Pharmacologically induced stereotyped locomotory movements in the pelecypod mollusc, Anodonta Cygnea

Camp. Biochem. Physiol. Printed in Great Britain

Vol. 92C,

No. 2, pp. 343-347,

1989 0

0306~4492/89 S3.00 + 0.00 1989 Pergamon Press plc

PHARMACOLOGICALLY INDUCED STEREOTYPED LOCOMOTORY MOVEMENTS IN THE PELECYPOD MOLLUSC, ANODONT’A CYGNEA D. A. *Institute

SAKHAROV,*

L. HIRIPI?

and J.

SALANKI?

of Developmental

Biology of the USSR Academy of Sciences. Moscow, USSR, and $Balaton ~imnoiogical Research Institute of the Hungarian Academy of Sciences, T&any, Hungary (Received 9 May 1988)

Bathing freshwater mussels in water containing ergotamine or methylergometrine induced rhythmic coordinated movements of the foot and shell maintained in a stereotyped form for hours. The effect was weak at 10-9mol/l, moderate to strong at 14 x iO-smol]l, and irreversible to lethal at S-8 x LO-*mol/I. 2. Bathing mussels in serotonin (10-20 rmoljl) induced similar movements. They closely correspond to the well-known digging cycles exhibited by pdecypod molluscs during burrowing locomotion. 3. The serotonin content of central ganglia was not changed significantly in mussels exhibiting the Abstract-l.

ergot-induced locomotory movements. Ergotamine, up to 10-5-10-6 mol/l, did not affect the spontaneous and evoked liberation of serotonin. as well as the serotonin uptake. There is thus little indication of any direct influence of ergots on serotonergic mechanisms.

MATERIALS

INTRODUCTION It has been demonstrated previously that pod molluscs shell movements associated feeding are under serotonergic excitatory minergic inhibitory control (Salitnki et

The animals

in pelecywith filter and dopa-

al., 1974); however, the neurotransmitter mechanisms for regulation of pelecypod locomotion are still unknown. Our approach to this problem is based on knowledge obtained in studies of chemical control of locomotion in gastropod molluscs. We have found earlier that ergometrine and ergotamine produce long-lasting stereotyped locomotory movements in the pulmonate snail, He& pomatia (Sakharov and Salanki, 1982). These observations have been confirmed on the pteropod mollust, Clione Iimacina: inhibitory episodes in intermittent swimming of this pelagic mollusc have been completely eliminated by treatment with ergometrine or a related ergot and, further on, a similar stereotyped locomotion was induced by 5HT (Sakharov and Kabotyanski, 1986). The ergots in question are widely regarded in invertebrate neuropharmacology as antagonists of dopamine receptors mediating a K+-dependent synaptic inhibition (Walker et al., 1968; Ascher, 1972; Gospe and Wilson, 1981; Shozushima, 1984). Locomotory behaviours disinhibited by the ergots in gastropods differ in modes of motion and, correspondingly, in central programs for body movement. This might imply that divergent molluscs possess a common, phylogenetically conservative neurochemical mechanism of inhibitory control of locomotion. We report here the results of experiments unfixing this suggestion. The results demonstrate that digging movements of the Anodo~a foot-which in fact are locomotory movements of this pelecypod mollusc+an be easily induced in a stereotyped form by ergot alkaloids. Investigations were also conducted in respect to a possible role of serotonin in this locomotor stereotypy.

343 C.B.P. PXX--L

AND METHODS

Fresh-water mussels (Anocfonra cvgneu) were obtained from a fish pond and kept in running water of Lake Balaton until use. Recording of motor activity

For observing and recording the movements of the animal, one of the valves was attached to a stand stuck to the bottom of a 3 I vessel provided with a perfusion system. The other valve was attached to a lever of the recording apparatus (mussel actograph, see Salanki and Balla, 1964) using a flexible thread. The Lake Balaton water in the vessel was continuously aerated. Continuous recording of movements of five mussels could be achieved simultaneously, each in its independent vessel. To investigate the effects of a drug, the water was permanently circulated for one day, then it was stopped and the aqueous stock solution of the drug was added to the water after 40min in a quantity to reach the desirable final concentration. Estimation of serotonin (5HT)

content

The cerebral, pedal and visceral ganglia were prepared and homogenized separately in 20 ~1 of a.1 M HCiO,-solution. The homogenate was centrifuged at 30,OOOgfor 20 min at 4°C and 10 ~1 of the clear supernatant was injected into the high pressure liquid chromatograph. Chromatography was performed with Waters Model 6OOOAsolvent delivers system, Model U6K universal injector and PBondapak C;, reverse-phase column. The mobile phase was 0.01 M acetate buffer (pH 4.7) containing 6% methanol. The flow rate was 1ml/min and the column temperature was set at 40°C during the analysis. Fluorimetric detection was accomplished using Aminco Bowman spectrophotofluorimeter. Excitation and emission wavelengths were set at 300 and 340 nm. Measuremeni of 3H-5HT

uptake

Pooled cerebral, pedal and visceral ganglia prepared from a single animal after measuring the wet wt were incubated in 1ml of physiological solution for IOmin at 25°C. The solution contained 3H-5HT (3.4 x IO-*moi/l) and er-

D. A. SAKHAROV et al.

344

Fig. 1. Change of the slow rhythm to the locomotory rhythm in an animal treated with methylergomet~ne, 4 x IO-smol/l. Record of shell movements (redrawn from actogram). Time calibration 1 hr. A. The activity characteristic of the slow rhythm, 12 hr prior to adding methylergometrine to the bath. Between active periods the shell was closed, as in the beginning and in the end of A (lower horizontal line). EJ. The same animal, continuous record. Methylergometrine was added to the bath 9 hr prior to B. In B, the shell can be seen opening for the first time since adding the drug. In C, the digging activity appears, characterized by fast adduction-relaxation. The shell is not closed between the periods of the digging activity. After the 5th period, closing can be seen for the first time in H, indicating the beginning of the recovery from the methyIergomet~ne action.

gotamine (lo-‘, 10m6 or 10-smol/l). At the end of the incubation the tissue was filtered on Whatman GF/B glass microfibre filters and rinsed twice with lOm1 of physiological solution. It was then dissolved in I ml of tissue solubilizer and, after adding 10 ml of toluene scintillator, the radioactivity was measured. Determinationof 5HT liberation Pooled cerebral, pedal and viscera1 ganglia were prepared from two animals. The ganglia were incubated for 30 min at 25°C in 2m1 of physiological solution containing 1.7 x lOA molji 3H-SHT and 0.1% ascorbic acid. Following incubation the ganglia were washed 3 times in 15 ml of physiological solution, then put into a 2ml chamber and perfused with (I) physiological solution, (2) physiological solution with 10 times increased potassium concentration and (3) physiological solution containing 10-7-10-6 mol/l ergotamine. The flow rate of the solutions was 1 ml/min, and 1 ml fractions were collected, The radioactivity of the fractions was counted by liquid ~intillation s~ctrometry in a Triton X-lOO/toiuene based solution. Drugs The drugs used were ergotamine tartrate (Sigma), methylergometrine (Spofa) and serotonin creatinine sulfate (Sigma). RESULTS Eflects of ergotamine and methylergometrine on motor activity In all animals used in these experiments periods of activity and quiescence were

alternate recorded

During the periods of activity, due to the force of the ligament connecting them, the two valves were separated but from time to time, as a result of the phasic contraction of the adductor muscles, a rapid and not full adduction occurred followed by a slow separation. Distribution of these contractions within the active periods was in general inhomogeneous. At the phase of quiescence the valves were kept closed for hours. This patterned activity of adductors corresponds to the so-called slow rhythm described originally by Barnes (1955) and investigated from different aspects by Salinki and co-authors (Salanki, 1966; Salanki et al., 19’70). The foot does not take part in these movements, which are believed to be functionally associated with respiration and filter feeding. Bathing the mussel in an effective concentration of ergot solution brought forth a complete change in the patterned motor activity. The new pattern was established rather abruptly after latency, which depended on the concentration of the ergot and was generally rather long (hours). The periods of rest were eliminated from this new pattern, fast movements of the continuously separated valves assumed a regular periodicity and higher frequency (Fig. 1). The foot produced cyclic movements being extended from the shell at a particular phase of the cycle or, later, continuously. Upon closer examination, we found that the rapid movements of the valves and the cyclic movements of the foot were coordinated. A rapid adduction of the prior to treatment.

345

Drug-induced locomotory movements in Anodonta

Content, uptake and release of 5HT in animals treated with ergotamine

Fig. 2. Movements of the foot in animals treated with ergot alkaloid or 5HT. Four consecutive steps of one cycle are shown, as described in the text.

valves occurred invariably when the foot rested after full extension, and was immediately followed by the foot retraction. The retraction itself was also patterned: the foot was first seen to be rapidly moving in anterior direction, then it moved more slowly in posterior direction and, finally, a general withdrawal towards the shell was manifest. This was followed by the extension of the foot, then it rested for tens of seconds in an extended state, until being clamped again by a rapid adduction of the valves (Fig. 2). There were no noticeable differences regarding the effects of ergotamine and methylergometrine. They were effective at concentrations ranging from 1 to 4 x 10-8mol/l, exhibiting stereotyped movements of the foot and valves for tens of hours. These movements were accompanied by a partial or complete loss of responsiveness to tactile stimulation. It was, however, possible to wash the drug out and restore the slow rhythm and responsiveness to tactile stimulation. Higher concentrations (5-8 x lo-‘mol/l) acted irreversibly and were lethal, while at 1.25-2 x 10m9mol/l the effect-if any-was rather weak and disappeared spontaneously. Efect of serotonin SHT in 10-20 pmol/l induced stereotyped cyclic movements of the foot coordinated with movements of the valves. There were overwhelming similarities between the stereotypy induced by 5HT, on the one hand, and that induced by ergots, on the other. The differences were as follows: (1) the latency of the effect was much shorter for 5HT (within 5min at 20 pmol/l, and 10 min at 10 pmol/l) than in the case of ergotamine and methylergometrine (hours); (2) rapid adduction of the valves was not preceded by the resting, immobile stage in the foot cycle (instead, the stage of foot extension was immediately followed by valve adduction which made the dilated foot clamped); (3) the animals were responsive to tactile stimuli. A similar effect, though in a weak and transient form, was observed at 5 pmol/l 5HT.

The results of behavioural experiments thus show that a similar motor stereotypy can be induced in Anodonta by 5HT, on the one hand, and by ergotamine or methylergometrine, on the other. Can it be that these ergots activate somehow endogeneous SHTergic mechanism; or is their SHT-like action due to another cause? To answer this question the 5HT content, uptake and release in animals treated with ergotamine were estimated. The 5HT content was measured separately in the cerebral, pedal and visceral ganglia of 5 animals kept for 20 hr in 5 1 of 3 x lo-* mol/l ergotamine solution at 18°C. The control group consisting of 5 animals of the same size was kept under similar conditions in water. When the nervous tissue was taken for analysis, the animals treated with ergotamine had their food characteristically extruded, whereas the control animals did not. Interestingly, there was a considerable difference in the wt of flesh between the two groups: the cumulated wt of the 5 control animals was equal to 80.1 g, while that of the ergotamine treated group was 122 g. There were, however, no significant differences in the 5HT level, though a tendency to lower values could be seen in the ergotamine-treated group in all three pairs of ganglia (Table 1). The results of the study on 5HT liberation are shown in Figs 3 and 4. A continuous background loss of 3H-5HT by incubated ganglia was observable throughout the experiment. An approximately 50% increase in 5HT liberation occurred when the ganglia were perfused with high K+ solution. Neither spontaneous nor evoked by high K+ release of 5HT was changed in the presence of ergotamine. The results of the uptake measurements are demonstrated in Fig. 5. Only an insignificant decrease in the level of 3H-5HT uptake was detectable in the presence of ergotamine, as compared to the control level. DISCUSSION

We have found that the freshwater mussels Anodonta cygnea immersed in water containing ergotamine or methylergometrine produce stereotyped, periodically repeated patterned movements of the foot and valves. This motor pattern closely corresponds to the digging cycle, the basic element of locomotory behaviour in pelecypod molluscs (Trueman, 1975). The investigated species of pelecypod molluscs has thus turned out to be similar in this respect to divergent gastropods, exhibiting a stereotyped locomotion after treatment with ergotamine,

Table I. Serotonin

Animals treated with ergotamine (N=4) Control animals (N = 5)

content in ganglia of A. cygnea (in pmol 5HT per Cerebral

Pedal

Visceral

417 f 39.9

626 + 60.9

501 f 42.7

457 f 43.2

715 + 62.1

581 k 57.9

D. A.

346

SAKHAROV

et a/.

control

ergota

rnin e

t ml/l I Fig. 5. Effect of ergotamine on the in uitro uptake of 3H-SHT (in % of control). 10

20

30 &a 50 number of fraction

60

Fig. 3. Effect of ergotamine on the spontaneous 3H-5HT release. Fractions: (a) physiological solution, (b) 1W6 mol/l ergotamine in physiological solution, (c) physiological solution containing increased (x IO) K+, (d) physiological solution.

ergometrine or related drugs (Sakharov and Salanki, 1982; Sakharov and Kabotyanski, 1986). We may, firstly, suggest that not only gastropods but also other molluscs might be provided with a phylogenetically conservative neurochemical mechanism for inhibitory control of locomotion, The digging cycle, as described by Trueman (197.Q consists of six stages. Stage III of this descriptionrapid adduction of the valves-occurs after a major probe of the foot downwards (stage I) at the maximal

10

20

30 40 50 60 number of fraction

Fig. 4. Effect of ergotamine on the release evoked by high K+ solution, Fractions: (a) physiological solution, (b) physiological solution containing increased ( x 10) K+, (c) as (b) plus 10m6mol/l ergotamine, (d) physiological solution.

pedal extension

and initial dilation

of the foot (stage

II). The adduction of the valves is described as being cause of a pressure pulse which is responsible for further dilation of the foot to form a terminal anchor. The stage III is followed by contraction of at first the anterior then the posterior retractors, resulting in the shell being drawn into the sand and, ~~espondingly, the foot being drawn into the shell (stage IV), Then follows relaxation of adductors and opening of the shell (stage V), and a period in which the shell is static and the foot is protracting (stage VI). This description seems to be easily applicable to movements induced in our experiments by SHT. It appears that 5HT elicits a normal locomotor behaviour, and that fast movements of the valves recorded previously in SHT-treated mussels (Saianki, 1963; SalAnki et al., 1974) are in fact an element of this behaviour. Ergotamine and ergometrine seem to release locomotory movements with a minor change in phasing of the digging cycle: there is a delay just prior to the stage III. Interestingly, in the swimming pteropod mollusc Clione limacina, these and related ergots also produced a delay at a particular phase of the locomotory cycle (Sakharov and Kabotyanski, 1986). It seems possible that molluscs differing in modes of locomotion may have conservative neurochemical elements not only at higher levels of control of locomotory behaviour, but within their respective central pattern generators for locomotory movements as well. The behavioural findings raise the question, why are the effects of ergots similar to those of 5HT? This is why we investigated the possibility of direct activation of 5HTergic mechanisms by the ergots. The reuptake of the released transmitter substances is a generally recognized way of their inactivation in the nervous system. Since the monoamine oxidase has a low affinity in the ganglia of A. cygnea (Hiripi and Salanki, 1971), the reuptake mechanism which exists for 5HT in the CNS of Anodonta (Hiripi et al., 1975) may represent the main way of inactivation for 5HT in this animal. The inhibition of this mechanism would result in a SHT-like effect. Our results suggest, however, that the behavioural effect of ergotamine is hardly caused by inhibition of the 5HT uptake. Neither the mechanisms for SHT release seem to be

Drug-induced locomotory movements in Anodonta affected by ergotamine, as far as short-term action is concerned. At the same time, the content of SHT was somewhat lower in all three pairs of ganglia of the animals treated with ergotamine in comparison with those of the control animals. These experiments do not suggest the idea that ergots would activate directly 5HTergic mechanisms

in the brain of mussels. There remained another possibility, that was raised in the studies on the pteropod Clione limacina by Sakharov and Kabotyanski (1986) who tended to interpret their results in terms of functional antagonism of 5HT and dopamine. According to this conception the ergots in question block the dopaminergic transmission and therefore the effect of endogeneous 5HT appears enhanced. Due to the serotonindopamine antagonism in mussels (Salanki et al., 1974) this idea may explain our findings, although a direct proof to that is still lacking. Therefore in this stage of investigations a nonconventional mechanism for ergot-induced locomotor stereotypes in molluscs still cannot be ruled out. REFERENCES

Ascher P. (1972) Inhibitory and excitatory effects of dopamine on Aplysia neurones. J. Physiol., Lond. 225, 173-209.

Barnes G. E. (1955) The behaviour of Anodonta cygnea L. and its neurophysiological basis. J. exp. Biol. 32, 158-l 74. Gospe M. S. and Wilson W. A. (1981) Pharmacological studies of a novel dopamine-sensitive receptor mediating burst-firing inhibition of neurosecretory cell R15 in Aplysia californica.

J. Pharmac.

exp. Ther. 216, 368-377.

Hiripi L., Rakonczay Z. and Nemcsok J. (1975) The uptake

341

kinetics of serotonin, dopamine and noradrenaline in the pedal ganglia of the freshwater mussel (Anodonta cygnea L., Pelecypoda). Ann. Biol. Tihany 42, 21-28. Hiripi L. and Salanki J. (1971) The role of monoamine oxidase in the inactivation of serotonin in the nervous system and other tissues of Anodonta cygnea L. Ann. biol. Tihany 38, 31-38.

Sakharov D. A. and Kabotyanski E. A. (1986) Integration of behavior of a pteropod mollusc by dopamine and serotonin. Zh. obshch. Biol. 47, 234-245 (in Russian). Sakharov D. A. and Salanki J. (1982) Effects of dopamine antagonists on snail locomotion. Experientia 38, 109(rlO91.

Salanki J. (1963) The effect of serotonin and catecholamines on the nervous control of periodic activity in fresh-water mussel (Anodonta cygnea). Comp. Biochem. Physiol. 8, 163-171.

Salanki J. (1966) Comparative studies on the regulation of the periodic activity in marine Lamellibranchs. Camp. Biochem.

Physiol.

18, 829-843.

Salanki J. and Balla Z. (1964) Ink-lever equipment for continuous recording of activity in mussels. Ann. Biol. Tihany 31, 117-121. Salanki J., Glaizner B. and Labos E. (1970) On the temporal organization of periodic and rhythmic activity in freshwater mussel (Anodonta cygnea L.). J. interdiscipl. Cycle Res. 1, 123-134. Salanki J., Hiripi L. and Nemcsok J. (1974) Regulation of periodicity by monoamines in the mussel Anodonta cygnea L. J. interdiscipl.

Cycle Res. 5, 277-285.

Shozushima M. (1984) Blocking effects of serotonin on inhibitory dopamine receptor activity of Aplysia ganglion cells. Jap. J. Physiol. 34, 225-243. Trueman E. R. (1975) The Locomotion of Soft-bodied Animals. Edward Arnold, London. Walker R. J., Woodruff G. N., Glaizner D., Sedden C. D. and Kerkut G. A. (1968) The pharmacology of dopamine receptors of specific neurones in the snail Helix aspersa. Camp. Biochem.

Physiol. 24, 455469.