Structure and Function of the Lateral Giant Neurone of the Primitive Crustacean Anaspides Tasmaniae

In mountain streams and tarns of southwest Tasmania lives a syncarid crustacean, Anaspides tasmaniae, whose family arose more than 200 million years ago in the late Palaeozoic, 100 million years earlier than the now dominant advanced crustaceans, the Decapoda (cf. Brooks, 1962a; and Glaessner, 1969). The animal, 3–4 cm in length and caridoid in form, has many features that are considered primitive; unfused free thoracic segments, biramous homonomous appendages and leaf-like epipodite gills (cf. Siewing, 1959; Smith, 1909; and Snodgrass, 1952). Behaviourally, this animal makes a rapid flexion of its body when touched. In the decapod crayfish similar behaviour is known to be mediated by giant fibres (Wiersma, 1947; Wine & Krasne, 1972). Since the Syncarida and the Eucarida, the malacostracan divisions to which Anaspides and the crayfish respectively belong, seemingly arose from common stock (Brooks, 1962b) we examined the neural basis of the evasive response in the primitive Anaspides in order to gain an insight into the origin of the neural organization of the decapod escape response.

Anaspides, we will show, has two giant fibres in each half of its ventral nerve cord and we have concentrated on the lateral of the two for comparison with the much studied lateral giant fibre of the crayfish. Since the thoracic body segments are not fused in Anaspides as they are in the crayfish, the thorax can contribute to the flexion that propels the animal away from a disturbance. Little attention has been given to the structure and function of the lateral giant fibre in the thorax of the crayfish, probably because its thoracic body segments are fused and covered with a carapace and thus do not participate in the escape reaction. Nevertheless, the crayfish lateral giant fibre is segmented in the thorax (Johnson, 1924) as well as in the abdomen, and one might expect that the thoracic segments of this fibre are organized homologously to those in the abdomen (Remler, Selverston & Kennedy, 1968). The organization of Anaspides prompted us to investigate the function of the lateral giant neurone in the thorax.

Anaspides 20–35 mm long were collected from small pools in streams above 500 m on Mt Wellington, near Hobart. After isolating them in small containers of stream water they were stored at 5°C, which is within the temperature range most commonly found in their natural environment (0–8°C; Reid, 1975). Our morphological and physiological results are taken from animals without regard to the duration of their captivity. However, the animals that were involved in the behavioural decrement experiments were tested within 2 days of their capture.

In behavioural experiments the animals were maintained in stream water cooled to 10–12°C. Evasive behaviour was recorded on film with a 16 mm Bolex ciné camera at 64 frames per second. For the behavioural decrement studies each animal was fastened to the bottom of the dish with a staple across the anterior segments of the body, so that it could flex its abdomen and thorax but could not escape. To give a constant intensity stimulus, a forceps was used with the gap between the tips adjusted by a set screw so that when the forceps was squeezed its tips just touched each side of the pleurite of the fourth abdominal segment. The strength of stimulation chosen was the setting which just provoked a rapid flexion.

For recordings from the lateral giant axon the ventral nerve cord was exposed from above and the tissue bathed in saline composed of (g/1): NaCl, 6·3; KC1, 0·34; CaCl₂.2H₂O, 1·035; MgCl₂.6H₂O, 0·213; NaHCO₃, 0·2. The ionic composition of the saline was the same as that of the blood, as determined by flame photometry (R. Swain, personal communication). Saline flowed continuously through the experimental chamber, and was oxygenated and cooled to 6−7°C. After fastening the animal to the floor of the chamber by a staple over the head-thorax joint and a pin through the abdomen-telson joint the dorsal cuticle was slit lengthwise, the underlying ventral wall of the pericardium was severed along one side of the heart and the gonads were removed. Minutien pins were inserted through the sternites on either side of the ventral nerve cord to hold the body walls apart. The muscles of the body wall in the thorax separated neatly into bilateral bundles; muscles in the abdomen, however, required severance of cross anastomoses before they could be spread to reveal the cord. The gut was sometimes pinned aside, sometimes partly removed with the ends ligatured, and sometimes removed entirely. As far as possible the supraneural artery was kept intact.

To record from single cells in the exposed cord, lateral giant fibres were penetrated, under visual observation, with 20–30 MΩ microelectrodes filled with 3 M-KCI. Penetrations were usually made in a ganglion rather than a connective. Electrical stimulation was made through the microelectrode and recorded potentials were amplified with FET input amplifiers. To record externally from the cord a suction electrode was coupled to an AC pre-amplifier. Flexing movements were monitored with a phonograph cartridge by attaching one end of a light-weight probe to the stylus holder and placing the other end upon the dorsum of the abdomen. When the animal flexed it deflected the probe and oscilloscope trace upwards. Signals were displayed conventionally and recorded on moving paper film.

Tactile stimuli in physiological experiments were applied with forceps by sharp proddings with the closed tips or by quick pinches of the cuticle. The instant of contact with the cuticle was not monitored.

To determine the morphology of lateral giant neurones, cells were filled with cobalt chloride (Pitman, Tweedle & Cohen, 1972). Axons of lateral giant neurones were penetrated under visual observation with microelectrodes filled with 1 M-COC1₂. The tips of these electrodes were broken off and the dye expelled using pressure from a micrometer syringe. We could tell when a fibre was injected by the sudden onset of an opacity around the tip of the electrode and along the fibre. After injection in situ at just one point in any segment of the fibre the cord was removed to a dish of saline. Cobalt was precipitated with a few drops of 10% ammonium sulphide. The cord was fixed in Carnoy’s fluid, cleared in methyl benzoate, mounted, and immediately sketched by means of a camera lucida. To examine giant fibres in transverse section the cord was removed from the body and fixed in alcoholic Bouin’s, and embedded in paraffin. Serial sections were cut at 8–10 μm and stained with Mallory’s Triple. A set of serial sections prepared in the same way by Dr S. Lake and one of us (I. S. W.) was also used.

We found Anaspides to be a very difficult and sensitive animal with which to work. Its small size precluded manipulation and access to nerves. Viability following exposure of the ventral nerve cord was highly variable and typically brief. Even in those animals in which interneuronal activity in the cord and postsynaptic activity in the lateral giant neurone persisted for 3–4 h the lateral giant axon would spike for only several minutes after the cord was exposed. During intracellular recording from the lateral giant fibre, spikes progressively and rapidly decremented in amplitude from overshooting down to several millivolts. In chronic and direct extracellular recordings from the cord the amplitude of giant fibre discharge was indistinguishable from action potentials of other neurones. These features of the animal prevented us from obtaining answers to certain questions and they will undoubtedly hinder further productive pursuit into neural circuitry.

The evasive action of Anaspides, as evoked by tactile stimulation, involves a fast flexion of every free segment of the body. Thoracic and abdominal segments bend one upon the other as illustrated by the records of Fig. 1 The records show that Anaspides, once touched upon the side (frame 1), adducts its appendages and bends the ends of its body ventrally (frame 3) so that its rostrum nearly touches its telson (frame 4). It adducts all its appendages but swimmerets are moved caudad, the more anterior legs rostrad and the middle legs extended ventrally as well. The flexion and adductions reach their maximum within about 40–50 ms (frames 3–4) and then the animal straightens its body (frames 5–7), returns its appendages to their normal position and uses them in swimming fashion (Wilson, Macmillan & Silvey, 1978) to move further away from the stimulus (frame 8). The force of these bodily actions against the water propels the animal upwards off the substrate.

The fast flexion response we attribute to lateral giant mediation. However, we have no direct evidence that the lateral giant fired during the sequences portrayed in Fig. 1 nor that selective firing of the lateral giant in an intact animal would lead to the movements displayed. Below, however, we will show (Fig. 6) that tactile stimulation applied similarly to that in the experiment of Fig. 1 and to the same regions of the body touched in the experiment of Fig. 1 provokes both lateral giant fibres to fire. We will also show that direct excitation of the lateral giant provokes a rapid and full flexion of the body (Fig. 5). On these grounds we assume that the behaviour, at least the rapid flexion, displayed in Fig. 1 is mediated by the lateral giant neurone.

In the evasive response the animal also moves away from the side on which it was touched, that is, there is a directional component in the response. In fact, the direction in the movement, namely away from the probe (frame 2 of Fig. 1), begins prior to complete development of the flexion (frame 4). Since we were unable to distinguish lateral giant spikes in chronic recordings we cannot specify the source of this directionality. Certainly it is due to activation of motor neurones ipsilateral to the side touched because muscles on this side of the body contract to produce the curvature of the body away from the probe (frame 2 of Fig. 1). Whether in fact directionality is due in a behaving animal to the firing of only one giant, namely, that ipsilateral to the side touched, or to the ipsilateral giant firing slightly before the contralateral giant relative to the side touched, or to the activation of other interneurones, we are unable to say.

Touches to parts of the body other than the sides also evoke rapid evasive responses and these too show a directional component. A touch to the antennae or head elicits an upward, usually forward movement containing some lateral component. These responses are presumably due to the medial giant interneurone (cf. Fig. 3). A touch to the telson or uropods of the abdomen leads predominantly to an upward and forward movement. Occasionally Anaspides may move backwards; it does so by an upward and backward somersault or twist but not by a full flexion of its abdomen as does the crayfish (Larimer et al. 1971; Wine & Krasne, 1972).

Evasive responses to tactile stimulation can be repeated frequently and do not decrement to the degree that the tail flip escape reaction habituates in the crayfish (Wine, Krasne & Chen, 1975). It is not unusual in the pursuit of an Anaspides to find that it rapidly flexes its body to each contact made with it.

To determine whether the response showed behavioural decrement, several animals were stimulated tactilely on the right fourth abdominal pleurite as described in Materials and Methods. Tactile stimulation of this pleurite leads to firing of the lateral giant as we shall show below (Fig. 6). However, we were not able to substantiate by chronic recordings that each stimulus which evokes an evasive response also excited a lateral giant. Nevertheless we assumed that fast flexions were due to lateral giant excitation. Initially we stimulated at the rate of 1 touch per min for 10 successive trials (Fig. 2 A). One group was then allowed to rest for an hour before retesting. This group served as controls for the other animals, which were subjected to a test of successive stimuli at 4 per second until individuals failed to respond to ten consecutive trials. This was our criterion for complete decrement. Once an animal was decremented to criterion it was immediately retested at 1 touch per min for 10 min and again 1 h later. These are the tests at 0 h and 1 h in Fig. 2B for the experimental animals. They are compared to a retest of the controls at 1 h.

Fig. 2A indicates that the animals do show behavioural decrement. After ten touches at 1 per min only 70% of the animals responded. There is no significant difference between experimental and control animals (analysis of variance double classification, P > 0·20). The crayfish, when stimulated to give a lateral giant mediated escape response with taps at 1 per 5 min, habituates to 40% and some animals fail to respond during the first two trials (Wine et al. 1975).

Following decrement to criterion the experimental animals retested immediately showed a recovery to 30% (0 h, Fig. 2B). One hour later 80% of the experimental animals responded to the first stimuli. At 1 h the control animals responded as they did initially (P > 0·20). The difference between the experimental animals at 0 h and the experimental animals at 1 h is significant (P < 0·001). The experimental animals at 1 h decreased their responses slightly more rapidly and completely than did the controls at 1 h (P < 0·001). Nevertheless, by the end of 1 h Anaspides nearly recovers its ability to respond to a stimulus that decremented its evasive reaction. The lateral giant mediated escape response of the crayfish, on the other hand, when habituated to a criterion of ten successive failures, does not recover for more than a day (Wine et al. 1975).

To determine the site of decrement we touched the contralateral pleurite after each test sequence and after complete decrement (criterion), and recorded whether the animal flexed. Only two animals failed to elicit a rapid body flexion to stimulation of the contralateral pleurite. This means that the lateral giant to motor circuit was still operating. Unfortunately, the test does not clearly show whether the behavioural decrement was due to sensory adaptation or to changes between sensory and lateral giant pathways. Nevertheless, the test does indicate that the source of behavioural decrement is on the input side of the circuit.

In the matter of behavioural response decrement it is difficult to compare Anaspides with the crayfish because it was impossible to give identical intensity and quality of tactile stimulation to the small, soft-bodied Anaspides as has been used on the larger, hard-bodied crayfish. Moreover, certain refinements in recording possible with the crayfish and which would have given our conclusions greater certitude were not possible with Anaspides. Nevertheless, there are differences between the two species with respect to rates of entrance into and recovery from decrement, both those noticed in the field and those measured in the laboratory, and we believe these to be expressions of differences in circuitry in the lateral giant system of each animal.

Cross-sections of the ventral nerve cord reveal a pair of large diameter fibres in each connective and hemiganglion (Fig. 3). Each giant fibre in the connective between the eighth thoracic and first abdominal ganglia is about 40 µm in diameter in a 35 mm long animal. The medial fibre is somewhat narrower posteriorly than anteriorly. In contrast the lateral fibre is broader posteriorly than anteriorly. In their passage through a ganglion both fibres narrow somewhat. This is apparent from cross-sections (Fig. 3 A) and from dye injected fibres (Fig. 4). The medial fibre is continuous through the length of the body and dye passes through it unobstructed. The lateral fibre is discontinuous, made up of fused processes from single nerve cells. It is formed from axons which arise from cell bodies in each of the six abdominal ganglia and the last five thoracic ganglia. Between two segments the posterolateral membrane of the more anterior cell forms a septum. This is largely impermeable to cobalt ions: in stained cells, only faint indication of precipitated cobalt could be seen on the uninjected side of the septum and along the membrane of the unfilled fibre. The septate organization of the crayfish lateral giant is similar (Johnson, 1924; wiersma, 1947).

Each segment of the lateral giant fibre arises from a cell in the contralateral hemiganglion. The example shown in Fig. 4 is representative of each of the 11 segments of the fibre. The cell body of each segment of the fibre is stituated near the posterolateral border of its hemiganglion and ventral to the level of the giant fibres. The neurite of each lateral giant soma rises upwards, crosses beneath the ipsilateral giant fibres and descends into the commissure. Within the commissure the neurites from opposite hemiganglia cross over one another. Each neurite sends dendrites into both ipsilateral and contralateral neuropiles. Within the opposite hemiganglion the neurite ascends, broadens and forms the axon of the fibre that passes forward in the cord. The axon that arises from the fourth thoracic ganglion passes uninterrupted into the circumoesophageal connective before entering the brain. Thus it traverses six pairs of ganglia: the first three thoracic and three suboesophageal. The lateral giant neurones in the sixth and last ganglion differ from those in the other ganglia in that the dendrites which invade the neuropile contralateral to the soma are longer and not directed laterally. Instead, one branch passes dorsally and two others posteriorly.

When the lateral giant neurone spikes it excites interneurones and motor neurones and causes a fast body flexion, as shown in Fig. 5. A just subthreshold stimulus to the fibre (A) produces neither action potentials in the cord (bottom trace) nor movement of the body (top trace). A slight increment in current passed through the intracellular electrode within the fibre (B) elicits an action potential (first dot) followed immediately by neuronal activity in the cord, and, within approximately 10 ms, a flexion of the body, as indicated by the upward deflexion of the top trace. In addition, the thoracic and abdominal appendages are sharply adducted. Thus, the lateral giant is competent to release the fast flexion component of the behaviour described above. As for the crayfish (Wiersma, 1947) it is a true command fibre: a single interneurone which by its firing releases a behavioural repertoire that in this case is the fast body flexion.

Of further interest in Fig. 5 are the two additional spikes of the same neurone (second and third dots). The waveform and size of these potentials are the same as those of the lateral giant spike (first dot). They are followed by a complex change in the movement trace which indicates further muscular contractions. We believe that the flexion which was triggered by electrical stimulation of the lateral giant re-excited the lateral giant orthodromically because the probe of the monitor acted as a tactile stimulus.

Only slightly more than 40 ms separates the electrically generated spike (first dot) from the spikes (second and third dots) generated by re-excitation. Normally a fast flexion-recovery cycle lasts 75 ms; at least this is the briefest interval we have recorded of successive evasive responses. In the crayfish an electrically generated spike in the lateral giant inhibits it from firing again for 80 ms (Roberts, 1968), which is in the order of time required for completion of a flexion-extension cycle (Wine & Krasne, 1972). The occurrence in Anaspides of spiking following an electrically generated spike before the animal would have extended from flexion suggests that it does not possess a recurrent inhibitory mechanism.

The multiple firing associated with the second flexion in Fig. 5 was commonly observed in this neurone; two further examples are shown in Fig. 6. At other times brief bursts of up to eight spikes were recorded, with intervals between spikes ranging from 5 to 15 ms. Multiple firing to tactile stimulation also occurs in the crayfish lateral giant although at a higher frequency and in shorter bursts, complete within 20 ms (Wine & Krasne, 1972).

Tactile stimulation is adequate to provoke the lateral giant to fire. Each of the records in Fig. 6 shows two spikes recorded intracellularly in a lateral giant, and coincident firing in the cord. These firings were elicited by touch to one of the abdominal segments. We have, however, recorded firing of the lateral giant to tactile stimulation of the thorax and its appendages as far forward as the fourth segment. Nevertheless, lateral giant firing is most readily elicited by touching the abdominal pleurites. The more posterior are the more sensitive. This leads us to assume that the fast flexions (Fig. 2) are mediated by the lateral giant.

The records in Fig. 6 also show that tactile input to either side of the body, and thus input to either side of a ganglion, is capable of exciting the same lateral giant. This means that both giants can fire to a single touch of the abdomen or thorax.

Spikes of the lateral giant fibre are often masked in extracellular recordings with many potentials being as large as those that appear to correlate with the spikes in the giant fibre (Fig. 6 A). Spikes recorded intracellularly within the lateral giant progressively decrement in amplitude as the preparation ages. Initially the intracellularly recorded spikes from the preparation in Fig. 6 were overshooting from a resting potential of about 60 mV but had attenuated to only 25–30 mV by the time of photographing.

Within a lateral giant a continuous bombardment of postsynaptic potentials can be recorded when the animal is at rest. These appear along the length of the fibre but are more readily recorded from the fibre near the septum within a ganglion. There is no synchrony in these potentials at two points along the length of the fibre (Fig. 7 A) but within contralateral fibres at the same segmental level (Fig. 7B) there is some correlation.

Postsynaptic potentials become highly synchronous, however, in response to touch. Tactile stimulation evokes depolarizations that arise coincidently in both fibres within the same segment (Fig. 8). The responses shown in Fig. 8 are to tactile stimulation rather than to visual stimulation or to vibration transmitted through the saline in the chamber because the potentials occurred only when the points of the forceps actually touched the animal; never when the forceps were moved about the saline even quite close to the restrained animal. Stimulation of the head (Fig. 8A) as well as of the thorax (Fig. 8B) and abdomen (Fig. 8C) is effective in eliciting these potentials. Thus, the receptive field of the lateral giant is the whole of the body and not just the abdomen as in the crayfish (Wine & Krasne, 1972). Moreover, postsynaptic potentials which arise to a touch of the body appear identically in each segment of the same fibre. There is some delay, as one would expect, in the time at which potentials arise in different segments. Potentials arise progressively later in segments further away from the site of afference.

Since spikes arise in both giants in response to input from one side of the body and since postsynaptic potentials are coordinated in the same ganglion the question arose of whether this is due to afferent neurones synapsing with each lateral giant or to current spread between the giants. The crossover of the neurites in a ganglion suggests that this is a site for current transfer as it is in the crayfish (Wiersma, 1947; Watanabe & Grundfest, 1961). We tested this by passing current into one fibre while recording in the other fibre at the same level on the opposite side of the body. Never did we record a spread of current, either depolarizing or hyperpolarizing, whether passed across a bridge (Fig. 9 A) or directly through a microelectrode, despite introducing up to one microamp of current. We could cause the lateral giant to fire but a spike so initiated never generated a spike or altered the level of polarization within the contralateral fibre (Fig. 9B). Thus, the cells do not appear to be electrically coupled. We also conclude that in order to produce synchronous postsynaptic potentials (Fig. 8) afferents must synapse identically with the processes of both fibres in each hemiganglion.

In many respects the lateral giant fibre system in Anaspides is similar to that in the crayfish. Morphologically the fibre is composed of 11 segments (cf. Johnson, 1924), each arising from a soma in the last 5 thoracic ganglia and all 6 abdominal ganglia. Each soma lies contralateral to its fibre (cf. Remler et al. 1968). Behaviourally the lateral giant mediated response consists of a rapid contraction of muscles of the body wall (cf. Wine & Krasne, 1972). The response decrements (cf. Wine et al. 1975) and the source of the decrement is on the input side of the circuit (cf. Krasne & Bryan, 1973). Tactile input causes the lateral giant fibre on both sides of the body to fire and this firing alone is sufficient to elicit the fast contraction of the flexor muscles (cf. Wiersma, 1947). The production of the flexion may be generated by multiple firing of the lateral giant (cf. Kao, 1960; Wine & Krasne, 1972).

In other respects the lateral giant systems are quite different. In Anaspides the neurite of the soma gives off processes which invade the neuropiles of both ipsilateral and contralateral hemiganglia. Only one process, that ipsilateral to the giant axon, invades the crayfish neuropile (Remler et al. 1968). Although the giant fibre mediated response in both animals decrements to tactile stimulation, the decrement in Anaspides begins much less rapidly than in the crayfish and recovers in slightly more than an hour compared with more than a day for the crayfish (Wine et al. 1975). The neurites of contralateral somata, despite coming close to each other where they cross over in the commissure of a ganglion of Anaspides, are apparently not physiologically coupled; current does not spread from one fibre to the other nor do antidromic spikes in one fibre excite the other as they do in the crayfish (Watanabe & Grundfest, 1961; Wiersma, 1947). This means that the mechanism by which a single touch on one side of the body can excite both lateral giants of Anaspides is dependent on synapses between afferents and both giants. Finally, Anaspides appears to lack a mechanism of recurrent inhibition such as that in the crayfish (Roberts, 1968). The lateral giant in Anaspides can apparently be re-excited during the course of a flexion-extension cycle but to what effect and advantage is unknown.

In two respects the lateral giant system of Anaspides shows novel features. One is that the lateral giant receives input from tactile receptors of the head and thorax as well as of the abdomen, although only input from the fourth thoracic and posterior segments was shown to stimulate the giant. Because the fast flexion developed to rostral stimulation is different from that to abdominal and thoracic stimulation we suspect that the input either recruits the medial giants alone or medial and lateral giants together. The second novel feature, which is related to the first, is the flexion of all segments of the body during an evasive response. Output from the lateral giant is effective in recruiting thoracic muscles as well as abdominal muscles.

One significant feature of the system is the absence of electrical coupling between giants. Tactile afferents, however, synapse separately with both lateral giants, and thereby assure a mechanism by which both fibres can fire to input from any one side. Nevertheless, in nature only the fibre ipsilateral to the side stimulated might fire. If so, then, in addition to contraction of the flexor muscles of the body, certain muscles ipsilateral to the excited giant could also contract and cause the directional component of the evasive response.

The features described in this paper provide Anaspides with a useful means of defence. Although it behaves somewhat cryptically, squeezing between and under rocks and amongst roots, stems and debris, individuals walk about exposed at all times of the day (Smith, 1909, and our observations). Also, Anaspides has a soft and easily penetrated cuticle, and bears no chelae nor other offensive/defensive morphological modifications (Manton, 1930). The evasive reaction seems to be its only defence mechanism. That it can repeatedly flex to subsequent approaches of a predator enables it to avoid continuous advances and the likelihood of being damaged if it stood its ground. The crayfish does not give repeated flips to successive advances (Wine et al. 1975). But the crayfish has a relatively hard cuticle with heavily sclerotized crushing and tearing chelae. When startled it may escape with one or more fast flexions but advances of the predator are repelled by active application of the chelipeds rather than further retreat.

The similarities in morphology and physiology of the lateral giant neurone in Anaspides and the crayfish suggest that the lateral giant system in both animals is homologous. Since the major groups to which Anaspides and the crayfish belong, the Syncarida and Eucarida respectively, differentiated in the Carboniferous (Brooks, 1962 a, b) and considering the phylogenetic closeness of the Syncarida to the Eucarida (Siewing, 1963), the lateral giant system probably arose in the stem line of these two groups if not in the Cambrian stem line of the Malacostraca. The system, however, is not exactly the same in each animal. In each, it is structured and functions slightly differently and seemingly to separate advantage for Anaspides and the crayfish. In both animals the system has been commensurate with evolutionary survival, namely, the ascendancy of the crayfish within relatively recent time and the persistence of Anaspides since the late Palaeozoic.

We are grateful to Dr Sam Lake for a set of slides of the nerve cord of Anaspides and to Dr Roy Swain for the ion analysis of the blood that permitted derivation of the saline for Anaspides.