such as the corpus cardiacum (CC) of insects and the sinus gland of crustaceans, but also a great number of cells that send axons to terminate in a variety of peripheral areas. In many arthropods, morphologically identifiable neurosecretory perikarya are widely distributed throughout the ventral ganglia (Raabe, 1965; Del-

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phin, 1963, 1965; Fletcher, 1969; Obenchain, 1974) and occasionally along peripheral nerves (Maynard, 1961; Finlayson and Osborne, 1968). Terminals from which hormones may be liberated into the circulation are often diffusely distributed along the margins of nerve trunks (Maddrell, 1966; Johnson, 1966; Brady and Maddrell, 1967; Weber and Gaude, 1971). In molluscs, the scattered distribution of secretory neurons (Frazier et al., 1967) is paralleled by diffuse release areas in the sheaths surrounding ganglia or along nerve margins. Also included in the basic definition are cells which directly control the activity of nonneural endocrine glands such as the corpora allata of insects. Recent accumulating evidence suggests that the two functional criteria cited above may be too restrictive. Scharrer (1975; Scharrer and Weitzman, 1970) in particular has emphasized findings that cells that fulfill many of the ultrastructural and biochemical criteria for neurosecretion may deliver their products more or less directly to some nonendocrine target organ. A list of such “private peptidergic inputs” in insects has been compiled by Maddrell(l974). Possible target organs for such cells include epidermis (Maddrell, 1965), Malpighian tubules (Maddrell and Gee, 1974), hindgut and reproductive tissue (Johnson, 1963), muscle (see below), nonendocrine glands (Quennedey, 1969), and possibly other neurons (Schooneveld, 197413; Scharrer, 1975). In the case of some of these targets, the effect of released material appears to be restricted to tissue near the endings: A nervous factor plasticizing the epidermis is effective only on one half of the abdomen of an insect if the other half has been denervated (Maddrell and Reynolds, 1972). In molluscs, direct delivery of neurosecretion to the kidney has been proposed as an osmoregulatory mechanism (Wendelaar Bonga, 1972), although the same cells appear to have branches which also release product into the general circulation. In planarians gonad maturation appears to be under the direct influence of secretory neurons (Grasso and Quaglia, 1971). Neurons have frequently been considered neurosecretory solely because they contain, among other inclusions, a number of electrondense granules, despite evidence that nonsecretory granules may have a similar appearance (Bern, 1966). In all the major groups of annelids a large percentage of the cells of the central nervous system contain dense granules, with no evidence for hormonal function, peptide secretion, or other-than-typical neuronal function (Hagadorn et al., 1963; Coggeshall, 1965; Dhainaut-Courtois, 1968). Neurons in several invertebrate and vertebrate systems which do not ordinarily contain granules may acquire them during regeneration (Lentz, 1965a; Pellegrino de Iraldi and de Robertis, 1968; Boulton and

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Rowell, 1969; Sauzin-Monnot, 1972), including cells identified as cholinergic or aminergic. In crustaceans, some dense granules are found in ordinary axons known to secrete y-aminobutyric acid (Atwood et al., 1971).Granules have also been reported in neurons in which synaptic transmission is thought to be purely electrical (Smith, 1971), in the sensory terminals of receptor cells (Smith, 1971), and in developing nervous systems. Such granules may be involved, in some cases, in mediating trophic effects ofnerve on other tissues (Atwood e t al., 1971), rather than effecting synaptic transmission. However, caution should be observed in applying the term neurosecretory to any neuron that contains dense granules; if actual secretion of granular material (release into the extracellular medium) is not demonstrated, it is possible that some intracellular role is subserved. The so-called neurosecretory innervation of arthropod skeletal muscle raises particularly difficult questions. Some of the granulecontaining cells are likely to secrete monoamines (Hoyle e t al., 1974; Hoyle, 1975).In some insects systems, all axons innervating particular muscles may contain granules (Osborne e t al., 1971; Anwyl and Finlayson, 1974), and there is some ultrastructural evidence for the release of granule contents into the extracellular medium. In the hemipteran Rhodnius prolixus, neurons to the ventral abdominal intersegmental muscles may release material both into the hemolymph and directly adjacent to the muscles (Anwyl and Finlayson, 1973). These muscles degenerate soon after each larval molt and in the adult. Axonal endings remain in the connective tissue after the muscles have degenerated and appear to have a higher granule content during the periods of degeneration. In several species of crustaceans a similar ultrastructural picture is observed in the nerves to some skeletal muscle, with large dense granules representing a minor fiaction of the total vesicle content (Atwood et al., 1971). In Carcinus claw muscle, however, a distinct neuron with only dense granules may form its own specialized endings on the muscle (Huddart and Bradbury, 1972). The physiological significance of such innervation remains obscure.

111. Chemical Nature of Secretory Products A.

HISTOLOGY AND HISTOCHEMISTRY OF THE SECRETORY PRODUCT

It is generally accepted that the material that reacts with the conventional neurosecretory stains [paraldehyde-fuchsin (PAF), chrome-

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hematoxylin-phloxin, azanl is in some manner closely related to the secreted active product. Intense staining is assumed to indicate a high content ofhormone but, in some cases in which an active principle can be extracted and assayed, this assumption is not valid (Highnam and Goldsworthy, 1972; Meola and Lea, 1971).It is also not always possible to demonstrate a correlation between staining intensity and the content of elementary neurosecretory granules as observed in electron micrographs. In alternate thick and thin sections of neurosecretory cells, highly stained areas are often associated with regions of granule accumulation. In Locustu rnigrutoriu, however, well-stained regions often appear, in adjacent thin sections, to be almost devoid of granules (Girardie, 1973). Such observations may be explained in part by the irregular distribution of granules in these cells. The likelihood of obtaining by chance a section without granules is greater for the thin sections processed for electron microscopy. Bern (1966) has emphasized the evidence that material and structures totally unrelated to the secretory product can occasionally react with neurosecretory stains, and suggests that such reactivity should not be utilized as the sole criterion for neurosecretory function. Lysosoma1 material (Schooneveld, 1970) and lipofuscin pigments (Wendelaar Bonga, 1970) appear to be reactive in some systems. In the cerebral neurosecretory system of the Colorado potato beetle, cycles of stainability are observed in the B cells, which appear to be more closely correlated with changes in the number of free ribosomes than with changes in neurosecretory granule content (Schooneveld, 1974a). Histochemical procedures utilized to elucidate the biochemical nature of NSM do not always give results that are of value in analyzing the nature of the secreted product. Different groups of investigators using different stains and slightly different techniques have asserted that NSM may be rich in phospholipoprotein, lipid or phospholipid, mucopolysaccharide or mucoprotein, sulfhydryl-rich lipoprotein, glycolipid, glycoprotein, lipofuscin, sulfhydryl-rich glycoprotein, or sulfhydryl-rich protein (Hinks, 1971). In all such histochemical studies the relationship between the stainable material and hormone is unclear. Even if it can be assumed that stains interact with hormone-associated molecules, complexes, or substructural elements, a direct association with hormone cannot be assumed. The granule membrane most likely represents a large commitment of lipid or phospholipid material, and the membranes or structural elements of granules may contain proteins, enzymes, and other molecular components only peripherally related to hormone content or production.

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The likelihood that stains react mainly with a carrier substance is discussed in detail in Section II1,C. Detailed histochemical studies using many stains on a single species show that the constituents of different neurosecretory cells within a single nervous system may vary widely. In the lepidopteran Triphaena pronuba, for example, nine cell types can be differentiated, in four main groups (Hinks, 1971). The cell type classification is based on position, size, and reactivity to 37 different histochemical techniques. All the groups appear to have NSM with a high protein content and little lipid. Glycoproteins are prominent in several cell types, but the sugadprotein ratio is variable. In the pond snail Lymnaea stagndis, seven types of neurosecretory cells can be distinguished by using alcian blue-alcian yellow, all of which react similarly to PAF or to chrome-hematoxylin (Wendelaar Bonga, 1970).The assumption that cells that appear identical by one technique secrete the same product is thus clearly invalid on the basis of these observations. The results of studies employing stains specifically reactive with sulfur-containing amino acids [performic acid-alcian blue, performic acid-Victoria blue (Dogra, 1968)], originating with a report from Sloper (1957) on insects, suggest that neurosecretory material in invertebrates is rich in sulfur, as are some vertebrate products. It is likely that the reactivity of several standard stains (particularly after oxidation) is partially based on sulfur content as well (Gabe, 1966).PAF in particular appears to react strongly with sulfonates resulting from the oxidation of cysteine (Gabe, 1966; Prenta, 1969). Hinks (1971), however, reports a very low content of the sulfur-containing amino acids cysteine and cystine in cells that stain with PAF. The presence of a high sulfur content in the NSM of some cells has been confirmed in many invertebrate phyla (cf. Bianchi, 1969), but it is by no means a distinguishing characteristic of such material. Within the Insecta, the prominent lateral neurosecretory cells of the brain of some species do not react with sulfur reagents (Dogra, 1968). In the brain of the lepidopteran Triphaena, cysteine and cystine are virtually absent from all neurosecretory cells (Hinks, 1971). Some of the presumptive neurosecretory cells in the optic lobe of PeripZaneta (Beattie, 1971a) and the ventral nerve cord of phasmids and other orthopterans also appear to contain little sulfur (Raabe, 1965; Raabe and Monjo, 1970). The reasons for the prominence of sulfur in many protein-secreting systems are not at all clear. In secretory cells of the vertebrate pancreas, a prominent proportion of the cellular protein consists of a

TABLE I SELECTEDINVERTEBRATENEUROHORMONES

Animal Coelenterata Hydra attenuata

5

Annelida Nereis diversicolor Mollusca Aplysfa calffornica octopus WlgadS

Echinodermata Asterias amurensis

Arthropods Crustacea Pandalus borealis Pandalus borealis

Activity

Source

Chemical nature

Approximate molecular weight

Comments

Reference

Activates head and bud formation

Stalk

Peptide

900

Active at
Schaller, 1973

Inhibitor of sexual development

Cerebral ganglion

Pentide

Small

Low aromatic acid content

Cardon. 1970

Egg-laying hormone Cardioexcitor

Bag cells

Peptide

See text

Arch, 1976

Nearosecretory system of vena cava

Glycoprotein

4800 13000 1300-1400

Gonad-stimulating substance

Radial nerves

Peptide

2100

Amino acid content known; may be species variability

Kanatani et al., 1971

Red-pigmentconcentrating hormone Distal retinal pigment hormone

Eyestalk

Octapeptide

1000

Structure knowna

Fernlund and Josefsson, 1972

Eyestalk

Octodecapeptide

2200

Structure known*

Femlund (cited in Kleinholz, 1976)

-

Loh et al., 1975 Blanchi and De Prisco, 1971; Blanchi et al., 1973

r (D 4

Three species

Hyperglycemic

Eyestalk

Peptide

6300-7400

Gecarcinus lateralis Uca pugilator

Limb growth inhibition Melanin-dispersing hormone

Eyestalk

Peptide

1000

Eyestalk

?

Cancer borealis

Cardioexcitor

Pericardial organ

Lipoproteinpeptide complex Peptide

Insecta Bombyx mod

Ecdysiotropic

Brain

Glycoprotein

20,000

Bombyx mori

Ecdysiotropic

Brain

Protein

Periplaneta americana

Ecdysiotropic

Brain

Protein

Periplaneta americana Periplaneta americana Periplaneta americana Periplaneta americana

Cardioaccelerator

cc

Peptide

15% sugar content 9000,12,000, Heterogeneous 31,000 20,000A healt accelerator; may have 40,000 activity Small Heterogeneous

Hyperglycemic

cc

Peptide

Small

Antidiuretic

Terminal ganglion Terminal ganglion

Peptide

8000

Protein

>30,000

May be same as bursicon

Periplaneta americana

Bursicon

Protein

40,000

Purified from whole animal homogenate

Diuretic

Ventral nerve cord

1000

May be species variability

Kleinholz and Keller, 1973 Bliss and Hopkins, 1974 Bartell et al.. 1971

May be two active peptides

Belamarich and Terwilliger, 1966

Heterogeneous

Yamazaki and Kobayashi, 1969 Ishizaki and Ichikawa, 1967 Gersch and Stkebecher, 1968 Migliori-Natalizi et al., 1970 Migliori-Natalizi et al., 1970 Golbard et al., 1970 Golbard et al., 1970;Mills and Whitehead, 1970 Mills and Nielsen, 1967 (Continued)

TABLE I (Continued) ~~

~

Animal

Activity

Sarcophaga bullata Sarcophaga bullata Sarcophaga bullata Schistocerca gregaria Three species of orthopterans

~

~

a

Chemical nature

Source

Bursicon Puparium factor (ARF) Puparium factor (pTF) Adipokinetic Myogenic (hindgut)

~

Approximate molecular weight

Brain, ventral ganglia, blood Hemolymph

Protein

40,000

Protein

CNS, hemolymph CC and hemolymph Proctodeal nerves, hindgut, head, terminal ganglion

Protein

90,000 (subunits?) 26,000 (subunits)

~

~

Peptide Peptide

?

400-600

Comments

May be larger in CNS

-

Not present in a dipteran or a lepidopteran

~

Structure of the red-pigment-concentrating hormone from P. borealis:

1 2 3 4 5 6 7 8 pyroGlu-Leu-Asn-Phe-Ser-Pro-Gly-TrpNHz

* Structure of one version of the light-adapting distal retinal pigment hormone: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pr~-Arg-Val-Met-Thr-Glu-AlaNH~

Reference Fraenkel et al., 1966 Sivasubramanian et al., 1974 Fraenkel. 1975 Mayer and Candy, 1969 Holman and Cook, 1972

INVERTEBRATE NEUROSECRETION

181

polysulfated anionic species. This highly negative molecule is thought to participate in the concentration, by ionic binding, of positively charged hormones within the membrane-bound fractions (Tartakoff e t al., 1974). The protein is heavily labeled by incubation with radioactive sulfate, but not with cysteine. In contrast, radioisotopic studies in invertebrate neurosecretory systems (Section V) have demonstrated incorporation of sulfur from amino acids, rather than from sulfate. The presence of sulfur as sulfhydryl groups or disulfides, as opposed to sulfates, is well established for the neurophysins and octapeptides of the vertebrate neurohypophysis. B. ISOLATION AND CHARACTERIZATION O F INVERTEBRATE NEUROHORMONES Biochemical information about the nature of invertebrate hormones, as opposed to the NSM, is still limited to an indication of the peptidic nature of most factors (based primarily on enzyme inactivation studies) and approximate estimates of molecular weight (derived from gel filtration or electrophoretic separation). Complete analyses of the structure of two hormones from the eyestalk of a crustacean have recently been reported. The nature of selected invertebrate neurohormones is outlined in Table I.

1. Crustaceans Progress reports on the isolation, purification, and characterization of crustacean eyestalk hormones have appeared periodically ( Kleinholz, 1970, 1976). Two hormones have been completely characterized, and their structures confirmed by synthesis (Fernlund and Josefsson, 1968, 1972; Fernlund, 1974a,b; Kleinholz, 1976). The redpigment-concentrating hormone from the shrimp Pandalus borealis is an octapeptide with a pyroglutamate residue in place of the Nterminal, a terminal amide group (see Table I for structure), and a molecular weight of approximately 1000. The synthetic product is active in doses as low as 2 x lo-'* gm injected into an intact animal, and also causes pigment concentration in isolated chromatophores (Josefsson, 1975). It may cause migration of pigment in leukophores (white-pigment-containing cells) as well as in erythrophores in natantians (Josefsson, 1975),but is apparently active only on erythrophores in a brachyuran (Uca) and a macruran (Cambarellis) (Fingerman,

1973).

Studies (reviewed b y Kleinholz, 1976) on the eyestalk factor promoting light adaptation of the distal retinal pigment have culminated

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ALLAN BERLIND

in its recent synthesis by Fernlund. This hormone is an octadecapeptide of molecular weight 2200, with a terminal amide group (Table I). The synthetic product is effective not only on the distal retinal pigment, but causes dispersal of pigment in melanophores and migration of pigment in erythrophores and leukophores as well (Kleinholz, 1976). It is possible that there is considerable variability in the structure of equivalent active principles of diverse crustacean species. By gel filtration and electrophoresis of extracts of the eyestalks of five species, Kleinholz (1972) detected seven molecular variants which possess melanin-dispersing activity. These factors are all of similar molecular weight and are not likely to result from the breakdown of a common larger component during isolation procedures. The hyperglycemic hormone of the eyestalk also seems to have a somewhat different structure in Cancer, Orconectes, and Pandalus, as judged by a limited number of observations of variability in molecular weight (Kleinholz and Keller, 1973). 2. Insects Gel filtration of extracts of the CC of Periplaneta americana reveals the presence of at least six peaks with activity when tested for cardioacceleratory effects, hyperglycemic activity, and influence on the electrical activity of the nerve cord ( Migliori-Natalizi et al., 1970). All six peaks are trypsin-sensitive and appear to represent small peptides. Cardioacceleration is caused by four different components derived from the chromatographic column; several of the peaks are active in more than one of the three assay systems tested. Paper chromatographic separation of CC extracts of the same species shows six to nine active regions when tested for cardioacceleratory and myogenic effects, with three peptidic components accounting for almost all the heart-exciting activity (Brown, 1965). Many of the effects are seen with low doses of extract, representing approximately 0.01 CC per milliliter of test solution. The significance of this multiplicity of factors is not yet clear. To some degree the presence of several peaks active on the same target tissue may represent the incomplete dissociation of a large molecule into active subunits. Since other factors now known or thought to be stored in the CC (e.g., ecdysiotropin) were not assayed in these experiments, it is possible that some of the effects tested are subsidiary to untested roles. The controversy concerning the chemical nature of the ecdysiotropic hormone in the brains of insects is discussed in the review by Goldsworthy and Mordue (1974b).

INVERTEBRATE NEUROSECRETION

183

3. Molluscs In some molluscs, neurons with the ultrastructural characteristics of neurosecretory cells can be identified as individuals. These neurons show a standard location within a particular ganglion from animal to animal, as well as consistent branching patterns and physiological properties (Frazier et al., 1967). The relative ease of identification of such cells, coupled with the large size of the perikarya within the ganglion, has allowed analysis of the patterns of protein content and synthesis in individual cells or populations of homogeneous cells. In the sea hare Aplysia californica the bag cells of the parietovisceral ganglia are the only neurons for which a neurosecretory role has been conclusively demonstrated, but cells L2 to L6, R3 to R13, R14, and R15 ( Frazier et aZ., 1967)are characterized by a high content of neurosecretory granules. Protein content patterns in individual cells or homogeneous clusters have been analyzed by staining after gel electrophoresis. The patterns of peptide synthesis in identified perikarya have been analyzed by incubating ganglia in medium containing radioactive amino acid and subjecting cell homogenates to gel electro(usually l e ~ c i n e - ~) H phoresis. Although the general staining pattern (with Coomassie blue) of protein bands separated from presumptive secretory ( R15) and nonsecretory (R2) neurons is similar, the patterns of amino acid incorporation are markedly different, particularly with regard to lowmolecular-weight components (Wilson, 1971). The incorporation of leucine into high-molecular-weight proteins (40,000-60,000) is similar in R2 and R15, but labeling in peptides below 15,000 is much more prominent in the secretory cell. In R15,25% ofthe total leucine incorporation is in a 12,000-MWpeak (Gainer and Barker, 1975). Similarly, prominent synthesis of small peptides is observed in other putative secretory neurons R 3 to R13, R14, and L2 to L6 (Loh and Peterson, 1974; Gainer and Wollberg, 1974; Loh and Gainer, 1975a). In R14, for example, a 12,000-MW peak represents 15% of the total labeled product, and 6000- to 9000-MW, material represents 18% (Gainer and Wollberg, 1974). It has become clear on the basis of a more extensive analysis of < 12,000-MW components in some cells that they are not identical from cell to cell, and that even within a single cell they may be heterogeneous. In studies involving the coelectrophoresis of extracts from cells previously incubated with two different labels the 12,000-MWpeaks of R 3 to R13 comigrate, but ( l e ~ c i n e - ~orH-IT), that of R15 appears to be somewhat different (Wilson, 1974). The final products in bag cells, L2 to L6, R14, and R15, are mainly smaller than 12,000 MW (Loh and Peterson, 1974; Loh and Gainer, 1975a,b).

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ALLAN BERLIND

It has been suggested that low-molecular-weight peptides may be related to electrophysiological properties of neurons, particularly to the capacity for endogenous activity, rather than to secretory function (Strumwasser, 1973). In R15 in Aplysia incorporation of labeled leucine into peptides of 6000-9000 MW is reduced under conditions that abolish the pacemaker function of the cell membrane. More recent results, however, suggest that the presence of small peptides is more closely correlated with the presence of secretory granules than with endogenous or pacemaker activity. Cells L7 to L9 in Aplysia, which are spontaneously active but contain no secretory granules, synthesize no appreciable amount of protein of 12,000 MW or less, whereas cell L5, which is normally silent but contains granules, incorporates considerable leucine into peptides in this range (Berry, 1975). The identity of the low-molecular-weight components as hormones has not yet been established. Analysis of released products by gel electrophoresis has confirmed that the small labeled peptides can be released from bag cells by stimuli known to cause the liberation of an egg-laying hormone. The molecular weights of released products have been estimated at 6000 (Arch, 1972a),4800 (Arch, 1976), and as a heterogeneous mixture of material of 12,000, 6000, and 5 3000 (Loh and Peterson, 1974). Which component actually corresponds to the egg-laying hormone has not been determined. 4. Echinoderms

A peptidic factor extracted from radial nerves in several groups of starfish induces oocyte maturation and spawning when injected into the body cavity (Chaet, 1966a,b). The gonad-stimulating substance (GSS) is not confined to the radial nerve but can also be extracted in smaller amounts from other areas of the body containing nervous elements which stain with PAF (Kanatani and Ohguri, 1966; Atwood, 1973; Atwood and Simon, 1973). Within the radial nerve GSS is found mainly in the ventral layer (Chaet, 196613) and appears to be the product of supporting cells rather than neurons (Kanatani and Shirai, 1970).The active factor is a peptide of 22 amino acids (Kanatani et al., 1971). It can be detected in the body fluids during spawning (Kanatani and Ohguri, 1966)and is clearly of a hormonal nature, but perhaps should not be considered a neiirosecretory product sensu strictu because of its source.

5. Multiple Effects of Peptide Hormones The number of discrete hormones secreted by any nervous system is unknown. In insects, the brain-CC complex is most likely responsi-

INVERTEBRATE NEUROSECRETION

185

ble for the control of many physiological functions (Maddrell, 1974),

including oocyte development (either directly or indirectly via the control of the corpus allatum), ecdysis, hydromineral balance, protein, lipid, and glucose metabolism, heart function, internal muscular activity (Girardie and Lafon-Cazal, 1972), motor activity patterns, sexual behavior (Pener et al., 1972), and color change ( Moreteau-Levita, 1972). Some of the effects reported may be of limited physiological importance. The significance of the effect of extracts (often active only in high doses) on spontaneous nerve activity, for instance, is difficult to evaluate without more detailed information on whether or not specific nerve pathways are altered. Recent reports have appeared on the effects of putative hormonal factors on well-defined sensory, reflex, and motor pathways in molluscs, crustaceans, and insects (Truman and Sokolove, 1972; Arechiga et al., 1974; Berlind, 1976). A study on Aplysia suggests that the egg-laying hormone, which causes behavioral changes associated with oviposition, alters the function of specific synapses within the central nervous system (Mayeri and Simon,

1975).

Some of the activities ascribed to neurosecretory hormones may be subsidiary to other effects of the same molecule. Cardioexcitors are frequently detected in neurohemal organ extracts, often in several biochemically separable fractions from a single gland. This observation raises the possibility that some hormonal molecules with major functions other than circulatory control may include a cardioexcitor moiety to facilitate their dispersion or to promote the spread of metabolites related to their main functions. 1t.has been suggested, for instance, that at least one cardioexcitor in Periplaneta, neurohormone D, also has ecdysiotropic activity as measured by its ability to stimulate RNA production and fatty acid synthesis in prothoracic glands (Gersch and Sturzebecher, 1968). Indications of multiple roles of single molecules have come from most of the major invertebrate groups that have been analyzed. In several insect species water balance hormones appear to be associated with other activities. The diuretic hormone and tanning factor (bursicon) from P. americana have similar molecular weights (about 30,000), and partially purified diuretic hormone increases the permeability of the epidermis to monoamines serving as substrates for tanning ( Mills and Whitehead, 1970). Locust diuretic and antidiuretic factors partially purified by chromatographic techniques can alter both heart rate and lipid mobilization (Mordue and Goldsworthy, 1969; Mordue, 1972). Only two peaks of activity attributable to peptides are reported after paper chromatographic separation of extracts

186

ALLAN BERLIND

of the locust CC when heart-exciting, diuretic, phosphorylaseactivating, and hyperglycemic effects are assayed. Only one of the peaks is attributed to extrinsic cell terminals (Mordue and Goldsworthy, 1969). The only areas of the chromatograms tested, however, were those corresponding to ninhydrin-positive peaks, and it is possible that other active factors were overlooked. (It appears that the small peptide fractions extracted from mollusc neurosecretory cells, and presumed to have hormonal activity, stain poorly with proteindetecting agents applied to gels and are recognized as prominent constituents of the cells only when more sensitive radiotracer techniques are used (Wilson, 1971)). The number of active factors in the locust may therefore be considerably greater than what is suggested by the results cited. In other studies of the locust a single cell type (the AB cell) has been implicated in physiological control of corpus allatum activity, pigmentation, protein metabolism related to oogenesis, hemocyte control, hydromineral balance, and muscle contractility (Girardie, 1972). Only two fuchsinophilic bands in gel electrophoretic separations of brain homogenates are attributable to the AB cells, since they disappear after cautery of the cells. A direct correspondence between the staining bands and the proposed active factor(s) has not been established. Until more extensive purification has been accomplished and chemical structures determined, it will be difficult to assess the extent to which the apparent overlap of activities is due to incomplete separation. Only in the case of the recently synthesized chromatophorotropins from crustacean eyestalks is it possible to have greater confidence that multiple physiological effects may be exerted by a single molecule (Kleinholz, 1976).

6 . Nonpeptide Factors in Neurohemal Organs Both the pericardial organs of crabs and the CC of insects contain significant amounts of nonpeptidic neurohumors. In pericardial organs of several species, axons containing 5-hydroxytryptamine and dopamine, detected by fluorescence microscopy, end within the neurohemal organs, as do peptide secretory cells (Cooke and Goldstone, 1970).Electron micrographs of the neurohemal structures show the presence of endings which contain dense-cored (haloed) vesicles typical of monoamine-secreting cells (Knowles, 1962). There is no convincing microscopic or physiological evidence ( Berlind et a1., 1970) that the monoaminergic endings form synapses on other elements within the neurohemal organ. In Carcinus it has been

INVERTEBRATE NEUROSECRETION

187

suggested that a monoamine (6-hydroxytryptamine) may account for the cardioexcitor activity of pericardial organ extracts (Kerkut and Price, 1964), but in Cancer and Libinia the amount of extractable monoamine appears to be too small to account for such activity (Maynard and Welsh, 1959).The possibility that these substances act as neurohormones in crabs therefore has not been proved. In the CC of some insects, 5-hydroxytryptamine and aminergic substances are probably present and can mimic some of the effects of peptides (Barton-Browne et al., 1961; Brown, 1965; Lafon-Cazal et al., 1973). The low levels of these substances present might indicate a local role within the neurohemal organ rather than hormonal activity (Brown, 1965).The presence of cholinesterase in the C C of Rhodnius suggests that cholinergic transmission mechanisms may be present as well (Beaulaton, 1967). Aminergic and cholinergic neurons in the C C may synaptically activate neurosecretory terminals to promote the release of peptide secretions (Gersch, 1972; Normann, 1974). The presence within the neurohemal structures of ordinary neurons, presumably containing neurotransmitters, is well established. C. CARRIERPROTEINSAND HORMONEPRECURSORS

In the hypothalamoneurohypophysial system of vertebrates, the small peptide hormones vasopressin and oxytocin are loosely bound to larger proteins, the neurophysins. The active factors, with molecular weights of about 1000,are synthesized as part of a larger molecule which is apparently broken down enzymically within neurosecretory granules as they are transported away from the Golgi apparatus (GA) toward storage and release areas (Pickering et al., 1975). It is likely that binding protein and hormone both result from the cleavage of the same protein precursor.

1. Carrier Proteins The existence of similar mechanisms for binding peptide hormones

in invertebrates has long been assumed, almost exclusively on the basis of indirect evidence. The term neurophysin has been applied to such hypothetical carriers in a variety of invertebrate systems, in analogy with the situation in vertebrates. Evidence for the existence of hormone-associated molecules in invertebrates comes from several types of observations. a. Cells that stain similarly with classic neurosecretory agents may

188

ALLAN BERLIND

give very different reactions to other stains. In the pond snail L. stagnalis, for example, seven different cell types can be detected by the use of alcian blue-alcian yellow, all of which react nearly identically to PAF (Wendelaar Bonga, 1970). In P . americana, it has been claimed that all neurosecretory cells stain red with acridine orange (Beattie, 1971b). Gabe (1966) and others have hypothesized that the

common staining component in such systems is a ubiquitous carrier protein which can bind diverse small molecules. Direct evidence for different factors secreted by cells that are all PAF-positive has recently been derived from immunohistochemical studies in Periplaneta. Three antisera produced in rabbits to partially purified extracts of the retrocerebral complex react with different PAF-positive cells of the pars intercerebralis (Eckert, 1973). b. The NSM that reacts with classic stains does not appear to be identical to the hormone, even when it is proteinaceous elements that account for the reactivity. This assertion has almost always been made without complete knowledge of hormone structure but seems to be valid in the few cases in which a partial molecular characterization is available. The clearest case involves a factor that stimulates the hindgut of the cockroach. The effector is present in high concentrations in the proctodeal nerves, terminal ganglion, and brain of several species of orthopterans (Holman and Cook, 1972). The myogenic factor, which is concentrated in a granule fraction, has been isolated and partially purified by gel filtration, ion exchange, and thin-layer chromatography. It is a basic peptide of molecular weight 400-600 which contains no sulfur (Holman and Cook, 1972). Cells in the proctodeal nerve, however, stain intensely with Victoria blue, a sulfurspecific reagent ( Dogra, 1968). The brain of Leucophaea also contains the myogenic factor, which accumulates in uitro. Freshly excised brains contain little stainable material and little assayable hormone activity, but brains incubated for several days show a parallel accumulation of Victoria blue-stainable material and assayable myogenic activity (Marks, 1971: Marks e t al., 1973; Holman and Marks, 1974). The close parallelism between the intensity of staining for sulfur-rich protein and the activity of a non-sulfur-containing peptide hormone is presented as evidence for a protein carrier. Additional evidence suggesting biochemical differences between a stained component and hormone is derived from studies involving the chromatographic separation of peptides fTom insect brains and CC after the incorporation of radioactive cysteine. Cysteine incorporation, which probably accounts for the intense sulfur staining of some brain cells, is found

INVERTEBRATE NEUROSECRETION

189

mainly in large protein fractions, while the smaller peptides with several types of hormonal activity contain little sulfur (Mordue and Goldsworthy, 1969). c. Neurosecretory cells monitored by two different techniques may show fluctuations in staining intensity that are out of phase. Cells in the pars intercerebralis of Oncopeltus fasciatus, the milkweed bug, show a cycle of PAF or alcian blue staining over the first 8 days of imaginal life. The peak of reactivity to alcian blue precedes that for PAF by several days (Mahon and Nair, 1975). It is suggested that the two stains monitor different events, and that the synthesis of hormone mainly stainable with alcian blue precedes the synthesis of a carrier. This interpretation is based on two questionable assumptions: first, that the hormone contains a higher proportion of sulfur than the hypothetical carrier and, second, that synthesis of carrier and peptide can occur independently. The results may be as easily explained on the basis of biochemical alteration of a primary product of the type discussed in Section 111,C,2. Schreiner (1966) has proposed a model for the same species, based on differential solubility in water and alcohol and differential sensitivity to fixation of discrete components of the NSM. In this model a core carrier molecule is surrounded by a shell of hormones. Such models remain highly speculative. d. Studies of the ultrastructure of neurosecretory granules in several species have revealed the existence of a structured matrix, often of low electron density, to which densely staining material appears to adhere. Under conditions that cause hormone release, or possibly during the fixation process, the dense material may be leached off the matrix (Finlayson and Osborne, 1975). In the blowfly Calliphora, material extruded from granules can be detected in the extracellular space. When such figures are observed, irregular patches of dense material are occasionally found still adhering to the matrix (Normann, 1970). It is possible that such figures reflect the dissociation of granular components. e. Direct biochemical demonstration of the binding of small hormonal molecules to a larger carrier is not available in invertebrates, with the possible exception of a study of the eyestalk neurosecretory system in crustaceans. Comparisons by gel filtration of ethanol and distilled-water extracts of a melanin-dispersing hormone ( MDH ) from the fiddler crab Uca pugi2ator suggest the association of a small peptidic factor with a larger lipoprotein moiety (Bartell et al., 1971). Ethanol extraction of eyestalks yields a highly active component, a substantial amount of which migrates with the high-molecular-weight

190

ALLAN BERLIND

fractions in simple gel filtration studies. In water or saline extracts, however, the activity resides mainly in a smaller (MW, -3750) fraction retained on the column, Activity from the high-molecular-weight fraction of ethanol extracts can be shifted to the smaller component by a variety of physical treatments including heating and cooling, stirring, and lyophilization. The large but not the small component appears to be partially inactivated by phospholipase A, whereas the activity of both fractions is destroyed by proteases. Although the form in which MDH is present in the hemolymph is not known, it is somewhat surprising that the dissociation of peptide from the lipoprotein complex appears to result in a great overall loss of activity. The question whether the lipoprotein represents a true carrier, a structural component of the tissue (e.g., microvesicles), or a nonphysiological aggregate promoted by the conditions of ethanol extraction, remains unanswered. Studies of the possible binding of the pericardial organ cardioexcitor peptide (MW, ca. 1000) in crabs have been reported. In Cancer borealis treatment of centrifuged aqueous extracts with acid releases more active material than does reextraction with distilled water (Terwilliger et al., 1970), which may indicate a separation of active hormone from an inactive complex. In gel filtration studies on extracts of pericardial organs of Libinia emarginata, no evidence for binding of the cardioexcitor to a larger component was obtained (Berlind and Cooke, 1970). The active factor in neurohemal organ extracts and that released from the cells by electrical stimulation in uitro both migrate identically in a low-molecular-weight fraction. No active material is found in large-protein fractions from tissue extracts, and no additional activity can be liberated from large proteins by a variety of mild treatments known to promote release of the vertebrate hormone from the neurophysins. If binding to a carrier exists in the pericardial organ system, it must be a different type of interaction than that between the neurophysins and vasopressin or oxytocin.

2. Hormone Precursors In contrast to the paucity of direct evidence for carrier molecules in invertebrates, there is strong evidence for the biochemical maturation of the neurosecretory product as it is transported. Particularly in molluscan neurosecretory cells, active peptides appear to be synthesized as part of a larger protein which is then cleaved to smaller components before release. The idea that the neurosecretory product matures in some manner

INVERTEBRATE NEUROSECRETION

191

during its cellular transit derived originally from histochemical and electron microscope results, and from occasional reports of a lack of correspondence between the hormone content of secretory cells as estimated by staining intensity and by bioassay. In many neurosecretory systems, from essentially all invertebrate groups, the electron density of the contents of elementary granules changes at some time after the granules are formed in the perikaryon (Section IV,B). While an increase in electron density may represent additional accumulation of material by the formed granule, it has frequently been interpreted as indicating a biochemical change in the nature of the granule contents. Decreases in granule density are also common and may occur in the cell body or closer to the terminal regions (e.g., Scharrer, 1963). Histochemical diversity of different regions of individual neurosecretory cells has been noted in insects and crustaceans. Using the alcian blue-alcian yellow technique, which involves the reaction of weakly acid groups to form a blue-green product, and of stronger acids to produce a yellowish hue, Gabe (1967) detected a decrease in the acidity of the neurosecretory product with distal transport in the cerebral neurosecretory cells of 10 species of pterygote insects. This observation is consistent with a progressive increase in the number of 1-2 glycol groups during migration (Gabe, 1972). In the eyestalk system of a crustacean exactly the reverse change in reactivity has been reported, with a brighter-yellow staining of the terminals as compared to the greener tracts indicating an increase in acidity of the product with transport (Lake, 1969). Such changes have been sought, but not found, in other species of insects ( PrentQ, 1972) and molluscs (Wendelaar Bonga, 1970). There also may be a decrease in the disulfide reactivity of a PAF-positive product as it is transported in the crustacean eyestalk ( Lake, 1970). Histochemical heterogeneity of NSM within individual nerve cells in the Daphnia central nervous system may be indicative of biochemical alterations (Van den Bosch de Aguilar, 1972). In neurosecretory cells of the ventral ganglia of phasmids differential staining of newly elaborated granules, as compared to older (stored?) material, has been reported (Raabe, 1965). Azan and PAF apparently stain newly synthesized material preferentially, while the larger accumulations of material in the periphery of the perikaryon react relatively more strongly with chromehematoxylin-phloxin. A discrepancy between the amount of stainable material in a secretory system and the amount of assayable hormone has been reported in locusts (Highnam and Goldsworthy, 1972) and in mosquitos

192

ALLAN BERLIND

(Meola and Lea, 1971). In L. migratoria, the hyperglycemic hormone is apparently synthesized by brain neurosecretory cells and released from the CC [regenerated neurohemal tissue formed at the end of sectioned nervus corpus cardiacum (NCC) do not contain intrinsic cells but do contain large amounts of hyperglycemic activity]. The neurosecretory cell axons form a complex storage area (neuropilar reserve) before the tracts exit from the brain (Highnam and West, 1971). The neuropilar reserve and perikarya stain intensely with PAF but contain little extractable hyperglycemic hormone as compared to the CC. The presence of intensely staining material without corresponding assayable hormone has been interpreted as indicating some type of processing of an inactive precursor molecule as the NSM is transported into the CC (Highnam and Goldsworthy, 1972). Such a conclusion seems hazardous, however, since it is possible that brain neurosecretory cells synthesize more than one factor, and that the stainable material may be related to hormone(s) other than that controlling blood sugar. The best biochemical evidence for the maturation of a secretory product derives from studies of neurosecretory cells in the gastropod mollusc Aplysia. Extracts of the bag cells, two clusters of approximate,ly 400 cells each in discrete groups at the margin of the parietovisceral ganglion, promote egg laying in mature animals (Kupfermann, 1970).I n early gel electrophoretic studies (Toevs and Brackenbury, 1969) the distribution of egg-laying hormone activity in cell bodies and in the sheath of the ganglion (neurohemal areas) was found to parallel the presence of a rapidly moving peptide band. The synthesis of the small, specific molecule (the molecular weight of which was originally estimated at 6000) was followed by combining gel electrophoresis studies with the incorporation of tritiated amino acids (Arch, 1972b, 1976; Gainer and Wollberg, 1.974; Loh et al., 1975). The pattern of incorporation of l e ~ c i n e - ~into H proteins of different sizes is markedly dependent on the elapsed time after a pulse application of the amino acid. The primary product appearing soon after a short pulse label is a component with a molecular weight of 25,000-29,000. With increasing time of chase after the l e ~ c i n e - ~pulse, H there is a redistribution of labeled material into smaller molecules, including a component of approximately 12,000 and a peak (probably heterogeneous) in the region of 6000 and smaller. Conversion of the large precursor to smaller components proceeds normally in the presence of the protein synthesis inhibitor anisomycin (Arch, 1972b; Loh and Peterson, 1974). Conversion is slowed by low temperature and probably occurs through enzymic cleavage of the primary product. The

193

INVERTEBRATE NEUROSECRETION A Rat hypotholomoneurohypophysial neuron

n

>

@ -precursor

Q - neurophysin-vasopressin complex

. --

G

B.li~)&s&

neurophysin vasopfessin

bag cells

n

a

-11 K fragment (not transported) --6K 4.8K 0 -r\J 0.9K

9

-

-*

C Aplysia bag cell

@ - 25K p r e c u r s w - 12K fragment (released in small

.

-6K -<3K

amounts)

FIG.1. Models for the processing of a precursor to hormone and other molecules in the rat hypothalamoneurohypophysial system (A) (Pickering e t al., 1975), and in Aplysia bag cells according to Arch (1976) (B) and Loh et al. (1975) ( C ) .In the rat, a precursor is cleaved within granules during transport and probably gives rise to both the active octapeptide and binding protein (neurophysin). In Arch’s model of bag-cell processing a 29,000-MW precursor is cleaved, possibly before packaging of the product in the CA; it may give rise to an 11,000-MW component which is not transported, and to three 6000-MW peptides. Further cleavage of the latter to 4800- and 900-MWfragments occurs before release. According to Loh et al., a 25,000-MW precursor ultimately gives rise to 12,000-, 6,000- and 53000-MW fragments, all of which are transported and released. The smallest peptide results from the breakdown of the 6000-MW molecule, but the stoichiometric relations between the components are not defined. Which molecules correspond to egg-laying hormone is not known. [As a result of research completed subsequent to the symposium which gave rise t o this model Arch now feels (personal communication) that the heterogeneity of small molecular fractions may represent a phosphorylation step rather than a cleavage.]

194

ALLAN BERLIND

6000-MW fragment appears to be bound to a granule fraction isolated

from other cell fractions (Gainer and Wollberg, 1974), and the final conversion to yet smaller molecules probably occurs within the granule during transit. Details concerning the intracellular distribution and molecular weights of the small molecules resulting from cleavage vary according to different investigators (Fig. 1). Arch (1972b) claims that a 12,000-MW product remains in the cell body while smaller fragments are transported, which would indicate that at least one cleavage step occurs before packaging of the neurosecretory product by the GA. According to Loh e t al. (1975), essentially all radioactivity disappears from the perikaryon by 20 hours after a pulse label, unless axonal transport is blocked by the application of colchicine. There is general agreement that the final products are a mixture of peptides, including some smaller than 6000 MW (Arch, 1976; Loh e t al., 1975). Some or all of the small components are released into the bathing medium by depolarization of the neurohemal area or by electrical stimulation of the bag cells (Arch, 1972a; Loh et al., 1975). Egg-laying hormone is also released by these treatments, but the components responsible for the activity have not been determined. Similar synthetic pathways for the production of small peptides are observed in other presumptive neurosecretory cells in Aplysia (R14, R15, and L2 to L6) (Loh and Gainer, 1975a,b). The sequence of synthesis of hormone as part of a larger molecule, and the subsequent cleavage to smaller active components, reported in Aplysia are similar in outline to the synthetic mechanisms for vasopressin and oxytocin in the mammalian hypothalamoneurohypophysialsystem, for insulin in the /3 cells of the pancreas, and possibly for other small peptides as well (cf. Steiner et al., 1974). The significance of such a mechanism is not yet clear. There may be difficulties involved in directly synthesizing small peptides from the information contained in small fragments of mRNA; or the active fragments may have evolved from macromolecules that originally had unrelated functions. In the case of insulin formation, primary incorporation of the active fragment into a larger molecule may, by proper folding of the precursor, bring into proximity different regions of the hormone moiety to allow the formation of secondary (e.g., disulfide) bonds. Whether or not such a role is likely for prohormones in Aplysia or in other invertebrate systems cannot be determined without additional detailed information on chemical structure. The large size of hormone precursors may also be necessitated by the permeability characteristics of the membranes of the endoplasmic reticulum ( ER). In some protein-secreting cells, these membranes appear to be permeable to molecules with a diame-

INVERTEBRATE NEUROSECRETION

195

ter of less than 20 A, and it is only in the GA that the limiting membrane becomes relatively less permeable to small molecules ( Palade, 1975). The synthesis of the small peptide hormone as part of larger precursors may therefore be a mechanism to ensure that it will remain sequestered within the cisternae of the ER during the early stages of its synthesis.

3. Summary The term carrier protein carries with it connotations of a mode of interaction between a hormone and a larger component for which there is no good direct evidence in invertebrates. In detailed studies of hormonal synthesis in molluscs, there is no indication that the final products are bound to other molecules with which they are associated. There is no evidence for the other functional roles of these molecules that have been suggested in vertebrates (Robinson et al., 1975). Applying the term neurophysins to hypothetical binding proteins in invertebrates seems rather inappropriate in view of the current state of knowledge. IV. Neurosecretory Granules A.

GRANULEISOLATION

Neurosecretory granules can be separated from other cellular constituents by differential or density gradient centrifugation. Although the number of cases in which these procedures have been applied in invertebrates is not large (Table 11), the conclusions derived are no doubt widely applicable to invertebrate neurosecretory systems as well as to those of vertebrates: The hormones produced by such cells are, to a large degree, if not entirely, sequestered in the granule fraction. The nature of the isolation procedure makes it impossible to determine precisely whether or not a significant proportion of the hormone content of cells is normally free in the cytoplasm. This question is especially relevant with regard to the terminal regions, where, according to some hypotheses concerning the release mechanism, a significant amount of extragranular hormone would be expected (Section VI1,C). Electron microscope surveys show that fractions isolated b y current methods are still too impure to allow accurate biochemical characterization. It is therefore not known in any case' what constituents other than hormones are present in granules. In the vertebrate neurohypophysis, the neurophysins are located in granules, as are other molecules of uncertain function (Pickering et al., 1975). In

TABLE I1 GRANULEISOLATION Group Coelenterates Hydra littoralis, H . attenuata Crustaceans Uca pugilator Cancer borealis Insects Periplaneta americana Leucophaen maderue Molluscs Eledone cirrosa Aplysia calijomia Echinoderms Asterina pectinifera

Tissue

Hormone

Reference

Comments

Stalk

Head-inducing

Lentz, 196%; Schaller and Gierer, 1973

Eyestalk

Melanophorotropic

Perez-Gonzalez, 1957; Bartell et al.,

Pericardial organ

Cardioexcitor

Terwilliger et al.,

cc

Cardioexcitor

Evans, 1962

Brain

Myogenic

Sowa and Borg, 1975

Neurosecretory system of vena cava Bag cells

Cardioexcitor?

Berry and Cottrell,

Bioassay not reported

Small peptides

Gainer and Wollberg,

Egg-laying hormone and other small peptides; too little material for EM study

Radial nerve

Gonad stimulating substance

DeAngelis et al.,

Especially pronounced in hypostomal extracts

1968 1970

1970

1974

1972

INVERTEBRATE NEUROSECRETION

197

Aplysia bag cells and white cells several classes of small peptides resulting from the breakdown of the large precursor are likely to b e present in granules (Gainer and Wollberg, 1974). Normann (1974) has suggested that the reasons for sequestration of hormones in a membrane-bound fraction may include the following: (1) Granules may provide protection for the hormone against destruction by cytoplasmic factors; (2) incorporation into granules may lead to more efficient transport of small molecules from the cell body to terminals; (3) sequestration may prevent the hormones from interfering with normal cell function. B. FORMATION OF SECRETORY GRANULES Neurosecretory material in invertebrates is normally synthesized and packaged in the same manner as protein secretory products in general ( Palade, 1975). The early ultrastructural description of the formation of secretory granules in annelids (Scharrer and Brown, 1961) has been reconfirmed in innumerable studies from all groups that have been analyzed. Neurohormones or protein precursors are synthesized at the membranes of the rough endoplasmic reticulum (RER) and carried into its cisternae. The synthetic products are transferred to the GA through more-or-less permanent connections (Borg and Marks, 1973) or via transitional clear vesicular elements. Concentration of the material, or biochemical changes that lead to increased electron density, may occur within the lamellae of the GA, in the granules budding from the ends of the Golgi plates, or within formed granules. The basic scheme suggested b y analysis of electron micrographs has been confirmed by electron microscope autoradiography in two classes of cells of L. migratoria (A. Girardie and Girardie, 1972; J. Girardie and Girardie, 1972). I n the A cells of the pars intercerebralis, label is confined to the RER and Golgi 15 minutes after the application of c ~ s t e i n e - ~after ~ s ; 30 minutes grains appear mainly over the Golgi; at 1-2 hours granules in the perikaryon are the most heavily labeled. Between 4 and 20 hours after the cysteine pulse the number of labeled granules in the perikaryon decreases from its peak value, indicating transport away from the synthetic areas. Marked granules first appear in the axons at about 1 hour. In the C cells of the same animals, the same sequence is observed, but over a slower time course. After formation of the granules by the GA, further changes in density or size may occur. Both increases and decreases in density of contents have been reported. In the intrinsic cells of the C C of the cockroach Leucophaea maderae, the primary granules are highly

198

ALLAN BERLIND

electron-dense, but the contents may become paler within both the perikaryon and cell processes (Scharrer, 1963). More typically, immature granules appear to be formed from the Golgi and acquire increased density during transport or storage (Hagadorn et al., 1963; Bassurmanova and Panov, 1967; Borg and Marks, 1973; Golding and Whittle, 1974; Schooneveld, 1974a). In some cells, neurosecretory granules may aggregate to form much larger inclusions (fusion bodies) for storage in the perikaryon (Golding, 1967c; Morris and Steel, 1975). In the pars intercerebralis of Lampyris noctiluca, the primary product of one class of cells appears to be granules of 800 to 1300-A diameter, which aggregate to form larger storage bodies of 8000 to 10,000 A (Raddoux-Crowet and Naisse, 1974). Before release, the large aggregates are broken down into smaller components along the axon. Other large membrane-bound aggregates with granular inclusions, the multivesiculate bodies, have been called storage bodies by some investigators, but are thought by others to represent autophagosomes which destroy excess material. The question whether or not the presence of granules of diverse appearance within single cells represents a similar diversity of secretory products has occasionally been raised ( Huddart and Bradbury, 1972; Stratton and Booth, 1975). In the cricket (Acheta domesticus) different granule types may appear together near the GA, in axons, or in other cellular regions, and there is little evidence for intermediate types (Geldiay and Edwards, 1973). Such evidence cannot, however, be regarded as strongly supporting the secretion of more than one product, because numerous alternative explanations for differences in granule appearance are currently impossible to rule out. Several reports based on ultrastructural studies have suggested that neurosecretory granules (often electron-lucent) may arise directly from elements of the ER (Bassurmanova and Panov, 1967; Takeuchi, 1968; Schooneveld, 1974a).Whether these elements represent immature secretion granules, or transitional elements for the transfer of material to the GA (Smith, 1975), is not clear. The transitional elements in other protein synthetic systems may represent a critical control point in cellular processing of the secretory product (Palade, 1975). It is therefore of interest that, in some systems, arrest of secretion seems to lead to an accumulation of inclusions of this sort. In neurosecretory cells of the frontal ganglion of diapausing pupae of the tobacco hornworm (Manduca sexta) clear vesicles apparently arising directly from the ER accumulate, but the GA is not active in forming dense secretory granules (Borg et al., 1973). In the wasp Polistes, physiological alterations which arrest egg development cause a similar accumulation of transitional elements, suggesting that processing

INVERTEBRATE NEUROSECRETION

199

has been arrested at a stage before the synthetic product is transferred to the GA (Strambi and Strambi, 1973). Similarly, in mutant Drosophila melanogaster lacking functional ovaries, NSM accumulates within clear vesicular elements from the RER (King et al., 1966). If normal ovaries are implanted, these prominent elements disappear, suggesting that further stages of processing can occur. Formation of secretory granules from whorls of membranous material (originally derived from mitochondria?) has been suggested by Knowles (1964) in axons within the pericardial organ of Squilla mantis. This report has frequently been cited as evidence for axonal synthesis of NSM, but was presented to demonstrate the accumulation and concentration of the secretory product, rather than its synthesis, within the axon. RER, the presumed site of most, if not all, peptide hormone synthesis in invertebrate cells appears to be absent from secretory axons in essentially all cases studied. It must be noted, however, that nonmitochondrial protein synthesis has been reported in some giant ordinary axons, despite the apparent absence of ribosomes in micrographs (Edstrom, 1969).

c.

ACTIVE AND INACTIVE SYSTEMS ( ELECTRON MICROSCOPEEVIDENCE) 1. Molluscs The clearest ultrastructural evidence distinguishing between active and inactive secretory neurons in invertebrates derives from quantitative electron microscope studies of several cell types in the pond snail L. stagnalis. To assess the activity of the synthetic process, perikarya of identified types were analyzed for the extent of RER membranes, number of GA, percentage of active (accumulating dense material) Golgi zones, number of granules, and volume fraction of mitochondria and cytosomes (autophagic vesicles). In the dark-green cells of the pleural ganglia, most of these parameters change dramatically as a result of exposure of the animal to altered environmental salinity (Wendelaar Bonga, 1971b) (Fig. 2). Transferring the animal from pond water to deionized water induces an increase in the total length of the RER and in the percentage of Golgi zones that are active (from 48% in pond water to 60-70% in deionized water); there is no change in the total number of GA. The number of granules observed in the cell body varies with the length of exposure to deionized medium; after 1 day the granule content falls by about 30%,but thereafter increases until it exceeds the original content. The results suggest that granules are transported out of the cell soma rapidly under these conditions, and

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FIG.2. Active and inactive Golgi zones in caudodorsal cells of the snail L. stagnalis. Top: Active zone characterized by dense material within the Golgi lamellae which are often dilated; elementary granules (eg) and other (immature?) vesicular inclusions near the Golgi zone. Bottom: Inactive zone with flattened lamellae devoid of dense material. cv, Clear vesicles; er, granular endoplasmic reticulum; Gs, Golgi saccules; ts, profiles of tubular system. From Wendelaar Bonga (1971a), reprinted by permission of SpringerVerlag and the author.

INVERTEBRATE NEUROSECFWTION

20 1

that the synthetic rate, which also increases, does not begin to override transport until several days have elapsed. If an animal is transferred back to pond water after 15 days in deionized water, the RER rapidly shrinks, and the percentage of active GA falls below the levels of controls maintained for the entire period in pond water. The number of autophagic particles rises during this regression, reflecting the destruction of excess granules. Transferring animals from pond water to hypertonic saline induces changes in the opposite direction. The proportion of active GA falls to 17% in 1 day. The RER is reduced in length, and ribosomes tend to detach from the ER membranes. There is at first an increase in the number of granules in the perikaryon (decreased transport?), followed by a decrease as the number of autophagic particles increases dramatically. In the few Golgi regions that remain active after 15 days’ exposure to saline, the content of acid phosphatase is high, indicating that the cells may have shifted in part to the synthesis of lysosomal rather than secretory products. The hypothesis that the dark-green cells synthesize and secrete a diuretic hormone is supported by ultrastructural evidence for the enhanced release of material from the axonal terminals in deionized medium (Section VIII). The changes in the perikaryon and terminals are observed in ganglia with all connections to the rest of the nervous system severed, indicating that the control mechanisms for synthesis and transport reside entirely within the pleural ganglion (Roubos, 1973).The yellow cells ofthe parietal ganglia show similar changes in response to osmotic alteration, but several other cell types do not (Wendelaar Bonga, 1972). The caudodorsal cells in the cerebral ganglion of the same species of snail exhibit a diurnal rhythm of synthesis and transport which can be entrained by cycles of illumination. As in the case of the dark-green cells, the total number of Golgi bodies appears to be constant, but the percentage that is active varies from a peak of 85% shortly after dark to 35% during the light phase (Wendelaar Bonga, 1971a). The number of granules in the cell body is inversely related to the synthetic activity as judged by the state of the GA. At night there are few granules, mostly localized close to the sites of production, while in the light numerous granules are scattered in clumps throughout the perikaryon. It is likely that transport of granules away from the synthetic areas is very slow during the day (since they accumulate despite decreased synthetic activity) and rapid during the night. In the dark phase, many ofthe small number of granules observed appear to be immature, with irregular outlines and density, while chains of mature granules con-

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nected by bridges are observed within the axons (Roubos, 1975). The rhythm of synthesis and transport exhibited by the caudodorsal cells is abolished by blinding. Since the quantitative methods of analysis used involve accumulated data from many individuals, it is possible that the rhythms are endogenous to the cells but become desynchronized in different individuals when photoperiodic cues are eliminated (Roubos, 1975).

2 . Insects More qualitative assessments of secretory activity in insect cells have come from studies attempting to correlate ultrastructural changes in the perikaryon with extrinsic light cycles, reproductive state, or developmental changes. The overall picture of active and inactive A cells in the pars intercerebralis of the potato beetle Leptinotarsa appears to be similar to that described above for a mollusc. During reproductive diapause the perikarya have many free ribosomes, inconspicuous RER, a large number of dispersed granules, and many lysosomes; during reproductive development the RER is much more conspicuous, and the small population of granules is located mainly close to the synthetic sites (Schooneveld, 1974a) (Fig. 3). The medial brain cells in the blowfly Calliphora erythrocephala also show stages of activity correlated with reproductive state (Bloch et al., 1966). The total number of Golgi complexes increases early during adult development. If the fly is maintained for several days on sugar (a nonreproductive diet), neurosecretory granules accumulate in the perikaryon, the RER becomes disorganized, the number of GA falls, and multivesiculate (lysosomal) bodies are more frequently observed. The changes are indicative of reduced production of hormone and storage in the perikaryon. In meat-fed flies, well-developed ER and numerous GA are maintained; although the granule population is not as high as in sugar-fed flies, it is significant. Ultrastructural signs suggesting increased synthetic activity are paralleled by an increase in the size of the nuclei. In several neurosecretory systems studied during the course of developmental changes, ultrastructural results suggest a more-or-less continuous (uncontrolled?) synthesis of new granules with regulation possibly being provided by intracellular breakdown of the product under conditions in which the hormone is not being secreted. The cells of the pars intercerebralis of R . prolixus have been separated into six ultrastructural types, mainly on the basis of granule morphology (Morris and Steel, 1975). During developmental arrest (caused by starvation) several types of cells contain prominent lysosomal or mul-

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tivesiculate bodies, with images suggestive of extensive granule breakdown. In at least one cell type, the lysosomes disappear abruptly within 1Y2 hours of feeding, suggesting that a brake on secretory activity has been removed. In the silkmoth Bombyx mori, medial cells thought to be involved in controlling development show prominent Golgi bodies at all stages, including diapause (Bassurmanova and Panov, 1967). In another lepidopteran, Pieris rupue, the photoperiod to which the larva is exposed determines whether or not the pupa enters diapause. During the last larval instar, the brain ultrastructure in early stages is similar in animals that do and do not enter diapause, and both are marked by a lack of GA. The GA appear to be reassembled during the second day of the instar and to resume granule formation in animals exposed to either a long day (nondiapause) or a short day (diapause) (Kono, 1975). Cells from the two types of individuals may, however, exhibit different transport and release kinetics. Shortly after the pupal molt, the cells from the two developmental types diverge more markedly. The perikarya of animals entering diapause become packed with granules and contain lysosomal-type breakdown bodies (Kono, 1973), while granules are sparsely distributed in nondiapause individuals. RER and GA are prominent in both types, but it is not clear whether or not they are correspondingly active in both situations. Neurosecretory cells in the pars intercerebralis of the house cricket A. domesticu exhibit ultrastructural and biochemical changes with a daily cycle. Shortly after the beginning of the photophase, when RNA synthesis is high (Cymborowski and Dutkowski, 1969), the cells show enlarged nuclei. The RER is well developed in plates distributed in concentric arrays about the nucleus. The GA are active in granule formation; there are few granules in the perikarya, but many in the axons. At night, there are only disrupted fragments of RER and many free ribosomes. The Golgi bodies appear to be inactive, and no dense material is seen within their lamallae. Granules are numerous within the perikaryon, but absent from the axon (Dutkowski et al., 1971). The brain cells thus seem to actively synthesize and transport a secretory product during the light phase. Most of the studies cited above, and several others (Scharrer and Brown, 1961; Geldiay and Edwards, 1973; Bell et al., 1974), suggest that inactive cells typically store a large population of granules and NSM, while active cells transport material rapidly so that it does not accumulate. In contrast are results on the A and B cells in the pars intercerebralis of L. migratoria. The two cell types, distinguished on the basis of different affinities for standard neurosecretory stains

FIG. 4. Synthetically active and inactive cells in L. mgratoria. Left An A cell (active) in the pars intercerebralis with elongate parallel arrays of rough endoplasmic reticulum (E);the Golgi apparatus (G) are often curved, with dense material accumulating in budding granules (arrow); there are many granules (NS) in the perikaryon. Right: An inactive (B)cell and a cell of intermediate activity (AB);the endoplasmic reticulum of the B cell is disordered and dispersed; Golgi zones are devoid of dense material; there are very few granules, but lysosomes (L) are prominent; CM, multivesiculate body, N, nucleus, M, mitochondrion. From Girardie and Girardie (1967), reprinted by permission of Springer-Verlag and the authors.

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(Nayar, 1955), are thought in many insects to represent two distinct functional classes of cells, which are not interconvertible ( Highnam, 1961b; Delphin, 1965; Gupta, 1970). In Locusta, however, and perhaps in other species as well, the two staining types may represent phases of activity of a single class of cell (Girardie and Girardie, 1967; Girardie, 1973). Interconversion of types is suggested by the apparent constancy of the total number of A and B cells, but the variable ratio of A to B cells in different stages of development or in different physiological states. In the A cells, thought to represent the more active state, the RER is well organized in long, parallel plates, the GA actively condense material and form granules, and granules are prominent in the perikaryon. In the B cells the ER is irregular and dispersed, with poorly developed cisternae, and Golgi lamellae are flattened and mostly devoid of dense material. There are fewer granules, but a more prominent population of lysosomal particles than in A cells (Girardie and Girardie, 1967) (Fig. 4). A and B cells may be involved in the control (via the corpus allatum) of sexual maturation. It is of interest that, after maturation is complete, cells that are well stocked with granules (A cells) may again exhibit higher levels of lysosomal inclusions than during development. A lack of secretory activity may therefore be associated with low granule production or with rapid destruction. D. SUMMARY Ultrastructural studies of invertebrate secretory systems suggest that control of the synthesis of protein hormones and of secretory activity may be exercised at several different points. A cell that is inactive may have a low level of synthetic activity, as indicated by ER or GA in disarray or uonexistent; it may have a permanent Golgi structure but block processing at the transitional membrane systems between ER and GA; or it may continually form granules, only to break them down within the perikaryon.

V. Control of Synthesis and Transport-Radiotracer Studies The incorporation of radioactive precursors into macromolecules in invertebrate secretory neurons has been followed by autoradiographic techniques (in insects and in annelids) and by electrophoretic analysis of specific labeled proteins (in molluscs).

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A. MOLLUSCS The use of labeled amino acids to follow the synthesis and metabolism ofpeptides in neurosecretory cells was discussed in Section II1,A and B. Control of synthesis has been analyzed in the snail Otala lactea and in Aplysia. In Otala, the incorporation of leucine into specific low-molecularweight components in an identified secretory neuron depends on the physiological state of the animal. The snail becomes dormant in response to a decrease in humidity or to a large rise or fall in temperature. Cell 11, in which the electrical properties change markedly during dormancy, also exhibits an altered pattern of protein synthesis. The cell incorporates label into proteins during dormancy, but complete processing to low-molecular-weight components is not observed. A 5000-MW component, which accounts for 20-25% of the total synthetic activity ofthe cell in active snails (Gainer, 1972a), is not formed at all during dormancy (Gainer, 197213). In Aplysia, the protein synthesis patterns in a uniquely identifiable cell can be altered selectively by synaptic or neurohumoral input. Cell R15 is hyperpolarized by the stimulation of specific presynaptic pathways or by the application of dopamine, a putative neurotransmitter at synapses on the cell. Both treatments cause a selective decrease in incorporation of label into the specific low-molecular-weight components produced by this cell, especially the 12,000-MW peptide (Gainer and Barker, 1974). Similar hyperpolarization induced by ionic alteration of the medium does not change the protein synthetic pattern (Gainer and Barker, 1975). The decrease in incorporation is apparently due to the synaptic input rather than to the induced decline in electrical output of cell R15, since abolishing this output by the use of tetrodotoxin (TTX) does not alter the synthetic pattern.

B. INSECTS The fact that the secretory products of many insect cells are rich in sulfur has fostered valuable autoradiographic studies utilizing the incorporation of labeled cysteine as a monitor of synthetic and secretory activity. Interpretations of the results of such studies are not devoid of difficulty. When ~ y s t e i n e - ~is~ S applied to isolated storage lobes of the CC of locusts, very rapid labeling occurs, which may be due largely to nonspecific adsorption (Highnam and Mordue, 1970; Mordue and Highnam, 1973).I n intact systems, the transport of newly synthesized material into the CC, release of labeled peptides, and passive loss must all be assessed before the dynamics of the terminal

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regions can be understood. Only where accurate data on turnover rates after labeling are available can the results be accepted with confidence. If turnover rates are not determined, the extent of radiotracer incorporation monitored autoradiographically may be as difficult to interpret as histological data; an increase in the amount of cysteine fixed in the perikaryon at a unit time after an injection may indicate an increased synthetic rate or decreased transport or proteolytic activity. Finally, conclusions about secretory dynamics based on the comparison of labeling in perikarya and terminals in the CC, seem to assume a homogeneity of cell type which may be misleading. Synthesis and metabolism of sulfur-containing peptides may be different in diverse cell types whose endings in the CC may be difficult to distinguish from one another. The contribution of different types of cells to the overall labeling pattern may therefore be difficult to sort out. Most tracer studies have attempted to relate labeling changes in brain neurosecretory cells to long-term physiological changes, particularly those involved in oocyte development (Gillott and Dogra, 1972; J. Girardie and Girardie, 1972). The amount of label in histologically identified A and B cells in the pars intercerebralis of L. migrutoria has been followed at various times after injection of cy~teine-~'S into the hemolymph. At all times, the A cells exhibit a higher level of incorporation than the B cells, and this is interpreted as lending support to the suggestion that the two types are phases of activity of a single cell (J. Girardie and Girardie, 1972). The cells of the pars intercerebralis of young females with rapidly maturing oocytes incorporate more label than older animals with mature eggs, at all times. Since the old females accumulate stainable material (as the release of a gonadotropic factor ceases?), their low level of incorporation implies that synthetic rates are lower than those of younger females. A brief period of electrical stimulation to the pars intercerebralis induces prolonged developmental changes, as well as changes in the pattern of incorporation of labeled cysteine (Girardie et al., 1975). If the stimulus is applied after a cysteine pulse, labeled material disappears from the perikaryon and CC more rapidly than in unstimulated controls. If the label is provided shortly after the stimulation period, however, incorporation into stimulated cells is about twice as high as in unstimulated controls. It therefore appears likely that stimulation enhances both synthesis of new protein and transport of sulfur-containing material. The locust brain contains other neurosecretory cells (in the subocellar zone) which also incorporate sulfur, but whose metabolism of label does not change with the progression of egg development (A. Girardie and Girardie, 1972). Since many studies have examined only

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correlations between neurosecretory cell metabolism and ovarian development, one is often left with the impression that the activity of all brain neurosecretory cells is intimately related to this function. It is reassuring to find evidence to the contrary. Subocellar cells may be specifically involved in the. regulation of water balance. Exposure to a long photoperiod promotes ovarian development in the Egyptian grasshopper Anacridium aegyptium. During reproductive diapause neurosecretory cells of the brain are characterized by the storage of large amounts of stainable material. Exposure to a 15-hour photophase terminates diapause, and during the early stages of ovarian development the stainability of brain neurosecretory cells decreases. This decrease in NSM is accompanied by an increase in nuclear diameter, possibly indicating that both increased transport and synthesis of NSM occurs. The incorporation of label into A and B cells in the pars intercerebralis has been followed at intervals after injection of ~ y s t e i n e - ~in~animals S exposed to either a 15-hour or 9-hour photophase (Geldiay, 1970).Long-day exposure results in greatly enhanced incorporation in both cell types. Labeling is intense in longday animals at 3-9 hours after injection but, by 18 hours, most of the newly synthesized material has disappeared from the brain. In brains of short-day animals the amount of cysteine increases much more slowly, and peaks later. The radioautographic results are therefore consistent with histological observations indicating rapid synthesis and transport by both A and B cells during ovarian development, and the slow accumulation of material during reproductive arrest. Similar conclusions have been derived from autoradiographic studies of A and B cells of the beetle Galeruca tanaceti. The time of complete turnover of label in the perikaryon is about 72 hours in females in reproductive diapause, and the specific labeling of neurosecretory cells is highest about 24 hours after injection of ~ y s t e i n e - ~(Siew, ~ S 1965).In females with maturing oocytes turnover occurs in 18-24 hours, and peak labeling (which is greater than that during diapause) at 9 hours. During oviposition turnover is complete by 18 hours, and maximum labeling is seen at 3 hours. Calculations from these observations suggest that the synthetic rate is about 10 times faster during oviposition than during diapause. This increase in synthetic rate is correlated with increased nuclear diameter during oviposition. Labeling of the C C was also followed in these experiments, and in diapausing females radioactivity in the neurohemal area builds up slowly over 120 hours. I n ovipositing females the CC are labeled most heavily at 18 hours but have discharged most of this material by 24 hours. More rapid transport and release, as well as synthesis, therefore occurs at this stage.

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The effect of feeding on brain neurosecretory cell metabolism has been monitored in several species of orthopterans and in the hemipteran Rhodnius ( Dogra, 1973). In starved grasshoppers (Melanoplus sanguinipes) cells of the pars intercerebralis stain intensely with ordinary neurosecretory agents. Within 20-40 minutes after feeding, stainable material is greatly depleted in the perikarya, but begins to reaccumulate after 90 minutes. The incorporation of labeled cysteine in the first 30 minutes after injection is not significantly different 90 minutes after feeding than in starved animals, but begins to increase after that time (Dogra and Gillott, 1971).The histological and incorporation studies are interpreted together as indicating a sudden discharge of stored material from the perikaryon, and delayed resumption of synthesis. The technique used, however, did not analyze turnover rate, since incorporation was studied at only one fixed time after injection. The low amount of labeling during the rapid discharge phase may therefore be explained alternatively as indicating rapid synthesis coupled with rapid transport out of the brain. In the locust Schistocerca, feeding after a period of starvation causes similar rapid depletion of PAF-positive material, correlated with the onset of vitellogenesis. Studies of the dynamics of labeling of the CC (analyzed by liquid scintillation counting of extracts rather than autoradiography) suggest a rapid transport of material under conditions that promote egg development. In young females with developing eggs, newly synthesized material arrives in the CC within 1Y2 hours after injection of labeled cysteine. In mature females with completely developed oocytes transport rates are slower, with labeled material arriving only after 3 hours, and in starved locusts slower yet (9 hours) (Mordue et al., 1970; Mordue and Highnam, 1973). When the CC of starved locusts were prelabeled with cysteine, feeding resulted in a massive loss of radioactive material starting about 10 minutes after the meal (Highnam and Mordue, 1974). The results suggest strongly that transport as well as release of neurohormone can be enhanced by the intake of food. Studies on Schistocerca females have also provided direct confirmation of the hypothesis that histologically empty neurosecretory systems may result from low synthetic rates or from rapid turnover. Newly emerged females without developing ovaries and with little stainable material in their perikarya incorporate cysteine slowly; somewhat older females which are rapidly maturing eggs also stain weakly, but synthesize and transport material much more rapidly (Highnam and Mordue, 1970). Circadian cycles of incorporation of cysteine and uridine into macromolecules have been monitored in the house cricket A. domes-

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21 1

ticus. Two classes of neurosecretory cells, one in the brain and one in the subesophageal ganglion, show daily cycles of RNA and protein

synthesis correlated with periods of motor activity. In the pars interH cerebralis neurosecretory cells, the incorporation of ~ r i d i n e - ~into RNA peaks after the onset of photophase. In presumptive secretory neurons of the subesophageal ganglion, RNA synthesis peaks twice, late in photophase and during the dark (Cymborowski and Dutkowski, 1969). Changes in nuclear volume parallel the changes in RNA synthesis. If the brain cells are destroyed, RNA incorporation in the subesophageal cells ceases, suggesting that a factor &om the brain normally stimulates the cyclic production of secretory products in the ventral ganglion ( Dutkowski and Cymborowski, 1971). Protein synthesis in the brain and subesophageal neurosecretory cells (monitored by the incorporation of labeled arginine, leucine, phenylalanine, and serine) appears to peak several hours later than RNA synthesis (Cymborowski and Dutkowski, 1970), although turnover rates were not measured. As indicated at several points above, the histological variable that often correlates best with isotopic measures of synthesis in insects is the size of the nucleus. The value of this parameter as a semiquantitative measure of synthesis has also been suggested by histological studies (Thomsen, 1965; Thomsen and Lea, 1968; Mordue et al., 1970), although nuclear size may not be a valid indicator of this process in all species (Schooneveld, 1970). C. ANNELIDS

A single autoradiographic study utilizing the incorporation of labeled c y ~ t e i n e - ~into H well-defined regions of the brain has been reported for the polychaete Platynereis dumerilii (Muller, 1973). The results are difficult to interpret in terms of the known endocrine function of the brain. During the course of polychaete development a juvenile hormone ( J H ) is secreted. The titer of this factor in the brain and the secretion rate fall as the animal ages (Golding, 1974). A single

factor probably prevents gonadal maturation, promotes segment proliferation, and allows regeneration to occur. If the number of cells labeled after application of c y ~ t e i n e - ~isHmonitored at different developmental stages, an inverse correlation with secretory activity of the brain is obtained. As sexual maturation proceeds (i.e., as brain hormone secretion decreases, there is a sharp increase in the number of cells that incorporate and retain cysteine after a short pulse (Muller, 1973). If part of the animal is amputated during the maturation phase, a treatment that could be expected to increase the secretion of brain

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hormone, the number of labeled cells decreases. Studies of the turnover of labeled material in young but developing worms indicate that newly synthesized material disappears from the cells within 1-3 days after a 2-hour pulse. Muller (1973) therefore feels that the labeling of cells does not indicate storage and decreased release of material. Turnover rates were, however, measured only at one stage of development, and it would be of interest to have corresponding information on earlier and later stages. The neurons monitored are in a region of the brain critical for the control of maturation. Two possible explanations for the inverse relationships observed are: (1)the cells produce JH when they are not involved in producing a hormone regulating some other function; (2) the cells produce a suppressor of JH release, and a rise in their secretory activity induces a fall in JH secretion. It must be noted that these experiments were originally based on the assumption that cells producing JH are sulfur-rich. If they are not (and there is no evidence that they are), then their activity would escape detection by the methods employed.

D. SUMMARY The use of radioactive tracers has provided more direct information on the synthesis, transport, and release phases of the secretory process than have histological data alone. The most striking conclusion derived from these studies is that turnover rates may be extremely rapid under conditions in which hormone is being released. Although radiotracer studies have been of great value in determining the effects of environmental changes on the synthesis of presumptive hormones, they have as yet given little information on the mechanisms by which these factors exert their effects at the level of the secretory perikarya. The recent experiments on transynaptic modulation of peptide synthesis in Aplysia (Gainer and Barker, 1974, 1975), which are a first step in answering questions of this sort, are therefore of profound significance not only to an understanding of secretory processes in invertebrates, but also to studies of changes in nerve function in general.

VI. Transport of Neurosecretory Material The synthetic products of neurosecretory cells, as of ordinary neurons, are transported from the cell soma to axonal terminals. Although evidence from neurosecretory cells provided some of the clearest early examples of such a phenomenon (Scharrer, 1952; Carlisle, 1953), the mechanisms of transport in such cells have not been investigated in detail. Extensive analyses of the transport process in

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neurons (Heslop, 1975) have derived mainly from longer or more homogeneous nerve tracts (optic nerves and sympathetic postganglionic trunks) than are available in invertebrate neurosecretory systems. It is clear from studies of molluscs that colchicine and other agents that alter microtubule function inhibit the transport of labeled peptides from the perikaryon (Loh et al., 1975), as they d o in ordinary neurons. Granules observed in axons often appear to be linked together in trains, bridged by electron-dense material which may be structurally continuous with the granule membrane ( McLaughlin and Howes, 1973; Roubos, 1975).The only estimates of rates of transport derived from radiotracer studies (Highnam and Mordue, 1974) give values that are somewhat slower than those of the fast transport observed in poikilothermic vertebrates ( Heslop, 1975). Histological evidence for transport comes from many studies which show a sudden change in the stainability of axons after activation of the system. Transfer of material from perikarya to axons is especially prominent in many insects that are fed after a period of starvation (Thomsen, 1965; Steel and Harmsen, 1971). In other groups, where storage occurs in the axon as well as in the cell body during inactivity, the tracts may be depleted very rapidly after an appropriate stimulus is supplied (Highnam, 1965; Girardie and Granier, 1973; Highnam and West, 1971; McCaffery and Highnam, 1975a). Conclusions based on changes in axon stainability should be somewhat tempered, however, b y findings which suggest that the reactivity of this region may be more susceptible to the vagaries of techniques than are storage areas in the cell body or terminals (Meola, 1970). Additional histological evidence for transport comes from damming experiments, in which NSM accumulates on the proximal (cell body) side of a ligature across a secretory axon; such studies have proved useful in outlining the pathways taken by particular neurosecretory axons (Scharrer, 1952; Delphin, 1963, 1965; Nair, 1973). There is a considerable amount of circumstantial evidence that the rates of synthesis and transport can be independently controlled to some degree. This is almost certainly the explanation of the many observations that NSM can accumulate in the perikaryon under conditions in which the system as a whole is inactive. Conversely, an increase in the rate of transport relative to that of synthesis must be invoked to explain the rapid emptying of the soma after activation in some systems. Direct evidence that synthesis and biochemical processing can occur in the absence of transport derives from molluscan neurosecretory cells treated with colchicine (Loh et al., 1975)or separated from their axons (Loh and Gainer, 1975b).

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It is less clear that transport and release of NSM from the terminals can be separately controlled, except where the neurohemal area serves as a major storage zone. The most detailed data relating to this question come from the histological studies of Schooneveld (1970) on the cerebral neurosecretory system of Leptinotarsa. When the A cells of this species are analyzed, no correspondence can be found in different individuals between the amount of stainable material in the perikarya and that in their terminals in the CC. This is true even within groups of animals raised under identical conditions and therefore assumed to be in similar physiological and reproductive states. Differences in the relative amount of secretion in different regions of the cell in animals that presumably synthesize and release material at the same average rate suggests that transport may be controlled independently. Some of the discrepancies in the staining of cell bodies and terminals of individual animals may, however, be alternatively explained if discharge and/or synthesis are highly phasic phenomena (Schooneveld, 1970). The mechanisms by which the transport of material within neurosecretory cells is controlled are totally unknown. Even in much more extensively studied neurons, there is still considerable question as to whether transport rates are affected by changes in the electrical activity of the cell (Heslop, 1975). It would be of interest to determine whether there are biochemical changes in the granule membrane, induced by appropriate stimuli, which alter the affinity of the granules for the transport system. VII. Release of Neurosecretory MaterialMicroscopic Studies A.

SPECIALIZATIONS OF THE

NEUROHEMALAREA

In the classic neurosecretory cell, hormone synthesized in the cell body is transported to a neurohemal organ where release into the circulatory system occurs. Easy access to the hemolymph in these areas is often ensured by loss of cellular sheathing elements (glia) from secretory cell terminals and by deep indentations of channels or lacunae of the circulatory system into the secretory tissue (Gabe, 1966; Maddrell, 1974).The cellular stromal sheath that surrounds many neurosecretory terminals appears to be freely permeable to hormones and perhaps to much larger complexes as well. A large surface area for the exchange of material often results from extensive branching of the secretory axons. In some cases such branching is minor: In the segmental neurohemal organs of Schistocerca approximately 100

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branches are formed by 20 axons (Brady and Maddrell, 1967). More extensive branching is observed in the axons of the caudodorsal cells of the pond snail L. stagnalis: 150 cells form approximately 800 branches each, to yield a total of 120,000 terminals in the neurohemal area at the surface of the intercerebral commissure (Wendelaar Bonga, 1971a). The effective surface area for the release of secretion is increased in the case of the million or so terminals ofthe neurosecretory system of the vena cava in octopods by the complex ridgework into which the wall of the vessel is thrown (Alexandrowicz, 1965). B. NONTERMINALRELEASE SITES There is histological and ultrastructural evidence from some insects and crustaceans purporting to show the release of NSM from nonterminal areas. In the vicinity of the neurosecretory cells of the brain of Periplaneta dense accumulations of cysteine-rich material are occasionally observed in distinct extracellular tracts (Dogra, 1968). Such tracts are found only in gravid females and might indicate release of material from neurosecretory perikarya directly into the protocerebrum. Release from perikarya has been suggested in the brains of a homopteran (Srivastava, 1969), a coleopteran (Abraham, 1974), and a lepidopteran (Takeda, 1972). Release of secretion within the brain of insects may also occur at the ends of collaterals normally assumed to be dendritic (Schooneveld, 1974b).Whether material released at such sites affects nerve targets within the brain or leaches into the general circulation is not known. In the eyestalks of a crab, one class of neurosecretory cell body appears to be in intimate contact with the hemolymph and may release its secretion directly from the perikaryon (Smith and Naylor, 1972). In several species of insects, crustaceans, and molluscs axonal release is not restricted to terminals in a neurohemal organ, but occurs preteminally as well (Johnson, 1966; Scharrer, 1968, Bunt and Ashby, 1968). In the NCC of Lepinotarsa some neurosecretory axons form tiny collaterals which end blindly at the surface of the nerve (Schooneveld, 197413). Functionally equivalent “lacunar release sites” occur along the NCC of Locusta (Cassier and Fain-Maurel, 1970b). In the milkweed bug Oncopeltus most of the NSM is probably released from terminals within the aorta, but the same axons may also release material preterminally directly into the hemocoel (Unnithan et al., 1971). Within the neurohemal areas, release often, but not always, occurs at regions of the cell terminal that are well exposed to hemolymph. Electron micrographs of the CC of cockroaches and blowflies reveal

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release sites which are frequently found adjacent to other cells. Secretory material is apparently extruded into narrow intracellular clefts opposite other axons or nonneural tissue (Scharrer, 1968; Normann, 1974).

C. RELEASE MECHANISMS It is universally accepted that most of the hormone content of invertebrate secretory neurons is sequestered intracellularly within the granule fraction (Section IV). Systems presumed or known to release hormone show common ultrastructural characteristics which must be taken into account in explanations of the release mechanism. In addition to populations of elementary granules, such endings typically contain large numbers of clear microvesicles and may contain granule profiles of variable density and/or size. Three basic mechanisms have been proposed for the release of invertebrate neurohormones: (1) exocytosis, (2) intracellular breakdown of neurosecretory granules to yield smaller vesicles or soluble hormones which must then cross the cell surface, and (3)holocrine secretion, in which an entire cell ruptures and its contents spill directly into the surrounding medium.

1. Exoc2/tosis The most frequently reported release mechanism in invertebrate neurosecretory cells, and one consistent for a variety of other protein secretory cells, involves Eusion of the granule membrane with the cell membrane and dissolution of the entire granule contents in the extracellular medium. Excellent micrographs showing stages of the exocytosis process have been produced by many laboratories, including studies on insects (e.g., Normann, 1970; Finlayson and Osborne, 1975),crustaceans (Weitzman, 1969), and molluscs (Wendelaar Bonga, 1970; Roubos, 1975). Profiles of granule membranes attached to the plasmalemma (omega figures) may be observed with electron-dense contents still within, or devoid of contents. The dense former contents, often still in spherical form, are not infrequently observed in the extracellular spaces or in the acellular sheath surrounding the terminals (Bunt and Ashby, 1968; Smith and Smith, 1966; Smith, 1970). Exocytosis involves the addition of a granular membrane to the cell membrane. If a large increase in the total surface area of the cell is to be prevented, a mechanism must exist for taking membrane fragments back into the cell after release has occurred. Such a process provides one explanation for the origin of the prominent population of clear vesicles observed in releasing terminals (Douglas et al., 1971). If the granule membrane is recaptured more or less intact after extrusion of

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its contents, large, clear vesicles would result. However, there is evidence that the membrane can be recaptured in the form of clear microvesicles. If small electron-dense particles (ferritin or thorium dioxide) are placed in the extracellular medium, they can be observed a short time later within terminals enclosed in ordinary clear microvesicles or in acanthosomes (coated microvesicles) (Bunt, 1969; Smith, 1970). In the sinus gland of crayfish coated pits attached to the plasmalemma may be observed with the dense marker within, indicating that acanthosomes may represent the primary uptake step (Bunt, 1969). However, microvesicles may also arise from the ends of deep invaginations of the cell membrane into the terminal cytoplasm. In actively releasing (stimulated) cells of the blowfly CC, clusters of microvesicles appear to arise directly from the membrane of the elementary granule while it is still fused to the cell surface (Normann, 1970). The membrane of the elementary granules formed in the GA is most likely of a composition different than that of the general cell surface. The apparent direct uptake of the former granule membrane, or the formation of acanthosomes, might thus represent reutilization of specialized patches of membrane rather than nonspecific portions of the plasmalemma. It is by no means certain, however, that all the clear microvesicles are identical, or that the entire population is derived from the cell membrane by such uptake processes (Bunt, 1969). The release phase of exocytosis (omega figures) is not usually observed with high frequency in a group of terminals, although a recent report on the cephalic neurohemal organ in a myriapod shows omega figures occurring in essentially all endings without applied stimulation (Juberthie-Jupeau and Juberthie, 1973). Clusters of microvesicles localized around a release point (synaptoid figures), or crenellations of the cell membrane, are more frequently observed. In most quantitative studies of release, it is the frequency of occurrence of microvesicle clusters that is monitored. Electrical stimulation, which can release hormone from neurosecretory cells, can also cause an increase in the frequency of occurrence of omega figures or synaptoid complexes (Normann, 1969, 1970; see also Section VIII), as can experimentally applied variation in the external environment (Wendelaar Bonga, 1971b, 1972; Normann, 1970). The experiments of Wendelaar Bonga (1971a), demonstrating a diurnal rhythm of release of a neurosecretory hormone in the snail Lymnaea, are of great interest. The terminals of the caudodorsal cells exhibit a large increase in total granule content during the normal period of exposure to light. Shortly after the onset of darkness the number of granules decreases to about 25% of its peak daytime level. The de-

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cline in granule content is associated with an increase in the occurrence of synaptoid figures, providing ultrastructural evidence that the stores of granules are being released; during the day less than 1% of the axon profiles are likely to show such figures, while the percentage rises to 25%during the night. After the peak release phase, the terminals remain depleted of granules until resupplied later in the day by transport from the perikaryon. During the interim period between release and restocking the ending is characterized by the prominence of clear vesicular and tubular elements which may represent later stages of reuptake and reorganization of membrane. Convincing biochemical evidence for exocytosis has been obtained from studies of other secretory systems (most clearly in the release of catecholamines from the adrenal medulla) in which other granule constituents, including large proteins, are released with the active factors (Douglas, 1968). Such evidence has not been obtained for any invertebrate neurosecretory system, since detailed information on the contents of granules is not yet available.

2. lntracellular Breakdown In many crustacean and insect neurohemal areas, cell terminals contain not only a population of more-or-less dense elementary granules, but also large granules of low or intermediate density and/or a range of various-sized granules which may exhibit a gradient down to the size of typical microvesicles. The presence of such heterogeneous pDpulations of inclusions is usually more pronounced close to the cell surface, indicating that release phenomena may be involved in their origin (Scharrer, 1968; Shivers, 1969; Simpson, 1969; Andrews et al., 1971). Inclusions of variable density can also, however, occur more proximally within the axons, or even within perikarya (Scharrer, 1963). Electrical stimulation of crustacean pericardial organs (Knowles, 1963) leads to an increase in the proportion of large, irregularly shaped, “empty” granules, although the occurrence of such figures is prominent in unstimulated cells as well (Andrews,

1973).

Large, pale granules may be derived from elementary neurosecretory granules by the leaching out of small osmiophilic components (hormones?) into the cell cytoplasm (Andrews et al., 1971). Increased electron density of the cytoplasmic background has been reported in conjunction with the paling of granules (Weitzman, 1969; Scharrer and Kater, 1969; Cassier and Fain-Maurel, 1970a; Andrews, 1973). The conclusion that loss in electron density implies intracellular release of hormones must, however, betreated with caution. In endings

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with a heterogeneous population of granules, the pale bodies are often swollen, irregular in shape, or have ruptured membranes (Scharrer, 1963; Shivers, 1969; Cassier and Fain-Maurel, 1970a). Although such figures may be indicative of a normal breakdown process during release, it is also possible that they result from a change in the properties of the granule membrane prior to release, or a biochemical change in the stored material, which decreases its affinity for osmium. In addition, the appearance of the granular content is critically dependent on fixation procedures, and differences in apparent content of granules may to some degree be due to fixation artifacts. In crayfish sinus glands, for instance, exposure to lead tartrate for 3 minutes results in a dense-cored appearance of most granules. If the tissue is stained for 5 minutes, the granules appear to be washed out (Bunt and Ashby, 1967). The appearance of granules in vertebrate systems is likewise critically dependent on the duration and chemical conditions, especially the pH of fixation (Morris and Cannata, 1973). Even in cells thought to release their products by exocytosis, paling of granules and membrane rupture may occur if fixation procedures are inadequate. In the snail Lyrnnaea ruptured granules may be observed in actively releasing cells fixed in osmium tetroxide only, but do not appear if treatment with glutaraldehyde is included (Wendelaar Bonga, 1970). If material is leached out of granules into the cell cytoplasm, it must still cross the plasmalemma before it can reach the circulatory system. The difficulty of transporting molecules even considerably smaller than most peptide hormones across a cell membrane without special carrying mechanisms has been stressed by Smith (1971). Such problems have not been discussed adequately by proponents of the hypothesis that the direct supply of releasable hormone is in a cytoplasmic, rather than a granular, pool. A release mechanism of this sort implies that a significant level of hormone must be extragranular during the release process. Although in most granule isolation procedures reported to date substantial extragranular hormone has been found, it is not yet possible to have confidence that it does not arise from the disruption of granule membranes during the isolation procedure. Intracellular breakdown of secretory granules may alternatively lead to the production of smaller vesicles which could then serve as the direct source of exocytotic release. Apparent vesiculation of the membranes of elementary granules to yield clear microvesicles has been reported in the roach CC (Scharrer, 1968) and is especially prominent in actively releasing cells (Scharrer and Kater, 1969). In

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FIG.5. Intrinsic cells of the corpus cardiacum of the Cecropta silkmoth pupa at different stages of initiation of adult development, (A) Immediately after emergence from the cold the intrinsic (IC) cells contain numerous granules. (B) By day 5 of adult development the cells appear to be disintegrating, showing few granules, prominent vacuolization (V),and degenerating mitochondria. The area is invaded by macrophages (MP),

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some cases in which the formation of small granules from large ones has been proposed, at least some of the population of microvesicles have dense cores similar to those of the larger inclusions (Johnson, 1966; Shivers, 1969). The formation of microvesicles directly from neurosecretory granule membranes has also been proposed in crustacean pericardial organs (Andrews, 1973), in endings in arachnid paraganglionic plates ( Juberthie and Juberthie-Jupeau, 1974), in secretory endings in the ganglionic sheath of the snail Helix (Fernandez and Fernandez, 1972), and in the neural lobes of several vertebrates (e.g., LaBella and Sanwal, 1965; Lederis, 1964; Bindler e t al., 1967). 3. Holocrine Secretion A release mode in which the entire secretory cell ruptures during the process has rarely been proposed recently. Such a mechanism was suggested for secretory cells in the earthworm brain (Aros et al., 1965), where cells showing intensive vacuolization and other signs of degeneration were observed at some stages. Disintegrated cells may be replaced by development from a population of undifferentiated cells in the brain. In crab pericardial organs it has been suggested that terminal expansions become disconnected from the axons and break down completely during the release process (apocrine secretion) (Maynard and Maynard, 1962). Recent studies of changes in the intrinsic cells of the CC of the silkmoth Cecropia during development may also indicate a disintegrative process. At the end of diapause the intrinsic cells are full of dense granules. By the third to fifth day after the initiation of development the cells are vacuolated, have swollen and deformed mitochondria, and are devoid of secretory material (Johnson et al., 1976) (Fig. 5). No normal intrinsic cells can be found at this stage, although they reappear by day 7. In preparations in which the application of methylxanthines has arrested development by preventing the proper functioning of the neurosecretory system (McDaniel and Berry, 1974), the ultrastructural changes described above are not observed. 4. Summary Although exocytosis has been clearly demonstrated and most likely represents a prominent mechanism for the release of invertebrate and no normal intrinsic cells are detected. During this period, brain hormone is released from the corpus cardiacum, but the role of the intrinsic cells is not clear. (C) If development is inhibited by the application of methylxanthines ( McDaniel and Berry, 1974), signs of degeneration do not occur at day 5. (Unpublished micrographs supplied by S. J. Berry and E. Johnson.)

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neurohormones, it is not necessarily the only process. It is possible that different mechanisms exist in different cells of a single organism (Cassier and Fain-Maurel, 1970a; Juberthie and Juberthie-Jupeau, 1974), or possibly even within the same cell under different conditions of release (Weitzman, 1969). Conclusions based on changes in density of granule contents or gross morphology of granules in the terminals should be made conservatively, however, since such changes may result from a variety of nonphysiological factors.

VIII. Electrical Activity of Invertebrate Neurosecretory Cells and the Release of Neurosecretory Material A. INTRODUCTION

Neurosecretory cells of invertebrates have many of the electrical properties of ordinary neurons. They can conduct action potentials and can be controlled synaptically by other neurons. The release of active factors from neurosecretory terminals is dependent on the depolarization of the cell membrane that results from the action potential, as is the release of neurotransmitters from ordinary nerve cells. The ionic dependencies for release appear to be identical. In addition, the range of patterns of activity that neurosecretory cells exhibit encompasses a great portion of the range shown by ordinary neurons; there are normally silent cells which must be activated synaptically, pacemaker cells which are spontaneously active, and cells in which the normal pattern of activity consists of discrete bursts of action potentials. Action potentials have recently been recorded from insect neurosecretory cells in tissue culture (Seshan and Levi-Montalcini, 1973). A general property that seems to distinguish the electrical activity of invertebrate neurosecretory cells from that of ordinary neurons is the longer duration of action potentials (2 to 10 times longer than in nearby nonsecretory cells (Yagi et al., 1963; Kupfermann and Kandel, 1970; Normann, 1973; Wilkens and Mote, 1970; Finlayson and Osborne, 1975). This may not be the case in all vertebrate neurosecretory cells (cf. Finlayson and Osborne, 1975). Extracellular recordings from tracts containing neurosecretory as well as ordinary axons do not always provide evidence for distinct differences in this property of the action potential. In crab pericardial organs, for instance, extracellular records from the neurohemal area show no prominent elongation of the action potentials in a large group of small-diameter neurons whose activity is associated with the release

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of cardioexcitor (Berlind, 1969). In contrast, the depolarization recorded intracellularly from neurosecretory terminals in the sinus gland of the crab Cardisoma guanhumi shows an extremely long duration as compared to the action potential in normal crab axons (Cooke, 1967).

B. ELECTRICALACTIVITY AND THE RELEASE OF NEUROSECRETORYMATERIAL 1. Electrical Activity and Release The role of electrical activity of neurosecretory cells in the release of neurosecretory products has been analyzed by studying histological and ultrastructural changes in the cells after electrical stimulation, and by bioassay for the presence of released hormone in the bathing medium. Histological changes following electrical stimulation were first described by Hodgson and Geldiay (1959), who observed a rapid decrease in the stainability of the cockroach CC after a 15-minute period of stimulation of the brain. More recent reports (Gosbee et al., 1968; Girardie et al., 1974) have confirmed this finding in other species. In many cases in which the phenomenon has been described, lack of precise knowledge of the relation between staining material and hormone content precludes a conclusive statement about the effect of such stimulation on hormone release. If electrical stimulation results in changes in membrane properties ofthe cell, granule membranes, or the chemical properties of cell constituents, any of which may alter the accessibility or reactivity of the NSM to stain, a decrease in staining intensity may not be an accurate measure of release. Electrically induced changes in the histological properties of secretory cells have rarely been correlated with direct measurement of the appearance of a hormone in the circulatory system, although they have been related to the occurrence of hormonally mediated events. In mature Schistocerca gregaria, for example, electrical stimulation of the optic tracts leads to a decrease in staining intensity of the pars intercerebralis neurosecretory complex, which is correlated with an enhancement of oocyte growth (Highnam, 1961a, 1962). Copulation causes a similar decrease in stainability and may be the natural stimulus for the release of a factor inducing oocyte maturation. More conclusive structural evidence for a role of conducted action potentials in release derives from electron microscope comparisons of stimulated and unstimulated cells. Electrical stimulation of the NCCl in P. americana induces release of hormone from the CC as judged by

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an increased frequency of occurrence of synaptoid figures, described previously (Section VII,C), of vesiculated granules, and of dense material within the cytoplasm of the endings (Scharrer and Kater, 1969). Ultrastructural evidence for release in this system has been supported by bioassay for released cardioexcitor. Electrical stimulation of isolated CC in the blowfly Calliphora results in the more frequent appearance of omega figures and microvesicle clusters in the intrinsic cells (Normann, 1974). In neither of the cases cited above is there a notable decrease in the total granule content of the terminals. This is probably due to the fact that in these systems only a small fraction of the glandular content may be sufficient to cause measurable biological effects. Detailed information on the effects of electrical activity on release as judged by bioassay of released material is available from crustaceans and insects. Isolated pericardial organs fiom crabs release a peptide cardioexcitor when the axon tracts entering the neurohemal structure are stimulated (Cooke, 1964). Extracellular recordings from the terminal show the presence of a small number of rapidly conducting fibers whose activation does not result in hormone release, and a large number of small, slowly conducting fibers. The action potentials of this second category summate in the extracellular record to give a delayed voltage change, the amplitude of which increases with the number of fibers activated. The slowly conducted compound potential probably represents the activity of secretory cells, since these are the only large class of small fibers in the structure. The amount of cardioexcitor released increases in proportion to the number of slowly conducting fibers activated by the stimulus (i.e., to the amplitude of the recorded voltage change). The amount of material released in as little as 15 seconds of stimulation at 10 per second can be detected by the most sensitive assay hearts (Berlind, 1969). Electrical stimulation causes the release of hyperglycemic hormone and cardioexcitor from the insect CC. Stimulation in situ of the brain of the blowfly Calliphora for 15-20 minutes at 5-10 per second results in the release of measurable hyperglycemic factor from the CC as long as the nervous connections between the brain and neurohemal organ are intact (Normann and Duve, 1969). Direct stimulation of isolated CC also causes release. The specific nerve pathways involved in the release of particular hormones fiom the CC have been analyzed in P. americana. A cardioexcitor is released after brain stimulation as long as the NCCl is intact (Kater, 1968). Direct stimulation of the NCC1, but not the NCC2, releases cardioexcitor activity (Gersch, 1969), while NCC2 stimulation releases both hyperglycemic factor

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and an ecdysiotropic factor (Gersch et al., 1970). Activation of release by electrical stimulation of the brain or tracts may indicate the presence within the tracts of the appropriate neurosecretory axons, but may alternatively be due to the presence in the stimulated areas of nonsecretory axons which form synapses on secretory neurons at their terminals and thus induce release indirectly. The results of Gersch (1972) suggest that the second explanation may be correct in the release of cardioexcitor and hyperglycemic factor from the roach CC. The effect on cardioexcitor release of stimulating the NCCl is abolished in the presence of atropine and enhanced by eserine, indicating that a cholinergic step is involved in the release mechanism. The release of hyperglycemic hormone, however, may involve a monoaminergic step (Gersch, 1972).It may be significant in this regard that there is ultrastructural evidence for synaptic interactions between monoamine-secreting cells containing B-type granules (Knowles, 1962) and the intrinsic cells of the blowfly CC (Normann, 1974), which are thought to synthesize and release a hyperglycemic factor in this animal. Direct depolarization of secretory cell terminals brought about by elevated external potassium concentration also results in the release of hormone from the insect CC (Normann, 1974) and other invertebrate secretory organs. A diuretic hormone is released from neurohemal tissue along the surface of the abdominal nerves of the hemipteran R. prolixus (Maddrell and Gee, 1974). The amount of hormone released by potassium treatment during a 30-second exposure is much larger than that normally released in 3 hours by a maximal natural stimulus (stretching of the abdomen) to the intact animal. This phenomenon is probably the result of the constant and prolonged nature of the depolarization caused by potassium ions, as compared with the brief and episodic nature of depolarizations produced by conducted action potentials. Potassium-induced release is not effected by treatment of the preparation with TTX; the depolarization produced by altering the potassium concentration gradient across the membrane should not be altered by interfering with action potential mechanisms. If cell bodies in the ganglionic mass that synthesize diuretic hormone are separated from their endings in the abdominal nerves, and the ganglion is treated with potassium, no release occurs unless the sheath of the ganglion has been previously split. Depolarization can apparently cause release of hormone from nonterminal areas of the secretory cell if potassium is allowed to penetrate into the ganglion. In the gastropod mollusc A. californica, potassium depolarization

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leads to the release of egg-laying hormone from bag cells (Arch, 1972a).If the protein components of the cells are prelabeled by incubation in tritiated leucine, depolarization of the terminals results in the release of small labeled peptides which can be separated into several fractions by gel electrophoresis (Loh et aZ., 1975). At least three components are present in the external medium, with molecular It is not yet known which moleweights of 12,000,6000, and ~3000. cule corresponds to the egg-laying hormone, or the nature of the significance of released molecules that do not promote egg laying. Release in response to potassium occurs only from terminal areas in the sheath; naked cell bodies separated from axonal processes do not release labeled peptides. 2 . Ionic Dependency of Release The ionic dependencies for the release of invertebrate neurohormones appear to be similar to those for the release of neurotransmitters from ordinary nerves, and for the release of hormones and other protein secretory products from nonnervous tissue. The release of cardioexcitor from isolated crab pericardial organs following nerve stimulation depends on the presence of calcium ions in the medium and is inhibited by excess magnesium (Berlind and Cooke, 1968, 1971).In low-calcium or high-magnesium medium, release of peptide hormone decreases without a corresponding decrease in the amplitude of the conducted compound action potential normally associated with release. Barium ions can substitute for calcium in allowing release (Berlind and Cooke, 1971). The dependence of secretion on calcium ions has been confirmed for the electrically elicited release of cardioexcitor from the cockroach CC (Gersch et al., 1970), for the potassium-induced release of diuretic hormone in Rhodnius (Maddrell and Gee, 1974), and for egg-laying hormones and small labeled peptides from Aplysia bag cells (Arch, 1972a; Loh e t al., 1975). Electron microscopy confirms the necessity for calcium ions in the release of neurohormones from the intrinsic cells of the CC of Calliphora. The increase in frequency of occurrence of omega figures and small vesicle clusters that normally follows potassium treatment is not observed if the medium contains no calcium or elevated magnesium (Normann, 1974). One exception to the general phenomenon of calcium dependency has been reported but not confirmed. The release of hyperglycemic factor from cockroach CC following stimulation of NCC2 does not seem to be calcium-dependent (Gersch et aZ., 1970). Depolarization most likely causes release by making the cell membrane transiently permeable to calcium ions which flow down their electrochemical gradient into the terminal regions. In some neurose-

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cretory cells, there is evidence that calcium ions can carry a significant proportion of the depolarizing current, since lowering the external sodium concentration (Sakharov, 1972) or treating with TTX (Iwasaki and Satow, 1971) does not abolish spikes in some regions of the cell membrane. In the eyestalk of crabs, the soma1 action potential is partially dependent on calcium current, but the axonal spike appears to be exclusively sodium-based; terminal regions were not analyzed with regard to this question (Iwasaki and Satow, 1971). Calcium currents in the soma suggest that release can occur fiom this level of the cell, as well as from the terminals in the sinus gland. The precise mechanisms by which calcium acts to cause release are not known in this or in other systems. The effect of this ion may be exerted on the granule membrane, on a calcium-dependent interaction between components in the granule membrane and the cell membrane, or on cytoplasmic proteins. Normann (1974) has suggested that calcium may act by liquefying the cytoplasm in the terminal areas, allowing the granules greater mobility and access to the cell membrane. 3. Readily Releasable Pools In several systems in which hormone release can be evoked by electrical stimulation or potassium depolarization, not all the available hormone can be released rapidly, even if stimulation is maintained. The rate of release may decline sharply, even when only a small fraction of the total content of the neurohemal area has been released. If an isolated crab pericardial organ is stimulated for several sessions, interspersed with periods without stimulation, release of cardioexcitor tends to decline, with no decrease in the associated nerve activity (Fig. 6) (Berlind, 1969). A prolonged period without stimulation allows partial recovery of the release mechanism. An analysis of the contents remaining in the pericardial organ after “exhaustion” of release has occurred shows that on the average only 6% of the total extractable activity has been released. This phenomenon is also seen in the potassium-induced release of Rhodnius diuretic hormone (Maddrell and Gee, 1974) and ofAplysia bag cell peptides (Arch, 1972a). The failure of release under conditions of maintained depolarization may be due to failure of calcium entry (Maddrell and Gee, 1974)or some other change in the properties of the cell membrane; alternatively, it may be due to depletion of granules adjacent to the membrane, or to a physiological or biochemical change in the properties of stored granules. In vertebrate neurosecretory systems, the newly synthesized granules enter a readily releasable pool but, if not released, are soon shunted to a storage pool (Pickeringet al., 1975). It is possible that granules in storage may require some type of priming

L

e

0.10

TIME (hours)

0.19

0.3

0.75

1.5

3

DOSE-percent of total activity (log. scale)

FIG. 6. Left: release of cardioexcitor peptide from an isolated crab pericardial organ during sequential periods of electrical stimulation (solid bars) separated by periods without stimulation (open bars). Hormone released from the neurohemal organ during 3-minute incubation periods is assayed for its effect on the frequency of beating of an isolated perfused crab heart. Hormone release declines during sequential stimulus periods (A to B), without a decline in the amplitude of the slow phase of the compound action potential (arrow in inset) associated with release. After a prolonged period without stimulation, partial recovery of the release mechanism occurs (period C)(action potentials retouched). Right: A dose-response cuwe of hormone extracted from the pencardial organ after period D. The amount of hormone released during the first period of stimulation was about 0.75% of the original content, and over the course of the entire experiment less than 4% of the total content was lost.

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before they can be released, and that the priming step can become rate-limiting under conditions of intense stimulation. Evidence for such a priming mechanism may be available from observations on crab pericardial organs. In this system the rapid phase of release appears to be relatively independent of sources of metabolic energy, whereas the restocking of the releasable supply is highly energydependent (Berlind, 1969). 4 . Hormone Release without Action Potentials? Williams (1969)has suggested that conducted action potentials are not necessary for release of ecdysiotropic hormones during the pupal-adult molt in the silkmoth Cecropia. In this species secretion from the medial cells of the pars intercerebralis is initiated by a period of chilling of the brain. If a pupa is treated with TTX, which blocks action potentials in normal and most neurosecretory neurons tested by its effect on the sodium channels of the membrane, metamorphosis ensues normally. Activation of the secretory process occurs in the presence of the drug, which remains in the hemolymph throughout metamorphosis at a level high enough to paralyze the animal by its effect on the peripheral nervous system. Proof was not presented that TTX had penetrated into the brain at sufficient levels to block conduc tion, nor has the possibility been tested that the neurons secreting ecdysiotropic hormone utilize a calcium-spiking mechanism resistant to TTX rather than a sodium-based action potential. The possibility that trophic factors in general might be released by mechanisms different from those for more rapidly acting hormones must, however, be considered. Most of the tests correlating nerve activity with release of hormone have been made on rapidly acting factors (e.g., cardioexcitors, hyperglycemic factors) which exert their effects at concentrations representing a minute fraction of the total glandular content. Trophic factors in general act in a more sustained fashion over a long period of time. It will be of interest to elucidate the release mechanisms of such factors.

c.

DETAILSOF ELECTRICAL ACTIVITY OF SELECTEDNEUROSECRETORYCELLSIN MOLLUSCS AND INSECTS

1. Molluscs Information on the electrical activity of neurosecretory cells comes mainly from three species of gastropods, A . californica, Helix pomatia, and 0. lactea.

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a. Bag Cells in Aplysia (Kupfermann and Kandel, 1970). The bag cells, which secrete an egg-laying hormone in mature animals, are normally electrically silent. A single shock to either pleuroabdominal connective induces repetitive spike activity in all cells of the entire group, which continues for many minutes. There is no obvious correlation between intensity of the triggering stimulus and the duration of the ensuing activity. All the 400 or so cells of a single cluster are activated simultaneously, and there are excitatory connections between the cells of one cluster and those on the opposite side. Neither the mechanism of synchronization of the cells nor the mechanism of sustenance of the long-duration activity has been clearly defined. There is no evidence for the existence of a common interneuron which might simultaneously activate all cells, but evidence for electrical interactions between the cells is ambiguous. The duration of activity is to some degree due to reverberatory stimulation between the two groups, but prolonged activity continues even when the groups are surgically separated. The system seems designed electrophysiologically to ensure that a brief natural stimulus (nature unknown) releases a large, and perhaps prefixed, amount of hormone in a relatively short time (Kupfermann and Kandel, 1970). b. Cells R3 to R13, R14 (White Cells) in Aplysia. The functional role of the white cells is unclear. Their neurosecretory nature is assumed from the prominent content of neurosecretory granules and the short processes that terminate blindly in the ganglionic sheath (Coggeshall et al., 1966). The cells also send a longer process into the branchial nerve, which ends in contact with a patch of specialized sensory tissue, the osphradium. The cells tend to fire spontaneously at low frequency and are relatively free of synaptic input within the ganglion (Coggeshall et al., 1966; Jahan-Parwar et al., 1969). Their pattern and frequency of firing can be influenced by chemical stimuli applied to the osphradium. Spikes resulting from osphradial stimulation, unlike those originating spontaneously, appear to be initiated at some distance from the soma (Jahan-Parwar e t al., 1969); although they have been termed antidromic axonal spikes, it is possible that they are functionally dendritic-type events. It is not known whether release of the secretory product can occur at the end of the axon, or only in the sheath. The cells R3 to R13 and the displaced R14 are generally considered a homogeneous population, but the details of spontaneous activity vary considerably for different members of the group, as do the effects of different types of stimuli applied to the osphradium (Jahan-Panvar et al., 1969). c. Cell R15 in Aplysia. Considerable electrophysiological informa-

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tion has accumulated on this cell, but its neurosecretory nature is not yet clear. The cell has a significant population of granules but does not form endings in a sheath as do most of the other secretory cells in Aplysia. The site of release of its products is not known. Recent studies of the effects of extracts of the cell, and of the effects of environmental osmotic changes on its electrical activity, suggest that R15 may be involved in the regulation of hydromineral balance. Extracts of R15 induce an increase in wet weight (water content?) of an animal kept in either normal sea water or slightly hypertonic or hypotonic medium (Kupfermann and Weiss, 1976). Dilute medium applied to the osphradium suppresses the normal spontaneous activity of the cell if the branchial nerve is intact (Stinnarke and Tauc, 1969). R15 is spontaneously active and fires in short bursts separated by intervals of hyperpolarization. The burst pattern exhibits both a diurnal cycle, which can be entrained by light, and a seasonal modulation (Lickey, 1969). The relationship of this pattern of activity to a possible role in osmoregulation is obscure. d. Ce2l 11in 0.lactea, and Other Bursters. A burst pattern of activity has also been reported in neurosecretory cells of the snails Otala (Gainer, 1972a,c)and Helix (Sakharov and Salanki, 1971). In cell 11of OtaZa, as in R15, the burst mechanism is endogenous, and bursts occur in completely isolated cell bodies. Cell 11, which is quiescent during a normal seasonal period of dormancy can be activated by lowering the calcium concentration of the medium, an event that probably occurs normally after estivation (Barker and Gainer, 1973). The resting potassium conductance ofthe cell is also higher in the dormant snail (Gainer, 1972b). The observation that cell 11can be activated to the normal burst pattern by application of lysine vasopressin at low dosage (Barker and Gainer, 1974) has prompted speculation that the peptide may be functionally important in invertebrates as well as in vertebrates. Recent evidence suggests that vasopressin may alter the activity of neurons in the hypothalamus of mammals (Nicoll and Barker, 1971). The burst pattern of such cells, which has been of great interest to neurophysiologists [Strumwasser (1973) discusses the mechanisms underlying burst formation], is somewhat problematical in terms of neurosecretory function. Any fine temporal pattern of nerve activity, and an associated fine pattern of hormone release, would almost certainly be lost during the relatively long time and in the diffuse pathways by which hormone in the circulation reaches its target organ. The burst pattern may rather be a mechanism for increasing the amount of hormone released by a given number of action potentials (Gainer,

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1972~).Facilitation of release by trains of closely grouped spikes, as compared to regularly spaced spikes of the same average frequency, has been reported for the release of neurotransmitter from ordinary nerves in crayfish (Gillary and Kennedy, 1969). 2. Znsects

Intracellular records of nerve activity have been obtained from medial neurosecretory cells in the brains of P . arnericana (Gosbee et al., 1968; Cook and Milligan, 1972) and Sarcophaga bullata (Wilkens and Mote, 1970), and from intrinsic cells in the CC of C. ewthrocepha2a (Normann, 1973). In both Periplaneta and Sarcophaga, the neurosecretory cells tend to be spontaneously active. As in many arthropod neurons, action potentials may not completely invade the soma (Wilkens and Mote, 1970). Spontaneously occurring excitatory and/or inhibitory postsynaptic potentials have been recorded (Cook and Milligan, 1972; Wilkens and Mote, 1910), but convincing data on the modulation of neurosecretory cell activity by specific natural inputs are not available. The intrinsic cells of the blowfly CC have short (50- to 100-p) axons with bulbous ends. The membrane of the perikaryon probably does not support action potentials, but axonal spikes can be recorded in intact CC or in operated glands in which the activity of extrinsic cells has been eliminated by previous severance of the nerves connecting the CC to the brain (Normann, 1973).The normal secretion of intrinsic cells may be controlled via axoaxonal synapses made with the intrinsic cell processes by aminergic fibers from the brain. Electrical activity has been recorded extracellularly from peripheral neurohemal areas in the stick insect C. rnorosus (Finlayson and Osborne, 1975). Burst activity can be recorded from the neurohemal region of the transverse nerve. Impulses in these cells can apparently be initiated either centrally or peripherally in the terminal areas (Finlayson and Osborne, 1970,1975) and are conducted in both directions within the nerve trunk. Four to six neurosecretory axons are involved, and it has not yet been determined whether or not centrihgal and centripetal action potentials can normally occur within the same cell. According to Finlayson andosborne (1975), the frequency of peripherally originating spikes can be modulated by the application of acetylcholine and norepinephrine. These investigators speculate that electrical activity originating in the periphery may convey information to the cell bodies about the level of hormone in the hemolymph, or about release initiated at the terminals by humoral factors.

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IX. Modes of Control of Neurosecretory Cell Activity

A.

SPONTANEOUSLYACTIVE SYSTEMS

Spontaneous electrical activity (i.e., nerve impulses recorded without a specific stimulus being applied by the experimenter) recorded in situ from neurosecretory cells may be of two types. It may be of autogenic origin, or the result of continuous (tonic) excitatory input. Autogenic (pacemaker) activity has been demonstrated conclusively only in several molluscan neurons (Section VI1,C). The physiological role of hormones produced by these cells is not known, so it is impossible to draw any conclusion about the significance of the autogenic mechanism. Hormones that have a tonic inhibitory function, in which a continuous supply of newly secreted active factor is needed to suppress a physiological or morphological change, are prime candidates for control by an autogenic system. The moltinhibiting hormone of crustaceans and the JH of the brain in polychaete annelids are factors of this type. The hormones must be secreted continuously into the circulation to prevent a molt or sexual maturation from occurring; if the source of the hormones is removed by extirpation of the secretory tissue, suppression no longer occurs (Golding, 1974). The precise locations of the cells controlling these phenomena are not known, so that no information is available on their electrical activity. Spontaneously active cells are also likely to be involved in homeostatic control mechanisms (e.g., blood sugar regulation, hydromineral balance), if hormone is rapidly inactivated once it has been released into the circulation. A steady release of hormone, finely modulated by both inhibitory and excitatory inputs would keep the concentration in the hemolymph at an appropriate level for physiological regulation. There are few invertebrate hormones for which accurate information is available on release rates, inactivation rates, and level of circulating hormone in intact animals. In several species of insects (Curuusius, Dysdercus, Locusta) the blood level of a diuretic hormone is controlled by a balance between secretion from the CC and inactivation by the Malpighian tubules (Mordue, 1969, 1972).Cauterization of the medial neurosecretory cells of feeding locusts leads to water retention and swelling (Mordue, 1972), indicating that, under normal conditions, the secretion rate of the diuretic factor must be significant. In the species noted, the hormone is probably secreted continuously as long as the animal is feeding (Berridge, 1966; Pilcher, 1970), to compensate for inactivation rates which are high

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[the equivalent of 0.05 to 0.15 pair of CC disappears from the hemolymph of a locust in 1 hour (Mordue, 1969)l. A surge of diuretic hormone release occurs in locusts fed after a period of starvation, with the equivalent of 0.3pair of CC per hour being liberated into the hemolymph. Evidence for the tonic release of a molluscan neurosecretory factor comes from quantitative electron microscope studies on the pond snail L. stagnalis. When the animal is in its normal environment (pond water), the secretory terminals of the dark-green cells exhibit moderate frequency of release profiles (27% of the axons). The frequency of occurrence of these figures can be modulated in either direction; placing the animal for 1 day in more concentrated medium (0.1 M sodium chloride) decreases the figures to 8%, while exposure to deionized water increases the figures to 50% or more (Wendelaar Bonga, 1971b). A diuretic factor from the pleural ganglion is most likely secreted at a moderate rate when the animal is in its normal environment. It is implicitly assumed in the above discussion that a high rate of spontaneous nerve activity is correlated with a high resting rate ofhormone release. From biochemical and electron microscope studies on Aplysia there is some evidence that the turnover rate (synthesis, transport, and secretion) of small peptides is to some degree correlated with the normal level of spontaneous electrical activity. Cell R15 usually shows a high average frequency of action potentials and a low content of secretory granules; newly synthesized small peptides disappear rapidly from the cell body (Loh and Gainer, 1975a,b). It is likely that newly formed granules are transported from the perikaryon as rapidly as they are synthesized and therefore never accumulate. Cell R14,however, is normally electrically silent and is characterized b y a large content of secretory granules. Newly synthesized labeled peptides disappear much more slowly from the perikaryon.

B. MONONEURONAL NEUROENDOCRINE REFLEXES A neurosecretory cell could theoretically encompass within its confines an entire neuroendocrine reflex pathway if one part of it were specialized for sensory function. There is no unequivocal evidence that such mononeuronal pathways exist. The work of Lees (1964) on the photoperiodic control of offspring type in aphids has frequently been cited as an example of such a phenomenon. Although receptors involved in the response are located in a small region of the brain which includes neurosecretory cells, Lees did not in fact claim that he

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had demonstrated that the secretory cells themselves were the photoreceptive elements. Developmental changes in the silkmoth Antherea pernyi are dependent on the presence of both lateral and medial clusters of neurosecretory cells (Williams, 1969). Ecdysiotropin secretion is initiated in this species by a photoperiod of appropriate length, and the day length can be monitored by brains disconnected from the eyes and from the rest of the nervous system (Williams, 1969). The photoperiodic response appears to be dependent on the presence of the region of the brain containing the lateral neurosecretory cells, and Williams has hypothesized that these elements are the photoreceptors. “Convincing evidence” that lateral cells control transport of ecdysiotropin within the medial cell axons has not yet been presented. In Locusta, lesions of the lateral cells retard or alter the nature of the molting and may control the activity of the medial cells (Girardie and Girardie, 1974). Two preparations in molluscs merit further investigation with regard to the question of mononeural pathways. The control by environmental salinity of the secretory activity of the dark-green cells in Lymnaea appears to be totally normal (with regard to both synthesis and release) in ganglia surgically isolated from the rest of the nervous system (Roubos, 1973). It is possible that there exist within the ganglion ordinary sensory neurons with an osmoreceptor function that control the dark-green cells synaptically. No microscopic evidence has been obtained, however, for synapses on either cell bodies or axons of the secretory cells (Roubos, 1973). The dark-green cells themselves may therefore function as receptors monitoring the osmolarity of the hemolymph. The architecture of the white cells in Aplysia has been mentioned previously (Section VII1,F). The electrical activity of the cell can be modulated by chemical stimuli to the osphradium. It is not clear whether the “axonal” processes of the cells are activated synaptically by primary receptors in the sensory patch, or whether they form sensory endings within the osphradium, which are activated directly (Jahan-Panvar et aZ., 1969). Microscopic evidence for a dual sensory-secretory function of individual cells has also been described. In the accessory genital mass of Aplysia, cells that produce elementary granules in typical fashion extend across the secretory epithelium. The luminal pole of the cell is devoid of granules but possesses sensorylike cilia extending into the lumen; the opposite pole consists of granule-filled processes which

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may play a secretomotor role in the liberation of mucus by adjacent cells (Coggeshall, 1971). C. SYNAPTICCONTROLIN NEUROENDOCRINE REFLEXES The normal pathways for nervous control of the activity of neurosecretory cells undoubtedly involve synaptic input from other neurons and the subsequent modulation of action potential frequency in the secretory axon. The medial cells of the pars intercerebralis of Periplaneta and Leptinotarsa form elaborate dendritic trees which can be detected by conventional staining techniques (Adiyodi and Bern, 1968; Schooneveld, 1970). In the terminal ganglion of Rhodnius, the cells secreting a diuretic hormone give off a dendritic-type process which branches extensively in the neuropile (Maddrell, 1966). The branches are presumably areas of synaptic input. The monopolar nature of the cells in these species is typical of insect neurons in general. Recent morphological studies of the brain and CC in Schistocerca, in which cobalt chloride was applied to individual medial cells or to neurosecretory tracts, and cobalt sulfide precipitated within the cells, showed the presence of similar branches near the perikaryon (Mason and Nishioka, 1974). The cobalt staining technique has also proved useful in providing morphological information on the existence and location of nonsecretory tracts or axons which might form axoaxonal synapses with neurosecretory elements (Mason, 1973). It is suggested, for instance, that axons within the ocellar nerve carry photic information directly to certain of the medial neurosecretory cells. In Mantis religiosa similar results have been obtained by the use of conventional nerve staining (Brousse-Gaury, 1968a). Second-order neurons connect the neuropile at the base of the ocellus to medial and lateral neurosecretory cells of the pars intercerebralis. Unilateral cauterization of one ocellus, or application of opaque lacquer, induces unilateral changes in the staining properties of the neurosecretory cells, indicating that photic input from the ocelli are involved in the normal control of secretory activity (Brousse-Gaury, 1968b). Electrophysiological evidence for synaptic interactions of other elements of the nervous system has been obtained from crustaceans (Iwasaki and Satow, 1971), insects (Cook and Milligan, 1972; Wilkens and Mote, 1970), and molluscs. Excitatory and/or inhibitory postsynaptic potentials resulting from stimulation or occurring spontaneously (i.e., without an experimentally applied stimulus) have been observed in intracellular records obtained from cells in these groups. The sources of naturally occurring synaptic inputs are unknown in most cases. It is not the intent to review here the multitude of factors in the in-

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ternal and external environments of invertebrates which, acting through the nervous system, control the activity of neurosecretory cells. Information of this sort is available in the more physiologically oriented reviews of Maddrell (1974), Goldsworthy and Mordue (1974b), and Golding (1974). The approximate location and nature of receptors involved in particular neuroendocrine reflexes are occasionally known [e.g., abdominal stretch receptors are involved in the control of diuretic hormone release from abdominal neurohemal areas in Rhodnius (Maddrell, 1966) and Periplaneta (Mills, 1967), and the release of ecdysiotropic hormones in Rhodnius (Wigglesworth, 1954)], but the nervous pathways by which receptors exert their control usually cannot be traced within the ganglionic neuropiles. More typically, t&e general environmental or physiological changes that control secretion are defined, but details about receptor function as related to secretion and about control pathways are lacking. The functional pathways for the release of adipokinetic hormone and diuretic hormone in locusts are perhaps the most thoroughly studied examples of control in insects. The secretion of adipokinetic hormone in Schistocerca is greatly enhanced by flight (Goldsworthy et al., 1972a) and serves to increase the lipid concentration in the hemolymph (Mayer and Candy, 1969; Goldsworthy et al., 1972b). The hormone is probably a product of the intrinsic cells of the CC, since regenerated stumps of the NCC do not contain the hormone (Goldsworthy and Mordue, 1974a).Axons entering the CC through both the NCCl and NCC2 seem to participate in release of the factor, since only if both are severed is the flight-induced increase in blood lipid abolished. The mechanism by which flight stimulates release is not known. Injection of trehalose, the blood sugar, into flying locusts prevents the rise in hemolymph diglyceride normally observed (Houben and Beenakkers, 1973), which perhaps indicates that a lowering of metabolic substrates in the hemolymph initiates secretion. It is not clear, however, that such a decrease occurs early enough to account for the rapid time course of hormone secretion after flight is initiated. In other insects the maintenance of flight may be more influenced by metabolic substrates other than diglycerides (Goldsworthy and Mordue, 1974b). It is of interest that, in Calliphora, the maintenance of blood levels of trehalose during flight also seems to involve a product of intrinsic cells, in this case a hyperglycemic factor (Vejbjerg and Normann, 1974). A diuretic hormone in Schistocerca is apparently the product of brain cells which release their product from the storage lobe of the CC after the animal is fed. After cautery of the medial neurosecretory

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cells, release of diuretic hormone after feeding can occur for a limited time, but resupply of the factor to the CC does not occur (Goldsworthy et al., 1973). It seems likely that the secretomotor center of the brain lies outside the secretory area itself, but the precise stimuli that affect this center are not clear. Davey (1962) located labral receptors which evoked, via the frontal ganglion, release of a cardioexcitor from the CC of Periplaneta. It is possible that the diuretic hormone also affects heart rate (Mordue, 1972), but the presence of the frontal ganglion may not be necessary for diuretic hormone release in response to feeding. Foregut chemo- or mechanoreceptors are possibly involved, but hemolymph osmoreceptors appear to have been eliminated as the source of input (Mordue, 1972).

D. CONTROL OF RELEASEAT NEUROSECFWTORYTERMINALS General reviews of cellular aspects of neurosecretion have often illustrated model neurons with synaptic or humoral input impinging directly on the terminals of the secretory cell. Pharmacological and physiological evidence that ordinary nerves synapse with secretory elements within the CC of insects has been presented above. The clearest evidence for control exercised at the secretory terminal derives from studies of an egg development hormone in the mosquito Aedes sollicitans. The hormone is produced by cells in the pars intercerebralis and stored in the CC (Meola et al., 1970). A blood meal evokes the release of material from the storage area (Lea, 1972). If all nervous connections between the brain and CC are severed, the egg development hormone can still be released normally. CC transplanted into animals from which the medial cells synthesizing the hormone have been ablated can likewise release hormone in response to a blood meal (Meola and Lea, 1971). It is likely that a humoral factor is involved in the control of release, since an unfed mosquito whose nervous system is intact releases hormone when parabiotically joined to an animal whose entire neurosecretory complex has been removed by decapitation immediately after it has been fed a blood meal (Lea, 1972).The CC ofA. sollicitans contains no intrinsic secretory cells but is richly endowed with ganglionic cells of ordinary neuronal nature, which appear to form synapses with the secretory elements (Meola and Lea, 1972). The blood-borne factor promoting egg development hormone release may therefore exert its effect either by activating neurons within the CC that synaptically control the secretory terminals, or by itself directly affecting the terminals. The latter explanation seems more likely, since regenerated endings at the base of the

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brain can release hormone in response to a blood meal; it does not appear likely that the nervous connections observed in the intact CC would regenerate normally (A. 0. Lea, personal communication). Humoral control over release from secretory terminals has also been proposed in the case of puparium formation factors in the fly Sarcophaga. The two large proteins promoting tanning (PTF) and retraction of the anterior segments (ARF) are widely distributed within the central nervous system where they are synthesized. They can be released, however, from peripheral endings (Fraenkel, 1975). Ecdysone, which makes target tissues competent to react to A R F and PTF, apparently also promotes the release of the factors from terminals and can do so in animals in which the central nervous system has been entirely ablated (Sivasubramanian et aZ., 1974; Fraenkel, 1975).

E. FEEDBACK CONTROL OF NEUROSECRETORY SYSTEMS The products released from a neurosecretory cell may, directly or through their effects on other tissues, influence further secretory activity of the cell. Experimental evidence for such feedback effects in invertebrates comes mainly from studies of development in polychaete annelids and insects. In nereid worms sexual maturation and regenerative competence are controlled by the declining titer of a hormone produced by the cerebral ganglion. In young worms the brain synthesizes and secretes at a rapid rate JH which prevents gonadal development; the same factor probably promotes segment proliferation and allows regeneration of segments to occur following amputation (Golding, 1972, 1974). Experiments involving the transplantation of brains from mature or juvenile worms into recipients of varying age demonstrates that secretion of the brain hormone decreases as maturation proceeds. Direct measures of inhibitory (juvenile) hormone content (Durchon and Porchet, 1971), and cultures of isolated brains with target tissue (isolated parapodia in which spermatogonia are maturing) (Porchet, 1972), confirm the results obtained in transplant experiments. The rate of secretion at any stage is probably the result of two components, an autogenic brain mechanism which may itself change with age, and negative feedback from maturing gonadal products. Brains from mature worms, which are normally inactive (in terms of repressing gametogenesis or allowing regeneration), can be activated by transplantation into immature hosts, and a brain that is serially transplanted into very young hosts may continue to secrete hormone indefinitely

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(Golding, 1967a,b). The artificial introduction of maturing or mature ovaries into young worms results in precocious development and loss of regenerative ability (Porchet, 1967; Porchet and Durchon, 1968). The intensity of the effect is a function of the maturity of the gonadal tissue implanted, with older oocytes more effectively shutting off the secretion of brain hormone (Porchet and Durchon, 1968). The feedback is mediated by a chemical fraction from maturing gametes and is not due to mechanical distension caused by gonadal products, since introduction of glass balls into the coelom does not shut off brain activity (Porchet and Cardon, 1972). The site at which feedback control is exercised is not known. The infracerebral gland, a structure with an uncertain role, is located at the base of the brain on the opposite side of a neural lamella from the endings of the main neurosecretory tracts (Golding, 1973). Baskin (1974) has speculated that the infracerebral cells may mediate feedback by monitoring the coelomic medium and conveying information regarding it to the nearby secretory cells. Feedback mechanisms, both positive and negative, are also suggested by results of studies on the control of secretion from the insect brain-CC complex. In R . prolixzrs, the staining pattern and intensity of PAF-positive cells in the pars intercerebralis have been followed from the time of feeding a blood meal to the fifth instar larva until after ecdysis of the adult (Steel and Harmsen, 1971). In the first 6-8 days following feeding, during which period the presence of the brain is required for further development, interconversions between several cell stages occur which, according to the interpretation provided, suggest that the rate of discharge of material from the cell soma exceeds the rate of synthesis. During this period, the axon tracts leading to the CC become more heavily stained, indicating an increase in distal transport. After the eighth day, material begins to reaccumulate in the perikarya. Feedback aspects of this system were analyzed by joining parabiotically an intact insect l day after feeding and an animal 7 days more advanced which was decapitated at the time of parabiosis (Steel, 1973). The exposure of the younger neurosecretory cells to physiologically older hemolymph seems to promote the synthesis of NSM or to inhibit its discharge, so that the restocking of cell body contents occurs much more rapidly than in a normal animal. Changes occurring during days 8 to 15 in the normal course of development are observed in the parabiotic system by day 7. Steel has suggested that the changes in synthesis and/or release may result from the activity of ecdysone which is secreted as a result of brain hormone release and is normally present in the hemolymph at high levels during days 7 to 12.

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A possible role of ecdysone in feedback control of neurosecretory cells has been studied in Leucophaea brains under culture conditions. In isolated brains cultured under normal conditions, alcian bluestainable material accumulates in the perkarya and tracts over a 2 to 3-day period. If the culture medium includes B-ecdysone, no such accumulation occurs (Marks et al., 1972; Marks and Ittycheriah, 1971), indicating that ecdysone either depresses the synthesis or enhances the release of the staining factor. There is no indication that the alcian blue-staining factor is directly related to ecdysiotropin, but the direction ofthe effect seems to be opposite that reported by Steel (1973). In dragonfly nymphs removal of the ventral gland, which is the source of ecdysone, results in the accumulation of material in secretory cells of the ventral ganglia (Schaller and Charlet, 1970). The gonadotropic effects of brain neurosecretory products in insects, often exercised through the mediation of endocrine factors from the corpus allatum, may also be subject to feedback from hormones from the corpus allatum or the developing gonads. In the fly Musca domestica, medial neurosecretory cells show cycles of PAF staining intensity correlated with the time course of oogenetic development. In normal animals material accumulates in the cell body toward the completion of egg development. The results of a series of experiments involving ovariectomy, ring gland extirpation, and/or the application of JH (gonadtropic) mimics suggest that the presence of a gonadal factor is necessary for the accumulation of material to occur (Adams et al., 1975). Negative feedback from the maturing gonad may therefore cut off the release ofhormone from secretory cells. Neither JH nor the activity of cells in the ring gland accounts for the feedback effects observed. In Schistocerca, however, ovariectomy leads to an increase in the stainability of brain neurosecretory cells (Highnam, 1962), suggesting that in this species the lack of a gonadal factor leads to NSM accumulation. The presence of a corpus allatum in Calliphora appears to be necessary for the increase in synthetic activity of medial neurosecretory cells (monitored by changes in nuclear volume) resulting from feeding a “reproductive diet” (Thomsen and Lea, 1968);the possibility of indirect effects operating through the gonads has not been eliminated, Basically similar results have been obtained from L. migratoria. Starvation or allatectomy of adult locusts leads to a decrease in synthetic activity of the pars intercerebralis cells, and to a moderate accumulation of material in the neuropilar reserve (McCaffery and Highnam, 1975a).Implantation of active corpora allata, or the application of JH (or its mimics famesyl methyl ether or famesol) increases the synthetic activity of the repressed brain cells, causes a rapid de-

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pletion of material from the neuropilar reserve, and apparently increases distal transport (McCafferyand Highnam, 1975a,b). It is therefore likely that both synthesis and movement of NSM, and also its release, are under the positive control of a gonadotropic factor. It is not clear whether these processes are directly controlled by JH, or indirectly via a change in electrical activity of the cells. To some degree, transport and synthesis seem to be under separate control, since farnesyl methyl ether stimulates synthesis less effectively than JH, but transport more effectively (McCaffery and Highnam, 1975b). Positive effects by allatal products on brain secretory effects are also suggested by other data from diverse species: (1)The application of JH or analogs to beetles (Leptinotarsa)in reproductive diapause can lead to an immediate termination of diapause, with the cerebral neurosecretory cells becoming activated within a day (Schooneveld, 1973); (2) farnesyl methyl ether accelerates egg development in Rhodnius, but only if the cerebral neurosecretory effects are intact (Pratt and Davey, 1972). It is of interest with regard to the question of positive feedback that a brief period of electrical stimulation of the pars intercerebralis ofA. aegypturn can set in motion long-term processes ofvitellogenesis and egg development (Girardie et al., 1974). It is possible that the initial activation of the medial cells, operating through the corpus allatum, establishes a positive feedback loop which sustains the neurosecretory activity of the brain as long as is necessary to complete ovarian development.

X. General Summary and Perspectives The studies reviewed here point to several areas in which continuing research on invertebrate systems should lead to results of importance to the future development of the concept of neurosecretion. Many of the problems suggested by cellular studies will require concurrent analysis on other levels of biological organization. Biochemistry: Analysis of the complete structure of invertebrate neurohormones has been limited, and is likely to remain limited, by the small amounts of material available. The use of the chromatographic and electrophoretic microtechniques currently available will no doubt provide more complete information on the range of active factors and their distribution in individual species. Assays of synthetic crustacean chromatophorotropins tend to confirm the impression derived from many partial purification schemes that unique molecules have the potential to affect several target organs. It is likely, however, that organisms possess mechanisms to control separately the

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responsiveness of individual targets. Such mechanisms might include: local control of target sensitivity mediated through some aspect of the intrinsic metabolism (unrelated to hormone action) of a particular target (Golding, 1967d); the antagonistic or synergistic control of some targets of a particular hormone by blood-borne factors from other sources, or by nervous input; local differences in hormone concentration occasioned by direct delivery of a hormone to some tissues and broadcast dispersal through the circulatory system to others. Release heterogeneity: Studies on mollusc neurosecretory cells have demonstrated clearly that several peptides are released by the activation of a homogeneous population of neurosecretory cells. The question whether more than one molecule released by a neurosecretory cell can have physiological activity remains to be answered. It will be of particular interest to determine whether or not different factors produced by individual cells have effects which differ greatly in time course [e.g., whether some factors exercise kinetic effects while others are involved in trophic action (Goldsworthy and Mordue, 1974b)l. This question is relevant to the function of ordinary neurons as well. Electron microscopy of cellular dynamics: Dramatic changes in cell ultrastructure after experimental manipulation are often qualitatively clear. There are, however, many reports of changes in activity or modes of granule processing in which the interpretation of micrographs is considerably more obvious to the investigator than to the reader. The careful quantitative studies of Wendelaar Bonga should establish a standard for further work with this technique, not only because synthetic activity can be estimated more accurately, but also because changes in the function of different regions of individual cell types are analyzed. The techniques ofelectron microscope autoradiography have been used only sparingly in studies of invertebrate neurosecretory systems and have the potential to provide valuable information on granule processing and transport, and on the possibility of an extragranular pool of hormone or hormone-associated molecules in the axon terminals. Control mechanisms: Some invertebrate neurosecretory cells present many of the same experimental advantages for electrophysiological studies as ordinary invertebrate neurons over their counterparts in vertebrates. Molluscs in particular, but perhaps other groups as well, should provide preparations suitable for detailed investigation of the integrative abilities of secretory neurons, of neuronal and/or humoral pathways which influence the electrical activity of such cells (neuroendocrine reflexes), of the effects of neurosecretory peptides on nerve activity [possible neurotransmitter or modulatory

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function, in the sense of Florey (1967)], and of the mechanisms that control the synthetic machinery and biochemistry of secretory neurons. The answers derived from such studies should be of interest not only to neuroendocrinologists but also to neurophysiologists of all persuasions. ACKNOWLEDGMENTS

I thank H. A. Bern and S. J. Berry for their helpful criticism of the manuscript. I am grateful to H. Schooneveld, S. E. Wendelaar Bonga, A. Girardie, and S. J. Berry who provided me with copies of micrographs, and to Denise Lensing who translated many articles from German and Italian.

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