Crustacean muscles are known to produce powerful contractions without propagated action potentials (Wiersma, 1941a; Katz & Kuffler, 1946 ; Katz, 1949). It has been suggested (Wiersma, 1941a) that this type of response is made possible by the presence of nerve endings which are widely distributed along the surface of the muscle fibres. When nerve impulses arrive at the endings, a local depolarization is produced in the muscle fibre and in turn gives rise to contraction. This depolarization will be referred to as ‘end-plate potential’ (e.p.p.) by analogy with the e.p.p. of vertebrate muscle. A sufficiently large e.p.p. initiates a spike potential (Katz & Kuffler, 1946), which serves to strengthen the muscle response and, probably, to level out local inequalities in the fibre rather than to conduct excitation (cf. Fatt & Katz, 1953).
If this picture of crustacean muscle response is correct, then the e.p.p. should be much more widely and uniformly distributed along individual fibres than, for instance, in frog or cat muscle where e.p.p.’s are localized at one or a few discrete junctional points of each fibre. Previous recordings of crustacean e.p.p.’s were open to criticism on various grounds: they were complicated by the pennate arrangement of the fibres within the shell and the usual difficulties of interpreting external records made in a volume conductor. To simplify conditions, Katz & Kuffler (1946) reduced the nerve supply by section of axon branches, so that only a small bundle of exposed muscle fibres remained active. Under these conditions they showed that simple, sharply localized, e.p.p.’s could be obtained. But it is possible that their reduced active bundle had also been partly denervated and deprived of adjacent nerve foci. To obtain evidence which is free from these objections, the intracellular recording technique was applied, and the e.p.p. of crustacean muscle mapped out along individual muscle fibres.
The muscle used most frequently was the extensor of the carpopodite of Portunus depurator. Dissection, mounting and application of the micro-electrode have been described in detail by Fatt & Katz (1953), where further references to the intracellular recording technique are given. In some experiments, the flexor of the dactylopodite of Carcinus maenas was used, its muscle fibres being locally exposed by opening the shell, and the limb being ‘perfused’ antidromically with crab Ringer (see Pantin, 1934; Fatt & Katz, 1953), flowing through the tip of the dactylopodite.
Stimulation and recording
The nerve was stimulated in the saline bath by applying an external capillary electrode. A platinized platinum wire was used inside the capillary tube, and a platinum sheet was affixed to the wall of the bath to serve as an indifferent electrode. The stimuli consisted of repetitive rectangular current pulses of less than 1 msec duration.
When the extensor of the carpopodite was used, the nerve was stimulated in situ lying on the surface of the muscle. A recording electrode was inserted into one of the more distally situated muscle fibres, selecting successively several points along some 5–6 mm. of its length. The stimulating electrode was placed on the nerve near its proximal entry into the meropodite, well away from the muscle fibres to be examined. The frequency of stimulation was 20–30 per sec., at which little or no movement occurred. According to Wiersma (1941b), the fibres of the extensor muscle are supplied by two motor and one inhibitor axons. These axons must have been stimulated together, and no attempt was made to discriminate between them. The response, however, was always of all-or-none type: the e.p.p. occurring above a certain threshold and showing no increase as the stimulus was strengthened.
Each record resulted from many successive sweeps (synchronized with the frequency of stimulation), and was obtained a few seconds after the commencement of stimulation when individual e.p.p.’s had built up to a steady amplitude.
During preliminary experiments nerve-muscle preparations of crabs, lobsters and hermit crabs were explored by inserting a recording electrode into exposed muscle fibres and stimulating the nerve repetitively. It appeared at the outset that ‘e.p.p.’s’ could be seen at every point of the muscle which was impaled. The potentials built up in amplitude with repetitive stimuli, and summated at high enough frequencies as previously described (Katz & Kuffler, 1946). Typical ‘e.p.p.’s’ recorded with an internal electrode are shown in Figs. 1 and 2. There were large differences in amplitude in different fibres, but it was never necessary to search for a ‘focus’, as is the case in most vertebrate muscles, and it appeared from the ubiquitous presence of e.p.p.’s that the innervation of crustacean muscles must indeed be widely distributed along all the fibres.
Further observations were made on the extensor muscle of the carpopodite which possesses long, and easily accessible, fibres. Resting and ‘end-plate’ potentials were measured at several points of the fibres covering as much of their length as possible. Examples are shown in Fig. 3, where the amplitude and duration of the rising phase of the e.p.p. have been plotted. The amplitude of the e.p.p. varied to some extent along each fibre, but in only one out of fifteen fibres did the e.p.p. vanish near one end. In any one of the other fourteen fibres, the range of variation of e.p.p. amplitudes did not exceed a factor of 2, the mean range (for a given fibre) being 1·4 (s.E. ± 0·045). It should be noted that the time of rise of the e.p.p. was approximately constant at every point.
In two muscle fibres, the recording electrode was also moved transversely across the fibre surface, and amplitude variations of the same order were found. Thus, in a 500 fi fibre the amplitude varied, across the fibre, by a factor of 1·2, in a 285μ fibre by 1·4.
To interpret these findings, one must distinguish between two factors which cause e.p.p.’s to spread along muscle fibres: (a) electrotonic currents, and (b) distributed nerve endings. The electrotonic spread in these muscle fibres has been investigated previously (Fatt & Katz, 1953), the average length constant being 0·9 mm. (the time constant 4·6 msec.). It is clear that the much more extensive spread of the e.p.p. in these fibres cannot be attributed to their electrotonic ‘cable properties ‘and must mainly be due to a distributed nerve supply. The constancy of the time course of the e.p.p. recorded at different points of a fibre, also suggests that electrotonic potential spread plays no important role. The results indicate, however, that there are appreciable differences in the density of innervation, or in the sensitivity of the muscle receptors, along the length and across the width of the fibre surface.
The following experiment supported these conclusions. In one muscle, after e.p.p.’s had been mapped, a block occurred in part of the axon supply (probably as a result of prolonged tetanization). When the experiment was repeated on one fibre the e.p.p. was found to have suffered a sharp ‘cut-off’ at a point about 2 mm. from the shell (Fig. 4). Beyond this point, the amplitude now declined sharply, and the time of rise increased, showing that the residual spread in this region was electrotonic, this part of the muscle fibre having now been deprived of a functioning nerve supply. Incidentally, the peak size of the e.p.p. was greater than before, but it is not clear whether this was a ‘potentiating’ after-effect of the prolonged tetanus, or due to blocking of an accompanying inhibitory axon.
In several experiments, the flexor of the dactylopodite of Carcinus maenas was used, and the two motor axons supplying it were stimulated separately. It was found that both motor axons produced distributed e.p.p.’s in the same muscle fibres (e.g. Fig. 5). The e.p.p.’s due to the two axons usually differed in amplitude, and sometimes also in time course and frequency-dependence (Fig. 6), but their spatial distribution along the fibres appeared to be the same.
The present experiments support the view held by Wiersma (1941a) and van Harreveld (1939), viz. that crustacean muscles are supplied with a widespread innervation enabling them to develop powerful responses without necessarily involving propagated action potentials in the muscle fibres. As has been pointed out (Fatt & Katz, 1953), the excitatory mechanism of the membrane (the ‘spike process’) which becomes operative when the depolarization exceeds some 30 mV., may serve merely to increase and equalize forces along the contractile chain, when a sudden maximum effort is required.
The experiment illustrated in Fig. 4 suggests that ‘focal’ e.p.p.’s found in ‘cut-down’ nerve-muscle preparations (Katz & Kuffler, 1946) may have been obtained from muscle fibres which had suffered partial denervation. In addition, however, the conditions of external recording in a crustacean limb muscle give rise to ‘spatial differentiation’, and a sharpening of the observed contours of the e.p.p. (cf. the volume-conductor effect, described by Bishop (1937)). With the use of an intracellular recording electrode both difficulties have been overcome, because (a) the membrane potential of a single muscle fibre is recorded directly, and (b) there is no need to reduce the number of responding elements by micro-dissection of the nerve.
The importance of the spatial distribution of motor nerve endings in crustacean muscle is discussed.
The spread of the ‘end-plate potential’ (e.p.p.) has been mapped out in individual crustacean muscle fibres using intracellular recording.
The e.p.p. is distributed over the whole length of the fibre with relatively small variations of its local amplitude. In any one of fourteen individual fibres, the size of the e.p.p. varied by less than a factor of two along 5-6 mm. of fibre length, the average range of variation being 1.
A sharp focal e.p.p. was only seen in a partially ‘denervated’ muscle fibre.
These experiments support the view that motor nerve endings are widely distributed along the length of crustacean muscle fibre.
We wish to thank Mr J. L. Parkinson for his unfailing help. This work was supported by a grant for scientific assistance made by the Medical Research Council.