Corny. Biochum. Phwd. Vol. 70A, pp 397 lo 403, 1981 0300.9629/81:011397-07SO2 00/O Prmted m Great Britam All rxghts reserved Copyright 0 1981 Pergamon Press Ltd

ELECTRICAL ACTIVITY DURING A “SIMPLE” BEHAVIOUR: SPINE-POINTING IN A SEA URCHIN

B. H. PETERS and G. A. B. SHELTON

Department of Zoology and Comparative Physiology, Queen Mary College, University of London, Mile End Road, London El 4NS, and Department of Zoology, South Parks Road,

Oxford OX1 3PS. U.K.

(Rrceiued 9 March 1981)

Abstract-l. A description is given of “spine-pointing” behaviour in the regular echinoid Echinus escu- lentus L.

2. Extracellular electrophysiological records from spine bases show bursts of impulses correlated with spine-pointing. These are interpreted as action potentials from a large population of small muscle fibres.

3. Mechanical stimuli evoke both behavioural and electrical responses but only very large electrical stimuli are effective.

4. Pharmacological experiments suggest the presence of two drug receptor types at the spine base: excitatory cholinergic receptors with nicotinic properties, and inhibitory receptors with a-adrenergic properties.

5. The demonstration of both peripheral excitation and inhibition suggests that the control of spine- pointing is more complex than supposed previously.

INTRODUCTION

Studies of the behaviour and physiology of echino- derms have often been undertaken to elucidate “simple” methods of co-ordination in a deuterostome (Romanes, 1885; von Uekiill, 1900; Kinosita, 1941; Smith, 1950; Kerkut, 1954; Bullock, 1965). Electro- physiological techniques offer an obvious approach, but have had only limited success when applied to echinoderms. Takahashi (1964) Sandeman (1965), Millott & Okumura (1968), Binyon & Hasler (1970) and Podol’skii (1972) have all recorded electrical ac- tivity in radial nerve cords, but only Brehm (1977) has recorded through-conducting unitary potentials. Muscle potentials have been recorded by Prosser et

al. (1951) and Cobb (1968) (see also Pentreath & Cobb, 1972). An alternative to the purely electrophy- siological approach is offered by pharmacological studies. These have been reviewed by Pentreath & Cobb (1972); more recent work includes that of Florey et al. (1975) Tsuchiya & Amemiya (1977) Shelkovnokov et al. (1977) and Prosser & Mackie (1980). Whilst providing valuable data on possible transmitter substances, these studies have relied on indirect, non-electrophysiological methods. Neither approach has been used in conjunction with the other in any detailed published echinoderm study to date. Our knowledge is thus fragmentary. In this paper we report our attempt to combine these methods in an investigation of a “simple” echinoderm behaviour: “spine-pointing” in an echinoid.

The responses of echinoid test appendages vary in complexity from the jaw closure reflex in pedicellariae (Campbell & Laverack, 1968) to localized whole appendage responses (Bullock, 1965; Campbell, 1973) and finally, complex centrally co-ordinated move- ments e.g. in locomotion and photic responses (Mil- lott, 1966). There is evidence (Romanes, 1885; Bul- lock, 1965) that echinoid spine-pointing in response to stimuli applied to the test epithelium is co-ordinated

by the basiepithelial plexus described in the last cen- tury (see Smith, 1965, for review). Bullock (1965) has shown that the convergence of spines in response to a light tactile stimulus (spine-pointing) spreads decre- mentally, (Fig. 1) and is not conducted around cuts in the nerve plexus and epithelium. Neuroid conduction has not been demonstrated in adult echinoids.

Peripherally-mediated spine responses offer an ac- cessible system for investigation by the behavioural neurobiologist (Shelton, 1975). We report here the first successful electrical recordings from spine muscles of the regular echinoid Echinus esculentus L., together with preliminary pharmacological data. The combination of these approaches yields new insights into the control of test appendages in echinoids.

MATERIALS AND METHODS

Live, adult E. esculentus were supplied by the Marine Biological Station, Millport, Isle of Cumbrae, and main- tained at 12°C in a closed-circuit seawater system.

Experiments were performed both on whole animals and on dissected preparations. Most of our observations were performed on the latter since they were easier to manipu- late but produced results indistinguishable from those obtained from whole animals. Coelomic fluid was drained carefully during dissection to prevent it from touching the test epithelium. The viscera were removed and, after thorough washing in clean seawater, 2 cm squares of test were excised from the interambulacra. The spine tips were trimmed to reduce mechanical interference with the record- ing electrodes.

Extracellular polythene suction electrodes (tip diameter 150 pm) were used in conjunction with conventional differ- ential preamplifiers and visual display. All experiments were carried out with preparations immersed in a bath containing 300 ml of seawater at 1&15”C.

Drugs were applied by syringe in 0.1 ml aliquots in the immediate vicinity of the spine muscles on which the recording electrode was placed. The concentrations given refer to the solutions expelled from the syringe and are therefore maximum concentrations. A fresh preparation

397

398 B. H. PETERS and G. A. B. SHELTON

I 1 Fig. 1. Spine-pointing in Ethinus c~~lentu.s: side view of spines on a portion of test showing decremen- tal spread of the spine convergence response following mechanical, eiectricai or chemical (ACh) stimu-

lation applied at the point marked by the arrow. Scale bar = 15 mm.

was used for each experiment. The following drugs were used: acetylcholine chloride (BDH), adrenaline tartrate BP (Boots), y-amino-n-butyric acid (Sigma), L-arterenoi hydro- chloride (Sigma), atropine sulphate (Sigma), decametho- nium iodide (ICN). eserine (Sigma). L-alutamic acid (Sigma). glycine hydrochloride (&ma)_ ~-hydroxytryp- tamine (Sigma), 3-hydroxytyramine (Sigma), or-isoprotere- nol hydrochloride (Sigma), m-muscarine chloride (Sigma), nicotine sulphate (Sigma), oL-octopamine hydrochloride (Sigma). r+-phenylephrine hydrochloride (Sigma), pilocar- pine hydrochloride (Sigma), oL-propranolol hydrochloride (Sigma), Rogitine (phentolamine mesylate BP, Ciba), ~~-tubocu~~rine chloride (Sigma) tyramine hydrochloride (Sigma).

Material for electron microscopy was fixed for 90 min in 3”,, glutaraldehyde and post-fixed for 60 min in lo,, osmium tetroxide, both in 0.1 M Sori;nsen’s phosphate buffer (pH 7.2) containing 0.45 M sucrose. Specimens were decalcified by agitation for 18-24 hr in a 1: 1 mixture of 2”,, ascorbic acid and 0.3 M sodium chloride (Dietrich & Fontaine, 1975). TAAB resin was used as embedding medium: ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Siemens Eimiskop.

Anatoq RESULTS

The gross anatomy of a spine base in E. ~scM~~ntus is shown diagrammatically in Fig. 2 and in the low-

power electron micrograph, Fig. 3. A cylinder of smooth muscle, divided into radial sheets, lies just below the epithelium and is inserted at either end into the calcareous skeleton of the test and the spine ossi- cle. A broad ring of nervous tissue, continuous with the basiepithelia~ plexus, is associated closely with the muscle cylinder. The “catch apparatus” (Takahashi, 1967a, b) which lies within the smooth muscle cylinder is composed largely of extracellular collagen fibres.

Preliminary experiments showed that electrical ac- tivity could not be recorded reliably from the basi- epithelial plexus overlying the test. The fleshy spine bases, however, proved an effective site for extracellu- lar recording. When the electrode was positioned on a spine base, variable numbers of impluses were detected. These were thought to arise in response to the mechanical irritation due to the electrode place- ment and declined to a low frequency within 2min. Figure 4a shows an impulse recorded in this way. The potentials were typically biphasic, 50-100 msec in duration and up to 150~1V in amplitude. Extensive tests were performed to distinguish between electrical depolarizations and movement artifacts. Even the

! 1 I

Fig. 2. Diagrammatic representation of a vertical section through the base of a single spine, showing the arrangement of nerve and muscle. The positions of the recording electrode and drug applicator are also indicated. a-spine ossicle; bb-ball-and-socket joint: c-catch apparatus (collagen); d-smooth muscle; e-nerve ring; f--epithelium; g--test ossicle; h-nerve plexus; i&recording electrode: j-drug appli-

cator. Scale bar = 1 mm.

Electrical activity in a sea urchin 399

I I Fig. 3. Low power transmission electron micrograph showing a slightly oblique vertical section through a decalcified spine base. Lettering as Fig. 2. N.R. collagen fibres of catch apparatus are extracellular.

Scale bar = 11 p. For a full description of the ultrastructure of the spine base see Peters (1981).

most rapid movements of the electrode tip produced “responses” with a much longer time course than the potentials described above.

Preparations were rarely electrically-silent and “spontaneous” impulses occurred at irregular inter- vals. Bursts of impulses were usually associated with spine-pointing but single impulses were not ac- companied by visible movement. Simultaneous be- havioural observations showed a low level of unco- ordinated spine and pedicellaria movement.

Coordinated spine-convergence was evoked by mechanical stimulation of the test (Fig.l). Pointing of the spine towards the recording electrode (Fig. 2) was always associated with a burst of impulses (Fig. 4b). Much smaller and variable numbers of impulses (not in bursts) were recorded when the spine pointed in other directions. The level of “spontaneous” activity was enhanced for up to 1 min following mechanical stimulation.

Electrical stimulation evoked spine-pointing only

I I

Fig. 4. (a) “Spontaneous” action potential recorded extracellularly from the spine base. Time scale = 425 msec. (b) Excitation: response of spine muscles to a light mechanical stimulus to the test at a distauce of 1Omm from the recording electrode. The moment of stimulation is marked by the arrow.

Time scale = 4 sec.

400 B. H. PETERS and G. A. B. SHELTON

7-

6-

IJ 0 / I

I I I

0 I I

10 20 30 CO Time kl

50 0-a

6b 76

M L I

Fig. 5. (a) Graph showing impulse frequency vs time following the application (arrow) of lo-’ M ACh. (b) Response of spine muscles to the application of a lo-’ M solution of ACh (arrow marks moment of application). N.B. The burst length is prolonged compared with that produced by mechanical stimu-

lation (see Fig. 4(b)). Time scale = 5 sec.

when very large (50 V, 20 msec) stimuli were applied. Small spines responded to lower intensity stimulation than l_arge spines and the ophiocephalous pedicellar- iae had a still lower threshold. In all cases, however, behaviourally effective electrical stimuli produced unacceptably large stimulus artifacts on electrical records.

Acetylcholine and cholinergic agonists

The use of drugs proved completely reliable in evoking spine-pointing and did not produce large stimulus artifacts. 0.1 ml of 10m6 M acetylcholine (ACh) applied gently to the preparation always evoked a strong spine convergence response towards the point of application (Fig. 1). Control experiments in which drops of seawater were applied in a similar way evoked no spine movements. The electrical re- sponse to ACh solutions applied as shown in Fig. 2 was comparable to that evoked by strong mechanical stimulation: a high frequency burst of spikes (Fig. 5). Each burst comprised activity from a large number of small units. The following mean spike frequencies were obtained from the summed results of 9 separate experiments in which 0.1 ml 10e6 M ACh was applied. The mean spike frequency during the 20 set preceding application was 0.19 Hz. During the 20 set following, this increased to 2.95 Hz. During the next 20 set it remained at a high level (2.22 Hz). The appli- cation of ACh caused a 15-fold increase in spike fre- quency. Spike frequency declined subsequently to pre- stimulus levels (Fig. 5). Within the tested range of applied drug concentration (10d3 to lo-‘M) spike frequency and duration of response was a function of concentration. A latency of 3-5 set between the drug

application and the onset of the electrical response was always present.

The actions of ACh were mimicked by nicotine at concentrations down to lo-’ M and the response of preparations to ACh was desensitized by pre-treat- ment with nicotine. Decamethonium at concen- trations of 10e5 M similarly desensitized preparations to subsequent doses of either ACh or nicotine, and at higher concentrations elicited long-lasting and sub- stantial current flow at the recording site. Neither muscarine nor pilocarpine in physiological concen- trations evoked changes in impulse frequency or spine-pointing.

Chohnergic antagonists

d-Tubocurarine was inconsistent in its actions, reducing the response to ACh and nicotine in 4 out of 8 trials but rarely abolishing it. Atropine failed to block the response to ACh.

Anticholinesterase

Eserine (physostigmine) strongly potentiated elec- trical responses to the application of ACh and to mechanical stimuli. Even in the absence of externally- applied ACh, eserine caused a gradual but marked increase in the level of “spontaneous” activity (Fig. 6).

Adrenaline and related drugs

Application of lo-’ M adrenaline solution to the epithelium elicited a spine divergence response. This was associated with a reduction in spike frequency usually culminating in up to 1 min of electrical “silence” from the preparation (Fig. 7). The following

mean spike frequencies were obtained from the

Electrical activity in a sea urchin 401

10Hz 1OHz t . !

. . . .

. .

. . a . . . .

. l

; c . . 2 l-

l 2, .*

n ? l . oz.. .

5 .

. . . . . . . l * . . :. : .*** ,* . 0.0 l ..**.. l * : . . .

I I 100 150 20

I 250

I

Tome (s) 303 & 310

Fig. 6. Graph showing instantaneous impulse frequency vs time following the application of 0.1 ml 10m4 M eserine at time zero. Note the gradual increase in impulse frequency with time. This is inter-

preted as arising from the build-up of endogenously-released ACh in the tissue.

9

8

7

6

I

0 10 20 30 LO 50 60 Time (5)

b) T 1 I

Fig. 7. (a) Graph showing impulse frequency vs time following the application uf 3 x lo-’ M adrena- line. Inhibition of spiking leading to a prolonged electrical silence in the muscles was only seen in the presence of applied adrenaline but was completely reversible when the drug was washed out of the tissue. (b) Response of spine muscles to the application of a 3 x lo-’ M solution of adrenaline (arrow marks moment of application). There was a progressive and prolonged inhibition of spiking activity in

the muscle. Time scale = 9 sec.

402 B. H. PETERS and G. A. B SHELTON

summed results of 6 separate experiments in which 0.1 ml 3 x 10mh M adrenaline tartrate solution was applied. In each case a preparation showing a high level of spontaneous activity was used. In the 20sec preceding application of the drug, the mean spike fre- quency was 31.17 Hz. In the next 20 set this declined to 25.08 Hz and in the 20 set following it declined further to 5.08 Hz. The application of adrenaline caused a 6-fold reduction in spike frequency within 40 sec. Within 60 set the spiking had been reduced to zero in four preparations and to 0.1 Hz in the other two. The rate of reduction of spike frequency and the duration of the ensuing “silence” depended on drug concentration. The smallest effective applied adrena- line concentration was 3 x lo-’ M.

15Opm extracellular suction electrode would be expected to record from many myofibrils simul- taneously and it is not surprising, therefore, that the recordings show many different-sized units.

Noradrenaline was also effective in inhibiting im- pulses. but did so less strongly than adrenaline. The lowest eff‘ective concentration was 1O-6 M. No con- sistent inhibitory effects were produced by isoprena- line or dopamine. Octopamine at approximately 10mh M caused a reduction in spike frequency but at higher concentrations it was excitatory, as was phenylephrine.

The possibility that specific receptor sites may be present in the tissue is raised by the results of the pharmacological experiments. Caution is needed in the interpretation of these, however, since the drugs were not applied directly to isolated muscle cells. The tissue consisted of an epithelium, which is likely to be impermeable, overlying a population of muscle cells, nerve cells, supporting cells and collagen fibres. The substantial delay between the application of drugs and the muscle response suggests that a permeability barrier may be present which restricts entry of chemi- cals into the tissue (Pentreath & Cobb, 1972). Our recordings, moreover, were made from the surface of the epithelium. Bearing in mind the foregoing, our results are consistent with the following conclusions.

Adrenrrgic anrugonists

The inhibitory effects of adrenaline were blocked by pretreatment with phentolamine or ergotamine but not by propranolol.

5_Hydroxytryptamine, 7 -aminobutyric acid, glycine and tyramine produced no effect on spike frequency of spine-pointing. r-Glutamic acid evoked complex electrical responses unlike those observed during nor- mal behaviour : corresponding behaviour was uncoordinated and there was no maintained spine convergence.

DISCUSSION

The presence of ACh receptors with nicotinic properties is probable. ACh and its agonist nicotine were equivalent in their actions and although the sys- tem was blocked only weakly by d-tubocurarine, this is consistent with results from other “cholinergic” nerve/muscle preparations from echinoderms (Pen- treath & Cobb, 1972; Florey et cd.. 1975). ACh receptors in echinoderms could well have a different molecular configuration to those in vertebrates (Mendes ct trl.. 1970) which is less susceptible to block by d-tubocur- arine. That ACh is present in the tissue is suggested by the potentiating effect of eserine both on “sponta- neous” activity and on the responses to mechanical stimuli in the absence of applied ACh. ACh is known to be present both centrally and peripherally in the echinoderm nervous system and appears to be located in small, clear “synaptic” vesicles (Pentreath & Cobb, 1972). Vesicles of this type have been found in axon terminals innervating spine muscle blocks in E. rsctc- lentus (Peters, 1981).

Impulses recorded from spine bases during the spine-pointing response in E. esculentus are consistent with those recorded from muscles in other echinoid species. In their shape and time-course, the potentials resemble those recorded from the lantern retractor muscles of E. esculentus by Cobb (1968). The delayed response of the lantern muscle, and the activity recorded from ampullae of Strongylocentrotus by San- deman (1965), take the form of bursts of spikes similar to those we recorded from spine bases. A nervous origin for the impluses seems unlikely since the muscle innervation is confined to a region at their bases (Fig. 2: see Takahashi, 1967a), but impulses could be recorded with equal facility from all parts of the muscle. Though the “catch apparatus” is impli- cated in the control of spine position (Takahashi, 1967b). there is no evidence to suggest that it ever depolarizes or hyperpolarizes during spine move- ment; the collagen fibres which comprise this tissue are extracellular (Fig. 3 and Takahashi, 1967a). We conclude that the impulses recorded from our prep- aration were action potentials from the spine muscles.

The inhibitory actions of adrenaline on the spine system were prevented specifically by drugs, such as phentolamine, known to block a-adrenergic receptors in other systems. Related momoamines have been detected in the echinoderm central nervous system (Cobb, 1969) and Cobb suggested that they were con- fined to the interneurones. More recent work (Bach- mann & Goldschmid, 1978 ; Cobb & Raymond. 1979), however. has indicated that aminergic motor inner- vation may exist in echinoderms. Axon terminals con- taining small granular vesicles of the type character- istically associated with monoamines (Pentreath & Cobb, 1972) have been located in spine muscle blocks (Peters, 1981). The presence of such vesicles at these sites indicates that monoamines in motorneurones may act directly on the muscles but our electrophy- siological results do not rule out the possibility that monoamines act on the nerves associated with the spine muscles. Fluorescence histochemical studies are required to locate precisely the monoamines sus- pected in the spine system. More detailed data on the distribution of drug receptor sites could be obtained by the use of labelled antibodies.

Peters (1981) has shown that the spine muscles are The presence of an adrenergic peripheral inhibitory arranged in blocks, each of which consists of 2c-50 system which is indicated by our results has been myofibrils up to lO,~m in diameter. There is no evi- unsuspected hitherto. Cobb (1978) pointed out that dence for electrical connection between them. A there was no evidence that echinoderm muscle is sen-

Electrical activity in a sea urchin 403

sitive to any transmitter other than ACh. Although DIETRICH H. F. & FONTAINE A. R. (1975) A decalcification monoamines have a relaxing effect on echinoid lan- method for the ultrastructure of echinoderm tissues. tern muscle (Boltt & Ewer, 1963), the mechanism of Stain Techno/. so, 351-354.

their action is uncertain (Pentreath & Cobb, 1972). FLOREY E., CAHILL M. A. & RATHMAYER M. (1975) Excita-

Bullock (1965) suggested the presence of an inhibitory tory actions of GABA and of acetylcholine in sea urchin

system to explain divergence responses of echinoid tube feet. Camp. Biochenr. Physioi. 51C, 5-12.

spines but could give no data on its chemical basis. KERKUT G. A. (1954) The mechanisms of coordination of

Our results lead us to conclude that spine-pointing is the starfish tube feet. Behariour 6, 206-233.

controlled by a peripheral system utilizing both exci- KINOSITA H. (1941) Conduction of impulse in superficial

nervous system of sea urchin. Jap. J. ZooL 9, 221-232. tation and inhibition in a complex way. The apparent MII.LOTT N. (1966) Coordination of spine movements in neuroanatomical simplicity of the animal is mislead- echinoids. In Ph~siokog.r of Echinodermata (Edited by inn BOOLOOTIAN R. A.) DD. 465487. Interscience. New York. “Lb’

We suggest that the ultimate decision on the extent and direction of pointing in each spine is taken by the spine nerve ring. This receives input from the central nervous system, the basiepithelial nerve plexus and the epithelial sense organs of the spine itself. Spine position is a function of muscle tension and the visco- elastic properties of the catch apparatus; the spine nerve ring influences these parameters by the release of ACh and a monoamine.

Acknowledgement-We are grateful to Dr J. L. S. Cobb for his valuable criticism of the manuscript.

III

MILLOTT N. & OKUMURA H. (1968) The electrical activity of the radial nerve in I)iadema antillurttm Philippi. and certain other echinoids. J. exp. Biol. 48, 279-287.

PENTREATH V. W. & COBB J. L. S. (1972) Neurobiology of echinodermata. Biol. Rer. 47, 3633392.

PETERS B. H. (1981) An ultrastructural study of spine and pedicellaria bases in Echinus escuientus L., with special reference to the nervous and effector systems (in prep- aration).

PO~OL’SKII 0. G. (1972) Responses of the radial nerve of the starfish Asterin rubens to single and rhythmical elec- tric shocks, J. evol. Biochem. Physiol. 8, 454459 (in Russian).

PROSSER C. L., CURTIS H. J. & TRAVIS D. M. (1951) Action

REFERENCES potentials from some invertebrate non-striated muscles. J. cell. camp. Phrsiol. 38, 299-319.

BACHMANN S. & GOLDSCHMID A. (1978) Ultrastructural, fluorescence microscopical and microfluorimetric study of the innervation of the axial complex in the sea urchin Sphaerechinus granuluris (Lam.). Cell Tiss. Res. 194, 3 15-326.

B~NYOX J. & HASLER B. (I 970) Electrophysiology of the star- fish radial nerve cord. Camp. B~uchem. Physiol. 32, 747-753.

Borrr R. E. & EWER D. W. (1963) Studies on the myo- neural physiology of Echinodermata V. The lantern retractor muscle of Parechinus; responses to drugs. J. esp. Biof. 40, 727-733.

BREHM P. (1977) Electrophysiology and luminescence of an ophiuroid radial nerve. J. exp. Binl. 71, 213-227.

BIJLL~CK T. H. (1965) Comparative aspects of superficial conduction systems in echinoids and asteroids. Am. Zool. 5, 545-562.

CAMPBELL A. C. (1973) Observations on the activity of echinoid pedicallariae 1. Stem responses and their sig- nrficance. Mar. Behav. Physiol. 2, 33-61.

CA~~PBELL A. C. & LAVERACK M. S. (1968) The responses of pedicellariae from Echinus esculenrus L. J. exp. mar. Biol. Ecol. 2, 191-214.

COBB J. L. S. (1968) Observations on the electrical activity of the lantern of Echinus esculentus using extracellular recording electrodes. Camp. Biochem. Physiol. 24,311-315.

COBR J. L. S. (1969) The distribution of monoamines in the nervous system of echinoderms. Comp. Biochem. Physiol. 28, 9677971.

COBB J. L. S. (1978) An ultrastructural study of the dermal papullae of the starfish Asterias rubens. with special reference to the innervation of the muscles. Ceil Tiss. Res. 187, 515-523.

COBB J. L. S. & RAYMOND A. M. (1979) The basiepithelial nerve plexus of the viscera and coelom of Eleutherozoan Echinodermata. Cell Tiss. Res. 202, 155.-163.

PR~~SER C. L. & .MACKIE G. 0. (1980) Contractions of holothurian muscles. J. camp. Physiol. 136, 103-I 12.

ROMANES G. J. (1885) Jellrjsh, Srarfish und Sea Urchins. Kegan Paul, London.

SANDEMAN D. C. (1965) Electrical activity in the radial nerve cord and ampullae of sea urchins. J. e.up. Bioi. 43, 247-256.

SHEL~OVNIKO~ S. A., STARSHI~OVA L. A. & ZEIMAL E. V. (1977) Two kinds of cholinoreceptors on the non-visceral muscle of some Echinodermata. Comp. Biochem. Physiol. 58C, 1-12.

SHELTON G. A. B. (1975) Colonial behaviour and electrical activity in the Hexacorallia. Proc. R. Sot. Lond. B190, 239-256.

SMITH J. E. (1950) Some observations on the nervous mech- anisms underlying the behaviour of starfishes. Stnrp. Sot. exp. Bioi. 4, 196220.

SMITH J. E. (1965) Echinodermata. In Structure and Func- tion in the Nervous SJstrms of Incrrtebrates (Edited b) BULLOCK T. H. & HORRIDGE G. A.). pp. 1519-1558. Freeman, San Francisco.

TAKAHASHI K. (1964) Electrical responses to light stimuli in the isolated radial nerve of the sea urchin urchin Dia- dema setosum. Nature, Land. 201, 1342-1344.

TAKAHASHI K. (1967) The catch apparatus of the sea urchin spine I. Gross histology. J. Far. Sci. c/nil,. Tokyo IV 11, 1099120.

TAE;AHASHI K. (1967) The catch apparatus of the sea urchin spine II. Responses to stimuli. J. Fat. Sci. Univ. Tokyo IV 11, 121-130.

TSUCHIYA T. & AMEMIYA S. (f977) Studies on the radial muscle of an echinothuriid sea urchin, Asthenosoma 1. Mechanical responses to electrical stimulation and drugs. Comp. Biochem. Physiol. 57C, 69-73.

UEXK~~LL J. VON (1900) Die physiologie des Seeigelstachels. Z. Biol. 39, 73-112.