Cytobiology of the Ovulation-Neurohormone Producing Neuroendocrine Caudo-Dorsal Cells of Lymnaea stagnalis

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 89

Cytobiology of the Ovulation-Neurohormone Producing Neuroendocrine Caudo-Dorsal Cells of Lymnaea stagnalis E. W. R o u ~ o s Department of Biology, Free University, Amsterdam, The Netherlands 1. 11. 111.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of the CDC in Reproductive Activity Chemical Nature of CDC Secretory Materi A. Cytochemical Aspects B. Purification and Chara ......................... Cytology of the CDC A. Topology and Gross ogy . . . . . . . . . . . . . . . . . . . . . . . . . B . Nucleus C. Soma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Axon.. . . E. Neurohem ...................................... States of Electrical Excitability of the CDC. . . . . . . . . . . . . . . . . . . . . A. Electrophysiological Characterization of the States . . . . . . . . . . . B . Relation betwecn States, CDCH Release, and Egg Laying Exocytosis of CDC Secretory Material. . . . . . . . . . . . . . . . . . . . . . . . . A . Demonstration of Exocytosis B. Relation between Exocytosis C. Exocytosis-Coupled Membrane Recapture and Processing.. . . . D. Role of Calcium in the Control of Exocytosis.. . . . . . . . . . . . . . Synchrony between Individual CDC ... Control of CDC Activity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Evidence for the Existence of Neural and Nonneurdl Inputs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. External and Internal Controlling Factors. . . . . . . . . . . . . . . , , . , C. Role of CAMP in the Cellular Response . . . . . . . . . . . . . . . . . . . Nonsynaptic Communication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction For a long time it has been common use in neurobiology to distinguish two types of nerve cell, viz. “conventional neurons,” which release neurotransmitters (e.g., acetylcholine, bioamines, and some amino acids) at synapses, and “neurosecretory cells,’’ which release peptidergic neurohormones into the blood 295 Cap)right ii-’ l Y X 4 by Academic PIC%. Inc. All right5 III rcprcnlucuon i n any lorn1 rcwvcd

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or lymph via neurohemal axon terminals. However, to date the usefulness of this distinction is disputable since evidence is rapidly accumulating that the characteristics of the functioning of neurosecretory cells do not basically differ from those of conventional neurons (e.g., de Vlieger, 1981) and, more specifically, that peptides do not exclusively function as neurohorniones but occur in various types of conventional neuron and can act as neuromodulator or neurotransmitter. As a result peptidergic neurons are gaining considerable attention of both neurobiologists and endocrinologists. One of the most important aims is to elucidate the nature of the mechanisms that control synthesis, storage, transport, release, and the function of the secretory peptides. Thus, nerve cells of various animal species are being investigated with morphological, (neuro)physiological, pharmacological, endocrinological, or biochemical techniques. However, multidisciplinary studies of a particular type of neuron are relatively scarce. This is partly because in most animals neurons of special interest cannot be identified as they are located deeply within the brain (vertebrates) or ganglia (e.g., annelids) and are intermingled with numerous other types of neuron. Furthermore, many neurons are too small for intracellular recording of membrane potentials or for intracellular injection of dyes or tracers. Some molluscs form fortunate exceptions to this rule; their central nervous system (CNS) contains a rather low number of neurons, many of which are well accessible to various experimental approaches since they have giant size (e.g., up to 1 mm diameter in the marine opisthobranch snail Aplysia californica). They can be easily identified because of their characteristic white, yellow, orange, or red pigmentation and superficial, specific location in the ganglia, just beneath the often thin, transparant connective tissue sheath. Therefore, it is not surprising that two of the most investigated peptidergic systems are found in molluscs. The one is formed by the bag cells of A . califovnica. These cells are particularly known from neurophysiological, biochemical, and behavioral studies (for reviews see Kandel, 1979; Strumwasser et a / ., 1980). The other peptidergic model system consists of the caudo-dorsal cells (CDC) of the freshwater pulmonate snail Lymnaea stagnalis. During the past 25 years the CNS of L . stugnalis has been studied extensively in the Department of Biology of the Free University in Amsterdam by research workers from various disciplines. At first neurosecretory neurons were distinguished from conventional ones on the basis of cytochemical staining properties (e.g., Gomori’s chrome-hematoxylin-phloxin method: Lever et al., 1961; Joosse, 1964, and the Alcian blue/Alcian yellow with oxydation technique: Wendelaar Bonga, 1970; Boer et al., 1977b; van Minnen et al., 1977; Wijdenes et al., 1980) and morphological characteristics (presence of neurohemal axon terminals: Joosse, 1964, and of large-sized elementary (secretory) granules: Boer et ul., 1968). In that way 10 types of peptidergic neurosecretory cell were identified. More recently, about 20 additional peptidergic neurons were found by

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FIG. 1. Diagram of cerebral ganglia and cerebral conunissure of L. stagnalis, with CDC ( I ) , lateral lobes (2), medio- and latero-dorsal bodies ( 3 and 4),and medial and lateral light green cells ( 5 and 6).

light and electron microscopic immunocytochemistry, using antisera raised to biologically active peptides from vertebrates and invertebrates (Boer et al., 1979; Boer and Schot, 1983; Schot et al., 1981; Schot and Boer, 1982). Of some of the peptidergic neurons the function has been elucidated (see Geraerts and Joosse, 1984) and of these the CDC are now being investigated most thoroughly. The 100 CDC are located in two clusters, one in each cerebral ganglion. They release an egg laying stimulating neurohormone from the neurohemal area in the periphery of the intercerebral commissure (Figs. I and 2). This review gives a survey of the present state of knowledge of the morphological, neurophysiological, endocrinological, biochemical, and parmacological characteristics of the CDC, with particular reference to structure-function relationships.

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11. Role of the CDC in Reproductive Activity

L . srugnulis is a simultaneous hermaphrodite, which means that sperms and oocytes mature simultaneously. This occurs within the same acini of the ovotestis (for reviews see Joosse et al., 1982; Joosse and Geraerts, 1983; de JongBrink et al., 1983; Geraerts and Joosse, 1984). Copulation occurs frequently, but isolated snails can perform self-fertilization. Female reproduction starts at a shell height of 23 mm and includes the ovulation of numerous (up to 200) oocytes from the follicles, fertilization in the hermaphroditic duct, and packaging into an egg mass which consists of several layers of secretions produced by a number of accessory sex glands (Plesch et al., 1971). Oviposition occurs - 2-3 hours after ovulation. The control of female reproductive activity is primarily exerted by two centers. One is formed by the paired medio- and latero-dorsal bodies, which possibly are the only true nonneural endocrine organs of L. stagnalis. The bodies are situated upon the cerebral ganglia (Fig. 1) and produce the dorsal body hormone, which stimulates vitellogenesis in the oocyte, and cellular differentiation, growth, and synthetic activity of the female accessory sex organs (Geraerts and Joosse, 1975; Geraerts and Algera, 1976; Veldhuijzen and

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Fit;. 2. CDC stained with Gomori's chrome-heniatoxylin-phloxin method. (a) Somata with Nissl disks. Note thin perineurium (arrow) !hat facilitates in virw visualization and iinpalcinent of CDC with niicroelectrodcs. Large51 cell in the section is 70 piii i n diameter. X475. (b) Closely packed ncurohemal axon terminal\ (arrows) just underneath perincurium of cerebral coniniissurc. Near the origin of the commissurc protilcs of the inedio-dorsal bodies ( M D H ) can be bccn. X 125. From Joosse I -

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Cuperus, 1976; Wijdenes et ul., 1983). The CDC constitute the other center that controls female reproductive activity. The neuroendocrine character of the CDC has been established by classical endocrinological experiments: cauterization of the CDC results in cessation of ovulation whereas injection of homogenates of their neurohemal area induces ovulation in CDC-cauterized snails (Geraerts and Bohlken, 1976). Apparently, the CDC produce a neurohormone (CDCH) that stimulates ovulation. Possibly, the hormone acts on muscle cells near the oocyte follicles, but as yet this has not been proven beyond doubt (cf. de Jong-Brink and Goldschmeding, 1983; de Jong-Brink et al., 1983). In vifro studies with extracts of the CDC neurohemal area (Wijdenes et al., 1983) suggest that the CDC stimulate the synthetic activity of the albumen gland, an accessory sex gland that secretes the galactogen- and protein-rich perivitellin fluid that serves as a nutrient for the embryo (de Jong-Brink et al., 1983). Lymnaea shows a characteristic egg laying behavior, which consists of four stereotyped phases, characterized as follows (Goldschmeding et al., 1983). (1) “Resting phase” (- 25 minutes): stop of locomotion, and posture change. (2) “Turning phase” (- 95 minutes): bending of the head/foot, turning of the shell, and frequent eating movements to clean the substrate before attaching the egg mass to it. (3) “Oviposition phase” (- 12 minutes): oviposition. (4) “Inspection phase” (less than 2 minutes): brushing over and along the egg mass, probably to flatten and anchor it. Obviously, the CDC are involved in the control of this behavior since all phases, except the resting phase, can be induced by injection of extracts of the cerebral cornrnissure. The absence of the resting phase in injected snails may be explained by assuming that this phase is triggered by a local, i.e., not hormonal, action of the CDC (Goldschmeding et al., 1983). The precise way the CDC control egg laying is unknown. It has been shown that the CDC can modulate electrical activity of the cerebral Ring-neuron, which synaptically regulates pedal motorneurons involved in headlfoot movement control (Jansen and Bos, 1983, 1984; see also Section VIII,B,7). Furthermore, the CDC affect interneurons and motorneurons of the central neuronal feeding network (Goldschmeding et al., 1983). It remains to be seen whether the CDC exert these different functions by the release of only one substance (CDCH?) or by releasing, possibly at different times, several biologically active products (cf. Section 111,B). Female reproductive activity of L . stagnalis strongly depends on external factors, such as photoperiod (Bohlken and Joosse, 1982, 1983), temperature (Joosse and Veld, 1972), nutritive state (Scheerboom, 1978; Dogterom et al., 1983c, 1984), quality and oxygen contents of the water (de Vlieger et a/., 1980; Kits and ter Maat, 1983), and tactile stimulation of the skin (ter Maat et al., 1983a,b). At least some of these factors affect reproduction by influencing the activity of the CDC (see Section VIII).

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111. Chemical Nature of CDC Secretory Material A. CYTOCHEMICAL ASPECTS At the light microscope level the secretory material in the CDC somata and axons is visible as small granules, larger drops, or floccules. The proteinergic nature of the material appears from the positive staining with bromophenol blue, which is counteracted by protein digestion procedures involving trypsin or pepsin (Boer, 1965). The material is basic since it stains with acid dyes such as phloxin, azocarmine, orange G, eosine, and acid fuchsin (Joosse, 1964; Boer, 1965; Wendelaar Bonga, 1970). The sulfur-containing amino acids cystine, cysteine, and methionine do not seem to occur in appreciable amounts as negative reactions have been obtained with Gomori’s chrome-hematoxylin, paraldehydefuchsin, Alcian blue/Alcian yellow with oxydation, and performic acid-Alcian blue (Boer, 1965; Wendelaar Bonga, 1970). On the other hand, tyrosine can be demonstrated with Millon’s method (Boer, 1965). No carbohydrate component is detectable using PAS, Best’s azocarmine reaction for glycogen, or staining methods for acid mucopolysaccharides (Steedman, Hale, Muller) (Boer, 1965; Wendelaar Bonga, 1970). Although the material is not extracted by lipid solvents, it is weakly stained by Sudan black B and Lux01 fast blue. Possibly, it contains lipid that is very closely associated with protein (Boer, 1965). Recently, light microscopic immunocytochemistry showed that the CDC axon terminals positively react with an antiserum raised to metenkephalin (Schot et ul., 1981). This staining may be the result of a cross-reaction since the structure of CDCH is completely different from that of metenkephalin (see Section ILI,B). On the other hand, injection of metenkephalin upto a (rather high) final hemolymph concentration of lop4M leads to ovulation (W. P. M. Geraerts, personal communication). Therefore, it may well be that the CDC contain metenkephalin in addition to CDCH. The absence of metenkephalin positivity in the soma may be a consequence of the relatively low number of elementary granules in the soma (see Section IV,B) or it indicates that metenkephalin is formed during or after axonal transport of elementary granules toward the axon terminals.

B . PURIFICATION AND CHARACTERIZATION CDCH has been partially purified, starting with a hydrochloric extract of cerebral commissures. Biological activity was tested with the rapid in vivo assay of Dogterom et ul. (1983a): 30 minutes after injection of a fraction into recipient snails the presence of egg cells and eggs in the spermoviduct and female duct is chequed. Purification was carried out by gel permeation chromatography on BioGel P-6 followed by cation exchange on SP-Sephadex C-25. With stability tests, proteolytic enzymes, SDShrea polyacrylamide electrophoresis, and iso-

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electric focusing, (partially) purified CDCH was characterized as a stable peptide with a molecular weight (MW) of 4700 and an isoelectric point (PI) of 9.3 (Geraerts et al., 1983a). These properties are strikingly similar to those of the egg laying hormone of A . californica (MW, 4385; pl, 9.0-9.2; Chiu et al., 1979), which suggests that the peptides are chemically closely related. However, it has to be doubted whether the hormones are identical, since it has been shown that both among opisthobranch and among basommatophoran snail species ovulation hormones are different (Ram et af., 1977; Dogterom and van Loenhout, 1984). The above described two-step procedure resulted in a low purification (purification factor - X22). Therefore, recently another procedure has been applied (Ebberink et al., 1983). A hydrochloric acid extract of cerebral commissures was subjected to chromatofocusing with a PBE94 anion exchange column. Then the fractions with biological activity were separated on the basis of molecular size, using high-performance gel permeation chromatography (TSK200 column). Finally, pure CDCH (- 1 pmol per snail) was obtained with reverse phase highperformance liquid chromatography. Preliminary amino acid analysis indicated that CDCH consists of 40 amino acids. Among these arginine, aspartate, and glutamate occur frequently; arginine and lysine are mainly responsible for the high pZ of the hormone (R. H. M. Ebberink, personal communication). Probably, the CDC synthesize more than one biologically active peptide. Electrically active CDC (“active state”; see Section V,A) preincubated with radioactive amino acids, release at least four labeled compounds, presumably peptides. One of these, probably, is CDCH. The nature and functions of the other compounds are as yet unknown (Geraerts ef al., 1983b).

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IV. Cytology of the CDC A. TOPOLOGY AND GROSSMORPHOLOGY The CDC somata are located in the caudo-dorsal part of each cerebral ganglion. CDC can already be observed in juvenile snails with a shell height of 7 mm; then they are rather small (- 10 pm in diameter) and occur in low numbers (- 5-15 cells per ganglion) (van Winkoop and Roubos, 1984; E. W. Roubos, unpublished research). In adult snails with a shell height of 30 and more mm the CDC form compact clusters in which three cell layers can be distinguished. The cluster in the left ganglion contains 20-40 cells, that in the right 50-100 cells. Rostrad a cluster adjoins the medial cluster of growth neurohormone producing LGC (Geraerts, 1976a), dorsad it is partly situated under the medio-dorsal bodies (Fig. 1). At the light microscope level the CDC appear ovoid shaped (Joosse, 1964), a

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few cells showing flattening at the side(s) where they contact each other. Within a cluster the diameters of the somata range from 15 to 90 p m (Joosse, 1964), the largests cells taking the most peripheral position in the ganglion (Boer, 1965). In each cluster three volume classes of somata are present. The ratio between the means of these classes is approximately 1:2:4 (see below). In addition to the clustered CDC, in many snails 1-3 large CDC (diameters - 80 pm) occur in the central part of the commissure (Joosse, 1964; E. W. Roubos, unpublished research).

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B . NUCLEUS Like the size of the somata, nuclear size of the CDC depends not only on the size of the animal. Thus, in adult specimens nuclear diameters range from 10 to 60 p m and, like cell body volume, three nuclear volume classes can be distinguished, of which the ratio between the means is 1:2:4. This suggests that the size classes represent different degrees of polyploidy, which is probably the result of repeated endomitosis during postembryonic development (Boer, 1965). More recently, the existence of different degrees of polyploidy was confirmed for various cell types in L . .stagna/i,s,including central neurons, by cytophotometry of nuclear DNA contents (Boer ef a / ., 1977a). During the life of the snail the DNA contents of neurons apparently double a number of times, upto adulthood (shell height 30 mm) even 1 I times. The rather dense and dispersely located nuclear and paranucleolar chromatin is stained by gallocyanin, toluidine blue, and the Feulgen reaction, but not by pyronin (Joosse, 1964; Boer, 1965), showing its DNA nature. The nucleoli of the CDC contain KNA as they are stained by pyronin, gallocyanin, and toluidine blue. Their total number per cell is rather low (ranging between 4 and 14 in a 30 mm snail) compared to that of other central neurons with equal cell volumes (e.g., light green cells: 13-80), A close relationship exists between nucleolus numbers on the one hand and cell body and nuclear volume classes on the other. The ratios between the mean nucleolus numbers per volume class approximate the 1 :2:4 sequence, which indicates that during development not only nuclear DNA but also KNA contents duplicate (Boer, 1965). Up to now the functional significance of neuron polyploidy is unclear. In spite of their relative scarcity the nucleoli of the CDC are larger (between 5 and 10 pm; Joosse, 1964) and more complex than in other neurons because of thc prcscncc of two types of inclusion: “vacuoles” and “refractile bodies” (Boer, 1965). Light microscopic cytochemistry shows that the vacuoles, which may occupy a large part of the nucleolus, hardly take any stain, whereas the smaller refractile bodies weakly react to a number of cytochemical stains such as PAS, toluidine blue, and performic acid-Alcian blue. Both structures are negative to pyrnnin and, therefore, do not appear to contain RNA. Their presence is

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not specific for the CDC or for peptidergic neurons of L. .~tagnalisin general, so that their function, which still awaits elucidation, may be a general one (cf. Boer, 1 965). In addition to nucleoli, small particles and globules occur in the nucleoplasm. The particles hardly take any stain, but the globules are basophilic and stain by pyronin, gallocyanin, and toluidinc blue. Therefore, they probably contain RNA. The globules are closely apposed to the inner nuclear membrane, suggesting that they represent RNA that is to be transported toward the cytoplasm (Boer, 1965). The nuclei of the CDC have in common with many other neurons of the cerebral ganglia that the larger the cells the more polymorphous they are. The nuclear envelope of small cells is rather smooth, whereas that of large CDC locally shows a strong lobulation (Joosse, 1965). The envelope contains many nuclear pores (Roubos et d., 1976b). C. SOMA The CDC exhibit the typical characteristics of protein synthesizing cells. Light microscopically they are particularly characterized by the presence of large numbers of regularly distributed disks of Nissl-substance (Fig. 2a). The disks are preferentially located around the nucleus and are absent from axon hillock and axon. They have a diameter of 3-6 Fm and are 1 pm thick (Joosse, 1964). The Nissl substance stains intensely with pyronin, gallocyanin, and toluidine blue (Boer, 1965), and appears ultrastructurally to consist of large stacks of granular endoplasmic reticulum studded with ribosomes (Boer et al., 1968) (Fig. 3). At some places continuities between the reticulum and the cytoplasmic membrane of the nuclear envelope occur, suggesting formation of new cisterns (Roubos er al., 1976b). In addition to this extensive reticulum, many free ribosomes and polyribosomes occur, accounting for the light microscopic observation (Boer, 1965) of RNA-rich, fine granular material in perikaryon and axon hillock. The existence of a well-developed Golgi apparatus, forming a filamentous network around the nucleus, was first indicated at the light microscope level with Cajal’s silver impregnation technique (Boer, 1965). Ultrathin serial section studies showed that the Golgi apparatus consists of Golgi zones formed by 3-6 saccules (Wendelaar Bonga, 1971). The saccules measure 1 p,m and are bent semicircularly, the convex side often facing the granular endoplasmic reticulum. Where the membranes of both organelles are close to each other the reticulum budds off small electron-lucent vesicles (diameter 60-80 nm), which apparently migrate to the Golgi apparatus and fuse with the outer Golgi saccule (Wendelaar Bonga, 1971; Roubos, 1975). Most probably, in this way proteinergic material synthesized in the reticulum is transferred to the Golgi apparatus, where it is condensed, possibly modified, and finally budded off as elementary granules. The granules are round, have a regular outline, an electron-lucent halo of uni-

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150 form width surrounding the electron-dense core, and a mean diameter of nm (Wendelaar Bonga, 1970). This granule morphology is a conclusive criterion to identify the CDC at the ultrastructural level, even when other characteristic cellular structures are absent (e.g., in axon profiles). Recently, autoradiographic and biochemical studies have provided strong evidence that the elementary granules contain CDCH. First, the CDC appear to be capable of incorporating tritiated arginine, a main amino acid of CDCH, in a much stronger and faster way than any other neuron type in the cerebral ganglia. In fact, when cerebral ganglia are incubated for 2 hours in snail Ringer containing labeled arginine, autoradiography showed that silver grains appear specifically located above the Golgi apparatus and elementary granules associated with the Golgi zones (Roubos, 1984, unpublished research). Second, when homogenates of the commissure are subjected to sucrose gradient centrifugation, one fraction consisting almost entirely of elementary granules contains most (- 55%) of the ovulation inducing activity present in the entire gradient (Geraerts et al., 1984b). With regard to granule formation two types of Golgi zone can be distinguished (Wendelaar Bonga, 1971; Fig. 3). (1) “Inactive Golgi zones”; usually the saccules are flattened and do not show electron-dense contents. No formation of elementary granules can be observed. ( 2 ) ‘‘Active Golgi zones”; some saccules, especially the inner ones, contain evenly distributed electron-dense material. At the distal parts of the saccules elementary granules with homogeneous, electrondense contents are budded off. These granules seem to be immature since the outline is irregular and the mean diameter is clearly smaller than that of the granules located elsewhere in the cytoplasm (Wendelaar Bonga, 197 I ; Roubos, 1984, unpublished research). Numerical and volumetric determinations of these two types of Golgi zone are routinely used to assess the rate of synthesis of elementary granules (e.g., Wendelaar Bonga, 1971; Roubos, 1975, 1976; Roubos et ul., 1980; see also Section VIII,A,B). Mature elementary granules are rather evenly distributed throughout the cytoplasm, but they are scarce between the cisterns of the rough endoplasmic reticulum. Generally, their number is not very high, but variations occur depending on the secretory state of the cells, i.e., on the balance between synthesis, transport, and release. In addition to elementary granules, the CDC contain large (mean diameter 0.4-0.5 pm) highly electron-dense granules (Fig. 3, LG) with an irregular outline (Boer et al., 1968). They are budded off from the concave side of the Golgi zone and are nearly always located in the vicinity of the Golgi apparatus (Roubos, 1976). Most likely, they contain secretory material. Generally, they are scarce, but in experimentally inactivated CDC they may be numerous. ProbaFIG. 3 . Soma of CDC showing granular cndoplasmic reticulum (GER), Golgi zones (active: aGZ, inactive; iGZ), elementary granules (EG), and large clectron-dense granules (LG). Arrow indicates formation of immature EG. x 25,000.

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bly, LG are involved in lysosomal breakdown of secretory material (see Section VIII,A,2). At the concave side Golgi zones show a number of small vesicles, sacs, and tubular structures (Wendelaar Bonga, 1971). In view of their acid phosphatase contcnts (Roubos, 1976) some of the vesicles may be primary lysosomes. Acid phosphatase activity is also present in the “Golgi tubule,” a flattened sac situated near the innermost Golgi saccule. The significance of this structure, which has also becn observed in the peptidergic dark green cells located in the visceral, parietal, and pleural ganglia of the CNS of Lymiiueu (Roubos, 1973) and in the paraventricular nucleus of the rat (Kalinio, 1971), in unknown. Furtherrnorc, the CDC soma shows the usual cell organellcs, such as (elongated) mitochondria, microtubulcs, and (some) lysosomes.

D. AXON ’The axonal pattern of the CDC has been studied in detail by light and electron microscopy (Wendelaar Bonga, 1970, 197 I ) , in some cases after intracellular injection of horseradish peroxidase (HRP; de Vlieger et ul., 1980; ter Maat et ul., 1983a). The axon emerges excentrically from the soma and initially has a diameter of 3- I2 pm depending on the size of the soma. Axons of all CDC run to the anterior part of the cerebral ganglion and form a loop (“loop area”). Here they come into close contact with each other, which probably plays an important role in establishing electrical coupling between individual CDC (see Section VII). After leaving the loop area the axons run toward the cerebral commissure and form many 0.2-2 p m thin branches. The branches end blindly in the periphery of the ipsilateral half of the commissure and form the neurohemal area (Figs. 2b, 4, and 5). Although the CDC are unipolar, in each cluster 5-8 more ventrally located CDC form an additional axon branch at some hundreds of micrometers from the soma (de Vlieger et ul., 1980). This branch runs straight to and through the intercerebral commissure and loops in the contralateral loop area. Then it returns to the contralateral half of thc commissure and forms, like the first axon branch, neurohenial axon terminals, though branching less extensively (Figs. 4 and 5 ) . These ventral CDC also differ from the other CDC in that the proximal, unbifurcated part of the axon is studded with lateral branches ( I -2 pni in diameter) and thin side branches (diameter 0.3-2 pm).The side branches have lengths of more than 30 p m and end blindly. They are involved in the reception of synaptic input (see Section Vlll,B,4). Besides terminals, the CDC axons in the cerebral commissure form lateral swellings, which may be involved in hydrolytical breakdown of elementary granules in inactive CDC (cf. Koubos, 1984). The internal axon structure is mainly characterized by the presence of microtubules and varying numbers of elementary granules. The mechanism that ac-

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FIG. 4. Whole mount preparation of HRP-filled ventral CDC. Two montagcs of interference contrast micrographs taken at different planes of focus. X90. (a) Additional axon branch ( I ) running through cerebral commissure. (b) Ipdateral axon branch (2) with beads, which probably represent axon terminals. From de Vlieger ef nl. (1980).

counts for granule transport is unclear. Probably, microtubules are involved, since in some cases association of granules with microtubules has been observed. Of particular interest is the finding that many granules are connected with each other via two or three very thin bridges, which might facilitate granule transport (Roubos, 1975). E. NEUROHEMAL AREA

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The cerebral commissure measures 1 x 0.2 mm. The central part of the commissure consists of many thousands of relatively thin (0.2-2 pm) axons, some of which are surrounded by glial cells. Various types of axon can be distinguished on the basis of diameter and morphology of the elementary granules, and particularly of the immunocytochemical reaction of the granules with antisera raised to biologically active peptides. For instance, axons are present containing vasopressin-, oxytocin-, vasotocin-, or insulin-like substances (Schot et al., 1981). The axon terminals of the CDC occur in the periphery of the commissure, in a layer with a thickness of 10-30 pm. On the basis of counts of axon terminal profiles and reconstructions of axons in ultrathin serial sections, it has been estimated that a CDC possesses more than 800 terminals (Wendelaar

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FIG. 5 . Schematic representation of cerebral ganglia (C) of L . srugnulis, with dorsal (D) and ventral ( V ) CDC. Each CDC has an axon branch that runs through the ipsilateral loop area ( I ~ A ) . Ventral CDC have an additional axon branch running via the cerebral commissure (C) through the contralatcral loop area. All axon branches form ncurohemal terminals in the periphery of the commissure. Note the thin latcral side branches on proximal part of axtin3 of ventral CDC ( B ) . Modified after Joosse ri t r l . (19x2).

Bonga, 1971). The size of the terminals ranges from - 1 to 10 pm. The terminals contain varying numbers of elementary granules, depending on the secretory state of the CDC. Especially under conditions of increased storage of granules some axons penetrate and terminate within the connective tissue sheath (perineurium) which normally surrounds the ncurohemal axon terminals (Fig. 6a). FIG. 6 . Neurohemal axon terminals ( A ) of CDC with elementary granules (EG), and glial cells tCC). (a) Periphery of cerebral commissure studded with terminals, some of which (arrows) have penctrated thc perineurium ( P ) . M muscle cell. X 2500. (b) Exocytosis (E). cmpty membrane indentation (arrow). vacuole ( V ) , cup-shaped vesicle (CSV). double-nicmbraned vesicles (DMV). clear and clear vesicle-like stnicturc (CVS). Routine fixation. X60.000. (c) Whorl ( W ) . vesicle (0'). Arrow indicates exocytosir. TACO fixation. x 25,000.

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The CDC secretory material is released by exocytosis, i.e., after fusion of the granule membrane with the axolemma the granule contents are extruded into the hemolymph (Fig. 6b). Ultrastructurally this process is visible as omega-shaped membrane indentations filled with electron-dense material (Wendelaar Bonga, 1971; Koubos, 1975, 1976; Roubos et al., 1976a,b, 1980, 1981a,c; Roubos and Buma, 1982; Buma et af., 1983). The frequency of exocytosis is related to the mode of electrical activity of the CDC and to the rate of neurohormone release (see Section V1,B). The released secretory material can easily reach the hemolymph since the neurohemal area is separated from the cephalopedal hemolymph sinus (Bekius, 1972) only by a thin basement membrane and the thin perineurium. The perineurium consists of one or two layers of various cell types (e.g., fibroblasts, pigment cells, amebocytes, calcium cells, and smooth muscle cells) embedded in a loosely structured, electron-lucent matrix of collagen (Wendelaar Bonga, 1971; Srninia, 1972). Injection studies with India ink have shown that there is an intimate contact between the neurohemal area and the hemolymph sinus via an extensive network of blood capillaries and blood spaces in the perineurium (Wendelaar Bonga, 197 I). In addition to elementary granules, the axon terminals contain various types of vesicular structure, which are preferentially located near the axolemma (Fig. 6b). Six types can be distinguished (Boer et al., 1968; Wendelaar Bonga, 1970, 197 I ; Roubos e t a / . , 1 9 8 1 ~Roubos ; and Burna, 1982; Buma and Koubos, 1983), viz. small clear vesicles, vacuoles, cup-shaped vesicles, double-membraned vesicles, clear vesicle-like structures, and multivesicular bodies. These vesicle types are thought to be involved in the process of recapture and intraaxonal sequestration of parts of the axolemma after exocytosis of granule contents. Furthermore, they seem to play a role in the intracellular calcium dynamics during and after hormone release. Such a role has also been proposed for the mitochondria that are contained within the terminals. Finally, large concentrically arranged membranous whorls (Fig. 6c) occur in the terminals, especially under conditions of activated neurohormone release. These structures may also be involved in recapture and resorption of the axolemma (see Sections VI,B and C).

V. States of Electrical Excitability of the CDC A . ELECTROPHYSIOLOCIICAL CHARACTERIZATION OF

THE STATES

In electrophysiological terms the CDC have been considered for a long time as “dull” cells, exhibiting electrical silence and showing no synaptic input or spontaneous fluctuations of the membrane potential (cf. Benjamin et al., 1976). However, a few years ago detailed studies of the electrical characteristics of the CDC have shown that the cells exhibit three different states of electrical excit-

NEUROENDOCRINE CAUDO-DORSAL CELLS IN LYMNMA

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-

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FIG. 7. The (after)dischargc of the CDC: Bars under upper trace indicate periods of repetitive intracellular stimulation (St). Below: shape of four action potentials of different parts of the discharge. Broadening of spikes 2 and 3 is duc to a calcium-dependent channel. Based on Kits (1980a). modified after Joosse (’1 ul. (1982).

ability, viz. the resting, the active, and the inhibited state (de Vlieger and Roubos, 1978; ter Maat, 1979a; de Vlieger et al., 1980; Kits, 1980a,b). In principle, the distinction between the states is based upon responses to repetitive intracellular stimulation of single cells, using the central ganglion ring as preparation (Fig. 7). However, simultaneous recordings of pairs of cells have demonstrated that all CDC exhibit the same state at a given time. In fact, all CDC can be considercd to behave as one network. The states are characterized as follows. 1. Resting State

In preparations taken from laboratory bred adult snails kept under a standard light/dark regime (lights on at 7.00 hours, off at 19.00 hours: “medium day,” MD) CDC are usually silent, having a membrane potential that varies among preparations from -68 to -58 mV. Orthodromic and antidromic action potentials can be evoked by stimulation of CDC somata or of the cerebral commissure, respectively. Spikes measure 80-100 mV, last 10-15 msec at half amplitude, and have an undershoot of 10 mV. Stimulation with a train of short intracellular stimuli results in one action potential per stimulus and a slow depolarization which spreads through the network. This process of facilitation may vary in strength among preparations. Weak facilitation is characterized by slow depolarization only; if facilitation is stronger the depolarization of the network results in an additional action potential not directly related to the stimuluscoupled potentials. The additional spike, which often arises first in other CDC, has a pronounced undershoot. Facilitation can be repeated several times when

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stimulation is continued. Generally, when stimulation is stopped the membrane potential returns slowly to its original value and the cells remain in the resting state. However, when the cells are prolonged stimulated with repetitive depolarizing pulses (e.g., 1 Hz) they will enter the active state. 2 . Active State

Upon termination of repetitive stimulation additional spiking persists but the spikes lose the undershoot, resulting in a strong depolarization: the active state starts, during which the CDC display a characteristic firing pattern (Fig. 7) called the afterdischarge (if the active state starts without experimental stimulation the pattern is called the discharge). The (after)dischargc consists of three parts. The first part lasts 5 minutes and consists of bursts separated by hyperpolarized waves. During a burst the cells show a high firing rate (up to 2 spikesisecond) and an increase in spike duration (up to 150 msec at half amplitude). At the end of this part the spikes have a height of up to 70 mV and show a shoulder. In the second part, which has a variable duration, the membrane repolarizes slowly, firing occurs regularly (“beat”), thc firing rate decreases slowly to 1 spike14 seconds, and the spikes narrow. In the last part, which lasts some minutes, firing occurs only irregularly and the firing rate declines considerably. Furthermore, spike duration at half amplitude shortens again to 15-25 msec, and the spikes lack a shoulder. Eventually, firing stops and the membrane potential returns to the original value. The duration of the active state is variable, ranging from 7 to 120 minutes with a mean of - 45 minutes (Kits, 1980b). The discharge is based upon an endogenous pacemaker. because ( I ) it can be evoked by repetitive stimulation of one single CDC (Kits, 1980b), and ( 2 ) blocking of synaptic input with Ca2 -free or high MgZ ’ Ringers does not inhibit the initiation or the progress of the discharge. The pacemaking mechanism appcars to involve a voltage-dependent Na iCa’ channel and a Ca2 -dependent K + channel (Kits and Bos, 1981).

-

+

+

+

+

3. Itzhibited Stcrtr After termination of the evoked afterdischarge the CDC show a period, varying from a few minutes to more than I hour, of some facilitation which gradually diminishes. Then the cells enter the inhibited state: they lack any spontaneous activity, and the membrane potential, which is about -65 mV, shows virtually no fluctuations. The cells respond to ortho- and antidromic stimulation with action potentials of XO-100 mV and 10-15 msec at half amplitude, as in the resting state, but no facilitation or additional action potentials occur upon repetitive stimulation. Thc inhibited state lasts 4-6 hours. In isolated preparations the inhibited state normally follows the active state, but also direct transitions from the resting to the inhibited state have been found. Transition from the inhibited to the resting state has never been observed irz vitro, but does take place in vivo (Kits, I9XOb).

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B. RELATION BETWEEN STATES, CDCH RELEASE, AND EGG LAYING In mature specimens of L . stagnulis kept under standard laboratory conditions (MD photoperiod) egg laying takes place with a periodicity of 1-3 days (e.g., Steen et al., 1969). The first indication for a correlation between the electrical CDC states and the egg-laying cycle was obtained by de Vlieger et al. (1980). They inhibited egg laying by keeping snails for some days in dirty water; then the snails were supplied with fresh and oxygenated water, which resulted in egg laying by a high percentage of the snails within 3 hours after stimulation (cf. Steen, 1967). Upon inspection within 2 hours after stirnulation, CDC were found to be in discharge only in animals that had just ovulated or were ovulating; snails that had not reacted to the ovulation inducing stimulus showed silent CDC. In more detailed experiments with animals selected at specific phases of the egg laying cycle, a clear correlation between these phases and the three states of excitability could be established (Kits, 1980b). CDC appear to be in the active state when ovulation occurs; 2 hours thereafter, when oviposition takes place, the cells are inhibited. About 4-6 hours later transition to the resting state occurs. The CDC remain in the resting state until the next discharge starts. The characteristics and succession of the three states found in vivo do not differ from those in isolated preparations (see Section VIII,A, 1). These findings strongly suggest that the CDC induce ovulation by releasing CDCH during the active state. This is substantiated by an experiment in which CDC incubated in physiological saline were brought to afterdischarge. Fifteen minutes after the onset of the discharge the saline contained ovulation inducing activity, indicating that CDCH release had taken place (Kits, 1981). No ovulation inducing activity could be detected after incubation of CDC that remained in the resting state. Recently, the kinetics of CDCH release were studied in more detail (Geraerts et al., 1984a). About 15 minutes after fresh water stimulation only little CDCH is found in the hemolymph. Then the titer increases rapidly and peaks at about the end of the active state. Subsequently, it decreases to zero at 4 hours after stimulation. These studies suggest that ovulation occurs already at a rather low titre of CDCH and that the high titer of CDCH at about the end of the active state is related to the control of other aspects of egg laying activity, e.g., elements of the egg laying behavior (cf. Section V1,B).

-

-

VI. Exocytosis of CDC Secretory Material A. DEMONSTRATION OF EXOCYTOSIS For many years there has been much debate as to the mechanism by which neurons release their secretory products. Several mechanisms have been proposed, ranging from extrusion of intact secretory granules to molecular diffusion

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across the axolemma (see, e.g., Normann, 1976). At present the insight is growing that exocytosis is the common mechanism by which release takes place, not only in neural cells but in secretory cells in general. Exocytosis in the CDC was firstly described by Wendelaar Bonga (1970) who used a standard procedure for tissue fixation involving simultaneous fixation with glutaraldehyde and osmium tetroxide. The disadvantage of such a procedure, as has been shown for numerous other secretory cell types (see, e.g., Normann, 1976). is that it is very ineffective in capturing the rapidly fleeting exocytosis phenomena. This explains why at first exocytosis phenomena in the CDC were only scarcely observed. In fact, the occurrence of exocytosis was concluded from the rare observations of the presence of omega-shaped indentations of the axolemma in which, only occasionally, some electron-dense material could be seen (Wendelaar Bonga, 1971) (cf. Figs. 6b and 8a). Some improvement was obtained by staining prior to embedding with 1% aquous uranylacetate for 30 minutes: granule contents are stained highly electron dense, rendering the detection of exocytosis somewhat more easy (Roubos, 1975). This approach showed that the CDC axon terminals release their secretory material not only close to the perineurium, but also intercellularly, i.e., into the space between two terminals or between a terminal and a glial cell ( “intercellular exocytosis”). Subsequently, a more selective method for the visualization of exocytosis was developed, involving simultaneous fixation with tannic acid and glutaraldehyde, followed by postfixation with osmium tetroxide and staining of the ultrathin sections with lead citrate (TAGO method: Koubos, 1975; Roubos and van der Wal-Divendal, 1980). Since tannic acid strongly binds to proteinergic materials (e.g., basic proteins, glycoproteins, and polypeptides) as well as to heavy metal ions (osmium, lead) but does not penetrate glutaraldehyde-fixed plasma membranes, only extracellular material, e.g., released contents of secretory granules, are stained highly electron dense (Fig. 8b). The general usefulness of the TAGO method appears from the fact that it has also enabled the unequivocal demonstration of exocytosis in neural and endocrine cells of various animals, e.g., in the anterior and posterior pituitary lobes of the rat, and in the gut and in the glandular and storage lobes of the corpora cardiaca of various insects (Roubos and van der Wal-Divendal, 1980; Endo and Nishiitsutsuji-Uwo, 1982). As to the CDC, exocytotic release of the elementary granule contents appears to be a very common phenomenon, especially in electrically active cells (see Section VI,B). BETWEEN EXOCYTOSIS AND B. RELATION

THE

ELECTRICAL STATES

A number of recent studies have shown the close relation between exocytosis of CDC secretory material, release of CDCH, and electrical activity of thc CDC. First, this relation was studied in vitro, in isolated cerebral ganglia, with quantitative electron microscopy (Kits, 1981; Roubos et a l ., 1981a). The number of

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FIG. 8. Ultrastructural demonstration of exocytosis (arrows). (a) Routine fixation with mixture of glutaraldehyde and osmium tetroxide. X80,OOO. (b) TACO fixation. Note specific electron’ density of released granule contents. X65,OOO.

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exocytosis phenomena, visualized with the TACO method, was counted in cross-sections half-way along the cerebral commissure. Resting state CDC showed low exocytotic activity, viz. 59 exocytosis figures per cross-section. However, after the cells had been brought electrically into the active state, the number of exocytoses rapidly increased: 5 minutes after the onset of the discharge the number was 9 times as high as in resting CDC, after 15 minutes of discharge even 36 times (cf. Fig. 9). This strongly suggests that the CDC release their secretory material at a high rate during the active state, by the process of exocytosis. This is substantiated by recent in vivo experiments in which CDC were excited by the physiological fresh water stimulus (Roubos and Buma, 1982; Buma and Roubos, 1983) (cf. Fig. 10). It appears that in the resting state the axon terminals are large and filled with many elementary granules; signs of exocytosis are rare. In contrast, in the active state (30 minutes after stimulation) the terminals are smaller and contain about 3 times as few elementary granules. Exocytosis phenomena are very abundant, viz. 140 times as frequent as in resting CDC. Numerous granules appear to fuse, releasing their contents simultaneously (“multiple exocytosis”; Fig. 9). In the subsequent inhibited state (3 hours after stimulation) the terminals are somewhat enlarged again and the number of elementary granules increases again as compared to the active state, which is probably a result of transport to the terminals of recently synthesized granules. The number of exocytoses is lower than in the active state (-7 I%) but considerably higher than in the resting state (X40). This indicates that CDCH release proceeds, although at a lower rate than during the active state, during (the first part 011 the inhibited state. Since ovulation already occurs 10 minutes after the start of the discharge (Kits, 1980b), the secretory material released after ovulation will have an(other) function(s), e.g., control of the synthetic activity of the female accessory sex organs (see also Geraerts et d.,1984a). In this respect also the possibility has to be raised that the cxocytoses observed in the different states are not exclusively related to the release of CDCH but also of other biologically active peptides (cf. Geracrts, 1983b, 1984a).

-

-

-

MEMBKANE A N D PROCESSING C. E X ~ ~ Y T ~ S I S - C ~ U P L E I , RECAPTURE Evidently, high exocytotic activity, not only in the active state but also, though to lower degrees, in the other states, prompts the CDC to remove large parts of the axolemnia in order to keep their normal shape. The following proccsses sccm to play a role (Fig. I I ) .

FIG Y . Kelatioii between electrical states and exocytosis in CDC axon tcrininals. TACO fixation. x35.000. ( a ) Resting state: no exocytosis. (b) Active atatc: iiunierous exocytoscs (highly electron-dcnsc contents). including multiple exocytoaca (arrows).

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-Rest-

kctid

Inhibited

1

Rest

t Fic. 10. Morphometry of CDC axon terniinals. Storage of elementary granules, expressed as numbers of granules (EG) per axon terminal profile (AT). and release of granule contents, expressed as numbers of exocytoses (Exo’s) per outline of cerebral commissure (100 pm). Five snails per state of electrical activity. Fresh water stimulation was started at time 0 (arrow). Based on Roubos and Bunid (1982) and Roubos (unpuhlished research).

1. Bulk Resorption by Whorls (Fig. I l f 2 )

Whorls, first described in the CDC axon terminals as “multilamellar bodies” (Wendelaar Bonga, 1971), are large (0.3-1 p m diameter), concentrically arranged membranous structures that often show continuities with the axolemma (Roubos and Buma, 1982; Buma and Roubos, 1983b). Preliminary ultrastructurd studies involving morphometry and application of extracellular markers (e.g., HRP, lanthanum, and tannic acid) showed that whorls are formed particularly during the active state, by invagination of the axolemma, and become intracellular during the subsequent inhibited state. This way of membrane recapture adequately accounts for the fact that in the active state, when exocytosis occurs at a very high rate, the axon terminals do not increase but, on the contrary, considerably decrease in size (Roubos and Buma, 1982; Buma and Roubos, 1983b). The fate of whorls is not fully clear. Some of them may be released from the axon terminal as whorl-like structures (Roubos and Buma, 1982). However, the observed number of such structures seems too low to explain the removal of all whorls. Possibly, whorls can desintegrate and form the smaller membranous structures which surround them in some cases, or they may broken down by lysomal action.

NEUROENDOCRINE CAUDO-DORSAL CELLS IN L Y M N A W

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FIG. 1 1 . Possible mechanisms of membrane sequestration (a, f) and transport of extracellular (b, g) and intracellular (c,d,e,h) calcium (black dots) during and after exocytosis of secretory material from CDC axon terminals. CSV, Cup-shaped vesicle; CV, clear vesicle; CVS, clear vesicle-like structure; DMV, double-membraned vesicle; E, exocytosis of elementary granule; L, lysosome; ME, microexocytosis of clear vesicle; MP, micropinocytosis; MVB, multivesicular body; VC, vacuole; W, whorl. After Buma and Roubos (1983b).

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2. Macropirzocytotic Resorption by V~cuoles(Fig. I l a ) Vacuoles are large, electron-lucent vesicles with a diameter between 70 and

- 140 nm (Koubos et a / . , 1 9 8 1 ~Roubos ; and Buma, 1982; Buma and Roubos,

1983b). They are preferentially located near the axolemma. HRP incorporation studies indicate that vacuoles are formed by macropinocytotic resorption of parts of the axolemma. They are not frequent in the resting and active states, but during thc inhibited state their number doubles. Consequently, they may be concerned with exocytosis-coupled membrane recapture during inhibition, when exocytosis occurs at a moderate rate and the parts of the axolemma that have to be resorbed are, as compared to the active state, relatively small. Morphometry and HRP-incorporation studies (Roubos and Buma, 1982; Buma and Roubos, 1983b) strongly suggest that the vacuoles are transformed in the following complex fashion. First they invaginate to form cup-shaped vesicles with a diameter of 70-150 nm. These structures then transform into doublernembraned (“doughnut-shaped”; Koubos and Buma, 1982) vesicles, consisting of a large outer (60-130 nm) and a small inner (50 nni) vesicle (Fig. 1 la). Two mechanisms have been proposed for the turnover of double-membraned vesicles. ( 1 ) They would fuse and form multivesicular bodies that subsequently release their vesicular contents by exocytosis into the extracellular space (Fig. I la2); this mechanism would account for the finding of clear vesicle-like structures (mean diameter - 50 nm) located extracellularly in invaginations of the axolemma. On the other hand, this mechanism does not seem to be very important since niultivesicular bodies are rarc i n the CDC axon terminals and d o not show uptake of extracellularly administered HRP, which would be expected if they were from niacropinocytotic, vacuolar, origin. (2) They would fuse exocytotically with the axolemma, giving rise to extraccllular clear vesicle-like structures (Fig. 1 l a l ) .

3 . Micropinocvrotic Rcsoryliori by Clear Vesicles (Fig.1 If) For several years small, electron-lucent vesicles (mean diameter 40-50 nm; also called “clear vesicles”) have been considered as the main resorption products of the CDC axolemma following exocytosis. This was because their appearance coincides with the occurrence of exocytosis and because ultrastructural signs of their formation by micropinocytosis (coated invaginations of the axolemma; Fig. I lf3) have been observed in some cases (Wendelaar Bonga, 1971; Roubos, 1973, 1975, 1976; Roubos eral., 1 9 8 1 ~ )The . vesicles are most numerous in the inhibited state, which may mean that they play a role in membrane resorption during this particular state. On the other hand, it has been recently shown that only a small fraction of the vesicles take up extracellularly applied HKP, suggesting that most of the vesicles are not derived from the axolemma, but possibly from other membranous structures such as whorls (Fig. 1 lf2) (Roubos and Burna, 1982; Buma and Roubos, 1983b).

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32 1

D. ROLEOF CALCIUM I N THE CONTROL OF EXOCYTOSIS Calcium ions form an important link between the electrical discharge and high exocytotic activity. When CDC are transferred to snail Ringer containing a high concentration of potassium (K stimulation) their axolemma depolarizes (Kits and Bos, 1981) and they start to release CDCH at a high rate (Geraerts et ul., 1984a) by massive exocytosis (exocytosis phenomena are 500 times as numer. any increase in ous as in unstimulated CDC; Roubos et al., 1 9 8 1 ~ ) Hardly exocytosis is induced by K stimulation when (1) the Ringer lacks calcium ions (Roubos et al., 1981c) or ( 2 ) the Ringer contains 5 X M CoCI,, a blocker of calcium channels (Buma at ul., 1983). This strongly suggests that the large increase of exocytotic activity during the active state is caused by calcium influx into the axon terminals via calcium channels that open as a result of depolarization of the axolemma. As mentioned, the CDC show exocytosis not only in the active state but also, though to lower degrees, in the inhibited and resting states, when they are electrically silent. Since, in comparison to the active state, the CDC are hyperpolarized during inhibition and rest, it seems likely that influx of calcium will hardly or not take place. This raises the question of how exocytosis is controlled during these states. If we leave out of consideration the theoretical possibility that during these states exocytosis is independent of calcium, the simplest answer to the problem is that calcium that enters during the active state is removed from the cells so slowly that its concentration during inhibition and rest is still high enough to induce some exocytoses. Ultracytochemical studies involving tissue fixation with K-pyroantimonate have shown that, in addition to calcium pumps (which have not been studied up to now in the CDC), mitochondria and vesicular structures may play an important role in controlling the axoplasmic calcium concentration in the CDC terminals (Roubos and Buma, 1982; Buma and Roubos, 1983b). During the active state mitochondria are preferentially located near the axolemma where exocytosis takes place, and compared to the resting state they contain a high number of pyroantimonate-positive and EGTA-sensitive calcium deposits (Fig. 12). This indicates that a large amount of calcium that enters the terminals during the discharge is taken up by the mitochondria. In this way the mitochondria may protect the more central parts of the terminals against an excessive rise of the axoplasmic calcium concentration, an event which would severely impair the cell’s functioning (e.g., by leading to precipitation of calcium phosphates). In view of the rather low affinity for calcium of mitochondrial transport systems, calcium will again leave the mitochondria1 reservoir when the axoplasmic calcium concentration falls below a critical level (e.g., by pumping action). Obviously, such a mechanism may well contribute to maintaining a relatively high level of axoplasmic calcium during the inhibited state and, though to a lower +

-

+

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Fici. 12. CDC axon terminal in active state, showing elementary granules (E), exocytosis (ar-

row), and mitochondria containing many K-pyroantimonate-positive calcium contents (M). x 90.000.

degree, during the first hours of the resting state. The role of mitochondrial calcium has been studied by exposing CDC containing calcium-rich mitochondria (taken 5 hours after start of the discharge) to M carbonylcyanide-ptrifluormethoxyphenylhydrazone (FCCP). FCCP is a potent uncoupler of oxydative phosphory lation and induces the release of mitochondrial calcium. After 30 minutes of FCCP treatment the number of exocytoses is about 10 times as high as in untreated CDC, and a considerable amount of CDCH has been released into the incubation medium. This finding strongly favors the idea that mitochondrial calcium can play a role in the induction of exocytosis (Buma et d.,1983; personal communication). Calcium deposits also occur in various vesicular structures, viz. in clear vesicles, vacuoles, cup-shaped vesicles, double-membraned vesicles, and extracellular clear vesicle-like structures (Fig. 1 I). As to the origin and fate of this calcium a number of possibilities have been considered (Buma and Roubos, 1983b), including (1) calcium uptake from the extracellular fluid (by pinocytotic formation of vacuoles; Fig. 1 1b,g), (2) calcium uptake from intracellular sources (by transport across the membrane of vacuoles, double-membraned vesicles and clear vesicles; Fig. 1 lc,e,h, or across the inner vesicle of double-membraned vesicles; Fig. 1 lc2, and by uptake of calcium-rich cytoplasm into cup-shaped vesicles; Fig. 1 le), (3) calcium release into the extracellular fluid (as a result of extrusion of clear vesicle-like structures; Fig. 1 Ic,e, and by microexocytosis of

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clear vesicles; Fig. I Ig,h), and (4) calcium release into the axoplasm (by the intracellular breakdown of clear vesicles; Fig. 1lg3).

VII. Synchrony between Individual CDC Studying the effects of intracellular current injection on the membrane potential of simultaneously recorded CDC de Vlieger el ul. ( 1 980) showed that all individual CDC are electrotonically coupled (Fig. 13a). During the discharge the broad, shouldered action potentials (Fig. 7) are transmitted in a relatively efficient way through the electrotonic junctions, thus ensuring close synchrony between individual CDC. Between the two CDC clusters there is a delay of 2060 msec; either the left or the right cluster is leading. All CDC of a cluster fire almost synchronously during the entire discharge (Kits, 1980a,b). Within a cluster the ventral cells lead the dorsal ones (Kits, 1980a, 1982). Electron microscope studies have been carried out to establish the morphological identity and the locations of the electrotonic junctions. Various structures have been considered as the possible morphological correlates of the electrical coupling between cells, and some years ago the following structures were suggested (Roubos, 1975): subsurface cisterns (small cisterns of the rough endoplasmic reticulum, closely apposed at the axolemmas of two adjacent axons), desmosome-like structures, interdigitations of apposing axolemmas, and specific release sites. However, in view of recent studies an involvement of these structures in electrical coupling is unlikely. Subsurface cisterns occur in a variety of cells that do not show electrical coupling, whereas the other structures obviously are absent in the light green cells (LGC) of L. stagnalis, which are also clearly electrotonically coupled (Roubos et al., 1983a). Desmosome-like structures and membrane interdigitations may rather be involved in cell-to-cell adhesion, and specific release sites may be the correlates of nonsynaptic chemical communication (see Section IX). Actually, gap junctions are the only relevant structures CDC and LGC have in common. Recent studies involving phosphotungstic acid staining or freezefracturing of glutaraldehyde-fixed ganglia (Fig. 13b and c), revealed that gap junctions are particularly present in the loop area (Fig. 13d). Additional junctions occur between the axons of the ventral CDC that pass through the cerebral commissure. Finally, small gap junctions have been found between some of the axon terminals in the neurohemal area (Roubos, 1984, unpubl. res.). So, it seems that these junctions are the morphological correlates of electrical coupling between the CDC (and the LGC), the loop area being particularly important for coupling between CDC within a cluster, the overcrossing axons for that between clusters. The functional significance of electrical coupling obviously lies in the synchronous firing activity during the discharge, which results in a massive release of secretory material during a relatively short period. Furthermore, cou-

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pling enables all cells to become activated upon stimulation of one or only a few CDC. Probably, also the observed synchrony of cellular processes such as synthesis, storage, and transport (Wendelaar Bonga, 1971; Roubos, 1975) is a result of electrical coupling. Thus, due to electrical coupling all CDC can act as one functional network.

VIII. Control of CDC Activity Under field conditions, and to some extent also in the laboratory, various internal and external factors determine whether and when L . stagnalis will lay an egg mass. Obviously, this complex control ensures that egg laying occurs under conditions that are optimal for the development of the snail’s offspring. There is now much evidence that this control is particularly exerted via the regulation of CDC activity. In this section attention will be given to the three main aspects of this control: the effects of external and internal stimuli on the CDC, the pathways by which stimuli reach the CDC, and the cellular mechanisms by which the CDC respond to stimuli. A . GENERAL EVIDENCE FOR THE EXISTENCE OF NEURALAND NONNEURALINPUTS

I . Neurul Control Direct evidence that the CDC system receives neural inputs comes from the ultrastructural demonstration of four types of synaptic contact (Roubos, 1975; Roubos and Moorer-van Delft, 1979), viz. one type of “true” synapse and three types of synapse-like structure (SLS) (Fig. 14). The true synapse type contains a mixed population of electron-dense granules (mean diameter 100 nm) and electron-lucent granules (60 nm), the latter being mainly located close to the thin (35 A) presynaptic membrane. The synaptic cleft is straight and widened (30 nm). The synapses occur in small numbers and are axo-axonic. The SLS, common in molluscan CNS, closely resemble synapses of the vertebrate peripheral nervous system. The presynaptic element generally contacts the postsynaptic element intensely and a cluster of granules is located near the presynaptic membrane. The Fic,. 13. Elcctrotonic coupling between the CDC. (a) Simultaneous recording of afterdischarge in two CDC. St: repetitive intracellular stimulation of CDC of lower trace. Arrow indicates start of afterdischargc Modificd after de Vlieger (1981). (b) Gap junction between CDC axons in loop area. Glutaraldchydc fixation and phoaphotungatic acid staining. X90,OOO. (c) Gap junction in freeze-ctch replica o f two CDC axons in ccrcbrul cornmissure. X200.000. (d) Schcme of localization of gap junctions bctwcen CDC in loop area (I),cerebral cominiswre (2). and neurohenial area ( 3 ) . Dots indicate axon terminals.

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SLS -A

SLS-B

SLS-c True synapse

FIG. 14. Diagram of ventral C D C with four types of synaptic contact. Type B and Type C synapse-like structiires (SLS) on lateral axon hi-anches presumably iii-e cholincrgic. Modified after Joossc e/ ul. [ 1982).

synaptic clcft is not widened and specializations of the synaptic membranes are inconspicuous. The SLS types on the CDC have been classified on the basis of the morphology of the presynaptically located granules. Type A: the granules ( 100 nm) possess a core of moderate electron density surrounded by an electronlucent halo. The type is axo-somatic and axo-axonic. Type B: the granules are electron lucent (60 nm). Type C: granules of type A and type B SLS are present. Type C SLS are found most frequently. Type B and type C are probably cholinergic; they abound the fine lateral branches of the axon root of the ventral CDC (Roubos et u l . , 1981a; ter Maat et ul., 1983a; see also Section VIII,B,4). In the first experimental study of the significance of external neural input to the CDC the effect of complete isolation of the cerebral ganglia was investigated

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(Roubos, 1976; Roubos et ul., 1976a,b). Since blocking of afferent input to the ganglia by nerve transsection greatly reduces the viability of the snail (Hekstra and Lever, 1960), a ganglion implantation method was applied: isolated cerebral ganglia were implanted into acceptor snails. Such implants are not rejected and remain viable for long periods. Quantitative electron microscopy showed a clear increase in the activity of the CDC of 2-week-old implants compared to CDC of snails that had not received an implant. A stimulated formation of elementary granules appeared from the higher number of active (granule forming) Golgi zones (+ 37%) and of elementary granules (nearly 3 times as high) in the somata. Also in the axons in the neurohemal area many granules were present. The rate of release seemed to be stimulated as well: omega-shaped indentations of the axolemma of the terminals were twice as frequent, whereas also the number of clear vesicles was considerably higher (+3 1%). These results can be taken as the first indication that the CDC receive inhibitory neural input from outside the cerebral ganglia. The physiological significance of this input may well be related to the delay of the onset of egg laying under adverse environmental conditions (see also Section VIII,8,4). Further experimental evidence for neural input to the CDC comes from an in vitro study in which CDC somata together with the proximal parts of the axons were completely isolated (Fig. 15) and subsequently kept in vitro for 7 days (Roubos et al., 1976a,b). Ultrastructural morphometry suggested that isolation does not affect general cell functions such as protein synthesis and respiration. However, compared to nonisolated CDC, the isolated cells showed a strongly decreased rate of formation of elementary granules: the volume of the Golgi apparatus (number of Golgi zones per unit of surface area) was 46% below control level, whereas active Golgi zones and immature elementary granules were completely absent. This result has led to the idea that a neural input, originating within the cerebral ganglia, is necessary for the stimulation of the synthesis of CDC elementary granules.

2. Nonneurul Control The implantation studies mentioned above (Roubos, 1976) also showed that implantation of cerebral ganglia affects the own CDC of the acceptor snail. Although in these acceptor snails the volume of the Golgi apparatus is not clearly changed, formation of elementary granules occurs only rarely and immature granules are scarce. Moreover, many Golgi zones form LG (see also Section IV,B) that appear to fuse with acid phosphatase containing vesicles (probably primary lysosomes) and seem to be broken down within the soma (Fig. 16). This process of degradation of secretory material ( ‘‘crinophagy”) apparently takes place at a high rate since the number of LG is 5 times as high as in the shamoperated controls. Also release activity in acceptor CDC is obviously decreased

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b FIG. 15. Mechanical isolation of CDC. (11) Scanning electron micrograph of CDC (arrows) after carefully tearing open the conncctive tissue sheath above a CDC cIu
since the number of exocytosis phenomena is 40% below that of implanted CDC. It is reasonable to assume that the acceptor CDC are inhibited by a factor released by the implant, but the identity of this Pictor is unclear. It might act on the CDC directly but also via other neural or (neuro)endocrine centers. In this respect the possibility of a hormonal negative feedback via the ovotestis (Roubos, 1976) should not be ruled out. It can be concluded that the activity of the CDC is controlled in a very complex way, in which various inputs, originating from different sources and reaching the cells via different pathways, are involved. In the following section this picture will be further developed by reviewing the results of studies of the effects of various external and internal factors on CDC activity and the interactions between the CDC and other neural and (neuro)endocrine centers.

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Fiti. 16. Proposed sequence of breakdown of secretory material contained in large clectrondense granules (LG) of CDC. In addition to elementary granules (EG) the Golgi apparatus (GA) forms LG, which subsequently fuse with primary lysosonies (PL) and are broken down. During this degradation process EG may fuse with LG. Finally the LG membrane ruptures. GER. Granular cndoplasmic reticulum; GT, Golgi tubule. Modified after Roubos ( I 976).

A N D INTERNAL CONTROLLING FACTORS B. EXTERNAL

1. Fresh Wuter Stimulation

Keeping snails for more than 3 days in our standard jars without water change leads to a stop of oviposition (see also Section V,B). Electrophysiological inspection of such “polluted snails” indicates that their CDC remain in the resting state and do not produce discharges (de Vlieger ef a)., 1980). Water change is a complex environmental stimulus that appears to consist of at least 3 components: supply of a clean jar, supply of clean water, and elevation of oxygen pressure in the water. Each of these components appeared to stimulate polluted snails to lay eggs, by inducing a CDC discharge within 10 minutes. Clean water and a rise of oxygen pressure are the most effective stimulants (Kits and ter Maat, 1983). In nature, sensing these aspects of water quality is relevant for Lyrnnaeu in finding a suitable place for egg deposition. The way by which these stimuli reach the CDC remains to be shown.

330

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Sturvntiorz

Another environmental factor that has an important effect on CDC activity is the availability of food. L. srugrzalis kept under MD conditions completely ceases oviposition within I week of starvation. Since the responsiveness of the ovotestis to injected CDCH does not decrease during the first 9 days of starvation (Dogterom et a / ., 1 9 8 3 ~the ) stop of oviposition must be the result of a decrease or complete stop of CDCH release. This is corroborated by electrophysiological (ter Maat et ul., 1982) and ultrastructural studies (Dogterom et al., 1984). It is shown that the CDC of starved snails contain ample CDCH but are mainly in the resting state and never enter the active state. Morphometry shows that the number of exocytosis phenomena in axon terminals of starved snails is 5 times as low as in fed ones. Also the membrane potential is considerably lower (- -71 vs - -63 mV). Apparently, this hyperpolarization results in an increased threshold of the CDC for extracellular input, forming a blockade for transition from the resting to the active state. Since, as has been shown experimentally, the CDC of starved snails are in fact more excitable than in fed animals, they can, in principle, enter the active state whenever they receive a stimulus that is strong enough to overcome the blockade. Apparently this is the case when starved snails are kept under long-day (lightldark 16:8) photoperiodic conditions (“LD snails”) since such snails continue egg laying (Bohlken and Joosse, 1982). The way starvation acts upon the CDC is not known. Possibly, changed ionic contents of the hemolymph are involved (cf. de With, 1978). However, the hyperpolarizing effect of starvation is not found in other types of neuron in the CNS of Lymnaeu (ter Maat et d., 1982), so that starvation may act on the CDC in a more specific way. 3. Temperature Bringing snails from 20 to 8°C causes a rapid reduction of ovipository activity, whereas oviposition ceases completely upon exposure to 4°C (Dogterom rt al., 1983c; see also Joosse and Veld, 1972). Lowering temperature results in a decreased response of the ovotestis to injected CDCH, but also the CDC are affected: both at 4 and at 8°C the CDC neurohemal area is well filled with CDCH but exocytotic activity is clearly below control level (Dogterom et al., 1 9 8 3 ~E. ; W. Roubos, unpublished research). Electrophysiological studies have shown that at 8°C no afterdischarge can be elicited. Furthermore, an ongoing discharge at 20°C is arrested by cooling to 8°C (A. ter Maat, personal communication). On the other hand, a rise of temperature (e.g., from 15 to 20°C) may stimulate egg laying within some hours, whereas such an increased temperature also stimulates egg laying activity on the long-term (e.g., van Nieuwenhoven and Lever, 1946; van der Steen, 1967; Joosse and Veld, 1972). Possibly, these stimulating effects of temperature are also exerted via the CDC.

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4. Tactile Stimulation Behavioral studies (see Kits and ter Maat, 1983) show that tactile stimulation of the skin postpones the beginning of egg deposition induced by fresh water stimulation. The biological significance of this behavior may well be the prevention of oviposition during adverse external conditions (e.g., presence of predators or parasites). The inhibitory effect of tactile stimulation on egg laying appears to be exerted via the CDC. Tactile stimulation of the skin elicits a biphasic postsynaptic potential (PSP) in the CDC, that is able to stop the discharge. The PSP also occurs after stimulation of peripheral nerves (ter Maat, 1979b; ter Maat et ul., 1983a). It consists of a rapid excitatory (EPSP) and a slow inhibitory (IPSP) component. The IPSP has the largest amplitude when evoked during the active state. Ion-substitution experiments indicate that the PSP underlies a conventional ionic mechanism, involving conductance increases for Na+ (EPSP) and K + (IPSP). The following arguments support the idea that the response is evoked by cholinergic input (ter Maat and Lodder, 1980). (1) Upon in vitro application, out of a large number of putative transmitters only acetylcholine (ACh) evokes a biphasic response: at low concentrations (below 10 M ) the response is inhibitory, at higher concentrations a depolarizing wave precedes hyperpolarization. (2) If ACh is topically applied (microiontophoresis) the response follows the same time course as the response evoked by peripheral nerve stimulation. (3) Blockers of the ACh-induced response as well as cholinomimetics interfere selectively with each phase of the synaptic potential evoked by peripheral nerve stimulation. Thus, two types of ACh receptor appear to be present on the CDC, one mediates the IPSP, the other the EPSP. The location of the presumed cholinergic synapses was firstly indicated by microiontophoresis studies (ter Maat and Lodder, 1980): the complete response can be found only when ACh is administered onto the proximal part of the ventral CDC axons. More recently, ACh-esterase activity was demonstrated ultrastructurally in the synaptic cleft of two types of synaptic contact located on the fine lateral axon branches of the ventral cells: the type €3 and type C SLS (Roubos et al., 1981a; ter Maat et ul., 1983a). Most likely, via these SLS the biphasic PSP is generated in the ventral CDC only and is conveyed electrotonically to the other CDC (Fig. 14). Concluding, it seems very likely that the biphasic PSP is responsible for the stop of the discharge upon tactile stimulation. ~

5 . Parasitical Iilfection

The early stages of many digenean trematodes live in molluscs, the adults in vertebrates. L . stugnalis is the intermediate host of the avian schistosome Trichobilharzia ocellata. When juvenile ( 10 mm shell height) snails are infected

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with miracidia of this parasite, body growth rate increases (“giant growth”), growth of the gonad and of the male and female accessory sex organs is retarded, and egg laying hardly occurs (McClelland and Bourns, 1969; Sluiters, 1981). These effects become distinct - 40 days after infection, i.e., at the moment the daughter sporocysts actively multiply to form cercariae (Joosse and van Elk, 1983). Bioassays show that CDC of infected snails contain CDCH. However, at electrophysiological inspection they always have been found to be in the inhibited state. Therefore, a major cause of the reduced egg laying activity of infected snails seems to be inability of the CDC to enter the active state (de Vlieger et al., 1983), possibly because infection retards the maturation of the CDC system. Presumably, T . ocellata influences the CDC in a humoral way, since the daughter sporocysts move freely in the blood lacunae between the lobes of the digestive gland (Joosse and van Elk, 1983). Infection with T . orellata also leads to a reduction of the activity of the male and female accessory sex glands. It is not known whether the parasite affects reproduction via the CDC or via (an)other ccntcr(s) that control(s) reproduction (Joosse and van Elk, 1983). 6. Rhythmiciry and Photoprriotl

Some years before the discovery of the electrical states ofthe CDC Wendelaar Bonga (1971) studied the secretory activity of CDC in LD snails kept under natural conditions outside the laboratory (June; light period 17 hours). Quantitative electron microscopy of groups of snails fixed at a number of intervals during a 24-hour period showed a clear diurnal cycle of exocytotic release activity. From 6.00 to 14.00 hours release occurred in less than 1% of the axon terminals. During this period the terminals became filled with elementary granules. At 18.00 hours release had increased to about 2%. In the early evening a steep rise to about 25% was noted; release phenomena were particularly obvious between 19.00 and 2 1 .OO hours and the number of granule-filled axons decreased rapidly. Also the volume of the tcrniinals diminished. From 20.00 hours onward “tubular and vesicular structures” appeared in an increasing number of terminals. At 2.00 hours these structures were present in the majority of the terminals. Up t o this time release activity remained rather high (16%). Subsequently, it rapidly decreased to less than 0.5%) at 6.00 hours. By then about 75% of the tcrniinals were empty. Also the tubular and vesicular structures had disappeared from nearly all terminals. At 10.00 hours most of the axons had started again to accumulate elementary granules. Wendelaar Bonga concluded that the CDC exhibit a diurnal rhythm, starting high release activity around the end of the afternoon. If thcsc results are reconsidered now, it is not hard to conceive that Wendelaar Bonga actually described the morphological aspects of the three electrical states; apparently, the CDC he studied were in the active state around the end of the afternoon, were inhibited during the evening and early night, and were resting during the rest of the 24-hour period. Furthermore, Wendelaar

-

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333

Bonga showed a clearly diurnal rhythmicity with respect to elementary granule formation and axonal transport (maximal during the evening and early night) and storage in the soma (maximal during the day). It seems feasible that these rhythms are also related to the electrical states. As would be expected, the activity rhythm of the CDC is reflected by a diurnal rhythm of egg laying activity. Thijssen (1983) showed such a rhythm, with a maximum around 18.00 hours, for LD snails kept under natural conditions. The question arises as to the control of the diurnal CDC rhythmicity. In view of the above presented evidence for the induction of the discharge by fresh water stimulation and the stimulating effect of high temperature on oviposition it would seem that these external factors sustain the rhythm by inducing the CDC to discharge. This view, however, does not explain why rhythms of CDC activity and egg laying also occur under laboratory conditions where quality and temperature of the water are kept constant (see Wendelaar Bonga, 1971; van der Steen et af., 1969; van der Steen, 1970). The explanation may be that CDC rhythmicity is not only exogenously but also endogenously controlled. This may be illustrated by morphometrical studies of the role of the eyes in the control of CDC rhythmicity (Roubos, 1975). In contrast to sham-operated snails blinded snails did not show rhythmicity for any of the ultrastructural parameters for CDC activity (Fig. 17). Apparently, the shams had synchronous CDC whereas blinded snails exhibited considerable individual differences, those of some snails being very inactive, those of others highly active at the same hour of the day. The best explanation for this phenomenon is that the CDC of blinded snails were rhythmically active but that the rhythms of individual snails were desynchronized. Such interanimal desynchronization occurs when circadian rhythms are not entrained by external synchronizing stimuli. Thus presumably, the CDC activity rhythm is, at least partly, controlled by an endogenous, circadian factor that is entrained via the eyes by the environmental light/dark cycle. Interestingly, it was found that blinding has no effect on the diurnal rhythmicity of cerebral neurosecretory LGC. Therefore, the eyes may influence the CDC via a specific, probably neural, pathway (Roubos, 1975). It is not known how exogenous and endogenous factors cooperate in entraining the CDC rhythm. From a number of theoretical possibilities only one will be given here. The function of the endogenous circadian “clock” may be to control the threshold of the CDC for external induction of the discharge, forming a blockade for stimulation during almost the whole 24-hour period and a “gate” for external factors (oxygen, temperature) around the end of the afternoon. The clock may be located either in the CDC or in other neurons that conpol CDC activity. Neither in the field nor in the laboratory all snails of a population show egg laying at the very same moment of the day. Thijssen (1983) demonstrated that the peak of egg laying is rather broad, some snails laying eggs up to several hours before or after the maximum at 18.00 hours; no egg laying occurs between

-

A Elementary granules /

SHAMS

4000r

BLINDED SNAILS

/

iooopm2

B Golgi zones (total) / 1000

I

pm2

-

c

0

C Golgi zones

""'4

iooopm

40 -

P 0 Perineurium axons

1

1000p m

F

400r

r

T

F Clear iooo p m

1000

I0

16

22

4

10 10 Time of day

16

22

4

10

Hrr.

FIG. 17. Morphometry showing rhythmicity of various ultrastructural parameters in CDC of sham-operated snails. Blinded snails do not show rhythmicity; note high standard errors of the mean (vertical bars; 5 snails per group) as compared to shams. From Roubos (1975).

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7.00 hours and noon. This may be explained in different ways, e.g. (1) the “gate” is rather wide, allowing external stimuli to “enter” over a relatively long period of the day, ( 2 ) the blockade is not absolute; very strong stimuli (local high oxygen pressure, sudden rise of temperature, etc.) may overcome it and induce a discharge, and/or (3) also in populations of nonblinded snails some degree of interanimal desynchronization may occur, either determined endogenously or caused by exogenous factors (tactile stimulation, temperature, nutritive state, etc.). Moreover, interanimal differences in egg laying may also appear because of interanimal differences in the latency between ovulation and egg laying, which amount upto some hours and may result from differences in feeding condition and (local) temperature (cf. Dogterom et al., 1983~). It should be noted that the occurrence of interanimal differences with respect to the start of the discharge is fully compatible with the morphometric observations of rhythmical CDC activity by Wendelaar Bonga (1971) and Roubos (1975): apart from the possibility that all the snails these authors studied actually were discharging at the very same moment (as a result of simultaneous external stimulation), the experimental set ups (only 5 snails per group fixed at relatively large intervals: Wendelaar Bonga: up to 4 hours, Roubos: 6 hours) may well have obscured that in fact the starts of the (relatively short lasting) discharges of individual snails differed considerably in time. Ultrastructural morphometry has shown that also under MD conditions in the laboratory the CDC exhibit a diurnal rhythm of release activity. However, this rhythm is not very pronounced since a fair number of snails do not show a peak of exocytosis activity every day (Roubos, 1975, unpublished research). CDC of MD snails are more often found to be in the inhibited state than those of LD snails (de Vlieger et al., 1980), which signifies that their chance to be excited to a discharge is also decreased. This explains why MD snails lay eggs at a much lower rate, viz. once per 2 or 3 days, than LD snails (once a day) (van der Steen, 1967; van der Steen et af., 1969; Bohlken and Joosse, 1983). Another factor that contributes to the lower egg laying activity under MD conditions is the lowered sensitivity of the ovotestis to CDCH (Dogterom et al., 198313). Furthermore, the possibility should be kept in mind that LD conditions reduce the activity of the endocrine dorsal bodies, which would inhibit the maturation of eggs and the formation of egg mass material. No egg laying occurs under MD conditions (in spring and fall) in the field, probably because both CDCH release and CDCH sensitivity of the ovotestis are strongly reduced. In the field CDC release clearly less CDCH during a discharge than in the laboratory: morphometry has indicated that CDC of snails in the field release about 4 times less secretory material in October than in June (Wendelaar Bonga, 1971). Possibly, this low release is due to a decreased rate of synthesis of secretory material: in spring and fall the amount of rough endoplasmic reticulum is clearly reduced in comparison to that in summer (Joosse, 1964). Obviously, the lower temperature of the water in these seasons may well be responsible for

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the reduced synthetic activity of the CDC as well as for the decreased sensitivity of the ovotestis to CDCH (Dogterom et al., 1983b).

I . The Ring Neuron The single Ring neuron (RN; Jansen and Bos, 1983, 1984) is located in the right cerebral ganglion. Light microscope studies of HRP-filled neurons show that the RN axon consists of two main branches. One branch crosses the cerebral commissure, where it forms lateral branches. Then it runs via the left cerebral ganglion to the left pedal ganglion. The other branch runs to the right pedal ganglion. In the pedal ganglia the branches divide up into a large number of small fibers some of which cross the pedal commissure. I n this way the axon branches form an almost complete ring. Intracellular stimulation of the RN evokes an inhibitory response in all CDC, though a larger one in ventral than in dorsal cells. When the RN is stimulated at high frequency a CDC discharge may stop. However, most probably, the RN is not involved in the determination of the duration of the discharge since in v i m it never fires at such a high rate that it can stop the discharge (Jansen and Bos, 1983, 1984). Not only the role of this inhibition but also the way in which the RN inhibits the CDC is still unclear. The RN seems to make a monosynaptic contact with the CDC since a 1:1 relationship exists between its spiking rate and the postsynaptic CDC potentials, and, moreover, the input cannot be blocked by high Mg2+/high Ca2 Ringer. On the other hand, the input is not classically synaptic, since the delay is remarkably long (varying from 0.5-1 second among preparations) (Jansen and Bos, 1983, 1984). Ultrathin serial section studies of the HRP-filled RN show that the RN axon branch in the cerebral commissure forms small side branches, which run into the direction of the CDC neurohemal area and the overcrossing CDC axons but end nonsynaptically at some tenths of micrometers distance from the CDC axons (E. W. Roubos, unpublished research). Therefore, it seems likely that the RN influences the CDC by releasing its secretory material in a diffuse way into the intercellular space in the commissure (“nonsynaptic release”; see Section 1X). +

8 . The Laterul Lobes L . stagnalis shows a conspicuous antagonism between body growth and reproductive activity. For instance, in contrast to sexually immature snails, which grow at a fast rate, adult snails show a growth rate that is reduced in favour of reproduction (Geraerts, 1976a). Furthermore, LD photoperiodic conditions stimulate reproduction at the cost of body growth (Joosse, 1964; Bohlken and Joosse, 1982), and parasitic infection leads to giantism and reduction of reproductive activity (see Section VIII,B,S). Evidently, the lateral lobes (LL), small ganglionic appendices of the cerebral ganglia, play a particular role in the control of this antagonism, since extirpation of the LL results in giant growth and in

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inhibition of reproductive activity (Geraerts, 1976b). Ultrastructural morphometry (Roubos et al., 1980) shows that LL extirpation leads to hyperactivity of the growth stimulating LGC and to inactivation of the gonadotropic dorsal bodies and CDC. As to the CDC, compared to sham-operated snails, animals without LL have a Golgi apparatus that is clearly less extensive (-23%) and only rarely buds off elementary granules. Furthermore, release of secretory material appears to be reduced as the number of release phenomena is much lower (-- 35% fewer omega-shaped figures and clear vesicles, complete absence of multiple exocytosis phenomena). These results support the idea rhat the LL retard body growth by reducing LGC activity and stimulate female reproductive activity by stimulating the activities of the dorsal bodies and the CDC. Since the effects of LL extirpation on growth and reproduction are completely abolished by implantation of complete cerebral ganglia with LL but not by implantation of these ganglia without LL (Geraerts, 1976b), the LL probably influence the CDC (and the LGC and the dorsal bodies) via blood-borne factors. In this respect it is relevant to note that the LL contain in addition to a large number of unidentified neurons, various types of peptidergic cell as well as a putative endocrine structure, the follicle gland (Lever et al., 1959; Brink and Boer, 1967; Wendelaar Bonga, 1970; van Minnen and Reichelt, 1980; Schot et a / . , 1981), which may be responsible for such a nonneural control. Possibly, also the ovotestis is involved in the antagonism between growth and reproduction, as in the related hermaphroditic basommatophoran snail Bulinus truncatus castration and LL extirpation have the same effects: giantism (and inhibition of female reproduction) (Boer et al., 1976; Geraerts and Mohamed, 1981). C. ROLEOF cAMP

IN THE

CELLULAR RESPONSE

As has been shown above, various external and internal factors influence CDC activity. In this section evidence will be presented that, at least in some of these processes, cAMP is the cellular mediator between stimulus and CDC response. Apparently, the CDC are capable of producing CAMP, since ultracytochemical studies have demonstrated the occurrence of the key enzyme for cAMP production, adenylate cyclase (Fig. 18a). Enzyme activity occurs at the axolemma of the axon terminals and is higher in the active than in the other states (Roubos er al., 1981b; Roubos and Buma, 1982). Indeed, preliminary radioimmunoassay studies indicate that the cAMP contents of the cerebral commissure are about twice as high in the active as in the resting state (Buma et al., 1984). The relation between cAMP and the discharge clearly appears when cerebral ganglia are incubated in snail Ringer containing 1 mmol8-parachlorophenylthiocAMP (8-CPT-CAMP) (In contrast to CAMP, this CAMP-analog readily penetrates the axolemma and is not broken down by cytoplasmic phosphodiesterase; Meyer and Miller, 1974.): within half a minute after the start of incubation a

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FIG. 18. Role of cAMP in release of CDC secretory matcrial. (a) Electron-dense reaction product (arrows) indicating adenylate cyclase activity at axolcrnrna of CDC axon terminals (A) in active state. C , Connective tissue of perincurium. X20.000. From Roubos et ul. (1981b). (b) High exocytotic activity in axon terminals during discharge induced with 1 mino1 IBMX. x30,OOO. (Courtesy of P. Buma.)

discharge starts, accompanied by high exocytotic activity and massive release of CDCH. Most likely, this incubation mimics the effects of an intracellular rise of CAMP,since the same effects (Fig. I8b) are found by incubation with 1 mmol3isobutylmethyl-1-xanthine (IBMX), a potent inhibitor of phosphodiesterase (Buma et a [ . , 1984) (Fig. 19b). Presumably, a rise of the cAMP concentration is a component of the mechanism that initiates the discharge and leads to phos-

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phorylation of proteins that control the permeability of the axolemma for particular ions. Obviously, CAMP may also play a role in various other aspects of CDCH release, such as exocytotic fusion, intracellular sequestration of calcium, and sequestration of the axolemma after exocytosis. Moreover, preliminary morphometric studies suggest that the adenylate cyclase-CAMP system also influences other cellular processes, viz. synthesis and transport of elementary granules (Roubos et d.,1984). Since various peptidergic axons have been localized immunocytochemically in the neighborhood of the CDC somata (Schot et a [ . , 1981) it seems worthwhile to test a possible role of peptides in CAMP-mediated internal and external control of CDC activity. It is not known what stimulates the CDC to produce CAMP. The stimulus may either be of intracellular origin (electrical activity?) or come from outside the cells, being mediated by synaptic or nonsynaptic input (cf. Section IX).

IX. Nonsynaptic Communication During recent years physiological data have indicated that chemical interneuronal communication within the CNS does not only occur via synaptic contacts but also nonsynaptically. In this view (Dismukes, 1977; Beaudet and Descarrier, 1978; Mayeri and Rothman, 1981) messengers, especially peptides, are released into the intercellular space, diffuse throughout the CNS, but act only upon those target neurons that possess the appropriate receptors. Since this type of communication does not involve (myelinated) axon pathways, it “would either result in economy of space or in an increased capacity of the CNS to process information” (Mayeri and Rothman, 1981). The concept also accounts for the abundancy of different peptides in the CNS, as peptide diversity adequately meets the need of this mode of “wireless” communication for “chemical addressing.” Possibly, the first morphological support for the concept of nonsynaptic communication lies in the demonstration of “specific release sites” on the proximai parts of CDC axons (Roubos, 1975). The structures constitute morphologically unspecialized areas of axolemma, where the presence of omega figures and clear vesicles suggests that secretory material is exocytotically released into the intercellular space. Recently, the demonstration of possible morphological correlates of nonsynaptic communication has been facilitated by the development of the TARI method (tannic acid-Ringer incubation; Buma and Roubos, 1983a; Roubos et al., 1983), a modification of the TAG0 method. The TARI method involves tissue incubation for some hours in Ringer to which 1% tannic acid has been added. During incubation exocytotic fusion of secretory granules with the plasma membrane proceeds but extrusion of the granule contents does not occur since

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the contents are immediately fixed by tannic acid (Fig. 19a). As a result exocytosis phenomena accumulate so that, after a sufficiently long incubation time, exocytosis can be visualized even at sites where release takes place with irregular intervals, at a very low frequency, or at a very high speed. With the TARI

FIG. 19. TARI method showing accumulation of exocytosis phenomena (arrows). (a) CDC axon terminal. x 100,000. (Courtesy of P. Buma.) (b) Proximal parts of CDC axons with NSRS. Exocytosis occurs at various sites of the axon, suggesting multidirectional, diffuse (nonsynaptic) release. x20,OOO.

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34 1

method at various places in the CNS nerve fibers can be observed releasing their secretory material by exocytosis in a nonsynaptic way. Exocytosis takes place at large areas of the axolemma where morphological specializations are absent. The phenomena principally occur almost all around the fiber. Apparently, at these nonsynaptic release sites (NSRS) neuronal messengers are not released locally or into one direction but diffusely and multidirectionally. Up to now these NSRS have been found in the CNS of various species of molluscs, insects, and in the rat (Buma et al., 1984; Buma and Roubos, 1983a; Roubos et al., 1983), illustrating their general importance. In the CNS of L. stagnalis NSRS are common. As to the CDC NSRS occur on the soma, on the proximal axon parts, on the axons (Fig. 19b) of the ventral cells that pass the cerebral commissure, and on axon branches running toward the neurohemal area (Roubos et al., 1983, unpublished research). Possibly, some of these NSRS are involved in the presumably nonsynaptically RN-induced inhibition of firing of the CDC (see section VIII,B,7) during a discharge (cf. Jansen and Bos, 1983).

X. Conclusions and Perspectives Three main factors have contributed to the present knowledge of the nature of the peptidergic CDC of L . stagnalis: the suitability of the cells for experimental studies, the multidisciplinary approach with which they have been investigated, and the detailed knowledge of various related aspects of the snail’s biology, such as endocrinology (e.g., Joosse and Geraerts, 1983), reproduction (e.g., Geraerts and Joosse, 1984), the sensory system (e.g., de Vlieger, 1968; Janse, 1974), the muscular system (Plesch, 1977), the blood and connective tissue (Sminia, 1975), and hydromineral regulation (de With, 1980). Some of the results may (have) increase(d) the insight into structure and functioning of nervous systems in general. Of these results the following should be mentioned. 1 . Of neuronal systems the CDC system is among the first of which the topological, morphological, cytochemical, biochemical, electrophysiological, and functional characteristics have been established in some detail. 2 . The quantitative electron microscopic analyses of the cyclic dynamics of CDC activity show how a neuroendocrine system can meet the physiological demand for a sudden high titer of neurohormone (to induce ovulation) by synthesizing, transporting, and, possibly, degrading secretory material in a timed, cyclic, and quantitative fashion. 3. The clear relation between the electrical discharge and massive CDCH release demonstrates the significance of the simultaneous neural and endocrine character of the neuroendocrine cell. 4. The electrophysiological and morphological establishment of electrical and

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functional synchrony between individual CDC explains how neurons can act together as one network in order to respond adequately to physiological requirements (CDC: high CDCH output during a relatively short period). 5 . The ultrastructural description of complex membrane events during and after exocytosis illustrates how neurons, and secretory cells in general, can release their products in a calcium-dependent way without losing their structural and physiological characteristics. 6. Apparently, a variety of environmental factors, acting via neural and nonneural pathways, exert short-lasting (tactile stimulation, water pollution, sudden rise of temperature) and long-lasting (starvation, photoperiod, sustained lowered or elevated temperatures, parasitic infection) effects on the endogenously (by pacemaker and circadian mechanisms) controlled states of CDC activity, which ensures that egg laying occurs under the ecophysiologically most favorable conditions. This enlightens how external and internal factors can alter neuronal functioning on the short- and the long-term. In addition, the CDC studies have rendered some technical and methodological modifications and developments (e.g., quantitative electron microscopy, TAG0 and TAR1 methods) that have general applicability. On the other hand, the CDC system obviously has some disadvantages, particularly at the biochemical level. Thus, for instance, in spite of the large size of the CDC, in the cerebral commissure (the source for purification of CDCH) at most 3 ng CDCH is present (Ebberink et al., 1983), which seriously hampers purification and subsequent analysis of the hormone. Similarly, much effort will be needed to elucidate the biochemical characteristics of, e.g., ion channels in the axolemma or CAMP-related processes in the cytoplasm, unless highly sensitive methods can be applied. Meanwhile, the following topics of current research seem to be very promising. 1. The relation between synthesis, transport, and release of secretory material, as well as the relation between these processes and electrical activity. 2. The role of the second messenger CAMP in exogenous and endogenous control of CDC activity. 3. The significance of different peptides within the CDC, with particular reference to their biosynthesis (cellular compartimentalization; storage within one type of elementary granule?), differential release (at different times? from different axon terminals?), and function (ovulation, egg mass formation, egg laying behavior). 4. The significance of NSRS in view of the concept of nonsynaptic communication. 5. The way various external and internal factors are integrated by the CDC, leading to a final response of the network.

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ACKNOWLEDGMENTS The author is greatly indebted to his colleagues for helpful discussions, and especially to Prof. H. H. Boer, Prof. J. Lever, Dr. W . P. M. Geraerts, and Dr. A. ter Maat for critically reading and commenting on the manuscript. Thanks are also due to Ms. A. M. H. van de Ven for preparing photomicrographs and Ms. T. Laan for typing parts of the manuscript. Considerable parts of the work described in this article have been made possible by grants of the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).

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