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Studies on the ionic basis of the action potential in the brittle-star, Ophiura ophiura
Comp. Biochem. Physiol. Vol. 91A, No. 4, pp. 821-825, 1988
0300-9629/88 $3.00 + 0.00 © 1988 Pergamon Press plc
Printed in Great Britain
STUDIES ON THE IONIC BASIS OF THE ACTION POTENTIAL IN THE BRITTLE-STAR, OPHIURA OPHIURA J. L. S. COBB and A. MOORE Gatty Marine Laboratory, University of St Andrews, Fife, Scotland, UK, KYI6 8LB. Telephone: 0334-76161
(Received 3 May 1988) Abstract--1. The ionic basis of the action potential was investigated in neurones of the brittle-star Ophiura ophiura using intra- and extracellular recording electrodes. 2. The use of ion substituted salines, and inorganic and organic blockers provided strong evidence for a Ca 2+ spike without the involvement of Na + ions. 3. Choline chloride was shown to be an unsatisfactory substitute for Na + ions since it had varied direct effects on the function of the nervous system.
INTRODUCTION
MATERIALS AND METHODS
There is less known about the nervous system of echinoderms than any other major phylum, and almost nothing about physiological mechanisms. There are a number of papers that described compound action potentials recorded to gross stimulation of the radial nerve cord which were conducted decrementally (Sandeman, 1965; Millott and Okumura, 1968; Binyon and Hailer, 1970). These recordings are very difficult to interpret but two in particular hinted that a calcium spike was involved. In 1977 Brehm showed that it was possible to record small unitary potentials just above noise from a class of giant fibres in a brittle-star, Ophiopsilis californica. This work also suggested the involvement of a calcium spike. Subsequently two papers have been published that present conflicting results concerning the ionic basis of the action potential. Tuft and Gilly (1984) recorded c o m p o u n d action potentials from the radial nerve cord of another species of brittle-star and presented evidence which they interpreted as showing two classes of neurone; one, with a fast conduction velocity they suggested had a calcium spike and for another class, with a slower conduction velocity, they proposed a sodium spike. Berrios et al. (1985) and Hernandez et al. (1987), however, have recently developed a quite different preparation using the fortuitously long spines of the sea urchin, Diadema antillarurn. In this preparation again only a complex compound action potential was recorded to electrical stimulation. These authors provide evidence that a purely calcium spike is present. Intra-cellular capability has now been achieved from a number of different classes of neurones in another species of brittle-star, Ophiura ophiura (see Cobb, 1985) allowed more reliable evidence to be obtained. This work provides further evidence for a calcium spike without the involvement of sodium ions and shows choline chloride, used by Tuft and Gilly (1984) as an ion substitute for Na ÷, to be unsatisfactory.
Large specimens of the brittle-star Ophiura ophiura were obtained from Millport, Isle of Cumbrae. Single arms were detached and placed in organ baths kept at a temperature of 10°C and plastic suction electrodes with a tip of diameter 0.5 mm were gently placed on the radial nerve cord. Intracellular electrodes containing 2 M KCI with a resistance of 60 M~ were used to impale various classes of neurones intracellularly concomittantly with extracellular monitoring. The neurones recorded from in particular were large longitudinal neurones, the cell body region of which is nonexcitable. A reflected action potential of almost full size is, however, normally recorded as well as synaptic potentials. Hyponeural motor neurones were also recorded from. The cell bodies of these neurones are also non-excitable but the action potential does not penetrate to the area normally impaled by the micro-electrode. Numerous excitatory and inhibitory junction potentials up to 15 mV are, however, recorded routinely. All information was recorded on tape for subsequent analysis or photographic reproduction. Control experiments were carried out in natural sea-water but the experimental media described by Tuft and Gilly (1984) and Berrios et al. (1985) were exactly duplicated. These artificial salines are summarized in Table 1. Previous studies using electrical stimulation by implanted electrodes (unpublished) have shown highly atypical activity within the nervous system and were not used during these experiments other than as early confirmatory studies on their general effectiveness in producing recordable action potentials. Four more natural stimulus regimes were used. A dilute food stimulus of ground Mytilus edulis as a filtrate (this causes activity in a class of longitudinally orientated neurones that conduct at about 30 cm/sec), a photic shadow stimulus (this causes activity of fast -75cm/seclongitudinal neurones which are a class of giant fibres), see Cobb (1987). 10-4M cysteine was applied as a noxious stimulus which causes the same class of giant neurones to fire as a photic stimulus and finally when all other responses were without effect 10-3 KCI was applied. Such a stimulus with KCI in normal sea-water causes high frequncy firing of large and small diameter neurones and almost invariably the autotomy of the arm. All stimuli were applied to the tip of a single arm at least 15 segments (and often much more) from the recording electrode which was placed within the 10 821
822
J.L.S.
COBB and A. M o o g t
Table I. Ionic concentration of bathing solution Constituent concentration (mol/1) After Tuft and Gilly (1984) Berrios et al. (1985) 0-Ca* ~Na 0-Ca 0-Na + Sr( - Ca) NaC1 CaC12 SrCI 2 BaCl 2 KC1 MgCI 2 Tris Choline-CI Sucrose NaHCO 3
440
450
+ Ba( - Ca)
450
I0
450
I1 11
10 40 10
10 20
10 53
10 53
25 2
752
10 53
11 10 53
2
2
420
*Buffered with 2 mmol-i EGTA. most proximal segments to the disc. The distance between
recording electrode and stimulus was up to 12 cm. RESULTS
Sodium free solutions Berrios et al. (1985) used a sucrose substituted saline and found it to have no effect on the level of response to stimuli. Our results exactly confirm their findings including a much increased size of extracellular potential due to the increased resistivity of the bathing sucrose solution (Fig. 1). All neurones recorded intracellularly showed normal action potentials in size, duration and frequency to maximum stimulation 3 hr after the preparation was immersed in the saline (Fig. 2). Responses to all stimuli were identical to control responses with the exception of the noxious stimulus of 10 -4 cysteine which was abolished. Presumably, in this case it is interference of sucrose with chemoreceptors since the photic
response was normal and the same giant interneurones conduct information about both types of stimuli. Tuft and Gilly 0984) used a choline chloride substituted Na-free saline. The effects of immersion in this were immediate and consistent. There was a very substantial induced activity in the nervous system of both large and small diameter neurones and this was recorded from all parts of the nervous system. This subsides over about l0 min. The nervous system becomes inexcitable to both cysteine and a shadow photic stimulation after about 5 min; however KCl still causes firing of large and small diameter neurones but autonomy does not occur (as it almost invariably does in a control preparation). Intracellular recordings show an action potential normal in all respects and there is no evidence that any particular class of neurone is no longer functioning (Fig. 2). One interesting effect of choline is that after about 5 rain the photic response to shadow ceases but
l Fig. 1. Extracellular recording from the radial nerve cord in a saline with Na + substituted by sucrose. This has no effect on the neural activity but there is a much greater signal-to-noise ratio due to the increased resistivity of the saline. The rapid conducting giant fibres and other smaller units are shown in the response recorded to a photic shadow stimulus (arrow). Scale = 1 sec.
F
G
Fig. 2. Intracellular recordings from longitudinal giant fibres of the radial nerve cord. Action potentials recorded to a photic shadow stimulus in all cases. (A) control in normal sea-water; (B) sucrose substituted for Na+; (C) choline substituted for Na+; (D) 10 -5 T T X in normal sea-water; (E) Sr 2+ substituted for Ca 2+, (note multiple firing); (F) Ba 2+ substituted for Ca2+; (G) Normal artificial sea-water. Horizontal scale = 0.1 sec, vertical scale = 20 mV.
Action potentials in brittle-stars the preparation becomes highly sensitive to light on (as opposed to light off) with information transmitted through the same neurones as those that normally carry the light off response. Thus choline effectively reverses the effects of light as recorded from the same class of interneurone. In the presence of 10 -5 gaUamine this effect is reversed back to the normal situation of light off stimulus being effective. A 100-fold dilution of the choline used in normal sea-water produces exactly the same effects and substituting acetylcholine chloride at the same concentration also produces identical effects. The addition of 5 x 10 -5 tetrodotoxin has no effect over periods up to 3 hr and no change in the shape of action potentials or other characteristics can be detected when recording intracellularly (Fig. 2). As a test against the failure of the various solutions to penetrate between the tightly packed, glial-less and bloodspace-less nerve cord, a saline with 3 times normal level of K + ions was added (there have been previous suggestions that the failure of many neuropharmacological agents to affect the nervous system are due to problems of penetration; Pentreath and Cobb, 1982). This caused immediate and massive activity of neurones, rapidly followed by complete failure of the nervous system. Calcium -free saline Calcium-free salines gradually abolish almost all nervous activity and muscular action. The effects are never total and may take up to 30 min to reach maximum effect. All feeding behaviour and escape responses are lost after 20min though throughconducted activity is present in a small number of both the largest sized longitudinal neurones and some smaller ones. Normal action potentials can be recorded from a very few axons but the vast majority of neurones penetrated show neither action or synaptic potentials and this includes lack of synaptic activity in hyponeural motor neurones. Small arm movements are noted after 20min to the direct application of test solutions of KC1 but these cease after 1 hr. After 2 hr, one or two neurones can still be found which respond to application of test KCI using extracellular recording. If Ca z+ free saline is used with 5 mM EGTA the effects are more rapid but some neural activity is still present after ½hr and some neurones still respond to test KC1 at 1 hr. The preparation gradually returns to normal after being washed out with natural sea-water. Inorganic calcium blockers All action potentials are blocked by 2 mM cadmium within 5 min, and this effect is irreversible. Cobalt produced an irreversible blockade at 5 mM but not at lower concentrations; lanthanum produced a similar effect at the same concentration. Manganese produced no effect at 15 mM even after 20 min. Magnesium produced a total suppression of action potentials at 10 -2 M which was reversible and partial blockage at 10 mM. Organic blockers The phenylalkylamine, Verapamil, blocks all activity in all neurones immediately at 1 mM and after
823
10min at 200#M. At 100#M there is still a small response to test KCI but little other activity. Calcium substitute ions Substituting strontium for calcium produces immediate and massive activity within the nervous system which gradually subsides over about 5 min (Fig. 2). Intracellular recordings show many neurones firing very high frequency trains of spikes and neurones that normally respond to the cessation of hyperpolarization with a single rebound spike, now often show a burst of three or four (Fig. 2). The shape, size and duration of all spikes are indistinguishable from those in normal sea-water (Fig. 2). Stimuli that produce feeding or escape movements cause much higher concomitant levels of activity within the nervous system and often exaggerated levels of response. The other effect apart from this general increase in responsiveness is a 5-fold increase in the lag between stimulus and onset of activity in the giant fibre sysem of longitudinal neurones, this effect is abolished if 5 mM EGTA is added to the saline. Substituting barium has variable effects which can be similar to either that o f a Sr 2÷ saline with increased activity or that of a Ca2+-free saline where activity is slowly abolished. In some cases where a Ca2+-free effect is noted there is evidence that Ba 2÷ is precipitating out onto the tissue selectively, particularly on calcite ossicles and it is possible that this effect is largely due to an effective Ca2+-free saline being produced.
DISCUSSION The major finding of this paper is that it is clear that the evidence presented by Tuft and Gilly (1984) for Ca 2÷ and Na ÷ spikes in separate classes of neurones is not valid even apart from the difficulties in interpreting complicated compound action potentials that they described. The effect that they obtained with Na+-free salines with choline substituted are due to the direct action of the choline on the nervous system. Indeed, the effects are so consistent in changing activity in the nervous system in a predictable, but as yet inexplicable way, that they may have some potential in analysing the neural basis of behaviour in this phylum. The present results are entirely consistent with the findings of Berrios et al. (1985), Hernandez et al. (1987). It is nevertheless worth remembering that Berrios et al. appear to have been recording from a class of sensory neurone. This is in fact the only preparation where this has been consistently achieved and is valuable for that alone. Previous studies have all shown that sensory axons arise from receptor cell bodies so that a class of interneurones in this situation is unlikely. In the ease of the present work and that of Tuft and Giily (1984) various interneurones are being recorded from. It is possible that there are classes that do have a Na + action potential that is too small to record from in a unitary fashion with any technique. There is no evidence to support this idea however. The hyponeural motor neurones, which are mesodermal, were recorded from the present study but it is difficult to do this without a damaging dissection
824
J.L.S. COBBand A. MOORE
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Fig. 3. (A) Extracellular recording showing response from the radial nerve cord to photic shadow; (B) identical stimulus 5 min after preparation bathed in Sr2+ substituted saline, note the multi-spike bursts compared to the control; (C) Identical stimulus with preparation bathed in normal sea-water with intracellular recording electrode in longitudinal giant fibre; (D) identical stimulus with Sr2+ substituted l0 min previously. Note the increased number of spikes in the response. Scale = l sec.
to the distal parts of the axons and the cell bodies only show synaptic potentials. It is possible that these neurones contain a Na + based action potential but it is unlikely. This is because muscle contraction is normal in sucrose Na+-free salines and thus presumably the axons must still be conducting. Echinoderm muscle cells show an intracellular spike (Pentreath and Cobb, 1975) and there has long been evidence to suggst that this is a Ca 2+ spike. The extracellular records of Tuft and Gilly (1984) are compound waveforms which are very difficult to interpret or associate with particular types of neurone. These authors suggested one part of the compound potential was associated with one type of nerve cell and another part with different neurones. Although the separate parts of the waveform appear to have different conduction velocities (and there are neurones which conduct at different velocities) this evidence is too tenuous to be satisfactory. Berrios et
al. (1985) recording from a much less complex area
of the nervous system show that there may be up to six parts to the complex compound action potential that they record but these authors accept the likely explanation that it is due to recording conditions. There are a number of previous studies recording these compound action potentials from the radial nerve cords of various sea urchins (Echinoidea) but they have shown little of lasting value (Sandemen, 1965; Millott and Okumura, 1968; and Binyon and Hasler, 1970). The evidence that is now available suggests that all neurones, in brittle-stars at least of the echinoderms, have a purely calcium-driven inward current generating the rising phase of their action potentials and our findings completely complement those of Berrios et al. (1985) particularly since they have now been able to reverse their findings that Ba 2+ does substitute for Ca :+ as it does in other Ca 2+ spike situations in
Action potentials in brittle-stars other groups of animals (Hernandez et al., 1987). It also agrees with the findings that Ca2÷-free saline blocks at least some neurones reported by Tuft and Gilly (1984). The reason that a Ca2+-free saline does not always block action potentials and can have an inconsistent effect seems certain to be the explanation put forward by Berrios et al. (1985) that Ca 2÷ ions are continually coming into solution from the calcite skeleton in Ca2+-free salines and the experiments with E G T A and E D T A seem to confirm this. No doubt the effect can vary since some neurones are surrounded by calcite (e.g. those of Berrios et aL) and others are not. A Ca2÷-free saline blocks conduction of what can be recorded intracellularly or extracellularly when the nerve cord is dissected out into an organ bath but then natural stimulus regimes cannot be used, so this evidence is not unequivocal. It is not possible to claim unequivocal p r o o f of a purely Ca 2+ spike and the non-involvement of Na ÷ ions even in the light of the current work using intracellular recording in conjunction with the considerable knowledge we have developed about the origins and function of the unitary potentials recorded from various neurones extracellularly. What is required in the first place is to impale active cells and maintain that impalement while solutions are changed. Regrettably, at least for the ectoneural neurones, this at present is technically very difficult. Beyond this it is necessary to study the currents themselves carried by the particular ions using voltage clamp and this too would be technically difficult. The most interesting question is why does there appear to be a calcium spike? It has been suggested that the primitive ion channel was a Ca 2÷ channel that carried the inward current but that it evolved into a Na + channel with the further evolution of the nervous system because of interference of an influx of Ca 2+ ions with other intracellular messenger and regulatory functions of calcium (Hille, 1984). Anderson (1988) has recently shown that ctenophores produce action potentials in Na÷-free or Ca2÷-free solutions and although this could be due to two types of channel this seems unlikely. A simpler explanation is that one channel can accept either ion but that it is normally selective for Ca 2÷ over Na ÷. These channels also occur elsewhere, for example in vertebrate smooth muscle. TTX insensitive channels are known that are Na ÷ channels, for example in cnidarians (Anderson, 1987) but since it now seems unlikely that a Na ÷ action potential is present in echinoderms, the finding that the supposed Na ÷ channel of Tuft and Gilly was TTX-insensitive is not significant. The finding that Verapamil blocks the echinoderm channel is also not absolute p r o o f that it is acting on a Ca 2÷ channel since some Na ÷ channels are also blocked by that drug (Adams and Gage, 1979). Platyhelminthes show a TTX-sensitive Na ÷ channel (Koopowitz and Keenan, 1982) and this would confirm an evolutionary sequence towards this state with the ctenophores and cnidarians being intermediate (see Anderson, 1987). The echinoderm Ca 2+ channel does not fit this scheme but it is possible that larval echinoderms do have a Na ÷ action potential; however recording from these nervous systems will be even more difficult than in the adults.
825
Echinoderm nervous systems are highly enigmatic even beyond their non-centralized layout. They have no glial cells, and cell bodies are packed with glycogen, not endoplasmic reticulum; there are no blood spaces within tightly packed nervous tissue. Furthermore, ectodermal nerves cannot penetrate mesodermal effector tissues, and transmitter substances diffuse across substantial distances of the connective tissue basement membrane sheaths, and a separate mesodermal motor system has evolved (see Cobb, 1987, for review). Finally the non-cellular connective tissue is mutable and innervated (see Wilkie, 1984, for review). The present evidence does not prove, but is strongly indicative of a Ca 2÷ spike in all neurones without the involvement of Na ÷ ions, but the reason for this must at present be one more echinoderm enigma to add to the list. Acknowledgements--The authors would like to thank Drs Wendy Nightingale and Robert Pitman for helpful discussion.
REFERENCES
Adams D. J. and Gage P. W. (1979) Ionic currents in response to membrane depolarization in an Aplysia neurone. J. Physiol. 289, 115-142. Anderson P. A. J. (1987) Properties and pharmacology of a TTX-insensitive Na ÷ current in neurones of the jellyfish Cyanea capillata. J. exp. Biol. 133, 231-248. Berrios A., Brink D., del Castillo J. and Smith D. S. (1985) Some properties of the action potentials conducted in the spines of the sea urchin Diadema antillarum. Comp. Biochem. Physiol. 81A, 15-23. Binyon J. and Hasler B. (1970) Electrophysiology of the starfish radial nerve cord. Comp. Biochem. Physiol. 32, 747-753. Brehm P. (1977) Electrophysiology and luminescence of an ophiuroid radial nerve. J. exp. Biol. 71, 213-227. Cobb J. L. S. (1985) The neurobiology of the ectoneural/ hyponeural synaptic connection in an echinoderm. Biol. Bull. mar. biol. Lab., Woods Hole 165, 432-446. Cobb J. L. S. (1987) The echinodermata. In Invertebrate Nervous Systems (Edited by Ali M. A.), pp. 483-526. Plenum Press, New York. Hernandez Z. M., Morales M., Smith D. S. and del Castillo J. (1987) Barium spikes are generated in the spines of the sea-urchin Diadema antillarum. Comp. Biochem. Physiol. 86A, 355-359. Hille B. (1984) Ionic Channels of Excitable Membranes. Sinauer Associates. Sunderland, Massachusetts. Koopowitz H. and Keenan L. (1982) The primitive brains of platyhelminthes. Trends Neurosci. 5, 77-79. Millott N. and Okumura H. (1968) The electrical activity of the radial nerve in Diadema antillarum and certain other echinoids. J. exp. Biol. 48, 279-289. Pentreath V. W. and Cobb J. L. S. (1982) Echinodermata, in Electrical Conduction and Behaviour in Simple Animals (Edited by Shelton G. A. B.), pp. 440-472. Clarendon Press, Oxford. Sandeman D. C. (1965) Electrical activity in the radial nerve cord and ampullae of sea urchins. J. exp. Biol. 43, 247-256. Tuft P. J. and Gilly W. M. (1984) Ionic basis of action potential propagation along two classes of "giant" axons in the ophiuroid, Ophiopteris papillosa. J. exp. Biol. 113, 337-349. Wilkie I. C. (1984) Variable tensility in echinoderm collagen tissues. Mar. Behav. Physiol. 11, 1-34.
0300-9629/88 $3.00 + 0.00 © 1988 Pergamon Press plc
Printed in Great Britain
STUDIES ON THE IONIC BASIS OF THE ACTION POTENTIAL IN THE BRITTLE-STAR, OPHIURA OPHIURA J. L. S. COBB and A. MOORE Gatty Marine Laboratory, University of St Andrews, Fife, Scotland, UK, KYI6 8LB. Telephone: 0334-76161
(Received 3 May 1988) Abstract--1. The ionic basis of the action potential was investigated in neurones of the brittle-star Ophiura ophiura using intra- and extracellular recording electrodes. 2. The use of ion substituted salines, and inorganic and organic blockers provided strong evidence for a Ca 2+ spike without the involvement of Na + ions. 3. Choline chloride was shown to be an unsatisfactory substitute for Na + ions since it had varied direct effects on the function of the nervous system.
INTRODUCTION
MATERIALS AND METHODS
There is less known about the nervous system of echinoderms than any other major phylum, and almost nothing about physiological mechanisms. There are a number of papers that described compound action potentials recorded to gross stimulation of the radial nerve cord which were conducted decrementally (Sandeman, 1965; Millott and Okumura, 1968; Binyon and Hailer, 1970). These recordings are very difficult to interpret but two in particular hinted that a calcium spike was involved. In 1977 Brehm showed that it was possible to record small unitary potentials just above noise from a class of giant fibres in a brittle-star, Ophiopsilis californica. This work also suggested the involvement of a calcium spike. Subsequently two papers have been published that present conflicting results concerning the ionic basis of the action potential. Tuft and Gilly (1984) recorded c o m p o u n d action potentials from the radial nerve cord of another species of brittle-star and presented evidence which they interpreted as showing two classes of neurone; one, with a fast conduction velocity they suggested had a calcium spike and for another class, with a slower conduction velocity, they proposed a sodium spike. Berrios et al. (1985) and Hernandez et al. (1987), however, have recently developed a quite different preparation using the fortuitously long spines of the sea urchin, Diadema antillarurn. In this preparation again only a complex compound action potential was recorded to electrical stimulation. These authors provide evidence that a purely calcium spike is present. Intra-cellular capability has now been achieved from a number of different classes of neurones in another species of brittle-star, Ophiura ophiura (see Cobb, 1985) allowed more reliable evidence to be obtained. This work provides further evidence for a calcium spike without the involvement of sodium ions and shows choline chloride, used by Tuft and Gilly (1984) as an ion substitute for Na ÷, to be unsatisfactory.
Large specimens of the brittle-star Ophiura ophiura were obtained from Millport, Isle of Cumbrae. Single arms were detached and placed in organ baths kept at a temperature of 10°C and plastic suction electrodes with a tip of diameter 0.5 mm were gently placed on the radial nerve cord. Intracellular electrodes containing 2 M KCI with a resistance of 60 M~ were used to impale various classes of neurones intracellularly concomittantly with extracellular monitoring. The neurones recorded from in particular were large longitudinal neurones, the cell body region of which is nonexcitable. A reflected action potential of almost full size is, however, normally recorded as well as synaptic potentials. Hyponeural motor neurones were also recorded from. The cell bodies of these neurones are also non-excitable but the action potential does not penetrate to the area normally impaled by the micro-electrode. Numerous excitatory and inhibitory junction potentials up to 15 mV are, however, recorded routinely. All information was recorded on tape for subsequent analysis or photographic reproduction. Control experiments were carried out in natural sea-water but the experimental media described by Tuft and Gilly (1984) and Berrios et al. (1985) were exactly duplicated. These artificial salines are summarized in Table 1. Previous studies using electrical stimulation by implanted electrodes (unpublished) have shown highly atypical activity within the nervous system and were not used during these experiments other than as early confirmatory studies on their general effectiveness in producing recordable action potentials. Four more natural stimulus regimes were used. A dilute food stimulus of ground Mytilus edulis as a filtrate (this causes activity in a class of longitudinally orientated neurones that conduct at about 30 cm/sec), a photic shadow stimulus (this causes activity of fast -75cm/seclongitudinal neurones which are a class of giant fibres), see Cobb (1987). 10-4M cysteine was applied as a noxious stimulus which causes the same class of giant neurones to fire as a photic stimulus and finally when all other responses were without effect 10-3 KCI was applied. Such a stimulus with KCI in normal sea-water causes high frequncy firing of large and small diameter neurones and almost invariably the autotomy of the arm. All stimuli were applied to the tip of a single arm at least 15 segments (and often much more) from the recording electrode which was placed within the 10 821
822
J.L.S.
COBB and A. M o o g t
Table I. Ionic concentration of bathing solution Constituent concentration (mol/1) After Tuft and Gilly (1984) Berrios et al. (1985) 0-Ca* ~Na 0-Ca 0-Na + Sr( - Ca) NaC1 CaC12 SrCI 2 BaCl 2 KC1 MgCI 2 Tris Choline-CI Sucrose NaHCO 3
440
450
+ Ba( - Ca)
450
I0
450
I1 11
10 40 10
10 20
10 53
10 53
25 2
752
10 53
11 10 53
2
2
420
*Buffered with 2 mmol-i EGTA. most proximal segments to the disc. The distance between
recording electrode and stimulus was up to 12 cm. RESULTS
Sodium free solutions Berrios et al. (1985) used a sucrose substituted saline and found it to have no effect on the level of response to stimuli. Our results exactly confirm their findings including a much increased size of extracellular potential due to the increased resistivity of the bathing sucrose solution (Fig. 1). All neurones recorded intracellularly showed normal action potentials in size, duration and frequency to maximum stimulation 3 hr after the preparation was immersed in the saline (Fig. 2). Responses to all stimuli were identical to control responses with the exception of the noxious stimulus of 10 -4 cysteine which was abolished. Presumably, in this case it is interference of sucrose with chemoreceptors since the photic
response was normal and the same giant interneurones conduct information about both types of stimuli. Tuft and Gilly 0984) used a choline chloride substituted Na-free saline. The effects of immersion in this were immediate and consistent. There was a very substantial induced activity in the nervous system of both large and small diameter neurones and this was recorded from all parts of the nervous system. This subsides over about l0 min. The nervous system becomes inexcitable to both cysteine and a shadow photic stimulation after about 5 min; however KCl still causes firing of large and small diameter neurones but autonomy does not occur (as it almost invariably does in a control preparation). Intracellular recordings show an action potential normal in all respects and there is no evidence that any particular class of neurone is no longer functioning (Fig. 2). One interesting effect of choline is that after about 5 rain the photic response to shadow ceases but
l Fig. 1. Extracellular recording from the radial nerve cord in a saline with Na + substituted by sucrose. This has no effect on the neural activity but there is a much greater signal-to-noise ratio due to the increased resistivity of the saline. The rapid conducting giant fibres and other smaller units are shown in the response recorded to a photic shadow stimulus (arrow). Scale = 1 sec.
F
G
Fig. 2. Intracellular recordings from longitudinal giant fibres of the radial nerve cord. Action potentials recorded to a photic shadow stimulus in all cases. (A) control in normal sea-water; (B) sucrose substituted for Na+; (C) choline substituted for Na+; (D) 10 -5 T T X in normal sea-water; (E) Sr 2+ substituted for Ca 2+, (note multiple firing); (F) Ba 2+ substituted for Ca2+; (G) Normal artificial sea-water. Horizontal scale = 0.1 sec, vertical scale = 20 mV.
Action potentials in brittle-stars the preparation becomes highly sensitive to light on (as opposed to light off) with information transmitted through the same neurones as those that normally carry the light off response. Thus choline effectively reverses the effects of light as recorded from the same class of interneurone. In the presence of 10 -5 gaUamine this effect is reversed back to the normal situation of light off stimulus being effective. A 100-fold dilution of the choline used in normal sea-water produces exactly the same effects and substituting acetylcholine chloride at the same concentration also produces identical effects. The addition of 5 x 10 -5 tetrodotoxin has no effect over periods up to 3 hr and no change in the shape of action potentials or other characteristics can be detected when recording intracellularly (Fig. 2). As a test against the failure of the various solutions to penetrate between the tightly packed, glial-less and bloodspace-less nerve cord, a saline with 3 times normal level of K + ions was added (there have been previous suggestions that the failure of many neuropharmacological agents to affect the nervous system are due to problems of penetration; Pentreath and Cobb, 1982). This caused immediate and massive activity of neurones, rapidly followed by complete failure of the nervous system. Calcium -free saline Calcium-free salines gradually abolish almost all nervous activity and muscular action. The effects are never total and may take up to 30 min to reach maximum effect. All feeding behaviour and escape responses are lost after 20min though throughconducted activity is present in a small number of both the largest sized longitudinal neurones and some smaller ones. Normal action potentials can be recorded from a very few axons but the vast majority of neurones penetrated show neither action or synaptic potentials and this includes lack of synaptic activity in hyponeural motor neurones. Small arm movements are noted after 20min to the direct application of test solutions of KC1 but these cease after 1 hr. After 2 hr, one or two neurones can still be found which respond to application of test KCI using extracellular recording. If Ca z+ free saline is used with 5 mM EGTA the effects are more rapid but some neural activity is still present after ½hr and some neurones still respond to test KC1 at 1 hr. The preparation gradually returns to normal after being washed out with natural sea-water. Inorganic calcium blockers All action potentials are blocked by 2 mM cadmium within 5 min, and this effect is irreversible. Cobalt produced an irreversible blockade at 5 mM but not at lower concentrations; lanthanum produced a similar effect at the same concentration. Manganese produced no effect at 15 mM even after 20 min. Magnesium produced a total suppression of action potentials at 10 -2 M which was reversible and partial blockage at 10 mM. Organic blockers The phenylalkylamine, Verapamil, blocks all activity in all neurones immediately at 1 mM and after
823
10min at 200#M. At 100#M there is still a small response to test KCI but little other activity. Calcium substitute ions Substituting strontium for calcium produces immediate and massive activity within the nervous system which gradually subsides over about 5 min (Fig. 2). Intracellular recordings show many neurones firing very high frequency trains of spikes and neurones that normally respond to the cessation of hyperpolarization with a single rebound spike, now often show a burst of three or four (Fig. 2). The shape, size and duration of all spikes are indistinguishable from those in normal sea-water (Fig. 2). Stimuli that produce feeding or escape movements cause much higher concomitant levels of activity within the nervous system and often exaggerated levels of response. The other effect apart from this general increase in responsiveness is a 5-fold increase in the lag between stimulus and onset of activity in the giant fibre sysem of longitudinal neurones, this effect is abolished if 5 mM EGTA is added to the saline. Substituting barium has variable effects which can be similar to either that o f a Sr 2÷ saline with increased activity or that of a Ca2+-free saline where activity is slowly abolished. In some cases where a Ca2+-free effect is noted there is evidence that Ba 2÷ is precipitating out onto the tissue selectively, particularly on calcite ossicles and it is possible that this effect is largely due to an effective Ca2+-free saline being produced.
DISCUSSION The major finding of this paper is that it is clear that the evidence presented by Tuft and Gilly (1984) for Ca 2÷ and Na ÷ spikes in separate classes of neurones is not valid even apart from the difficulties in interpreting complicated compound action potentials that they described. The effect that they obtained with Na+-free salines with choline substituted are due to the direct action of the choline on the nervous system. Indeed, the effects are so consistent in changing activity in the nervous system in a predictable, but as yet inexplicable way, that they may have some potential in analysing the neural basis of behaviour in this phylum. The present results are entirely consistent with the findings of Berrios et al. (1985), Hernandez et al. (1987). It is nevertheless worth remembering that Berrios et al. appear to have been recording from a class of sensory neurone. This is in fact the only preparation where this has been consistently achieved and is valuable for that alone. Previous studies have all shown that sensory axons arise from receptor cell bodies so that a class of interneurones in this situation is unlikely. In the ease of the present work and that of Tuft and Giily (1984) various interneurones are being recorded from. It is possible that there are classes that do have a Na + action potential that is too small to record from in a unitary fashion with any technique. There is no evidence to support this idea however. The hyponeural motor neurones, which are mesodermal, were recorded from the present study but it is difficult to do this without a damaging dissection
824
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Fig. 3. (A) Extracellular recording showing response from the radial nerve cord to photic shadow; (B) identical stimulus 5 min after preparation bathed in Sr2+ substituted saline, note the multi-spike bursts compared to the control; (C) Identical stimulus with preparation bathed in normal sea-water with intracellular recording electrode in longitudinal giant fibre; (D) identical stimulus with Sr2+ substituted l0 min previously. Note the increased number of spikes in the response. Scale = l sec.
to the distal parts of the axons and the cell bodies only show synaptic potentials. It is possible that these neurones contain a Na + based action potential but it is unlikely. This is because muscle contraction is normal in sucrose Na+-free salines and thus presumably the axons must still be conducting. Echinoderm muscle cells show an intracellular spike (Pentreath and Cobb, 1975) and there has long been evidence to suggst that this is a Ca 2+ spike. The extracellular records of Tuft and Gilly (1984) are compound waveforms which are very difficult to interpret or associate with particular types of neurone. These authors suggested one part of the compound potential was associated with one type of nerve cell and another part with different neurones. Although the separate parts of the waveform appear to have different conduction velocities (and there are neurones which conduct at different velocities) this evidence is too tenuous to be satisfactory. Berrios et
al. (1985) recording from a much less complex area
of the nervous system show that there may be up to six parts to the complex compound action potential that they record but these authors accept the likely explanation that it is due to recording conditions. There are a number of previous studies recording these compound action potentials from the radial nerve cords of various sea urchins (Echinoidea) but they have shown little of lasting value (Sandemen, 1965; Millott and Okumura, 1968; and Binyon and Hasler, 1970). The evidence that is now available suggests that all neurones, in brittle-stars at least of the echinoderms, have a purely calcium-driven inward current generating the rising phase of their action potentials and our findings completely complement those of Berrios et al. (1985) particularly since they have now been able to reverse their findings that Ba 2+ does substitute for Ca :+ as it does in other Ca 2+ spike situations in
Action potentials in brittle-stars other groups of animals (Hernandez et al., 1987). It also agrees with the findings that Ca2÷-free saline blocks at least some neurones reported by Tuft and Gilly (1984). The reason that a Ca2+-free saline does not always block action potentials and can have an inconsistent effect seems certain to be the explanation put forward by Berrios et al. (1985) that Ca 2÷ ions are continually coming into solution from the calcite skeleton in Ca2+-free salines and the experiments with E G T A and E D T A seem to confirm this. No doubt the effect can vary since some neurones are surrounded by calcite (e.g. those of Berrios et aL) and others are not. A Ca2÷-free saline blocks conduction of what can be recorded intracellularly or extracellularly when the nerve cord is dissected out into an organ bath but then natural stimulus regimes cannot be used, so this evidence is not unequivocal. It is not possible to claim unequivocal p r o o f of a purely Ca 2+ spike and the non-involvement of Na ÷ ions even in the light of the current work using intracellular recording in conjunction with the considerable knowledge we have developed about the origins and function of the unitary potentials recorded from various neurones extracellularly. What is required in the first place is to impale active cells and maintain that impalement while solutions are changed. Regrettably, at least for the ectoneural neurones, this at present is technically very difficult. Beyond this it is necessary to study the currents themselves carried by the particular ions using voltage clamp and this too would be technically difficult. The most interesting question is why does there appear to be a calcium spike? It has been suggested that the primitive ion channel was a Ca 2÷ channel that carried the inward current but that it evolved into a Na + channel with the further evolution of the nervous system because of interference of an influx of Ca 2+ ions with other intracellular messenger and regulatory functions of calcium (Hille, 1984). Anderson (1988) has recently shown that ctenophores produce action potentials in Na÷-free or Ca2÷-free solutions and although this could be due to two types of channel this seems unlikely. A simpler explanation is that one channel can accept either ion but that it is normally selective for Ca 2÷ over Na ÷. These channels also occur elsewhere, for example in vertebrate smooth muscle. TTX insensitive channels are known that are Na ÷ channels, for example in cnidarians (Anderson, 1987) but since it now seems unlikely that a Na ÷ action potential is present in echinoderms, the finding that the supposed Na ÷ channel of Tuft and Gilly was TTX-insensitive is not significant. The finding that Verapamil blocks the echinoderm channel is also not absolute p r o o f that it is acting on a Ca 2÷ channel since some Na ÷ channels are also blocked by that drug (Adams and Gage, 1979). Platyhelminthes show a TTX-sensitive Na ÷ channel (Koopowitz and Keenan, 1982) and this would confirm an evolutionary sequence towards this state with the ctenophores and cnidarians being intermediate (see Anderson, 1987). The echinoderm Ca 2+ channel does not fit this scheme but it is possible that larval echinoderms do have a Na ÷ action potential; however recording from these nervous systems will be even more difficult than in the adults.
825
Echinoderm nervous systems are highly enigmatic even beyond their non-centralized layout. They have no glial cells, and cell bodies are packed with glycogen, not endoplasmic reticulum; there are no blood spaces within tightly packed nervous tissue. Furthermore, ectodermal nerves cannot penetrate mesodermal effector tissues, and transmitter substances diffuse across substantial distances of the connective tissue basement membrane sheaths, and a separate mesodermal motor system has evolved (see Cobb, 1987, for review). Finally the non-cellular connective tissue is mutable and innervated (see Wilkie, 1984, for review). The present evidence does not prove, but is strongly indicative of a Ca 2÷ spike in all neurones without the involvement of Na ÷ ions, but the reason for this must at present be one more echinoderm enigma to add to the list. Acknowledgements--The authors would like to thank Drs Wendy Nightingale and Robert Pitman for helpful discussion.
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Adams D. J. and Gage P. W. (1979) Ionic currents in response to membrane depolarization in an Aplysia neurone. J. Physiol. 289, 115-142. Anderson P. A. J. (1987) Properties and pharmacology of a TTX-insensitive Na ÷ current in neurones of the jellyfish Cyanea capillata. J. exp. Biol. 133, 231-248. Berrios A., Brink D., del Castillo J. and Smith D. S. (1985) Some properties of the action potentials conducted in the spines of the sea urchin Diadema antillarum. Comp. Biochem. Physiol. 81A, 15-23. Binyon J. and Hasler B. (1970) Electrophysiology of the starfish radial nerve cord. Comp. Biochem. Physiol. 32, 747-753. Brehm P. (1977) Electrophysiology and luminescence of an ophiuroid radial nerve. J. exp. Biol. 71, 213-227. Cobb J. L. S. (1985) The neurobiology of the ectoneural/ hyponeural synaptic connection in an echinoderm. Biol. Bull. mar. biol. Lab., Woods Hole 165, 432-446. Cobb J. L. S. (1987) The echinodermata. In Invertebrate Nervous Systems (Edited by Ali M. A.), pp. 483-526. Plenum Press, New York. Hernandez Z. M., Morales M., Smith D. S. and del Castillo J. (1987) Barium spikes are generated in the spines of the sea-urchin Diadema antillarum. Comp. Biochem. Physiol. 86A, 355-359. Hille B. (1984) Ionic Channels of Excitable Membranes. Sinauer Associates. Sunderland, Massachusetts. Koopowitz H. and Keenan L. (1982) The primitive brains of platyhelminthes. Trends Neurosci. 5, 77-79. Millott N. and Okumura H. (1968) The electrical activity of the radial nerve in Diadema antillarum and certain other echinoids. J. exp. Biol. 48, 279-289. Pentreath V. W. and Cobb J. L. S. (1982) Echinodermata, in Electrical Conduction and Behaviour in Simple Animals (Edited by Shelton G. A. B.), pp. 440-472. Clarendon Press, Oxford. Sandeman D. C. (1965) Electrical activity in the radial nerve cord and ampullae of sea urchins. J. exp. Biol. 43, 247-256. Tuft P. J. and Gilly W. M. (1984) Ionic basis of action potential propagation along two classes of "giant" axons in the ophiuroid, Ophiopteris papillosa. J. exp. Biol. 113, 337-349. Wilkie I. C. (1984) Variable tensility in echinoderm collagen tissues. Mar. Behav. Physiol. 11, 1-34.