The Functioning of the Giant Nerve Fibres of the Squid¹

Study of the structure and connexions of the giant fibres which occur in the nerves of cephalopods has led to the supposition that they are motor nerve fibres (Young, 1936 a, b, c). However, no experimental investigation of their functioning has yet been published, and even the nervous nature of such curious structures still requires to be demonstrated. The experiments reported in this paper have shown that these fibres are in fact motor axons, and that on account of their enormous size they provide unique opportunities for study of the functioning of single neuromuscular units.

A squid is capable of moving through the water either slowly by means of its fins or very fast by the expulsion of a jet of water through its funnel. It is this rapid movement which is produced by the activity of the giant nerve fibres. The muscles concerned in the movement are circular fibres of the mantle, serving to make its cavity smaller, and longitudinal fibres in the retractor muscles of the head and of the funnel, serving to pull these parts of the body back towards the mantle cavity. All of these groups of fibres can be activated from the single pair of giant nerve cells in the central nervous system which are termed the primary or first order giant cells (Young, 1936 a, b). These do not innervate the muscles directly but branch and make synaptic contact with second order giant fibres, of which there are on each side five running to the retractor muscles of the head and funnel, and two to the stellate ganglion. In the latter ganglion the fibres branch and make synaptic contact with about ten third order giant fibres which pass to the muscles of the mantle. There is, therefore, one giant synapse on the path to the retractors but two on that leading to the muscles of the mantle.

The full description of these synapses and their functioning must be left to a later date. Here we are concerned only with the final motor fibres, namely, the second order axons running to the retractors and the third order axons running to the mantle muscles.

The first aim of the investigation was to discover whether these large structures are in fact motor nerve fibres. This having been shown the second aim became to discover whether each fibre with its muscles behaves as a single neuromotor unit in the manner of vertebrate motor neurons (see Sherrington, 1929; Kato, 1936). This latter problem is of particular interest since the third order giant axons have an unusual syncytial structure, being formed by fusion of the processes of numerous nerve cells (Young, 1936a).

The mantle of Loligo is used for respiration as well as for locomotion. It is a deep sack, closed at the hind end, water being taken in anteriorly at the sides and expelled through a narrow tube, the funnel, also at the front end, suitable valves operating to close the lateral inlets during expulsion. During life a continual slow rhythmical expansion and contraction of the cavity of the mantle is kept up, providing a flow of water over the gills. In addition to this rhythmical movement the sack can also be suddenly contracted, causing expulsion of the powerful jet which propels the animal rapidly along, either forwards or backwards according to the position of the funnel, a stream of “ink “being sometimes ejected at the same time to provide cover for the animal’s escape.

The muscles which produce the movements of the mantle include circular fibres running around the sack and radial fibres running through its thickness from the inside to the outside (Fig. 1). There is also a thin layer of longitudinal muscles on the outer surface of the very front end of the mantle, but this layer extends only over a very small part of the surface and has been neglected in the present account. The circular muscles have been described by Marceau (1906) and Burian (1908), but the radial fibres appear to have been overlooked by previous workers.

The circular fibres are uninucleate and very long. They are joined to each other end to end, the whole mass being attached dorsally to the pen. The radial fibres form sheets which divide the circular fibres into blocks. These sheets of radial fibres, however, are not continuous, but broken up, especially laterally, as shown in Fig. 1, in which the section is thick enough to include both circular and radial fibres.

The presence of two sets of antagonistic fibres mixed in the same mass of muscles appears to be characteristic of the cephalopods, since it is found also in the retractor muscles of the head and funnel (see p. 181). For proper working of such a system particular conditions of rigidity and elasticity are necessary and these are apparently supplied by a very complex system of connective tissue fibres found in these muscles. There is one set of such fibres lying in the plane of the circular muscles, but running obliquely in all directions across the latter. A second set of connective tissue fibres lies in the plane of the radial muscles.

According to Plenk (1933), who investigated the fine structure of the muscle fibres of various organs in cephalopods, a true cross striation is present in all of them.

It appears from the structure of the mantle, therefore, that water is taken into the sack by contraction of the radial muscles, serving to make the wall thinner and the cavity greater. The mantle is like a balloon which has the power of blowing itself up by stretching the wall. Such a mechanism of intake is highly peculiar, and does not seem to have been reported from any other group of animals. There is in this paper very little evidence as to the functions of the radial fibres (see p. 178), but their direction of action, and the absence of any other antagonists for the circular muscles, leaves little doubt as to their mode of action.

Whatever may be the mechanism of expansion of the mantle cavity it is certain that contraction of the circular muscles expels the water, either gently, as in respiration, or violently in sudden movements. It is the nerves producing this contraction of the circular muscles which have been principally studied in the present work.

In Loligo pealii (Leseuer), the species of squid most commonly found at Wood’s Hole, the mantle is supplied by between nine and eleven stellar nerves, which radiate from the stellate ganglion (Fig. 2). Each of these nerves contains one giant fibre and numerous smaller ones, the diameters of the giant fibres increasing progressively from 100μ or less in the anterior and shorter stellar nerves to 800μ or more in the hindmost and longest nerve. The giant fibres branch repeatedly among the muscle fibres but the nature of their final terminations has not yet been made out.

For investigation of the contractions of the circular muscles of the mantle the hindmost stellar nerve and the territory which it innervates proved to be convenient (Fig. 3). The animal is opened by cutting with scissors along the mid-ventral line and the pallial nerve, which joins the stellate ganglion to the central nervous system, severed on both sides. With fine scissors or scalpel the hindmost stellar nerve is then freed from the visceral mass on each side, after which the whole of the viscera can readily be removed, starting from the anterior end. This leaves only the stellate ganglia, mantle and pen, which latter can then also be carefully lifted away, starting from the hind end.

The portion of the hindmost stellar nerve which is to be stimulated can next be isolated by dissecting the fin nerve away from it, tying it with cotton close to the stellate ganglion and cutting it away from the latter. About 3 cm. of nerve can very easily be prepared in this manner, and if required a further 5 cm. or more can be added without great difficulty by further dissection of the nerve as it runs through the muscles.

The piece of muscle chosen for recording is finally cut free with a scalpel as shown in Fig. 3 and attached to a lever with glass or silver hooks, or, quite satisfactorily, by threading a piece of cotton through it with a needle. The preparation is pinned down to a piece of cork, and the thread attached so that the circular fibres of the mantle pull vertically. Both isotonic and semi-isometric levers were used, recording on a smoked drum.

Stimulation of the nerve was usually by means of single condenser discharges through chlorided silver electrodes placed under the nerve. Make and break induction shocks were also tested. Sea water was used throughout as a medium.

The whole preparation can be completed in about 10 min. Unfortunately the muscles do not last well after isolation and the responses obtained gradually decline. However, with animals in good condition preparations which last for half an hour or more can be obtained.

If an entire stellar nerve be stimulated at 15 sec. intervals with discharges of a 0·04μF. condenser and progressively increasing voltage, then it is found that at about 1 V., after a small increase of voltage, a very large twitch of the circular fibres of the muscle appears (Figs. 4, 5 and 6). Lesser voltages produce no contraction at all, and further increase of the voltage applied does not lead to any increase of the tension developed isometrically or the amplitude of contraction isotonically. The only exception to this rule is that at about ten times threshold there is often a slight increase in the tension above that produced at lower voltages (Figs. 4 and 6).

The experiments described in the next section will show that this contraction of the circular muscles is due to the activity of the giant fibre in the nerve. Since at all voltages above threshold a constant response is produced, it seems probable that the effect of stimuli such as those used is to set up a single impulse in the giant fibre which in turn excites all the muscle fibres which it reaches. Presumably the increased response obtained at about ten times threshold voltage is due to the setting up of two or more impulses in the nerve, but the proof of this requires investigation of the action potentials in the nerve. Preliminary studies (Bronk, Gerard, Hartline and Young unpublished) have shown that condenser discharges usually set up single impulses in the nerve, but the conditions for repetitive firing have not yet been investigated. Kato (1936) found closely similar increases in response with strong stimuli during his investigation of single neuro-motor units in the frog.

Using break induction shocks exactly similar results are obtained. At the threshold, which is very low, the stimulus produces a large response, which is not further increased even by great increase in the intensity of stimulation.

By careful preparation under a low-power binocular microscope all the smaller nerve fibres can be removed from a stretch of stellar nerve, leaving only the giant fibre. Stimulation of this with condenser discharges gives results similar to those from stimulation of the intact stellar nerve (Fig. 6), there being a threshold at which nearly maximal contractions appear. This threshold can be determined very accurately so that an increase of 0·01 V. applied to the condenser will produce the response. With further increase in applied voltage the tension developed remains constant except for a slight increase at voltages greatly above threshold, presumably due to repetitive firing.

To avoid the rather difficult task of cleaning a long section of the giant fibre without damaging it, all the smaller fibres can be cut at some point between the stimulating electrodes and the muscle. In this way single fibre preparations of the giant fibre can very easily be obtained.

If the giant fibre be destroyed, by pricking with a needle, at some point between the stimulating electrodes and the muscle, leaving the other fibres in the nerve intact, then stimulation will still produce contraction of some of the circular muscles of the mantle. The tension which can be developed in this way is always very much less than that produced by the activity of the giant fibre, and unlike the latter it can be produced in graded steps, increasing voltages producing increased tension up to a maximum (Figs. 7 and 8). Presumably increasing numbers of nerve fibres are being brought into action. The threshold for stimulation of the smaller fibres in the nerve is usually higher than that for stimulation of the giant fibre, but in a few cases a small graded contraction appears on stimulation of the whole stellar nerve, at voltages lower than that at which the large response due to the giant fibre appears (Fig. 8). Presumably in these cases the large fibre is unfavourably placed for receiving the current, so that enough of the small fibres are stimulated to produce some contraction at voltages still too low to stimulate the giant fibre.

It cannot at present be decided whether these graded contractions produced by stimulation of the smaller fibres in the nerve are due to the shortening of muscle fibres which are also innervated by the giant fibre, or to a different set of muscles. It has not yet been possible by histological studies to differentiate two types of circular muscle fibre or to decide whether any one fibre receives nerve endings both from the giant and from smaller nerve fibres. However, the physiological analysis has already been sufficient to show that the neuromuscular mechanism allows of the production of two types of response on the part of circular fibres in the mantle, one graded and smaller and the other much larger, and total, involving the simultaneous contraction of all the fibres. The presumption is that the lesser contraction, operated by the smaller fibres in the nerve, is that used in the gentle respiratory movements, while the maximum contraction produced by a single impulse in the giant fibre ejects a propulsive stream of water.

No thorough investigation has yet been made of the time course of the contractions produced by either set of fibres. The twitches set up through the giant fibre are quick (Fig. 9), the maximum tension being reached about 60 ms. after the beginning of the contraction and relaxation being over in about 200 ms. under the conditions of the experiment.

After stimulation either of the whole stellar nerve or of the smaller fibres only, the quick twitch is sometimes followed by a small further contraction which lasts for some seconds (Fig. 8). It remains uncertain whether this persisting response is due to sustained activity on the part of the nerve or muscle fibres. Probably it is of no significance in the normal life of the animal.

It has not yet been found possible to obtain a preparation in which the activity of the muscle fibres which run through the thickness of the mantle is recorded directly. However, in a few cases after stimulation of the whole stellar nerve it was found that with increase of the applied voltage above threshold the tension or amplitude of contraction of the circular fibres decreased progressively until it was reduced by as much as one-fifth in extreme cases. Lowering of the strength of stimulus then caused an increase in the response to its original threshold value (Fig. 10). This progressive reduction at higher voltages can be interpreted as the result of the stimulation of more and more of the nerve fibres which activate the radial muscles and thus decrease the effect of the circular muscles set in action through the giant fibre.

For purposes of maximal contraction, therefore, each side of the mantle is divided into about 10 motor units each controlled by a single nerve fibre. It must be remembered, however, that each of these giant fibres in the stellar nerves is a syncytium, produced by the fusion of the axons of a great number of cells in the stellate ganglion (Young, 1936a, b). Histological investigation has led me to suppose that this fusion is complete, producing a true single neural unit, provided with a single continuous surface membrane, and a single myelin-like sheath (Bear et al. 1937). This conclusion is now amply confirmed by the demonstration that each giant fibre is a functional unit which can only be set into action maximally.

The fact that the territory innervated by each stellar nerve constitutes functionally a single neuromotor unit can also readily be demonstrated in various other ways without recording its contraction with a lever. Thus if condenser discharges are applied with an electrode placed in various parts of the territory a stimulus which is strong enough to cause any contraction will always be found to cause contraction of the whole unit, irrespective of the position of the electrode, whether near to the ganglion or to the edge of the piece. In the latter case the impulse is presumably set up in the finer branches of the giant fibre and conducted antidromically towards the ganglion. By cutting up the muscle in various ways it can be shown that if one part of a unit is stimulated all other parts will always contract provided suitable connexions are left to allow the impulse to run back up the nerve and down again (Fig. 11). In this way an exact counterpart of the gracilis experiment can be performed with the branches of a single nerve fibre. When an electrode is placed in the muscle itself it is occasionally possible to produce local contractions by direct stimulation of the muscle fibres themselves, but usually stimulation of the finer branches of the giant fibre occurs before that of the muscle fibres, the whole unit being thus set into action.

This experiment also shows that there is no functional connexion in the ganglion between the individual giant nerve fibres, since stimulation in one unit never produces contractions in its neighbours. There has been much debate about the possibility of obtaining reflex responses or axon reflexes through the isolated stellate ganglion (see Sereni & Young, 1932). No evidence of them has been seen during the present work, but since the ganglia of the squid are even more susceptible than those of octopods to oxygen lack, the caution of Frohlich (1910) that such reflexes must only be expected in very fresh preparations applies with especial force.

A further demonstration of the action of whole masses of muscle as single units can be given by making use of the fact that the muscles rapidly become opaque when stimulated (Sereni & Young, 1932). Under favourable conditions this opacity can be seen to develop after the application of only a few stimuli to the nerve, and if the stimulation be continued for a short while the whole unit which is contracting will become quite opaque and sharply demarcated from the still transparent muscles on either side of it (Fig. 12). The converse of this experiment can be performed by cutting one of the stellar nerves of the animal shortly before it is killed. During the process of death of the animal the muscles become opaque, presumably because of the imperfect removal of products liberated during contraction. But when one of the stellar nerves has been cut the unit which it innervates does not thereafter contract at all, and hence remains as a strikingly transparent window in the otherwise opaque mantle.

The retractor muscles of the head and funnel are very suitable for myographic purposes. The apparatus by means of which the head is drawn backwards includes two sets of muscles, first, the short m. retractor capitis anterior, running from the cephalic to the nuchal cartilage and innervated by the n. retractor capitis anterior which does not contain any very large fibres, and secondly, the much larger m. retractor capitis posterior. This latter muscle runs from the nuchal cartilage to the pen and consists predominantly of longitudinal fibres, though in its lateral part there are also transverse fibres running through the thickness of the muscle, the contraction of these presumably causing protrusion of the head (Fig. 1).

Particularly suitable for experimental purposes is the muscle which draws in the funnel and is usually known as the m. depressor infundibuli, though m. retractor infundibuli is a more suitable name in view of the function of the muscle and of its affinity with the retractors of the head (see below). The muscle consists mainly of parallel longitudinal fibres, but, as in the case of the mantle muscles and head retractors, antagonistic fibres are combined in the same muscle mass, being in this case transverse fibres running across the muscle (Fig. 1).

The innervation of the retractors of the head and funnel is somewhat complex since m. retractor (depressor) infundibuli, in addition to two giant fibres which it receives through branches of the visceral nerve, receives one also through n. retractor capitis posterior (Fig. 13). This latter nerve contains altogether three giant fibres, two smaller ones to the anterior parts of m. retractor capitis posterior and a large one which sends branches to the hind parts of these muscles, and also, as mentioned above, to m. retractor infundibuli. The fact that portions of the head and funnel retractors are innervated by the same axon emphasizes their close functional relationship. Both of the muscles also receive numerous smaller nerve fibres.

To prepare these muscles for myographic purposes the animal is opened ventrally and the whole of the posterior visceral mass removed. A transverse cut is made across the mantle and pen behind the attachment of m. retractor infundibuli, and a portion of the pen, with the required muscles attached, can then be separated completely from the mantle. The digestive gland must be carefully removed, exposing the visceral nerve and n. retractor capitis posterior (a branch of the large pallial nerve). Finally the whole of the head region is cut away to the level of the hind end of the cephalic cartilage, the retractors of one side are completely removed and those of the other pinned down at the anterior end, and hooks attached either to the remainder of the pen or to the muscle itself. This whole preparation can be done in a few minutes, and the muscle will continue to show good responses for a further half-hour or longer.

The preliminary investigation which has been made with this preparation, and especially of m. retractor infundibuli, has shown responses to stimulation of the visceral nerves closely similar in a general way to those obtainable from the circular muscles of the mantle. There is, however, the important and interesting difference that, when stimulating the visceral nerve with condenser discharges of increasing voltage, the tension developed by the muscle increases in two well-marked steps (Fig. 14). This corresponds with the fact that there are two giant fibres of slightly differing diameters in this nerve. The thresholds for the two lie very close together, and in some cases it proved impossible to separate them, even by increasing the applied voltage by very small amounts, though in many cases the two steps could be clearly distinguished.

In some preparations evidence of small graded contractions similar to those produced by stimulation of the small fibres in the stellar nerves was seen. Presumably these small fibres are responsible for the movements of the muscles during respiration.

The experiments in which single isolated giant fibres were stimulated supply proof that these structures are motor axons. It has also been shown that in spite of their syncytial nature they conduct in an all-or-nothing manner, constituting, with the muscle fibres which they innervate, single motor units, fully comparable with those into which the vertebrate muscular system is divided (Lucas, 1909; Pratt, 1917; Adrian, 1933). Each unit, however, is capable of developing an enormous tension. Even the small strips used in the above experiments develop over 100 g. and the whole of the unit of which they form a part must develop nearly a kilogram. This may be compared with the largest tension per unit found by Eccles & Sherrington (1930) in the cat, namely 30·1 g. in the median head of gastrocnemius, which is composed of 430 units. The ease with which the single nerve fibres can be isolated in the squid, and the large number of muscle fibres which each innervates, make this system ideal for showing the working of such single neuromotor units. The demonstration is particularly clear since preparations with one, two and many units can be obtained, and the all-or-nothing, two-step and graded twitches which they produce compared.

No evidence has been seen in the squid for variations in the response of the single nerve fibres such as those which are supposed by Jordan & Lullies (1933) to occur in the nerves of another mollusc, Aplysia, contrary to the all-or-nothing “law”. There are, however, undoubtedly great differences between animals in the number of nerve impulses which are needed to set the effector cells into action. In the frog and in mammals a single impulse is normally adequate to excite all of the striped muscle fibres which it reaches. In the muscles of Crustacea and coelenterates, however, repetitive impulses produce contraction in muscle fibres which fail to respond to single impulses, the tension developed therefore varying with the frequency of stimulation (Pantin, 1934–6; Katz, 1936).

The circular mantle muscles of the squid resemble vertebrate striped muscle in that a single impulse normally produces a twitch in all of the muscle fibres which it reaches, and it has been shown (Prosser & Young, 1937) that repetitive stimulation of a giant fibre, even at high frequencies, does not increase the tension developed by the muscle.

The value of systems of giant nerve fibres to the animals possessing them lies presumably either in the greater speed of conduction in larger axons, or in that such large fibres make it possible to call into action a large number of effector agents nearly simultaneously. Studies of the conduction rates in the giant fibres of the squid are being undertaken in order to discover whether these axons make possible a significant saving of time for the animal. The investigations reported in the present paper show that the second of the above factors is certainly significant since each of the large nerve fibres connects with great numbers of muscle fibres, all of which are caused to contract by a single nerve impulse. Such a system is very well suited to the performance of the sudden movements by means of a jet of water which play such a large part in the life of the squid, and are no doubt partly responsible for the evolutionary success of this type of animal.