the nervous control of autotomy in carcinus maenas - Journal of ...

J. Exp. Biol. (1974). 60, 423-436

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With 1 plate and 10 text-figures Printed in Great Britain

THE NERVOUS CONTROL OF AUTOTOMY IN CARCINUS MAENAS BY ALISTAIR McVEAN Marine Science Laboratories, Menai Bridge, North Wales* {Received 30 August 1973) INTRODUCTION

Autotomy is the process by which a part of the body of an animal may be discarded at a point where there are structural adaptations which facilitate severance and reduce subsequent bleeding. Usually it is a damaged limb that is lost, as in the case of spiders (Parry, 1957), mantids (Bordage, 1905), starfish (Abeloos, 1932) and the gastropods Helixarian and Harpa (Abeloos, 1932) while lizards are well known for their ability to drop their tails (Morgan, 1932). Carcinus can autotomize any limb subjected to damage. The mechanism by which this happens has been documented several times with the most convincing accounts given by Fr6dericq (1892) and Paul (1915). Both basi-ischiopodite levators are involved, operating together to break the limb at a preformed plane situated in the fused basiischiopodite of each limb. The posterior levator is much the smaller of the two muscles. This muscle contracts when the limb is grossly stimulated, breaking a preformed plane in the tendon of the anterior levator (McVean, 1973). This action switches the application of the large levator so that its tension is concentrated onto a small skeletal plug crossing the limb breakage plane. A powerful contraction of the anterior levator withdraws the plug from its socket distal to the breakage plane so that a relatively small force applied externally to the limb is enough to sever it. It follows from the above account that autotomy in Carcinus is a precise act involving co-ordination of at least two muscles. While the nature of the stimulus can vary, the neural output to these two muscles is standardized. Thus the central nervous system must be programmed to produce the co-ordinated contraction of these two muscles whenever a particular sensory threshold is achieved. It seems probable that this threshold is centrally determined and can be varied. An animal that has already lost several limbs shows an increased resistance to the loss of yet another (Hoadley, 1934; Gomez, 1964), while Carlisle (1957) found that Maia will not autotomize after terminal anecdysis has been reached. What is not clear is how the motor commands to the two levators differ from normal walking and elevator commands. There is the choice, as far as the animal is concerned, between employing normal motor neurones at higher firing rates or invoking special motor neurones reserved for this purpose only. • Present address: Bedford College, Regent's Park, London N.W. 1.

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Text-fig. 1. Diagram illustrating the form, situation and innervation of the basi-ischiopodite levators of the fifth left limb from a ventral viewpoint. The basi-ischiopodite depressor has been removed. allt al2, anterior and posterior branches of the anterior levator; bp, limb breakage plane; dr, dorsal coxopodite remoter; vr, ventral coxopodite remotor; n, main nerve to limb; n alt, nerve to posterior branch of the anterior levator; pi, posterior levator.

MATERIALS AND METHODS

Carcinus maenas (L.) were obtained locally. For straightforward light microscopy, material was fixed in Bouin's solution and treated with Palmgren's (1948) silver staining technique. The nerve supplying the anterior levator wasfixedin 2-5 % gluteraldehyde at o °C, buffered with 0-05 M cacodylate to pH 7-4, rinsed in buffer and postfixed in 1 % buffered osmium tetroxide. After alcoholic dehydration it was infiltrated with epoxypropane and embedded in Araldite. From this were cut sections for light microscopy which were stained in a solution of 1 % borax and toluidene blue. Areas requiring greater resolution were cut from the same block and examined in the transmission electron microscope. Electrophysiological investigations were performed with the crab held firmly upside down in a tailor-made clamp. The fifth limbs provide the greatest access with least dissection damage by virtue of their expanded sternal plates. To expose the anterior levator muscle and its nerve the sternal plate of the left side was removed and the ventral surface of the coxopodite was cut away. The basi-ischiopodite depressor and ventral remotor were cleanly removed, exposing the ventral face of the large anterior basi-ischiopodite levator (Text-fig. 1). The nerve supplying this muscle runs ventrally from the limb nerve as soon as it enters the box formed by the endophragmal skeleton around the muscles, but is accessible for only a short distance before it enters the body of the muscle. Activity in this nerve was recorded with glass suction electrodes while the immediate area was bathed in saline. Good preparations lasted several hours. Intracellular muscle activity was recorded with glass micropipettes filled with 3 M-KC1, suspended by heat-shrinkable plastic tubing so that the tip of the electrode could move with the muscle. To record the tension produced by the anterior levator muscle when the nerve to it was stimulated, the tendon was cut at its insertion onto the basi-ischiopodite and held at rest length with forceps attached to the anode of an RCA 5734 tube.

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Text-fig. 2. Diagram showing the application of each thoracic muscle on the coxopodite and basi-ischiopodite of the fifth right limb viewed from the right-hand side. The arrows indicate the direction in which each muscle moves the segment to which it is attached while the length of the arrow is proportional to the number of muscle fibres in each muscle. The shaded portion represents the basi-ischiopodite.

Electromyograms were obtained from both levators with silver wires of 50 jum diameter, insulated except for the tip, inserted through small holes bored through the skeleton in the region of the muscle. The position of the tip was confirmed by dissection after recording. All signals were displayed on either a Tektronix 565 or 502A oscilloscope. RESULTS

General anatomy

Within the endophragmal chamber associated with each of the fifth limbs are five separate muscles. Three of these operate the coxopodite, two as remotors while the third promotes the limb. These muscles form discrete bundles and tend to have their origins restricted to within a small area. The large basi-ischiopodite levator and depressor muscles also have their origins within the thorax. Distally they penetrate the coxopodite to attach to the dorsal and ventral rim of the basi-ischiopodite respectively. These are powerful muscles containing a large number of fibres all of which attach to a central tendon, thus giving a bifurcated form to the muscle. Such muscles, as in the claw closer, are extremely powerful although their optimum tension range may be restricted because of the shorter length of the individual fibres. Sectioning these muscles gave an estimate of the number of fibres each contained. The results are expressed in Text-fig. 2. The number of fibres in the small posterior levator was estimated by dissection. This is the only muscle in this complex that has its origin

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outside the thorax, spanning from the inner dorsal face of the coxopodite to the dorsal rim of the basi-ischiopodite. It is instructive to compare the capabilities of the basi-ischiopodite depressor and its antagonist, the anterior levator. This can be done by calculating the force required in each muscle to fulfil its particular function. Thus the depressor, when contracted, depresses the limb until the tip of the dactylopodite touches the ground. Further contraction will now raise the body of the crab, the dactylopodite acting as the fulcrum. By considering the weight of the crab and by taking moments about the coxo-basal joint, the force exerted by the depressor muscle to hold the animal off the ground can be calculated. For an animal weighing 60 gms this works out at about 130 g wt if all eight walking limbs share an equal load. This figure is calculated for the first two segments being in line. For an emarginated articulation as this is, the force required to hold the animal off the ground varies with the angle subtended between the coxopodite and basi-ischiopodite. If the limb should be imparting an acceleration to the body, then the force achieved by this muscle will be greater than in the static situation. In any case 150 gwt must be a conservative estimate. Under water, the fraction of this force devoted to combating gravity is considerably less. Assuming that the contribution of each muscle fibre is more or less the same, the force exerted by each fibre of the depressor is in the region of 1 g wt. By a similar argument it can be shown that the force exerted by each fibre in the anterior levator muscle when the limb is held in the same position but with the dactylopodite off the ground is only o-i g wt. Forces of these magnitudes are within the range possible for single musclefibresof crabs (Atwood, 1967). When the crab is submerged, the depressor muscle fibres have to mount a tension of about 0-2 g wt when all eight walking limbs contribute evenly in supporting the weight. The transition for a crab of this size from walking unsupported in air to walking under water must entail a remarkable re-setting of tension and motor output to the basi-ischiopodite depressor muscles. It seems likely that the disproportionate number of muscle fibres of the anterior levator, in comparison with the depressor, are used to effect autotomy. If the muscle fibres of the anterior levator are capable of attaining the same order of tension as do the fibres of the depressor when they support the crab out of water, then nine-tenths of the potential power of the levator is reserved for securing a sufficient force to break the plug across the preformed breakage plane when the limb is autotomised. The larger the levator muscle the greater can be the strength of the plug across the breakage plane and the safer the limb from accidental autotomy. There must be a nice balance of strengths between the plug and the levator muscle allowing a reasonable safety factor to prevent the limb being broken off accidentally, yet the muscle must be strong enough to rupture the plug in autotomy. Two kinds of forces produce limb severance; a tensile force along the axis of the plug withdraws it from its socket while a shearing force performs the final separation along the preformed part of the breakage plane. Because the force exerted by the anterior levator during autotomy is applied at the point of insertion, there is a one-to-one mechanical advantage. Similarly, the breakage plane is sited immediately beyond the insertion so that once again the force elicited in the shearing plane is almost equal to that exerted by the muscle.


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Text-fig. 3. Graph showing the relationship between the cross-sectional area of each muscle and the average cross-sectional area and diameter of each muscle fibre contained within any one muscle. 1, basi-ischiopodite depressor; 2, ventral coxopodite remoter; 3', anterior branch of the anterior basi-ischiopodite levator 53*, posterior branch of the anterior levator; 4, coxopodite promotor; 5, dorsal coxopodite remotor.

Structure of the muscles

All five muscles contained fibres whose structure ranged from the two classic extremes of 'felderstruktur' and 'fibrillenstruktur'. The anterior levator contained a majority of 'fibrillenstruktur' fibres, with diameters ranging from 580 to 775 /tm. A few fibres showing 'felderstruktur' and having diameters ranging from 290 to 370 /tm are found towards the centre of the muscle. The posterior branch also contained a few fibres with an apparently exaggerated 'felderstruktur'. Their diameters of about 680 /tm are twice as large as any of the other fibres. In Text-fig. 3 the cross-sectional area of each muscle is shown to increase with the number of fibres contained in it. If the cross-sectional area and the diameter of all the musclefibreswere uniform, then there should be a one-to-one correspondence between the cross-sectional area of the whole muscle and the number of muscle fibres contained within it. Thus if it was assumed that fibre diameter is independent of the crosssectional area of the whole muscle, then a muscle containing half the number of muscle fibres should have half the cross-sectional area of another muscle with twice the number of fibres. Text-fig. 3 shows that this relationship does not hold. Instead, the average cross-sectional area of each muscle fibre diminishes as the area in cross28

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section of the whole muscle increases. Inspection of individual muscles reveals that there is no uniform diameter for each muscle fibre, so that to be consistent with the described trend, the larger muscles must contain a greater proportion of small-diameter fibres than do the smaller muscles. Bittner (1968), in a table of distinguishing characteristics often attributed to crustacean phasic and tonic fibres, lists the former as having diameters of 300-800 /on and tonic fibres as having diameters of 50-800/tm. It is possible that the larger muscles contain a greater proportion of tonic fibres than do the small muscles. Both the basi-ischiopodite depressor and anterior levator have to be able to exert considerable tonic forces, so this interpretation is consistent with their function. Conversely, during fast walking on land these muscles have to work at rates of up to 1 Hz. Both muscles therefore need to display both phasic and tonic properties. Innervation of the levator muscles

The gross anatomy of the nerves supplying both levators is shown in Text-fig. 1. In cross-section the nerve supplying the posterior branch of the anterior levator, at the point where it leaves the main nerve to the limb, contains twenty-one axons (PI. 1, fig. 1). The smaller-diameter axons seen in the light microscope were confirmed by electron microscopy (PI. 1, fig. 2). The five largest axons conform to the concept of a giant axon (Bullock & Horridge, 1965). Their diameters lie between 33 and 50 /im. The diameter of the remaining axons ranges from 24 to 3 jim. On the basis of diameter these axons fall into three groups, a conclusion which is supported by the recordings from the nerve. Tonic and phasic activity in the nerve to the anterior levator

Two units could regularly be seen to be active. Each had a distinctive spike height. The unit with the smaller spike was tonic in character, with a strong tendency to continue firing whatever the position of the CB joint. Both units displayed the resistance reflex demonstrated by Bush (1965). Activity in the larger unit could always be arrested instantaneously by the smallest elevatory movement, but the tonic unit would continue firing though at a lower frequency. If the limb was rotated about the CB articulation and held at successively more depressed positions, followed by a similar traverse back towards an elevated position, the discharge in the tonic unit was not identical for a repeated position, being consistently less pronounced when the limb was being elevated step by step. Thus the discharge followed a hysteresis curve. The reflex discharge frequency was therefore not absolute for any given limb position, but was influenced by the immediately preceding movement or position. Bush showed that the resistance reflex in this case was generated by the CB chordotonal organ. In addition, passive movement of both ipsilateral and contralateral limbs evoked a similar but reduced discharge in both units. Tactile stimulation to either the experimental limb, the carapace or the abdomen produced particularly vigorous activity in both units, although the tonic unit had the lower threshold and continued to be active after the stimulus was withdrawn, whereas the phasic unit stopped immediately. Gentle to moderate stimulation thus produces the same kind of motor output to the anterior levator as is produced by stretching the CB organ. Both share the same motor neurones to the muscle.


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2 sec Text-fig. 4. Action potentials in three motor axons in n a/2 in response to damaging stimuli applied distally to the limb. The tonic (a) and phasic (b) units fire almost continuously from the moment the stimulus is applied, while the giant axon (c) produces a coherent burst some seconds after the application of the stimulus. During autotomy this burst lasts for up to 20 sec.

Autotomy of a limb in an intact crab can be produced by a variety of stimuli. The most effective of these and the one that probably occurs in natural conditions is crushing of any limb segment except the dactylopodite. Autotomy can be produced in a more controlled manner by bringing a hot iron near to the limb. This has the advantage of not disturbing any electrodes monitoring activity in the nerves. If either such stimuli is applied to the limb, both tonic and phasic units fire at high frequencies. If the stimulus is prolonged, a third, large spiking unit, becomes active (Text-fig. 4). Initially this third unit fires at a relatively slow rate, but stops if stimulation to the limb is removed. If the stimulation is maintained the frequency of the third unit increases to a critical level beyond which it continues irrespective of the presence or absence of any stimulus. In all cases this unit stopped firing after a few seconds. Neuromuscular transmission and muscle tension

Preparations mounted with minimum dissection damage showed a variety of junction potentials in the anterior levator muscle. Text-fig. 5 a shows continuous tonic activity of small junction potentials. The resting potential is seen to fluctuate but not obviously in response to the frequency of the junction potentials which individually averaged about 0-5 mV. depolarization. Other fibres sampled showed junction potentials of about this size but with a much lower background frequency. Tactile stimulation of the carapace served to increase the frequency, and these junction potentials then showed some facilitation with marked summation (Text-fig. $b). These potentials were also produced by gentle stretching of the CB organ. Activity in the nerve to the muscle simultaneously with the junction potentials suggests that the tonic and phasic motor neurones respectively are responsible for the two kinds of muscle response. The intracellular response of the muscle fibres to stimuli which in an intact crab 28-2

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20 mV f 1 sec Text-fig. 5. Intracellularly recorded junction potentials from the anterior levator muscle take three forms. Tonic junction potentials (a) at a steady frequency barely disturb the resting potential whereas phasic potentials (6) produced in response to scratching the carapace, show some facilitation and marked summation. Crushing or heating the limb elicits bursts of overshooting potentials of some 60-75 m V , (c).

produce autotomy are shown in Text-fig. 5 c. The junction potential gave rise to overshooting spikes of some 60-70 mV. By monitoring the tension of the muscle and the post-synaptic response to stimulation of the nerve, three systems could be separately distinguished. The tonic system was elicited by reducing the intensity of stimulation until there was no longer any mechanical response to individual stimulus pulses. Initially no mechanical response could be seen, but by increasing the duration of stimulation a slow contraction became apparent. Tension varied with stimulation frequency, the lowest effective rate (20 Hz) requiring half a second to produce any measurable tension, while a stimulation frequency of 100 Hz produced the maximum rate of increase of tension as well as the maximum final tension (Text-fig. 6). This tonic system operates at high frequency, but within a comparatively narrow range of frequency, with a fivefold increase of tension from the minimum to the maximum effective rates. At all effective frequencies tension was maintained for a further 250 ms after stimulation had stopped, then declined slowly. Within the same neuromuscular unit there are present two twitch systems (Textfig. 7). Stimulation at gradually increasing intensities elicitsfirstthe smaller of the twitch

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Text-fig. 6. By reducing the intensity of stimulation to n a/s until individual twitches of the anterior levator are abolished, the slow tension response becomes evident for high and prolonged stimulation frequencies. Stimulation frequency: 20, 50, 100, 200 Hz.

systems which shows summation of both junction potentials and tension. This unit differs from the tonic system in that it is considerably activated at frequencies that are only just beginning to produce tension in the tonic system. After stimulation has stopped, tension declines rapidly. At stimulation intensities above those that elicit this first twitch response, a further twitch system is seen which involves an active response of the muscle membrane. The active response follows the junction potential and is associated with a ' giant' twitch of the muscle. As expected, the tension of the muscle when this system is activated is frequency-related, so that between frequencies of 20 and 50 Hz an extremely powerful tetanus is produced.

DISCUSSION

It is suggested that non-autotomizing activity of the anterior levator muscle is mediated through the tonic unit and smaller of the two twitch units. The tonic neurone maintains a background discharge and thus a continual, gently fluctuating tension unless centrally or reflexly inhibited. This tonic activity may be reflexly excited by external stimuli such as touching the carapace or internally by stretching the CB organ. The small twitch system always operates in conjunction with the tonic unit but requires a higher threshold of excitation to activate it. The response of this unit declines rapidly with withdrawal of the stimulus and also exhibits habituation. Centrally originating activity is also mediated through both these units. The neurone producing the ' giant' twitch in the anterior levator is activated only by gross stimulation to the limb, such as crushing or heating it. This neurone is apparantly directly influenced by peripheral stimulation but has a much higher threshold

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3g 10 mV 200 msec Text-fig. 7. Intracellularly recorded junction potentials and tension produced by the whole of the anterior levator muscle in response to stimuli of the same frequency and duration applied at two intensities to n aL. In (a) the small junction potentials are associated with small but distinct twitches in the whole muscle. In (6) the intensity of stimulation was increased to the threshold at which the giant axons were excited, causing overshooting potentials in the muscle associated with much larger contractions.

than either the tonic or small phasic neurones. Once this neurone is excited and achieves a certain firing frequency, usually about 1-5 Hz, it becomes self generating and runs for a pre-determined period. It has been shown (McVean, 1973) that autotomy is brought about by the coordinated contraction of both basi-ischiopodite levators. The levators contract together, the posterior levator moving its tendon in such a way that it breaks the tendon of the anterior levator at a preformed breakage plane. Paul (1915) has already shown that both levators contract simultaneously when the limb is crushed distally. Electromyograms show that the posterior levator is innervated by two units (Text-fig. 8 a), the smaller of which is tonic in nature and responds to non-autotomizing stimuli. The larger, phasic unit, like the autotomizer neurone innervating the anterior levator, responds only to crushing or heating of the same limb. Clarac, Wales & Laverack (1971) reported that mechanical deformation of a cuticlestress detector, CSD 1 (Wales, Clarac and Laverack, 1971) whether by external means


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2 sec Text-fig. 8. Electromyogram from the posterior levator (a) showing the two units active when the limb was crushed distally. The small tonic unit was activated by non-autotomizing stimuli whereas the phasic unit responded to gross stimulation of the limb. In (6) the activity of the phasic unit to the posterior levator is shown to precede the low-frequency potentials of the anterior levator which produce the powerful tetanus employed to sever the limb. 40 o

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