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Role of cAMP in electrical and secretory activity of the neuroendocrine caudo-dorsal cells of Lymnaea stagnalis
Brain Research, 380 (1986) 26-33
26
Elsevier BRE 11900
Role of cAMP in Electrical and Secretory Activity of the Neuroendocrine Caudo-Dorsal Cells of Lymnaea stagnalis P. BUMA*, E.W. ROUBOS and K. BRUNEKREEF**
Department of Biology, Vrije Universiteit,Amsterdam (The Netherlands) (Accepted December 24th, 1985)
Key words: cyclic adenosine 3' ,5'-monophosphate (cAMP) - - neuronal electrical and secretory activity - exocytosis - - caudo-dorsal cell - - Lymnaea stagnalis
The peptidergic neuroendocrine caudo-dorsal cells (CDC) in the cerebral ganglia of the freshwater snail Lymnaea stagnalis L. produce an ovulation-stimulating neurohormone (CDCH). Release occurs by exocytosis in a calcium-dependent way from axon terminals in the periphery of the intercerebral commissure, particularly during a period of electrical activity (the 'discharge'). An important factor in electrical and, hence, secretory activity of the CDC appears to be cyclic adenosine 3' ,5'-monophosphate (cAMP). Incubation of cerebral ganglia in snail Ringer with the cAMP-analogue 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) or with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) leads to activation of the CDC: electrophysiological, quantitative electron microscopic and bioassay studies show that incubation results in the onset of intense electrical activity, a marked reduction in the number of secretory granules in the axon terminals, an enormous increase in the number of exocytosis phenomena and a strong stimulation of CDCHrelease. It is assumed that treatment with IBMX or with cpt-cAMP mimics a rise in the cytoplasmic level of cAMP when the CDC become activated by a physiological stimulus. This rise most likely effectuates a permeability change of the axolemma for ions involved in the discharge. As a consequence of the depolarization of the axolemma during the discharge, calcium ions would enter the axon terminal and induce exocytotic release of CDCH.
INTRODUCTION Particular n e u r o n e s may undergo long-lasting activity changes u p o n short-lasting stimuli (e.g. ref. 17). This p h e n o m e n o n receives much attention in neurobiology because it seems to underlie important neurally controlled processes such as learning, memory and behaviour (e.g. ref. 18). In this field of study molluscan n e u r o n e s are favourite study objects, as they are large and readily identifiable 12. A m o n g these n e u r o n e s the n e u r o e n d o c r i n e caudodorsal cells (CDC) of the freshwater snail, Lymnaea stagnalis, serve as a model for the study of neural and n e u r o e n d o c r i n e control of egg-laying and egg-laying behaviour 11'22'3°. The C D C somata are located in two clusters, one in the left (ca. 25 cells) and one in the
right cerebral ganglion (ca. 75 cells). Their axons form a n e u r o h a e m a l area in the periphery of the intercerebral commissure. The C D C exhibit 3 states of electrical activity 13. Generally, they are electrically inactive ('resting state', lasting 1 - 2 days). D u r i n g this state the axon terminals are filled with secretory granules and release of granule contents, visible at the ultrastructural level as exocytosis p h e n o m e n a , occurs at a very low level ('basal release'). U p o n appropriate physiological or electrical stimulation, all ceils in both clusters depolarize and start a ca. 45-min lasting period of spiking activity, the discharge ('active state'). During this state at least 9 different peptides are released, which are involved in the control of stereotyped egglaying behaviour 9'1°. One of these peptides is C D C H
* Present address: Department of Anatomy and Embryology, Medical Faculty, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. ** Present address: Organon International B.V., Kloosterstraat 6, 5340 BH Oss, The Netherlands. Correspondence: E.W. Roubos, Department of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam-Buitenveldert, The Netherlands. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
27 (MW 4300 kDa), which chemical structure has recently been elucidated 6. It is released into the haemolymph s'9'14'25 and induces ovulation 7. Ultrastructural studies have shown that the high secretory activity in the active state is concomitant with a very high rate of exocytosis in the axon terminals 22. Exocytosis depends on the entry of calcium ions into the axon terminals during the discharge 2'3'27. Excess calcium is taken up by mitochondria 2. Large, concentrically arranged, membranous structures ('whorls') in the terminals probably represent resorbed granule membranes after exocytosis 2'23'27. After the active state the CDC enter a period of electrical inactivity and inexcitability ('inhibited state', lasts ca. 5 h). During this state some secretion takes place - - this possibly reflects control of the last part of the egg-laying behaviour and of accessory sex gland activity - but secretory activity is much lower than during the active state 22. Little is known about the environmental and internal factors that control the transitions between the 3 CDC states. The physiologically most important transition, viz. that between the resting and the active state, can be effectuated in vivo by placing snails that have previously been kept in badly aerated, polluted water into fresh, aerated water ('freshwater stimulus') 19. The cellular mechanism by which the discharge is induced is unknown. The discharge is driven by an endogenous pacemaker that consists of a voltage-dependent Na+/CaZ+-channel and a Ca 2+activated K+-channe115. Calcium ions are not necessary for the induction of the discharge: electrical stimulation of the CDC in calcium-free Ringer evokes a characteristic long-lasting discharge (though the action potentials lack a calcium component and show an increased frequency15'16). Some years ago the presence of adenylate cyclase at the axolemma of the CDC axon terminals was shown by ultrastructural enzyme cytochemistry. The enzyme appeared to be inactive during the resting and the inhibited state, but highly active during the active state, suggesting that cAMP is involved in the control of high electrical and secretory activity during the discharge 23'26. In the present investigation this hypothesis has been tested. For that purpose the effects of the cAMP-analogue 8-(4-chlorophenylthio)cAMP (cpt-cAMP) and of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) on
CDC activity (electrical discharge, exocytosis activity and C D C H release) have been examined. In order to study whether calcium ions induce exocytosis directly or via cAMP, in one experiment a possible direct stimulatory effect of cAMP on exocytosis was investigated using calcium-free Ringer. MATERIALS AND METHODS Specimens of Lymnaea stagnalis L. with a shell height of ca. 33 mm, bred under standard laboratory conditions (photoperiod 04.00-20.00 h, fed on lettuce, water temperature 20 + 1 °C), were used. All experiments were started with CDC in the resting state. Such CDC were obtained from snails that had been kept in stagnant badly aerated water with lettuce ad libitum, for 3 days. After decapitation, tissues (cerebral ganglia with the intercerebral commissure), were collected in snail Ringer's solution consisting of (in mM): NaC130, KC1 1.5, MgC12 2, CaCI 2 4, NaH2PO 4 0.25 and N a H C O 3 18, pH 7.8. In a number of cases (see below) tissues were incubated in Ringer's solution containing 8-(4-chlorophenylthio)cAMP (cpt-cAMP) or 3-isobutyl-l-methylxanthine (IBMX). These agents were always used in a concentration of 1 mM. Incubations were performed at 20 + 1 °C. The following experiments were carried out.
Effects of cpt-cAMP and IBMX on electrical activity The CNS was pinned down on a Xantoprene layer (Bayer) in a perspex recording chamber and pre-incubated in Ringer's solution for 30 min. Subsequently, the solution was replaced by another Ringer's solution containing cpt-cAMP or IBMX. The electrical activity of the CDC system was studied by impalement with a microelectrode of one CDC soma (all CDC are electrotonically coupled). In this way 20 CNS were investigated, 10 for the effect of cptcAMP, 10 for that of IBMX. CDC of another 10 CNS served as controls: they were electrically stimulated to induce the active state, by repetitive intracellular stimulation with depolarizing pulses at 2/s. In all studies signals were fed into conventional electrophysiological apparatus and recorded on a Brush 2200 pen recorder. (For details of the electrophysiological procedures, see ref. 13.) Per group the patterns of (bursting) discharges of 6 CNS were analyzed quantitatively.
28
Effect of cpt-cAMP and IBMX on release of ovulation inducing material Three groups of 10 (pairs of) cerebral ganglia were pre-incubated in Ringer for 30 min. Then they were incubated for 1 h in Ringer (controls), cpt-cAMP, or IBMX (20 pairs per 500 ml). After incubation the 3 bathing media were assayed for ovulation-inducing activity with an in vivo bioassay4; one ovulation-inducing_unit (OIU) is defined as the threshold dose of ovulation hormone needed to induce ovulation in a recipient snail. (Note: control Ringer containing 1 mM cpt-cAMP or IBMX does not induce ovulation.) In addition, 10 intercerebral commissures were homogenized directly after dissection and assayed for ovulation-inducing activity.
or Zeiss EM 10A electron microscope. For morphometry counts of highly electron-dense (tannic acid-positive) exocytosis phenomena were made in a cross-section halfway along the intercerebral commissure, in all CDC axon terminals that were in direct contact with the basal lamina. Counts were made by direct electron microscopic examination, at magnifications ranging between x30,000 and x80,000. The data were analysed with a one-way analysis of variance (a = 5%) 1, followed by the multiple range test of Duncan 29. This analysis was preceded by tests for the homogeneity of variance (Bart-
Q
Ultrastructural effects of cpt-cAMP and IBMX Three groups of 6 (pairs of) cerebral ganglia were pre-incubated in Ringer for 30 rain and subsequently incubated in Ringer for 1 h (controls), cptcAMP, or IBMX. Then the ganglia were processed for electron microscopy (see below).
Ultrastructural effects of cpt-cAMP and IBMX in calcium-free Ringer Three groups of 6 (pairs of) cerebral ganglia were dissected and treated as follows. Group 1: incubation in Ringer for 2 h (controls). Group 2: pre-incubation in Ringer for 90 min, and subsequently in Ringer with cpt-cAMP for 30 rain. Group 3: pre-incubation in Ringer without CaC12 for 90 min, and subsequently in this Ringer with cpt-cAMP for 30 rain. After the incubations tissues were processed for electron microscopy (see below). An additional group of 3 cerebral ganglia was incubated as group 3 and electrical characteristics of the CDC were determined electrophysiologically as in the first experiment. The electron microscopic T A G O - m e t h o d (tannic acid-glutaraldehyde-osmium tetroxide method) for the ultrastructural demonstration of exocytosis was performed according to Roubos and van der WalDivenda124 in a slightly modified way: fixation in a 0.05 M Na-cacodylate-buffered (pH 7.2) solution of 1% glutaraldehyde and 1% tannic acid (BDH), for 18 h at 4 °C; postfixation in 1% OsO 4 in the buffer, for 2 h at 4 °C. Tissues were embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with a Philips EM 300
(9
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4sec
I
'
2Om~j/ 4sec
j
20mV J2omV 4sec 4sec Fig. 1. Effect of cpt-cAMP and IBMX on CDC electrical activity. a: discharge in CDC evoked by intracellular stimulation of one CDC. After cessation of stimulation (not shown) the membrane potential depolarizes, followed by a few initial spikes, intense bursts and regular beating activity, b, c: beating and bursting firing patterns after intracellular stimulation of a discharge, d, e: bursting firing patterns after induction of a discharge by cpt-cAMP and IBMX, respectively.
29
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t
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,. ,
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.it
i. Fig. 2. TAGO-fixation. CDC axon terminals in periphery of intercerebral commissure in resting (a, b) and active state (c-e). a: axon terminals (A) filled with many secretory granules (S) (× t5,000), b: arrow indicates electron-dense (tannic acid-positive) exocytosis phenomenon (x65,000). c: note many exocytoses in axon terminal of active CDC (x40,000). d: multiple exocytosis (arrows) (x40,000). e: whorl (W) in intimate contact with the axolemma (arrow) (x30,000).
30 TABLE I
burst hyperpolarizations (mean duration: 3.6 s; Fig.
Effects of treatment with 1 mM cpt-cAMP and IBMX on characteristics of CDC discharge
Discharges of controls were evoked by electrical stimulation. Values are means _+standard deviations of 6 preparations. For none of the parameters does a statistically significant difference exist between any of the groups. Parameter
Controls
cpt-cAMP
IBMX
Duration of discharge in s 26.2 + 16.1 36.1 + 21.5 32.2 + 14.4 Number of spikes per burst 7.4 + 1.3 7.9 _ 3.7 6.7 + 4.0 Duration of interburst hyperpolarization 3.6 + 1.6 3.2 + 0.7 3.8 + 0.8
lc). Treatment with cpt-cAMP results into a rather rapid depolarization (ca. 0.5 mV/s). Durations and patterns of (bursting) discharges are not clearly different from those found after electrical stimulation (Table I), but regular beating activity never occurs (Fig. ld). Treatment with I B M X leads to a slow depolarization (ca. 0.01 mV/s). After ca. 5 - 2 0 min the discharge starts. Discharge durations and patterns are not essentially different from those in C D C treated with cpt-cAMP (Table I; Fig. le). Release o f ovulation-inducing material and ultrastructure
lett'stest; cf. ref. 1) and for the joint assessment of normality 28. RESULTS The results presented with respect to incubations with cpt-cAMP also apply to incubations with I B M X , unless stated otherwise. Electrical activity
During pre-incubation all preparations show electrical silence with a resting potential of ca. - 7 0 inV. U p o n repetitive electrical stimulation C D C rapidly (ca. 0.3 mV/s) depolarize. After cessation of stimulation a long-lasting discharge occurs (5-45 min; Table I). A discharge consists of a few initial intense bursts of action potentials followed by either a regular beating firing pattern (found in 3 CNS; Fig. la, b) or bursting pattern (mean n u m b e r of spikes: 7.4 per burst) intermitted by more or less pronounced interTABLE II Effects of treatment with 1 mM cpt-cAMP and IBMX on exocytosis activity of CDC
Controls are unstimulated CDC in the electrical resting state. Exocytosis activity is expressed as number of tannic acid-positive exocytosis phenomena per cross-section of the cerebral commissure. Values are means _+ standard deviations of 6 preparations. Per parameter each group differs from the other groups (statistically significant, P < 0.01). Parameter
Controls
Number of exocytosis phenomena 27 _+8
cpt-cAMP
IBMX
623 + 397
1030 _+605
After incubation of resting C D C in control Ringer no ovulation-inducing activity in the bathing medium could be demonstrated. Bioassays of homogenates of the intercerebra! commissure show that the axon terminals of such C D C contain ca. 130 O I U . A t the ultrastructural level the terminals appear large (ca. 2 ~tm in diametei') and filled with numerous secretory granules (Fig. 2a). D u e to the application of the T A G O - m e t h o d exocytotic release of the contents of secretory granules can readily be observed, because the contents are stained selectively and are highly electron-dense (tannic acid-positive; Fig. 2b). Generally, exocytozed contents of secretory granules are present in omega-shaped indentations of the axolemma (fused granule membranes). At some places the exocytosis p h e n o m e n a are visible as 'caps', i.e. the opening of the granule with the extracellular space is not present in the plane of the section 27. In resting C D C , however, exocytosis p h e n o m e n a are rare (mean number: 27 per cross-sectioned neurohaemal area; Table II). Simultaneous release of contents of more than one granule ('multiple exocytosis') was never encountered. Also whorls, indicating resorption of the axolemma after exocytosis, appear to be absent. U p o n treatment with cpt-cAMP, the C D C of one pair of cerebral ganglia release ca. 60 O I U into the bathing medium. The axon terminals of these C D C are rather small (ca. 0.5 # m ) and only partly filled with secretory granules (Fig. 2c). Exocytosis phen o m e n a are very numerous (ca. 25x as many as in unstimulated CDC). Exocytosis particularly takes place in a multiple fashion; at some places up to 20
31 TABLE III Effect of incubation in Ringer, Ringer + 1 mM cpt-cAMP and Ca2+-free Ringer + 1 rnM cpt-cAMP, on exocytosis activity of CDC
This activity has been expressed as number of exocytosis phenomena per cross-section of the cerebral commissure. Values are means _+standard deviations of 6 preparations. No statistically significant difference exists between any of the groups. Parameter
Ringer
Number of exocytosis phenomena 10.0 + 4.6
cpt-cAMP in Ringer
cpt-cAMP in Ca2+-free Ringer
433.3 + 88.5 37.8 + 12.1
granules appear to be fused with each other while releasing their contents (Fig. 2d). The axolemma of the terminals shows deep foldings and many terminals contain a whorl. Some whorls show a continuity with the axolemma, indicating their formation by membrane invagination (Fig. 2e). Whorls are considered to represent a process of membrane resorption during and after high exocytosis activity 2. Electrical activity and ultrastructure in calcium-free Ringer's solution In calcium-free Ringer's solution, resting CDC show the same electrical silence and similar membrane potential as found in resting CDC in normal Ringer. Upon application of cpt-cAMP the cells exhibit the basic characteristics of the discharge, viz. as to duration and pattern (action potentials are increased in frequency and lack the calcium component; see also ref. 16). However, whereas the'discharge in normal Ringer is concomitant with an enormous increase in exocytosis activity (×43; Table III), in calcium-free Ringer only a small increase occurs (x3.8). Moreover, CDC in calcium-free Ringer treated with cpt-cAMP do not reveal any of the other ultrastructural changes characteristic of CDC stimulated in normal Ringer (partial loss of secretory granules, presence of multiple exocytosis phenomena and of whorls).
DISCUSSION The electrical, morphological and secretory characteristics of the active state of the CDC have been described extensively 13'22'25. The present study shows
that treatment with cpt-cAMP or with IBMX brings resting CDC into a state of high activity of which the characteristics are basically the same: a long-lasting spiking activity (discharge), a high frequency of exocytosis (particularly in the multiple form), depletion of secretory granules from the axon terminals, occurrence of whorls and strong release of ovulation-inducing material. Therefore, it can be concluded that treatment with cpt-cAMP as well as with IBMX brings the C D C into the active state. This supports the view that under physiological conditions the start of the active state is related to an intracellular rise of cAMP, for the following reasons. (1) It is well-known that cAMP-dependent processes can be stimulated with cAMP-analogues. The rapid and effective action of cpt-cAMP is due to the strongly hydrophobic character of this compound which promotes its rapid movement across the axolemma El. Moreover, this analogue of cAMP is not broken down by phosphodiesterase El. (2) In view of the well-established specific, inhibitory effect of IBMX on phosphodiesterase (which breaks cAMP down), the induction of the active state by IBMX is apparently based on an intracellular accumulation of cAMP. The period of IBMX treatment passing before the start of the active state is relatively long. This indicates that the rate of cAMP production in the resting state is rather low. This is in agreement with the observation that adenylate cyclase activity in the axon terminals is lower in the resting than in the active state 26. (3) Previous cytochemical studies of adenylate cyclase activity in the CDC 23'24 have indicated that cAMP levels are high in CDC in the active state. The electrical discharge of the CDC depends on a voltage-dependent Na+/Ca2+-channel and a Ca 2+activated K+-channe115. cAMP-stimulated phosphorylation of membrane proteins seems to be an important step in the control of the activity of ion channels in the axolemma of various neurons 5. Consequently, cAMP possibly promotes the electrical discharge by inducing activity changes of Na+/Ca 2÷ and/or K + ion channels. After a few initial action potentials the electrical discharge reveals a constant pattern of firing activity 13. The character of this pattern, however, varies among preparations, but also depends to some degree on the type of discharge-inducing stimulus. Thus, after freshwater stimulation and after intracel-
32 lular electrical stimulation, generally a regular beating p a t t e r n is found a3, but some p r e p a r a t i o n s m a y reveal a bursting p a t t e r n (present study). A bursting p a t t e r n is f u r t h e r m o r e generally found after stimulation with high potassium or with b a r i u m (substituting calcium) 15. W h e t h e r these different patterns represent different physiological states of the C D C and w h e t h e r they r e p r e s e n t different rates of s e c r e t o r y activity is not known. Meanwhile, the possibility that c A M P plays a role in the d e t e r m i n a t i o n of the discharge p a t t e r n should be envisaged: whereas the present results indicate that 10 -3 M c p t - c A M P induces a bursting pattern, p r e l i m i n a r y studies show that lower concentrations of this analogue (e.g. 10 -4 M) result into a beating p a t t e r n (P.J. M o e d and A . ter M a a t , p e r s o n a l communication). Extracellular calcium ions are crucial for the induction of secretion during the active state. A p p a r ently, they e n t e r the axon terminals as a result of depolarization of the a x o l e m m a and induce exocytosis from an axoplasmic site 22'27. The p r e s e n t study confirms this role of calcium: whereas t r e a t m e n t of C D C with c p t - c A M P or with I B M X in calcium-free Ringer's solution results in an electrical discharge (concomitant with a x o l e m m a depolarization), no induction of high exocytosis activity (as takes place in normal Ringer) occurs. This indicates that c A M P does not have a direct stimulatory effect on the process of
REFERENCES 1 Bliss, C.J., Stat&tics in Biology, Vol. L McGraw-Hill, New York, 1967. 2 Buma, P. and Roubos, E.W., Calcium dynamics, exocytosis. and membrane turnover in the ovulation hormone-releasing caudo-dorsal cells of Lymnaea stagnalis, Cell Tissue Res., 233 (1983) 143-159. 3 Buma, P. and Roubos, E.W., Involvement of cAMP, calcium and calmoduline in membrane dynamics of ovulationhormone releasing neurones in Lymnaea stagnalis, Cell Biol. Intern. Reports, 8(1984)277-278, 4 Dogterom, G.E., Bohlken, S. and Geraerts, W.P.M., A rapid in vivo bioassay of the ovulation hormone of Lymnaea stagnalis, Gen. Comp. Endocrinol., 50 (1983) 467-482. 5 Drummond, A.H., Benson, J.A. and Levitan, I.B., Serotonine-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5013-5017. 6 Ebberink, R.H.M., Van Loenhout, H,, Geraerts, W.P.M. and Joosse, J., Purification and amino acid sequence of the ovulation hormone of Lymnaea stagnalis, Proc. Natl. Acad. Sci. U.S.A., in press. 7 Geraerts, W.P.M. and Bohlken, S., The control of ovula-
exocytosis and, hence, that calcium does not induce exocytosis via stimulation of c A M P production. Nevertheless, a c o m b i n e d role of calcium and c A M P in the control of exocytosis can not be excluded. The only physiological trigger known to induce the active state is the freshwater stimulus, but how it acts upon the C D C is not known. Recently it was found 2° that the C D C p r o d u c e a small (1500 D) p e p t i d e (the C D C - a u t o t r a n s m i t t e r , C D C A ) that has an auto-excitatory effect, stimulating the C D C in vitro to a discharge 2°. C D C A is p r o b a b l y released during the initial part of the discharge. W e speculate that freshwater stimulation induces (some) C D C to release C D C A , which subsequently induces a d e n y l a t e cyclase in the axon terminals of all C D C to increased c A M P production. In this way the physiological stimulus is amplified by c A M P , leading to high electrical and secretory activity of all C D C .
ACKNOWLEDGEMENTS This research was m a d e possible by a grant from the F o u n d a t i o n for F u n d a m e n t a l Biological Research ( B . I . O . N . ) , which is subsidized by the Netherlands Organization for the A d v a n c e m e n t of Pure Research ( Z . W . O . ) . T h e authors wish to thank Prof. Dr. H . H . B o e r for critically reading the manuscript.
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33 Amsterdam, 1982, pp. 380-404. 12 Kandel, E.R., Neuronal plasticity and the modification of behavior. In J.M. Brookhart, V.B. Mountcastle, E.R. Kandel and S.R. Geiger (Eds.), Handbook of Physiology, Sect. I, Vol. I, Part I, Am. Physiol. Soc., Bethesda, 1977, p.
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26
Elsevier BRE 11900
Role of cAMP in Electrical and Secretory Activity of the Neuroendocrine Caudo-Dorsal Cells of Lymnaea stagnalis P. BUMA*, E.W. ROUBOS and K. BRUNEKREEF**
Department of Biology, Vrije Universiteit,Amsterdam (The Netherlands) (Accepted December 24th, 1985)
Key words: cyclic adenosine 3' ,5'-monophosphate (cAMP) - - neuronal electrical and secretory activity - exocytosis - - caudo-dorsal cell - - Lymnaea stagnalis
The peptidergic neuroendocrine caudo-dorsal cells (CDC) in the cerebral ganglia of the freshwater snail Lymnaea stagnalis L. produce an ovulation-stimulating neurohormone (CDCH). Release occurs by exocytosis in a calcium-dependent way from axon terminals in the periphery of the intercerebral commissure, particularly during a period of electrical activity (the 'discharge'). An important factor in electrical and, hence, secretory activity of the CDC appears to be cyclic adenosine 3' ,5'-monophosphate (cAMP). Incubation of cerebral ganglia in snail Ringer with the cAMP-analogue 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) or with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) leads to activation of the CDC: electrophysiological, quantitative electron microscopic and bioassay studies show that incubation results in the onset of intense electrical activity, a marked reduction in the number of secretory granules in the axon terminals, an enormous increase in the number of exocytosis phenomena and a strong stimulation of CDCHrelease. It is assumed that treatment with IBMX or with cpt-cAMP mimics a rise in the cytoplasmic level of cAMP when the CDC become activated by a physiological stimulus. This rise most likely effectuates a permeability change of the axolemma for ions involved in the discharge. As a consequence of the depolarization of the axolemma during the discharge, calcium ions would enter the axon terminal and induce exocytotic release of CDCH.
INTRODUCTION Particular n e u r o n e s may undergo long-lasting activity changes u p o n short-lasting stimuli (e.g. ref. 17). This p h e n o m e n o n receives much attention in neurobiology because it seems to underlie important neurally controlled processes such as learning, memory and behaviour (e.g. ref. 18). In this field of study molluscan n e u r o n e s are favourite study objects, as they are large and readily identifiable 12. A m o n g these n e u r o n e s the n e u r o e n d o c r i n e caudodorsal cells (CDC) of the freshwater snail, Lymnaea stagnalis, serve as a model for the study of neural and n e u r o e n d o c r i n e control of egg-laying and egg-laying behaviour 11'22'3°. The C D C somata are located in two clusters, one in the left (ca. 25 cells) and one in the
right cerebral ganglion (ca. 75 cells). Their axons form a n e u r o h a e m a l area in the periphery of the intercerebral commissure. The C D C exhibit 3 states of electrical activity 13. Generally, they are electrically inactive ('resting state', lasting 1 - 2 days). D u r i n g this state the axon terminals are filled with secretory granules and release of granule contents, visible at the ultrastructural level as exocytosis p h e n o m e n a , occurs at a very low level ('basal release'). U p o n appropriate physiological or electrical stimulation, all ceils in both clusters depolarize and start a ca. 45-min lasting period of spiking activity, the discharge ('active state'). During this state at least 9 different peptides are released, which are involved in the control of stereotyped egglaying behaviour 9'1°. One of these peptides is C D C H
* Present address: Department of Anatomy and Embryology, Medical Faculty, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. ** Present address: Organon International B.V., Kloosterstraat 6, 5340 BH Oss, The Netherlands. Correspondence: E.W. Roubos, Department of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam-Buitenveldert, The Netherlands. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
27 (MW 4300 kDa), which chemical structure has recently been elucidated 6. It is released into the haemolymph s'9'14'25 and induces ovulation 7. Ultrastructural studies have shown that the high secretory activity in the active state is concomitant with a very high rate of exocytosis in the axon terminals 22. Exocytosis depends on the entry of calcium ions into the axon terminals during the discharge 2'3'27. Excess calcium is taken up by mitochondria 2. Large, concentrically arranged, membranous structures ('whorls') in the terminals probably represent resorbed granule membranes after exocytosis 2'23'27. After the active state the CDC enter a period of electrical inactivity and inexcitability ('inhibited state', lasts ca. 5 h). During this state some secretion takes place - - this possibly reflects control of the last part of the egg-laying behaviour and of accessory sex gland activity - but secretory activity is much lower than during the active state 22. Little is known about the environmental and internal factors that control the transitions between the 3 CDC states. The physiologically most important transition, viz. that between the resting and the active state, can be effectuated in vivo by placing snails that have previously been kept in badly aerated, polluted water into fresh, aerated water ('freshwater stimulus') 19. The cellular mechanism by which the discharge is induced is unknown. The discharge is driven by an endogenous pacemaker that consists of a voltage-dependent Na+/CaZ+-channel and a Ca 2+activated K+-channe115. Calcium ions are not necessary for the induction of the discharge: electrical stimulation of the CDC in calcium-free Ringer evokes a characteristic long-lasting discharge (though the action potentials lack a calcium component and show an increased frequency15'16). Some years ago the presence of adenylate cyclase at the axolemma of the CDC axon terminals was shown by ultrastructural enzyme cytochemistry. The enzyme appeared to be inactive during the resting and the inhibited state, but highly active during the active state, suggesting that cAMP is involved in the control of high electrical and secretory activity during the discharge 23'26. In the present investigation this hypothesis has been tested. For that purpose the effects of the cAMP-analogue 8-(4-chlorophenylthio)cAMP (cpt-cAMP) and of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) on
CDC activity (electrical discharge, exocytosis activity and C D C H release) have been examined. In order to study whether calcium ions induce exocytosis directly or via cAMP, in one experiment a possible direct stimulatory effect of cAMP on exocytosis was investigated using calcium-free Ringer. MATERIALS AND METHODS Specimens of Lymnaea stagnalis L. with a shell height of ca. 33 mm, bred under standard laboratory conditions (photoperiod 04.00-20.00 h, fed on lettuce, water temperature 20 + 1 °C), were used. All experiments were started with CDC in the resting state. Such CDC were obtained from snails that had been kept in stagnant badly aerated water with lettuce ad libitum, for 3 days. After decapitation, tissues (cerebral ganglia with the intercerebral commissure), were collected in snail Ringer's solution consisting of (in mM): NaC130, KC1 1.5, MgC12 2, CaCI 2 4, NaH2PO 4 0.25 and N a H C O 3 18, pH 7.8. In a number of cases (see below) tissues were incubated in Ringer's solution containing 8-(4-chlorophenylthio)cAMP (cpt-cAMP) or 3-isobutyl-l-methylxanthine (IBMX). These agents were always used in a concentration of 1 mM. Incubations were performed at 20 + 1 °C. The following experiments were carried out.
Effects of cpt-cAMP and IBMX on electrical activity The CNS was pinned down on a Xantoprene layer (Bayer) in a perspex recording chamber and pre-incubated in Ringer's solution for 30 min. Subsequently, the solution was replaced by another Ringer's solution containing cpt-cAMP or IBMX. The electrical activity of the CDC system was studied by impalement with a microelectrode of one CDC soma (all CDC are electrotonically coupled). In this way 20 CNS were investigated, 10 for the effect of cptcAMP, 10 for that of IBMX. CDC of another 10 CNS served as controls: they were electrically stimulated to induce the active state, by repetitive intracellular stimulation with depolarizing pulses at 2/s. In all studies signals were fed into conventional electrophysiological apparatus and recorded on a Brush 2200 pen recorder. (For details of the electrophysiological procedures, see ref. 13.) Per group the patterns of (bursting) discharges of 6 CNS were analyzed quantitatively.
28
Effect of cpt-cAMP and IBMX on release of ovulation inducing material Three groups of 10 (pairs of) cerebral ganglia were pre-incubated in Ringer for 30 min. Then they were incubated for 1 h in Ringer (controls), cpt-cAMP, or IBMX (20 pairs per 500 ml). After incubation the 3 bathing media were assayed for ovulation-inducing activity with an in vivo bioassay4; one ovulation-inducing_unit (OIU) is defined as the threshold dose of ovulation hormone needed to induce ovulation in a recipient snail. (Note: control Ringer containing 1 mM cpt-cAMP or IBMX does not induce ovulation.) In addition, 10 intercerebral commissures were homogenized directly after dissection and assayed for ovulation-inducing activity.
or Zeiss EM 10A electron microscope. For morphometry counts of highly electron-dense (tannic acid-positive) exocytosis phenomena were made in a cross-section halfway along the intercerebral commissure, in all CDC axon terminals that were in direct contact with the basal lamina. Counts were made by direct electron microscopic examination, at magnifications ranging between x30,000 and x80,000. The data were analysed with a one-way analysis of variance (a = 5%) 1, followed by the multiple range test of Duncan 29. This analysis was preceded by tests for the homogeneity of variance (Bart-
Q
Ultrastructural effects of cpt-cAMP and IBMX Three groups of 6 (pairs of) cerebral ganglia were pre-incubated in Ringer for 30 rain and subsequently incubated in Ringer for 1 h (controls), cptcAMP, or IBMX. Then the ganglia were processed for electron microscopy (see below).
Ultrastructural effects of cpt-cAMP and IBMX in calcium-free Ringer Three groups of 6 (pairs of) cerebral ganglia were dissected and treated as follows. Group 1: incubation in Ringer for 2 h (controls). Group 2: pre-incubation in Ringer for 90 min, and subsequently in Ringer with cpt-cAMP for 30 rain. Group 3: pre-incubation in Ringer without CaC12 for 90 min, and subsequently in this Ringer with cpt-cAMP for 30 rain. After the incubations tissues were processed for electron microscopy (see below). An additional group of 3 cerebral ganglia was incubated as group 3 and electrical characteristics of the CDC were determined electrophysiologically as in the first experiment. The electron microscopic T A G O - m e t h o d (tannic acid-glutaraldehyde-osmium tetroxide method) for the ultrastructural demonstration of exocytosis was performed according to Roubos and van der WalDivenda124 in a slightly modified way: fixation in a 0.05 M Na-cacodylate-buffered (pH 7.2) solution of 1% glutaraldehyde and 1% tannic acid (BDH), for 18 h at 4 °C; postfixation in 1% OsO 4 in the buffer, for 2 h at 4 °C. Tissues were embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with a Philips EM 300
(9
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4sec
I
'
2Om~j/ 4sec
j
20mV J2omV 4sec 4sec Fig. 1. Effect of cpt-cAMP and IBMX on CDC electrical activity. a: discharge in CDC evoked by intracellular stimulation of one CDC. After cessation of stimulation (not shown) the membrane potential depolarizes, followed by a few initial spikes, intense bursts and regular beating activity, b, c: beating and bursting firing patterns after intracellular stimulation of a discharge, d, e: bursting firing patterns after induction of a discharge by cpt-cAMP and IBMX, respectively.
29
l
t
P b
,. ,
@
.it
i. Fig. 2. TAGO-fixation. CDC axon terminals in periphery of intercerebral commissure in resting (a, b) and active state (c-e). a: axon terminals (A) filled with many secretory granules (S) (× t5,000), b: arrow indicates electron-dense (tannic acid-positive) exocytosis phenomenon (x65,000). c: note many exocytoses in axon terminal of active CDC (x40,000). d: multiple exocytosis (arrows) (x40,000). e: whorl (W) in intimate contact with the axolemma (arrow) (x30,000).
30 TABLE I
burst hyperpolarizations (mean duration: 3.6 s; Fig.
Effects of treatment with 1 mM cpt-cAMP and IBMX on characteristics of CDC discharge
Discharges of controls were evoked by electrical stimulation. Values are means _+standard deviations of 6 preparations. For none of the parameters does a statistically significant difference exist between any of the groups. Parameter
Controls
cpt-cAMP
IBMX
Duration of discharge in s 26.2 + 16.1 36.1 + 21.5 32.2 + 14.4 Number of spikes per burst 7.4 + 1.3 7.9 _ 3.7 6.7 + 4.0 Duration of interburst hyperpolarization 3.6 + 1.6 3.2 + 0.7 3.8 + 0.8
lc). Treatment with cpt-cAMP results into a rather rapid depolarization (ca. 0.5 mV/s). Durations and patterns of (bursting) discharges are not clearly different from those found after electrical stimulation (Table I), but regular beating activity never occurs (Fig. ld). Treatment with I B M X leads to a slow depolarization (ca. 0.01 mV/s). After ca. 5 - 2 0 min the discharge starts. Discharge durations and patterns are not essentially different from those in C D C treated with cpt-cAMP (Table I; Fig. le). Release o f ovulation-inducing material and ultrastructure
lett'stest; cf. ref. 1) and for the joint assessment of normality 28. RESULTS The results presented with respect to incubations with cpt-cAMP also apply to incubations with I B M X , unless stated otherwise. Electrical activity
During pre-incubation all preparations show electrical silence with a resting potential of ca. - 7 0 inV. U p o n repetitive electrical stimulation C D C rapidly (ca. 0.3 mV/s) depolarize. After cessation of stimulation a long-lasting discharge occurs (5-45 min; Table I). A discharge consists of a few initial intense bursts of action potentials followed by either a regular beating firing pattern (found in 3 CNS; Fig. la, b) or bursting pattern (mean n u m b e r of spikes: 7.4 per burst) intermitted by more or less pronounced interTABLE II Effects of treatment with 1 mM cpt-cAMP and IBMX on exocytosis activity of CDC
Controls are unstimulated CDC in the electrical resting state. Exocytosis activity is expressed as number of tannic acid-positive exocytosis phenomena per cross-section of the cerebral commissure. Values are means _+ standard deviations of 6 preparations. Per parameter each group differs from the other groups (statistically significant, P < 0.01). Parameter
Controls
Number of exocytosis phenomena 27 _+8
cpt-cAMP
IBMX
623 + 397
1030 _+605
After incubation of resting C D C in control Ringer no ovulation-inducing activity in the bathing medium could be demonstrated. Bioassays of homogenates of the intercerebra! commissure show that the axon terminals of such C D C contain ca. 130 O I U . A t the ultrastructural level the terminals appear large (ca. 2 ~tm in diametei') and filled with numerous secretory granules (Fig. 2a). D u e to the application of the T A G O - m e t h o d exocytotic release of the contents of secretory granules can readily be observed, because the contents are stained selectively and are highly electron-dense (tannic acid-positive; Fig. 2b). Generally, exocytozed contents of secretory granules are present in omega-shaped indentations of the axolemma (fused granule membranes). At some places the exocytosis p h e n o m e n a are visible as 'caps', i.e. the opening of the granule with the extracellular space is not present in the plane of the section 27. In resting C D C , however, exocytosis p h e n o m e n a are rare (mean number: 27 per cross-sectioned neurohaemal area; Table II). Simultaneous release of contents of more than one granule ('multiple exocytosis') was never encountered. Also whorls, indicating resorption of the axolemma after exocytosis, appear to be absent. U p o n treatment with cpt-cAMP, the C D C of one pair of cerebral ganglia release ca. 60 O I U into the bathing medium. The axon terminals of these C D C are rather small (ca. 0.5 # m ) and only partly filled with secretory granules (Fig. 2c). Exocytosis phen o m e n a are very numerous (ca. 25x as many as in unstimulated CDC). Exocytosis particularly takes place in a multiple fashion; at some places up to 20
31 TABLE III Effect of incubation in Ringer, Ringer + 1 mM cpt-cAMP and Ca2+-free Ringer + 1 rnM cpt-cAMP, on exocytosis activity of CDC
This activity has been expressed as number of exocytosis phenomena per cross-section of the cerebral commissure. Values are means _+standard deviations of 6 preparations. No statistically significant difference exists between any of the groups. Parameter
Ringer
Number of exocytosis phenomena 10.0 + 4.6
cpt-cAMP in Ringer
cpt-cAMP in Ca2+-free Ringer
433.3 + 88.5 37.8 + 12.1
granules appear to be fused with each other while releasing their contents (Fig. 2d). The axolemma of the terminals shows deep foldings and many terminals contain a whorl. Some whorls show a continuity with the axolemma, indicating their formation by membrane invagination (Fig. 2e). Whorls are considered to represent a process of membrane resorption during and after high exocytosis activity 2. Electrical activity and ultrastructure in calcium-free Ringer's solution In calcium-free Ringer's solution, resting CDC show the same electrical silence and similar membrane potential as found in resting CDC in normal Ringer. Upon application of cpt-cAMP the cells exhibit the basic characteristics of the discharge, viz. as to duration and pattern (action potentials are increased in frequency and lack the calcium component; see also ref. 16). However, whereas the'discharge in normal Ringer is concomitant with an enormous increase in exocytosis activity (×43; Table III), in calcium-free Ringer only a small increase occurs (x3.8). Moreover, CDC in calcium-free Ringer treated with cpt-cAMP do not reveal any of the other ultrastructural changes characteristic of CDC stimulated in normal Ringer (partial loss of secretory granules, presence of multiple exocytosis phenomena and of whorls).
DISCUSSION The electrical, morphological and secretory characteristics of the active state of the CDC have been described extensively 13'22'25. The present study shows
that treatment with cpt-cAMP or with IBMX brings resting CDC into a state of high activity of which the characteristics are basically the same: a long-lasting spiking activity (discharge), a high frequency of exocytosis (particularly in the multiple form), depletion of secretory granules from the axon terminals, occurrence of whorls and strong release of ovulation-inducing material. Therefore, it can be concluded that treatment with cpt-cAMP as well as with IBMX brings the C D C into the active state. This supports the view that under physiological conditions the start of the active state is related to an intracellular rise of cAMP, for the following reasons. (1) It is well-known that cAMP-dependent processes can be stimulated with cAMP-analogues. The rapid and effective action of cpt-cAMP is due to the strongly hydrophobic character of this compound which promotes its rapid movement across the axolemma El. Moreover, this analogue of cAMP is not broken down by phosphodiesterase El. (2) In view of the well-established specific, inhibitory effect of IBMX on phosphodiesterase (which breaks cAMP down), the induction of the active state by IBMX is apparently based on an intracellular accumulation of cAMP. The period of IBMX treatment passing before the start of the active state is relatively long. This indicates that the rate of cAMP production in the resting state is rather low. This is in agreement with the observation that adenylate cyclase activity in the axon terminals is lower in the resting than in the active state 26. (3) Previous cytochemical studies of adenylate cyclase activity in the CDC 23'24 have indicated that cAMP levels are high in CDC in the active state. The electrical discharge of the CDC depends on a voltage-dependent Na+/Ca2+-channel and a Ca 2+activated K+-channe115. cAMP-stimulated phosphorylation of membrane proteins seems to be an important step in the control of the activity of ion channels in the axolemma of various neurons 5. Consequently, cAMP possibly promotes the electrical discharge by inducing activity changes of Na+/Ca 2÷ and/or K + ion channels. After a few initial action potentials the electrical discharge reveals a constant pattern of firing activity 13. The character of this pattern, however, varies among preparations, but also depends to some degree on the type of discharge-inducing stimulus. Thus, after freshwater stimulation and after intracel-
32 lular electrical stimulation, generally a regular beating p a t t e r n is found a3, but some p r e p a r a t i o n s m a y reveal a bursting p a t t e r n (present study). A bursting p a t t e r n is f u r t h e r m o r e generally found after stimulation with high potassium or with b a r i u m (substituting calcium) 15. W h e t h e r these different patterns represent different physiological states of the C D C and w h e t h e r they r e p r e s e n t different rates of s e c r e t o r y activity is not known. Meanwhile, the possibility that c A M P plays a role in the d e t e r m i n a t i o n of the discharge p a t t e r n should be envisaged: whereas the present results indicate that 10 -3 M c p t - c A M P induces a bursting pattern, p r e l i m i n a r y studies show that lower concentrations of this analogue (e.g. 10 -4 M) result into a beating p a t t e r n (P.J. M o e d and A . ter M a a t , p e r s o n a l communication). Extracellular calcium ions are crucial for the induction of secretion during the active state. A p p a r ently, they e n t e r the axon terminals as a result of depolarization of the a x o l e m m a and induce exocytosis from an axoplasmic site 22'27. The p r e s e n t study confirms this role of calcium: whereas t r e a t m e n t of C D C with c p t - c A M P or with I B M X in calcium-free Ringer's solution results in an electrical discharge (concomitant with a x o l e m m a depolarization), no induction of high exocytosis activity (as takes place in normal Ringer) occurs. This indicates that c A M P does not have a direct stimulatory effect on the process of
REFERENCES 1 Bliss, C.J., Stat&tics in Biology, Vol. L McGraw-Hill, New York, 1967. 2 Buma, P. and Roubos, E.W., Calcium dynamics, exocytosis. and membrane turnover in the ovulation hormone-releasing caudo-dorsal cells of Lymnaea stagnalis, Cell Tissue Res., 233 (1983) 143-159. 3 Buma, P. and Roubos, E.W., Involvement of cAMP, calcium and calmoduline in membrane dynamics of ovulationhormone releasing neurones in Lymnaea stagnalis, Cell Biol. Intern. Reports, 8(1984)277-278, 4 Dogterom, G.E., Bohlken, S. and Geraerts, W.P.M., A rapid in vivo bioassay of the ovulation hormone of Lymnaea stagnalis, Gen. Comp. Endocrinol., 50 (1983) 467-482. 5 Drummond, A.H., Benson, J.A. and Levitan, I.B., Serotonine-induced hyperpolarization of an identified Aplysia neuron is mediated by cyclic AMP, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5013-5017. 6 Ebberink, R.H.M., Van Loenhout, H,, Geraerts, W.P.M. and Joosse, J., Purification and amino acid sequence of the ovulation hormone of Lymnaea stagnalis, Proc. Natl. Acad. Sci. U.S.A., in press. 7 Geraerts, W.P.M. and Bohlken, S., The control of ovula-
exocytosis and, hence, that calcium does not induce exocytosis via stimulation of c A M P production. Nevertheless, a c o m b i n e d role of calcium and c A M P in the control of exocytosis can not be excluded. The only physiological trigger known to induce the active state is the freshwater stimulus, but how it acts upon the C D C is not known. Recently it was found 2° that the C D C p r o d u c e a small (1500 D) p e p t i d e (the C D C - a u t o t r a n s m i t t e r , C D C A ) that has an auto-excitatory effect, stimulating the C D C in vitro to a discharge 2°. C D C A is p r o b a b l y released during the initial part of the discharge. W e speculate that freshwater stimulation induces (some) C D C to release C D C A , which subsequently induces a d e n y l a t e cyclase in the axon terminals of all C D C to increased c A M P production. In this way the physiological stimulus is amplified by c A M P , leading to high electrical and secretory activity of all C D C .
ACKNOWLEDGEMENTS This research was m a d e possible by a grant from the F o u n d a t i o n for F u n d a m e n t a l Biological Research ( B . I . O . N . ) , which is subsidized by the Netherlands Organization for the A d v a n c e m e n t of Pure Research ( Z . W . O . ) . T h e authors wish to thank Prof. Dr. H . H . B o e r for critically reading the manuscript.
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