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Electrophysiology of the starfish radial nerve cord
Comp. Biochem. Physiol., 1970, Vol. 32, pp. 747 to 753. PergamonPress. Printedin Great Britain
ELECTROPHYSIOLOGY OF THE STARFISH RADIAL NERVE CORD JOHN BINYON and BASIL HASLER Royal Holloway College, Englefield Green, Surrey (Received 6 M a y 1969)
A b s t r a c t - - 1 . Electrically evoked propagated massed action potentials were recorded extracellularly from the radial nerve cord of starfish. 2. T h e conduction velocity (6.7-10.0 cm/sec) and absolute (60 msec) and relative refractory periods were approximately determined. 3. Drug action suggests that transmission is cholinergic. INTRODUCTION ELECTROPHYSIOLOGICAL s t u d i e s o n e c h i n o d e r m s are e x t r e m e l y scarce, p r i n c i p a l l y b e c a u s e t h e n a t u r e o f t h e tissues a n d t h e s m a l l size o f t h e cells s e e m to p r e c l u d e t h e u s e o f s o p h i s t i c a t e d i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s at t h e m o m e n t . ( T h e l a r g e s t a x o n s in t h e r a d i a l n e r v e s o f b o t h e c h i n o i d s a n d a s t e r o i d s are 0.1 tzm in d i a m e t e r ; C o b b & L a v e r a c k , 1966 a n d p e r s o n a l c o m m u n i c a t i o n . ) R e c e n t w o r k on t h e n e r v o u s s y s t e m has m o s t l y b e e n d e v o t e d to t h e E c h i n o i d e a ( T a k a h a s b i , 1964; S a n d e m a n , 1965; M i l l o t & O k u m u r a , 1968). T h e p r e s e n t s t u d y s h o w s t h a t t h e r a d i a l n e r v e c o r d s o f Asterias rubens a n d o t h e r c o m m o n B r i t i s h a s t e r o i d s have s i m i l a r p r o p e r t i e s to t h o s e d e s c r i b e d b y M i l l o t & O k u m u r a (1968) for Diadema antillarum. MATERIALS AND METHODS Specimens of Asterias rubens and other species were obtained mainly from Plymouth. T h e y were maintained in tanks of recirculated aerated sea water at 8-10°C and survived well, apart from the obvious difficulties experienced when animals spawned in such a comparatively small volume of water, (5-10 1 per animal). Lengths of radial nerve cord were dissected free from the arms and supported across cotton-wrapped electrodes of 0'010 in. dia. silver wire. Each pair of electrodes was usually spaced 2 m m apart. T h e preparation was kept moistened with sea water and excess water removed by blotting with strips of filter paper. A moist chamber made of perspex was used for some experiments, but proved inconvenient for the majority because the section of nerve cord could not easily be immersed in solutions without moving the electrodes relative to each other or the preparation. T h e pieces of nerve cord survived for up to 4 hr at room temperatures of 20-25°C and longer if kept cool. Electrical stimulation was by means of a Grass S.D.5 stimulator, and activity was recorded via a Tektronix RM122 preamplifier and a Tektronix 502A oscilloscope, using a Cossor 1428/2B oscilloscope camera. Suction electrodes of both glass and plastics were tried, but the small and unattached nerve cord did not readily lend itself to their use. T h e y were found to be inconvenient and 747
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JOHN BINYON AND BASIL HASLER
to give inconsistent results, and were not persisted with for this reason. A t t e m p t s to record f r o m the nerve cord in situ were not very successful for a n u m b e r of reasons. W h e n the radial nerve cord is touched, the ambulacral groove begins to close up, the tube feet contract and the whole arm is flexed away from the point of contact. Connection with the electrodes is therefore broken even w h e n using suction electrodes. T h e fragility of the tissues, combined with the lack of a rigid a m b u l a c r u m such as is found in echinoids, makes these animals almost impossible to restrain by means of pins, rubber bands or other devices. T h e noise level w h e n recording f r o m a whole animal was m u c h higher than with isolated preparations and this, coupled with the m o v e m e n t artefacts from the intact animal m a d e such recordings valueless. Pieces of radial nerve cord which had been excised and used were afterwards sectioned and examined microscopically in comparison with sections of whole arms. T h i s confirmed that the radial nerve and no other tissues were being removed. T h e preparation included varying amounts of hyponeural tissue, which because of its distribution, lateral and aboral to the bulk of the ectoneural tissue, was liable to be lost in dissection. H o w e v e r , experiments involving the longitudinal splitting of the cord showed that its absence had no effect on the responses recorded here.
RESULTS
Response of electrical stimulation In most preparations, stimulation by single square waves produced a typical biphasic potential (Fig. 1). T h e small size and long duration of this potential are remarkable. It is similar to those reported by Millot & Okumura (1968) from certain echinoids and, as they suggest, is presumably the sum effect of massed potentials in the very small axons which make up most of the echinoderm radial nerve cord (Kawaguti, 1965 etc.).
[ - w k15 V, 20 msec; normal polarity. Vertical scale = 800/zV; horizontal scale = 200 msec. FIG. 2. 15 V, 20 msec. Vertical scale - 800/~V; horizontal scale = 200 msec.
FIG. 1.
A stimulus of the order of 15 V for 20 msecs is necessary to produce a maximal response. In certain preparations a "shoulder" is to be seen on the rising phase of the potential (Fig. 3) and in several cases this was resolvable into a completely separate peak by recording over a sufficient distance (Fig. 5). This is presumably due to a group of axons which conduct significantly faster than the rest of the nerve cord. Unfortunately this "fast c o m p o n e n t " could not be seen or separated consistently and varied in its manifestation between identically prepared nerve cords from the same animal, using the same electrodes. T h e only way its presence could be demonstrated in most preparations was to increase the stimuli from just above threshold level, for the fast component has a lower threshold. Thus, in Fig. 4, the peak position of this component can be seen to move farther away from the stimulus artefact as the strength of stimulus is increased, and then to remain
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constant. This represents the fast component's appearance at its lower threshold followed by its occlusion by the rest of the potential as the latter grows in magnitude with increased strength of stimulation. Threshold values for the fast response, measured 4 m m from the point of stimulation, are of the order of 15 V, 0.3 msecs, and 0.5 V, 10 msecs. T h e threshold for the slower component is of course higher, but not clearly measurable because of the presence of the fast component.
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FIC. 3. 15 V, 20msec; stimuli at 0"5 sec intervals. Vertical scale = 800/zV; horizontal scale -- 400 msec. FIC. 4. 1, 2, 5 and 10 V, 10 msec; stimuli, superimposed. Vertical scale = 400/zV; horizontal scale -- 200 msec.
FIG. 5. 1 V, at 4, 8, 12, 16 and 20 msec; stimuli superimposed. Vertical scale -400/zV; horizontal scale = 200 msec. FIC. 6. 5 V, 1 msec; stimuli, 200/sec. Vertical scale = 400/zV; horizontal scale -200 msec. Conduction of the potential is strongly decremental, the decline in recordable size of the response with distance from the stimulating electrodes being almost logarithmic. T h e maximum distance at which any activity could be detected was about 25 mm. "Reverse polarity" stimulation, with the positive electrode nearer to the recording pair, produced an effect intermediate between that described by Millot & Okumura (1968) and that described by Sandeman (1965). Stimulation just above threshold produced a small potential of normal form. (Sandeman obtained normal, bus smaller, potentials.) As the strength of stimulation was increased, however, this was occluded by a positive potential change of form seen in Fig. 2. This prolonged hyperpolarization is as described by Millot & Okumura (1968), and is difficult to account for. We confirm their findings that such behaviour is a property of living nervous tissue only. It increases in size in proportion to the stimulating voltage up to 80 V at least, although a 15 V, 20 msec stimulus of normal polarity is sufficient to elicit a maximal response. When conduction has been blocked with eserine (see below), normal stimulation produces a long depolarization of similar form. After prolonged exposure to eserine, from which recovery would be incomplete, this response is not produced by "normal" or "reverse polarity" stimulation.
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The conduction velocity of the typical undivided potential was found to be 6.5-8.8 cm/sec. In a few preparations in which the two components could be clearly distinguished, the velocities of fast and slow components were found to be 5.0-5-5 and 6.9-8.8, respectively. One or two specimens of other asteroids were examined and similar responses to electrical stimulation of the radial nerve cords recorded. The mean conduction velocities were estimated to be as follows: Marthasterias glacialis, 6.7-8.0 cm/sec, Astropecten irregularis, 8"9 cm/sec, Solaster papposus, 10.0 era/see. Immersion in a bath of sea water cooled to O°C resulted in a reduction in the velocity of up to 25 per cent, but both components were affected similarly, and there was no useful separation of the two.
Multiple electrical stimulation There is an absolute refractory period of approximately 60 msecs. The relative refractory period is of indeterminate length, because of the slow recovery of the potential to its previous height towards the end of the period, but it is of the order of 6-7 sec. When a series of stimuli at high frequency is administered, a potential is transmitted only in response to the initial stimulus. At the end of the train of stimuli, however, there is a positive deflection of the trace, as is seen in the case of "reverse polarity" stimulation (Fig. 6). When a series of two or more stimuli, with a time interval greater than the relative refractory period, is administered, the decline in height of the resulting potentials is greater in the case of the slow component than in that of the fast (Fig. 3). This resembles the findings of Millot & Okumura (1968) in D. antillarum.
Mutilation of the nerve cord Vigorously crushing the nerve cord between the stimulating and recording pairs of electrodes eliminates all recordable nervous activity, as might be expected. Cutting partly across the cord in this position reduces the size of the potential and it is apparently immaterial whether the nerve is cut in the centre or as a hemisection from any side. If cuts are made from alternate sides so that they overlap, i.e. so that the nerve is interrupted in all parts of its cross-sectional area at some point between the stimulating and recording electrodes, the potential is reduced to about half of its original height. However, even a series of up to six overlapping cuts from alternate sides does not abolish the potential completely, although it is much reduced. SpiRting the nerve cord longitudinally or scraping away the oral surface, as described by Kerkut (1955), effects a reduction in the height of the potential, but the form is unaltered. The size of the potential in these cases is approximately in proportion to the size of the strip of nervous material remaining. There is, therefore, no evidence for the two components being associated with a division of the nerve cord into tracts on a macroscopic scale.
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Effect of artificial sea water and sea water of reduced salinity A simple artificial sea water was prepared using isotonic solutions of the chlorides of the four major metals according to the table given by Pantin (1946). Survival and behaviour of the preparation in this solution was just as in normal sea water. Echinoderms in culture are notorious for their intolerance of sea water substitutes. When the nerve cord is placed in dilutions of natural sea water, the survival time and response to electrical stimulation are unaltered at a concentration of 25~o. T h e potential shows an immediate decrease in height of about 10 per cent after immersion in sea water at 20~o, with further decrease as the preparation is kept. Lower concentrations still cause an immediate and rapid decrease to total extinction of the potential. T h e salinity tolerance of whole Asterias rubens from British shores was shown by Binyon (1961) to be 23~oo. The tolerance of the radial nerve cord, which is very exposed to the environment for such a major organ would therefore seem to be of the same order as that of the whole animal. Immersion in calcium-free artificial sea water and in artificial sea water containing ten times the normal potassium concentration both cause abolition of the potential within minutes, with a rapid recovery when the nerve cord is returned to water of normal composition (Fig. 7). The speed of action of such solutions is in great contrast to the slow reaction of the nerve cord to poisons and blocking agents.
Fio. 7. 20 V, 20 msec; stimuli administered: (a) to fresh nerve; (b) after 2 rain in calcium-free artificial sea water; (c) 2 min after return to normal sea water; (d) 5 min after return to normal sea water. Vertical scale = 800/zV; horizontal scale = 200 msec.
Effect of drugs Immersion of the nerve cord in a strong solution of potassium cyanide in sea water destroys any response in about 10 min. This is comparatively slow compared with the action of the artificial sea water solutions described above. Dilute hydrochloric acid, by contrast, is effective almost immediately. A similar slowness of reaction was found with pharmacological agents; very strong solutions or prolonged exposures were necessary. Immersion of the nerve cord in a 0.01 M solution of eserine sulphate in sea water blocked conduction of the potential completely after 30 min, apart from the long wave of depolarization referred to above. Recovery of the potential to its previous height was complete after 60 rain back in sea water. Longer exposures to such a solution also slowly deleted the remaining response, but recovery was slow and incomplete. Comparatively strong solutions of eserine are necessary to achieve this complete block, and solutions more dilute than 0.01 M have little effect in the life of the preparation. Strong solutions of veratrine alkaloids, of atropine, and of procaine exert a similar blocking effect to eserine, but recovery is slow and never complete.
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JOHN BINYON AND BASIL HASLER
The acetylcholinesterase inhibiting agent 62C47K (1,5-bis(4-trimethylammoniumphenyl)pentan-3-diiodide, Burroughs-Welcome) was found to irreversibly reduce the size of the potential, but not to extinguish it completely in the life of the preparation. The butyrylcholinesterase inhibitor iso-OMPA (Tetraisopropylpyrophosphoramide, Koch-Light) did not affect the performance of the preparation in any way. Animals exposed for up to 7 days to sea water containing 5 ~g/ml soluble reserpine showed the symptoms of inanition and slow righting reaction described by Cottrell (1967) but again the response of the nerve cords was unaffected. CottreU (1967) showed that exposure of animals to 3 ~g/ml soluble reserpine for 5 days depleted the dopamine content of the cords by 50-70 per cent and the noradrenaline content by 20-50 per cent. The detergent BP1002 at 25 p.p.m, is described by Smith (1968) as killing the majority of A. rubens in 24 hr. The nerve cords from two animals which had undergone such an exposure (and survived) were also quite normal. The detergent might have been expected to affect the exposed nervous tissue rapidly. DISCUSSION The records obtained from asteroid radial nerves resemble in many respects those obtained from echinoids by Millot & Okumura (1968). There is also a resemblance to those reported by Sandeman (1965), in as far as the two components of the potential recorded overlap more. The actual form differs somewhat from Sandeman's (1965) results, but as Millot & Okumura (1968) point out, the precise disposition of the recording electrodes may affect this, and Sandeman does not always give these details. The double character of the massed potential is presumably due to two conducting systems within the nerve of differing speed. No evidence exists so far that these forms of conduction occur in macroscopically recognizable tracts in the nerve cord. The inconsistent separation of the two peaks is difficult to account for, but may be related to the absence of any well-defined motor response of the type shown by Millot & Okumura (1968). In view of the general similarity in the results obtained from Asterias and Diadema it is a pity that a clear correlation of electrical stimulation of the radial nerve cord with motor activity has not been shown so far, although the work of Kerkut (1955) suggests that such a relationship exists. Blocking of the potential by eserine and 62C47K suggests that transmission of the potential is cholinergic, and predominantly mediated by acetylcholine. In view of the high concentrations of these agents necessary to produce a block, the lack of effect of iso-OMPA does not rule out transmission via butyrylcholine completely. The recent work of Pentreath & Cottrell (1968) has shown that acetylcholine is to be found in significant quantities in both the ectoneural and hyponeural regions of the nerve cord. The lack of effect of exposure to reserpine on the nerve cord, despite an obvious
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effect o n t h e a n i m a l as a whole, suggests t h a t c a t e c h o l a m i n e s play little part i n t h e t r a n s m i s s i o n of the r e s p o n s e r e c o r d e d here.
Acknowledgement--This work was supported by a grant from the Science Research Council. REFERENCES BINYON J. (1961) Salinity tolerance and permeability to water in the starfish Asterias rubens L. ft. mar. Biol. Ass. U.K. 41, 161-174. COBB J. L. S. & LAVERACKM. S. (1966) The lantern of Echinus esculentus ( L . ) - - I I . Fine structure of hyponeural tissue and its connections. Proc. R. Soc. B 164, 641-650. COTTRELL G. A. (1967) Occurrence of dopamine and noradrenaline in the nervous tissues of some invertebrate animals. Br. ft. Pharmac. Chemother. 29, 63-69. KAWAGUTI S. (1965) Electron microscopy on the radial nerve of the starfish. Biol. ft. Ohayama Univ. 11(1-2), 41-52. KEaKUT G. A. (1955) The retraction and protraction of the tube feet of the starfish. Behaviour 8, 112-129. MILLOT N. & OKUMURAH. (1968) The electrical activity of the radial nerve in Diadema antillarum Philippi and certain other echinoids, ft. exp. Biol. 48, 279-287. PANTIN C. F. A. (1946) Notes on Microscopical Techniques for Zoologists. Cambridge University Press. PENTREATH V. A. & COTTRELL G. A. (1968) Acetylcholine and cholinesterase in the radial nerve of Asterias rubens. Comp. Biochem. Physiol. 27, 775-785. SANDEMAN D. C. (1965) Electrical activity in the radial nerve cord and ampullae of sea urchins, ft. exp. Biol. 43, 247-256. SMITH J. E. (1968) (Editor) "Torrey Canyon" Pollution and Marine Life. Cambridge University Press. TAKAHASHIK. (1964) Electrical responses to light stimuli in the isolated radial nerve of the sea urchin Diadema setosum (Leska). Nature, Lond. 201, 1343-1344.
Key Word Index--Action potential; calcium-free sea water; cholinergic transmission; conduction velocity; detergent; dilute sea water; drug action; electrophysiology; fast component; radial nerve cord; reversed polarity; salinity; slow component; starfish; tracts.
ELECTROPHYSIOLOGY OF THE STARFISH RADIAL NERVE CORD JOHN BINYON and BASIL HASLER Royal Holloway College, Englefield Green, Surrey (Received 6 M a y 1969)
A b s t r a c t - - 1 . Electrically evoked propagated massed action potentials were recorded extracellularly from the radial nerve cord of starfish. 2. T h e conduction velocity (6.7-10.0 cm/sec) and absolute (60 msec) and relative refractory periods were approximately determined. 3. Drug action suggests that transmission is cholinergic. INTRODUCTION ELECTROPHYSIOLOGICAL s t u d i e s o n e c h i n o d e r m s are e x t r e m e l y scarce, p r i n c i p a l l y b e c a u s e t h e n a t u r e o f t h e tissues a n d t h e s m a l l size o f t h e cells s e e m to p r e c l u d e t h e u s e o f s o p h i s t i c a t e d i n t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s at t h e m o m e n t . ( T h e l a r g e s t a x o n s in t h e r a d i a l n e r v e s o f b o t h e c h i n o i d s a n d a s t e r o i d s are 0.1 tzm in d i a m e t e r ; C o b b & L a v e r a c k , 1966 a n d p e r s o n a l c o m m u n i c a t i o n . ) R e c e n t w o r k on t h e n e r v o u s s y s t e m has m o s t l y b e e n d e v o t e d to t h e E c h i n o i d e a ( T a k a h a s b i , 1964; S a n d e m a n , 1965; M i l l o t & O k u m u r a , 1968). T h e p r e s e n t s t u d y s h o w s t h a t t h e r a d i a l n e r v e c o r d s o f Asterias rubens a n d o t h e r c o m m o n B r i t i s h a s t e r o i d s have s i m i l a r p r o p e r t i e s to t h o s e d e s c r i b e d b y M i l l o t & O k u m u r a (1968) for Diadema antillarum. MATERIALS AND METHODS Specimens of Asterias rubens and other species were obtained mainly from Plymouth. T h e y were maintained in tanks of recirculated aerated sea water at 8-10°C and survived well, apart from the obvious difficulties experienced when animals spawned in such a comparatively small volume of water, (5-10 1 per animal). Lengths of radial nerve cord were dissected free from the arms and supported across cotton-wrapped electrodes of 0'010 in. dia. silver wire. Each pair of electrodes was usually spaced 2 m m apart. T h e preparation was kept moistened with sea water and excess water removed by blotting with strips of filter paper. A moist chamber made of perspex was used for some experiments, but proved inconvenient for the majority because the section of nerve cord could not easily be immersed in solutions without moving the electrodes relative to each other or the preparation. T h e pieces of nerve cord survived for up to 4 hr at room temperatures of 20-25°C and longer if kept cool. Electrical stimulation was by means of a Grass S.D.5 stimulator, and activity was recorded via a Tektronix RM122 preamplifier and a Tektronix 502A oscilloscope, using a Cossor 1428/2B oscilloscope camera. Suction electrodes of both glass and plastics were tried, but the small and unattached nerve cord did not readily lend itself to their use. T h e y were found to be inconvenient and 747
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JOHN BINYON AND BASIL HASLER
to give inconsistent results, and were not persisted with for this reason. A t t e m p t s to record f r o m the nerve cord in situ were not very successful for a n u m b e r of reasons. W h e n the radial nerve cord is touched, the ambulacral groove begins to close up, the tube feet contract and the whole arm is flexed away from the point of contact. Connection with the electrodes is therefore broken even w h e n using suction electrodes. T h e fragility of the tissues, combined with the lack of a rigid a m b u l a c r u m such as is found in echinoids, makes these animals almost impossible to restrain by means of pins, rubber bands or other devices. T h e noise level w h e n recording f r o m a whole animal was m u c h higher than with isolated preparations and this, coupled with the m o v e m e n t artefacts from the intact animal m a d e such recordings valueless. Pieces of radial nerve cord which had been excised and used were afterwards sectioned and examined microscopically in comparison with sections of whole arms. T h i s confirmed that the radial nerve and no other tissues were being removed. T h e preparation included varying amounts of hyponeural tissue, which because of its distribution, lateral and aboral to the bulk of the ectoneural tissue, was liable to be lost in dissection. H o w e v e r , experiments involving the longitudinal splitting of the cord showed that its absence had no effect on the responses recorded here.
RESULTS
Response of electrical stimulation In most preparations, stimulation by single square waves produced a typical biphasic potential (Fig. 1). T h e small size and long duration of this potential are remarkable. It is similar to those reported by Millot & Okumura (1968) from certain echinoids and, as they suggest, is presumably the sum effect of massed potentials in the very small axons which make up most of the echinoderm radial nerve cord (Kawaguti, 1965 etc.).
[ - w k15 V, 20 msec; normal polarity. Vertical scale = 800/zV; horizontal scale = 200 msec. FIG. 2. 15 V, 20 msec. Vertical scale - 800/~V; horizontal scale = 200 msec.
FIG. 1.
A stimulus of the order of 15 V for 20 msecs is necessary to produce a maximal response. In certain preparations a "shoulder" is to be seen on the rising phase of the potential (Fig. 3) and in several cases this was resolvable into a completely separate peak by recording over a sufficient distance (Fig. 5). This is presumably due to a group of axons which conduct significantly faster than the rest of the nerve cord. Unfortunately this "fast c o m p o n e n t " could not be seen or separated consistently and varied in its manifestation between identically prepared nerve cords from the same animal, using the same electrodes. T h e only way its presence could be demonstrated in most preparations was to increase the stimuli from just above threshold level, for the fast component has a lower threshold. Thus, in Fig. 4, the peak position of this component can be seen to move farther away from the stimulus artefact as the strength of stimulus is increased, and then to remain
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constant. This represents the fast component's appearance at its lower threshold followed by its occlusion by the rest of the potential as the latter grows in magnitude with increased strength of stimulation. Threshold values for the fast response, measured 4 m m from the point of stimulation, are of the order of 15 V, 0.3 msecs, and 0.5 V, 10 msecs. T h e threshold for the slower component is of course higher, but not clearly measurable because of the presence of the fast component.
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FIC. 3. 15 V, 20msec; stimuli at 0"5 sec intervals. Vertical scale = 800/zV; horizontal scale -- 400 msec. FIC. 4. 1, 2, 5 and 10 V, 10 msec; stimuli, superimposed. Vertical scale = 400/zV; horizontal scale -- 200 msec.
FIG. 5. 1 V, at 4, 8, 12, 16 and 20 msec; stimuli superimposed. Vertical scale -400/zV; horizontal scale = 200 msec. FIC. 6. 5 V, 1 msec; stimuli, 200/sec. Vertical scale = 400/zV; horizontal scale -200 msec. Conduction of the potential is strongly decremental, the decline in recordable size of the response with distance from the stimulating electrodes being almost logarithmic. T h e maximum distance at which any activity could be detected was about 25 mm. "Reverse polarity" stimulation, with the positive electrode nearer to the recording pair, produced an effect intermediate between that described by Millot & Okumura (1968) and that described by Sandeman (1965). Stimulation just above threshold produced a small potential of normal form. (Sandeman obtained normal, bus smaller, potentials.) As the strength of stimulation was increased, however, this was occluded by a positive potential change of form seen in Fig. 2. This prolonged hyperpolarization is as described by Millot & Okumura (1968), and is difficult to account for. We confirm their findings that such behaviour is a property of living nervous tissue only. It increases in size in proportion to the stimulating voltage up to 80 V at least, although a 15 V, 20 msec stimulus of normal polarity is sufficient to elicit a maximal response. When conduction has been blocked with eserine (see below), normal stimulation produces a long depolarization of similar form. After prolonged exposure to eserine, from which recovery would be incomplete, this response is not produced by "normal" or "reverse polarity" stimulation.
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The conduction velocity of the typical undivided potential was found to be 6.5-8.8 cm/sec. In a few preparations in which the two components could be clearly distinguished, the velocities of fast and slow components were found to be 5.0-5-5 and 6.9-8.8, respectively. One or two specimens of other asteroids were examined and similar responses to electrical stimulation of the radial nerve cords recorded. The mean conduction velocities were estimated to be as follows: Marthasterias glacialis, 6.7-8.0 cm/sec, Astropecten irregularis, 8"9 cm/sec, Solaster papposus, 10.0 era/see. Immersion in a bath of sea water cooled to O°C resulted in a reduction in the velocity of up to 25 per cent, but both components were affected similarly, and there was no useful separation of the two.
Multiple electrical stimulation There is an absolute refractory period of approximately 60 msecs. The relative refractory period is of indeterminate length, because of the slow recovery of the potential to its previous height towards the end of the period, but it is of the order of 6-7 sec. When a series of stimuli at high frequency is administered, a potential is transmitted only in response to the initial stimulus. At the end of the train of stimuli, however, there is a positive deflection of the trace, as is seen in the case of "reverse polarity" stimulation (Fig. 6). When a series of two or more stimuli, with a time interval greater than the relative refractory period, is administered, the decline in height of the resulting potentials is greater in the case of the slow component than in that of the fast (Fig. 3). This resembles the findings of Millot & Okumura (1968) in D. antillarum.
Mutilation of the nerve cord Vigorously crushing the nerve cord between the stimulating and recording pairs of electrodes eliminates all recordable nervous activity, as might be expected. Cutting partly across the cord in this position reduces the size of the potential and it is apparently immaterial whether the nerve is cut in the centre or as a hemisection from any side. If cuts are made from alternate sides so that they overlap, i.e. so that the nerve is interrupted in all parts of its cross-sectional area at some point between the stimulating and recording electrodes, the potential is reduced to about half of its original height. However, even a series of up to six overlapping cuts from alternate sides does not abolish the potential completely, although it is much reduced. SpiRting the nerve cord longitudinally or scraping away the oral surface, as described by Kerkut (1955), effects a reduction in the height of the potential, but the form is unaltered. The size of the potential in these cases is approximately in proportion to the size of the strip of nervous material remaining. There is, therefore, no evidence for the two components being associated with a division of the nerve cord into tracts on a macroscopic scale.
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Effect of artificial sea water and sea water of reduced salinity A simple artificial sea water was prepared using isotonic solutions of the chlorides of the four major metals according to the table given by Pantin (1946). Survival and behaviour of the preparation in this solution was just as in normal sea water. Echinoderms in culture are notorious for their intolerance of sea water substitutes. When the nerve cord is placed in dilutions of natural sea water, the survival time and response to electrical stimulation are unaltered at a concentration of 25~o. T h e potential shows an immediate decrease in height of about 10 per cent after immersion in sea water at 20~o, with further decrease as the preparation is kept. Lower concentrations still cause an immediate and rapid decrease to total extinction of the potential. T h e salinity tolerance of whole Asterias rubens from British shores was shown by Binyon (1961) to be 23~oo. The tolerance of the radial nerve cord, which is very exposed to the environment for such a major organ would therefore seem to be of the same order as that of the whole animal. Immersion in calcium-free artificial sea water and in artificial sea water containing ten times the normal potassium concentration both cause abolition of the potential within minutes, with a rapid recovery when the nerve cord is returned to water of normal composition (Fig. 7). The speed of action of such solutions is in great contrast to the slow reaction of the nerve cord to poisons and blocking agents.
Fio. 7. 20 V, 20 msec; stimuli administered: (a) to fresh nerve; (b) after 2 rain in calcium-free artificial sea water; (c) 2 min after return to normal sea water; (d) 5 min after return to normal sea water. Vertical scale = 800/zV; horizontal scale = 200 msec.
Effect of drugs Immersion of the nerve cord in a strong solution of potassium cyanide in sea water destroys any response in about 10 min. This is comparatively slow compared with the action of the artificial sea water solutions described above. Dilute hydrochloric acid, by contrast, is effective almost immediately. A similar slowness of reaction was found with pharmacological agents; very strong solutions or prolonged exposures were necessary. Immersion of the nerve cord in a 0.01 M solution of eserine sulphate in sea water blocked conduction of the potential completely after 30 min, apart from the long wave of depolarization referred to above. Recovery of the potential to its previous height was complete after 60 rain back in sea water. Longer exposures to such a solution also slowly deleted the remaining response, but recovery was slow and incomplete. Comparatively strong solutions of eserine are necessary to achieve this complete block, and solutions more dilute than 0.01 M have little effect in the life of the preparation. Strong solutions of veratrine alkaloids, of atropine, and of procaine exert a similar blocking effect to eserine, but recovery is slow and never complete.
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The acetylcholinesterase inhibiting agent 62C47K (1,5-bis(4-trimethylammoniumphenyl)pentan-3-diiodide, Burroughs-Welcome) was found to irreversibly reduce the size of the potential, but not to extinguish it completely in the life of the preparation. The butyrylcholinesterase inhibitor iso-OMPA (Tetraisopropylpyrophosphoramide, Koch-Light) did not affect the performance of the preparation in any way. Animals exposed for up to 7 days to sea water containing 5 ~g/ml soluble reserpine showed the symptoms of inanition and slow righting reaction described by Cottrell (1967) but again the response of the nerve cords was unaffected. CottreU (1967) showed that exposure of animals to 3 ~g/ml soluble reserpine for 5 days depleted the dopamine content of the cords by 50-70 per cent and the noradrenaline content by 20-50 per cent. The detergent BP1002 at 25 p.p.m, is described by Smith (1968) as killing the majority of A. rubens in 24 hr. The nerve cords from two animals which had undergone such an exposure (and survived) were also quite normal. The detergent might have been expected to affect the exposed nervous tissue rapidly. DISCUSSION The records obtained from asteroid radial nerves resemble in many respects those obtained from echinoids by Millot & Okumura (1968). There is also a resemblance to those reported by Sandeman (1965), in as far as the two components of the potential recorded overlap more. The actual form differs somewhat from Sandeman's (1965) results, but as Millot & Okumura (1968) point out, the precise disposition of the recording electrodes may affect this, and Sandeman does not always give these details. The double character of the massed potential is presumably due to two conducting systems within the nerve of differing speed. No evidence exists so far that these forms of conduction occur in macroscopically recognizable tracts in the nerve cord. The inconsistent separation of the two peaks is difficult to account for, but may be related to the absence of any well-defined motor response of the type shown by Millot & Okumura (1968). In view of the general similarity in the results obtained from Asterias and Diadema it is a pity that a clear correlation of electrical stimulation of the radial nerve cord with motor activity has not been shown so far, although the work of Kerkut (1955) suggests that such a relationship exists. Blocking of the potential by eserine and 62C47K suggests that transmission of the potential is cholinergic, and predominantly mediated by acetylcholine. In view of the high concentrations of these agents necessary to produce a block, the lack of effect of iso-OMPA does not rule out transmission via butyrylcholine completely. The recent work of Pentreath & Cottrell (1968) has shown that acetylcholine is to be found in significant quantities in both the ectoneural and hyponeural regions of the nerve cord. The lack of effect of exposure to reserpine on the nerve cord, despite an obvious
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effect o n t h e a n i m a l as a whole, suggests t h a t c a t e c h o l a m i n e s play little part i n t h e t r a n s m i s s i o n of the r e s p o n s e r e c o r d e d here.
Acknowledgement--This work was supported by a grant from the Science Research Council. REFERENCES BINYON J. (1961) Salinity tolerance and permeability to water in the starfish Asterias rubens L. ft. mar. Biol. Ass. U.K. 41, 161-174. COBB J. L. S. & LAVERACKM. S. (1966) The lantern of Echinus esculentus ( L . ) - - I I . Fine structure of hyponeural tissue and its connections. Proc. R. Soc. B 164, 641-650. COTTRELL G. A. (1967) Occurrence of dopamine and noradrenaline in the nervous tissues of some invertebrate animals. Br. ft. Pharmac. Chemother. 29, 63-69. KAWAGUTI S. (1965) Electron microscopy on the radial nerve of the starfish. Biol. ft. Ohayama Univ. 11(1-2), 41-52. KEaKUT G. A. (1955) The retraction and protraction of the tube feet of the starfish. Behaviour 8, 112-129. MILLOT N. & OKUMURAH. (1968) The electrical activity of the radial nerve in Diadema antillarum Philippi and certain other echinoids, ft. exp. Biol. 48, 279-287. PANTIN C. F. A. (1946) Notes on Microscopical Techniques for Zoologists. Cambridge University Press. PENTREATH V. A. & COTTRELL G. A. (1968) Acetylcholine and cholinesterase in the radial nerve of Asterias rubens. Comp. Biochem. Physiol. 27, 775-785. SANDEMAN D. C. (1965) Electrical activity in the radial nerve cord and ampullae of sea urchins, ft. exp. Biol. 43, 247-256. SMITH J. E. (1968) (Editor) "Torrey Canyon" Pollution and Marine Life. Cambridge University Press. TAKAHASHIK. (1964) Electrical responses to light stimuli in the isolated radial nerve of the sea urchin Diadema setosum (Leska). Nature, Lond. 201, 1343-1344.
Key Word Index--Action potential; calcium-free sea water; cholinergic transmission; conduction velocity; detergent; dilute sea water; drug action; electrophysiology; fast component; radial nerve cord; reversed polarity; salinity; slow component; starfish; tracts.