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Cellular Mechanisms of Neurohormone Release in the Snail Lymnaea stagnalis
Cellular Mechanisms of Neurohormone Release in the Snail Lymnaea stagnalis ERIC W ROUBOS and PETER BUMA Biological Ltrhorator,
V r ~ j t Utzrvet ,
citerr Atnstrrdtrnl, (The Nerherlutids)
INTRODUCTION Mechanisms that control the release of peptidergic neurosubstances (hormones, transmitters) are being studied particularly in the vertebrate neurohypophysis and in some neurosecretory systems of invertebrates (see e.g., Normann, 1976). Such studies have indicated that an increase of the electrical activity of the neuron induces an influx of calcium into the axon terminal, after which the release of the peptide contents of the secretory granules takes place by exocytosis. Subsequently the membrane remnants of the granules are resorbed into the axoplasm by pinocytosis (e.g. Normann, 1976; Roubos and Van der Wal-Divendal, 1980). Recent findings suggest that in addition to electrical activity and calcium, CAMPis involved in the control of peptide release (e.g. Mathison and Lcderis, 1978). However, the mechanisms by which electrical activity, calcium and CAMP would control the processes of exocytosis and membrane resorption are far from being understood. In our laboratory attention has been focused on the mechanisms controlling the release of the ovulation-stimulating neurohormone produced by the (peptidergic) neuroendocrine CaudoDorsal Cells (CDC) of the freshwater snail Lyrnn~ieustugnnlis. The CDC appear to be suitable objects for such studies for the following reasons. ( 1 ) The location of the CDC is very specific : their approximately 100 cell bodies occur in two clusters, one in each cerebral ganglion, and the approximately 80,000 axon terminals all lie at the periphery of the intercerebral commissure, adjacent to the cephalopedal haemolymph sinus (Wendelaar Bonga, 1971). (2) Since the CDC are large (approximately 50 p m in diameter) they can be easily studied with endocrinological and neurophysiological techniques. Generally the CDC are electrically silent (“resting state”). However, after appropriate stimulation they start an approximately 45 min period of spiking activity (“active state”), during which the ovulation-stimulating hormone (CDCH) is released. After this state the cells start a period of about 5 h during which no electrical activity can be evoked (“inhibited state”). These 3 states can be easily induced experimentally. CDC are brought into the resting state by keeping snails in stagnant water for 6 days. Then the active state can be induced either in vivo by transfer of such snails to fresh, oxygenated water, or in vitro (in a preparation of the central nervous system) by repetitive electrical stimulation of the cells. Within 1 h after induction of the active state the CDC enter the inhibited state (Kits, 1980, 1981). (3) During the last decade much attention has been paid to the ultrastructural aspects of CDCH release (e.g. Wendelaar Bonga, 1971 ; Roubos, 1975). Observations on exocytosis
186 were particularly favoured by the use of the tannic acide-glutaraldehyde-osmium fixation method (TAGO-method; Roubos and Van der Wal-Divendal, 1980; Roubos et al., 1981b). In this paper the present state of knowledge of the dynamics and mechanisms of CDCH release is considered. Special attention will be given to some recent experiments on the relation between the “states” of the CDC on the one hand, and of exocytosis, membrane resorption, calcium dynamics and CAMP on the other. EXOCYTOSIS There is much ultrastructural evidence for the occurrence of exocytosis in the CDC terminals (Wendelaar Bonga, 1971 ;Roubos, 1975; Roubos and Van der Wal-Divendal, 1980; Roubos et al., 1980, 198 1b). CDC that are electrically active in vitro do not only show CDCH release, but also a high exocytotic activity (Kits, 1981; Roubos et al., 1982). This indicates that release of CDCH is triggered by electrical activity and takes place by exocytosis during the active state. The results of an ultrastructural, morphometrical (cf. Roubos, 1975 ; Roubos et al., 1981 b) study of the relation between the CDC states and the dynamics of exocytosis in vivo can be summarized as follows. Resting state. In the neurohaemal area numerous, large CDC tenninals, filled with many secretory granules, are present (Fig. 1a). Signs of exocytotic release of secretory material are scarce. Active stute. The axon terminals are smaller and contain fewer secretory granules (- 80%) than during the resting state. Exocytosis occurs very frequently (Fig. lb,c,d). Numerous secretory granules appear to be fused, and release their contents simultaneously (multiple exocytosis; Fig. 1 d). The number of exocytozed granule contents in the part of the axolemma facing the haemolymph is about 140 times that in the resting state. Inhibited state. Compared to the active state the neurohaemal area contains approximately twice as many secretory granules, but the degree of filling as found in the resting state is not attained. The number of exocytoses is lower than that in the active state (- 71 W ) , but still considerably higher than in the resting state ( X 40; Fig. le). The finding that only few exocytotic phenomena occur in the resting state is in line with previous observations, and indicates that CDCH release occurs at a low rate during this state (Kits, 1981). Furthermore, the results confirm the conclusion drawn before (Roubos et al., 198 1 a) that during the active state CDCH release occurs at a high rate, and often by multiple exocytosis. Remarkably, in the inhibited state the CDC terminals show a considerable number of exocytoses, i.e. during electrical inactivity. This fact indicates that CDCH release still proceeds, albeit at a lower rate than in the active state. The mechanism by which the CDC release CDCH during electrical inactivity is obscure. A possibility would be that the exocytotic phenomena observed in the inhibited state had started already in the active state. However, this is not very plausible, since calculations of the speed of exocytosis in the CDC have indicated that, although the extrusion of a granule content may proceed rather slowly, it will take no more than some minutes (Roubos et al., 1981b). Therefore, it is more likely that, during the inhibited state, the axoplasmic concentration of calcium is still high enough to induce a considerable number of exocytoses. Evidently, the CDCH that is released during the inhibited state does not induce ovulation, since ovulation takes place during the active state only (Kits, 1980). In this respect it is noteworthy that the activity of the albumen gland, afemale accessory sex gland which secretes nutrients for the developing embryo. can be stimulated in vitro by
187
Fig. I .a-h: axon terminals of CDC. B. basal lamina: C. connective tissue; S. secretory granule. a : resting state. No exocytosis. x 35.000. b : active state. Numerous TAG(>-positive. electron-dense granule contents are released by exocytosis (arrows). x 20,000. c : active state. Intercellular release by exocytosis (arrow). G, glial cell process. X 65.000. d : active state. Multiple exocytosis (asterisk). X 65.000. e : inhibited state. Exocytosis (arrows). X 35,000. f : resting state. Some clear vesicles (CV) and vacuoles ( V ) . C , glial cell. X40.000. g : inhibited 5tate. Possible formation of vacuoles (arro~c).X 50.000. h : inhibitcd state. Possible formation of clear vesicle (arrow). X 75,000. a-e. TAGO-fixation: f-h. routine fixation.
188 homogenates of the neurohaemal area of the CDC (Wijdenes, 1981). Thus, the CDCH released during the inhibited state may stimulate the activity of the albumen gland. This idea is in line with the observation that the activity of the albumen gland in vivo shows a peak some hours after ovulation (M. de Jong-Brink, personal communication). MEMBRANE RESORPTION In view of the high exocytotic activity in the active state, which continues at a lower level in the inhibited state, it is clear that the CDC terminals have to resorb many parts of the axolemma in order to keep this in normal shape. The following observations deal with the mechanism of this resorption. Resting stute. The terminals contain two types of electron-lucent vesicle, viz. “clear vesicles” (CV ; diameter approximately 50 nm), and “vacuoles” (diameter 70-120 nm) (Fig. If). Both types occur in low numbers. In a few terminals a ‘‘whirl’’ consisting of concentrically arranged, double membranes was found (cf. Wendelaar Bonga, 197I ) . Active stute. Compared to the resting state the numbers of CV and vacuoles are unchanged. However, the number of whirls is clearly higher (+ 70 %). In some cases the membranes of a whirl seem to be continuous with the axoleinma (Fig. 2a). Inhibited stute. Compared to the active state the numbers of CV and vacuoles are much higher ( x 3 and x 2, respectively). Indications of the formation of CV (micropinocytosis) and of vacuoles (macropinocytosis) were obtained occasionally (Fig. I g, h). The number of whirls is also considerably higher ( X 3). Some whirls are surrounded by CV, vacuoles and other vesicular structures (Fig. 2b). Furthermore, doughnut-shaped vesicles (DSV ; diameter approximately 100 nm; Fig. 2d) and multivesicular bodies (MVB; Fig. 2c) are encountered. Of particular interest is the finding of many clear vesicle-like structures (CVL) lying together at the outside of the terminal, in invaginations of the axolemma (cf. Fig. 3c). Finally some axons seem to release membranous, whirl-like structures (WS; Fig. 3c). The observations suggest that CV and vacuoles are formed by resorption of parts of the axolemma (cf. Wendelaar Bonga, 1971 ; Roubos, 1975; Normann, 1976; Morris and Nordmann, 1980; Roubos et al., 1981b). Since CV and vacuoles are most numerous in the inhibited state, it would seem that membrane resorption occurs mainly in this period. In other words, membrane resorption would be delayed with respect to maximum exocytotic activity (active state). Such a delay is not uncommon in secretory cells (e.g. Gemmel and Stacy, 1979), but it is inconsistent with the observation that the size of the CDC terminals is smaller after the active state (see experiment on exocytosis) : an increase would be expected if high exocytotic activity is not followed immediately by a strong resorption of membrane. Possibly, not only CV and vacuoles but also whirls play a role in membrane resorption: the observed associations of the whirl membranes with the axolemma suggest that whirls are formed by strong invagination of the axolemma. Since whirls increase in number during the active state, it may be that they represent “bulk resorptions” of the axolemma during high (multiple) exocytotic activity. CV and vacuoles may be components of a more subtle mechanism of membrane resorption mainly operating during the inhibited state, when exocytosis occurs less intensely. As to the fate of the “resorption structures” (CV, vacuoles, whirls) the present study permits some speculations. Some of the whirls may be partially broken down by lysosomal action and subsequently released from the terminals as whirl-like structures (“residual bodies”). However, the number of these structures seems too low to account for the complete removal of all whirls. Therefore, possibly, some of the whirls are transformed into vesicular structures
189
Fig. 2.a-h: axon terminals of CDC. a: active state. Whirl (W). closely apposed to axolemma (arrow). x 35,000. b: inhihitcd state. Whirls (W), surrounded by vesicular structures (VS). X 30,000. c : inhibited state. Multivesicular body. S . secretory granules. x 75,000. d : inhibited state. Doughnut-shaped vesicles (DSV). X 80.000. e : inhibited state. Possible formation of DSV by invaginating vacuoles (arrows). X 100.000. f : resting state. Mitochondrion (M) without calcium deposits. X 70,000. g: activc state. Mitochondria (M) with numerous calcium deposits (arrows). x 70.000. h: inhibited state. Some calcium deposits in mitochondria ( M ) . X 70,000. a-e, routine fixation; f-h, pyroantimonate method.
190
-_ Fig. 3.a-e: axon terminals of CDC. a : inhibited state. Calcium (arrows) in clear vesicles. X60,OOO. b : calcium (arrows) in doughnut-shaped vesiclcs (DSV). X 80,000. c : inhibited statc. Clear vesicle-like structures containing calcium (arrows) and whirl-like structurc (WS) outside terminal (T). X 80,000. d : active state. Numerous electrondense reaction products of adenylate cyclasc activity (arrows). X 20,000. e : inhibited statc. Low adenylatc cyclasc activity. X 15,000. a-c. pyroantimonate method; d and e, adenylate cyclase method.
191
(including CV and vacuoles) that were seen to surround them in some cases. The finding of extracellular CVL can be interpreted as misdirected pinocytosis (cf. Douglas et al., 1971). On the other hand, vacuoles might transform by invagination into DSV (Fig. 2e), which. in turn, might fuse to form MVB. Next, these MVB could fuse with the axolemma and release their contents as CVL into the extracellular space. At present experiments are in progress to test these and other hypothetical mechanisms of resorption and disposal of membranes of the CDC terminals (Fig. 4). CALCIUM DYNAMICS Calcium plays a crucial role in many secretory processes. It is involved in, for instance, initiation of exocytosis, control of stability and permeability of the axolemma, and activation of enzymes (e.g. Normann, 1976). The axoplasmic concentration of calcium will be strictly regulated, since it is much lower ( 10p7-10-s M) than the extracellular concentration ( 1 0-j M). This control is particularly needed during electrical activity (active state), when calcium enters the axoplasm, and afterwards, when superfluous calcium has to be removed again from the axoplasm. The importance of calcium for electrical activity and for exocytosis in the CDC has been recently demonstrated (Kits, 198 1 ; Roubos et al., 198 1b). The first results are given of a study of the localization and the transport of calcium in the CDC terminals. Using the
A
E Fig. 3 . Diagram of CDC axon terminal (A). Possible mechanisms of resorption (thick arrows) and turnover (thin arrows) of parts of axolemma. and of calcium dynaniics (dotted arrows; asterisks indicate calcium contents). Inner circle: resorption structures (W. whirl: V , vacuole; CV, clear vesicles); Cal, calmodulin; CVL, clear vesicle-like structures ; DSV, doughnut-shaped vesicles ; E, exocytosis of secretory granule ; S , secretory granule contents ; M , mitochondrion; MVB, multivesicular body; WS, whirl-like structure.
192 pyroantimonate technique (Weakly, 1979) calcium could be demonstrated at the ultrastructural level as highly electron-dense deposits. Resting state. In this state only few, small deposits of calcium are occasionally found in the mitochondria. Most mitochondria show no deposits at all (Fig. 20. Positive reactions are found in some CV located near the axolemma (Fig. 3a). Active state. All mitochondria contain much calcium, located in numerous, large deposits (Fig. 2g). Calcium also occurs in some CV. Inhibited state. The number of calcium containing mitochondria is approximately 50 % lower than in the active state. The deposits are moderate in number and size (Fig. 2h). Furthermore, calcium is present in DSV and in CVL located outside the terminals (Fig. 3b, c). The observations indicate that mitochondria take up calcium during the active state. Calcium uptake by mitochondria in neurosecretory terminals has been described previously in insects (e.g. Normann and Hall, 1978). The present study strongly suggests that during the inhibited state calcium is released again from the mitochondria. Futhermore, calcium appears to be present in CV located near the axolemma. Although (some?) CV seem to be involved in membrane resorption (see above), it is not unlikely that they also take up superfluous calcium from the axoplasm (cf. Shaw and Morris, 1980) and subsequently release it from the terminal by exocytosis (Fig. 4). In the inhibited state calcium occurs in DSV, MVB and CVL. This observation is in line with the hypothesis (see above) that a vacuole can transform via a DSV and a MVB into a CVL, if it is assumed that calcium is taken up from the axoplasm into a DSV during the process of invagination of a vacuole (Figs. 2e, 4). This would be another mechanism by which superfluous calcium is removed from the terminals during the inhibited state. Evidently, the proposed mechanisms are particularly suited for the removal of bound calcium (e.g. to calmodulin?); most likely, free calcium is removed from the terminal by “conventional” membrane pumps (Fig. 4). cAMP It has been suggested that CAMPis involved in the control of calcium-dependent release of secretory materials (e.g. Rasmussen, 1970). Adenylate cyclase activity (indicating the production of CAMP) has been demonstrated cytochemically on the axolemma of neurosecretory axons in the neurointermediate lobe of the rat pituitary (Santolaya and Lederis, 1980), where it may play a role in the modulation of the release of neurohypophyseal hormones (Mathison and Lederis, 1978). In the marine opistobrach mollusc Aplysia californica cAMP seems to trigger the release of the ovulation-stimulating hormone of the neuroendocrine bag cells (Kaczmarek et al., 1978). Here the results are given of an ultracytochemical study of the presence of adenylate cyclase in the CDC terminals, using the method of Reik et al. (1970). Preliminary results were reported previously (Roubos et al., 1981a). Resting stute. Some adenylate cyclase activity occurs on the external surface of the axolemma of a small number of teiminals. Active state. Strong adenylate cyclase activity is present on the external surface of nearly all terminals, at sites where the terminals contact the basal lamina or face other CDC terminals or glial cells (Fig. 3d). Inhibited state. The situation is very similar to that in the resting state. Only few CDC terminals show adenylate cyclase activity (Fig. 3e). Apparently, adenylate cyclase activity is low in the resting and in the inhibited states and high in the active state. This probably means that in the active state there is also a high
193 intracellular concentration of CAMP. Thcrefore, it may well be that CAMP is involved in the control of one or more secretory-linked processes, such as exocytosis, membrane resorption and calcium dynamics. SUMMARY The neuroendocrine Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater snail Lymnaea stagnalis produce a peptidergic ovulation hormone. They are good models for the study of the mechanisms that control secretory processes in neuronal terminals. The cells show 3 states of electrical activity, viz. the resting, active, and inhibited states. CDCH release occurs by exocytosis and is clearly related to electrical activity (active state), although some release takes place during the inhibited state, when the CDC are electrically inactive. Presumably, membrane resorption after exocytosis takes place during the active and the inhibited states. Mechanisms for this resorption process are proposed. Calcium is necessary for exocytosis. Mitochondria and various vesicular structures appear to contain calcium and seem to be involved in the control of the dynamics of intracellular calcium. Mechanisms are suggested by which calcium is transported intracellularly and is removed from the terminals after secretory activity. Finally, indications have been obtained that CAMP plays a role in one or more secretory-linked processes in the CDC. ACKNOWLEDGEMENTS The authors wish to thank Prof. Dr. H.H. Boer for his stimulating interest and comments on the manuscript, Prof. Dr. J . Lever for critically reading the manuscript, and Mr. Th. van der Woude and Mr. A.N. de Keijzer for participation in some of the experiments. REFERENCES Douglas, W.W., Nagasaua, J . and Schulz, R . (1971) Electron microscopic studies on the mechanism of secretion of posterior pituitary hormones and significance of mici-civesicles(“synaptic vesiclcs”) : evidence of secretion by exocytosis and formation of microvesicles as a by-product of this process. M e m . Soc. Endocrinol.. 19: 353-378. Gemmcl, R.T. and Stacy, B.D. (1979) Granule secretion by the luteal cell of the sheep: the fate of the granule membrane. Cell Tissue R P S . . 197: 413419. Kaczmarek, L.K., Jennings, K. and Slrumwasser, F. (1978) Neurotransmitter modulation, phosphodiesterase inhibitor effects, CAMP correlates of afterdischarge in peptidergic neurites. Proc. nor. Acnd. Sci. (U.S.A.), 75 : 5 2 0 c S 2 0 5 . Kits, K.S. ( 1980) States of excitability in ovulation hormone producing neuroendocrine cells of Lymntrrcr srczgndis (Gastropoda) and their relation to the egg-laying cycle. J . Neurohiol., I I : 397410. Kits, K . S . (1981j Electrical activity and hormonal output of ovulation hormone producing neuroendocrine cells in Lynnnerr sragrlrrlis (Gastropoda). In Advnric. P h ~ t i o lSci.. . Vol. 23. Naurohiology offnverrebrntes, J. Salanki (Ed.), AkadCmiai Kiadb, Budapest. pp. 35-54, Mathison, R.D. and Lederis, K. ( 1978) Modification of CAMPand phosphodiesterase inhibitors of potassium-stimulated vasopressin release from the isolated neural lobe and the hypothalamo-neurohypophysial system in vitro. In Current Studies ofHypothdamic Func,/iori. Vol. 1 , K . Lederis and W.R. Veale (Eds.), Karger, Basle pp. 88-97. Morris, J.F. and Nordmann, J . J . (1980) Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland. Neuroscicvwe. 5 : 639-649. , 1-77. Norniann, T.C. (1976) Neurosecretion by exocytosis. Int. R m . C ~ t o l .46:
194 Normann, T.C. and Hall. T . A . ( 1 078) Calcium and sulphur in neurasecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis. Cell Tiss. Res., 186 : 453-463. Rasmussen, H. ( 1 970) Cell communication, calciuni ion and cyclic adenosine monophosphate. Science, 170: 40UI2. Reik, L . , Petzold, G.L., Higgins. J.A., Greengard, P.. Barnett, R.J. (1970) Hormone-sensitive adenyl cyclase: cytochemical localization in rat liver. Science. 168 : 382-384. Roubos, E. W. (197.5) Regulation of neurosecretory activity in the freshwater pulmonate snail Lyninaeu stugriulis (L.) with particular reference to the role of the eyes. Cell Tiss. Res., 160: 291-314. Roubos, E.W. andVan der Wal-Divcndal, R.M. ( 1980) Ultrastructural analysis of peptide release by exocytosis. Cell Tiss. Res.. 207: 261-275. Roubos, E.W. Geracrts, W.P.M., Boerrigter, G.H. and Kampen, G.P.J. van (1980) Control of the activities of the neurosecretory light green and caudo-dorsal cells and of the endocrine dorsal bodies by the lateral lobes in the freshwater snail Lytnnum sfngriulis (L.). Get?. Conip. Errdoc,ririo/., 40 : 446-454. Roubos, E.W., Keijzer, A.N. de and Buma, P. (198 la) Adenylatc cyclasc activity in axon terminals of ovulation-hormonc producing neuroendocrine cells of Lynitiueu srtigriuli.v (L.). Cell Tiss. Res., 220: 665-668. Roubos, E.W., Schmidt, E.D. and Moorer-van Delft, C.M. (1981b) Ultrastructural dynamics of exocytosis in the ovulation neurohormone producing caudo-dorsal cclls of the freshwater snail Lymnaeci stu,ynali.s (L. ). Cell T i n . Res.. 21.5: 63-73. Roubos, E. W . , Boer, H.H. and Schot, L.P.C. (1982) Peptidergic neurones and the control ofneuroendocrine activity in the freshwater snail Lyrnfiueu stagridis (L.). In Nrurosecwtion -Molecules, Cells, Sysfenzs, D.S. Farner and K . Lederis, (Eds.), Plenum Press, New York, pp. II%l27. Santolaya, R. and Lederis, K. (1980) Localization of adenylate cyclase in the neurointermediate lobe of the rat pituitary. Cell Tiss. Res.. 207: 387-394. Shaw, F.D. and Moms, J.F. (1980) Calcium localization in the rat neurohypophysis. Nature (Lond.). 287: 56-58. Weakly, B.S. (1979) A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue. J . Histuchen. Cvtochenr . , 27 : 10 17- 1028. Wendelaar Bonga, S.E. (107 I)Forination, storage. and release of neurosecretory material studied by quantitative electron microscopy in the freshwater snail Lymnueu stugidis ( L . ) . Z . Zel(for.sch., 113 : 49Cb.517. Wijdenes, J . ( 198 I ) A Compurutive Srudy on the Neuro-Endocrine Control of Gruwrh and Reproduction in Pulrnonute Snails und Slugs. Thesis, Free University, Amsterdam.
V r ~ j t Utzrvet ,
citerr Atnstrrdtrnl, (The Nerherlutids)
INTRODUCTION Mechanisms that control the release of peptidergic neurosubstances (hormones, transmitters) are being studied particularly in the vertebrate neurohypophysis and in some neurosecretory systems of invertebrates (see e.g., Normann, 1976). Such studies have indicated that an increase of the electrical activity of the neuron induces an influx of calcium into the axon terminal, after which the release of the peptide contents of the secretory granules takes place by exocytosis. Subsequently the membrane remnants of the granules are resorbed into the axoplasm by pinocytosis (e.g. Normann, 1976; Roubos and Van der Wal-Divendal, 1980). Recent findings suggest that in addition to electrical activity and calcium, CAMPis involved in the control of peptide release (e.g. Mathison and Lcderis, 1978). However, the mechanisms by which electrical activity, calcium and CAMP would control the processes of exocytosis and membrane resorption are far from being understood. In our laboratory attention has been focused on the mechanisms controlling the release of the ovulation-stimulating neurohormone produced by the (peptidergic) neuroendocrine CaudoDorsal Cells (CDC) of the freshwater snail Lyrnn~ieustugnnlis. The CDC appear to be suitable objects for such studies for the following reasons. ( 1 ) The location of the CDC is very specific : their approximately 100 cell bodies occur in two clusters, one in each cerebral ganglion, and the approximately 80,000 axon terminals all lie at the periphery of the intercerebral commissure, adjacent to the cephalopedal haemolymph sinus (Wendelaar Bonga, 1971). (2) Since the CDC are large (approximately 50 p m in diameter) they can be easily studied with endocrinological and neurophysiological techniques. Generally the CDC are electrically silent (“resting state”). However, after appropriate stimulation they start an approximately 45 min period of spiking activity (“active state”), during which the ovulation-stimulating hormone (CDCH) is released. After this state the cells start a period of about 5 h during which no electrical activity can be evoked (“inhibited state”). These 3 states can be easily induced experimentally. CDC are brought into the resting state by keeping snails in stagnant water for 6 days. Then the active state can be induced either in vivo by transfer of such snails to fresh, oxygenated water, or in vitro (in a preparation of the central nervous system) by repetitive electrical stimulation of the cells. Within 1 h after induction of the active state the CDC enter the inhibited state (Kits, 1980, 1981). (3) During the last decade much attention has been paid to the ultrastructural aspects of CDCH release (e.g. Wendelaar Bonga, 1971 ; Roubos, 1975). Observations on exocytosis
186 were particularly favoured by the use of the tannic acide-glutaraldehyde-osmium fixation method (TAGO-method; Roubos and Van der Wal-Divendal, 1980; Roubos et al., 1981b). In this paper the present state of knowledge of the dynamics and mechanisms of CDCH release is considered. Special attention will be given to some recent experiments on the relation between the “states” of the CDC on the one hand, and of exocytosis, membrane resorption, calcium dynamics and CAMP on the other. EXOCYTOSIS There is much ultrastructural evidence for the occurrence of exocytosis in the CDC terminals (Wendelaar Bonga, 1971 ;Roubos, 1975; Roubos and Van der Wal-Divendal, 1980; Roubos et al., 1980, 198 1b). CDC that are electrically active in vitro do not only show CDCH release, but also a high exocytotic activity (Kits, 1981; Roubos et al., 1982). This indicates that release of CDCH is triggered by electrical activity and takes place by exocytosis during the active state. The results of an ultrastructural, morphometrical (cf. Roubos, 1975 ; Roubos et al., 1981 b) study of the relation between the CDC states and the dynamics of exocytosis in vivo can be summarized as follows. Resting state. In the neurohaemal area numerous, large CDC tenninals, filled with many secretory granules, are present (Fig. 1a). Signs of exocytotic release of secretory material are scarce. Active stute. The axon terminals are smaller and contain fewer secretory granules (- 80%) than during the resting state. Exocytosis occurs very frequently (Fig. lb,c,d). Numerous secretory granules appear to be fused, and release their contents simultaneously (multiple exocytosis; Fig. 1 d). The number of exocytozed granule contents in the part of the axolemma facing the haemolymph is about 140 times that in the resting state. Inhibited state. Compared to the active state the neurohaemal area contains approximately twice as many secretory granules, but the degree of filling as found in the resting state is not attained. The number of exocytoses is lower than that in the active state (- 71 W ) , but still considerably higher than in the resting state ( X 40; Fig. le). The finding that only few exocytotic phenomena occur in the resting state is in line with previous observations, and indicates that CDCH release occurs at a low rate during this state (Kits, 1981). Furthermore, the results confirm the conclusion drawn before (Roubos et al., 198 1 a) that during the active state CDCH release occurs at a high rate, and often by multiple exocytosis. Remarkably, in the inhibited state the CDC terminals show a considerable number of exocytoses, i.e. during electrical inactivity. This fact indicates that CDCH release still proceeds, albeit at a lower rate than in the active state. The mechanism by which the CDC release CDCH during electrical inactivity is obscure. A possibility would be that the exocytotic phenomena observed in the inhibited state had started already in the active state. However, this is not very plausible, since calculations of the speed of exocytosis in the CDC have indicated that, although the extrusion of a granule content may proceed rather slowly, it will take no more than some minutes (Roubos et al., 1981b). Therefore, it is more likely that, during the inhibited state, the axoplasmic concentration of calcium is still high enough to induce a considerable number of exocytoses. Evidently, the CDCH that is released during the inhibited state does not induce ovulation, since ovulation takes place during the active state only (Kits, 1980). In this respect it is noteworthy that the activity of the albumen gland, afemale accessory sex gland which secretes nutrients for the developing embryo. can be stimulated in vitro by
187
Fig. I .a-h: axon terminals of CDC. B. basal lamina: C. connective tissue; S. secretory granule. a : resting state. No exocytosis. x 35.000. b : active state. Numerous TAG(>-positive. electron-dense granule contents are released by exocytosis (arrows). x 20,000. c : active state. Intercellular release by exocytosis (arrow). G, glial cell process. X 65.000. d : active state. Multiple exocytosis (asterisk). X 65.000. e : inhibited state. Exocytosis (arrows). X 35,000. f : resting state. Some clear vesicles (CV) and vacuoles ( V ) . C , glial cell. X40.000. g : inhibited 5tate. Possible formation of vacuoles (arro~c).X 50.000. h : inhibitcd state. Possible formation of clear vesicle (arrow). X 75,000. a-e. TAGO-fixation: f-h. routine fixation.
188 homogenates of the neurohaemal area of the CDC (Wijdenes, 1981). Thus, the CDCH released during the inhibited state may stimulate the activity of the albumen gland. This idea is in line with the observation that the activity of the albumen gland in vivo shows a peak some hours after ovulation (M. de Jong-Brink, personal communication). MEMBRANE RESORPTION In view of the high exocytotic activity in the active state, which continues at a lower level in the inhibited state, it is clear that the CDC terminals have to resorb many parts of the axolemma in order to keep this in normal shape. The following observations deal with the mechanism of this resorption. Resting stute. The terminals contain two types of electron-lucent vesicle, viz. “clear vesicles” (CV ; diameter approximately 50 nm), and “vacuoles” (diameter 70-120 nm) (Fig. If). Both types occur in low numbers. In a few terminals a ‘‘whirl’’ consisting of concentrically arranged, double membranes was found (cf. Wendelaar Bonga, 197I ) . Active stute. Compared to the resting state the numbers of CV and vacuoles are unchanged. However, the number of whirls is clearly higher (+ 70 %). In some cases the membranes of a whirl seem to be continuous with the axoleinma (Fig. 2a). Inhibited stute. Compared to the active state the numbers of CV and vacuoles are much higher ( x 3 and x 2, respectively). Indications of the formation of CV (micropinocytosis) and of vacuoles (macropinocytosis) were obtained occasionally (Fig. I g, h). The number of whirls is also considerably higher ( X 3). Some whirls are surrounded by CV, vacuoles and other vesicular structures (Fig. 2b). Furthermore, doughnut-shaped vesicles (DSV ; diameter approximately 100 nm; Fig. 2d) and multivesicular bodies (MVB; Fig. 2c) are encountered. Of particular interest is the finding of many clear vesicle-like structures (CVL) lying together at the outside of the terminal, in invaginations of the axolemma (cf. Fig. 3c). Finally some axons seem to release membranous, whirl-like structures (WS; Fig. 3c). The observations suggest that CV and vacuoles are formed by resorption of parts of the axolemma (cf. Wendelaar Bonga, 1971 ; Roubos, 1975; Normann, 1976; Morris and Nordmann, 1980; Roubos et al., 1981b). Since CV and vacuoles are most numerous in the inhibited state, it would seem that membrane resorption occurs mainly in this period. In other words, membrane resorption would be delayed with respect to maximum exocytotic activity (active state). Such a delay is not uncommon in secretory cells (e.g. Gemmel and Stacy, 1979), but it is inconsistent with the observation that the size of the CDC terminals is smaller after the active state (see experiment on exocytosis) : an increase would be expected if high exocytotic activity is not followed immediately by a strong resorption of membrane. Possibly, not only CV and vacuoles but also whirls play a role in membrane resorption: the observed associations of the whirl membranes with the axolemma suggest that whirls are formed by strong invagination of the axolemma. Since whirls increase in number during the active state, it may be that they represent “bulk resorptions” of the axolemma during high (multiple) exocytotic activity. CV and vacuoles may be components of a more subtle mechanism of membrane resorption mainly operating during the inhibited state, when exocytosis occurs less intensely. As to the fate of the “resorption structures” (CV, vacuoles, whirls) the present study permits some speculations. Some of the whirls may be partially broken down by lysosomal action and subsequently released from the terminals as whirl-like structures (“residual bodies”). However, the number of these structures seems too low to account for the complete removal of all whirls. Therefore, possibly, some of the whirls are transformed into vesicular structures
189
Fig. 2.a-h: axon terminals of CDC. a: active state. Whirl (W). closely apposed to axolemma (arrow). x 35,000. b: inhihitcd state. Whirls (W), surrounded by vesicular structures (VS). X 30,000. c : inhibited state. Multivesicular body. S . secretory granules. x 75,000. d : inhibited state. Doughnut-shaped vesicles (DSV). X 80.000. e : inhibited state. Possible formation of DSV by invaginating vacuoles (arrows). X 100.000. f : resting state. Mitochondrion (M) without calcium deposits. X 70,000. g: activc state. Mitochondria (M) with numerous calcium deposits (arrows). x 70.000. h: inhibited state. Some calcium deposits in mitochondria ( M ) . X 70,000. a-e, routine fixation; f-h, pyroantimonate method.
190
-_ Fig. 3.a-e: axon terminals of CDC. a : inhibited state. Calcium (arrows) in clear vesicles. X60,OOO. b : calcium (arrows) in doughnut-shaped vesiclcs (DSV). X 80,000. c : inhibited statc. Clear vesicle-like structures containing calcium (arrows) and whirl-like structurc (WS) outside terminal (T). X 80,000. d : active state. Numerous electrondense reaction products of adenylate cyclasc activity (arrows). X 20,000. e : inhibited statc. Low adenylatc cyclasc activity. X 15,000. a-c. pyroantimonate method; d and e, adenylate cyclase method.
191
(including CV and vacuoles) that were seen to surround them in some cases. The finding of extracellular CVL can be interpreted as misdirected pinocytosis (cf. Douglas et al., 1971). On the other hand, vacuoles might transform by invagination into DSV (Fig. 2e), which. in turn, might fuse to form MVB. Next, these MVB could fuse with the axolemma and release their contents as CVL into the extracellular space. At present experiments are in progress to test these and other hypothetical mechanisms of resorption and disposal of membranes of the CDC terminals (Fig. 4). CALCIUM DYNAMICS Calcium plays a crucial role in many secretory processes. It is involved in, for instance, initiation of exocytosis, control of stability and permeability of the axolemma, and activation of enzymes (e.g. Normann, 1976). The axoplasmic concentration of calcium will be strictly regulated, since it is much lower ( 10p7-10-s M) than the extracellular concentration ( 1 0-j M). This control is particularly needed during electrical activity (active state), when calcium enters the axoplasm, and afterwards, when superfluous calcium has to be removed again from the axoplasm. The importance of calcium for electrical activity and for exocytosis in the CDC has been recently demonstrated (Kits, 198 1 ; Roubos et al., 198 1b). The first results are given of a study of the localization and the transport of calcium in the CDC terminals. Using the
A
E Fig. 3 . Diagram of CDC axon terminal (A). Possible mechanisms of resorption (thick arrows) and turnover (thin arrows) of parts of axolemma. and of calcium dynaniics (dotted arrows; asterisks indicate calcium contents). Inner circle: resorption structures (W. whirl: V , vacuole; CV, clear vesicles); Cal, calmodulin; CVL, clear vesicle-like structures ; DSV, doughnut-shaped vesicles ; E, exocytosis of secretory granule ; S , secretory granule contents ; M , mitochondrion; MVB, multivesicular body; WS, whirl-like structure.
192 pyroantimonate technique (Weakly, 1979) calcium could be demonstrated at the ultrastructural level as highly electron-dense deposits. Resting state. In this state only few, small deposits of calcium are occasionally found in the mitochondria. Most mitochondria show no deposits at all (Fig. 20. Positive reactions are found in some CV located near the axolemma (Fig. 3a). Active state. All mitochondria contain much calcium, located in numerous, large deposits (Fig. 2g). Calcium also occurs in some CV. Inhibited state. The number of calcium containing mitochondria is approximately 50 % lower than in the active state. The deposits are moderate in number and size (Fig. 2h). Furthermore, calcium is present in DSV and in CVL located outside the terminals (Fig. 3b, c). The observations indicate that mitochondria take up calcium during the active state. Calcium uptake by mitochondria in neurosecretory terminals has been described previously in insects (e.g. Normann and Hall, 1978). The present study strongly suggests that during the inhibited state calcium is released again from the mitochondria. Futhermore, calcium appears to be present in CV located near the axolemma. Although (some?) CV seem to be involved in membrane resorption (see above), it is not unlikely that they also take up superfluous calcium from the axoplasm (cf. Shaw and Morris, 1980) and subsequently release it from the terminal by exocytosis (Fig. 4). In the inhibited state calcium occurs in DSV, MVB and CVL. This observation is in line with the hypothesis (see above) that a vacuole can transform via a DSV and a MVB into a CVL, if it is assumed that calcium is taken up from the axoplasm into a DSV during the process of invagination of a vacuole (Figs. 2e, 4). This would be another mechanism by which superfluous calcium is removed from the terminals during the inhibited state. Evidently, the proposed mechanisms are particularly suited for the removal of bound calcium (e.g. to calmodulin?); most likely, free calcium is removed from the terminal by “conventional” membrane pumps (Fig. 4). cAMP It has been suggested that CAMPis involved in the control of calcium-dependent release of secretory materials (e.g. Rasmussen, 1970). Adenylate cyclase activity (indicating the production of CAMP) has been demonstrated cytochemically on the axolemma of neurosecretory axons in the neurointermediate lobe of the rat pituitary (Santolaya and Lederis, 1980), where it may play a role in the modulation of the release of neurohypophyseal hormones (Mathison and Lederis, 1978). In the marine opistobrach mollusc Aplysia californica cAMP seems to trigger the release of the ovulation-stimulating hormone of the neuroendocrine bag cells (Kaczmarek et al., 1978). Here the results are given of an ultracytochemical study of the presence of adenylate cyclase in the CDC terminals, using the method of Reik et al. (1970). Preliminary results were reported previously (Roubos et al., 1981a). Resting stute. Some adenylate cyclase activity occurs on the external surface of the axolemma of a small number of teiminals. Active state. Strong adenylate cyclase activity is present on the external surface of nearly all terminals, at sites where the terminals contact the basal lamina or face other CDC terminals or glial cells (Fig. 3d). Inhibited state. The situation is very similar to that in the resting state. Only few CDC terminals show adenylate cyclase activity (Fig. 3e). Apparently, adenylate cyclase activity is low in the resting and in the inhibited states and high in the active state. This probably means that in the active state there is also a high
193 intracellular concentration of CAMP. Thcrefore, it may well be that CAMP is involved in the control of one or more secretory-linked processes, such as exocytosis, membrane resorption and calcium dynamics. SUMMARY The neuroendocrine Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater snail Lymnaea stagnalis produce a peptidergic ovulation hormone. They are good models for the study of the mechanisms that control secretory processes in neuronal terminals. The cells show 3 states of electrical activity, viz. the resting, active, and inhibited states. CDCH release occurs by exocytosis and is clearly related to electrical activity (active state), although some release takes place during the inhibited state, when the CDC are electrically inactive. Presumably, membrane resorption after exocytosis takes place during the active and the inhibited states. Mechanisms for this resorption process are proposed. Calcium is necessary for exocytosis. Mitochondria and various vesicular structures appear to contain calcium and seem to be involved in the control of the dynamics of intracellular calcium. Mechanisms are suggested by which calcium is transported intracellularly and is removed from the terminals after secretory activity. Finally, indications have been obtained that CAMP plays a role in one or more secretory-linked processes in the CDC. ACKNOWLEDGEMENTS The authors wish to thank Prof. Dr. H.H. Boer for his stimulating interest and comments on the manuscript, Prof. Dr. J . Lever for critically reading the manuscript, and Mr. Th. van der Woude and Mr. A.N. de Keijzer for participation in some of the experiments. REFERENCES Douglas, W.W., Nagasaua, J . and Schulz, R . (1971) Electron microscopic studies on the mechanism of secretion of posterior pituitary hormones and significance of mici-civesicles(“synaptic vesiclcs”) : evidence of secretion by exocytosis and formation of microvesicles as a by-product of this process. M e m . Soc. Endocrinol.. 19: 353-378. Gemmcl, R.T. and Stacy, B.D. (1979) Granule secretion by the luteal cell of the sheep: the fate of the granule membrane. Cell Tissue R P S . . 197: 413419. Kaczmarek, L.K., Jennings, K. and Slrumwasser, F. (1978) Neurotransmitter modulation, phosphodiesterase inhibitor effects, CAMP correlates of afterdischarge in peptidergic neurites. Proc. nor. Acnd. Sci. (U.S.A.), 75 : 5 2 0 c S 2 0 5 . Kits, K.S. ( 1980) States of excitability in ovulation hormone producing neuroendocrine cells of Lymntrrcr srczgndis (Gastropoda) and their relation to the egg-laying cycle. J . Neurohiol., I I : 397410. Kits, K . S . (1981j Electrical activity and hormonal output of ovulation hormone producing neuroendocrine cells in Lynnnerr sragrlrrlis (Gastropoda). In Advnric. P h ~ t i o lSci.. . Vol. 23. Naurohiology offnverrebrntes, J. Salanki (Ed.), AkadCmiai Kiadb, Budapest. pp. 35-54, Mathison, R.D. and Lederis, K. ( 1978) Modification of CAMPand phosphodiesterase inhibitors of potassium-stimulated vasopressin release from the isolated neural lobe and the hypothalamo-neurohypophysial system in vitro. In Current Studies ofHypothdamic Func,/iori. Vol. 1 , K . Lederis and W.R. Veale (Eds.), Karger, Basle pp. 88-97. Morris, J.F. and Nordmann, J . J . (1980) Membrane recapture after hormone release from nerve endings in the neural lobe of the rat pituitary gland. Neuroscicvwe. 5 : 639-649. , 1-77. Norniann, T.C. (1976) Neurosecretion by exocytosis. Int. R m . C ~ t o l .46:
194 Normann, T.C. and Hall. T . A . ( 1 078) Calcium and sulphur in neurasecretory granules and calcium in mitochondria as determined by electron microscope X-ray microanalysis. Cell Tiss. Res., 186 : 453-463. Rasmussen, H. ( 1 970) Cell communication, calciuni ion and cyclic adenosine monophosphate. Science, 170: 40UI2. Reik, L . , Petzold, G.L., Higgins. J.A., Greengard, P.. Barnett, R.J. (1970) Hormone-sensitive adenyl cyclase: cytochemical localization in rat liver. Science. 168 : 382-384. Roubos, E. W. (197.5) Regulation of neurosecretory activity in the freshwater pulmonate snail Lyninaeu stugriulis (L.) with particular reference to the role of the eyes. Cell Tiss. Res., 160: 291-314. Roubos, E.W. andVan der Wal-Divcndal, R.M. ( 1980) Ultrastructural analysis of peptide release by exocytosis. Cell Tiss. Res.. 207: 261-275. Roubos, E.W. Geracrts, W.P.M., Boerrigter, G.H. and Kampen, G.P.J. van (1980) Control of the activities of the neurosecretory light green and caudo-dorsal cells and of the endocrine dorsal bodies by the lateral lobes in the freshwater snail Lytnnum sfngriulis (L.). Get?. Conip. Errdoc,ririo/., 40 : 446-454. Roubos, E.W., Keijzer, A.N. de and Buma, P. (198 la) Adenylatc cyclasc activity in axon terminals of ovulation-hormonc producing neuroendocrine cells of Lynitiueu srtigriuli.v (L.). Cell Tiss. Res., 220: 665-668. Roubos, E.W., Schmidt, E.D. and Moorer-van Delft, C.M. (1981b) Ultrastructural dynamics of exocytosis in the ovulation neurohormone producing caudo-dorsal cclls of the freshwater snail Lymnaeci stu,ynali.s (L. ). Cell T i n . Res.. 21.5: 63-73. Roubos, E. W . , Boer, H.H. and Schot, L.P.C. (1982) Peptidergic neurones and the control ofneuroendocrine activity in the freshwater snail Lyrnfiueu stagridis (L.). In Nrurosecwtion -Molecules, Cells, Sysfenzs, D.S. Farner and K . Lederis, (Eds.), Plenum Press, New York, pp. II%l27. Santolaya, R. and Lederis, K. (1980) Localization of adenylate cyclase in the neurointermediate lobe of the rat pituitary. Cell Tiss. Res.. 207: 387-394. Shaw, F.D. and Moms, J.F. (1980) Calcium localization in the rat neurohypophysis. Nature (Lond.). 287: 56-58. Weakly, B.S. (1979) A variant of the pyroantimonate technique suitable for localization of calcium in ovarian tissue. J . Histuchen. Cvtochenr . , 27 : 10 17- 1028. Wendelaar Bonga, S.E. (107 I)Forination, storage. and release of neurosecretory material studied by quantitative electron microscopy in the freshwater snail Lymnueu stugidis ( L . ) . Z . Zel(for.sch., 113 : 49Cb.517. Wijdenes, J . ( 198 I ) A Compurutive Srudy on the Neuro-Endocrine Control of Gruwrh and Reproduction in Pulrnonute Snails und Slugs. Thesis, Free University, Amsterdam.