The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis

Brain Research, 476 (1989) 298-306

298

Elsevier

BRE 14137

The role of cAMP in regulation of electrical activity of_the neuroendocrine caudodorsal cells of Lymnaea stagnalis P.J. Moed, A.W. Pieneman, N.P.A. Bos and A. ter Maat Department of Biology, Vrije Universiteit, Amsterdam (The Netherlands) (Accepted 28 June 1988)

Key words: Cyclic adenosine monophosphate; Neuroendocrine cell; Egg laying; Mollusc

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaeastagnalisrelease a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-diseharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclaseand phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an afterdischarge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs followingan afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.

INTRODUCTION The neuroendocrine Caudodorsal Cells (CDCs) of the pond snail Lymnaea stagnalis play a central role in the animal's stereotyped egg-laying behavior, because they synthesize the ovulation hormone ( C D C H ) 6.s. Besides C D C H the CDCs release several other peptides 9'1°, one of which affects Ca-influx in the albumin gland 5. The CDCs are a group of about one hundced electrotonically coupled cells. The intercerebral commissure forms the neurohaemal area of the CDCs 25'a2. CDCs are generally electrically silent and only rarely fire spontaneous action potentials 3°. Preceding egglaying, however, they are electrically active for 60 min 16, during which they release C D C H 12. A similar active state can be induced by a period (2-5 min) of intracellular electrical stimulation of one CDC. Dur-

ing such stimulation the whole CDC-system gradually depolarizes until all CDCs become synchronously active. This activity has been called the afterdischarge 3°. Recently it has been shown that the capability of making afterdischarges is preserved when CDCs are brought into dissociated primary cell culture, indicating that the generation of afterdischarges is an endogenous property of the CDCs 24. In situ, the spread of excitation from the stimulated cell over the C D C network is thought to be achieved by chemically mediated positive feedback in conjunction with electrotonic connections 2~. The CDCs do not always respond to electrical stimulatioa in the same way. In 35% of snails cultured under standard laboratory conditions, the CDCs respond upon intracellular electrical stimulation with an afterdischarge la. The state preceding the afterdischarge is termed resting state (R-state), and

Correspondence: A. ter Maat, Department of Biology, Vrije Universiteit, P.O. Box 7161, 1007MC Amsterdam, The Netherlands. 0006-8993/89/$03.50© 1989Elsevier SciencePublishers B.V. (Biomedical Division)

?99 the afterdischarge the active state (A-state) n. At the end of an afterdischarge induced in vitro by electrical stimulation, the CDCs b,,come refractory to stimulation (inhibited state or I-state) 12. This refractoriness is a common phenomenon, not only in CDCs but also in other cells in which afterdischarges can be induced 15. A majority (65%) of the preparations contains CDCs that only show some depolarization or none at all upon stimulation and can be considered Istate. The percentage of brains that contain CDCs that are in R-state can be increased by culturing the snails in polluted water or by starving them is,19. The state of the CDCs can be determined by eliciting a short train of suprathreshold depolarizing of current pulses at 3 Hz. In R-state CDCs this gives rise to a depolarizing afterpotential. The absence of such an afterpotential is indicative of the I-state 3. The events leading to the CDC-discharge are probably mediated by second messengers. For instance, calcium plays an important role in the release events of the CDCs 25 and is probably also involved in the activity of the pacemaker channels 13'14. During the afterdischarge an increase of adenylate cyclase activity in the CDC axon terminals occurs, while addition of the membrane permeable cyclic adenosine monophosphate (cAMP) analogue 8-chloropbenylthio (8-CPT)-cAMP induces exocytosis2'26 and release of several peptides 9. This suggests a role for cAMP in the regulation of the physiological state of the CDC system. This study aims at relating the states of excitability of CDCs to intracellular levels of cAMP u~ing a nondegradable cAMP analog and by maniputating the activities of the enzymes regulating its production (adenytate cyclase) and breakdown (phosl~hodiesterase). It was found that an increase of cAMP in CDCs, induced either by 8-CPT-cAMP or by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) together with the adenylate cyclase activator forskolin, resulted in initiation of discharges. IBMX alone did not affect the resting membrane potential but greatly reduced refractoriness in CDCs, whereas forskolin alone had a dual effect: although it depolarized the CDCs, it inhibited the response of the cells to electrical stimulation. The results are discussed in terms of transitions between the states of excitability.

MATERIALSAND METHODS

Animals Laboratory-bred adult snails (shell heights 27-33 mm) were used. They were kept paired in jars with continuous water change, under a 12/12 light/dark regime at 20 *C and were fed lettuce ad libitum. Solutions The composition of the standard buffered saline (HBS) was (in mM): NaC130, NaCH~ SO4 10, NailCO 3 5, KCi 1.7, MgC!2 1.5, CaC!2 4.0, and HEPES 10 (adjusted to pH 7.b with 3.5 mM NaOH). The culture medium of the isolated cells contained the following supplements: Eagle's Minimal Essential Amino Acids and vitamins, glucose (5 mM), streptomycin (100/~g/ml) and penicillin (100 U/ml). For electrophysiological recording of cells in the CNS, the ganglia ring including the buecal ganglia was dissected out and pinned down on a Xantopren layer (Bayer) at the bottom of the experimental chamber filled with HBS. The outer connective tissue was removed from the cerebral ganglia and the COM. Cell culture The identifiable CDC somata were removed from the cerebral ganglia after proteolytic treatment (60 min in 0.2% trypsin at 35 °C) and plated in culture Petri dishes (Costar) filled with the culture medium. The cells generally attached to the bottom of the dish within an hour. Petri dishes were stored in the dark at 20 °C in a humidified incubator. CDCs were used for experimentation 1-3 days after isolation. For electrophysiological experiments the culture dishes were placed on the stage of an inverted phase-contrast microcope and the culture medium was replaced with HBS before the start of the experiment. Intracellular electrical recordings were made using glass (Clark) microelectrodes filled with 0.5 M KCI (resistance: 30-90 MQ), connected to conventional electrophysi~logical apparatus. Recordings were stored on FM tape and displayed on a Gould pen recorder (Brush 2200). Electrical stimulation Standard repetitive stimulation consisted of suprathreshold square current pulses of 200 ms duration at

300 a frequency of 2 Hz. During stimulation, CDCs that are in resting state depolarize and start to fire additional action potentials during the stimulus intervals. When these action potentials reach a frequency of 0.5 Hz, stimulation can be stopped and the afterdischarge continues without further stimulation. When stimulation was ineffective within 15 min, or when no additional action potentials were found, stimulation was stopped.

_J IOldX

t

.1

Administration of chemicals Chemicals kept in stock solutions were dissolved to final concentration in HBS immediately before experimentation. The HBS in the experimental chamber was replaced with this solution within 30 s using a perfusion system (20 mi/min).

Chemicals 8-CPT-cAMP, Eagle's minimal essential amino acids and vitamins were obtained from Boehringer Mannheim, IBMX from Sigma, and forskolin from Calbiochem.

forskolln

t

rJ

.1

IBMX • forskoSln

9

Statistics Data were analyzed by ANOVA, followed by a posteriori comparisons of individual groups. Analysis was preceded by a test for homogeneity of variances (Fmax procedure). In the graphs and the text standard errors (S.E.M.) are given. RESULTS

Fig. 1. The response of CDCs in the isolated central nervous system to the application of 10-4 M 8-CPT-cAMP (A), 10-3 M IBMX (B), 10-4 M forskolin (C), and a combination of 10-3 M IBMX and 10-4 M forskolin (D). The agents were continuously present during the course of the experiment (arrow: point at which the chemicalswere added, calibration: 40 mV, 200 s).

Addition of the membrane-permeable and phosphodiesterase-resistant cAMP analogue 8-CPT-cAMP 2a into the bathing solution resulted in depolarization of the CDCs, the firing of action potentials and eventually in a discharge (Fig. 1A). The fraction of preparations that responded with a discharge and the latency of the response were dose-dependent (P < 0.01 in both cases) (Table I). At a concentration of 10-4 M 8-CPT-cAMP the response of the CDCs was maximal. The latency of the response at this concentration was 11.3 + 1.3 min. The latency was decreased to 2.5 _+ 0.6 rain at 10 -3 M 8-CPT-cAMP (Table I). This indicates that the latency of the response of CDCs to 10-4 M 8-CPT-cAMP was mainly determined by the diffusion gradient of 8-CPTcAMP across the cell membrane.

The firing pattern of a discharge initiated by 8CPT-cAMP was very regular and in this respect comparable to the afterdischarge induced by electrical stimulation. However, at the start of a discharge initiated by 8-CPT-cA~ IP the bursting phase, typical of the afterdischarge induced by electrical stimulation 3°, was often absent. At a concentration of 5.10 -5 M 8-CPT-cAMP the mean duration of the CDC-discharge was 98 + 11 min (n = 3), while higher concentrations resulted in discharges of 1.5-6 h (n = 5). The durations of afterdischarges induced by 8-CPTcAMP were much longer (P < 0.01) than those elicited by electrical stimulation in normal saline (38.9 + 3.0 min, n = 18). Intracellular levels of endogeneous cAMP may be

301 TABLE I In situ CDCs

Induction of discharges by 8-CPT-cAMP, IBMX, and forskolin. Means and S.E.M.s are given. Ranges are presented of the durations of the induced discharges. The numbers of preparations pertain to the percentage response. Numbers of preparations of which the deviations have been determined are given in brackets. Agent

Concentration

n

% showing discharge

Latency(min)

8-CPT-cAMP

10-5 5.10-5 10-4 3.10-a 10-3

5 12 15 3 4 2

0 50 93 100 100 100

18.8 + 1.6(6) 11.3 + 1.3 (14)~ 9.3 + 2.1 (3) | 7.5 _+2.6(4) [ 150-360 (5) 2.5 + -0.6 (2)J

IBMX

10-3

7

0

-

Forskolin

10-6-10 -4

21

0

-

IBMX (10-3) + forskolin

5.10-5 10-~

5 8

20 50

5.10 -4

increased by inhibition of the phosphodiesterase that breaks down cAMP. Such inhibition can be achieved by adding I B M X to the bathing solution (reviewed, ref. 31). We found that IBMX at concentrations of 10.4 and 10-3 M had no effect on the membrane potential (+1.1 + 0.8 mV (n = 7) a n d - 0 . 1 + 1.2 mV (n = 9) respectively, see also Fig. 1B and Table I). This suggests that the basal adenylate cyclase activity was not high enough to accumulate cAMP to an effective concentration within the time of incubation (30-90 rain). The activity of the adenylate cyclase in many different cells has been shown to increase in the presence the ditetpene forskolin (reviewed, ref. 27). Forskolin (10-6-10 -4 M) did not induce discharges in CDCs. The membrane potential only showed a small, but dose-dependent, depolarization of up to 6.4 + 1.2 mV (n = 13) at 10-4 M forskolin (Fig. 1C). When the phosphodiesterase was inhibited by 10 -3 M IBMX, however, 5.10 -5 M forskolin depolarized the cells by 12.2 + 1.2 mV (n = 5), and in one case a CDC-discharge was initiated (latency: 12 min). At a concentration of 10-4 M forskolin a CDC-discharge was induced in 4 out of 8 preparations, whereas in the remaining 4 preparations the CDCs depolarized by 16.0 + 2.0 mV. The latency for the induction of a discharge by IBMX plus forskolin was 44.5 + 3.0 rain (n = 3; see also Table I).

12.0 (1) 44.5 __3.9 (4)

Duration (min)

98 + 11 (3)

-

not determined 24-270 (4)

The first phase of discharges induced by IBMX plus forskolin consisted of a very rapid depolarization after the initial action potentials. This plateau phase, where no action potentials were observed, could last for several minutes (Fig. 1D). In contrast to the first phase of a 8-CPT-cAMP stimulated discharge, repolarization was slower. In two preparations this electrical behavior was repeated in the course of a single discharge, with a periodicity of 70.5 + 4.9 min (average of 7 periods). The durations of afterdischarges induced by IBMX and forskolin ranged from 24 min to more than 4.5 h. In conclusion, these results show that elevation of intracellular cAMP induces electrical activity in CDCs. In addition they indicate that to increase c A M P levels above the threshold for initiation of a discharge, both low activity of the phosphodiesterase and high activity of the adenylate cyclase are necessary. Are the responses of the CDCs to cAMP agonists and regulators a property of the individual cells? To answer this question we investigated the effects of the agents used above on CDCs maintained in dissociated cell culture. Cells were used for electrical recording 1-3 days after isolation. During this time they did not grow substantial new processes and consisted mainly of a soma with a small part of the original axon. Isolated CDCs maintain most of their elec-

302 tricai characteristics in dissociated cell culture, including the ability to express afterdischarges24. The results are summarized in Fig. 2 and Table II. 8-CPT-cAMP (10.4 M) induced discharges in nearly all impaled cells. IBMX (10 -3 M) alone did not significantly change the membrane potential (3V = -4.8 _+ 2.5 mV (n = 8)) and only induced a discharge in 1 out of 8 experiments. Forskolin appeared very effective in inducing discharges. At a concentration of 10-4 M, forskolin induced discharges in 2 out of 2 experiments, while 10-5 M forskolin induced a discharge in 4 out of 5 experiments. IBMX (10-3 M) together with forskolin (10-s M) induced a discharge in 5 out of 7 impaled cells. The percentage response in IBMX was significantly lower (P < 0.01) than with the 3 other treatments. As can be seen in Fig. 2, isolated CDCs responded earlier than CDCs in the CNS. Furthermore, the difA

8.CP~eAMP

/ db wash B

IBMX

/

t " _ _

C

r

-

I

I

forskolin

.lll!|l!ll!lllll!l!!!!l..

TABLE II

_J

db wash

D

ference in latency between the responses to 8-CPTo cAMP and IBMX plus forskolin, as was found for CDCs in the CNS, was no longer observed. Another difference with the results obtained in situ was that isolated CDCs responded with discharges upon low concentrations of forskolin without the simnRaneous inhibition of the phosphodiesterase by IBMX. These lower thresholds and shorter latencies might originate from the better accessibility of isolated CDCs for the pharmaca in the solution because they are no longer surrouded by connective tissue. The firing patterns of discharges in isolated CDCs induced by the various agents (Fig. 2) were variable, showing regular spiking, irregular or regular bursting, or sometimes prolonged depolarization in combination with irregular oscillations of the membrane potential occasionally interrupted by action potentials. These different firing patterns were not correlated with the type of agent used, and therefore probably reflect small differences in excitability and characteristics of individual cells. These differences between individual cells are probably much more difficult to detect in the intact cluster, because the cells are electrotonically coupled, and this synchronizes their firing pattern 2°. In conclusion, the results show that isolated CDCs respond to cAMP elevation by producing discharges, as in the intact CNS. Under f:he laboratory conditions used for culturing the snails, 36% of CNSs contained CDCs that were in R-state (n = 80). For these, the stimulation time for

IBMX + forskolin

/

Isolated CDCs

Discharge induction by 8-CPT-cAMP, IBMX, and forskolin. The percentage of cells (culture dishes) that showed a discharge after addition of the agent is given. The means and standard deviations of the responding cells are presented. IBMX alone was less powerful in eliciting CDC-discharges than the other agents. The latencies did not differ significantly. ,

4b wash

Fig. 2. The responseof isolated CDCs to the applicationof 10-4 M 8-CPT-cAMP(A), 10-3 M IBMX (B), 10-5 M forskolin (C), and a combination of 10-3 M IBMX and 10-5 M forskolin (D). The agents were washed out after a few minutes (arrow down: point at which the chemicals were added, calibration: 40 mV, 200 s).

8-CPT-cAMP (10-4 M) IBMX (10-3M) IBMX (10-3) + forskolin (10-s M) Forskolin (10-4-10-5 M)

n

% showing discharge

Latency (rain)

0 8

89 13

5 +_0.5 (8) 0.8 (1)

8

88

3.3 4- 1.3 (7)

7

86

6.7 4- 3.0 (6)

303 A

Control

TABLE III

In situ CDCs

B

IDMX

C

forokolln

J D

_l

Effects of IBMX and forskolin on the responsiveness of CDCs to repeated stimulation with suprathreshoid current pulses (at 2 Hz). Means and S.E.M.s are given. The number of preparations is shown in brackets. The percentage of preparations responding to electrical stimulation in IBMX (10 -3 M) alone and in IBMX (10-3 M) combined with forskolin (5.10-5-10 -4 M) was higher (P < 0.01) than in control, IBMX (10 -3 M) or forskolin (10-4 M). In forskolin this percentage was reduced as compared to control (P < 0.05). Duration of stimulation is lowest in the control group (P < 0.01). Within the group of agonists, IBMX (10-3 M) and forskolin (5.10-5-10 -4 M) combined yielded the shortest stimulation time. Durations of discharges were not significantly different.

IBMX + forskolln

_J Fig. 3. Responses of CDCs in the isolated central nervous system to a period of repetitive intracellular electrical stimula:ion of one CDC (bar: period of stimulation. A: control condition in HBS. B: response in the presence of 10-3 M IBMX. C: response in the presence of 10-4 M forskolin. In this particular experiment we stimulated the cell briefly before adding the forskolin, to show that the CDCs were capable of producing a depolarizing afterpotential, which is indicative for the R-state of the CDCs. After a 30-min incubation with forskolin no afterdischarge could be induced, despite some depolarization. D: response in the presence of a combination of 10-3 M IBMX and 10-4 M forskolin. The agents were continuously present during the course of the experiment. (Calibration: 40 mV, 200 s.)

the induction of the afterdischarge or A-state was 2.9 + 0.3 rain (n = 18). The mean duration of the A-~tate was 38.9 + 3.0 min (n = 18) (see Fig. 3A, Table III). When C D C s had not entered the A-state after 15 min, stimulation was stopped and C D C s were considered to be in I-state. We investigated the role of the phosphodiesterase in determining the state of excitability by inhibiting its activity with IBMX. A s we have shown, IBMX had no direct effect on the resting membrane potential (Fig. 1B). Also at a concentration of 10-4 M I B M X did not alter the proportion of R-state CDCs (Table III). However, it decreased the stimulation time to 0.8 + 0.2 min (n = 3) (P < 0.01). In the presence of 10 -3 M I B M X , however, a much larger percentage (92%) of the preparations responded to electrical stimulation by entering the A-state than in con-

Agent

% showing discharge

Durationof Duration of stimulation discharge (min) (min)

None (control) IBMX(10-4M) IBMX (10-3 M) IBMX + forskolin Forskolin (10-4 M)

36 (80) 43(7) 92(12)

2.9+0.3 (18) 38.9+3.0 (18) 0.8+0.2(3) 27.9+12.9(2) 0.6+0.1(8) 46.7+11.5(7)

92 (12)

0.2+0.1 (7)

51.4+26.3 (7)

11 (18)

5.3 and 0.1

18.7 and 9.1

trois ( P < 0.01). The average stimulus time was 0.6 + 0.1 rain (n = 19) (see also Table l i t ) . Apparently, inhibition of the phosphodiesterase not only increased excitability in R-state CDCs, as expressed by the decrease in stimulation time, but also induced a transition from I-state to R-state. The firing pattern during the first part of the A state in the presence of 10-3 M I B M X had a marked plateau phase (Fig. 3B), while the normal initial bursting phase was seen in the presence of 10-4 M I B M X (not shown). The duration of the A-state was similar to that of the controls (10 -3 M IBMX, 46.7 + 11.5 rain; n = 7). It was not possible to induce a second A-state in the same preparation, despite the continuous presence of IBMX. In conclusion, inhibition of the phosphodiesterase greatly increased excitability and, at higher concentration, induced a transition from I- to R-state in C D C s in the isolated CNSs. The A-state was not significantly longer than under control conditions and at the end of A-state the CDCs re-entered the I-state, indicating that inhibition of the phosphodiesterase is probably an important event in determinating the threshold for the A-state, but has no effect on the

304 prolongation and termination of the A-state. Does direct activation of the adenylate cyclase alter the state of excitability? We have shown in this study that forskolin alone can induce a small depolarization in CDCs in the intact CNS. We found that forskolin reduced the number of preparations that responded to electrical stimulation (P < 0.05; Table

III). In the continuous presence of IBMX, however, forskolin (5.10 -5 to 10-4 M) decreased the stimulus time for the induction of the A-state to 0.2 _+0.1 rain (n = 7) as compared to control and to IBMX alone (P < 0.01). This stimulus time might he affected by the depolarized state of the CDCs (see Fig. 1C). The number of preparations that responded to electrical stimulation was not significantly different from that responding to IBMX alone. The firing pattern during the A-state induced by electrical stimulation in the presence of IBMX plus forskolin showed an initial strong depolarization, followed by slow repolarizafion and return of spiking activity. The duration of the A-state varied from 39 to 270 rain. One of the firing patterns showed the periodic occurrence of plateaus similar to the pattern seen during discharges induced directly in IBMX + forskoliu (Fig. 1D). In one preparation, a second :xstate could be induced within 15 rain after the e~d of the first A-state. In conclusion, activation of adenylate cyclase by forskolin had a dual effect. It caused a slow depolarization, which was increased by simultaneous inhibition of phosphodiesterase by IBMX. On the other hand, without IBMX it decreased excitability as defined by the response to electrical stimulation. DISCUSSION In this paper it is shown that (1) the non-degradable cAMP analog 8-CPT-cAMP is an effective stimulus for the induction of electrical activity in the CDCs in a dose-dependent way, and (2) the same effect can be achieved by manipulating the cAMP-regulatory enzymes adenylate cyclase and phosphodiesterase. The response of CDCs to an increase in cAMP is probably an endogenous property of the cells, because isolated CDCs responded in a similar way as CDCs in the intact brain. When 8-CPT-cAMP levels were maintained at a

high level the duration of electrical activity outlasted the normal duration of afterdischarges. This suggests that during the normal afterdischarge the cAMP level is high initially, and then probably decreases again. This is in keeping with the results obtained from the bag cells of Aplysia by Kaczmarek et al. u, who found a two-fold increase of cAMP at 2 min after the start of the afterdischarge, whereafter it decreased to control levels within 5 rain. The fning pattern of 8-CPT-cAMP-induced discharges was very regular and comparable to the pattern of the normal CDC-discharge. However, when IBMX and forskolin were used the firing pattern frequently showed a periodicity. Interestingly, this periodicity had a relatively constant cycle time (70 + 12 min). The main difference between this type of stimulation and the administration of cAMP analogs, where no periodic plateaus were observed, is that forskolin stimulates the adenylate cyclase directly via the catalytic subunit27. Perhaps this periodicity can be explained by cyclic fluctuations of the substrate for cAMP, i.e. ATP 4. Basal adenylate cyclase activity is probably low in CDCs that do not show electrical activity, because inhibition of phosphodiesterase did not affect the resting membrane potential. Should adenylate cyclase activity have been high, then inhibition of phosphodiesterase would have resulted in the accumulation of cAMP and hence a CDC-discharge. This is in keeping with the results of ultracytochemical measurements of the adenylate cyclase activity26 where it was found that, before the discharge, adenylate cyclase activity was ~ow in the silent state cells. Phospho~iesterase activity is probably high in CDCs in ~itu. This conclusion is based on the fact that activation of adenylate cyclase by forskolin could not trigger CDC-discharges unless phosphodiesterase activity was blocked. In isolated cells, however, inhibition of the phosphodiesterase was not necessary for the induction of electrical activity by forskolin. Moreover, forskolin acted effectively at lower doses. This suggests that phosphodiesterase activity is lo~ in isolated cells. The egg-laying cycle of Lymnaea is reflected by a cycle of 3 distinct states of CDC excitability, the R-, A- and I-states. In situ, a transition between t~ie Rand the A-state can be induced by electrical stimulation. The A-state is followed bythe I-state, whe':e the

305 CDCs are refractory to electrical stimulation 12. In this study we have confirmed that the majority of the preparations (64%) are in the I-state. The non-degradable analog 8-CPT-cAMP induced A-states in all preparations, regardless of whether they were in the I-state as determined by electrical stimulation or following a CDC-discharge. This means that the regulation of the CDC states occurs at the level of the cAMP-regulating mechanisms, rather than the coupling of cAMP with the ionic channels underlying the electrical activity. In the presence of IBMX the percentage of l-state CDCs decreased to 8%. IBMX is thought to inhibit phosphodiesterase activity, although at higher concentrations (>0.5 mM) it can interfere with nicotinic cholinergic transmission at the neuromuscular junction m. As the CDCs receive cholinergic input, IBMX might interfere with this input. However, since the nicotinic component of the biphasic input in the CDC~ is excitatoryiT, a suppression of excitability would be expected instead of the elevation in excitability found in our study. We therefore feel that blockage of phospbodiesterase offers a more likely expla~lation for the effects of IBMX. Apparently, CDCs that were in I-state as defined by Kits et al. t2 could be changed to R-state by inhibition of the phosphodiesterase. This suggests that phosphodiesterase activity is an important factor in determining the transition from I- to R-state. The transition to the A-state can be achieved by activation of adenylate cyclase and simultaneous inhibition of phosphodiesterase. This occurs in 50% of the preparations. As this percentage is not statistically different from the percentage of preparations that are normally in the R-state, this indicates that the transition from R- to A-state involves activation of adenylate cyclase. The transition from A- to I-state was delayed in *he continuous presence of 8-CPT-cAMP. Inhibition of phosphodiesterase alone did not lengthen the Astate. This suggests that the CDCs do not maintain a continuous high level of cAMP, despite the fact that the breakdown of cAMP is inhibited. Assuming that

REFERENCES 1 Akasu, T. and Karczmat, A.G., Effects of l-methyl-3-isohutylxaathine (MIX) on amphibian neuromyal transmis-

adenylate cyclase is involved in the initiation of the CDC-discharge, its activity is probably transient. We propose that the refractoriness of CDCs to electrical stimulation originates primarily from the incapability to activate the adenylate cyclase and in the second place by a high phosphodiesterase activity. In contrast to its depolarizing effect, forskolin alone induced a decrease in the fraction of preparations responding to electrical stimulation. Although matter of some dispute 29, many studies on the mode of action of forskolin have revealed that forskolin probably acts directly on the catalytic subunit of the adenylate cyclase (review: ref. 28). However, nearly all studies on the effects of forskolin have been done in the presence of a phosphodiesterase inhibitor. An increase of cAMP production by forskolin without the presence of IBMX will probably induce an increased phosphodiesterase activity and hence restoration of normal resting cAMP concentration. In our experiments this increased turnover of cAMP might have counteracted the stimulating effect by electrical stimulation on cAMP production. Also the possibility of a direct, anaesthetic-like effect cannot be excluded 22. When phosphodiesterase was inhibited, and the high turnover circumvented, there was no inhibitory effect of forskolin en the response to electrical stimulation. We suggest, therefore, that this inhibitory effect of forskolin originates from an increased turnover of cAMP, emphasizing the importance of both cAMP-reguiating enzymes in determining the states of excitability of the CDCs.

ACKNOWLEDGEMENTS Thanks are due to Prof. T.A. de Vlieger and the members of his group for critically reading the manuscript, and to Mrs. T. Laan for her help in its preparation. This work was supported by the foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the advancement of Pure Research (ZWO).

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