J. Physiol. (1978), 285, pp. 113-128 With 6 text-figure8 Printed in Great Britain

113

TRANSMITTER RELEASE FROM NORMAL AND DEGENERATING LOCUST MOTOR NERVE TERMINALS

BY J. P. HODGKISS* AND P. N. R. USHERWOOD From the Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD (Received 30 November 1977) SUMMARY

1. An analysis has been made of spontaneous and evoked transmitter release from terminals of 'fast' excitatory motor axons on locust muscle fibres using intra- and extracellular recording together with a Ca-electrode technique for activating transmitter release from single nerve terminals on multiterminally innervated muscle fibres. 2. Spontaneous intracellular miniature excitatory junction potentials (m.e.j.p.s), recorded at active spots on these muscle fibres, occurred non-randomly with frequent bursts of m.e.j.p.s. 3. M.e.j.p.s of subnormal amplitude were also seen but contributed only a small fraction of the minature discharge. 4. The amplitude distribution of intracellularly recorded excitatory junction potentials (e.j.p.s) evoked during ionophoretic application of Ca onto single nerve terminals was adequately predicted by Poisson statistics. 5. During the course of nerve terminal degeneration m.e.j.p.s of subnormal amplitude became more frequent and eventually formed the major part of the miniature discharge. Transmitter quanta responsible for 'small' m.e.j.p.s did not contribute to evoked release either at normal or degenerating terminals. Evoked transmitter release from degenerating axon terminals before excitation-secretion coupling failure conformed to Poisson statistics. 6. It is concluded that more than one release mechanism operates on the transmitter pool or pools in locust motor nerve terminals. INTRODUCTION

Recent studies of spontaneous transmitter release from locust and cockroach motorneurones (Usherwood, 1972, 1973; Rees, 1974; Washio & Inouye, 1975) have suggested that it does not conform to the Poison process. The bursts of miniature potentials that occurat synapses on skeletal muscles of these insects suggest that limited interaction takes place between the release of one transmitter quantum and the next. In view of the hypothesis that evoked transmitter release is a transient acceleration of the spontaneous mode of release (Katz, 1969; Hubbard, 1970) it became of interest to see whether evoked transmitter release at the insect nerve-muscle junction is non-Poisson. A recent attempt to answer this question was largely inconclusive * Present address: Department of Pharmacology, University of Aberdeen, Aberdeen AB9 2ZD.

J. P. HODGKISS AND P. N. R. USHER WOOD because of reservations in the interpretation of the extracellularly-recorded data (Usherwood, 1972). The multiterminal innervation of insect muscle has hitherto precluded the use of intracellular recording techniques for accurate analyses of evoked transmitter release. However, in the studies described in this paper use has been made of the Ca-electrode technique (Katz & Miledi, 1965a, b, c) by means of which evoked transmitter release can be restricted to a single nerve terminal on a muscle fibre. This has been used in combination with intra- and extracellular recording techniques to study the transmitter release from normal and degenerating locust neuromuscular 114

junctions. METHODS

The experiments were made on the phasicc' muscle fibres (Usherwood, 1967, 1969; Cochrane, Elder & Usherwood, 1972) of the metathoracic extensor tibiae nerve-muscle preparation of the locust Schi8tocerca gregaria (Hoyle, 1955). The metathoracic leg was detached from the animal at the joint between the coxa and the thorax and placed in a perspex bath of 2 ml. replaceable volume to which it was secured by means of Takiwax (Usherwood & Machili, 1968). Tfe nerve-muscle preparation was exposed and equilibrated for 0.5-1 hr in locust saline of standard composition (mM): NaCi (170); KCl (10); CaCl2 (2); HEPES buffer (10); adjusted to pH 6X8. The extensor tibiae motor nerve which runs along the ventral surface of the muscle was cut at the proximal end of the muscle and sucked into the fine tip of a plastic suction electrode. The bathing solution was then exchanged for one similar to the above but containing 20-30 mm-Mg, isomotically substituted for Na, and a reduced Ca concentration (0-5-1 mM). Miniature excitatory post-synaptic potentials (min e.p.s.p.s) and excitatory post-synaptic potentials (e.p.s.p.s) were recorded intracellularly with micro-electrodes filled with 2 M-K citrate and having a resistance, measured in locust saline, of 8-15 MCI. Transmitter release from single nerve terminals was monitored extracellularly at active spots on the surface of the muscle fibres (del Castillo & Katz, 1956) by micro-electrodes filled with 2 M-NaCl, at pH 6-8, having resistances in the range 2-8 MO. Those intracellular min e.p.s.p.s accompanied by an extracellular potential are referred to in the text as m.e.j.p.s. M.e.j.p.s are identical to the 'marked' intracellular min e.p.s.p.s described by Tsherwood (1972). The Ca-electrode technique of Katz & Miledi (1965a, b, c) was used to investigate evoked transmitter release from single nerve terminals. Twin-barrel Na-Ca electrodes were used for this purpose. A barrel filled with 2 m-NaCl was used to record extracellularly from the nerve-muscle junction whilst the other barrel, filled with 0 3-0 5 M-CaCl2, was used to apply Ca ions to the nerve terminal in a controlled manner. Electrodes with barrel resistances of 5-20 MQL proved the most suitable. Active spots on the surface of the muscle fibres were located by trial and error (Usherwood, 1972). After location of such a spot the bathing solution was exchanged for one containing 5-10 mm-Mg but no added Ca. (The mean amplitude of the spontaneous min e.p.s.p.s was reduced by only about 10 % by this treatment.) This abolished evoked transmitter release within 10-20 min. It was possible to restore evoked transmitter release at the single nerve terminal under the extracellular micro-electrode by passing a small anodal current through the Ca electrode. This was signalled by the appearance of e.p.s.p.s which, since they resulted from the release of transmitter from one nerve terminal only, we shall refer to as e.j.p.s. Intracellular and extracellular recordings of these events were displayed on an oscilloscope and photographed on moving film. The amplitudes of potentials were determined by measuring from the top of the base-line to the peak of the potential. E.j.p.s with amplitudes greater than 2-5 mV were individually corrected using Martin's equation (Martin, 1955). The equilibrium potential for transmitter action at excitatory junctions on the phasic muscle fibres is about 0 mV (Anwyl & Usherwood, 1974; Hodgkiss, 1976). The properties of nerve-muscle junctions at various times after motor nerve section were also studied. Nerves 5 and 3b, which supply axons to the extensor tibiae muscle (Hoyle, 1955), were transacted in the thorax at a point close to the ganglion and also at a point as close as possible

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 115 to the coxa. Further details of the operation are given in an earlier study of this preparation (lfsherwood, 1963 a). Operated animals were maintained at 30 'C. The survival rate was about 90 %. The m.e.j.p. frequency data were tested for conformity to a Poisson process by comparing the mean number of events (Nt) occurring in equal, consecutive, non-overlapping sample periods (t) with the variance (at). For a Poisson process, irrespective of the nature of t, Vtshould equal Nt (Hubbard, Llinas & Quastel, 1969; Hubbard & Jones, 1973; Washio & Inouye, 1975). Because of experimental limitations estimates of Vt contained successively fewer samples as t was increased and therefore were less reliable for high values of t. For this reason a comparison of Vt and N. was made only when Nt was less than 20 % of the total number of m.e.j.p.s in the sample (125-500 in the present experiments). The Poisson analysis developed by del Castillo & Katz (1954) was used for the distribution of e.j.p. amplitudes. An estimate of quantal size was obtained from the mean amplitude (q) of the m.e.j.p.s recorded intracellularly at an active spot during Ca ionophoresis. One estimate (nib) of the quantal content (m) of the e.j.p. was obtained from

Q

mb= -

q e.j.p. recorded at the same active spot as intracellular of the mean amplitude is the where Q the m.e.j.p.s and under identical conditions. The other estimate (me) of m was obtained using the method of failures (del Castillo & Katz, 1954), i.e. a modification of Poisson's theorem with m. = log.N/no to the motor nerve and no is the number of such of stimuli number applied N total is the where stimuli which failed to elicit a response (e.j.p.). We have assumed that failures of nerve-muscle transmission resulted from failures of excitation-secretion coupling rather than conduction failure in preterminal regions of the motor axon. The deep location and structural organization at locust nerve-muscle junctions prevented us from monitoring nerve terminal potentials. The observed e.j.p. amplitude distribution was compared with that theoretically predicted by Poisson's theorem. Since the amplitudes of m.e.j.p.s werafound to be approximately normally distributed the distribution of e.j.p. amplitudes should be given by the following equation: x -- exp (-m)mX 1

where q and 2 are respectively the mean and variance of the m.e.j.p. amplitude. Another property of the Poisson distribution is that the mean should equal its variance. This can be restated as (CV)2 = 1/m where C(V is the coefficient of variation (Martin, 1966). The simplest way to apply this test is to separate the e.j.p. amplitude distribution into discrete quantal classes following the method of del Castillo & Katz (1954) and Wernig (1975a) and compute CV for the quantal distribution. To determine how well the theoretical predictions fitted the observed quantal distribution of the e.j.p. a x2 statistic was computed for each comparison. The calculated statistic was tested for significance in a x2 table with 2 degrees of freedom less than the number of classes. If the calculated x2 statistic was less than the corresponding figure in the table at P = 0-05 then the null hypothesis was retained and no significant difference between the observed and Poisson distributions was assumed. RESULTS

Spontaneo transmitter release The data referred to in this paper were obtained from active spots on extensor tibiae muscle fibres where m.e.j.p.s occurred at frequencies of 0-01-0-05 sec-' in standard locust saline. At many active spots the m.e.j.p.s did not always occur randomly, short bursts sometimes signalled the start of a period of elevated

116 J. P. HODGKISS AND P. N. R. USHERWOOD spontaneous transmitter release lasting in some cases up to 1-5 min (Fig. 1 B). Variancemean (Vt-Nt) plots for such data recorded at seven active spots indicated that spontaneous release of transmitter was non-Poisson (Fig. 2). Six of the Vt-Nt plots exhibited a characteristic deviation from the relationship expected for a Poisson process, with a tendency for Vt > Nt. At only one active spot studied did the relationship conform closely to that expected for a Poisson process (Fig. 2, filled circles). At this particular active spot m.e.j.p. frequency exhibited only slight variation over the 600 sec time interval studied (Fig. 1A). At active spots on locust retractor A 03 0 0 02 0 0. _ me 0 *Low

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Fig. 1. Variation in m.e.j.p. frequency with time at two active spots on different extensor tibiae nerve-muscle preparations. At one active spot the miniature discharge did not contain bursts of m.e.j.p.s (A). However, at the other active spot there were periods of elevated m.e.j.p. frequency when bursting occurred, one of which is represented in (B) at 50 msec. Each point in A and B represents the mean m.e.j.p. frequency determined for a 10 s interval. Variance-mean plots for these data are presented in Fig. 2. ,

unguis (Usherwood, 1972) and cockroach leg muscle (Rees, 1974; Washio & Inouye, 1975) there was also a tendency for Vt to be greater than Nt. The results obtained in the present study therefore support the idea that spontaneous transmitter release at some locust and cockroach neuromuscular junctions is non-Poisson. Amplitudes of spontaneous m.e.j.p.s Amplitude distributions of intracellular m.e.j.p.s were approximately normal (Fig. 3). Whilst some m.e.j.p.s were several times larger than the modal amplitude the multicomponent nature of such potentials was clearly indicated by concomitant extracellular events. Such potentials were therefore not included in the analyses to determine quantum size nor represented in the m.e.j.p. amplitude histograms. In contrast Usherwood (1972) found that the amplitude distributions of 'marked' min e.p.s.p.s (m.e.j.p.s) recorded at locust retractor unguis neuromuscular junctions exhibited a positive skew.

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 117 Evoked release of transmitter It is possible to abolish evoked transmitter release from all nerve terminals by reducing the extracellular Ca concentration, and to selectively restore evoked transmitter release at a single nerve terminal under the Ca electrode. The records in Fig. 4A illustrate such an experiment. The preparation was exposed to a Ca-free saline whereupon no e.p.s.p.s were recorded when the extensor tibiae motor nerve

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NC Fig. 2. Variance (Vt)-mean (Nt) plots for spontaneous m.e.j.p.s recorded at seven active spots (each designated by a different symbol) on extensor tibiae muscles. The dashed lines shows the relationship excepted for a Poisson process. Two of the plots i.e. filled circles and open circles were made from data presented in Fig. 1 A and B respectively.

was stimulated (Fig. 4Aa). Application of a small anodal current (5 x 10-8 A) to the Ca electrode critically placed above a neuromuscular junction then resulted in e.j.p.s which were recorded intracellularly and extracellularly (Fig. 4Ab). During the Ca-electrode studies the extensor tibiae nerve was usually stimulated at 1 Hz. All nerve terminals studies in this manner responded within less than 1 second during Ca application. The e.j.p.s which resulted fluctuated in amplitude during repetitive stimulation of the extensor tibiae motor nerve with occasional failures of response (Fig. 4B). Increasing the magnitude of the Ca ejection current led to an increase in the mean e.j.p. amplitude together with fewer response failures (Fig. 4Ac, d). As the Ca ejection current was further increased (5 x 1O-7-1 X 106 A) the

118 J. P. HODOKISS AND P. N. R. USHER WOOD intracellular e.j.p. further increased in amplitude but fluctuated to a smaller extent than before, whereas the mean amplitude of the extracellular e.j.p. exhibited no further increase. Presumably under such conditions transmitter release was being activated at sites outside the recording distance of the extracellular electrode. Reducing the Ca ejection current to zero or imposing a small Ca retaining current led to abolition of evoked transmitter release within 1-2 sec. Al

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Fig. 3. Histograms of amplitude distributions of m.e.j.p.s (filled histograms A-C) and e.j.p.s (open histograms A-C) recorded at single active spots on normal extensor tibiae muscle fibres. The evoked responses to nerve stimulation (at 0-5-1-5 Hz) were obtained during ionophoretic application of Ca to the nerve-muscle junction. The preparations were bathed in saline containing 5-6 mM-Mg but no Ca. The theoretical Poisson amplitude distribution of the e.j.p. (continuous curves, A2, B2, C2 and C3) was determined from the mean and variance of the m.e.j.p. amplitude distribution. Note the good agreement between the e.j.p. amplitude distributions and the theoretical curves. The results ofX-square tests were: 0-98 > P > 0-95 (A. and A2),P> 0-99 (B1 and B2), 0-98 > P > 0-95 (C1 and C2), 0-9 > P > 0-8 (C1 and C3); m,, mb, quantal content of

e.j.p.; no obs, no

exp,

observed and expected number of response failures respectively.

Analysis of e.j .p. amplitudes indicated good agreement between the two independent estimates of e.j.p. quantal content over a range of e.j.p. quantal contents (Fig. 5A). If only extracellular m.e.j.p. and e.j.p. data are usedthere is very little agreement between these estimates (Fig. 5C). A plot of CV against mb also conformed to the

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 119 a _

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I

msec msec Fig. 4. M.e.j.p.s and e.j.p.s recorded at two active spots on different extensor tibiae muscle fibres during ionophoretic application of Ca to the nerve-muscle junction. A, effect of increasing the Ca ejecting current on the response to nerve stimulation at 0-5 Hz. Each trace in a to d represents a series of five to seven superimposed responses of the muscle fibre recorded intracellularly (upper records) and extracellularly (lower records). With zero Ca ejection current (a) no e.j.p.s occurred although m.e.j.p.s still occurred (not clearly shown in a). In b a small Ca ejection current (5 x 10-8 A) led to the appearance of e.j.p.s, although not in response to every nerve stimulus. When the calcium ejection current was further increased to 1 x 10-7(Ac) and then to 5 x 10-7 (Ad) there were fewer response failures. Note spontaneous miniature potentials recorded intracellularly and extracellularly in b and c. Calibration: intracellular, 0.5 mV, extracellular, 0-26 mV; time, 100 msec (a, b), 200 msec (c, d). B, e.j.p.s and spontaneous m.e.j .p.s recorded intracellularly (lower records a to g) and extracellularly (upper records a to g). The evoked responses to neural stimulation (at 1 Hz) were obtained during ionophoretic application of Ca to the nerve terminal using a constant current. The records show that evoked transmitter release only occurs within the recording distance of the extracellular electrode as illustrated by the simultaneous occurrence on the intracellular and extracellular traces of either a response or a response failure. The upward mark on the extracellular records is the stimulus artifact. Note the slower rise times of the 'unmarked' intracellular miniature potentials. Calibration: intracellular, 1 mV; extracellular, 0-3 mV; time, 40 msec. Extracellular records retouched for clarity.

J. P. HODGKISS AND P. N. R. USHERWOOD relationship expected for a Poisson distribution of e.j.p. amplitudes (Fig. 5B). This type of distribution was found to reasonably accurately predict the observed e.j.p. amplitude distribution at low (Fig. 3B) and high (Fig. 3A) values of e.j.p. quantal content. Even when two shocks were applied to the motor nerve in quick succession at a rate of 0.5-1 Hz the observed e.j.p. amplitude distributions to the first and second impulses were both accurately predictable by Poisson statistics (Fig. 3C). If evoked transmitter release does conform to Poisson statistics then the binomial 120

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Fig. 5. A, plot of m. against mb for nineteen active spots on different extensor tibiae nerve-muscle preparations. Equality of the two estimates of m is represented by the line through the origin. Only intracellular 'marked' m.e.j.p. and e.j.p. amplitudes were included in data for analysis. In two experiments the nerve was stimulated repetitively by pairs of stimuli thus giving two estimates of m, and mb for each active spot. The data for one of these spots are represented by the filled circles whereas the filled squares represent data from the other active spot. B, when mb was plotted against CV (see text for details) for eighteen active spots there was good agreement with the relationship expected for a Poisson distribution (continuous line). C, a plot of m, against mt, using only extracellular data, revealed very little agreement with the relationship expected for a Poisson distribution (continuous line).

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 121 parameters n (the number of release sites) and p (average probability of quantal transmitter release) might not have any particular physiological correlate (Ginsborg, 1970). However, evoked transmitter release at the locust neuromuscular junction could be governed by binomial statistics which are approximated by Poisson statistics. This appears to be true for the frog neuromuscular junction when the quantal content of the end-plate potential is low (Wernig, 1975a; Miyamoto, 1975). If it is also the case at the locust extensor tibiae neuromuscular junction then p should be very small. When computed using the method of Johnson & Wernig (1971) p was approximately 0.1 for all but one junction where p - 0-2. When evoked transmitter release was facilitated by the second of two closely following nerve impulses Poisson statistics also provided a good description of the amplitude distribution of the e.j.p. for the second. In Fig. 3 it can be seen that the responses to the first impulse of pairs of impulses (Fig. 302) contained fifty-five response failures, i.e. three less than predicted, whilst the series of responses to the second impulse of such pairs (Fig. 3C3) contained thirty-three response failures i.e. five more than predicted. Clearly this does not suggest a significant deviation from a Poisson to a binomial distribution. Similar results were obtained at two other active spots studied. Even when the e.j.p. quantal content was as high as 5 05 the observed e.j.p. amplitude distribution still conformed to Poisson statistics (Fig. 3A). In this case Poisson's theorem predicted one response failure whilst only two were observed. The relationship between quanta released spontaneously and those released in response to nerve impulses at degenerating neuromuscular junctions The Ca-electrode technique was used to study the quantal release of transmitter from extensor tibiae motor nerve terminals at times up to 65 hr after motor nerve section. The location of active spots on muscle fibres at times > 40 hr after motor nerve section proved more difficult than on control muscle fibres. This was possibly due to the fact that some of the superficial nerve terminals on these fibres no longer responded to nerve impulses. Invariably excitation-secretion coupling at all the degenerating nerve terminals irreversibly failed in the interval 53-100 hr after motor nerve section. The amplitude distributions of m.e.j.p.s recorded at active spots at times up to 24 hr after motor nerve section were normally distributed (Fig. 6C). The e.j.p. amplitude distribution (Fig. 6C) at this time is accurately predicted by Poisson statistics (0.9 > P > 0.8). At times greater than about 40 hours after nerve section, however, the m.e.j.p. amplitude distribution were frequently multipeaked and positively skewed sometimes with many m.e.j.p.s just discernible above the base-line noise (Fig. 6A, B, E, F). As nerve terminal degeneration progressed 'giant' m.e.j.p.s became more evident (Usherwood, 1963b, 1973). Comparison of amplitude distributions for e.j.p.s and m.e.j.p.s at single active sites revealed a discrepancy between the size of the unit potentials evoked by nerve stimulation and those released spontaneously (Fig. 6D, E, F). This is particularly evident in Fig. 6D and E, where the mean amplitude of the evoked unit potentials is clearly larger than that of the m.e.j.p.s. This discrepancy appears to be due to the presence of a class of 'small' m.e.j .p.s, particularly evident in Fig. 6 F, represented

122 J. P. HODGKISS AND P. N. R. USHERWOOD by the first peak in the m.e.j.p. amplitude distribution. 'Small' m.e.j.p.s were a feature of the amplitude distribution 53-65 hr after motor nerve section, although the ratio of 'small' to 'normal' m.e.j.p.s varied from site to site and from preparation to preparation. Similar miniature potential amplitude distributions have A

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Fig. 6. Histograms of the amplitude distribution of m.e.j.p.s (A-F) and corresponding e.j.p.s (C-F only) obtained at six active spots on different extensor tibiae nerve-muscle preparations. The histograms were obtained during ionophoresis of Ca onto the nerve terminal at 57, (A); 55, (B); 24, (C); 51, (D); 53, (E) and 65 (F) hr after motor nerve section. The theoretical Poisson amplitude distribution of the e.j.p. (continuous curve, C, D) was determined from the mean and variance of the m.e.j.p. amplitude distribution. M.e.j.p.s represented by the unshaded portion of the histograms in D,, E1 and F1 were arbitrarily judged to be 'giant' m.e.j.p.s and were not used in such determinations. In C the m.e.j.p. amplitude distribution is normal and Poisson statistics provide a good description of the e.j.p. amplitude distribution (0-9 > P > 0-8). In D there is a small discrepancy between the sizes of the spontaneous and evoked unit potentials, although the observed and predicted distributions were not significantly different (0-3 > P > 0 2). In E and F where the discrepancy between the amplitude of spontaneous and evoked unit potentials is large, the observed and predicted distributions were significantly different, although the distributions predicted from mb are not shown. The motor nerve was stimulated at 1 Hz in C and E and 0-2 Hz in D and F. The separation between the first and second stimuli in F was 200 msec.

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 123 been described at normal (Bevan, 1976), regenerating (Miledi, 1960; Dennis & Miledi, 1971, 1974) and botulinum toxin-treated frog neuromuscular junctions (Harris & Miledi, 1971; Spitzer, 1972; Boroff, Del Castillo, Evoy & Steinhardt, 1974). One noteworthy feature concerned the relationship between the time to irreversible transmission failure and the proportion of 'small' m.e.j.p.s in the m.e.j.p. amplitude distribution. For example, at the active spot referred to in Fig. 6D evoked release of transmitter continued for many hours after the data presented in this Figure were collected. A similar observation was made at three other active spots 40-60 hr after motor nerve section. In all cases the presence of 'small' m.e.j.p.s was detectable only because of the discrepancy between the peaks of the observed and predicted evoked single quantum class. However, the data presented in Fig. 6E and F were obtained at two junctions where excitation-secretion coupling irreversibly broke down soon after sampling was complete. The times to irreversible transmission failure were 10 and 20 min respectively, at a stimulation frequency of 1 Hz. In both cases the small m.e.j.p.s constituted a sizeable proportion of the distribution of m.e.j.p. amplitudes. It was also established that after irreversible failure of evoked transmitter release spontaneous transmitter release invariably continued for many hours (see also Usherwood, 1963b, 1973). The reverse situation was, however, never encountered, i.e. e.j.p.s in the absence of m.e.j.p.s. DISCUSSION

The present study of the spontaneous release of transmitter at locust excitatory neuromuscular junctions, although not extensive, supports previous findings that this phenomenon does not completely fulfil the requirements for a Poisson process. At many active spots bursts of m.e.j .p.s were recorded, even at low m.e.j .p. frequencies. Such bursts varied in duration and occurred unpredictably. Bursts of miniature potentials have also been recorded at the frog neuromuscular junction (Katz & Miledi, 1965a; Boroff et al. 1974). However, these authors ascribe most of these bursts to local disturbances of the nerve terminal by the extracellular electrode. Although spontaneous fluctuations in potential difference across the presynaptic membrane cannot be ruled out by the present study it seems unlikely that disturbances by the extracellular electrode are responsible for the bursts of m.e.j.p.s recorded from locust muscle for two reasons. First, the bursts can be monitored by an intracellular electrode which is unlikely to deform the nerve terminals and, secondly, it has been established in the present study and elsewhere (Usherwood, 1972) that vigorous prodding of the nerve terminal by an extracellular electrode is remarkably unsuccessful in altering the m.e.j.p. discharge frequency. If such bursts represent an inherent instability in the nerve terminal membrane or transmitter release mechanism (Cooke & Quastel, 1973) such that there is interaction between the release of one quantum and the next then one might have expected that the number of synchronous releases would be higher than predicted on the basis of chance, due to 'drag' effects (Martin & Pilar, 1964). Such a feature was observed at the frog and crayfish neuromuscular junction where spontaneous transmitter release conforms to a branching Poisson process (Cohen, Kita & Van

J. P. HODGKISS AND P. N. R. USHER WOOD der Kloot, 1974a, b, c). In the present study no obvious positive skew was observed in the amplitude distributions of m.e.j .p.s recorded at normal neuromuscular junctions on extensor tibiae phasic muscle fibres although Usherwood (1972) obtained skewed distributions for amplitudes of 'marked' miniature potentials (i.e. m.e.j.p.s) for junctions on locust retractor unguis muscle. One plausible explanation for the present results is that 'drag' effects vary in intensity and only rarely produce synchronous quantal transmitter release. Another explanation could be that spontaneous release of quanta from locust extensor tibiae 'fast' axon terminals operates with a 'dead' time or refractory period (Hubbard & Jones, 1973; Cunningham, 1975). It seems reasonable to assume from the data presented in this paper that under normal conditions evoked transmitter release from 'fast' extensor tibiae motor nerve terminals conforms to a Poisson process. A similar conclusion was reached in studies of the larval neuromuscular- junction Drosophila melanogawter (Jan & Jan, 1976). This implies that either the number of quanta available for release or the number of release sites is very large, whereas the average probability of quantal transmitter release is very low (del Castillo & Katz, 1954; Wernig, 1975b; Zucker, 1973). If it is accepted that evoked release of transmitter is a transient acceleration of the spontaneous mode of transmitter release (Gage & Hubbard, 1965; Hubbard, Jones & Landau, 1968; Katz, 1969; Hubbard, 1970) then one might have expected a nonrandom distribution of e.j.p. amplitudes at the locust neuromuscular junction in view of the non-Poisson spontaneous release mode (Usherwood, 1972, 1976). That is not so suggests that the mechanisms underlying evoked and spontaneous transmitter release could be different. Indeed studies of the effects of various ions on transmitter release at the frog neuromuscular junction indicate that this might be a general phenomenon (Kajimoto & Kirpekar, 1972; Manalis & Cooper, 1973; Weakly, 124

1973). The idea that the mechanism of evoked transmitter release is distinct from that of spontaneous transmitter release is also suggested by the presence of 'small' m.e.j.p.s at normal (Hodgkiss, 1976; Walther & Reincke, 1977) and at degenerating locust neuromuscular junctions; the 'small' m.e.j.p.s apparently representing transmitter quanta not released by nerve impulses. 'Small' miniature potentials have also been described at normal (Bevan, 1976) and degenerating (Kriebel & Gross, 1974; see also Birks, Katz & Miledi, 1960) frog neuromuscular junctions. Dennis & Miledi (1974) considered various possible causes for the discrepancy between the sizes of spontaneous and evoked unit potentials recorded at frog neuromuscular junctions. One suggestion was that the 'small' m.e.j.p.s were due to transmitter release from the Schwann cell. At the extensor tibiae neuromuscular junctions transmitter release from the lemnoblast cannot be ruled out as being responsible for the 'small' m.e.j.p.s although at present there is no evidence to suggest that insect glial cells can store and release transmitter. Another possibility considered by Dennis & Miledi (1974) to account for such a discrepancy was that their evoked unit responses might be composed of two or more quanta. In the present studies it was assumed that all response failures were true Poisson failures to release transmitter. On this basis a Poisson series was generated using ma from -=N exp (- ma)amax

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J. P. HODGKISS AND P. N. R. USHER WOOD where ma = In N/no, x = 1, 2, 3 etc. Such a distribution could then be compared with that observed. Since the quantal contents of the e.j.p. were kept low in these experiments the amplitude distributions were multipeaked and the peaks were approximately integral multiples of the first peak. Thus it was possible to, rather coarsely, divide the amplitude distributions into quantal distributions. The results obtained (Table 1) indicate a reasonable agreement between the observed and expected quantal distributions suggesting that the evoked responses represent the release of single packets of transmitter. This is strengthened by the finding that when transmitter release is facilitated by repetitively applying two closely following stimuli to the motor nerve there is a reduction in the number of transmission failures without any change in the modal distribution of the e.j.p.s (Fig. 6F). By arbitrarily excluding 'giant' m.e.j.p.s and computing the Poisson series using mb for some active spots there is poor agreement between the observed and expected distributions except in one case where the m.e.j.p. amplitude distribution was approximately normal (Table 1, site 1). A number of possible explanations for the occurrence of small m.e.j.p.s at locust nerve-muscle junctions spring to mind. Apart from the possible release of transmitter from lemnoblast there is the likely presence of neurosecretory axon terminals on the phasic fibres of the locust extensor tibiae muscle to be considered. There are indications, in some insect skeletal muscles at least, that neurosecretory axons may make intimate contact with the muscle fibre membrane sometimes in the vicinity of a neuromuscular junction (Osborne, Finlayson & Rice, 1971). Perhaps, as has been suggested by Bevan (1976) and Katz (1977) 'small' transmitter quanta represent some labile stage in the packaging and release of transmitter. Another possibility is that 'small' transmitter quanta may be real quantal units, several of which are released synchronously to produce a miniature potential (Harris & Miledi, 1971; Kriebel & Gross, 1974; Kreibel, Llados & Matteson, 1976; Wernig & Stirner, 1977). Clearly further studies are needed to definitely establish the source of the 'small' transmitter quanta at locust nerve-muscle junctions and some caution must be exercised in extrapolating from observations on vertebrate preparations. 126

We are indebted to Drs M. D. Burns and R. T. Joy for assistance in writing the computer programmes, and to Dr R. Ramsey for invaluable technical assistance. REFERENCES ANwYIL, R. & USHERWOOD, P. N. R. (1974). Voltage-clamp studies of a glutamate synapse. Nature, Lond. 252, 591-593. BEVAN, S. (1976). Sub-miniature end-plate potentials at untreated frog neuromuscular junctions. J. Physiol. 258, 145-155. BiuKs, R., KATz, B. & MILEDI, R. (1960). Physiological and structural changes at the amphibian myoneural junction in the course of nerve degeneration. J. Phygiol. 150, 145-168. BOROFF, I. A., DEm CASTILO, J., Evoy, W. H. & STENHARDT, R. A. (1974). Observations on the action of type A botulinum toxin on frog neuromuscular junctions. J. Physiol. 240, 227-253. CocmtANE, D. G., ELDER, H. Y. & USHERWOOD, P. N. R. (1972). Physiology and ultrastructure of phasic and tonic skeletal muscle fibres in the locust Schitocerca gregaria. J. Cell Sci. 10, 419-441. Cooxx, J. D. & QUASTEL, D. M. J. (1973). Transmitter release by mammalian motor nerve terminals in response to focal polarization. J. Physiol. 228, 377-405.

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Transmitter release from normal and degenerating locust motor nerve terminals.

J. Physiol. (1978), 285, pp. 113-128 With 6 text-figure8 Printed in Great Britain 113 TRANSMITTER RELEASE FROM NORMAL AND DEGENERATING LOCUST MOTOR...
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