J. Physiol. (1978), 285, pp. 113-128 113With 6 text-figure8Printed in Great Britain

TRANSMITTER RELEASE FROM NORMAL AND DEGENERATINGLOCUST MOTOR NERVE TERMINALS

BY J. P. HODGKISS* AND P. N. R. USHERWOODFrom 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 fromterminals of 'fast' excitatory motor axons on locust muscle fibres using intra- andextracellular recording together with a Ca-electrode technique for activating trans-mitter release from single nerve terminals on multiterminally innervated musclefibres.

2. Spontaneous intracellular miniature excitatory junction potentials (m.e.j.p.s),recorded at active spots on these muscle fibres, occurred non-randomly with frequentbursts of m.e.j.p.s.

3. M.e.j.p.s of subnormal amplitude were also seen but contributed only a smallfraction of the minature discharge.

4. The amplitude distribution of intracellularly recorded excitatory junctionpotentials (e.j.p.s) evoked during ionophoretic application of Ca onto single nerveterminals was adequately predicted by Poisson statistics.

5. During the course of nerve terminal degeneration m.e.j.p.s of subnormalamplitude became more frequent and eventually formed the major part of theminiature discharge. Transmitter quanta responsible for 'small' m.e.j.p.s did notcontribute to evoked release either at normal or degenerating terminals. Evokedtransmitter release from degenerating axon terminals before excitation-secretioncoupling failure conformed to Poisson statistics.

6. It is concluded that more than one release mechanism operates on the trans-mitter pool or pools in locust motor nerve terminals.

INTRODUCTION

Recent studies of spontaneous transmitter release from locust and cockroachmotorneurones (Usherwood, 1972, 1973; Rees, 1974; Washio & Inouye, 1975) havesuggested that it does not conform to the Poison process. The bursts of miniaturepotentials that occurat synapses on skeletal muscles ofthese insects suggestthat limitedinteraction takes place between the release of one transmitter quantum and the next.In view of the hypothesis that evoked transmitter release is a transient accelerationof the spontaneous mode of release (Katz, 1969; Hubbard, 1970) it became of interestto see whether evoked transmitter release at the insect nerve-muscle junction isnon-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. USHERWOODbecause 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 transmitterrelease. However, in the studies described in this paper use has been made of theCa-electrode technique (Katz & Miledi, 1965a, b, c) by means of which evokedtransmitter release can be restricted to a single nerve terminal on a muscle fibre.This has been used in combination with intra- and extracellular recording techniquesto study the transmitter release from normal and degenerating locust neuromuscularjunctions.

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 thelocust 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 bymeans of Takiwax (Usherwood & Machili, 1968). Tfe nerve-muscle preparation was exposedand 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 whichruns along the ventral surface of the muscle was cut at the proximal end of the muscle andsucked into the fine tip of a plastic suction electrode. The bathing solution was then exchangedfor one similar to the above but containing 20-30 mm-Mg, isomotically substituted for Na, anda reduced Ca concentration (0-5-1 mM).

Miniature excitatory post-synaptic potentials (min e.p.s.p.s) and excitatory post-synapticpotentials (e.p.s.p.s) were recorded intracellularly with micro-electrodes filled with 2 M-Kcitrate and having a resistance, measured in locust saline, of 8-15 MCI. Transmitter release fromsingle nerve terminals was monitored extracellularly at active spots on the surface of themuscle 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 anextracellular 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 thispurpose. A barrel filled with 2 m-NaCl was used to record extracellularly from the nerve-musclejunction whilst the other barrel, filled with 0 3-0 5 M-CaCl2, was used to apply Ca ions to thenerve terminal in a controlled manner. Electrodes with barrel resistances of 5-20 MQL provedthe 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 containing5-10 mm-Mg but no added Ca. (The mean amplitude of the spontaneous min e.p.s.p.s wasreduced by only about 10% by this treatment.) This abolished evoked transmitter releasewithin 10-20 min. It was possible to restore evoked transmitter release at the single nerveterminal under the extracellular micro-electrode by passing a small anodal current through theCa electrode. This was signalled by the appearance of e.p.s.p.s which, since they resulted from therelease of transmitter from one nerve terminal only, we shall refer to as e.j.p.s. Intracellular andextracellular recordings of these events were displayed on an oscilloscope and photographedon moving film. The amplitudes of potentials were determined by measuring from the top ofthe base-line to the peak of the potential. E.j.p.s with amplitudes greater than 2-5 mV wereindividually corrected using Martin's equation (Martin, 1955). The equilibrium potential fortransmitter 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), weretransacted in the thorax at a point close to the ganglion and also at a point as close as possible

114

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 about90 %.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 sampleperiods (t) with the variance (at). For a Poisson process, irrespective of the nature of t, Vtshouldequal Nt (Hubbard, Llinas & Quastel, 1969; Hubbard & Jones, 1973; Washio & Inouye, 1975).Because of experimental limitations estimates of Vt contained successively fewer samples as t wasincreased and therefore were less reliable for high values of t. For this reason a comparison ofVt and N. was made only when Nt was less than 20% of the total number of m.e.j.p.s in thesample (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) ofthe 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

Qmb= -

q

where Q is the mean amplitude of the intracellular e.j.p. recorded at the same active spot asthe 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/nowhere N is the total number of stimuli applied to the motor nerve and no is the number of suchstimuli which failed to elicit a response (e.j.p.). We have assumed that failures of nerve-muscletransmission resulted from failures of excitation-secretion coupling rather than conductionfailure in preterminal regions of the motor axon. The deep location and structural organizationat 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 normallydistributed 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 toseparate the e.j.p. amplitude distribution into discrete quantal classes following the method ofdel 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 wastested 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 = 0-05then the null hypothesis was retained and no significant difference between the observed andPoisson distributions was assumed.

RESULTSSpontaneo transmitter releaseThe 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-' instandard locust saline. At many active spots the m.e.j.p.s did not always occurrandomly, short bursts sometimes signalled the start of a period of elevated

116 J. P. HODGKISS AND P. N. R. USHERWOODspontaneous transmitter release lasting in some cases up to 1-5 min (Fig. 1 B). Variance-mean (Vt-Nt) plots for such data recorded at seven active spots indicated thatspontaneous release of transmitter was non-Poisson (Fig. 2). Six of the Vt-Nt plotsexhibited a characteristic deviation from the relationship expected for a Poissonprocess, with a tendency for Vt > Nt. At only one active spot studied did the relation-ship conform closely to that expected for a Poisson process (Fig. 2, filled circles). Atthis particular active spot m.e.j.p. frequency exhibited only slight variation overthe 600 sec time interval studied (Fig. 1A). At active spots on locust retractor

A

03 -0 0 0 6 * 0

02 0 0 0 0 0

0._ me 0 0* 0 00

C.)

CDm

0)

*--..1-00-

0-5

Low *- * @0.00 *S *0 gop I pL l

200 400 600

B0

1 _

00** 0

* 0 **00 0

0000 0 0 0.00 0.0* 0 @ 00 00 0

@00 @ 0 0 00*00 0 00 go 00 0

L -I I L

200 400 600Time (sec)

Fig. 1. Variation in m.e.j.p. frequency with time at two active spots on differentextensor tibiae nerve-muscle preparations. At one active spot the miniature dischargedid not contain bursts of m.e.j.p.s (A). However, at the other active spot there wereperiods of elevated m.e.j.p. frequency when bursting occurred, one of which is repre-sented 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 pre-sented 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 inthe present study therefore support the idea that spontaneous transmitter releaseat 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 amplitudethe multicomponent nature of such potentials was clearly indicated by concomitantextracellular events. Such potentials were therefore not included in the analyses todetermine quantum size nor represented in the m.e.j.p. amplitude histograms. Incontrast Usherwood (1972) found that the amplitude distributions of 'marked' mine.p.s.p.s (m.e.j.p.s) recorded at locust retractor unguis neuromuscular junctionsexhibited a positive skew.

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 117

Evoked release of transmitterIt is possible to abolish evoked transmitter release from all nerve terminals by

reducing the extracellular Ca concentration, and to selectively restore evokedtransmitter release at a single nerve terminal under the Ca electrode. The recordsin Fig. 4A illustrate such an experiment. The preparation was exposed to a Ca-freesaline whereupon no e.p.s.p.s were recorded when the extensor tibiae motor nerve

100 2

80-

60-

40-

Nt~ ~ ~ tN

20/-

10 20 30

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

was stimulated (Fig. 4Aa). Application of a small anodal current (5 x 10-8 A) tothe Ca electrode critically placed above a neuromuscular junction then resulted ine.j.p.s which were recorded intracellularly and extracellularly (Fig. 4Ab). Duringthe 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 secondduring Ca application. The e.j.p.s which resulted fluctuated in amplitude duringrepetitive stimulation of the extensor tibiae motor nerve with occasional failures ofresponse (Fig. 4B). Increasing the magnitude of the Ca ejection current led to anincrease 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. USHERWOODintracellular e.j.p. further increased in amplitude but fluctuated to a smaller extentthan before, whereas the mean amplitude of the extracellular e.j.p. exhibited nofurther increase. Presumably under such conditions transmitter release was beingactivated at sites outside the recording distance of the extracellular electrode.Reducing the Ca ejection current to zero or imposing a small Ca retaining currentled to abolition of evoked transmitter release within 1-2 sec.

Al

11

W

0an._L-

0

0z

mb =5 05no obs. =2noexp. =1

1 2 3 4E.j.p. amplitude (mV)

C,

C2

C3

B. 20

k25 0-5 M.e.j.p. amplitude (mV)

ma=087hI), =0-87Q\ no obs. = 1 17

s no exp. =1 18

05 1

E.j.p. amplitude (mV)

05 1 15E.j.p. amplitude (mV)

Fig. 3. Histograms of amplitude distributions of m.e.j.p.s (filled histograms A-C) ande.j.p.s (open histograms A-C) recorded at single active spots on normal extensor tibiaemuscle fibres. The evoked responses to nerve stimulation (at 0-5-1-5 Hz) were obtainedduring ionophoretic application of Ca to the nerve-muscle junction. The preparationswere bathed in saline containing 5-6 mM-Mg but no Ca. The theoretical Poissonamplitude distribution of the e.j.p. (continuous curves, A2, B2, C2 and C3) was deter-mined from the mean and variance of the m.e.j.p. amplitude distribution. Note thegood 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 (B1and B2),0-98 > P > 0-95 (C1 and C2), 0-9 > P > 0-8 (C1 and C3); m,, mb, quantal content ofe.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 independentestimates 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 agreementbetween these estimates (Fig. 5C). A plot of CV against mb also conformed to the

B,

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 119

a

_ _ ~~~~~B1mV a

b r- --_b _

d

C

f

g

I msec msec

Fig. 4. M.e.j.p.s and e.j.p.s recorded at two active spots on different extensor tibiaemuscle 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 at0-5 Hz. Each trace in a to d represents a series of five to seven superimposed responsesof the muscle fibre recorded intracellularly (upper records) and extracellularly (lowerrecords). With zero Ca ejection current (a) no e.j.p.s occurred although m.e.j.p.s stilloccurred (not clearly shown in a). In b a small Ca ejection current (5 x 10-8 A) led tothe appearance of e.j.p.s, although not in response to every nerve stimulus. When thecalcium 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 recordedintracellularly 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 spontaneousm.e.j .p.s recorded intracellularly (lower records a to g) and extracellularly (upper recordsa to g). The evoked responses to neural stimulation (at 1 Hz) were obtained duringionophoretic application of Ca to the nerve terminal using a constant current. Therecords show that evoked transmitter release only occurs within the recordingdistance of the extracellular electrode as illustrated by the simultaneous occurrenceon 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 slowerrise times of the 'unmarked' intracellular miniature potentials. Calibration: intra-cellular, 1 mV; extracellular, 0-3 mV; time, 40 msec. Extracellular records retouchedfor clarity.

J. P. HODGKISS AND P. N. R. USHERWOODrelationship expected for a Poisson distribution of e.j.p. amplitudes (Fig. 5B). Thistype 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. quantalcontent. Even when two shocks were applied to the motor nerve in quick successionat a rate of 0.5-1 Hz the observed e.j.p. amplitude distributions to the first and secondimpulses were both accurately predictable by Poisson statistics (Fig. 3C).

If evoked transmitter release does conform to Poisson statistics then the binomial

A

41..

Co 1

8

1 2 3 4 01 1mb mb

10

3

2

C

00 0

0 /

1 2

mb

3

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

120

1

1

TRANSMITTER RELEASE FROM LOCUST MOTORNEURONES 121

parameters n (the number of release sites) and p (average probability of quantaltransmitter release) might not have any particular physiological correlate (Ginsborg,1970). However, evoked transmitter release at the locust neuromuscular junctioncould be governed by binomial statistics which are approximated by Poisson statistics.This appears to be true for the frog neuromuscular junction when the quantal contentof the end-plate potential is low (Wernig, 1975a; Miyamoto, 1975). If it is also thecase at the locust extensor tibiae neuromuscular junction then p should be verysmall. When computed using the method of Johnson & Wernig (1971) p was approxi-mately 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 theamplitude distribution of the e.j.p. for the second. In Fig. 3 it can be seen that theresponses to the first impulse of pairs of impulses (Fig. 302) contained fifty-fiveresponse failures, i.e. three less than predicted, whilst the series of responses to thesecond impulse of such pairs (Fig. 3C3) contained thirty-three response failuresi.e. five more than predicted. Clearly this does not suggest a significant deviationfrom a Poisson to a binomial distribution. Similar results were obtained at two otheractive spots studied. Even when the e.j.p. quantal content was as high as 5 05 theobserved 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 wereobserved.

The relationship between quanta released spontaneously and those released in responseto nerve impulses at degenerating neuromuscular junctionsThe 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 nervesection. The location of active spots on muscle fibres at times > 40 hr after motornerve section proved more difficult than on control muscle fibres. This was possiblydue to the fact that some of the superficial nerve terminals on these fibres no longerresponded to nerve impulses. Invariably excitation-secretion coupling at all thedegenerating nerve terminals irreversibly failed in the interval 53-100 hr after motornerve 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 Poissonstatistics (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 andpositively skewed sometimes with many m.e.j.p.s just discernible above the base-linenoise (Fig. 6A, B, E, F). As nerve terminal degeneration progressed 'giant' m.e.j.p.sbecame 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 nervestimulation and those released spontaneously (Fig. 6D, E, F). This is particularlyevident in Fig. 6D and E, where the mean amplitude of the evoked unit potentialsis clearly larger than that of the m.e.j.p.s. This discrepancy appears to be due tothe presence ofa class of 'small' m.e.j .p.s, particularly evident in Fig. 6F, represented

J. P. HODGKISS AND P. N. R. USHERWOODby 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, althoughthe ratio of 'small' to 'normal' m.e.j.p.s varied from site to site and from pre-paration to preparation. Similar miniature potential amplitude distributions have

A10

0-5 1 1-520 B

10 ;

05 1

20 - y El

0-5 1 1-5 2 2*5

Io I n E M.e.j.p. amplitude (mV)

no obs. =57

(AC

0

-o

.0

6z

10 E.j.p. amplitude (mV)

0-5 1

M.e.j.p. amplitude (mV)20 -D!!

i

[l~i no obs. =10310 | l o exp. =9110~~~~~~~

0*5 1 15 2 25E.j.p. amplitude (mV)

0-520 I F.

1 1 5 2 2-5E.j.p. amplitude (mV)

10

0-5 1 1.530 - F2 M.e.j.p. amplitude (mV)

20 -n obs. =77

10

0-5 1 1-5 2

F3 Amplitude 1 st e.j.p. (mV)

20-

no obs. =6310 5

0. 5 1 1-5

Amplitude 2 nd e.j.p. (mV)

Fig. 6. Histograms of the amplitude distribution of m.e.j.p.s (A-F) and correspondinge.j.p.s (C-F only) obtained at six active spots on different extensor tibiae nerve-musclepreparations. The histograms were obtained during ionophoresis of Ca onto the nerveterminal at 57, (A); 55, (B); 24, (C); 51, (D); 53, (E) and 65 (F) hr after motor nervesection. 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 werearbitrarily judged to be 'giant' m.e.j.p.s and were not used in such determinations. InC the m.e.j.p. amplitude distribution is normal and Poisson statistics provide a gooddescription of the e.j.p. amplitude distribution (0-9 > P > 0-8). In D there is a smalldiscrepancy between the sizes of the spontaneous and evoked unit potentials, althoughthe 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 andevoked unit potentials is large, the observed and predicted distributions were signifi-cantly different, although the distributions predicted from mb are not shown. The motornerve was stimulated at 1 Hz in C and E and 0-2 Hz in D and F. The separation betweenthe first and second stimuli in F was 200 msec.

122

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. amplitudedistribution. For example, at the active spot referred to in Fig. 6D evoked releaseof transmitter continued for many hours after the data presented in this Figure werecollected. A similar observation was made at three other active spots 40-60 hr aftermotor nerve section. In all cases the presence of 'small' m.e.j.p.s was detectableonly because of the discrepancy between the peaks of the observed and predictedevoked single quantum class. However, the data presented in Fig. 6E and F wereobtained at two junctions where excitation-secretion coupling irreversibly brokedown soon after sampling was complete. The times to irreversible transmissionfailure were 10 and 20 min respectively, at a stimulation frequency of 1 Hz. In bothcases the small m.e.j.p.s constituted a sizeable proportion of the distribution ofm.e.j.p. amplitudes.

It was also established that after irreversible failure of evoked transmitter releasespontaneous transmitter release invariably continued for many hours (see alsoUsherwood, 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 excitatoryneuromuscular junctions, although not extensive, supports previous findings thatthis 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 miniaturepotentials have also been recorded at the frog neuromuscular junction (Katz &Miledi, 1965a; Boroff et al. 1974). However, these authors ascribe most of these burststo local disturbances of the nerve terminal by the extracellular electrode. Althoughspontaneous fluctuations in potential difference across the presynaptic membranecannot be ruled out by the present study it seems unlikely that disturbances by theextracellular electrode are responsible for the bursts of m.e.j.p.s recorded from locustmuscle for two reasons. First, the bursts can be monitored by an intracellularelectrode which is unlikely to deform the nerve terminals and, secondly, it has beenestablished in the present study and elsewhere (Usherwood, 1972) that vigorousprodding of the nerve terminal by an extracellular electrode is remarkably unsuccess-ful in altering the m.e.j.p. discharge frequency.

If such bursts represent an inherent instability in the nerve terminal membraneor transmitter release mechanism (Cooke & Quastel, 1973) such that there is inter-action between the release of one quantum and the next then one might haveexpected that the number of synchronous releases would be higher than predictedon the basis of chance, due to 'drag' effects (Martin & Pilar, 1964). Such a featurewas observed at the frog and crayfish neuromuscular junction where spontaneoustransmitter release conforms to a branching Poisson process (Cohen, Kita & Van

J. P. HODGKISS AND P. N. R. USHERWOODder Kloot, 1974a, b, c). In the present study no obvious positive skew was observedin the amplitude distributions of m.e.j .p.srecorded at normal neuromuscular junctionson extensor tibiae phasic muscle fibres although Usherwood (1972) obtained skeweddistributions for amplitudes of 'marked' miniature potentials (i.e. m.e.j.p.s) forjunctions on locust retractor unguis muscle. One plausible explanation for the presentresults is that 'drag' effects vary in intensity and only rarely produce synchronousquantal transmitter release. Another explanation could be that spontaneous releaseof 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 undernormal conditions evoked transmitter release from 'fast' extensor tibiae motor nerveterminals conforms to a Poisson process. A similar conclusion was reached in studiesof the larval neuromuscular- junction Drosophila melanogawter (Jan & Jan, 1976).This implies that either the number of quanta available for release or the number ofrelease sites is very large, whereas the average probability of quantal transmitterrelease is very low (del Castillo & Katz, 1954; Wernig, 1975b; Zucker, 1973). If it isaccepted that evoked release of transmitter is a transient acceleration of the spon-taneous mode of transmitter release (Gage & Hubbard, 1965; Hubbard, Jones &Landau, 1968; Katz, 1969; Hubbard, 1970) then one might have expected a non-random distribution of e.j.p. amplitudes at the locust neuromuscular junction inview of the non-Poisson spontaneous release mode (Usherwood, 1972, 1976). That isnot so suggests that the mechanisms underlying evoked and spontaneous transmitterrelease could be different. Indeed studies of the effects of various ions on transmitterrelease at the frog neuromuscular junction indicate that this might be a generalphenomenon (Kajimoto & Kirpekar, 1972; Manalis & Cooper, 1973; Weakly,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.sat normal (Hodgkiss, 1976; Walther & Reincke, 1977) and at degenerating locustneuromuscular junctions; the 'small' m.e.j.p.s apparently representing transmitterquanta not released by nerve impulses. 'Small' miniature potentials have also beendescribed at normal (Bevan, 1976) and degenerating (Kriebel & Gross, 1974; seealso Birks, Katz & Miledi, 1960) frog neuromuscular junctions. Dennis & Miledi(1974) considered various possible causes for the discrepancy between the sizes ofspontaneous 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 fromthe Schwann cell. At the extensor tibiae neuromuscular junctions transmitter re-lease 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 cellscan store and release transmitter. Another possibility considered by Dennis &Miledi (1974) to account for such a discrepancy was that their evoked unit responsesmight be composed of two or more quanta. In the present studies it was assumedthat all response failures were true Poisson failures to release transmitter. On thisbasis a Poisson series was generated using ma from

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J. P. HODGKISS AND P. N. R. USHERWOODwhere ma = In N/no, x = 1, 2, 3 etc. Such a distribution could then be comparedwith that observed. Since the quantal contents of the e.j.p. were kept low in theseexperiments the amplitude distributions were multipeaked and the peaks wereapproximately integral multiples of the first peak. Thus it was possible to, rathercoarsely, divide the amplitude distributions into quantal distributions.The results obtained (Table 1) indicate a reasonable agreement between the ob-

served and expected quantal distributions suggesting that the evoked responsesrepresent the release of single packets of transmitter. This is strengthened by thefinding that when transmitter release is facilitated by repetitively applying twoclosely following stimuli to the motor nerve there is a reduction in the number oftransmission 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 Poissonseries using mb for some active spots there is poor agreement between the observedand expected distributions except in one case where the m.e.j.p. amplitude distri-bution 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 transmitterfrom lemnoblast there is the likely presence of neurosecretory axon terminals on thephasic fibres of the locust extensor tibiae muscle to be considered. There are in-dications, in some insect skeletal muscles at least, that neurosecretory axons maymake intimate contact with the muscle fibre membrane sometimes in the vicinity ofa neuromuscular junction (Osborne, Finlayson & Rice, 1971). Perhaps, as has beensuggested by Bevan (1976) and Katz (1977) 'small' transmitter quanta representsome labile stage in the packaging and release of transmitter. Another possibility isthat 'small' transmitter quanta may be real quantal units, several of which arereleased 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 beexercised in extrapolating from observations on vertebrate preparations.

We are indebted to Drs M. D. Burns and R. T. Joy for assistance in writing the computerprogrammes, and to Dr R. Ramsey for invaluable technical assistance.

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