J. Phyriol. (1986), 379, pp. 257-274 257With 7 plates and 10 text-figure8Printed in Great Britain

THE PROBABILITY OF QUANTAL SECRETIONALONG VISUALIZED TERMINAL BRANCHES AT AMPHIBIAN

(BUFO MARINUS) NEUROMUSCULAR SYNAPSES

BY MAX R. BENNETT, PHILLIP JONES AND NICKOLAS A. LAVIDISFrom the Neurobiology Research Centre, University of Sydney, Sydney,

New South Wales 2006, Australia

(Received 27 January 1986)

SUMMARY

1. The number of quanta secreted from selected sites along terminal branches attoad (Bufo marines) neuromuscular junctions was determined. Terminal brancheswere visualized by prior staining with the fluorescent dye, 3-3 Diethyloxardicarbo-cyanine iodide (DiOC2(5)); neither impulse conduction nor quantal release wereaffected by DiOC2(5) at concentrations less than 10 /LM.

2. The evoked quantal release recorded with an extracellular micro-electrode (me)at different sites along the length of terminal branches was determined in an externalcalcium concentration, [Ca]o, of 0 35-0 45 mm. For short branches (40-80 #um), meremained approximately constant for over 60% of the branches; for the rest, -medeclined approximately exponentially with an average length constant of 17 + 2 gum(mean + S.E. of mean). For both medium (81-120 gim) and long branches(121-160 jsm), ige declined in nearly all cases approximately exponentially withlength constants of 39+ 5 and 54+ 8 jpm respectively. These changes in me wereobserved at synapses having a wide range of terminal branching patterns.

3. Some DiOC2(5)-stained branches possessed discontinuous cholinesterasestaining. In general, me declined along these branches in the same way as alongDiOC2(5)-stained branches with continuous cholinesterase staining.

4. It is suggested that because of the decline in me along most medium and longterminal branches, many release sites have a very low probability for secretion in low[Ca].. Release sites near the point of nerve entry, which have a relatively highprobability, therefore make the main contribution to secretion recorded with anintracellular micro-electrode. As a consequence, transmitter secretion from the wholeterminal does not fluctuate from impulse to impulse as much as expected ifthere werea large number of release sites, each with a low probability of secretion. Transmittersecretion then follows binomial rather than Poisson statistics.

INTRODUCTION

Amphibian motor-nerve terminals consist of hundreds of release sites or activezones from which quanta of transmitter may be secreted on arrival of the nerveimpulse (Couteaux & Pecot-Dechavassine, 1968; McMahn, Spitzer & Peper, 1972;Dreyer, Peper, Akert, Sandri & Moore, 1973; Miller & Heuser, 1984). Although

PHY 3799

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

release sites have been assumed to possess the same probability for transmittersecretion, this is difficult to reconcile with the statistical description of transmittersecretion. The quantal content of the end-plate potential (e.p.p.), which has beenmade just subthreshold for initiation of the muscle impulse by lowering the externalcalcium concentration, [Ca]., does not fluctuate much during a low-frequency trainof impulses: it behaves as a binomial rather than a Poisson random variable (Bennett& Florin, 1974; Wernig, 1975; Bennett & Fisher, 1977). This is not the result to beexpected if each of the release sites along the length of terminal branches has thesame low probability for secretion of a quantum. It can be most easily explained ifsome release sites have a relatively high probability for secretion and many have a

very low probability (see Appendix in Bennett & Fisher, 1977).Bennett & Lavidis (1979) examined the possibility that transmitter secretion was

not uniform at amphibian neuromuscular junctions by comparing the average

quantal content for a portion of the nerve terminal using an extracellular micro-electrode with the average quantal content for the whole terminal using an intra-cellular micro-electrode. It was not unusual for the extracellular micro-electrode atsome sites to record 30 % of the average quantal content observed with theintracellular micro-electrode, suggesting considerable non-uniformity in transmittersecretion. However, as the position of the extracellular micro-electrode with respectto the branching terminal was not determined, the possibility remained that theextracellular micro-electrode was recording from a region of multiple branching inthe end-plate. As extracellular micro-electrodes can record over distances of about10-20,um (Katz & Miledi, 1965 a; see also the Discussion), a considerable proportionof the nerve terminal may have contributed to these estimates. In order to avoid thisproblem, the position of the extracellular micro-electrode in relation to the terminalbranches was determined following zinc-iodide staining of the terminals (Bennett &Lavidis, 1982). This allowed analysis of release probabilities along the length of singleterminal branches, well removed from other branches. At the seven end-platesexamined in this way, quantal release along terminal branches declined with distancefrom their origins; typically, the average quantal content 60,tm from the origin ofa branch was only 300 of that found at the origin in a [Ca]. of 0 35 mm. This indicatesconsiderable non-uniformity in the probability of quantal release (see also Fig. 10 inde Cino, 1981).More recently D 'Alonzo & Grinnell (1985) used a different approach to the problem

of determining release probabilities along terminal branches. They recordedintracellularly at both ends of a terminal and determined the ratios of theamplitudes of single-quantum e.p.p.s recorded at the two electrodes (Gunderson,Katz & Miledi, 1981); as the amplitude of these e.p.p.s decreases with distance, thendifferent ratios correspond to the different sites of origin of the e.p.p.s. This techniqueallows for transmitter secretion to be determined along individual terminal branchesif there are no parallel branches. It has allowed D 'Alonzo & Grinnell (1985), followingstaining of the terminal, to show that in general there is a reduced secretion near theends of long terminal branches(100-200 Itm); this may be as low as 5-10% of thatfound near the more proximal portions.

In the present work, use is made of the discovery that living nerve terminals can

be visualized using the fluorescent dye, 3-3 Diethyloxardicarbocyanine iodide

258

SECRETION AT VISUALIZED TERMINALS

(DiOC2(5); Yoshikami & Okun, 1984). This has enabled accurate placement ofextracellular micro-electrodes with respect to the visualized terminal branches. Itfacilitates avoidance of pressure effects on the terminal as well as the relocation ofthe micro-electrode along a chosen terminal branch. The results show that somebranches, especially short ones less than 80 gm long, show very little or only slightdecline in quantal secretion along their length. However, longer branches invariablyshow a decrease in secretion such that 60 gm from the origin of a branch the quantalsecretion is only about 30% of that found at the origin.

METHODS

The iliofibularis and sartorius muscles of mature toads (Bufo marinus) were used in theseexperiments. Animals were anaesthetized with tricaine methanesulphonate (Rural Chem. Ind.Australia); both muscles and their nerve supply were dissected free from surrounding connectivetissue and tendinous insertions, and mounted in a Perspex bath of 3 ml capacity. The muscle wasstretched to approximately 110% of its resting length on the limb. The bath was perfused at roomtemperature (18 + 2 0C) with a modified Ringer solution of the following composition (mM): Na+,1 7; K+, 3 0; Mg2+, 1-2; Cl-, 103 1; H2PO4-, 0-64; HP042-, 9 70; Ca2+, 0 35-0445; glucose, 7-8. [Ca].was changed by altering the amount of CaCl2 dissolved in the Ringer solution supplying the bath.Possible changes in the conduction of the nerve impulse, due to the divalent cation concentrationfalling below 0-7 mm (Frankehauser & Hodgkin, 1957) were avoided by maintaining the externalmagnesium concentration, [Mg]o, constant at 1-2 mm. The solution was gassed continuously with95% 02 and 5% C02; this maintained the pH between 7-2 and 7-4.The isolated nerve-muscle preparations were first bathed for 30 s in 1-0 ,UM of the fluorescent

dye DiOC2(5) (Yoshikami & Okun, 1984); they were then thoroughly washed with Ringer solution.End-plates were chosen by viewing the fluorescent terminals on a video monitor attached via animage intensifier camera (Panasonic National) to an Olympus (BH2) fluorescent microscope.Yoshikami & Okun (1984) have shown that low concentrations (0 5 ,#M) of DiOC2(5) do not alterneurosecretion. The effects of exposing the junctions to high concentrations of DiOC2(5) fordifferent times were investigated in the present work. The quantal content of the e.p.p. in a [Ca]oof 0 35 mm (me = 1 1 ±+ 04, n = 4) was not altered by adding 10 /SM-DiOC2(5) for an hour followedby fluorescence illumination for over 10 min (m9 = 1-2 ± 0-3, n = 4). However, the quantal contentof the e.p.p. could be altered by increasing the concentration of DiOC2(5) above 10 ,M and exposingthe terminal to fluorescence for long periods (> 10 min). Long periods of fluorescence (in1 /SM-DiOC2(5)) were avoided during the mapping of mWe along terminal branches by the followingmethod: first the fluorescent terminal branch was drawn on a transparent overlay on the videomonitor shortly after its fluorescent profile had been identified; the fluorescence was then turnedoff and transmitted light used whilst the micro-electrode was manipulated with respect to themuscle, connective tissue and the drawing of the terminal on the monitor. Occasional checks thatthe terminal had not shifted with respect to the drawing were made by refluorescing the terminal.Any distortion of the connective tissue during repositioning of the micro-electrode was apparentusing either transmitted light or fluorescence.

End-plates were studied which possessed at least one single primary branch greater than 40 ,umlong with no secondary branches. In addition, primary branches had to be separated from otherbranches by over 10 ,um outside their points of origin.An extracellular glass micro-electrode filled with 2 M-NaCl (tip o.d. 0-2-1-0 ,um) was placed within

1-2 ,um of a branch; the extracellular field potential of the nerve impulse in the nerve terminal couldbe observed under these conditions. The nerve was stimulated with suction glass-capillaryelectrodes using current pulses of 0-08 ms duration and 6 V amplitude. At least 200 samples ofevoked release in the form of the extracellular voltage generated by end-plate currents (e.p.c.s;del Castillo & Katz, 1956; Katz & Miledi, 1965b) were recorded at 1 Hz for each electrodeplacement (Fig. 1).The end-plate current density decays in the extracellular space such that an external electrode

records quantal release originating only over a distance of about 10-20 ,um along the length of aterminal branch to either side of the electrode tip (del Castillo & Katz, 1956; Katz & Miledi, 1965a;

9-2

259

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

Bieser, Wernig & Zucker, 1984). However, if the cholinesterase reaction product is not continuousalong a terminal branch, changes occur in the extracellular resistivity in the vicinity of thecholinesterase discontinuity (Bieser et al. 1984). As a consequence, terminal branches were onlyanalysed if the cholinesterase was continuous (except in the case of Fig. 10 and Pls. 6 and 7). Insome cases, observations were made of evoked release from branches which had other branchespassing in parallel and as close as 12 jsm away. It is then possible that the micro-electrode recordedevoked release from both branches. The spatial decay of the e.p.c. with distance was ascertained

A 1 B

2__

3 H ~~~~~34 , 4

5 5

6 6

5 ms 2 mVFig. 1. Recordings of the extracellular signs of quantal secretion at two different sites (Aand B) on a terminal branch (these sites are indicated by 1 and 2 in Fig. 5A). Successivetraces give the responses to successive stimuli (1-6) applied to the motor nerve at 1 Hz(stimuli of 0-8 ms duration and 6 V strength). AC coupling used in all records. [Ca]o is0 35 mM.

by first determining the amplitude-frequency distribution of e.p.c.s at about 1 ,um from a site ona terminal branch that did not possess any branches in parallel to it; the micro-electrode was thenmoved 1-2 Aim away at right angles to the terminal branch, and the amplitude-frequencydistribution of e.p.c.s again determined; this procedure was continued for distances up to 12 jamfrom the original recording site. Fig. 2 shows results for three such series of measurements. Thelargest amplitude e.p.c.s (about 2-3 mV), presumably those originating from the release sitesclosest to the micro-electrode (see Bieser et al. 1984), decay approximately exponentially withdistance of the micro-electrode from the sites giving an average length constant for the decay of6 8 +0 8 jam (+ S.E. of mean, n = 6). These observations indicate that if only e.p.c.s greater than1 mV are counted when a micro-electrode is about 1 sm from a terminal branch, then e.p.c.sgenerated by other terminal branches passing in parallel but over 7 jam away are unlikely to beincluded (Fig. 2); this procedure was therefore used. It should be noted that although exponentialcurves were reasonably fitted to the data (Fig. 2), this is not meant to imply that the e.p.c. spreadis one dimensional. However, it did not seem necessary for the purposes of the present work touse the more complex curve-fits required for current flow in a radial sheet (see, for example, chapter5 in Jack, Noble & Tsien, 1975).Primary terminal branches (see Davey & Bennett (1982) and Bennett (1983)) usually began near

the point of nerve entry and this was taken as the origin of the branch in constructing the secretionprofiles given in Figs. 4, 5, 6, 9, and 10. However, it was not unusual for a primary branch to beginas an offshoot of a small branch near the point of nerve entry; this point of division was then takenas the origin of the primary branch.

260

SECRETION AT VISUALIZED TERMINALS 261

2 0

\; ~~~~~A

1-0 ---:: . 1.

---- Ruin 4,

~0o2-0 2 4

X, 1-0

Fig~.2.Teapiueof e,~ reore at difrn ditace frmatemnlrnh

20 4

~~:. * \.~O*

0 2 4 6 8 10 12Distance (;Am)

Fig. 2. The amplitude of e.p.c.s recorded at different distances from a terminal branch.In each case (A, B and C) recordings were made at the distances indicated by arrows onthe abscissa by moving the micro-electrode at right angles to the terminal branch.Terminal branches were chosen that had no other branches passing in parallel. Each filledcircle gives the amplitude of an e.p.c. The continuous line gives the least-squaresexponential fit based on the three largest e.p.c.s observed at each recording site. Theseexponential fits all had a correlation coefficient of 0 95 and gave length constants in A,B and C of 8-8, 5-4 and 7-5 /um respectively.

The extracellular signs of quantal release were used to construct frequency histograms of thenumber of quanta released, over 200 trials. A binomial analysis of these histograms was then madeaccording to the method given in Johnson & Wernig (1971): estimates of the binomial parametersat the extracellular sites (Pe and We) were made from Pe = 1-S2/We and ke = me/Pe, where mie andS2 are the mean and variance of the observed quantal release. The histograms are subject to theerror in which early quantal releases tend to mask later quantal release (Katz & Miledi, 1965a;Barrett & Stevens, 1972) so that some quanta are missed. This leads to amongst other things, anunderestimate of me. The recording methods used in the present work allowed a distinction to bemade of quanta separated by at least 0 5 ms. In addition, most observations were made at a low[Ca]o (about 0 35 mM) in which the probability of quantal release is sufficiently low that it isunlikely that many quanta were missed as a consequence of near-synchronous release. For thesereasons, it is unlikely that me is significantly underestimated because of the difficulty ofunambiguously identifying the occurrence of second and third quanta.

262 M. R. BENNETT, P. JONES AND N. A. LA VIDIS

20

A

10

Cn0

.0030

0~

U-

20

10

0 10 20 30 40 50Deviation from the mean (%)

Fig. 3. Tests for stationarity in me at identified sites. In A the micro-electrode was leftat one site for about 30 min, during which time me was estimated every 5 min; thevariation in me during this period was expressed as a percentage deviation from the meanvalue of me; the frequency of these percentage deviations is given for all sites examined.In B, estimates ofme were determined at a site and the micro-electrode then moved morethan 20 #sm from the site; the micro-electrode was subsequently repositioned as close tothe original site as possible and another estimate of me determined; the frequency of thepercentage differences between the two estimates of -me is given for each of forty-threesites.

It is known that pressure of the external electrode on nerve-terminal release sites can give riseto an increase in the frequency of spontaneous m.e.p.p.s (Fatt & Katz, 1952) and may thereforealter the normal evoked release of quanta from these sites. The possibility of such pressure effectswas examined in the present work in the following way: first, the stationarity of evoked quantalrelease was determined for individual sites at 5 min intervals over 30 min; it was found that over800% of these estimates of me varied by less than 20% from the mean value of me at a site(Fig. 3 A). Next, me was estimated at a site and the micro-electrode then removed some 20 /tm awaybefore being returned as closely as possible to the original site for a further estimation of me; againthe percentage difference in the two estimates was less than 200% for over 800% of the sitesinvestigated in this way (Fig. 3B). These observations (Fig. 3) show that in most cases pressureeffects could cause only a 10% change in me.The sites of recording along each visualized terminal branch were random with respect to the

origins of the branch; this ensured that the peripheral parts of a branch did not have a low -me

SECRETION AT VISUALIZED TERMINALS 263

because of terminal damage due to recordings made at several sites on the more proximal partsof a branch.

HistochemistryAt the end of an experiment, the DiOC2(5)-stained fluorescent terminal was photographed

through a camera attached to the Olympus (BH2) microscope. The pinned-out muscle was thenfixed with 2-5% glutaraldehyde and stained for cholinesterase according to Karnovsky (1964) andphotographed. Comparison could then be made between the distribution of cholinesterase reactionproduct, the DiOC2(5)-stained nerve terminal and the positions at which recordings had been madeaccording to their location marked on an overlay of the video monitor. The drawings of the terminalbranching pattern given in Figs. 4, 5, 6, 9 and 10 were derived from those parts of the

A B04 \

Me 020.2~~~~~ 0~~~~~

0 20 40 60 0 20 40 60 80

Distance (jAm)

Fig. 4. Changes in mie along the length of short terminal branches (40-80 /am) at whichrelease either decreased along the branch (A) or remained relatively constant (B). In eachcase a drawing of the extent of the nerve terminal is given, derived from the DiOC2(5)-stained nerve and cholinesterase-stained post-synaptic membrane. The numbers on theterminal give the sequence in which the recordings were made; arrows indicate the positionof the micro-electrode. A least-squares exponential curve-fit to the data in A gives a lengthconstant of 14,um with correlation coefficient of 0-80. The DiOC2(5)-stained nerveterminal and cholinesterase-stained end-plates of A and B are given in Pls. 1 and 2respectively.

DiOC2(5)-stained terminal associated with cholinesterase; if there was a break in cholinesterasealong a DiOC2(5)-stained branch then this was clearly shown in the drawing (see Fig. 10). In somecases a thin trail of cholinesterase, unaccompanied by nerves, emerged from the terminals; theseare indicated in Figs. 6A, 9B and lOB by single thin lines leaving the DiOC2(5)-stained terminal.

It should be noted that although the toad iliofibularis muscle contains some slow fibres whichreceive a distributed innervation, they compose less than IO% of all fibres, and are only found inthe centre of the muscle (Lannergren & Smith, 1966; Davey & Bennett, 1982). These slow fibreswere avoided in the present work by recording from superficial muscle fibres only.

RESULTS

me along terminal branchesEstimates of me were made for different sites along the length of terminal branches

in a [Ca]. of 0 35-0 45 mm. Of the eighty primary terminal branches studied in thisway, only twenty-six were shown to fulfil the criteria for analysis following theirstaining for cholinesterase: namely to be separated from other branches by over10 ,um outside their point of origin. Three different length categories of terminal

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

Id0-6

0-4\

02

0 20 40 60 80

Distan

1 25 34-,I G-

B

.00 0 00iii i i

0 20 40 60 80ce (jm)

1 3 5

Fig. 5. Changes in me along the length of medium-size terminal branches (81-120 ,sm) atwhich the release either decreased along the branch (A) or remained relatively constant(B). The length of each of these branches was just sufficient for them to fall into themedium-size category. In each case a drawing of the extent of the nerve terminalbranching is given, derived from the DiOC2(5)-stained nerve and cholinesterase-stainedpost-synaptic membrane. The numbers on the terminal give the sequence in which therecordings were made; arrows indicate the positions of the micro-electrode. An exponentialcurve-fit to the data in A gives a length constant of 40 ,um with correlation coefficient of0-82. The DiOC2(5)-stained nerve and cholinesterase-stained end-plate of A and B aregiven in Pls. 3 and 4.

0-6

0-4me

02

A

0

0 20 40 60 80 100 120

B

0 20 40 60 80 100 120Distance (Mm)

1 2 4 3 5 6

Fig. 6. Changes in me along the length of long terminal branches (121-160 ,tm) at whichthe release showed a relatively steep decline along the branch (A) or a shallow decline (B).In each case a drawing of the extent of the nerve terminal branching is given, derivedfrom the DiOC2(5)-stained nerve and cholinesterase-stained post-synaptic membrane. Thenumbers on the terminal give the sequence in which the recordings were made; arrowsindicate the positions of the micro-electrode. Exponential curve-fits to the data in A andB give length constants and correlation coefficients (in parentheses) of 34 ,sm (0 90) and68 ,um (0-99) respectively. The DiOC2(5)-stained nerve and cholinesterase-stained end-plateof A is given in P1. 5.

264

me

SECRETION AT VISUALIZED TERMINALS 265

0-5A

0*4

030-3* 0@

0-2

*1B05-

0-40

me 0-3 U 20 0AL

0-2 AoA

A.0-1 *t| rC

0-4 i, A

0-3 0

00.2 -

o 20 40 60 80 100 120 140Distance (jm)

Fig. 7. Cumulative results for the changes in me along the length of short (40-80 #nm, A),medium-size (81-120,um, B) and long (121-160 #nm, C) terminal branches which showeda decline in quantal release. A least-squares exponential curve-fit to the decline in mie foreach terminal gave the following length constants and correlation coefficients (inparentheses). A: open circles, 24,m (080); filled circles, 19 #m (088); open triangles,23 4um (093); filled triangles, 12,m (0-99). B: filled circles, 21 ,um (099); open triangles,48,m (0 55); open circles, 38,um (0 83); filled squares, 45,sm (0 95); filled triangles, 37,m(093). C: filled circles, 36,m (086); open circles, 24,m (087); filled triangles, 50,m(10-89); open squares, 33,m (085); filled squares, 42 ,sm (096); crosses, 62,m (0-88);filled diamonds, 90 #um (0 98); open triangles, 91 4am (0 88); open diamonds, 59 Jim (0 87).The length constants and correlation coefficients (in parentheses) for the curves in A, Band C are 20 4m (070), 50,m (066) and 53 #m (074) respectively. The [Ca]O variedbetween 0 35 and 0 40 mm.

branches were studied: short branches, 40-80 ,tm; medium-size branches, 81-120 ,tmand long branches, 121-160 ,um.Most of the short branches showed no decline in me (Fig. 4B and P1. 2), whilst the

remainder showed a spatial decline to which an exponential curve was fitted(Fig. 4A and P1. 1). The length constants for these varied between 13 and 23,m(17+2 ,tm, mean+s.E. of mean; Fig. 7A). In general, the short branches which

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

03 r

* A0-2 *0

0.1 2-A me NoAAA Ap

C

me

C

B

)-2

0~~~~01 h * -

C

022

0.1 -U *

m 1.

I

0 20 40 60 80 100 120Distance (pm)

Fig. 8. Cumulative results for the changes in mie along the length of short (40-80 sum, A),medium-size (81-120,um, B) and long (121-160 /tm, C) terminal branches which showedno consistent decline in quantal release. The [Ca]O varied between 0 35 and 0 45 mM.

0-6 rA

0

0*4 F

0 2

0

0

0 20 40 60 80 100 0Distance (bum)

B

20 40 60 80

92 7836 4 1 5

-lf 1 53

-AgAFig. 9. Changes in me along the length of medium-size terminal branches (81-120 ,um) atsimple end-plates with two branches (A) and complex end-plates with several branches(B). In each case a drawing of the extent of the nerve terminal branching is given, derivedfrom the DiOC2(5)-stained nerve and cholinesterase-stained post-synaptic membrane. Thenumbers on the terminal give the sequence in which the recordings were made; arrows

indicate the position of the micro-electrodes. An exponential curve-fit to the data givesa length constant in A of 43 ,um (leaving out points below 20 #sm) and in B of 37 /um; boththese exponentials had correlation coefficients of 0 94.

266

me

u II

SECRETION AT VISUALIZED TERMINALS 267

showed no consistent decline in me had relatively smaller values of me than themaximum values of mie found along branches which showed a decline in me (compareFigs. 8A and 7 A).

Nearly all medium-size branches showed a decline in me (Fig. 5A and PI. 3)although one did not (Fig. 5B and PI. 4). Some of the medium-size branches showed

0-6 \ A B0-6~~~~~~~~~~~~~

ffe0.40-2

0 20 40 60 80 100 0 20 40 60 80 100Distance jurm)

/ 3 24 1

Fig. 10. Changes in mie along the length of terminal branches with discontinuouscholinesterase stain but continuous DiOC2(5) stain. In each case (A and B) a drawing ofthe extent of the cholinesterase-stained post-synaptic membrane is given. Pls. 6 and7 show that the DiOC2(5)-stained terminal branches of A and B respectively werecontinuous even though the cholinesterase was not. The numbers on the terminal give thesequence in which the recordings were made; arrows indicate the position of themicro-electrode. An exponential curve-fit to the data gives a length constant andcorrelation coefficients (in parenthesis) in A and B of 20 #um (0-79) and 37 ,um (0-88)respectively.

an increase in Ie near the origins of the branch followed by an approximatelyexponential decline in me; these had length constants between 21 and 48 4m(39+ 5 ,um; Fig. 7 B). The medium-size branch which showed no consistent declinein me had relatively low values of me compared with branches for which me declined(compare Figs. 8B and 7B).Most long branches showed a decline in me (Fig. 6A) although some showed a

very shallow decline (Fig. 6B and P1. 5). Many of the long branches showed anincrease in me near the origins of the branch followed by an approximatelyexponential decline in me (Fig. 7 C) as did the medium-size branches. Theseexponential had length constants between 24 and 91 ,tm (54+ 8 ,Im; Fig. 7 C). Theremaining long branch which showed no consistent decline in me for much of itslength (Fig. 8C) also had relatively low values of me compared with branchespossessing a steep decline in mie (compare Figs. 8C and 7C).

Relation between me along terminal branches and the complexity of the branching pattern

The possibility exists that terminal branches show a decline in me depending on

the relative complexity of the branching within the terminal. No such correlationswere observed when the change in me along medium-size branches was estimated for

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

terminal branches of increasing complexity. Medium-size branches of simpleterminals, consisting of only two primary branches, showed a decline in me along thebranches (Fig. 9A) as did medium-size branches of complex terminals consisting ofmany branches (Fig. 9B).

me along terminal branches with discontinuous cholinesterase stainingSome DiOC2(5)-stained branches possessed discontinuous cholinesterase staining

(Pls. 6 and 7). These were not included in the terminals analysed above. It was ofinterest to see if me declined along these branches in which interruptions may occurin the formation of release sites apposed to post-synaptic membrane (Bieser et al.1984). There was an over-all decline in me along these terminal branches (Fig. 10)similar to that along normal terminal branches.

DISCUSSION

Artifacts in estimating Wie at release sites

me recorded with an extracellular micro-electrode at a point on a terminal branchmay be increased by contributions from immediately surrounding terminal branches.To avoid this, secretion profiles were only determined for branches which did notthemselves branch and which were positioned outside their points of origin more than1O jum from any parallel branches. Micro-electrodes with tip diameters of 2-3 #umrecord e.p.c.s attenuated by about one-quarter of their amplitude when placed about10 jnm in the longitudinal direction from the source of the current (del Castillo &Katz, 1956; Katz & Miledi, 1965a, b); if an electrode is more than 15-20 jtm fromthe source then the e.p.c.s are not recorded (Katz & Miledi, 1965a; Wernig, 1975,1976; Bennett & Lavidis, 1982). In the present work it has been shown thatdisplacement of a micro-electrode in the transverse direction from a terminal branchalso leads to a decline in the amplitude of the e.p.c. of about one-quarter over adistance of 10 jam. Close apposition of the micro-electrode to within 1-2 jsm of aterminal branch gave e.p.c.s of about 2-3 mV. As e.p.c.s less than 1 mV were notcounted, it is likely that only quantal secretion over a distance much less than 10 jImwas included in the analysis; this would exclude any secretion from parallel branchesgreater than 10 ,um away. Furthermore, some terminal branches analysed had noother branches in parallel with them (see Fig. 9A); there was no difference in the rateof decline of me along these branches compared with those which did have parallelbranches.The placing of an external micro-electrode with respect to visualized terminal

branches using a video monitor allows any direct pressure of the micro-electrode onthe branches to be avoided (Katz & Miledi, 1973). In addition, using careful visualmonitoring allows detection of tissue distortion in the vicinity of the micro-electrode;if signs of this occurred as the micro-electrode was advanced, it was withdrawn anda different approach to the site utilized until a successful approach was obtained. Ifthis was not possible then the site was abandoned. The sequence in which themicro-electrode was placed at successive sites along each terminal branch wasrandom. As the decline in me along terminal branches occurred independently of thesequence of electrode placement, it is unlikely that the decline was related to

268

SECRETION AT VISUALIZED TERMINALS

subtleties associated with unobserved mechanical distortion of the tissue (Katz &Miledi, 1973). Furthermore, individual sites were recorded from more than once afterrepositioning the micro-electrode; there was no trend for the quantal secretion tochange at a site during successive recordings, which typically showed a variation ofless than 10% for successive estimates of me. These various considerations suggestthat the secretion profiles along terminal branches recorded using the presenttechnique accurately reflect the probability for secretion.

Changes in me along terminal branchesProfiles of quantal secretion per unit length of terminal have been determined with

two intracellular micro-electrodes (Gunderson et al. 1981; D'Alonzo & Grinnell,1985). Results from such studies show that for terminals with considerable branchingat the point of nerve entry there is relatively uniform probability of secretion perunit length in the central region but reduced secretion per unit length near the endsof longer terminal branches (D'Alonzo & Grinnell, 1985). These observations can beexplained in terms of the present results if the short branches (< 80 jtm) near thepoint of nerve entry frequently have a uniform probability for secretion, whereas themedium and long branches generally show a decline in secretion probability.The double intracellular electrode technique can be used to determine the decline

in probability of secretion along a single terminal branch if the branch exists inisolation on the fibre, without any parallel terminal branches. Such a condition existsin Figs. 4, 5 and 6 of the work of D'Alonzo & Grinnell (1985). A least-squaresexponential curve-fit to the decline in release probability along these branches gavecorrelation coefficients ranging from 0 7 to 0-9; the length constants varied from50 to 66 ,sm. Note also that when the probability of secretion per unit terminal lengthis most nearly uniform in the work ofD 'Alonzo & Grinnell (1985) (as between 50 and180 ,sm in Fig. 4; between 0 and 100 pm in Fig. 6 and between 0 and 400,/m inFig. 7), there are in general many short overlapping branches.A decline in the frequency of spontaneous m.e.p.p.s also occurs along the length

of motor-nerve terminal branches (Tremblay, Robitaille & Grenon, 1984; D'Alonzo& Grinnell, 1984). It is possible to determine the rate of decline of the frequency ofm.e.p.p.s along single branches in the work of Tremblay et al. (1984) as they usedthe two intracellular micro-electrode technique on end-plates consisting only ofunbranched primary branches with no other branches in parallel: the results can befitted with exponential curves (correlation coefficient > 0 8), that have lengthconstants of between 26 and 59 sm. This is within the range of length constantsdetermined in the present work for evoked release along medium to long terminalbranches.

Non-uniformity of the probability of quantal secretion at the release sites of synapsesIn 1954 del Castillo & Katz noted that 'when the average quantal content (mi)

of the e.p.p. is small (< 3) its amplitude fluctuates in a manner predictable byPoisson's law. At higher levels (m > 10) deviations occur which may be due ... toa variation in the probability of response among different units'. There is nowconsiderable evidence that the deviations from Poisson's law observed at theneuromuscular junction (Bennett & Florin, 1974; Wernig, 1975; Bennett, Florin &

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M. R. BENNETT, P. JONES AND N. A. LA VIDIS

Hall, 1975; Bennett, Fisher, Florin, Quine & Robinson, 1977; Bennett & Fisher,1977; Bennett & Raftos, 1977) can be attributed to some sites (or units) having verylow probability for secretion compared with other sites (Bennett & Lavidis, 1979,1982; Fig. 10 in de Cino, 1981; D'Alonzo & Grinnell, 1985).Non-uniformity in the probability of secretion occurs at different boutons of single

Ia afferent terminals synapsing on motoneurones (Redman & Walmsley, 1983;Henneman, LUscher & Mathis, 1984): transmission occurs in an all-or-none mannerat these boutons, each of which has a different probability for secretion; in some casesthis probability is effectively zero. In autonomic ganglia, the distal boutons ofterminals synapsing within the ciliary ganglion have a lower turnover of synapticvesicles, and are therefore likely to have a lower rate of quantal secretion, than moreproximal boutons of the terminals (Laurie & Tremblay, 1982). At the peripheralsympathetic neuromuscular synapse, nerve impulses only release transmitter froma small proportion of the terminal varicosities (Hirst & Neild, 1980); most of thetransmitter is secreted from a preferred set of varicosities (Cunnane & Stjarne, 1984).The exponential decrease in probability of secretion along medium-size and long

terminal branches may explain the inverse relationship between the level of trans-mitter secreted per unit length of terminal and the total terminal length found atamphibian neuromuscular synapses (Nudell & Grinnell, 1982). The terminals atsynapses in some muscles secrete greater amounts of transmitter per unit terminallength than do terminals in other muscles (for example terminals in the frogcutaneous pectoris muscle secrete 2-4 times the transmitter per unit length ofterminal than do those in the sartorius muscle; Grinnell & Herrera, 1980). It will beinteresting to see if the high-secreting synapses consist mostly of small branches withrelatively uniform profiles of secretion along most of their length; low-secretingsynapses may then consist of long branches with relatively low secretion along mostof their length.

The physical basis for non-uniformity in secretion at different release sitesThe question arises as to whether there are any structural differences between

nerve-terminal release sites which may be correlated with their differences insecretion probability. Release sites of fast-twitch fibres consist of two double rowsof 2 nm intramembranous particles (Heuser, Reese & Landis, 1974); it is speculatedthat these intramembranous particles are calcium channels (Llinas, 1982). The lengthof these active zones has been estimated by a number of workers with conflictingresults: Davey & Bennett (1982) found that active zones in the toad (Bufo marinus)iliofibularis muscle tended to shorten for release sites along terminal branches moredistal from the point of nerve entry; in contrast, Werle, Herrera & Grinnell (1984)failed to find any changes in active-zone size in the sartorius muscle. The post-synapticfolds in the terminal gutter are immediately opposed to active zones; the length ofthe zones may be estimated by determining the length of the folds after chemicalremoval of the nerve terminal. Fold length decreases along some terminal brancheswith distance from the point of nerve entry at least for the last quarter or so of theterminal (Shooton, Heuser, Reese & Reese, 1979; Desaki & Uehara, 1981; but seeVerma & Reese, 1984).

Comparisons have been made between the average length of active zones at

270

SECRETION AT VISUALIZED TERMINALS

terminals which are known to secrete small numbers of quanta with those atterminals which secrete large numbers of quanta. These investigations have shownthat terminals with a low average quantal secretion have active zones which are onaverage smaller than those at terminals with a high average quantal secretion(Fukunaga, Engel, Osame & Lambert, 1982; Herrera, Grinnell & Wolowske, 1985).Such observations are consistent with the idea that the length of active zones, andtherefore the number of intramembranous particles, is important in determining theprobability of secretion at a release site.A recent report indicates that voltage-activated sodium channels are only present

in high density along the first half of terminal branches at amphibian neuromuscularjunctions (Mallart, 1984); they are not detectable near the ends of the branches. Twoobservations argue against this providing an adequate explanation for the decreasein mie along terminal branches: first, the profiles of decline in mge along medium tolong terminal branches do not follow the distribution of sodium-channel densitydescribed by Mallart (1984); secondly, a decrease in sodium-channel density does notprovide an explanation for the decline in frequency of spontaneous m.e.p.p.s alongthe terminal branches (Tremblay et al. 1984). The physical basis for decline insecretion probability along terminal branches is yet to be determined.We thank Drs R. Malik and S. Redman for their very helpful comments on the manuscript.

N. A. Lavidis was in receipt of a N.H. & M.R.C. Post-doctoral Fellowship.

REFERENCES

BARRETT, E. F. & STEVENS, C. F. (1972). The kinetics of transmitter release at the frog neuro-muscular junction. Journal of Physiology 227, 691-708.

BENNETT, M. R. (1983). Development of neuromuscular synapses. Physiological Reviews 63,915-1048.

BENNETT, M. R. & FISHER, C. (1977). The effect of calcium ions on the binomial parameters thatcontrol acetylcholine release during trains of nerve impulses at amphibian neuromuscularsynapses. Journal of Physiology 271, 673-698.

BENNETT, M. R., FISHER, C., FLORIN, T., QUINE, M. & ROBINSON, J. (1977). The effect of calciumions and temperature on the binomial parameters that control acetylcholine release by a nerveimpulse at amphibian neuromuscular synapses. Journal of Physiology 271, 641-672.

BENNETT, M. R. & FLORIN, T. (1974). A statistical analysis of the release of acetylcholine at newlyformed synapses in striated muscle. Journal of Physiology 238, 93-107.

BENNETT, M. R., FLORIN, T. & HALL, R. (1975). The effect of calcium ions on the binomial statisticparameters that control acetylcholine release at synapses in striated muscle. Journal ofPhysiology 247, 429-446.

BENNETT, M. R. & LAVIDIS, N. (1979). The effect of Ca ions on the secretion of quanta evoked byan impulse at nerve terminal release sites. Journal of General Physiology 74, 429-456.

BENNETT, M. R. & LAVIDIS, N. A. (1982). Variation in quantal secretion at different release sitesalong developing and mature motor terminal branches. Developmental Brain Research 5, 1-9.

BENNETT, M. R. & RAFTOS, J. (1977). The formation and regression of synapses during there-innervation of axolotl striated muscles. Journal of Physiology 265, 261-295.

BIESER, A., WERNIG, A. & ZUCKER, H. (1984). Different quantal responses within single frogneuromuscular junctions. Journal of Physiology 350, 401-412.

COUTEAUX, R. & PEcOT-DEcHAVASSINE, M. (1968). Vesicules synaptiques et porches au niveau deles 'zones actives' de la junction neuromusculaire. Compete rendus hebdomadaire des s8ances del'Academie des Sciences 271 2346-2349.

CUNNANE, T. C. & STJARNE, L. (1984). Transmitter secretion from individual varicosities ofguinea-pig and mouse vas-deferens: higher intermittent and monoquantal. Neuroscience 13,1-20.

271

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D 'ALONZO, A. J. & GRINNELL, A. D. (1984). Profiles of spontaneous release along the length of frognerve terminals. Neuroscience Abstracts 268, 13.

D 'ALONZO, A. J. & GRINNELL, A. D. (1985). Profiles ofevoked release along the length offrog motornerve terminals. Journal of Physiology 359, 235-258.

DAVEY, D. F. & BENNETT, M. R. (1982). Variation in the size of synaptic contacts along developingand mature motor terminal branches. Developmental Brain Research 5, 11-22.

DEL CASTILLO, J. & KATZ, B. (1954). Quantal components of the end-plate potential. Journal ofPhysiology 124, 560-573.

DEL CASTILLO, J. & KATZ, B. (1956). Localization of active spots within the neuromuscular junctionof the frog. Journal of Physiology 132, 630-649.

DE CINO, P. (1981). Transmitter release properties along regenerated nerve processes at the frogneuromuscular junction. Journal of Neuroscience 1, 308-317.

DESAKI, J. & UEHARA, Y. (1981). The overall morphology of neuromuscular junctions as revealedby scanning electron microscopy. Journal of Neurocytology 10, 101.

DREYER, F., PEPER, K. M., AKERT, D. M., SANDRI, C. & MOORE, H. (1973). Ultrastructure of the' active zone' in the frog neuromuscular junction. Brain Research 62, 373-380.

FATT, P. & KATZ, B. (1952). Spontaneous subthreshold activity at motor nerve endings. Journalof Physiology 117, 109-128.

FRANKENHAUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical propertiesof squid axons. Journal of Physiology 137, 218-244.

FUKUNAGA, H., ENGEL, A. G., OSAME, M. & LAMBERT, E. H. (1982). Paucity and disorganizationof presynaptic membrane active zones in the Lambert-Eaton myasthenic syndrome. Muscle andNerve 5, 686-687.

GRINNELL, A. D. & HERRERA, A. A. (1980). Physiological regulation of synaptic effectiveness atfrog neuromuscular junctions. Journal of Physiology 307, 301-317.

GUNDERSON, C. B., KATZ, B. & MILEDI, R. (1981). The reduction of end-plate responses bybotulinum toxin. Proceedings of the Royal Society B 213, 489-493.

HENNEMAN, E., LUSCHER, H.-R. & MATHIS, J. (1984). Simultaneously active and inactive synapsesof single I a fibres on cat spinal motoneurones. Journal of Physiology 352, 147-161.

HERRERA, A. A., GRINNELL, A. D. & WOLOWSKE, B. (1985). Ultrastructural correlates of naturallyoccurring differences in transmitter release efficacy in frog motor nerve terminals. Journal ofNeurocytology 14, 193-202.

HEUSER, J. E., REESE, T. S. & LANDIS, D. M. (1974). Functional changes in frog neuromuscularjunction studies with freeze-fracture. Journal of Neurocytology 3, 108-131.

HIRST, G. D. S. & NEILD, T. 0. (1980). Some properties of spontaneous excitatory junction poten-tials recorded from arterioles of guinea-pigs. Journal of Physiology 303, 43-60.

JACK, J. J. B., NOBLE, D. & TSIEN, R. W. (1975). Electrical Current Flow in Excitable Cells. Oxford:Clarendon Press.

JOHNSON, E. W. & WERNIG, A. (1971). The binomial nature of transmitter release at the crayfishneuromuscular junction. Journal of Physiology 218, 757-767.

KARNOVSKY, M. J. (1964). The localization of cholinesterase activity in rat cardiac muscle byelectron microscopy. Journal of Cell Biology 23, 217-232.

KATZ, B. & MILEDI, R. (1965a). The measurement of synaptic delay, and the time course ofacetylcholine release at the neuromuscular junction. Proceedings of the Royal Society B161,483-495.

KATZ, B. & MILEDI, R. (1965b). Propagation of electric activity in motor nerve terminals.Proceedings of the Royal Society B 161, 453-482.

KATZ, B. & MILDEI, R. (1973). The binding of acetylcholine to receptors and its removal from thesynaptic cleft. Journal of Physiology 231, 549-574.

LANNERGREN, J. & SMITH, R. S. (1966). Types of muscle fibres in toad skeletal muscle. Actaphysiologica scandinavica 68, 263-274.

LAURIE, R. E. & TREMBLAY, J. P. (1982). Boutons originating from the same axon do notparticipate equally in synaptic transmission. Neuroscience Letters 29, 135-140.

LLINAS, R. R. (1982). Calcium in synaptic transmission. Scientific American 247, 56-65.MCMAHAN, U. J., SPITZER, N. L. & PEPER, K. (1972). Visual identification of nerve terminals in

living isolated skeletal muscle. Proceedings of the Royal Society B 181, 421-430.MALLART, A. (1984). Presynaptic currents in frog motor endings. Pfluigers Archiv 400, 8-13.

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MILLER, T. M. & HEUSER, J. E. (1984). Endocytosis of synaptic vesicle membrane at the frogneuromuscular junction. Journal of Cell Biology 98, 685-698.

NUIDELL, B. M. & GRINNELL, A. D. (1982). Inverse relationship between transmitter release andterminal length in synapses on frog muscle fibers of uniform input resistance. Journal ofNeuroscience 2, 216-224.

REDMAN, S. & WALMSLEY, B. (1983). Amplitude fluctuations in synaptic potentials evoked in catspinal motoneurones at identified group Ia synapses. Journal of Physiology 343, 135-145.

SHOOTON, D. M., HEUSER, J. E., REESE, B. F. & REESE, T. S. (1979). Post-synaptic membranefolds ofthe frog neuromuscular junction visualized by scanning electron microscopy. Neuroscience4, 427-435.

TREMBLAY, J. D., ROBITAILLE, R. & GRENON, G. (1984). Distribution of spontaneous release alongthe frog neuromuscular junction. Neuroscience Letters 51, 247-252.

VERMA, V. & REESE, T. S. (1984). Structure and distribution of neuromuscular junctions on slowmuscle fibres in the frog. Neuroscience 12, 647.

WERLE, M. J., HERRERA, A. A. & GRINNELL, A. D. (1984). Ultrastructural uniformity alongbranches of frog motor nerve terminals. Neuroscience Abstracts 10, 919.

WERNIG, A. (1975). Estimates ofstatistical release parameters from crayfish and frog neuromuscularjunctions. Journal of Physiology 244, 107-221.

WERNIG, A. (1976). Localization of active sites in the neuromuscular junction of the frog. BrainResearch 118, 63-72.

YOSHIKAMI, D. & OKUN, L. M. (1984). Staining of living presynaptic nerve terminals with selectivefluorescent dyes. Nature 310, 53-56.

M. R. BENNETT, P. JONES AND N. A. LA VIDIS

EXPLANATION OF PLATES

PLATE 1

Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate with a short terminal branch. This branch showed a decline in me along its length (seeFig. 4A).

PLATE 2Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate with a short terminal branch. This branch did not show a decline in mie along itslength (see Fig. 4B).

PLATE 3Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate with a medium-size terminal branch. This branch showed a decline in me along mostof its length (see Fig. 5A).

PLATE 4

Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate with a medium-size terminal branch. This branch did not show a decline in me alongits length (see Fig. 5B).

PLATE 5Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate with a long terminal branch. This branch showed a shallow decline in me along itslength (see Fig. 6B).

PLATE 6Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate in which discontinuities occur in the localization ofcholinesterase along a DiOC2(5)-stainedterminal branch. This branch showed a decline in me along its length (see Fig. 10A).

PLATE 7Distribution of DiOC2(5)-stained fluorescent nerve terminal (A) and cholinesterase (B) for anend-plate in which discontinuities occur in the localization ofcholinesterase along a DiOC2(5)-stainedterminal branch. This branch showed a decline in mie along its length (see Fig. lOB).

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