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Response of insect muscle to denervation—II. Changes in neuromuscular transmission
3. Ins. Physiol., 1963, Vol. 9, pp. 811 to 825. Pergamon Press Ltd.
Printed in Great Britain
RESPONSE OF INSECT MUSCLE TO DENERVATION-II. CHANGES IN NEUROMUSCULAR TRANSMISSION P. N. R. USHERWOOD Department of Zoology,
University of Glasgow
(Received 11 May 1963) Abstract-The effects of motor-nerve section on neuromuscular transmission in the locust extensor tibiae preparation have been investigated by intracellular recording. Impulse transmission failed between the ninth and twenty-fourth post-operative days, in the region of the nerve-muscle synapse. Before impulse transmission failed, abnormally large depolarizing and hyperpolarizing responses to stimulation of the ‘slow’ axons, and prolonged depolarizing responses to the ‘fast’ axon were recorded from many muscle fibres. At the time of impulse transmission failure the discharge of spontaneous miniature post-synaptic potentials underwent a number of complicated change’s leading to the appearance of ‘giant’ miniature potentials up to 10 mV in height. The discharge persisted apparently for a few days after the muscle became neurally inexcitable. When the discharge finally stopped the fibres became permanently electrically silent ; no evidence was obtained for a resumption of miniature activity during the later post-operative days. The cessation of the miniature discharge is apparently correlated to the decline of muscle-resting potential observed in denervated insect muscle and the possibility that the transmitter, or a substance released along with it, maintains the resting potential of the muscle at its normal level is discussed. INTRODUCTION
TFIE possibility that the motor nerves innervating the metathoracic extensor tibiae muscle of the locust exert a ‘trophic’ influence on this muscle was suggested by USHERWOOD (1963a) on the basis of the results of experiments designed to study the effects of motor-nerve section on the resting potential (R.P.) of insect skeletal muscle. These results showed that during the first two post-operative days the R.P. of denervated muscle falls b&low normal; a temporary recovery occurs during the next 2-6 days, but this is soon followed by a second, irreversible fall in the R.P. to a level about 25 per cent below normal. More recently, studies have been made on the effects of denervation on neuromuscular transmission in the locust leg and the results of these studies are made the subject of this paper. METHODS The procedure for denervation (i.e. severance of the motor innervation at its points of origin from the ganglion) of the metathoracic extensor tibiae muscle of the locust, Scf&ocerca gregaria, was the same as that described by USHERWOOD (1963a). The motor axons to the extensor muscle, which are contained in metathoracic nerves 5 and 3b, were stimulated through silver-silver chloride electrodes. 811
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The electrical activity occurring in response to direct and indirect stimulation, the spontaneous miniature activity, and the R.P’s of the denervated and control muscles were recorded with glass, intracellular micro-electrodes, together with conventional amplifying and display equipment. Obviously, it was not possible to examine the same preparation more than once and since all the changes in neuromuscular transmission, which result from denervation, cannot be seen in any one preparation, it was necessary to examine a large number of different preparations (ca. 200) on a variety of post-operative days in order to ensure that no changes of significance were overlooked. RESULTS Effects of denervation on the response to nerve 5 In the locust extensor tibiae muscle, electrical stimulation of nerve 5 by a single supramaximal square pulse evokes a single action potential (Fig. la) composed of a distributed, ‘fast’ post-synaptic potential (‘fast’ p.s.p.) and graded electrically excitable or spike response (DEL CASTILLOet al., 1953). In some denervated muscles the magnitude of the ‘fast’ response was slightly lower than normal during the first 2-3 post-operative days, but this is to be expected since the R.P. of denervated muscle is also usually subnormal during this period (USHERWOOD,1963a). The first consistent change in indirect excitability occurred on about the third post-operative day. The repolarization phase of the ‘fast’ potential became increasingly prolonged (Fig. lb) and by about the sixth day the typical ‘fast’ response consisted of a large p.s.p. and a large spike followed by a pronounced plateau. Oscillatory potentials often appeared on the plateau, and at times up to five spike responses to a single indirect stimulus have been recorded. Less frequently, the peripheral portion of the severed crural nerve responded repetitively to a single applied shock and thereby elicited a series of ‘fast’ p.s.p’s and spikes in the extensor muscle fibres (Fig. lc). A reversal of the trend towards increased muscle excitability to neural stimulation was first recorded on the seventh post-operative day. The p.s.p’s started to get smaller and consequently the electrically excitable component also declined in magnitude (Fig. Id). Soon only diminished pure ‘fast’ p.s.p’s were evoked (Fig. le) and as the excitability of the muscle deteriorated even further, ‘fast’ p.s.p’s, similar in magnitude to the spontaneous miniature potentials recorded from normal locust muscle fibres (USHERWOOD, 1963b), were recorded in many denervated fibres (Fig. If). Eventually, all traces of responsiveness to the ‘fast’ axon were lost. The timing of onset of impulse transmission failure varied from preparation to preparation. In some denervated muscles it occurred as early as the ninth postoperative day, whilst other muscles remained responsive to neural stimulation until as late as the twenty-fourth day. There was a slight increase in the responsiveness of the control muscles to stimulation of nerve 5 from about the fourth post-operative day, but apart from this the responsiveness of these muscles was relatively normal, even in muscles examined 84 days post-operatively.
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It was still possible to excite the denervated muscle for about 10 days after impulse transmission had failed, simply by placing the stimulating electrodes directly on the surface of the muscle (Fig. lg). However, during this period only the fibres under the cathodal electrode responded to the applied stimuli, the number of fibres involved and the extent to which they contracted being partly dependent on the stimulus intensity. The electrical responses of the fibres under both cathodal and anodal electrodes, depolarizing under the cathode and hyperpolarizing under the anode, were graded in amplitude according to the stimulus intensity. In view of the vanishingly small intervals between the application of the stimuli and the appearance of the responses, together with the graded nature of the responses, it is suggested that the responses were in fact direct. Direct stimulation of normal insect muscle is not possible, probably because the nerve endings have a much lower threshold than that of the membrane of the muscle fibre (HOYLE, 1957) and because excitation via the distributed nerve endings is propagated so rapidly that the direct response is blocked through refractoriness (ROEDER and WEIANT, 1950). These obstacles are obviated by denervation since the excitatory influence of the motor innervation is then removed. The only other detailed electrophysiological investigations of the excitability of denervated insect muscle have been made by ROEDER ,and WEIANT (1950). They found that the abdominal muscle of the cockroach becomes electrically inexcitable to neural stimulation about 3-5 days after nerve section, but they were unable to obtain a response to direct stimulation with extracellular electrodes at any time before or after denervation. On the basis of their results, they suggested that cockroach skeletal muscle is either electrically inexcitable or, alternatively, that ‘the muscle surface has such a high electrical resistance that externally applied current fails to reach the depolarizable regions of the muscle fibres’. The first of their suggestions is clearly untenable, since cockroach muscle fibres are in fact electrically excitable, as shown by stimulation with intracellularly applied depolarizing current pulses (USHERWOOD,unpublished), though in a graded rather than all-or-nothing manner. The alternative suggestion is more plausible and could also be invoked to account for the decline and eventual loss of direct excitability of the denervated locust muscle fibre to externally applied stimuli which occurs about 10 days after impulse transmission has failed. The fibres still respond to intracellularly applied stimuli at this time (Fig. lh) but they are considerably atrophied and this in itself could lead to the abnormally high resistance of the muscle surface postulated to account for the ineffectiveness of external stimuli. Apart from these considerations, perhaps one of the more significant features of these results is that denervated locust muscle is electrically excitable for a considerable time after transmission of impulses from the ‘fast’ motor axon has failed. Since the isolated peripheral portion of the crural nerve is often still able to conduct impulses during this period, it follows that the site of impulse transmission failure must be in the region of the nerve-muscle junction. A more detailed examination of the critical changes leading up to impulse transmission failure has been made possible by virtue of the pinnate arrangement
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of the fibres in the extensor muscle. The distal fibres in this muscle are separated from the proximal fibres by a distance of about 2 cm and there is usually a short delay of about 1-3 hr between the onset of indirect inexcitability in the distal fibres and its appearance in the proximal fibres. The distal fibres were not invariably affected first; in some denervated muscles the proximal fibres were involved first, whilst in others the first change in responsiveness to neural stimulation was recorded from fibres in the middle. In denervated muscles exposed at the critical transitional stage there was usually a gradient of responsiveness from the inexcitable to the excitable regions of the muscles, i.e. it was possible to record ‘fast’ electrical responses ranging from about 70 mV to less than O-5 mV according to the position in the muscle of the fibre in which the recording electrode was impaled. The excitable fibres often deteriorated quickly during the experiment, especially if they were stimulated intermittently through nerve 5. In one preparation, in which initially about 70 per cent of the fibres were responsive to stimulation of nerve 5, transmission to the remaining, indirectly excitable fibres deteriorated so rapidly that within the space of about 2 hr the muscle became completely inexcitable to neural stimulation. This indicates that the complete process of impulse transmission breakdown apparently takes no more than a few hours. However, the possibility that exposure of the preparation for examination accelerates or even triggers the change-over from indirect excitability to inexcitability cannot be ruled out. Effect of denervation on the response to nerve 3b Nerve 3b supplied the extensor muscle with three motor axons (USHERWOOD, 1962), most of the fibres innervated by these axons being located in a bundle at the proximal end of the muscle. The electrical responses of the innervated fibres to stimulation of nerve 3b are extremely varied, ranging from hyperpolarizing potentials of just under 1 mV to depolarizing potentials of from 2-50 mV. HOYLE (1955) included all of these under the general heading of ‘slow’ responses. The ‘slow’ electrical responses of muscles denervated for 3-9 days were often noticeably larger, and at times more prolonged, than normal. ,+bnormally large ‘slow’ responses were also recorded from fibres of the contralateral muscle examined at this time. The depolarizing ‘slow’ potentials often reach a magnitude of 65 mV and frequently overshot the zero membrane potential (Fig. 2a, b) ; overshooting ‘slow’ potentials have never been recorded from muscle fibres of normal locusts (HOYLE, 1955). The apparent increased excitability of the denervated muscles to nerve 3b was sometimes clearly due to repetitive firing of the ‘slow’ nerve, resulting in the appearance of a series of summated depolarizing ‘slow’ p.s.p’s. Usually, however, a single shock applied to the peripheral portion of nerve 3b set up only a single nerve action potential, although the electrical response of the extensor muscle was still enhanced. It is probable, therefore, that denervation leads to changes in the membrane properties of the extensor muscle fibres and that these changes are reflected in the apparent increased excitability to neural stimulation.
FIG. 1. Effect of denervation on response of extensor tibiae muscle to nerve 5. (a) Intracellularly recorded 'fast' response from a n o r m a l fibre. Records (b-f) illustrate typical fast response recorded (b) 5, (c) 6, (e) 8, (f) 9 days after denervation. Note plateau following spike in record (b), evidence of neural repetition in record (c), and step-wise fluctuations in amplitude of diminished 'fast' p.s.p's in record (f). Record (g) illustrates response of a neurally inexcitable fibre to stimulation with external electrodes 12 days after denervation. Record (h) illustrates response of a neurally inexcitable fibre to intracellular stimulation with depolarizing current pulses (upper trace) of graded intensity 20 days after denervation. T h i s fibre did n o t respond at this time to externally applied current pulses. Voltage calibration same for records (a), (b), (c), and (g), and for records (d) and (e). T i m e calibration same for records (a) a n d (b) and for records (c), (d), and (e).
FIC. 2. E n h a n c e d depolarizing responses to stimulation of nerve 3b. (a, b) Intracellular records of overshooting, depolarizing potentials in response to maximal stimulation of nerve 3b. These responses were obtained from fibres of a muscle denervated for 6 days. Note double nature of p.s.p, in record (a). (c) Response obtained to nerve 3b from a fibre denervated for 5 days. T h i s was the largest depolarizing 'slow' response recorded in the present experiments. Voltage calibration same for all 3 records. T i m e calibration same for records (a) and (b).
FIG. 3. Enhanced hyperpolarizing responses to stimulation of nerve 3b recorded from (a-d) denervated proximal extensor tibiae muscle fibres, 2-9 days after section of both nerves 3b and 5 and (e, f) partially denervated proximal fibres, 53 days after section of nerve 5 only. Voltage calibration same for all records.
FIG. 4. A, Attenuation of depolarizing 'slow' p.s.p's by hyperpolarizing 'slow' p.s.p's in denervated proximal fibres of eight different extensor tibiae muscles, 2-12 days after nerve section. All eight fibres were innervated by the hyperpolarizing and depolarizing axons. (a-d) The hyperpolarizing axon had the lower threshold. Each record illustrates the response to stimulation of the hyperpolarizing axon alone (i) and also the response to stimulation of the hyperpolarizing and depolarizing axons together (ii).
Fro. 4. B, (a-d) T h e depolarizing axon had the lower threshold. Each record illustrates response to stimulation of depolarizing axon alone (i) and also the response to simultaneous stimulation of both 'slow' axons (ii). Voltage calibration same for all records except (c).
FIG. 5. Facilitation and summation of hyperpolarizing potentials in a fibre (R.P. 58 mV) denervated for 6 days. T h e hyperpolarizing axon was stimulated at a frequency of (a) 5/sec, (b) 20/sec, (c) 30/sec, and (d) 50/sec. T i m e and voltage calibrations were the same for all records.
FIG. 6. Changes in pattern of spontaneous miniature activity after denervation (motor-nerve section). (a, b) Miniature potentials recorded from a normal fibre. (c, d) Potentials recorded from a fibre denervated for 14 days. Note normal appearance Of individual potentials but tendency for discharge to occur in bursts and for potentials in bursts to summate. (e, f) 'Giant' miniature potentials recorded from a fibre denervated for 14 days. Note incomplete summation of the potentials to give irregularly shaped compound responses. (g, h) 'Giant' miniature potentials recorded from a muscle fibre denervated for 16 days. Summation is complete and the compound nature of the potentials is no longer obvious. Note longer duration of 'giant' potentials compared with normal. Voltage calibration applies to all records. Time calibration same for (a), (c), (e), and (g) and for (b) and (d).
FIC. 8. (a-c) Small oscillatory potentials recorded from a neurally inexcitable fibre of a muscle denervated for 18 days, (a) 3 min, (b) 6 min, and (c) 13 min after the fibre had started contracting spontaneously. (d-f) Much larger oscillatory potentials recorded from a neurally inexcitable fibre of muscle denervated for 16 days, (d) 1 min, (e) 3 rain, and (f) 7 min after the first signs of spontaneous activity appeared in the fibre. Voltage calibrations same for (a-c) and for (d-f). Time calibration same for (e-f).
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Hyperpolarizing ‘slow’ p.s.p’s, up to 10 mV in magnitude, were often recorded from both denervated and control muscles during the third to seventh postoperative days (Figs. 3 and 4). These responses facilitated and summated as the stimulus frequency was increased (Fig. 5). As in muscles of unoperated locusts (HOYLE, 1955), the largest responses were usually recorded from fibres with relatively low R.P’s. It is accepted that muscle fibres with subnormal R.P’s would probably give abnormally large responses to stimulation of the hyperpolarizing axon, but there is no evidence that the R.P’s are, at this time, any lower in fibres of operated locusts than they are in fibres of unoperated locusts (USHERWOOD, 1963a). Considering that relatively large hyperpolarizing potentials were recorded from fibres with R.P’s as high as 65 mV as well as from fibres with much lower R.P’s, that they occurred in fibres which at the same time gave abnormally large responses to stimulation of the depolarizing axon, and that they were recorded from fibres of the contralateral control muscles as well as from fibres of the denervated muscles, it appears unlikely that the magnitude of the R.P. is the overriding determining factor. However, any explanation for the enhanced responsiveness to either the hyperpolarizing axon or the depolarizing axon in the muscles of operated locusts, based on the very small amount of information obtained in the present investigations, is purely speculative. For any interpretation to be of value it must take into account the membrane properties of the denervated extensor muscle, and so far these have not been investigated. In any future investigations it would probably be of value not only to examine the effects of denervation on the membrane constants of the extensor muscle, but also to record the effects of the hyperpolarizing axon on the ‘slow’ mechanical response of this muscle, since in some of the denervated proximal fibres normally innervated by both depolarizing and hyperpolarizing axons, the depolarizing p.s.p’s were often attenuated by the large hyperpolarizing p.s.p’s (Fig. 4). In muscles of unoperated locusts, the hyperpolarizing p.s.p’s are very small and completely overridden by the responses to the depolarizing axon. As a result, stimulation of the hyperpolarizing axon has no effect on the ‘slow’ mechanical response (HOYLE, 1955). The response of the completely denervated muscle to nerve 3b often, but not invariably, survived the response to nerve 5. The reason for this is unknown, but it is of interest to note that BIRKS et aZ. (1960) f ound that in the denervated ‘mixed muscles of the frog the indirect excitability of the ‘slow’ fibres invariably survived the indirect excitability of the ‘fast’ fibres.
Effects of denervation on spontaneous miniature activity Spontaneous miniature potentials have been recorded from a variety of insect muscle preparations (USHERWOOD, 1961, 1963b) and have been compared with the miniature p.s.p’s recorded at vertebrate nerve-muscle junctions. The miniature potentials are postulated to results from a randomized release of quanta1 ‘packets’ of a chemical transmitter agent from the pre-synaptic nerve terminals (e.g. FATT and KATZ, 1952). Those recorded from the locust extensor tibiae muscle have
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amplitudes ranging from 0.1-2.0 mV and appear at frequencies ranging from 0*2/set-15/set (USHERWOOD, 1963b). In the present investigations only the overall changes in spontaneous miniature activity of the extensor muscle which result from denervation have been noted. It is unlikely that a quantitative examination would add very much more in the way of new information, but it would, however, be extremely time-consuming in view of the large natural variations in the spontaneous activity of even adjacent fibres in a muscle. There was little change in the spontaneous miniature activity of the denervated muscle during the first few post-operative days, but at some time between the seventh and thirty-second days a number of complicated changes occurred, before the activity finally ceased completely (Fig. 6). The first change in the general pattern of activity was indicated by an increase in the discharge frequency and a tendency for the miniature potentials to appear in bursts interposed between periods of relative inactivity (Fig. 6d). Each burst typically contained a large number of potentials, which, on occasion, summated to give a single large ‘giant’ response up to 10 mV in magnitude and often four times the duration of the normal miniature potential (Fig. 6g, h). S ometimes, especially during the ealier stages, the summation, process was incomplete and then the compound nature of these abnormally large responses was clearly apparent (Fig. 6e, f). The appearance of the ‘giant’ potentials coincided with an overall reduction in the level of activity, which steadily continued thereafter until eventually the miniature potentials disappeared completely and the muscle fibres became electrically silent. (Fibres were considered to be electrically silent if no miniature potentials were recorded from them for a period of about 10 min after impalement.) In a few of the fibres examined just before they became electrically silent the miniature activity consisted mainly of the large ‘giant’ potentials and hardly any miniature responses of normal amplitude were recorded. No attempt was made to determine whether transmission breakdown occurred simultaneously at all the junctions on any one muscle fibre or, alternatively, if some of the junctions became non-functional before others. The timing of the onset of complete transmission failure varied considerably. In one muscle it occurred as early as the twenty-first post-operative day, whereas miniature activity was recorded from other denervated muscles as late as the thirtysecond post-operative day. It has already been shown that impulse transmission usually fails much earlier than this. It is probable, therefore, that the spontaneous miniature activity survives for a considerable time after the muscle becomes indirectly inexcitable. Evidence contrary to this and in favour of a more rapid disappearance of the miniature potentials comes mainly from denervated muscles which were at the critical stage of transmission failure when first exposed. In these muscles, impulse transmission failed during the experiment and the miniature activity survived for only about 1 hr afterwards. The disappearance of the miniature potentials was accelerated by stimulating the preparation just before the muscle fibres became completely inexcitable to indirect stimulation and it was often found that under these conditions disappearance of the miniature potentials almost
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coincided with the breakdown of impulse transmission. It is probable, therefore, that whereas in unexposed denervated muscles the miniature activity persists for some days after impulse transmission has failed, exposure of the muscle in some way accelerates the decline in miniature activity so that the fibres become electrically silent at the time of impulse transmission failure. BIRKS et al. (1960) showed that in denervated frog muscle the miniature activity ceases about 3-4 days after denervation, only to be resumed, in a modified form, about 1 week later. There is at present no evidence for a resumption of miniature activity in the denervated locust muscle fibre; even preparations examined 84 days post-operatively were completely inactive, but there are certain resemblances between the changes of miniature activity in denervated locust and frog muscles. ‘Giant’ miniature potentials similar in magnitude to those recorded from indirectly inexcitable denervated locust muscle fibres have been recorded at denervated frog end-plates (BIRKS et al., 1960). It is important to note, however, that ‘giant’ miniature potentials are also recorded from normal vertebrate end-plates (LILEY, 1957) although presumably they occur more frequently at denervated end-plates. The histogram for the frequency distribution of amplitudes of the miniature potentials recorded from denervated frog end-plates during the period of renewed activity is markedly asymmetrical and has a much wider dispersion than the approximately ‘Gaussian’ type of histogram for the miniature activity at normal end-plates (BIRKS et al., 1960). Fig. 7a illustrates that the histogram for frequency distribution of amplitudes of the miniature potentials recorded from a denervated locust muscle fibre, just after impulse transmission had failed, is also very asymmetrical and has a wide dispersion. Unfortunately, any direct comparison with frog ‘fast’ muscle fibres is made difficult by the fact that even the histogram of frequency distribution of amplitudes of the miniature potentials recorded from a normal locust fibre is positively skewed (Fig. 7b). This is attributed to the distributed innervation of the locust muscle fibre (USHERWOOD, 196313). However, there is little doubt that the histogram for the denervated locust muscle fibre which has just become indirectly inexcitable is more asymmetrical and has a much wider dispersion than the histogram for the normal fibre. Spontaneous mechanical activity Spontaneous contractions have been recorded from denervated insect muscles on a number of occasions. CASE (1956, 1957) noted spontaneous mechanical activity in the femoral muscles of the cockroach 4-6 days after motor-nerve section and BEFG~NEK and NOVOTN.~; (1958) recorded spontaneous mechanical and electrical activity in the denervated femoral muscles of this insect. In the present experiments spontaneous contractions of the denervated extensor tibiae preparation of Schistocerca occurred only rarely. In fact, activity of the type described by CASE (1956) was recorded in only seven out of a total of over 200 denervated muscles examined during these investigations. Even in these muscles the spontaneous activity, which appeared soon after exposure of the muscle, persisted for no longer than 15 min. The denervated and contralateral
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FIG. 7. {a) Frequency distribution of amplitudes of spontaneous miniature potentials recorded 17 days after nerve-section from a denervated extensor tibiae muscle fibre just after impulse transmission from the motor nerve stump had failed. (b) Amplitude distribution of spontaneous potentials recorded from a fibre of the innervated contralateral control muscle. (See text for furtber explanation.)
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control legs were always examined before the extensor muscles were exposed, to check for the possibility of spontaneous contractions of the intact muscle: it was assumed that any contractions of the extensor muscles would lead to movements of the respective tibia1 segments. However, rhythmical twitches similar to those seen in the extremities of the denervated locust leg as early as the first day after denervation (VOSKRENSENSKAYA, 1945) were never seen at any time in the present experiments. If spontaneous contractions did occur in the unexposed, denervated muscles, then they must have been too weak to move the extremities of the legs. The contractions seen in exposed denervated muscles were usually restricted to fibres which were already indirectly inexcitable and usually involved only a single fibre or a bundle of fibres; on no occasion was the entire muscle seen to contract spontaneously. On the two occasions that the mechanical activity persisted for some time after exposure of the muscles it was possible to record accurately, with intracellular electrodes, the electrical activity of the contracting fibres. The activity consisted of oscillations of the membrane potential similar to those recorded from fibrillating vertebrate muscle fibres (e.g. B~~LBRINGet al., 1956; CHOH-LUH et al., 1957). Apparently the oscillations occurred in phase with the contractions. In one fibre rhythmical, alternating slow and fast, oscillations were readily distinguished throughout the period of the contractions, although at times the fast oscillation was complicated by a step on its rising phase (Fig. 8a-c). The oscillations in this fibre never depolarized the membrane by more than about 8 mV from its resting value (about 45 mV). The activity in the other fibre (Fig. Sd, e) consisted initially of a very complicated series of rhythmical oscillations which depolarized the membrane by as much as 40 mV (R.P. ca. 58 mV), but the form of the oscillations soon became much simpler and it was then possible to recognize alternating slow and fast potentials as before. In both fibres the fast potential apparently occurred when the slow potential reached a critical level of depolarization, a level which was relatively constant for each of the fibres. In a few spontaneously active fibres slow potentials were recorded in the absence of fast potentials. Fast potentials were absent presumably because the slow potentials failed to depolarize the membrane potential to its critical level. The contractions of fibres which showed only slow oscillations were apparently weaker than those which showed both fast and slow oscillations. In all the spontaneously active fibres the frequency and amplitude of the potentials declined gradually during the experiment before the oscillations finally disappeared completely and the membrane potential stabilized, but over short periods the frequency and amplitude of the potentials were relatively constant (Fig. 8). A second type of spontaneous mechanical activity, which was recorded from about ten denervated and two control muscles, consisted of much slower rhythmical contractions of the extensor muscle. This activity was usually restricted to the proximal bundle of muscle fibres and each contraction often took more than 1 min. The membrane potentials of the contracting fibres apparently oscillated slowly, falling to peak depolarizations as great as 40 mV, in phase with the
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contractions, but fast potentials were absent. However, the possibility that these oscillations were in fact nothing more than mechanical artifacts cannot be ruled out, since the contractions, although very slow, were nevertheless rather vigorous at times. In this respect it is considered rather surprising that they were never ‘signalled’ in unexposed muscles by movements of the tibia1 segments. Perhaps they only occur on exposure of the muscle due to some form of interaction between a hyper-excitable muscle membrane and the locust saline. A similar explanation could also be invoked for the much faster contractions described earlier, since these too were never recorded from unexposed muscles. Response to partial denervation Part of the extensor muscle is innervated by branches of both nerves 5 and 3b and in the first paper of this series partial denervation, i.e. section of only one of the motor nerves, was shown to have no effect on the R.P. of the dually innervated fibres. Experiments to determine the effects of partial denervation on the responsiveness of these fibres to indirect stimulation have shown that section of either nerve 5 or 3b does not reduce the response of the extensor muscle to the motor nerve still connected to the metathoracic ganglion. In fact, the response to the intact nerve is increased slightly and in the present experiments the increased responsiveness to the intact axon persisted long after all traces of responsiveness to the sectioned axon had disappeared. The neurally excited responses to stimulation of the cut axon underwent the same sequence of changes outlined for the completely denervated muscle. When fibres of partially denervated muscles, which were originally innervated by both ‘fast’ and ‘slow’ axons, were examined just after impulse transmission from the severed axon had failed, they showed a characteristically mixed pattern of miniature activity. ‘Giant’ potentials were often seen as in the completely denervated fibres but these later disappeared and then the activity reverted to the pattern seen in normal fibres. Presumably, partial denervation leads to degenerative changes at the endings of the severed axon whilst the endings of the intact axon are unaffected by the operation. Correlation between neuromuscular traKcmission and resting potential in denervated muscle The possibility that the changes in transmission which result from motornerve section might be causally correlated to the changes of muscle R.P. became increasingly evident during the course of these investigations. To test this possibility the effects of denervation on the indirect excitability, R.P., and spontaneous miniature activity of the extensor muscle have been examined simultaneously in a number of different preparations on different post-operative days and in particular during the critical period of impulse transmission failure. The recording procedure consisted of inserting a microelectrode into a fibre of the denervated muscle to and recording the R.P., miniature activity, and electrical responsiveness nerve 5, in that order. The procedure was then,repeated for a fibre of the control muscle. About 20-50 fibres were examined in each muscle and the results from
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each pair of muscles were collated. As before, no attempt was made to examine the miniature potentials of the different fibres quantitatively; only the gross changes in activity were noted. The changes in R.P. and indirect excitability after denervation are illustrated graphically in Fig. 9. It is apparent from the graph that the second, irreversible fall in the R.P. of the denervated muscle usually occurred on the same post-operative day that impulse transmission failed, although in a few preparations there
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FIG. 9. Graph illustrating correlation between resting potential and neurally excited responses after denervation. Ordinates, mean R.P. (0) (‘fast’ electrical response (0)) of denervated muscle as a percentage of mean R.P. (‘fast’ electrical response) of contralateral control muscle. Abscissae, number of post-operative days. were indications
of a slight delay between impulse transmission failure and R.P. decline. Detailed analysis of the correlation between R.P. and impulse transmission in muscles, in which impulse transmission failed during examination, gave a much clearer picture of the timing of the various changes. In these muscles there was often a delay of about 3 hr from the time impulse transmission failed before the R.P. started to fall. The close correlation between miniature activity
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and R.P. was most clearly illustrated in one denervated muscle, which was completely inexcitable when first exposed. It was still possible to record miniature potentials from the proximal fibres and the mean R.P. of these fibres was about 20 per cent higher than the mean R.P. of the distal fibres, which were electrically silent, and only slightly lower (about 5 per cent) than the mean R.P. of the contralateral control muscle. At least in some denervated muscles, therefore, the decline in R.P. is correlated not with impulse transmission failure, but with the cessation of spontaneous miniature activity. The possibility of a correlation between the type of spontaneous miniature activity recorded from a denervated muscle fibre and the responsiveness of the fibre to neural stimulation was also investigated. In completely denervated muscles (i.e. muscles in which the central connexions of both nerves 3b and 5 were severed), which contained both neurally excitable and neurally inexcitable fibres when first exposed, ‘giant’ miniature potentials were often recorded from the inexcitable fibres but were never recorded from the fibres which were still neurally excitable. It is probable, therefore, that the appearance of these ‘giant’ compound miniature potentials coincides with the failure of impulse transmission. DISCUSSION There is little doubt that denervation of the locust extensor tibiae muscle results in the failure of impulse transmission presumably before any gross structural changes occur (USHERWOOD, to be published), but the exact site at which transmission first breaks down is unknown. The fact that the isolated peripheral portions of the severed motor nerves to the extensor muscle are still able to conduct impulses and that the muscle is still electrically excitable after transmission has failed indicates that the breakdown is in the region of the nerve-muscle junction. The survival of the spontaneous miniature activity for a considerable time after impulse transmission fails precludes the possibility that the breakdown results initially from a depletion of transmitter agent from the pre-synaptic nerve terminals, where it is presumably stored before release, and also rules out the possibility of some form of blockage at the post-synaptic receptor regions on the muscle fibre. Perhaps the mechanism which couples the excitation of the nerve terminals by the invading nerve impulse to the release of the transmitter, responsible for setting up the p.s.p., is impaired as a result of denervation. DEL CASTILLO and KATZ (1954) have shown that the frequency of transmitter discharge from the peripheral endings of the frog motor nerve is coupled to the magnitude of the potential differences across the axonal membrane at the pre-synaptic terminals. The arrival of the nerve impulse at the terminals, with the concomitant reduction of the membrane potential of that region, presumably results in a dramatic shortterm increase in the frequency of transmitter release. It follows, therefore, that although a breakdown in the mechanism which links the level of membrane potential at the terminals to the discharge frequency would lead to impulse transmission failure, it would not necessarily stop the spontaneous release of transmitter from the terminals. If such a breakdown did occur, subsequent depolarizations of
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the nerve endings either by applied current or by high external potassium should, according to the hypothesis, have no effect on the level of the miniature activity. The hypothesis has been crudely tested in the present experiments by increasing the potassium concentration of the saline bathing a denervated locust muscle fibre, which had just become indirectly inexcitable, to two to three times its normal value (HOYLE, 1955). Significantly, this change had no effect on the mean frequency of the miniature potentials of the denervated fibre, although the mean frequency of the miniature potentials recorded from a normal locust muscle fibre is markedly raised by an equivalent increase in the external potassium concentration (USHERWOOD, 1963b). It is perhaps of interest to note that the ‘resumed miniature activity at the denervated frog end-plate is similarly insensitive to the external potassium concentration (BIRKS et al., 1960). The gradual decline of the miniature activity to zero, which occurs after impulse transmission has failed, is explicable on the basis of transmitter depletion from the nerve terminals. There are a number of possible sites for the production of transmitter in the intact nerve cell. The site of production could be in the soma of the motor neurone, the transmitter being transported to the nerve terminals along the axon. Alternatively, the transmitter could be produced at sites situated either throughout the length of the axon or even restricted to the terminal regions of the axon. Whichever system operates, section of the ‘motor nerve and, therefore, isolation of the axon from the metabolic control centre, the cell body, would eventually curtail any further renewal of transmitter in the terminals. The fact that transmission does not fail for some time after nerve-section implies that either the production of transmitter persists for some time after nerve-section or that there is sufficient transmitter stored in the axon and its terminals to allow transmission to continue for some time. Concomitant with the decline of the miniature activity of the denervated muscle fibre, a second change leading to the appearance of ‘giant’ miniature potentials takes place. The present results indicate that a ‘giant’ potential results from summation of a large number of smaller miniature potentials. The requirement for complete summation would be met by the synchronous release of a large number of quanta of transmitter from the nerve terminals and their attachment as a multiple unit to the receptor sites of the post-synaptic membrane. Perhaps the quanta coalesce to form multiple units before they are released from the nerve terminals and as this process becomes more intimate, so attachment at the receptor sites becomes more synchronized and summation of the miniature p.s.p’s more complete. A release of transmitter agent in multi-quanta1 units has been invoked previously to account for the ‘giant’ miniature p.s.p’s recorded from the isolated rat diaphragm muscle (LILLEY, 1957), ‘since these potentials occurred more frequently than would be expected from random coincidences of miniature potentials. It would undoubtedly be of interest to know exactly what happens to the arrangement of the vesicles, which are found in the nerve terminals of the locust, after denervation, since they have been postulated as possible storage sites for the transmitter.
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The results of the present experiments lend further support to the possibility that the motor nerves innervating the locust extensor tibiae muscle exert a ‘trophic’ influence on this muscle, and at the same time give some indication of how this influence is exerted. For example, the second, irreversible fall in the R.P. of the extensor muscle has been shown to more or less coincide with transmission failure at the neuromuscular junction, thus suggesting a possible causal relationship between the two events. Recent electron micrographs of insect nerve-muscle junctions have clearly demonstrated the existence of a gap between the nerve terminals and the post-synaptic membrane of the muscle fibre (EDWARDS et al., 1958). This implies that there is no protoplasmic connexion between the axon and the muscle fibre and we are left with the possibility that the transmitter agent or some neurohumoral substance released along with it is responsible for maintaining the R.P. of the muscle fibre at its normal level. This hypothesis is supported by the fact that the R.P. presumably does not start to fall until after the release of transmitter from the nerve terminals has stopped completely. Acknowledgements-The author wishes to thank Professor G. HOYLE for his helpful comments on the manuscript. REFERENCES BE~~NEKR. and NOVOTNPI. (1958) Spontaneous electrical activity in the denervated muscles of the cockroach Periplaneta ame-ricana. Nature, Lond. 182, 957-958. BIRKS R., KATZ B., and MILEDI R. (1960) Physiological and structural changes at the ~5p~~~ myoneural junction, in the course of nerve degeneration. J. Physiol. 150, B~LBRINGE., HOLMANM., and L~LLMANNH. (1956) Effect of calcium deficiency on striated muscle of the frog. J. Physiol. 133, 101-117. CASEJ. F. (1956) Spontaneous activity in denervated insect muscle. Science 124, 1079-1080. CASE J. F. (1957) The median nerves and cockroach spiracular function. J. Ins. Physiol. 1,85-94. CHOH-LUH LI G., MILTON SHY G., and WELLS J. (1957) Some properties of mammalian skeletal muscle fibres with particular reference to fibrillation potentials. 7. PhysioE. 135, 522-535. DEL CASTILLOJ., HOYLE G., and MACHNE X. (1953) Neuromuscular transmission in a locust. 3. Physiol. 121, 539-547. DEL CASTILLO J.and KATZ G. (1954) Changes in end-plate activity produced by presynaptic polarization. J, Physiol. 124, 586-604. EDWARDSG. A., RUSKAH., and DE HARVENE. (1958) Electron microscopy of peripheral nerves and neuromuscular junctions in the wasp leg. r. Biophys. Biochem. Cytol. 4, 107-l 14. FATT P. and KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. HOYLE G. (1955) Neuromuscular mechanisms of a locust skeletal muscle. Proc. joy. Sot. (B) 143, 343-367: HOYLE G. (1957) Nervous control of insect muscles. In Recent Advances in Invertebrate Physiology (Ed. by SCHEERB. T.). University of Oregon Press. LILLEY A. W. (1957) Spontaneous release of transmitter substance in multiquantal units. r, Physiol. 136, 595-605. ROEDERK. D. and WEIANT E. A. (1950) Th e electrical and mechanical events of neuromuscular transmission in the cockroach Periplaneta americana. J. exp. Biol. 27, l-13.
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USHERWOODP. N. R. (1961) Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. USHERWOODP. N. R. (1962) The nature of ‘slow’ and ‘fast’ contractions in the skeletal muscles of insects. Ph.D. Thesis, University of Glasgow. USHERWOOD P. N. R. (1963a) Response of insect muscle to denervation-I. Resting potential changes. J. Ins. Physiol. 9, 247-255. USHERWOOD P. N. R. (1963b) Spontaneous miniature potentials from insect muscle fibres. J. Physiol. In press. USHERWOOD P. N. R. To be published. VOSKRENSENSKAYA A. K. (1945) The investigation of functional properties of locomotor muscles of insects. Works of Pavlov’s Physiol. Inst., Acad. Sci. U.S.S.R. 1, 29-43.
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RESPONSE OF INSECT MUSCLE TO DENERVATION-II. CHANGES IN NEUROMUSCULAR TRANSMISSION P. N. R. USHERWOOD Department of Zoology,
University of Glasgow
(Received 11 May 1963) Abstract-The effects of motor-nerve section on neuromuscular transmission in the locust extensor tibiae preparation have been investigated by intracellular recording. Impulse transmission failed between the ninth and twenty-fourth post-operative days, in the region of the nerve-muscle synapse. Before impulse transmission failed, abnormally large depolarizing and hyperpolarizing responses to stimulation of the ‘slow’ axons, and prolonged depolarizing responses to the ‘fast’ axon were recorded from many muscle fibres. At the time of impulse transmission failure the discharge of spontaneous miniature post-synaptic potentials underwent a number of complicated change’s leading to the appearance of ‘giant’ miniature potentials up to 10 mV in height. The discharge persisted apparently for a few days after the muscle became neurally inexcitable. When the discharge finally stopped the fibres became permanently electrically silent ; no evidence was obtained for a resumption of miniature activity during the later post-operative days. The cessation of the miniature discharge is apparently correlated to the decline of muscle-resting potential observed in denervated insect muscle and the possibility that the transmitter, or a substance released along with it, maintains the resting potential of the muscle at its normal level is discussed. INTRODUCTION
TFIE possibility that the motor nerves innervating the metathoracic extensor tibiae muscle of the locust exert a ‘trophic’ influence on this muscle was suggested by USHERWOOD (1963a) on the basis of the results of experiments designed to study the effects of motor-nerve section on the resting potential (R.P.) of insect skeletal muscle. These results showed that during the first two post-operative days the R.P. of denervated muscle falls b&low normal; a temporary recovery occurs during the next 2-6 days, but this is soon followed by a second, irreversible fall in the R.P. to a level about 25 per cent below normal. More recently, studies have been made on the effects of denervation on neuromuscular transmission in the locust leg and the results of these studies are made the subject of this paper. METHODS The procedure for denervation (i.e. severance of the motor innervation at its points of origin from the ganglion) of the metathoracic extensor tibiae muscle of the locust, Scf&ocerca gregaria, was the same as that described by USHERWOOD (1963a). The motor axons to the extensor muscle, which are contained in metathoracic nerves 5 and 3b, were stimulated through silver-silver chloride electrodes. 811
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The electrical activity occurring in response to direct and indirect stimulation, the spontaneous miniature activity, and the R.P’s of the denervated and control muscles were recorded with glass, intracellular micro-electrodes, together with conventional amplifying and display equipment. Obviously, it was not possible to examine the same preparation more than once and since all the changes in neuromuscular transmission, which result from denervation, cannot be seen in any one preparation, it was necessary to examine a large number of different preparations (ca. 200) on a variety of post-operative days in order to ensure that no changes of significance were overlooked. RESULTS Effects of denervation on the response to nerve 5 In the locust extensor tibiae muscle, electrical stimulation of nerve 5 by a single supramaximal square pulse evokes a single action potential (Fig. la) composed of a distributed, ‘fast’ post-synaptic potential (‘fast’ p.s.p.) and graded electrically excitable or spike response (DEL CASTILLOet al., 1953). In some denervated muscles the magnitude of the ‘fast’ response was slightly lower than normal during the first 2-3 post-operative days, but this is to be expected since the R.P. of denervated muscle is also usually subnormal during this period (USHERWOOD,1963a). The first consistent change in indirect excitability occurred on about the third post-operative day. The repolarization phase of the ‘fast’ potential became increasingly prolonged (Fig. lb) and by about the sixth day the typical ‘fast’ response consisted of a large p.s.p. and a large spike followed by a pronounced plateau. Oscillatory potentials often appeared on the plateau, and at times up to five spike responses to a single indirect stimulus have been recorded. Less frequently, the peripheral portion of the severed crural nerve responded repetitively to a single applied shock and thereby elicited a series of ‘fast’ p.s.p’s and spikes in the extensor muscle fibres (Fig. lc). A reversal of the trend towards increased muscle excitability to neural stimulation was first recorded on the seventh post-operative day. The p.s.p’s started to get smaller and consequently the electrically excitable component also declined in magnitude (Fig. Id). Soon only diminished pure ‘fast’ p.s.p’s were evoked (Fig. le) and as the excitability of the muscle deteriorated even further, ‘fast’ p.s.p’s, similar in magnitude to the spontaneous miniature potentials recorded from normal locust muscle fibres (USHERWOOD, 1963b), were recorded in many denervated fibres (Fig. If). Eventually, all traces of responsiveness to the ‘fast’ axon were lost. The timing of onset of impulse transmission failure varied from preparation to preparation. In some denervated muscles it occurred as early as the ninth postoperative day, whilst other muscles remained responsive to neural stimulation until as late as the twenty-fourth day. There was a slight increase in the responsiveness of the control muscles to stimulation of nerve 5 from about the fourth post-operative day, but apart from this the responsiveness of these muscles was relatively normal, even in muscles examined 84 days post-operatively.
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It was still possible to excite the denervated muscle for about 10 days after impulse transmission had failed, simply by placing the stimulating electrodes directly on the surface of the muscle (Fig. lg). However, during this period only the fibres under the cathodal electrode responded to the applied stimuli, the number of fibres involved and the extent to which they contracted being partly dependent on the stimulus intensity. The electrical responses of the fibres under both cathodal and anodal electrodes, depolarizing under the cathode and hyperpolarizing under the anode, were graded in amplitude according to the stimulus intensity. In view of the vanishingly small intervals between the application of the stimuli and the appearance of the responses, together with the graded nature of the responses, it is suggested that the responses were in fact direct. Direct stimulation of normal insect muscle is not possible, probably because the nerve endings have a much lower threshold than that of the membrane of the muscle fibre (HOYLE, 1957) and because excitation via the distributed nerve endings is propagated so rapidly that the direct response is blocked through refractoriness (ROEDER and WEIANT, 1950). These obstacles are obviated by denervation since the excitatory influence of the motor innervation is then removed. The only other detailed electrophysiological investigations of the excitability of denervated insect muscle have been made by ROEDER ,and WEIANT (1950). They found that the abdominal muscle of the cockroach becomes electrically inexcitable to neural stimulation about 3-5 days after nerve section, but they were unable to obtain a response to direct stimulation with extracellular electrodes at any time before or after denervation. On the basis of their results, they suggested that cockroach skeletal muscle is either electrically inexcitable or, alternatively, that ‘the muscle surface has such a high electrical resistance that externally applied current fails to reach the depolarizable regions of the muscle fibres’. The first of their suggestions is clearly untenable, since cockroach muscle fibres are in fact electrically excitable, as shown by stimulation with intracellularly applied depolarizing current pulses (USHERWOOD,unpublished), though in a graded rather than all-or-nothing manner. The alternative suggestion is more plausible and could also be invoked to account for the decline and eventual loss of direct excitability of the denervated locust muscle fibre to externally applied stimuli which occurs about 10 days after impulse transmission has failed. The fibres still respond to intracellularly applied stimuli at this time (Fig. lh) but they are considerably atrophied and this in itself could lead to the abnormally high resistance of the muscle surface postulated to account for the ineffectiveness of external stimuli. Apart from these considerations, perhaps one of the more significant features of these results is that denervated locust muscle is electrically excitable for a considerable time after transmission of impulses from the ‘fast’ motor axon has failed. Since the isolated peripheral portion of the crural nerve is often still able to conduct impulses during this period, it follows that the site of impulse transmission failure must be in the region of the nerve-muscle junction. A more detailed examination of the critical changes leading up to impulse transmission failure has been made possible by virtue of the pinnate arrangement
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of the fibres in the extensor muscle. The distal fibres in this muscle are separated from the proximal fibres by a distance of about 2 cm and there is usually a short delay of about 1-3 hr between the onset of indirect inexcitability in the distal fibres and its appearance in the proximal fibres. The distal fibres were not invariably affected first; in some denervated muscles the proximal fibres were involved first, whilst in others the first change in responsiveness to neural stimulation was recorded from fibres in the middle. In denervated muscles exposed at the critical transitional stage there was usually a gradient of responsiveness from the inexcitable to the excitable regions of the muscles, i.e. it was possible to record ‘fast’ electrical responses ranging from about 70 mV to less than O-5 mV according to the position in the muscle of the fibre in which the recording electrode was impaled. The excitable fibres often deteriorated quickly during the experiment, especially if they were stimulated intermittently through nerve 5. In one preparation, in which initially about 70 per cent of the fibres were responsive to stimulation of nerve 5, transmission to the remaining, indirectly excitable fibres deteriorated so rapidly that within the space of about 2 hr the muscle became completely inexcitable to neural stimulation. This indicates that the complete process of impulse transmission breakdown apparently takes no more than a few hours. However, the possibility that exposure of the preparation for examination accelerates or even triggers the change-over from indirect excitability to inexcitability cannot be ruled out. Effect of denervation on the response to nerve 3b Nerve 3b supplied the extensor muscle with three motor axons (USHERWOOD, 1962), most of the fibres innervated by these axons being located in a bundle at the proximal end of the muscle. The electrical responses of the innervated fibres to stimulation of nerve 3b are extremely varied, ranging from hyperpolarizing potentials of just under 1 mV to depolarizing potentials of from 2-50 mV. HOYLE (1955) included all of these under the general heading of ‘slow’ responses. The ‘slow’ electrical responses of muscles denervated for 3-9 days were often noticeably larger, and at times more prolonged, than normal. ,+bnormally large ‘slow’ responses were also recorded from fibres of the contralateral muscle examined at this time. The depolarizing ‘slow’ potentials often reach a magnitude of 65 mV and frequently overshot the zero membrane potential (Fig. 2a, b) ; overshooting ‘slow’ potentials have never been recorded from muscle fibres of normal locusts (HOYLE, 1955). The apparent increased excitability of the denervated muscles to nerve 3b was sometimes clearly due to repetitive firing of the ‘slow’ nerve, resulting in the appearance of a series of summated depolarizing ‘slow’ p.s.p’s. Usually, however, a single shock applied to the peripheral portion of nerve 3b set up only a single nerve action potential, although the electrical response of the extensor muscle was still enhanced. It is probable, therefore, that denervation leads to changes in the membrane properties of the extensor muscle fibres and that these changes are reflected in the apparent increased excitability to neural stimulation.
FIG. 1. Effect of denervation on response of extensor tibiae muscle to nerve 5. (a) Intracellularly recorded 'fast' response from a n o r m a l fibre. Records (b-f) illustrate typical fast response recorded (b) 5, (c) 6, (e) 8, (f) 9 days after denervation. Note plateau following spike in record (b), evidence of neural repetition in record (c), and step-wise fluctuations in amplitude of diminished 'fast' p.s.p's in record (f). Record (g) illustrates response of a neurally inexcitable fibre to stimulation with external electrodes 12 days after denervation. Record (h) illustrates response of a neurally inexcitable fibre to intracellular stimulation with depolarizing current pulses (upper trace) of graded intensity 20 days after denervation. T h i s fibre did n o t respond at this time to externally applied current pulses. Voltage calibration same for records (a), (b), (c), and (g), and for records (d) and (e). T i m e calibration same for records (a) a n d (b) and for records (c), (d), and (e).
FIC. 2. E n h a n c e d depolarizing responses to stimulation of nerve 3b. (a, b) Intracellular records of overshooting, depolarizing potentials in response to maximal stimulation of nerve 3b. These responses were obtained from fibres of a muscle denervated for 6 days. Note double nature of p.s.p, in record (a). (c) Response obtained to nerve 3b from a fibre denervated for 5 days. T h i s was the largest depolarizing 'slow' response recorded in the present experiments. Voltage calibration same for all 3 records. T i m e calibration same for records (a) and (b).
FIG. 3. Enhanced hyperpolarizing responses to stimulation of nerve 3b recorded from (a-d) denervated proximal extensor tibiae muscle fibres, 2-9 days after section of both nerves 3b and 5 and (e, f) partially denervated proximal fibres, 53 days after section of nerve 5 only. Voltage calibration same for all records.
FIG. 4. A, Attenuation of depolarizing 'slow' p.s.p's by hyperpolarizing 'slow' p.s.p's in denervated proximal fibres of eight different extensor tibiae muscles, 2-12 days after nerve section. All eight fibres were innervated by the hyperpolarizing and depolarizing axons. (a-d) The hyperpolarizing axon had the lower threshold. Each record illustrates the response to stimulation of the hyperpolarizing axon alone (i) and also the response to stimulation of the hyperpolarizing and depolarizing axons together (ii).
Fro. 4. B, (a-d) T h e depolarizing axon had the lower threshold. Each record illustrates response to stimulation of depolarizing axon alone (i) and also the response to simultaneous stimulation of both 'slow' axons (ii). Voltage calibration same for all records except (c).
FIG. 5. Facilitation and summation of hyperpolarizing potentials in a fibre (R.P. 58 mV) denervated for 6 days. T h e hyperpolarizing axon was stimulated at a frequency of (a) 5/sec, (b) 20/sec, (c) 30/sec, and (d) 50/sec. T i m e and voltage calibrations were the same for all records.
FIG. 6. Changes in pattern of spontaneous miniature activity after denervation (motor-nerve section). (a, b) Miniature potentials recorded from a normal fibre. (c, d) Potentials recorded from a fibre denervated for 14 days. Note normal appearance Of individual potentials but tendency for discharge to occur in bursts and for potentials in bursts to summate. (e, f) 'Giant' miniature potentials recorded from a fibre denervated for 14 days. Note incomplete summation of the potentials to give irregularly shaped compound responses. (g, h) 'Giant' miniature potentials recorded from a muscle fibre denervated for 16 days. Summation is complete and the compound nature of the potentials is no longer obvious. Note longer duration of 'giant' potentials compared with normal. Voltage calibration applies to all records. Time calibration same for (a), (c), (e), and (g) and for (b) and (d).
FIC. 8. (a-c) Small oscillatory potentials recorded from a neurally inexcitable fibre of a muscle denervated for 18 days, (a) 3 min, (b) 6 min, and (c) 13 min after the fibre had started contracting spontaneously. (d-f) Much larger oscillatory potentials recorded from a neurally inexcitable fibre of muscle denervated for 16 days, (d) 1 min, (e) 3 rain, and (f) 7 min after the first signs of spontaneous activity appeared in the fibre. Voltage calibrations same for (a-c) and for (d-f). Time calibration same for (e-f).
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Hyperpolarizing ‘slow’ p.s.p’s, up to 10 mV in magnitude, were often recorded from both denervated and control muscles during the third to seventh postoperative days (Figs. 3 and 4). These responses facilitated and summated as the stimulus frequency was increased (Fig. 5). As in muscles of unoperated locusts (HOYLE, 1955), the largest responses were usually recorded from fibres with relatively low R.P’s. It is accepted that muscle fibres with subnormal R.P’s would probably give abnormally large responses to stimulation of the hyperpolarizing axon, but there is no evidence that the R.P’s are, at this time, any lower in fibres of operated locusts than they are in fibres of unoperated locusts (USHERWOOD, 1963a). Considering that relatively large hyperpolarizing potentials were recorded from fibres with R.P’s as high as 65 mV as well as from fibres with much lower R.P’s, that they occurred in fibres which at the same time gave abnormally large responses to stimulation of the depolarizing axon, and that they were recorded from fibres of the contralateral control muscles as well as from fibres of the denervated muscles, it appears unlikely that the magnitude of the R.P. is the overriding determining factor. However, any explanation for the enhanced responsiveness to either the hyperpolarizing axon or the depolarizing axon in the muscles of operated locusts, based on the very small amount of information obtained in the present investigations, is purely speculative. For any interpretation to be of value it must take into account the membrane properties of the denervated extensor muscle, and so far these have not been investigated. In any future investigations it would probably be of value not only to examine the effects of denervation on the membrane constants of the extensor muscle, but also to record the effects of the hyperpolarizing axon on the ‘slow’ mechanical response of this muscle, since in some of the denervated proximal fibres normally innervated by both depolarizing and hyperpolarizing axons, the depolarizing p.s.p’s were often attenuated by the large hyperpolarizing p.s.p’s (Fig. 4). In muscles of unoperated locusts, the hyperpolarizing p.s.p’s are very small and completely overridden by the responses to the depolarizing axon. As a result, stimulation of the hyperpolarizing axon has no effect on the ‘slow’ mechanical response (HOYLE, 1955). The response of the completely denervated muscle to nerve 3b often, but not invariably, survived the response to nerve 5. The reason for this is unknown, but it is of interest to note that BIRKS et aZ. (1960) f ound that in the denervated ‘mixed muscles of the frog the indirect excitability of the ‘slow’ fibres invariably survived the indirect excitability of the ‘fast’ fibres.
Effects of denervation on spontaneous miniature activity Spontaneous miniature potentials have been recorded from a variety of insect muscle preparations (USHERWOOD, 1961, 1963b) and have been compared with the miniature p.s.p’s recorded at vertebrate nerve-muscle junctions. The miniature potentials are postulated to results from a randomized release of quanta1 ‘packets’ of a chemical transmitter agent from the pre-synaptic nerve terminals (e.g. FATT and KATZ, 1952). Those recorded from the locust extensor tibiae muscle have
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amplitudes ranging from 0.1-2.0 mV and appear at frequencies ranging from 0*2/set-15/set (USHERWOOD, 1963b). In the present investigations only the overall changes in spontaneous miniature activity of the extensor muscle which result from denervation have been noted. It is unlikely that a quantitative examination would add very much more in the way of new information, but it would, however, be extremely time-consuming in view of the large natural variations in the spontaneous activity of even adjacent fibres in a muscle. There was little change in the spontaneous miniature activity of the denervated muscle during the first few post-operative days, but at some time between the seventh and thirty-second days a number of complicated changes occurred, before the activity finally ceased completely (Fig. 6). The first change in the general pattern of activity was indicated by an increase in the discharge frequency and a tendency for the miniature potentials to appear in bursts interposed between periods of relative inactivity (Fig. 6d). Each burst typically contained a large number of potentials, which, on occasion, summated to give a single large ‘giant’ response up to 10 mV in magnitude and often four times the duration of the normal miniature potential (Fig. 6g, h). S ometimes, especially during the ealier stages, the summation, process was incomplete and then the compound nature of these abnormally large responses was clearly apparent (Fig. 6e, f). The appearance of the ‘giant’ potentials coincided with an overall reduction in the level of activity, which steadily continued thereafter until eventually the miniature potentials disappeared completely and the muscle fibres became electrically silent. (Fibres were considered to be electrically silent if no miniature potentials were recorded from them for a period of about 10 min after impalement.) In a few of the fibres examined just before they became electrically silent the miniature activity consisted mainly of the large ‘giant’ potentials and hardly any miniature responses of normal amplitude were recorded. No attempt was made to determine whether transmission breakdown occurred simultaneously at all the junctions on any one muscle fibre or, alternatively, if some of the junctions became non-functional before others. The timing of the onset of complete transmission failure varied considerably. In one muscle it occurred as early as the twenty-first post-operative day, whereas miniature activity was recorded from other denervated muscles as late as the thirtysecond post-operative day. It has already been shown that impulse transmission usually fails much earlier than this. It is probable, therefore, that the spontaneous miniature activity survives for a considerable time after the muscle becomes indirectly inexcitable. Evidence contrary to this and in favour of a more rapid disappearance of the miniature potentials comes mainly from denervated muscles which were at the critical stage of transmission failure when first exposed. In these muscles, impulse transmission failed during the experiment and the miniature activity survived for only about 1 hr afterwards. The disappearance of the miniature potentials was accelerated by stimulating the preparation just before the muscle fibres became completely inexcitable to indirect stimulation and it was often found that under these conditions disappearance of the miniature potentials almost
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coincided with the breakdown of impulse transmission. It is probable, therefore, that whereas in unexposed denervated muscles the miniature activity persists for some days after impulse transmission has failed, exposure of the muscle in some way accelerates the decline in miniature activity so that the fibres become electrically silent at the time of impulse transmission failure. BIRKS et al. (1960) showed that in denervated frog muscle the miniature activity ceases about 3-4 days after denervation, only to be resumed, in a modified form, about 1 week later. There is at present no evidence for a resumption of miniature activity in the denervated locust muscle fibre; even preparations examined 84 days post-operatively were completely inactive, but there are certain resemblances between the changes of miniature activity in denervated locust and frog muscles. ‘Giant’ miniature potentials similar in magnitude to those recorded from indirectly inexcitable denervated locust muscle fibres have been recorded at denervated frog end-plates (BIRKS et al., 1960). It is important to note, however, that ‘giant’ miniature potentials are also recorded from normal vertebrate end-plates (LILEY, 1957) although presumably they occur more frequently at denervated end-plates. The histogram for the frequency distribution of amplitudes of the miniature potentials recorded from denervated frog end-plates during the period of renewed activity is markedly asymmetrical and has a much wider dispersion than the approximately ‘Gaussian’ type of histogram for the miniature activity at normal end-plates (BIRKS et al., 1960). Fig. 7a illustrates that the histogram for frequency distribution of amplitudes of the miniature potentials recorded from a denervated locust muscle fibre, just after impulse transmission had failed, is also very asymmetrical and has a wide dispersion. Unfortunately, any direct comparison with frog ‘fast’ muscle fibres is made difficult by the fact that even the histogram of frequency distribution of amplitudes of the miniature potentials recorded from a normal locust fibre is positively skewed (Fig. 7b). This is attributed to the distributed innervation of the locust muscle fibre (USHERWOOD, 196313). However, there is little doubt that the histogram for the denervated locust muscle fibre which has just become indirectly inexcitable is more asymmetrical and has a much wider dispersion than the histogram for the normal fibre. Spontaneous mechanical activity Spontaneous contractions have been recorded from denervated insect muscles on a number of occasions. CASE (1956, 1957) noted spontaneous mechanical activity in the femoral muscles of the cockroach 4-6 days after motor-nerve section and BEFG~NEK and NOVOTN.~; (1958) recorded spontaneous mechanical and electrical activity in the denervated femoral muscles of this insect. In the present experiments spontaneous contractions of the denervated extensor tibiae preparation of Schistocerca occurred only rarely. In fact, activity of the type described by CASE (1956) was recorded in only seven out of a total of over 200 denervated muscles examined during these investigations. Even in these muscles the spontaneous activity, which appeared soon after exposure of the muscle, persisted for no longer than 15 min. The denervated and contralateral
818
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Amplitude,
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0
I
Amplitude,
mV
FIG. 7. {a) Frequency distribution of amplitudes of spontaneous miniature potentials recorded 17 days after nerve-section from a denervated extensor tibiae muscle fibre just after impulse transmission from the motor nerve stump had failed. (b) Amplitude distribution of spontaneous potentials recorded from a fibre of the innervated contralateral control muscle. (See text for furtber explanation.)
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control legs were always examined before the extensor muscles were exposed, to check for the possibility of spontaneous contractions of the intact muscle: it was assumed that any contractions of the extensor muscles would lead to movements of the respective tibia1 segments. However, rhythmical twitches similar to those seen in the extremities of the denervated locust leg as early as the first day after denervation (VOSKRENSENSKAYA, 1945) were never seen at any time in the present experiments. If spontaneous contractions did occur in the unexposed, denervated muscles, then they must have been too weak to move the extremities of the legs. The contractions seen in exposed denervated muscles were usually restricted to fibres which were already indirectly inexcitable and usually involved only a single fibre or a bundle of fibres; on no occasion was the entire muscle seen to contract spontaneously. On the two occasions that the mechanical activity persisted for some time after exposure of the muscles it was possible to record accurately, with intracellular electrodes, the electrical activity of the contracting fibres. The activity consisted of oscillations of the membrane potential similar to those recorded from fibrillating vertebrate muscle fibres (e.g. B~~LBRINGet al., 1956; CHOH-LUH et al., 1957). Apparently the oscillations occurred in phase with the contractions. In one fibre rhythmical, alternating slow and fast, oscillations were readily distinguished throughout the period of the contractions, although at times the fast oscillation was complicated by a step on its rising phase (Fig. 8a-c). The oscillations in this fibre never depolarized the membrane by more than about 8 mV from its resting value (about 45 mV). The activity in the other fibre (Fig. Sd, e) consisted initially of a very complicated series of rhythmical oscillations which depolarized the membrane by as much as 40 mV (R.P. ca. 58 mV), but the form of the oscillations soon became much simpler and it was then possible to recognize alternating slow and fast potentials as before. In both fibres the fast potential apparently occurred when the slow potential reached a critical level of depolarization, a level which was relatively constant for each of the fibres. In a few spontaneously active fibres slow potentials were recorded in the absence of fast potentials. Fast potentials were absent presumably because the slow potentials failed to depolarize the membrane potential to its critical level. The contractions of fibres which showed only slow oscillations were apparently weaker than those which showed both fast and slow oscillations. In all the spontaneously active fibres the frequency and amplitude of the potentials declined gradually during the experiment before the oscillations finally disappeared completely and the membrane potential stabilized, but over short periods the frequency and amplitude of the potentials were relatively constant (Fig. 8). A second type of spontaneous mechanical activity, which was recorded from about ten denervated and two control muscles, consisted of much slower rhythmical contractions of the extensor muscle. This activity was usually restricted to the proximal bundle of muscle fibres and each contraction often took more than 1 min. The membrane potentials of the contracting fibres apparently oscillated slowly, falling to peak depolarizations as great as 40 mV, in phase with the
820
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contractions, but fast potentials were absent. However, the possibility that these oscillations were in fact nothing more than mechanical artifacts cannot be ruled out, since the contractions, although very slow, were nevertheless rather vigorous at times. In this respect it is considered rather surprising that they were never ‘signalled’ in unexposed muscles by movements of the tibia1 segments. Perhaps they only occur on exposure of the muscle due to some form of interaction between a hyper-excitable muscle membrane and the locust saline. A similar explanation could also be invoked for the much faster contractions described earlier, since these too were never recorded from unexposed muscles. Response to partial denervation Part of the extensor muscle is innervated by branches of both nerves 5 and 3b and in the first paper of this series partial denervation, i.e. section of only one of the motor nerves, was shown to have no effect on the R.P. of the dually innervated fibres. Experiments to determine the effects of partial denervation on the responsiveness of these fibres to indirect stimulation have shown that section of either nerve 5 or 3b does not reduce the response of the extensor muscle to the motor nerve still connected to the metathoracic ganglion. In fact, the response to the intact nerve is increased slightly and in the present experiments the increased responsiveness to the intact axon persisted long after all traces of responsiveness to the sectioned axon had disappeared. The neurally excited responses to stimulation of the cut axon underwent the same sequence of changes outlined for the completely denervated muscle. When fibres of partially denervated muscles, which were originally innervated by both ‘fast’ and ‘slow’ axons, were examined just after impulse transmission from the severed axon had failed, they showed a characteristically mixed pattern of miniature activity. ‘Giant’ potentials were often seen as in the completely denervated fibres but these later disappeared and then the activity reverted to the pattern seen in normal fibres. Presumably, partial denervation leads to degenerative changes at the endings of the severed axon whilst the endings of the intact axon are unaffected by the operation. Correlation between neuromuscular traKcmission and resting potential in denervated muscle The possibility that the changes in transmission which result from motornerve section might be causally correlated to the changes of muscle R.P. became increasingly evident during the course of these investigations. To test this possibility the effects of denervation on the indirect excitability, R.P., and spontaneous miniature activity of the extensor muscle have been examined simultaneously in a number of different preparations on different post-operative days and in particular during the critical period of impulse transmission failure. The recording procedure consisted of inserting a microelectrode into a fibre of the denervated muscle to and recording the R.P., miniature activity, and electrical responsiveness nerve 5, in that order. The procedure was then,repeated for a fibre of the control muscle. About 20-50 fibres were examined in each muscle and the results from
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each pair of muscles were collated. As before, no attempt was made to examine the miniature potentials of the different fibres quantitatively; only the gross changes in activity were noted. The changes in R.P. and indirect excitability after denervation are illustrated graphically in Fig. 9. It is apparent from the graph that the second, irreversible fall in the R.P. of the denervated muscle usually occurred on the same post-operative day that impulse transmission failed, although in a few preparations there
100
SO
60
40
20
0
FIG. 9. Graph illustrating correlation between resting potential and neurally excited responses after denervation. Ordinates, mean R.P. (0) (‘fast’ electrical response (0)) of denervated muscle as a percentage of mean R.P. (‘fast’ electrical response) of contralateral control muscle. Abscissae, number of post-operative days. were indications
of a slight delay between impulse transmission failure and R.P. decline. Detailed analysis of the correlation between R.P. and impulse transmission in muscles, in which impulse transmission failed during examination, gave a much clearer picture of the timing of the various changes. In these muscles there was often a delay of about 3 hr from the time impulse transmission failed before the R.P. started to fall. The close correlation between miniature activity
a22
P. N. R. USHERWOOD
and R.P. was most clearly illustrated in one denervated muscle, which was completely inexcitable when first exposed. It was still possible to record miniature potentials from the proximal fibres and the mean R.P. of these fibres was about 20 per cent higher than the mean R.P. of the distal fibres, which were electrically silent, and only slightly lower (about 5 per cent) than the mean R.P. of the contralateral control muscle. At least in some denervated muscles, therefore, the decline in R.P. is correlated not with impulse transmission failure, but with the cessation of spontaneous miniature activity. The possibility of a correlation between the type of spontaneous miniature activity recorded from a denervated muscle fibre and the responsiveness of the fibre to neural stimulation was also investigated. In completely denervated muscles (i.e. muscles in which the central connexions of both nerves 3b and 5 were severed), which contained both neurally excitable and neurally inexcitable fibres when first exposed, ‘giant’ miniature potentials were often recorded from the inexcitable fibres but were never recorded from the fibres which were still neurally excitable. It is probable, therefore, that the appearance of these ‘giant’ compound miniature potentials coincides with the failure of impulse transmission. DISCUSSION There is little doubt that denervation of the locust extensor tibiae muscle results in the failure of impulse transmission presumably before any gross structural changes occur (USHERWOOD, to be published), but the exact site at which transmission first breaks down is unknown. The fact that the isolated peripheral portions of the severed motor nerves to the extensor muscle are still able to conduct impulses and that the muscle is still electrically excitable after transmission has failed indicates that the breakdown is in the region of the nerve-muscle junction. The survival of the spontaneous miniature activity for a considerable time after impulse transmission fails precludes the possibility that the breakdown results initially from a depletion of transmitter agent from the pre-synaptic nerve terminals, where it is presumably stored before release, and also rules out the possibility of some form of blockage at the post-synaptic receptor regions on the muscle fibre. Perhaps the mechanism which couples the excitation of the nerve terminals by the invading nerve impulse to the release of the transmitter, responsible for setting up the p.s.p., is impaired as a result of denervation. DEL CASTILLO and KATZ (1954) have shown that the frequency of transmitter discharge from the peripheral endings of the frog motor nerve is coupled to the magnitude of the potential differences across the axonal membrane at the pre-synaptic terminals. The arrival of the nerve impulse at the terminals, with the concomitant reduction of the membrane potential of that region, presumably results in a dramatic shortterm increase in the frequency of transmitter release. It follows, therefore, that although a breakdown in the mechanism which links the level of membrane potential at the terminals to the discharge frequency would lead to impulse transmission failure, it would not necessarily stop the spontaneous release of transmitter from the terminals. If such a breakdown did occur, subsequent depolarizations of
RESPONSE OF INSECT MUSCLE TO DENERVATION-II
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the nerve endings either by applied current or by high external potassium should, according to the hypothesis, have no effect on the level of the miniature activity. The hypothesis has been crudely tested in the present experiments by increasing the potassium concentration of the saline bathing a denervated locust muscle fibre, which had just become indirectly inexcitable, to two to three times its normal value (HOYLE, 1955). Significantly, this change had no effect on the mean frequency of the miniature potentials of the denervated fibre, although the mean frequency of the miniature potentials recorded from a normal locust muscle fibre is markedly raised by an equivalent increase in the external potassium concentration (USHERWOOD, 1963b). It is perhaps of interest to note that the ‘resumed miniature activity at the denervated frog end-plate is similarly insensitive to the external potassium concentration (BIRKS et al., 1960). The gradual decline of the miniature activity to zero, which occurs after impulse transmission has failed, is explicable on the basis of transmitter depletion from the nerve terminals. There are a number of possible sites for the production of transmitter in the intact nerve cell. The site of production could be in the soma of the motor neurone, the transmitter being transported to the nerve terminals along the axon. Alternatively, the transmitter could be produced at sites situated either throughout the length of the axon or even restricted to the terminal regions of the axon. Whichever system operates, section of the ‘motor nerve and, therefore, isolation of the axon from the metabolic control centre, the cell body, would eventually curtail any further renewal of transmitter in the terminals. The fact that transmission does not fail for some time after nerve-section implies that either the production of transmitter persists for some time after nerve-section or that there is sufficient transmitter stored in the axon and its terminals to allow transmission to continue for some time. Concomitant with the decline of the miniature activity of the denervated muscle fibre, a second change leading to the appearance of ‘giant’ miniature potentials takes place. The present results indicate that a ‘giant’ potential results from summation of a large number of smaller miniature potentials. The requirement for complete summation would be met by the synchronous release of a large number of quanta of transmitter from the nerve terminals and their attachment as a multiple unit to the receptor sites of the post-synaptic membrane. Perhaps the quanta coalesce to form multiple units before they are released from the nerve terminals and as this process becomes more intimate, so attachment at the receptor sites becomes more synchronized and summation of the miniature p.s.p’s more complete. A release of transmitter agent in multi-quanta1 units has been invoked previously to account for the ‘giant’ miniature p.s.p’s recorded from the isolated rat diaphragm muscle (LILLEY, 1957), ‘since these potentials occurred more frequently than would be expected from random coincidences of miniature potentials. It would undoubtedly be of interest to know exactly what happens to the arrangement of the vesicles, which are found in the nerve terminals of the locust, after denervation, since they have been postulated as possible storage sites for the transmitter.
824
P. N. R. USHERWOOD
The results of the present experiments lend further support to the possibility that the motor nerves innervating the locust extensor tibiae muscle exert a ‘trophic’ influence on this muscle, and at the same time give some indication of how this influence is exerted. For example, the second, irreversible fall in the R.P. of the extensor muscle has been shown to more or less coincide with transmission failure at the neuromuscular junction, thus suggesting a possible causal relationship between the two events. Recent electron micrographs of insect nerve-muscle junctions have clearly demonstrated the existence of a gap between the nerve terminals and the post-synaptic membrane of the muscle fibre (EDWARDS et al., 1958). This implies that there is no protoplasmic connexion between the axon and the muscle fibre and we are left with the possibility that the transmitter agent or some neurohumoral substance released along with it is responsible for maintaining the R.P. of the muscle fibre at its normal level. This hypothesis is supported by the fact that the R.P. presumably does not start to fall until after the release of transmitter from the nerve terminals has stopped completely. Acknowledgements-The author wishes to thank Professor G. HOYLE for his helpful comments on the manuscript. REFERENCES BE~~NEKR. and NOVOTNPI. (1958) Spontaneous electrical activity in the denervated muscles of the cockroach Periplaneta ame-ricana. Nature, Lond. 182, 957-958. BIRKS R., KATZ B., and MILEDI R. (1960) Physiological and structural changes at the ~5p~~~ myoneural junction, in the course of nerve degeneration. J. Physiol. 150, B~LBRINGE., HOLMANM., and L~LLMANNH. (1956) Effect of calcium deficiency on striated muscle of the frog. J. Physiol. 133, 101-117. CASEJ. F. (1956) Spontaneous activity in denervated insect muscle. Science 124, 1079-1080. CASE J. F. (1957) The median nerves and cockroach spiracular function. J. Ins. Physiol. 1,85-94. CHOH-LUH LI G., MILTON SHY G., and WELLS J. (1957) Some properties of mammalian skeletal muscle fibres with particular reference to fibrillation potentials. 7. PhysioE. 135, 522-535. DEL CASTILLOJ., HOYLE G., and MACHNE X. (1953) Neuromuscular transmission in a locust. 3. Physiol. 121, 539-547. DEL CASTILLO J.and KATZ G. (1954) Changes in end-plate activity produced by presynaptic polarization. J, Physiol. 124, 586-604. EDWARDSG. A., RUSKAH., and DE HARVENE. (1958) Electron microscopy of peripheral nerves and neuromuscular junctions in the wasp leg. r. Biophys. Biochem. Cytol. 4, 107-l 14. FATT P. and KATZ B. (1952) Spontaneous subthreshold activity at motor nerve endings. J. Physiol. 117, 109-128. HOYLE G. (1955) Neuromuscular mechanisms of a locust skeletal muscle. Proc. joy. Sot. (B) 143, 343-367: HOYLE G. (1957) Nervous control of insect muscles. In Recent Advances in Invertebrate Physiology (Ed. by SCHEERB. T.). University of Oregon Press. LILLEY A. W. (1957) Spontaneous release of transmitter substance in multiquantal units. r, Physiol. 136, 595-605. ROEDERK. D. and WEIANT E. A. (1950) Th e electrical and mechanical events of neuromuscular transmission in the cockroach Periplaneta americana. J. exp. Biol. 27, l-13.
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USHERWOODP. N. R. (1961) Spontaneous miniature potentials from insect muscle fibres. Nature, Lond. 191, 814-815. USHERWOODP. N. R. (1962) The nature of ‘slow’ and ‘fast’ contractions in the skeletal muscles of insects. Ph.D. Thesis, University of Glasgow. USHERWOOD P. N. R. (1963a) Response of insect muscle to denervation-I. Resting potential changes. J. Ins. Physiol. 9, 247-255. USHERWOOD P. N. R. (1963b) Spontaneous miniature potentials from insect muscle fibres. J. Physiol. In press. USHERWOOD P. N. R. To be published. VOSKRENSENSKAYA A. K. (1945) The investigation of functional properties of locomotor muscles of insects. Works of Pavlov’s Physiol. Inst., Acad. Sci. U.S.S.R. 1, 29-43.
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