- Email: [email protected]
The giant through conducting neuron of the brittlestar Ophiura ophiura—A key neuron?
Camp. Biochem. Physiol. Vol. IOSA, No. 4. pp. 697-703, 1993 Printed in Great Britain
0
THE GIANT THROUGH CONDUCTING BRITTLESTAR OPHIURA OPHIURA-A
0300-9629/93 $6.00 + 0.00 1993 Pergamon Press Ltd
NEURON OF THE KEY NEURON?
J. L. S. COBB and M. GHYCOT School
of Biology and Bre-Clinical Medicine, University of St Andrews, KY16 8LB, U.K. (Tel. 0334-76161; Fax 033478299) (Received 5 October
1992; accepted 1I
November
1992)
Abstract-l. The morphology of the giant through conducting neuron (GTC) in the brittlestar Ophiura ophiuru is described using dye injection. 2. These neurons show bursts of action potentials correlated with stimuli that are potentially threatening. 3. GTC neurons conduct in both directions non decrementally throughout the animal. 4. GTC is not a command neuron and there are no changes in motor output correlated with this neuron firing. 5. It is hypothesized that it is a key neuron in coordinating whole animal responses to local stimuli.
INTRODIJCIION
Evidence about function in the nervous system of echinoderms has accumulated slowly and there is no doubt they are difficult animals to work with. Much of their biology is still enigmatic when considered comparatively with other groups of animals. Original studies using methylene blue by Smith (1966) have been shown to be largely unreliable because the technique fails to distinguish between narrow axon like processes from muscles and neurons themselves. Brehm (1977) reported that extracellular suction electrodes could record unitary activity from the nerve cord of brittlestars and that coincident with this were giant fibres in the nervous system. This technique was applied to the species Ophiuru ophiuru and recordings with very well differentiated unitary activity obtained (see Moore and Cobb, 1985). Subsequently, intracellular electrodes were used to record from some ectoneural neurons and the main hyponeural motor neurons. Early studies showed that a particular class of neurons [which we have now called the giant through conducting (GTC neuron)] fired in response to a shadow (Moore and Cobb, 1985). GTC neurons have two unique properties, firstly, spike activity is conducted unchanged throughout all the radial nerve cords when the periphery of any one arm is stimulated and, secondly, spike activity is conducted at a greater velocity than in other neurons cu 80 cm/set compared to 35 cm/set for other neurons (see Cobb and Moore, 1989). GTC neurons have subsequently been shown to fire to any stimulus that is associated with a threat to the well being of the animals. Such stimuli include shadow, mechanical vibration or noxious chemicals. GTC neurons are thus anatomically the largest and also the fastest conducting and this suggests they may have a key
function in the nervous system. This investigation reports some preliminary findings on their structure and function.
MATERIALS
AND METHODS
Specimens of the brittlestar Ophiura ophiura were obtained from the Marine Laboratory, Millport and kept in aerated running sea water. The largest possible specimens were used with a disc diameter of at least 2cm. Specimens were either used whole or a single arm was cut off. In both cases the material was clamped inverted and the part to be dissected immobile. The oral arm plates were then removed as well as part of the lateral plates to expose the radial nerve cord. A small amount of methylene blue in sea water was added to highlight the sheaths which are formed by the epineural sinus and both sheaths then removed with two pairs of finest watchmakers forceps. In a small number of preparations, especially large animals with a natural ochre coloured pigmentation, a number of cell bodies were sometimes visible. Electrodes were prepared with a resistance of approximately 50 MR when filled with various different solutions. Initially Lucifer Yellow was used iontophoretically to inject cells but later 5,6-carboxyfluorescein was used since it produced more reliable fills. This was prepared according to Rao ef al. (1986). The electrode tips were filled with carboxyfluorescein by capillary action and then backfilled with 4 M potassium acetate. Iontophoretic injection was achieved using IOnA of hyperpolarizing current applied every second in half second pulses. Extracellular recordings were made using plastic suction electrodes which had an internal diameter four fifths of the width of a particular nerve cord.
697
J. L. S. COBB
698
Activity was amplifiers.
subsequently
recorded
via standard
RESULTS
Morphology Initial results were obtained through random impalements. Numerous impalements were made of various types of cell and gradually various categories of neurons were identified morphologically. Among these neurons, which are illustrated in Cobb (1985) some have axons that may run either peripherally or centrally, usually for three or more segments. There are also bipolar neurons with axons passing in both directions, again for several segments. H-shaped neurons with four processes running for two to four segments each way occur (Fig. lb) as well as neurons that run transversely in a single segment. The GTC cells are however the only ones which produce a burst of spikes to a shadow stimulus during axon impalement. The approximate position of the cell body was eventually localized which increased the success rate of GTC impalement. It was finally realised that in a small number of highly pigmented animals, the GTC neuron was visible using a binocular microscope. Approximately 150 of GTC neurons have been filled, although most of them incompletely. The morphology of all GTC cells has been the same and is illustrated diagrammatically in Fig. la. The cell body always lies in the ganglion about one fifth of the way out from the midline of the nerve cord and approximately in line with the edge of the intervertebral ossicles. At no time, have two cells bodies been filled in the same ganglion; however it is not known for certain whether a GTC cell body occurs in each ganglion. On a number of occasions two successful fillings in two succeeding ganglia showed that each of them contains one GTC cell body. Moreover, the cell bodies appear to be on opposite sides of the midline in the succeeding ganglia and thus alternate (Fig. 1b). The main axon runs first transversely to the opposite side of the nerve cord from the cell body before turning a sharp right angle, and then runs longitudinally towards the central disc of the animal; about one fifth of the way out from the midline of the nerve cord (the longitudinal part of the axon lies thus on the opposite side to the cell body). No neurons have ever been filled that are physiologically characteristic of the GTC cell that have a peripherally running axon (i.e. towards the arm tip). In all impalements close to the cell body, the axon runs for at least six full segments (not including the one containing the cell body). This distance represents nearly 2 cm in an arm of a large animal (ca 15 cm in diameter). There are three areas of substantial varicose processes arising from the main axon trunk. These varicose areas are present in the transverse and the initial longitudinal part of the axon as well as its
and
M. GHYOOT
terminal region. There are no other processes along the length of the axon. In all three areas, the varicosities are very fine and often arise from the axon without processes of intermediate diameter. Especially good fills suggest that many of them are too small to be resolved except under exceptional circumstances. Because of the length of the cell, the terminal varicosities were only rarely filled. However, axon impalements were useful since they could be made nearer the terminal region and more reliable fills of this area obtained. There are also substantial dendrite like processes arising from the cell body on the opposite side to the axon hillock. These form a substantial dendritic tree which covers an area of about one third of the ganglion on the side of the cell body. They show varicosities and many of these are of a very fine size. When the cell bodies are impaled there is much synaptic activity that is both excitatory and inhibitory but there is no morphological evidence to suggest which processes are pre-synaptic and which are post synaptic. Nor is there evidence as to whether there are functional differences between the fine processes arising from the axon and those from the cell body. GTC cells were impaled and filled in the ganglionic arm segments immediately adjacent to the circumoral nerve ring. The morphology is the same as previously described. Entering the circumoral ring, the axon does not branch but travels round the ring on the side the axon is occupying in the arm segment. The terminal connections of these circumoral axons have not been traced. Physiofog) Impalement of the axon of GTC is coincident with a drop of about 70mv and a rapid burst of overshooting spike potentials to a test stimulus of general shadow applied to one arm tip (Figs 2 and 3). Extracellular recording shows that this activity is conducted rapidly throughout the whole animal (Fig. 4, but see also Moore and Cobb, 1985). Passage of hyperpolarizing current causes spikes on rebound as does 2-3 nA of depolarizing current. There are no other electrical events detected in axon impalements other than spikes, whatever stimuli are presented to the animal or even when the arm is moving. Penetration of the cell body is normally accompanied by small spontaneous synaptic potentials that are both inhibitory and excitatory. In a good impalement with a resting potential of 6&70 mV, these synaptic potentials are 3-7 mV at maximum (Figs 5 and 6). Recordings were also made using intra- and extracellular electrodes in the same ganglionic arm segment simultaneously. It becomes clear during these experiments that there are the axons of several GTC neurons in each segment since there are more large extracellular spikes than can be correlated with intracellular spikes (Fig. 7). If the preparation is stimulated by shadow. noxious chemical or mechanical vibration then a burst of synaptic activity gives rise to a number of
Fig 1.(a) Camera tucida drawing af a GTC neuron filkd with carbxy&mescein. The ~11 body lies one fifth out from the midline and roughly in line with the edge of the vertebral ossicle (VO). Note typically mast of the processes arc fine snd varicose and run transversely. Small processes arise from the iongitudina1 section of the neuron only in the first segment (inset a sketch of a complete GTC WZ.W~FI), &) Montage of photo~i~~~g~a~h~ of two GTC neurom (large arrows) in three successive sementar gang&. The nerve cord Is not stxai$% but the midline Is ~e~~~at~ &J faint auto%.mrescece of the septum of tlte ~~~~e~~~~ &us (smalt +3scffopis) Pati of an E%&qed neuron fHj is a&o s2mwx-x in t&z third segment. SC&z lW j.8Rx.
J. L. S. COBB and M. GHYWT
700
isolated arm. A stimulus applied to the tip of the isolated arm results in a burst of spikes in the GTC impaled as does a stimulus to any part of the rest of the animal, and the spikes are thus conducted in opposite directions within the axon of GTC. In a whole animal preparation which is responding to stimulus of the whole animal and the isolated arm, if the nerve cord is severed in the single arm peripheral to the recording electrode, then, as expected, responses to stimuli of the single arm are abolished (Figs 12 and 13). Those cells where the axon was shown to conduct both orthodromically and antidromically were subsequently filled and the morphology shown to be that typical of a GTC neuron. An attempt was made to find ectoneural interneurons post-synaptic to GTC. Other types of neuron impaled and subsequently filled (i.e. H cells) show no electrical activity (i.e. synaptic potentials or spikes) that is concomitant either with the stimulus or activity in GTC. An effect of GTC on motor output was also sought. The main muscles of the arm are innervated by a completely separate nervous system-the hyponeural nervous system (see Cobb, 1985). The hyponeural neurons receive both excitatory and inhibitory input across the basement membrane that separates the two nervous systems. The motor neurons are easily impaled and show both synaptic activity and spikes to stimuli that cause arm movements (see Cobb, 1985). Despite very intensive search no activity, either excitatory or inhibitory of ongoing activity, have ever been noted when GTC fires. DISCUSSION It is now clear that the echinoderm nervous system is arranged on the basis of segmental ganglia in the radial nerve cord and that the circumoral nerve ring mainly serves as non ganglionated connectives between them. Each segmental ganglion appears to be
built on the same morphological pattern with the individual neurons becoming larger as the animal grows bigger (see review, Cobb, 1987). Much of this information has come from studies on the nervous system of the brittlestar Ophiuru where some nerve ceils are large enough to impale with a recording micro-electrode (see Cobb, 1990). The most easily impaled ectoneural neuron is the GTC since it is consistently the largest and has the largest axon. Co-related with this is the fact that it has the fast conduction velocity of 80cm/sec compared to 35 cm/see in other large neurons, and it is apparently the only neuron that is part of a chain which passes activity non~ecrementally throughout the animal to a given stimulus in any position. All this implies that this neuron has a key function in coordinating whole animal behaviour to threatening situations. Before considering the possible function of this neuron, it is well to consider some of the enigmas associated with it. First the size and uniquely fast speed of conduction imply that rapid function is a prime consideration and yet the terminal processes at each end are of small diameter and the synapses almost certainly chemical. Indeed there have been no morphological or physiological reports of electrical synapses of conventional type in any adult echinoderm tissue (see Cobb, 1987). Second, there is not an obvious reason for the fact that many GTC neurons appear to run for six full segments (it never runs for less but high quality fills of the entire neuron are relatively rare and it cannot be ruled out that in some places it may be longer). There must therefore be overlap if each segment contains one neuron which makes no further synaptic connections for six segments. There is evidence, apart from the morphological, for overlapping neurons since extracellular recordings carried out simultaneously with intracellular ones in the same segment show more than one GTC is firing. The exact number is not clear from extracellular records but there are only a few record-
Fig. 2. Action potentials recorded from a GTC neuron to a shadow stimulus. The spikes are about 60 mV above the resting potential. Time scale = 2 sec. Fig. 3. Extracellular record to a similar shadow in a different preparation. Time scale = 1sec. Fig. 4. Record obtained with 4 extracellular electrodes each on a different arm to a shadow stimulus. Time scale = 5 sec. Fig. 5. Impalement of GTC cell body showing spontaneous inhibitory and excitatory synaptic potentials. Vertical scale = 5 mV, time scale = 1 sec. Fig. 6. Similar above with faster time scale. Vertical scale = 3 mV, time scale = 100 msec. Fig. 7. ~irnul~n~~ intra and extracelhdar records from the same segment after stimuIation of GTC. It can be seen that more than one GTC is recorded from extracellufarly in each segment. Time scale = 1 sec. Fig. 8. Synaptic potentiats giving rise to a burst of action potentials on a shadow stimulus. Vertical scale = 3 mV, time scale = 1 sec. Fig. 9. Recording of response to fifth stimulus of a train of I set shadows every 5 sec. Note reduction in number of action potentials compared to full response in Fig. 8. Scale as Fig. 8. Fig. 10. As Fig. 8 but the seventh stimulus in train. Scale as in Fig. 8. Fig. 1I. The same preparation as in Figs 8-10 but a vibration stimulus applied 5 set after that of shadow given in Fig. IO. Note the habituation to shadow is overridden. Scale as in Fig. 8.
W
I .
702
J. L. S. COBB and M. GHYCKIT
Fig. 12. Shadow response of whole animal recorded from an intact animal. Vertical scale = 3 mV. time scale = I sec. Fig. 13. The same stimulus to the same preparation with the same cell impaled but with the nerve cord severed peripheral to the impalement. Note the reduction in response. Scales as in Fig. 12.
able in each segment. Since only a small proportion of GTCs are visible with the light microscope even in fortuitous preparations the exact number of GTCs can only be estimated. Recent studies using immunological probes against the peptides SALMFamide (see Elphick et al., 1991) and RFamide (Grimmelikhuijzen, 1985) show that there are usually only single giant neurons staining to these probes in each segment and that they alternate on either side of the midline (Ghyoot and Cobb, in preparation). This evidence of a widespread phenomenon of single alternate neurons taken with the evidence extracted from all fiiis obtained suggests the hypothesis that GTC is a single neuron in each segment with cell bodies alternating on either side is reasonable. Segments immediately adjacent to the circumoral ring show that some GTC run one way round the ring and others the opposite. It is known from extraceliular recording that severing the nerve ring in one place does not inhibit transmission of GTC activity to any of the arms and this information can be conveyed “the long way round” the nerve cord between adjacent arms (Moore and Cobb, 1985). Threatening stimuli that are mechanical or chemical are responded to by the rapid movement of the animal away from the stimulus, a shadow on the other hand causes the animal to freeze its position (Moore and Cobb, 1985). Simultaneous recording from the ectoneural system with extracellular electrodes and the hyponeural motor neurons intracellularly shows no excitatory or inhibitory potentials in the motor neurons to a shadow or indeed any increase or decrease in spontaneous synaptic activity already present. Cobb (1990) previously suggested this neuron was an “alert” neuron that cleared the way for a coordinated response to a threat. All
evidence suggests that any part of the nervous system is capable of directing a whole animal behavioural response and that this is certainly not done centrally by the nerve ring and immediately adjacent ganglia. Further in an animal cut up into either large or small pieces (and autotomy of a lot or a little is very much a general echinoderm strategy) then providing the skelet~muscular system remaining has any sort of integrity-the bits are capable of co-ordinated behaviour within themselves. As each part of an intact animal is clearly able to carry out independent movements a coordinated whole animal response requires suppression of the local motor output (associated with local sensory input) and replacement by an output coordinated by the area of the nervous system directing a response. This would imply that a primary function of GTC would be inhibition of local reflexes in classes of segmental interneuron and possibly facilitation of interneurons responsible for interganglionic transmission of motor command information. Regrettably, almost certainly because of small size, no neurons that are post synaptic to GTC have consistently been recorded from or dye filled. It is now clear that there is a complex neurochemical layout in each ganglion which is similar in each segment and it is possible that GTC may also exert a pre-synaptic influence on these (Ghyoot and Cobb, in preparation). What is still not understood is what the minimum unit of the nervous system is. Initially Cobb and Stubbs (1981) suggested that the nerve cord contained neurons that were always confined to single segments. Dye-fills of many hundreds of neurons has shown however that this is a simplification with many neurons spanning several segments, in some cases at least six segments and in the case of some H-shaped
Giant neurons in brittlestars with processes in each direction, eight segments. The answer may well eventually be shown to be that there is no morphological minimum but control is exercised by an area of nervous system that has overlapping connections and is in receipt of a particular stimulus input at any one time. It may well be that anti- and orthodromic conduction is a widespread phenomenon associated with this flexibility. This overlap in function may be valuable to an animal that can shed all or part of an arm at any time. The neurons of Ophiuru are fortuitously large but even here many of the interneurons and all sensory neurons have so far proved intractable to study with intracellular techniques. In other classes of echinoderm there have been no successful intracellular studies reported and the neurons are certainly very small (see Cobb, 1970). It is valuable that the input to the motor neurons can easily be monitored intracellularly and thus something of the consequences of intemeuronal processing appreciated. The GTC neuron at present offers the best prospect for beginning to unravel the interneuronal processing that is the basis of the non-centralized behaviour co-ordinating mechanism. neurons
REFERENCES Brehm P. (1977) Electrophysiology and luminescence of an ophiuroid radial nerve. J. exp. Biol. 71, 213-227.
CBRA,
IWC”
703
Cobb J. L. S. (1970) The significance of the radial nerve cords in asteroids and echinoids. Z. Zelljbrsch. 108, 457474. Cobb J. L. S. (1985) The neurobiology of the ectoneuralhyponeural synaptic connection of an echinoderm. Biol. Bull. 168, 432-446.
Cobb J. L. S. (1987) Neurobiology of the Echinodermata. In Nervous Sysrems of Inverrebrutes (Edited by Ah M. A.), pp. 483-527. Plenum, New York. Cobb J. L. S. (1990) Enigmas of the echinoderm nervous system. In Evolution of the First Nervous System (Edited by Anderson P. A. V.), pp. 329-337. Plenum Press, New York. Cobb J. L. S. and Moore A. (1989) Studies on the integration of sensory information by the nervous system of the brittlestar Ophiura ophiura. Mar. Behav. Physiol. 14, 21 l-222.
Cobb J. L. S. and Stubbs T. R. (1981) The giant neurone system in ophiuroids I. The general morphology of the radial nerve cords and circumoral ring. Cell Tiss. Res. 219, 197-207.
Elphick M., Reeve J. R., Burke R. D. and Thorndyke M. (I 991) Isolation of the neuropeptide SALMFamide- I from startish using a new antiserum. Peprides 12,455459. Grimmelikhuijzen C. J. P. (1985) Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid nol~os. Cell Tiss. Res. 241. 171-183. Moore A. and Cobb J. L. S. (1985) Neurophysiological studies on photic responses in Ophiuru ophiuro. Comp. Biochem. Physiol. SOA,
1I-16.
Rao G., Barnes C. A. and McNaughton B. L. (1986) Intracellular fluorescent staining with carboxytluorescein: a rapid and reliable method for quantifying dye-coupling in the mammalian central nervous system. J. Neurosci. Meth. 16, 251-263. Smith J. E. (1966) The form and functions of the nervous system. In Echinoderm Physiology (Edited by Boolootian R. A.), pp. 503-512. Wiley, New York.
0
THE GIANT THROUGH CONDUCTING BRITTLESTAR OPHIURA OPHIURA-A
0300-9629/93 $6.00 + 0.00 1993 Pergamon Press Ltd
NEURON OF THE KEY NEURON?
J. L. S. COBB and M. GHYCOT School
of Biology and Bre-Clinical Medicine, University of St Andrews, KY16 8LB, U.K. (Tel. 0334-76161; Fax 033478299) (Received 5 October
1992; accepted 1I
November
1992)
Abstract-l. The morphology of the giant through conducting neuron (GTC) in the brittlestar Ophiura ophiuru is described using dye injection. 2. These neurons show bursts of action potentials correlated with stimuli that are potentially threatening. 3. GTC neurons conduct in both directions non decrementally throughout the animal. 4. GTC is not a command neuron and there are no changes in motor output correlated with this neuron firing. 5. It is hypothesized that it is a key neuron in coordinating whole animal responses to local stimuli.
INTRODIJCIION
Evidence about function in the nervous system of echinoderms has accumulated slowly and there is no doubt they are difficult animals to work with. Much of their biology is still enigmatic when considered comparatively with other groups of animals. Original studies using methylene blue by Smith (1966) have been shown to be largely unreliable because the technique fails to distinguish between narrow axon like processes from muscles and neurons themselves. Brehm (1977) reported that extracellular suction electrodes could record unitary activity from the nerve cord of brittlestars and that coincident with this were giant fibres in the nervous system. This technique was applied to the species Ophiuru ophiuru and recordings with very well differentiated unitary activity obtained (see Moore and Cobb, 1985). Subsequently, intracellular electrodes were used to record from some ectoneural neurons and the main hyponeural motor neurons. Early studies showed that a particular class of neurons [which we have now called the giant through conducting (GTC neuron)] fired in response to a shadow (Moore and Cobb, 1985). GTC neurons have two unique properties, firstly, spike activity is conducted unchanged throughout all the radial nerve cords when the periphery of any one arm is stimulated and, secondly, spike activity is conducted at a greater velocity than in other neurons cu 80 cm/set compared to 35 cm/set for other neurons (see Cobb and Moore, 1989). GTC neurons have subsequently been shown to fire to any stimulus that is associated with a threat to the well being of the animals. Such stimuli include shadow, mechanical vibration or noxious chemicals. GTC neurons are thus anatomically the largest and also the fastest conducting and this suggests they may have a key
function in the nervous system. This investigation reports some preliminary findings on their structure and function.
MATERIALS
AND METHODS
Specimens of the brittlestar Ophiura ophiura were obtained from the Marine Laboratory, Millport and kept in aerated running sea water. The largest possible specimens were used with a disc diameter of at least 2cm. Specimens were either used whole or a single arm was cut off. In both cases the material was clamped inverted and the part to be dissected immobile. The oral arm plates were then removed as well as part of the lateral plates to expose the radial nerve cord. A small amount of methylene blue in sea water was added to highlight the sheaths which are formed by the epineural sinus and both sheaths then removed with two pairs of finest watchmakers forceps. In a small number of preparations, especially large animals with a natural ochre coloured pigmentation, a number of cell bodies were sometimes visible. Electrodes were prepared with a resistance of approximately 50 MR when filled with various different solutions. Initially Lucifer Yellow was used iontophoretically to inject cells but later 5,6-carboxyfluorescein was used since it produced more reliable fills. This was prepared according to Rao ef al. (1986). The electrode tips were filled with carboxyfluorescein by capillary action and then backfilled with 4 M potassium acetate. Iontophoretic injection was achieved using IOnA of hyperpolarizing current applied every second in half second pulses. Extracellular recordings were made using plastic suction electrodes which had an internal diameter four fifths of the width of a particular nerve cord.
697
J. L. S. COBB
698
Activity was amplifiers.
subsequently
recorded
via standard
RESULTS
Morphology Initial results were obtained through random impalements. Numerous impalements were made of various types of cell and gradually various categories of neurons were identified morphologically. Among these neurons, which are illustrated in Cobb (1985) some have axons that may run either peripherally or centrally, usually for three or more segments. There are also bipolar neurons with axons passing in both directions, again for several segments. H-shaped neurons with four processes running for two to four segments each way occur (Fig. lb) as well as neurons that run transversely in a single segment. The GTC cells are however the only ones which produce a burst of spikes to a shadow stimulus during axon impalement. The approximate position of the cell body was eventually localized which increased the success rate of GTC impalement. It was finally realised that in a small number of highly pigmented animals, the GTC neuron was visible using a binocular microscope. Approximately 150 of GTC neurons have been filled, although most of them incompletely. The morphology of all GTC cells has been the same and is illustrated diagrammatically in Fig. la. The cell body always lies in the ganglion about one fifth of the way out from the midline of the nerve cord and approximately in line with the edge of the intervertebral ossicles. At no time, have two cells bodies been filled in the same ganglion; however it is not known for certain whether a GTC cell body occurs in each ganglion. On a number of occasions two successful fillings in two succeeding ganglia showed that each of them contains one GTC cell body. Moreover, the cell bodies appear to be on opposite sides of the midline in the succeeding ganglia and thus alternate (Fig. 1b). The main axon runs first transversely to the opposite side of the nerve cord from the cell body before turning a sharp right angle, and then runs longitudinally towards the central disc of the animal; about one fifth of the way out from the midline of the nerve cord (the longitudinal part of the axon lies thus on the opposite side to the cell body). No neurons have ever been filled that are physiologically characteristic of the GTC cell that have a peripherally running axon (i.e. towards the arm tip). In all impalements close to the cell body, the axon runs for at least six full segments (not including the one containing the cell body). This distance represents nearly 2 cm in an arm of a large animal (ca 15 cm in diameter). There are three areas of substantial varicose processes arising from the main axon trunk. These varicose areas are present in the transverse and the initial longitudinal part of the axon as well as its
and
M. GHYOOT
terminal region. There are no other processes along the length of the axon. In all three areas, the varicosities are very fine and often arise from the axon without processes of intermediate diameter. Especially good fills suggest that many of them are too small to be resolved except under exceptional circumstances. Because of the length of the cell, the terminal varicosities were only rarely filled. However, axon impalements were useful since they could be made nearer the terminal region and more reliable fills of this area obtained. There are also substantial dendrite like processes arising from the cell body on the opposite side to the axon hillock. These form a substantial dendritic tree which covers an area of about one third of the ganglion on the side of the cell body. They show varicosities and many of these are of a very fine size. When the cell bodies are impaled there is much synaptic activity that is both excitatory and inhibitory but there is no morphological evidence to suggest which processes are pre-synaptic and which are post synaptic. Nor is there evidence as to whether there are functional differences between the fine processes arising from the axon and those from the cell body. GTC cells were impaled and filled in the ganglionic arm segments immediately adjacent to the circumoral nerve ring. The morphology is the same as previously described. Entering the circumoral ring, the axon does not branch but travels round the ring on the side the axon is occupying in the arm segment. The terminal connections of these circumoral axons have not been traced. Physiofog) Impalement of the axon of GTC is coincident with a drop of about 70mv and a rapid burst of overshooting spike potentials to a test stimulus of general shadow applied to one arm tip (Figs 2 and 3). Extracellular recording shows that this activity is conducted rapidly throughout the whole animal (Fig. 4, but see also Moore and Cobb, 1985). Passage of hyperpolarizing current causes spikes on rebound as does 2-3 nA of depolarizing current. There are no other electrical events detected in axon impalements other than spikes, whatever stimuli are presented to the animal or even when the arm is moving. Penetration of the cell body is normally accompanied by small spontaneous synaptic potentials that are both inhibitory and excitatory. In a good impalement with a resting potential of 6&70 mV, these synaptic potentials are 3-7 mV at maximum (Figs 5 and 6). Recordings were also made using intra- and extracellular electrodes in the same ganglionic arm segment simultaneously. It becomes clear during these experiments that there are the axons of several GTC neurons in each segment since there are more large extracellular spikes than can be correlated with intracellular spikes (Fig. 7). If the preparation is stimulated by shadow. noxious chemical or mechanical vibration then a burst of synaptic activity gives rise to a number of
Fig 1.(a) Camera tucida drawing af a GTC neuron filkd with carbxy&mescein. The ~11 body lies one fifth out from the midline and roughly in line with the edge of the vertebral ossicle (VO). Note typically mast of the processes arc fine snd varicose and run transversely. Small processes arise from the iongitudina1 section of the neuron only in the first segment (inset a sketch of a complete GTC WZ.W~FI), &) Montage of photo~i~~~g~a~h~ of two GTC neurom (large arrows) in three successive sementar gang&. The nerve cord Is not stxai$% but the midline Is ~e~~~at~ &J faint auto%.mrescece of the septum of tlte ~~~~e~~~~ &us (smalt +3scffopis) Pati of an E%&qed neuron fHj is a&o s2mwx-x in t&z third segment. SC&z lW j.8Rx.
J. L. S. COBB and M. GHYWT
700
isolated arm. A stimulus applied to the tip of the isolated arm results in a burst of spikes in the GTC impaled as does a stimulus to any part of the rest of the animal, and the spikes are thus conducted in opposite directions within the axon of GTC. In a whole animal preparation which is responding to stimulus of the whole animal and the isolated arm, if the nerve cord is severed in the single arm peripheral to the recording electrode, then, as expected, responses to stimuli of the single arm are abolished (Figs 12 and 13). Those cells where the axon was shown to conduct both orthodromically and antidromically were subsequently filled and the morphology shown to be that typical of a GTC neuron. An attempt was made to find ectoneural interneurons post-synaptic to GTC. Other types of neuron impaled and subsequently filled (i.e. H cells) show no electrical activity (i.e. synaptic potentials or spikes) that is concomitant either with the stimulus or activity in GTC. An effect of GTC on motor output was also sought. The main muscles of the arm are innervated by a completely separate nervous system-the hyponeural nervous system (see Cobb, 1985). The hyponeural neurons receive both excitatory and inhibitory input across the basement membrane that separates the two nervous systems. The motor neurons are easily impaled and show both synaptic activity and spikes to stimuli that cause arm movements (see Cobb, 1985). Despite very intensive search no activity, either excitatory or inhibitory of ongoing activity, have ever been noted when GTC fires. DISCUSSION It is now clear that the echinoderm nervous system is arranged on the basis of segmental ganglia in the radial nerve cord and that the circumoral nerve ring mainly serves as non ganglionated connectives between them. Each segmental ganglion appears to be
built on the same morphological pattern with the individual neurons becoming larger as the animal grows bigger (see review, Cobb, 1987). Much of this information has come from studies on the nervous system of the brittlestar Ophiuru where some nerve ceils are large enough to impale with a recording micro-electrode (see Cobb, 1990). The most easily impaled ectoneural neuron is the GTC since it is consistently the largest and has the largest axon. Co-related with this is the fact that it has the fast conduction velocity of 80cm/sec compared to 35 cm/see in other large neurons, and it is apparently the only neuron that is part of a chain which passes activity non~ecrementally throughout the animal to a given stimulus in any position. All this implies that this neuron has a key function in coordinating whole animal behaviour to threatening situations. Before considering the possible function of this neuron, it is well to consider some of the enigmas associated with it. First the size and uniquely fast speed of conduction imply that rapid function is a prime consideration and yet the terminal processes at each end are of small diameter and the synapses almost certainly chemical. Indeed there have been no morphological or physiological reports of electrical synapses of conventional type in any adult echinoderm tissue (see Cobb, 1987). Second, there is not an obvious reason for the fact that many GTC neurons appear to run for six full segments (it never runs for less but high quality fills of the entire neuron are relatively rare and it cannot be ruled out that in some places it may be longer). There must therefore be overlap if each segment contains one neuron which makes no further synaptic connections for six segments. There is evidence, apart from the morphological, for overlapping neurons since extracellular recordings carried out simultaneously with intracellular ones in the same segment show more than one GTC is firing. The exact number is not clear from extracellular records but there are only a few record-
Fig. 2. Action potentials recorded from a GTC neuron to a shadow stimulus. The spikes are about 60 mV above the resting potential. Time scale = 2 sec. Fig. 3. Extracellular record to a similar shadow in a different preparation. Time scale = 1sec. Fig. 4. Record obtained with 4 extracellular electrodes each on a different arm to a shadow stimulus. Time scale = 5 sec. Fig. 5. Impalement of GTC cell body showing spontaneous inhibitory and excitatory synaptic potentials. Vertical scale = 5 mV, time scale = 1 sec. Fig. 6. Similar above with faster time scale. Vertical scale = 3 mV, time scale = 100 msec. Fig. 7. ~irnul~n~~ intra and extracelhdar records from the same segment after stimuIation of GTC. It can be seen that more than one GTC is recorded from extracellufarly in each segment. Time scale = 1 sec. Fig. 8. Synaptic potentiats giving rise to a burst of action potentials on a shadow stimulus. Vertical scale = 3 mV, time scale = 1 sec. Fig. 9. Recording of response to fifth stimulus of a train of I set shadows every 5 sec. Note reduction in number of action potentials compared to full response in Fig. 8. Scale as Fig. 8. Fig. 10. As Fig. 8 but the seventh stimulus in train. Scale as in Fig. 8. Fig. 1I. The same preparation as in Figs 8-10 but a vibration stimulus applied 5 set after that of shadow given in Fig. IO. Note the habituation to shadow is overridden. Scale as in Fig. 8.
W
I .
702
J. L. S. COBB and M. GHYCKIT
Fig. 12. Shadow response of whole animal recorded from an intact animal. Vertical scale = 3 mV. time scale = I sec. Fig. 13. The same stimulus to the same preparation with the same cell impaled but with the nerve cord severed peripheral to the impalement. Note the reduction in response. Scales as in Fig. 12.
able in each segment. Since only a small proportion of GTCs are visible with the light microscope even in fortuitous preparations the exact number of GTCs can only be estimated. Recent studies using immunological probes against the peptides SALMFamide (see Elphick et al., 1991) and RFamide (Grimmelikhuijzen, 1985) show that there are usually only single giant neurons staining to these probes in each segment and that they alternate on either side of the midline (Ghyoot and Cobb, in preparation). This evidence of a widespread phenomenon of single alternate neurons taken with the evidence extracted from all fiiis obtained suggests the hypothesis that GTC is a single neuron in each segment with cell bodies alternating on either side is reasonable. Segments immediately adjacent to the circumoral ring show that some GTC run one way round the ring and others the opposite. It is known from extraceliular recording that severing the nerve ring in one place does not inhibit transmission of GTC activity to any of the arms and this information can be conveyed “the long way round” the nerve cord between adjacent arms (Moore and Cobb, 1985). Threatening stimuli that are mechanical or chemical are responded to by the rapid movement of the animal away from the stimulus, a shadow on the other hand causes the animal to freeze its position (Moore and Cobb, 1985). Simultaneous recording from the ectoneural system with extracellular electrodes and the hyponeural motor neurons intracellularly shows no excitatory or inhibitory potentials in the motor neurons to a shadow or indeed any increase or decrease in spontaneous synaptic activity already present. Cobb (1990) previously suggested this neuron was an “alert” neuron that cleared the way for a coordinated response to a threat. All
evidence suggests that any part of the nervous system is capable of directing a whole animal behavioural response and that this is certainly not done centrally by the nerve ring and immediately adjacent ganglia. Further in an animal cut up into either large or small pieces (and autotomy of a lot or a little is very much a general echinoderm strategy) then providing the skelet~muscular system remaining has any sort of integrity-the bits are capable of co-ordinated behaviour within themselves. As each part of an intact animal is clearly able to carry out independent movements a coordinated whole animal response requires suppression of the local motor output (associated with local sensory input) and replacement by an output coordinated by the area of the nervous system directing a response. This would imply that a primary function of GTC would be inhibition of local reflexes in classes of segmental interneuron and possibly facilitation of interneurons responsible for interganglionic transmission of motor command information. Regrettably, almost certainly because of small size, no neurons that are post synaptic to GTC have consistently been recorded from or dye filled. It is now clear that there is a complex neurochemical layout in each ganglion which is similar in each segment and it is possible that GTC may also exert a pre-synaptic influence on these (Ghyoot and Cobb, in preparation). What is still not understood is what the minimum unit of the nervous system is. Initially Cobb and Stubbs (1981) suggested that the nerve cord contained neurons that were always confined to single segments. Dye-fills of many hundreds of neurons has shown however that this is a simplification with many neurons spanning several segments, in some cases at least six segments and in the case of some H-shaped
Giant neurons in brittlestars with processes in each direction, eight segments. The answer may well eventually be shown to be that there is no morphological minimum but control is exercised by an area of nervous system that has overlapping connections and is in receipt of a particular stimulus input at any one time. It may well be that anti- and orthodromic conduction is a widespread phenomenon associated with this flexibility. This overlap in function may be valuable to an animal that can shed all or part of an arm at any time. The neurons of Ophiuru are fortuitously large but even here many of the interneurons and all sensory neurons have so far proved intractable to study with intracellular techniques. In other classes of echinoderm there have been no successful intracellular studies reported and the neurons are certainly very small (see Cobb, 1970). It is valuable that the input to the motor neurons can easily be monitored intracellularly and thus something of the consequences of intemeuronal processing appreciated. The GTC neuron at present offers the best prospect for beginning to unravel the interneuronal processing that is the basis of the non-centralized behaviour co-ordinating mechanism. neurons
REFERENCES Brehm P. (1977) Electrophysiology and luminescence of an ophiuroid radial nerve. J. exp. Biol. 71, 213-227.
CBRA,
IWC”
703
Cobb J. L. S. (1970) The significance of the radial nerve cords in asteroids and echinoids. Z. Zelljbrsch. 108, 457474. Cobb J. L. S. (1985) The neurobiology of the ectoneuralhyponeural synaptic connection of an echinoderm. Biol. Bull. 168, 432-446.
Cobb J. L. S. (1987) Neurobiology of the Echinodermata. In Nervous Sysrems of Inverrebrutes (Edited by Ah M. A.), pp. 483-527. Plenum, New York. Cobb J. L. S. (1990) Enigmas of the echinoderm nervous system. In Evolution of the First Nervous System (Edited by Anderson P. A. V.), pp. 329-337. Plenum Press, New York. Cobb J. L. S. and Moore A. (1989) Studies on the integration of sensory information by the nervous system of the brittlestar Ophiura ophiura. Mar. Behav. Physiol. 14, 21 l-222.
Cobb J. L. S. and Stubbs T. R. (1981) The giant neurone system in ophiuroids I. The general morphology of the radial nerve cords and circumoral ring. Cell Tiss. Res. 219, 197-207.
Elphick M., Reeve J. R., Burke R. D. and Thorndyke M. (I 991) Isolation of the neuropeptide SALMFamide- I from startish using a new antiserum. Peprides 12,455459. Grimmelikhuijzen C. J. P. (1985) Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid nol~os. Cell Tiss. Res. 241. 171-183. Moore A. and Cobb J. L. S. (1985) Neurophysiological studies on photic responses in Ophiuru ophiuro. Comp. Biochem. Physiol. SOA,
1I-16.
Rao G., Barnes C. A. and McNaughton B. L. (1986) Intracellular fluorescent staining with carboxytluorescein: a rapid and reliable method for quantifying dye-coupling in the mammalian central nervous system. J. Neurosci. Meth. 16, 251-263. Smith J. E. (1966) The form and functions of the nervous system. In Echinoderm Physiology (Edited by Boolootian R. A.), pp. 503-512. Wiley, New York.