Nerve Net Pacemakers and Phases of Behaviour in the Sea Anemone Calliactis Parasitica

J. exp. Biol. 104, 231-246 (1983) 231 Erinted in Great Britain © The Company of Biologists Limited 1983

NERVE NET PACEMAKERS AND PHASES OF BEHAVIOUR IN THE SEA ANEMONE CALLIACTIS PARASITICA

BY IAN D. McFARLANE Department of Zoology, University of Hull, Hull HU6 7RX

(Received 9 November 1982 —Accepted 31 January 1983)

SUMMARY Bursts of through-conducting nerve net (TCNN) pulses, 20—45 min apart, were recorded from Calliactis attached to shells. Within 15—25 min of the anemones being detached the TCNN bursts suddenly became more frequent (only 4—11 min apart). Such bursts continued for several hours if re-attachment was prevented. In an attached anemone simultaneous electrical stimulation of the TCNN and ectodermal slow system (SSI) with 20-30 shocks at one every 5 s also led to more frequent TCNN bursts, whether or not detachment took place. If, however, the anemone remained attached, the intervals between bursts returned to the normal resting duration after about 90 min. In all cases the decay of the 4—11 min interval TCNN bursts involved a reduction in pulse number, not an increase in burst interval. Partial activation of the TCNN pacemakers followed stimulation of the SSI alone. It is suggested that in sea anemones the change from one behavioural phase to another is associated with a change in the patterned output of nerve net pacemakers.

INTRODUCTION The most obvious behavioural response of a sea anemone is that it contracts when touched. These fast contractions are triggered by a through-conducting nerve net (TCNN), (e.g. Pantin, 1935a; Josephson, 1966; Pickens, 1969). Other more complex, but still obvious, behavioural responses such as swimming in Stomphia coccinea and shell climbing in Stomphia and Calliactis parasitica involve additional conducting systems, the slow systems SSI and SS2 (Lawn, 1976a; Lawn& McFarlane, 1976; McFarlane, 1976). These complex behaviour patterns are also evoked by external stimuli. Far less noticeable are the slow, rhythmical, spontaneous shape changes shown by all species of sea anemone. These contractions are important as they may be the basic primitive form of neuromuscular activity in anemones (Ross, 1957) and components of these rhythms are incorporated into the complex behaviour patterns. Slow contractions have received less attention than the fast reflexes because the time scale is so much longer. Techniques such as kymograph or time-lapse cine' recording have shown that a particular sequence of muscular contractions (a phase) may be repeated for

words: Pacemakers, nerve net, sea anemone. 232 I. D. MCFARLANE several hours but even a brief stimulus can trigger a new phase that may persist several more hours (Batham & Pantin, 1950). The responses to stimuli may v according to the phase the animal is in. The only neurophysiological analysis of the coordination of slow contractions to date has shown that bursts of TCNN pulses in half-animal preparations of Calliactis parasitica are followed by a sequence of parietal and circular muscle contractions (McFarlane, 1974a). In this paper, neurophysiological evidence is presented for a change in behavioural phase in intact Calliactis. It is shown that detachment of the pedal disc leads to a long-lasting activation of TCNN bursts.

MATERIALS AND METHODS Calliactis parasitica, attached to Buccinum shells, were supplied by the Plymouth Marine Laboratory and kept at 15-19 °C. They were starved for a week before use. Animals were detached from the shells by being gently pulled. The pedal disc of a detached anemone will quickly re-attach to any object it touches, so to prevent this the anemone was suspended free of such contact by a large diameter suction electrode attached to the mid-column region. Recordings were made with polyethylene suction electrodes attached to tentacles. To minimize mechanical stimulation, only one recording electrode was used. This made pulse identification difficult (previous studies always compared pulses at two electrodes) and required selection of in- dividuals where all pulse types were clearly recognizable. Electrodes sometimes remained attached for several hours but usually there was a gradual deterioration in signal size, presumably due to tissue damage, after about 1 h. Consequently the electrode had to be moved periodically and this caused a short break in the record as a few minutes must elapse before pulses can again be confidently identified. Pulse identification was facilitated by connecting the pre-amplifier output to the oscilloscope via a Datalab DL902 transient recorder in the roll mode with a sample rate of 1 KHz. This gave a 2s period in which to identify each pulse. When a pulse was seen a contact was made which deflected a slow moving plotter pen. Different pulse types were indicated by different sized deflections. SSI pulses are relatively large (around 10-30/iV) and easily recognized. TCNN pulses have a distinctive shape but during a TCNN burst they usually become smaller and some may have been missed. SS2 pulses are small (often only 5 fiV) and are often lost in background activity (Fig. 1). The results are based upon more than 160h of recording from 10 anemones.

RESULTS Detachment causes frequent nerve net bursts: a pre-settling phase The spontaneous electrical activity of an anemone attached to a shell could generally be predicted by the anemone's appearance. If it was 'alert' (i.e. well expanded with tentacles outstretched) a typical recording was as shown in Fig. 2A. This record shows the distribution of electrical activity in the three conducting systems (TCNN, SSI and SS2). For this, and subsequent Figures, the activities of the systems are sum- marized in Table 1. Nerve net pacemakers in Calliactis 233

Fig. 1. Single suction electrode recordings from a tentacle of Calliactis. The first deflection in each record is the stimulus artefact. (A) Single shock (40 V, lms) to base of column elicits through- conducting nerve net (TCNN) pulse (•), probably a muscle action potential, an ectodermal slow conduction system (SSI) pulse (A), and an endodermal slow conduction system (SS2) pulse (A). (B) Same size shock, 30s later, evokes same pulses but the SS2 pulse is lost in a burst of complex pulses. Such complex activity is usually localized and associated with a twitch contraction of the tentacle. The SS2 pulse often has an amplitude of only 5 fiV and many may be missed during continuous monitoring of spontaneous activity, particularly during and just after TCNN bursts, when complex activity is common. Time scale: 500 ms.

SSI pulses were rarely detected in an attached, unstimulated anemone. Generally SS2 activity was at a low frequency — about one pulse every 25 s. There were also occasional short bursts of TCNN activity, usually 20-45 min apart and rarely containing more than seven pulses. The pulse frequency in the bursts was about one pulse every 4 s. A small, slow sphincter muscle contraction was visible after most bursts. The pattern of electrical activity recorded from an unstimulated, attached anemone will be termed 'resting phase' activity in this paper. Occasionally, anemones looked 'limp' with flaccid tentacles. Recordings then showed a much higher level of SS2 activity — around one pulse every 8 s. TCNN bursts were rarely detected (less than 1 h"1). This is atypical and such anemones were not used in the experiments described below. Electrical stimulation of the SS2 can cause a similar loss of muscle tone (McFarlane, 1976). When an anemone was detached by being pulled off the shell there was a marked change in TCNN activity. After a delay of 15—20 min, the widely-spaced TCNN bursts seen in the attached anemone were suddenly replaced by more frequent bursts, often only 5 min apart (Fig. 2B) and rarely as much as 11 min apart (Fig. 2C). The bursts contained more pulses than resting phase bursts; containing 9-12 pulses on average in different anemones (Table 1). A wide range of pulse intervals was seen n the bursts (3—6 s). There was rarely any noticeable change in the overall level I. D. MCFARLANE

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Fig. 2. Electrical activity patterns of attached and detached Calhactis. In each section the records are continuous and each line of the record covers 260 s. Pulses are shown by different-sized deflections of the plotter pen: TCNN > SSI > SS2. Periods of complex activity, sufficiently large to mask SS2 pulses, are shown by long-duration marks at SS2 amplitude. The long-duration marks at TCNN amplitude show where fast contractions occurred. A break in the record signifies a period when electrode contact was lost. (A) Resting phase: activity in an anemone attached to a shell. Two short- duration TCNN bursts are shown. The spontaneous activity of the resting phase is characterized by widely-spaced TCNN bursts and regular firing of the SS2. (B) Pre-settlement phase: activity in an anemone pulled off a shell 10 mm before the start of the record. Note the frequent TCNN bursts that in this case start 17min after detachment. (C) Another detached anemone, showing TCNN bursts some 11 min apart. This record starts 20min after detachment. Time scale: 1 min. of SS2 activity but there were always more SSI pulses (Table 1), probably caused by mechanical stimulation of the detached pedal disc margin (McFarlane, 1976). TCNN bursts in detached anemones were followed by a small slow sphincter muscle contraction, a peristaltic wave which passed down the column, and Nerve net pacemakers in Calliactis 235

1. Changes in conducting system activity following mechanical detachment and electrical stimulation

TCNN TCNN burst interval(s) pulses/burst SS2 SSI Duration of (mean) (mean) pulses/h pulses/h record(8)

Attached to shell (Fig. 2A)» 1300 4 133 4 1650 Pulled off shell (Fig. 2B) 324 12 132 7 2080 Pulled off shell (Fig. 2C) 670 9 105 19 2340 In pre-settling phase (Fig. 4A, lines 1-10) 310 10 104 32f 2600 End of pre-settling phase (Fig. 4B, lines 1-5) 280 4 17 0 1300 Resting phase (Fig. 4C, lines 6-17) 2460 7 157 0 3120 TCNN + SSI stimulation (Fig. 5) Before stimulationj >900 0 144 3 900 After first burst 390 8 18§ 3 2600 TCNN stimulation (Fig. 6) Before stimulation >1300 0 33 14 1300 After stimulation 800 6 128 2 3140 SSI stimulation (Fig. 7) Before stimulation >780 0 14§ 5 780 After stimulation 495 7 160 2 4890

• Fig. 2A and 2B from the same anemone, Fig. 2C from a different anemone. •)• Most of these pulses came in 'bursts'. | Not shown on record. § SS2 pulses not clear in this recording period - actual value probably higher. Most TCNN pulses occur in bursts so TCNN activity is here expressed as the interval between bursts (measured from the start of one burst to the start of the following burst) and the number of pulses per burst. For SSI and SS2, activity is shown as the number of pulses per hour. of the pedal disc. This will be termed the 'pre-settlingphase' in this paper as it appears to increase the chances of contacting a surface on which to settle. Pre-settling phase TCNN bursts sometimes persisted for several hours if the anemone was prevented from re-attaching. A kymograph was used to record the rhythmic movements (Fig. 3) by connecting the anemone to a light isotonic lever attached to a pin hooked into the sphincter muscle. The movements shown in Fig. 3A occurred at 5 min intervals and were probably contractions of the sphincter and circular muscles. On rare occasions the rhythm stopped (here for 30 min) and then restarted. Fig. 3B shows contractions of the same anemone 2h after it had re-attached to a shell; contractions were fewer, smaller and less rhythmic. Fig. 3C shows pre-settling phase contractions in another detached anemone. Note that the contractions vary in size: this may reflect variations in pulse number or frequency in the TCNN bursts. In 15 trials only two animals failed to show frequent contractions when detached. In both cases more than 60 % of the pedal disc was still covered by periostracum from shell. In all other trials there were never more than a few scraps of periostracum 236 I. D. MCFARLANE still adhering. Perhaps contact with shell or periostracum inhibits activation of TCNN pacemakers.

Change from pre-settling phase to resting phase The onset of the TCNN bursts after detachment was delayed but sudden: there was no evidence for a gradual build-up of TCNN activity. By contrast, the termination of the pre-settling phase was far less abrupt. Fig. 4 shows what may be a transition between the pre-settling phase and the resting phase. Here the pre-settling bursts stopped about 5 h after the anemone was detached. Note that although the bursts at the end of the pre-settling phase contained far fewer pulses and were much shorter than earlier bursts, the interburst interval was not increased. In fact the last few bursts were about 280 s apart whereas the first few bursts were 310 s apart (Table 1). Ap- parently the termination of the phase involves reduction in TCNN pulse number and not a gradual increase in interburst interval. Possibly the processes which control burst interval and burst content are only loosely linked. In this recording SS2 activity was particularly clear and there was obviously very little SS2 activity towards the end of the pre-settling phase, but a marked increase on return to the resting phase (Table 1). It is not clear if the increase in SS2 activity was the cause or the result of the phase change. In half-animal preparations, evoked or spontaneous SS2 pulses during the TCNN bursts can cause an increase in subsequent nerve net pulse intervals (McFar- lane, 19746). This inhibitory effect was not obvious in the pre-settling phase bursts

Fig. 3. Kymograph records of spontaneous contractions of the sphincter and upper column circular muscles in attached and detached Cailiactis. (A) Detached anemone. (B) Same anemone after re- attachment to a shell. (C) Another detached anemone. Time scale: ISmin. Nerve net pacemakers in Calliactis 237

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Fig. 4. Decay of pre-settling phase TCNN activity in a detached Calliactis. (A) Record starts 20 min after anemone was pulled off shell. The TCNN bursts are unusually long, possibly due to an inhibitory action of SS2 activity during the bursts. (B) Continues 169 min later and shows what appear to be the last five bursts of the pre-settlement phase followed by a 40 min delay before the first TCNN burst of the resting phase. The return to the resting phase activity pattern is here associated with an increase in SS2 activity. Note that the last few bursts of the pre-settling phase still occur at 5-min intervals but they are shorter and contain fewer pulses than bursts earlier in the phase. Time scale: 1 min. 238 I. D. MCFARLANE but, as pointed out before, it is often difficult to identify SS2 pulses during a burst. Such an inhibitory action may explain some of the more irregular pulse i vals seen in some bursts (e.g. Fig. 4).

Electrical stimulation of the pre-settling phase Activity similar to the pre-settling phase TCNN bursts can be evoked by simultaneous electrical stimulation of the TCNN and SSI. Prolonged SSI stimulation causes pedal disc detachment (McFarlane, 1969), but in the present study the anemone had to remain attached to control for the possibility that detachment itself causes TCNN burst activation. This can be done by stimulating on the oral side of a shallow cut around the mid-column region: SSI activity cannot pass this cut and reach the pedal disc. This method, however, damages the anemone and was not used. Sometimes just 20 shocks, at one every 4 or 5 s were used, as this is insufficient to cause detachment. Alternatively, if anemones were repeatedly detached by electrical stimulation (say once a day for a week) they then failed to detach in response to 30 shocks,

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Fig. 5. Activation of the prc-setthng phase pacemakers by simultaneous electrical stimulation of the TCNN and SSI (30 shocks at one every 5 s). The anemone did not detach. The record starts immediately after stimulation. Stimulation is followed here by 14min with very little activity of any sort. The bursts, believed to be part of the pre-settlement phase, are numbered 1 — 11. The two unnumbered TCNN bursts are thought to be part of the resting phase. Note that SS2 pulses were very small and difficult to detect during most of the recording period: there were probably many more than shown here. Time scale: 1 min. Nerve net pacemakers in Calliactis 239 fcesibly due to depletion of the chemicals that cause shedding of the pedal disc cement f yer. In Fig. 5 the mid-column region was stimulated with 30 shocks at one every 5 s at an intensity sufficient to excite both the TCNN and the SSI, but not the SS2. The anemone remained attached. At the beginning of the record very little SS2 activity was detected, compared with a mean frequency of one pulse every 25 s in the 15-min period immediately prior to stimulation (Table 1). The first TCNN burst came 17 min after stimulation, but, judging by its duration and its relation to subsequent bursts, it was not part of the pre-settling phase. The first burst of the pre-settling phase came 28 min after stimulation. A total of 11 bursts was recorded, the last coming some 90 min after stimulation. In some replicates of this experiment the bursts lasted just over 2h, but never for as long as with detached anemones. Apparently detachment is necessary for the activity to be maintained over an extended period. As in Fig. 4 the recording shows the decay of the phase. Again the interburst intervals remained quite constant (for the 11 bursts they are 390, 360, 430, 365, 410, 370, 410, 400, 370, 390 s), but the bursts shortened and the number of pulses fell, again to a minimum of three or four. Unfortunately SS2 pulses were not very clear over the latter half of this recording period and it is not known if the return to the resting phase was accompanied by increased SS2 activity. Note that a possible resting phase TCNN burst appeared before the first pre-settling phase burst and also between bursts 9 and 10. This independence of the two types of burst implies that they

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Fig. 6. Stimulation of the TCNN alone (30 shocks at one every 5 a) is followed by a marked increase in SS2 activity. The two TCNN bursts are short and 800s apart: they are probably resting phase bursts. Stimulation took place during the first break in line 6. Time scale: 1 min. 240 I. D. MCFARLANE

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Fig. 7. Stimulation of the SSI alone (20 shocks at one every 5 8) is followed by a marked increase in SS2 activity and irregularly-spaced, short-duration TCNN bursts. They may be pre-aettling phase bursts as the interburst interval is less than lOmin. Stimulation took place during the first break in line 4. Time scale: 1 min. originate in separate pacemakers, rather than in a single pacemaker that switches from one type of output to another. Stimulation of the TCNN or SSI alone was only partially successful in activating the pre-settling phase burst rhythm. Stimulation of the TCNN alone, with 30 shocks at one every 5 s, always led to slow sphincter muscle contraction and loss of electrode contact. In cases where the electrode could be quickly replaced it was obvious that contraction was followed by a marked increase in SS2 activity (Fig. 6 and Table 1). This increase was not seen when the SSI and TCNN were stimulated together. It may be a sensory response to the muscular contractions that followed stimulation. Eventu- ally TCNN bursts appeared, 36 min after stimulation. In no case did the bursts contain more than six pulses and were thus more likely to be resting phase bursts than pre-settling phase bursts. Also the interval between bursts was quite long - just over 16 min in this case. Stimulation of the SSI alone was more successful in evoking TCNN bursts, but somewhat less effective than stimulation of both systems together. In Fig. 7 the SSI was stimulated by an electrode attached to a shallow ectodermal flap (McFarlane, 1969). Here just 20 shocks at one every 5 s were given — too few to evoke detachment. SSI stimulation causes circular muscle contraction (McFarlane, 1976) and this £ Nerve net pacemakers in Calliactis 241

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Fig. 8. Electrical activity patterns of a detached anemone change when a shell is placed against the pedal disc. The pre-settling TCNN bursts continue but as the anemone settles additional, short, high- frequency TCNN bursts are seen (arrows). Time scale: 1 min.

have led to the observed increase in SS2 activity (Table 1). This time the first TCNN burst did not appear until 58 min after stimulation. Several bursts followed but at irregular intervals (95 s, 610s, 735 s).

Electrical activity during settlement of the pedal disc The pre-settlement phase is somewhat unnatural in that the anemone is prevented from re-attaching. If the pedal disc is allowed to start to settle there is a further change in the electrical activity to what may be termed the settlement phase. If a shell is gently brought into contact with the pedal disc when it is expanded there is immediate adhesion, perhaps due to sticky secretions from the ectodermal cells. The area of contact is then gradually increased and cycles of pedal disc expansion and contraction ensue as the anemone adjusts its position on the shell. This is equivalent to the last stage of shell-climbing behaviour, after the tentacles have released their hold on the shell (Ross & Sutton, 1961). Recordings made during this settlement phase show that pre-settlement TCNN bursts continue unchanged but in addition there are short, high-frequency bursts preceding alternate pre-settlement bursts (Fig. 8). The last high-frequency burst seen here only contained two pulses but its position suggests it is related to the two preceding bursts. The high-frequency bursts come at 550 s intervals whereas the pre- settlement bursts are 260-290 s apart. Possibly the high-frequency bursts represent activation of another pacemaker. There is also a marked increase in SSI activity, the pulses tending to come in bursts, particularly in the interval between one pre- settlement TCNN burst and the subsequent high-frequency TCNN burst. Future work will attempt to relate this complex pattern of electrical activity to the behaviour flkthe anemone. 242 I. D. MCFARLANE

DISCUSSION The behavioural correlates of the spontaneous TCNN bursts have not been clearly established. Needier & Ross (1958) described a sequence of rhythmic contractions, presumably evoked by TCNN bursts, in attached Calliactis parasitica. A sequence of parietal and circular muscle contractions has been described following TCNN bursts recorded from half-animal preparations of Calliactis (McFarlane, 1974a). Rhythmic behaviour in Calliactis has also been detected as cyclical variations in oxygen consumption, with cycle lengths of llmin and 34min (Brafield, 1980); presumably this is directly related to contraction cycles. A 34 min respiratory rhythm also occurs m Metridium senile and Actinia equina (Jones, Pickthall & Nesbitt, 1977). Many rhythmic movements in animals are thought to be coordinated by patterned output from the central nervous system. The cells that produce the rhythm are called central pattern generators (CPGs). Phasic sensory inputs or tonic excitatory inputs sometimes maintain the rhythm (Delcomyn, 1980; Selverston & Miller, 1980). In the leech, identified CPGs are small groups of synaptically-linked interneurones (Friesen, Poon & Stent, 1978; Peterson & Calabrese, 1982). Methods which can distinguish peripheral sensory feedback from central rhythm origin (Delcomyn, 1980) cannot be applied easily to sea anemones. Nevertheless TCNN bursts are thought to have a central origin as an intercalated TCNN pulse resets the intraburst rhythm (McFarlane, 19746). Also the pre-settling bursts precede, rather than follow, visible movements. TCNN bursts probably arise in CPGs somewhere in the nervous system. The most likely site is within the network of endodermal multipolar nerve cells. These are particularly obvious in the column of the swimming sea anemone Stomphia cocdnea where the soma are up to 30 /im across and bear up to twelve processes; there may be more than 10 000 such cells in a small Stomphia (Robson, 1963). The network is less developed in Calliactis parasitica, where the column multipolars are 10-15 /zm across and have three to five processes (Robson, 1965). Even so, at the quoted density of 50-100 per mm2 in fixed sphincter muscle preparations one can expect well over 10000 even in a small Calliactis. These cells appear to connect with sense cells and with the bipolar neurites of the through-conducting system (Robson, 1961). Metridium senile has a poorly developed multipolar system consisting of scattered tripolar cells in the mesenteries (Pantin, 1952). Metridium does not swim like Stom- phia or somersault onto shells like Calliactis, so perhaps restricted behaviour can be correlated with restricted neural networks. Assuming that the multipolar cells are the CPGs and that their output appears as TCNN bursts, what is the switch that changes their output as behaviour moves from one phase to another? In the case of the pre-settling phase bursts it appears to be simultaneous stimulation of the SSI and TCNN. Both systems can be excited by touch (McFarlane, 1969), with the lower column being particularly sensitive, so both must be stimulated when the anemone is pulled off the shell. There is anatomical evidence (Robson, 1961, 1965) for connections between the bipolar cells of the TCNN and the multipolar cells. There is no such evidence of SSI connections onto the multipolar cells, indeed we still do not know the cellular basis of the SSI. There is, however, physiological evidence for links between the ectodermal SSI and endodermal regior^ Nerve net pacemakers in Calliactis 243 ;son & McFarlane (1976) showed that an endodermal conducting system, termed tDelayed Initiation System (DIS), connects with the SSI. It was suggested that the DIS is the multipolar nerve net. Also we know (McFarlane, 1976) that SSI stimulation causes contraction of endodermal circular muscles: possibly this action operates via the multipolar net. In Stomphia coccinea the SSI has transmesogloeal connections with the pacemaker system that coordinates swimming (Lawn, 1980). Possibly then the multipolar cells receive input from at least two sources — the SSI and the TCNN. The Results show that simultaneous SSI and TCNN stimulation is more effective in evoking pre-settlement bursts than stimulation of either system alone. This may be because the inputs summate or because the pacemakers are prevented from full expression by the increased SS2 activity that accompanies stimulation of the systems separately. Earlier work has shown that SS2 activity has an inhibitory action on TCNN pacemakers (McFarlane, 19746). A puzzling feature of the response is the long delay, usually 15-25 min, between stimulation and the start of bursting. Whilst this may not be long in sea anemone terms, representing only one TCNN cycle at the resting phase rate, it does seem difficult to reconcile with the proposed model for multipolar cell activation, and may imply that pacemaker activation is an indirect (hormonal?) action of the conducting systems. As Croll & Davis (1982) point out, animals can perform more than one behaviour with the same set of muscles so there must be a number of CPGs capable of feeding different outputs to the same set of neurones. In Calliactis parasitica there may be no completely independent motor supplies: a pulse in the TCNN reaches all muscle groups. There are many reasons why this arrangement does not lead to behavioural inflexibility. First, muscle groups differ in their speed of contraction and their stimulus - response delay (Pantin, 1935a; Batham & Pantin, 1954). Secondly, the TCNN has both excitatory and inhibitory actions (Ewer, 1960; Lawn, 19766). Third- ly, different muscle groups respond optimally at different stimulus frequencies (Pan- tin, 1935a; Ross, 1957; McFarlane, 19746). Finally, muscles are affected by other conducting systems. Local contractions, in only part of a muscle group, may be coordinated by a 'primary nerve net' (Pantin, 19356). The two slow systems, SSI and SS2, may inhibit inherent muscular activity (McFarlane & Lawn, 1972; McFarlane, 1974a). Clearly then, considerable behavioural flexibility can result from changes in the TCNN burst pattern (e.g. burst length, pulse interval and interburst interval). Presumably a given phase represents a given output pattern from the TCNN pacemakers and whatever alters the output of these CPGs can be regarded as a switch which redirects the anemone's behaviour. Switching between behavioural phases is seen in other cnidarians. In Hydra at- tenuata changes in light intensity modulate the rhythm of contraction pulse trains (Taddei-Ferretti & Cordelia, 1976). A number of swimming activity rhythms have been detected in the hydromedusan Sarsia tubulosa (Leonard, 1982) and it was suggested that the animal can 'choose' from the set of available rhythms. In the actinian Stomphia coccinea, contact with a starfish evokes a sensory response lasting only a few seconds, followed by swimming flexions lasting for several minutes (Lawn, 1976a). The pacemakers are probably the multipolar cells of the column endoderm (Robson, 1963) but here they feed activity directly to the muscles responsible for ^fc and not into the TCNN. Bursts of TCNN pulses are, however, recorded 244 I. D. MCFARLANE from Stomphia during the latter stages of another behavioural phase, that of s' settling (I. D. Lawn & I. D. McFarlane, unpublished observations). Perhaps th are at least two groups of pacemaker cells, only one of which connects with the TCNN. Alternatively there may be only one group of cells but the output could be switched: such capabilities are present in molluscan multipolars (Haydon & Winlow, 1982). It is probable that the rhythmic contractions of burrowing anemones are coordinated by TCNN bursts. In the burrowing anemone Calamactispraelongus, there may be three different pacemaker systems with outputs to the TCNN (Marks, 1976). The pacemakers are activated by light but none are concerned with burrowing. A burrowing pacemaker has, however, been proposed in Phyllactis concinnata (Man- gum, 1970) where the digging rhythm is similar to the pre-settling rhythm in Calliac- tisparasitica, the contractions coming every 4 min at 21 °C. Such contractions can be maintained for as long as 6 h. No form of electrical stimulation was found which would elicit burrowing in an inactive animal. In structural terms actinians lack a centralized nervous system but the proposed network of multipolar cell pacemakers, able to produce various types of patterned output in response to a variety of inputs, shows one of the main functions associated with central nervous systems. It seems reasonable therefore to say that actinians have a diffuse central nervous system. A network of multipolar cells, extending as a single cell layer, can be regarded as an extended ganglion. Indeed, rather than look for tasks that can be performed by this simple nervous system it might be more instructive to ask what the system cannot do. Perhaps it cannot make associations between stimuli. Although habituation is a feature of the nervous system in actinians (Logan, 1975) claims of associative learning (see review by Ross, 1965) have not been rigorously tested. Another limitation of the anemone central nervous system is that each species has a restricted behavioural repertoire. Although some can swim by body flexions, some swim by tentacle flexions, some climb onto shells, some crawl, some fight, and some burrow (see review by Shelton, 1982), no single species is capable of all of these activities even though all species have more or less the same arrangement of muscles. Of course some behaviour patterns require particular specializations of the muscles and not all available behaviour is appropriate to the life style of a given species. I propose that restrictions are placed upon the behavioural repertoire of individual species and that such restrictions are to be found within the controlling neural networks and the range of available sensory inputs. Whereas the density of neurones and their interconnections in a three-dimensional ganglion may allow for complex switching based upon a wealth of information provided about the state of the body and the environment, the simple two-dimensional central nervous system of sea anemones, with its restricted sensory input, has only a limited ability to switch between behaviour patterns. Sensory limitation can be seen in the case of the SSI. The SSI is mechanically sensitive but also responds to chemicals (different chemicals in different species). In Tealiafelina var. lofotensis, now to be known as Urticina eques (Manuel, 1981), SSI chemoreceptors respond to food (McFarlane, 1970; McFarlane & Lawn, 1972). Alow frequency discharge (about one pulse every 10 s) produces oral disc expansion Nerve net pacemakers in Calliactis 245 ^ parts of the pre-feeding response. In Calliactisparasitica the SSI again shows alow frequency response to food extracts and evokes a pre-feeding phase but it can also be activated by a chemical in molluscan shells (McFarlane, 1976). In this case, however, SSI pulses appear at a much higher frequency, about one pulse every 5 s, and are accompanied by SS2 pulses. The pulses lead to pedal disc detachment and other parts of shell-climbing behaviour. Apparently Calliactis achieves a degree of behavioural flexibility by using SSI pulses to activate two behavioural phases - one at low frequency, one at high frequency. In captivity this strategy is not always successful: a hungry anemone sometimes detaches when fed. The most developed use of the SSI sensory response is seen in Stomphia coccinea, where a short, high- frequency, burst of SSI pulses evoked by contact with certain starfish, triggers swimming behaviour (Lawn, 1976a). Swimming is an escape response and thus must be executed quickly to be effective: hence the small number of SSI pulses required. Presumably the SSI in Stomphia is relatively insensitive to food or touch, if this were not the case, inappropriate stimuli might too readily evoke swimming.

REFERENCES BATHAM, E. J. & PANTIN, C. F. A. (1950). Phases of activity in the sea anemone, Metridium senile (L.) and their relation to external stimuli. J. exp. Biol. 27, 377-399. BATHAM, E. J. & PANTIN, C. F. A. (1954). Slow contraction and its relation to spontaneous activity in the sea anemone, Metridium senile (L.). J. exp. Biol. 31, 84-103. BRAFIELD, A. E. (1980). Oxygen consumption by the sea anemone Calliactis parasitica (Couch). J. exp. Biol. 88, 367-374. CROLL, R. P. & DAVIS, W. J. (1982). Motor program switching mPleurobranchaea. II. Ingestion and egestion in the reduced preparation. .7- comp. Physiol. 147, 143—154. DELCOMYN, F. (1980). Neural basis of rhythmic behavior in animals. Science, N.Y. 210, 492-498. EWER, D. W. (1960). Inhibition and rhythmic activity of the circular muscles of Calliactis parasitica (Couch). J. exp. Biol. 37, 812-831. FRIESEN, W. O., POON, M. & STENT, G. S. (1978). Neuronal control of swimming in the medicinal leech. IV. Identification of a network of oscillatory interneurones. J. exp. Biol. 75, 25-43. HAYDON, P. G. & WINLOW, W. (1982). Multipolar neurones olLymnaea stagnalis. I. Multiple spike initiation sites and propagation failure allow neuronal compartmentalization. jf. comp. Physiol. 147, 503-510. JACKSON, A. J. & MCFARLANE, I. D. (1976). Delayed initiation of SSI pulses in the sea anemone Calliactis parasitica: evidence for a fourth conducting system. J. exp. Biol. 65, 539-552. JONES, W. C, PICKTHALL, V. J. & NESBITT, S. P. (1977). A respiratory rhythm in sea anemones. J. exp. Biol. 68, 187-198. JOSEPHSON, R. K. (1966). Neuromuscular transmission in a sea anemone. J. exp. Biol. 45, 305-320. LAWN, I. D. (1976a). Swimming in the sea anemone Stomphia coccinea triggered by a slow conduction system. Nature, Land. 262, 708-709. LAWN, I. D. (19766). The marginal sphincter of the sea anemone Calliactis parasitica. II. Properties of the inhibitory response. J. comp. Physiol. 105, 301-311. LAWN, I. D. (1980). A transmesogloeal conduction system in the swimming sea anemoneSfompAia.7. <*£. Biol. 83, 45-52. LAWN, I. D. & MCFARLANE, I. D. (1976). Control of shell settling in the swimming sea anemone Stomphia coccinea. J. exp. Biol. 64, 419-429. LEONARD, J. L. (1982). Transient rhythms in the swimming activity of Sarsia tubulosa (Hydrozoa). J. exp. Biol. 96, 181-193. LOGAN, C. (1975). Topographic changes in responding during habituation to water-stream stimulation in sea anemones (Anlhopleura elegantissima). J. comp. physiol. Psychol. 89, 105-117. MCFARLANE, I. D. (1969). Co-ordination of pedal-disk detachment in the sea anemone Calliactis parasitica. J. exp. Biol. 51, 387-396. MCFARLANE, I. D. (1970). Control of preparatory feeding behaviour in the sea anemone Tealiafelina.J. exp. Biol. 53, 211 -220. E, I. D. (1974a). Excitatory and inhibitory control of inherent contractions in the sea anemone arasitica. J. exp. Biol. 60, 397-422. 246 I. D. MCFARLANE MCFARLANE, I. D. (19746). Control of the pacemaker system of the nerve net in the sea anemone parasitica. J. exp. Biol. 61, 129-143. MCFARLANE, I. D. (1976). Two slow conduction systems coordinate shell-climbing behaviour in the sea anemone Calliactis parasitica. J. exp. Biol. 64, 431-446. MCFARLANE, I. D. & LAWN, I. D. (1972). Expansion and contraction of the oral disc in the sea anemone TeaJia fclina. J. exp. Biol. 57, 633-649. MANGUM, D. C. (1970). Burrowing behaviour of the sea anemone Phyllactis. Biol. Bull. mar. biol. Lab., Woods Hole 138, 316-325. MANUEL, R. L. (1981). British Anthozoa (Synopses of the British fauna : new series; 18). London: Academic Press. 241 pp. MARKS, P. S. (1976). Nervous control of light responses in the sea anemone, Calamactispraelongus.J. exp. Biol. 65, 85-96. NEEDLER, M. & Ross, D. M. (1958). Neuromuscular activity in the sea anemone Calliactis parasitica (Couch). J. mar. biol. Ass. U.K. 37, 789-805. PANTIN, C. F. A. (1935a). The nerve net of the Actinozoa. I. Facilitation. J. exp. Biol. 12, 119-138. PANTIN, C. F. A. (19356). The nerve net of the Actinozoa. II. Plan of the nerve net.7. exp. Biol. 12, 139-155. PANTIN, C. F. A. (1952). The elementary nervous system. Proc. R. Soc. Land. B 140, 147-168. PETERSON, E. L. & CALABRESE, R. (1982). Dynamic analysis of a rhythmic neural circuit in the leech Hirudo medicinalis.jf. Neurophysiol. 47, 256-271. PICKENS, P. E. (1969). Rapid contraction and associated potentials in a sand-dwelling anemone. J. exp. Biol. 51,513-528. ROBSON, E. A. (1961). A comparison of the nervous systems of two sea-anemones, Calliactis parasitica and Metridium senile. Q. Jl microsc. Sri. 102, 319-326. ROBSON, E. A. (1963). The nerve-net of a swimming anemone, Stomphia coccinea. Q. Jl microsc. Set. 104, 535-549. ROBSON, E. A. (1965). Some aspects of the structure of the nervous system in the anemone Calliactis parasitica. Am. Zool. 5, 403-410. Ross, D. M. (1957). Quick and slow contractions in the isolated sphincter of the sea anemone, Calliactis parasitica. J. exp. Biol. 34, 11-28. Ross, D. M. (1965). The behaviour of sessile coelenterates in the light of some conditioning experiments. Anim. Behav. Suppl. 1, 43-53. Ross, D. M. &SUTTON, L. (1961). The response of the sea anemone Calliactis parasitica to shells of the hermit crab Pagurus bemhardus. Proc. R. Soc. Land. B 155, 266-281. SELVERSTON, A. I. & MILLER, J. P. (1980). Mechanisms underlying pattern generation in lobster stomatogastric ganglion as determined by selective inactivation of identified neurons. I. Pyloric system. J. Neurophysiol. 44, 1102-1121. SHELTON, G. A. B. (1982). Anthozoa. In Electrical conduction and behaviour in 'simple' invertebrates, (ed. G. A. B. Shelton), Oxford: Oxford University Press, pp. 203-242. TADDEI-FERRETTI, C. & CORDELLA, L. (1976). Modulation of Hydra attenuala rhythmic activity: phase response curve. .7- exp. Biol. 65, 737-751.