The effect of the Crustastun on nerve activity in crabs and lobsters

Anatomy

Decapod crustaceans, the taxonomic group to which crabs and lobsters belong, have nervous systems with the characteristic arthropod plan (Brusca and Brusca, 2002). This involves a ladder-like arrangement of paired nerve cords, with a dorsal brain (supraoeophageal ganglia) separate circumoesophageal connectives and segmental ganglia in the thorax and (if present) in the abdomen, from which nerves arise to supply the segmentally-arranged muscles and sense organs. Lobsters exemplify all these features (Figure 1) whereas in crabs a distinct abdomen has been lost and the thoracic ganglia are condensed into a single thoracic mass, from which all the peripheral nerve roots emerge (Figure 2).

Figure 1. The arrangement of the nervous system in a clawed lobster such as the European lobster (Homarus gammarus).

Figure 2. The arrangement of the nervous system in a crab such as the shore crab (Carcinus maena).

Materials and Methods

Animal supply and holding

All animals used in these tests were obtained live from commercial suppliers (UMBSM Animal Supply Service Millport and Loch Fyne Sea Farms Ltd.) and were retained within a closed seawater circulating system for at least one week before experimentation. Intact and alert animals were held on ice for 30 min immediately prior to the experimental procedures, in order to reduce their metabolic rate.

Crustastunning

The ‘Crustastunning’ procedure was applied without prior anaesthesia using a machine supplied by Studham Technologies Ltd., according to the manufacturer’s operating instructions. The chamber was filled with a salt solution (~3g L-1). Individual crabs or lobsters were stunned by a 110 volt, 2-5 amp electrical charge for 10 s immediately after removing them from the holding aquaria.

Exposing the nervous systems

In order to expose the central nervous system of the crab for recording, the carapace was removed and the preparation was submerged in a balanced salt solution corresponding in composition and osmolarity to crab haemolymph, at a temperature of 10°C. The internal organs were then removed or displaced in order to expose the circumoesophageal connectives around the base of the stomach. A similar procedure was employed for the lobster, but prior to this the cephalothorax was separated from the abdomen. To expose the abdominal ventral nerve cord of the lobster for recording, after separating the abdomen from the cephalothorax the dorsal skeletal plates (terga) were detached, and the bulk of the underlying deep flexor musculature was removed. The preparation was then submerged in a balanced salt solution corresponding in composition and osmolarity to lobster haemolymph, at a temperature of 10°C. Selective removal of muscle blocks hen revealed the motor roots emerging from the ventral nerve cord.

In order to expose the leg nerve of crabs for recording and stimulating, the leg was first detached from the body of an intact crab by applying pressure to the basipodite segment, which caused the animal to shed its leg naturally by the process of autotomy (McVean, 1976), or by amputation in a stunned crab. The joint between the meropodite and carpopidite (M-C) was then disarticulated, and the muscle tendons spanning this joint were cut with fine scissors. The leg was separated gently at this point, revealing the leg nerve still attached to the distal portion. This isolated leg preparation was submerged in balanced salt solution at a temperature of 10oC until required, and remained viable for many hours.

Electrophysiological recordings

Electrophysiological recordings were made from the exposed nerves using various extracellular techniques. For recording from the circumoesophageal connectives of crabs and lobsters, and from the ventral nerve cord of lobsters, a suction electrode method was used. A fine-tipped polythene electrode containing salt solution was applied to the surface of the nerve, and a gentle suction was applied through attached tubing and a syringe. A silver wire positioned close to the tip of the electrode acted as the indifferent (reference) electrode. Such a recording configuration is termed ‘en passant’, as it involves attaching the suction electrode to an intact nerve, allowing both directions of nerve transmission to be recorded. However, in some cases the circumoesophageal connective was cut and the lectrode was attached to either its anterior or posterior cut end. In this way the presence of active neurones transmitting information in ascending or descending directions could be ascertained.

Results

The Crustastunning procedure was applied to 6 crabs and 6 lobsters, and the same number of intact animals was used as controls. The data are presented as traces of the original electrophysiological recordings and where appropriate also as plots of the muscle forces produced in relation to stimulus parameters.

Activity in the nervous system of intact crabs and lobsters

Recordings made from one or both circumoesophageal connectives in intact crabs indicated that there was a high level of spontaneous neuronal activity passing along the axons of this nerve, even in the absence of any imposed stimulation (Figures 5 and 6). Due to the variety of sizes of the extracellularly-recorded spikes, it can also be concluded that the signals arose from a large number of different individual nerve axons, of varying diameters.

Figure 5. Spontaneous nerve activity recorded extracellularly in the left and right circumoesophageal connectives (COCs) of a shore crab, Carcinus  maenas. Upper panel, intact animal; lower panel, animal after Crustastunning. Scale bar 1s.

Discussion and Conclusions

Activity in the nervous systems

The results obtained here are consistent with the literature on the neurophysiology of crustacean nervous systems (see, for example, the articles in Wiese, 2002) in showing that the central nervous systems of crabs and lobsters display continuous nerve activity, which in turn produces outputs in the motor nerves to the body and limb muscles. A large body of evidence, including studies conducted in this laboratory (Chachri et al., 1994; Holmes et al, 2002), indicates that this activity persists even when parts of the CNS are isolated from each other by severing the nerve cord at one or more levels (Larimer and Moore, 2003). Even isolated single ganglia of the abdominal nerve cord can produce patterned outputs (e.g. Chachri and Neil, 1993), and there is an extensive literature on the most-studied ganglion that can continue to operate in isolation, the stomatogastric ganglion (reviewed by Marder and Bucher, 2007).

A characteristic feature that is common in these isolated parts of the nervous system is the long-lived nature of continued activity and signal conduction. It is widely reported that, provided the structures are bathed in an appropriate solution, activity can continue for many hours, and indeed this was observed in the present study both with the central nervous preparations and with the isolated crab legs after autotomy. Such robustness makes it easier to interpret any loss of activity following an intervention as due to the intervention itself, rather than to any underlying decline in nervous system responsiveness.

The effects of Crustatunning

The findings obtained on the effect of Crustastunning on nerve activity in crabs and lobsters are relatively conclusive. As far as can be determined from the extracellular recording method used, the various forms of spontaneous activity within the central nervous system are completely arrested. Consistent with this, there are no outputs produced in the motor nerves supplying the abdominal muscles of lobsters, which are known to be synaptically driven from neurones in the CNS.

The recordings made on isolated crab legs allow some further conclusions to be drawn, namely that Crustastunning also has an effect on the functioning of the peripheral parts of the nervous system. There is both a loss of responsiveness to all three types of sensory stimulation, and also a failure in neuromuscular activation. The first of these effects renders the animals insensitive to external stimuli, while the second renders them paralysed and incapable of making movements. Thus it has been found that as a result of Crustastunning the nervous system is incapacitated simultaneously at two levels, i.e. both centrally and peripherally, which completely prevents all normal neuronal functioning.