546

J. Physiol. (I954) 125, 546-565

THE MEMBRANE CHANGE PRODUCED BY THENEUROMUSCULAR TRANSMITTER

BY J. DEL CASTILLO AND B. KATZFrom the Department of Biophysics, University College London

(Received 30 March 1954)

Until recently, it was generally believed that the action potential which anerve impulse sets up in a muscle fibre is identical with that produced by directstimulation. Recent work has shown that this is only true if the impulse isrecorded at a point remote from the neuromuscular junction. At the end-plate itself, the amplitude of the spike initiated by the synaptic transmitter issmaller than that due to a direct electric stimulus (Fatt & Katz, 1951, 1952 a, b;Nastuk, 1953). To explain this and other differences Fatt & Katz (1951)suggested that the chemical transmitter, acetylcholine (ACh), 'short-circuits'the end-plate membrane, i.e. produces a large increase of permeability to allions, in contrast with the selective change of sodium permeability whichoccurs during electric excitation (Hodgkin, 1951). The action potential at theend-plate region would be smaller than elsewhere because a local short-circuitwould merely reduce, and not reverse, the resting potential. But the changeof membrane conductance (i.e. of total ion permeability) would be greater atthe end-plate than at other parts of the fibre surface. The end-plate wouldthus become a focus of depolarization leading to a rapid discharge of adjacentparts of the fibre membrane and starting off a new wave of electric excitation.The short-circuit hypothesis is not the only way in which the features of

the junctional electric response might be explained. An alternative mechanismhas recently been proposed by Nastuk (1953), who suggests that ACh increasesthe end-plate permeability to sodium alone, but that the process becomes in-effective when the membrane potential reverses. Nastuk points out that thishypothesis would also explain a reduction of the spike potential but, in con-trast to the short-circuit theory, it requires that the membrane resistanceshould be higher during the crest of the reduced spike than during normalelectric activity. Measurements of the membrane resistance should thereforedecide between these alternative suggestions, and experiments of this kindwill be reported in the later part of this paper.

END-PLATE ACTIVITYThe initial object of the present work was to find the 'equilibrium level'

towards which the membrane potential is shifted by the neuromusculartransmitter. Previous experiments indicated that the equilibrium potentialof the active end-plate is well below that ofthe active muscle fibre and probablysomewhere near zero membrane potential (Fatt & Katz, 1951, p. 356). If thereaction between ACh and the receptor molecules produces a true short-circuit, then the end-plate membrane would cease to act as a selective ionbarrier, and the potential across it would decline, not immediately to zero,but towards a finite low level set by the free diffusion of ions across the end-plate surface. No direct measurements of this diffusion potential are available,but Nastuk & Hodgkin's (1950) observations indicate that the liquid junctionpotential between isotonic KCI and myoplasm is about 10 mV, and calculationsbased on either Henderson's (1907) or Goldman's (1943) treatment suggestthat the p.d. between Ringer and myoplasm would amount to 15-20 mV,the direction being the same as that of the resting potential.

Hence, if the short-circuit theory is correct, one would expect the end-platepotential (e.p.p.) to vanish if the resting membrane potential is lowered toabout 15-20 mV, and to reverse in sign if the membrane potential is displacedbeyond this point. To determine the reversal point experimentally, the fol-lowing procedure was used. A recording electrode was inserted into a musclefibre at the myoneural junction and a stimulus applied to the nerve-free endof the fibre. In addition an impulse was set up in the nerve so that its arrivalat the end-plate followed, or coincided with, that of the directly excitedmuscle spike. In this way the neuromuscular transmitter could be released atdifferent moments, and different levels, of the action potential, above or belowthe suspected equilibrium position of the e.p.p. If the instant of release occursbelow this level, we expect to find an e.p.p. which adds to the action potential;if the release occurs above this level, the e.p.p. should reverse and subtractfrom the potential wave.

METHODSThe method of intracellular recording at individual myoneural junctions has been described indetail by Fatt & Katz (1951). Frog's nerve-sartorius preparations were used, end-plates beinglocated by a preliminary recording of e.p.p.'s in curarized muscle. After removal of the drug, themuscle was soaked in a Ringer solution of high calcium content (usually 5.4 mm) to boost thesize of the e.p.p. (Fatt & Katz, 1952c; Castillo & Stark, 1952).The earlier recording technique was somewhat improved by shortening the connexions of the

microelectrode, and in some experiments the input capacity was further reduced by envelopingthe shaft of the electrode with a cathode-connected shield. During the experiment, a check waskept on the electrode resistance by observing the distortion of a voltage square-pulse applied tothe Ringer bath, and when necessary the microelectrode was replaced. The following modificationsof the previously described technique were used:

(a) Interaction of direct and nerve 8timvulation. In these experiments it was essential to comparethe action potential set up by direct stimulation alone (M spike) with one in which an e.p.p. wassuperimposed (MN potential). The aim was to obtain several records from the same end-plate,showing action potentials at different MN intervals as well as the M spike and the response to

35-2

547

548 J. DEL CASTILLO AND B. KATZa nerve impulse (N spike) alone. There were few experiments in which a long series could beobtained (as in Fig. 2 below); usually, after three or four twitches resting and action potentialsof the fibre had declined to a level at which it was useless to continue. Nevertheless, in manycases valid records were obtained for the analysis of at least one MN potential, and more completeinformation covering a range of intervals could be collected from different fibres.

Besides the usual difficulties encountered in recording intracellularly from twitching muscle,a source of special irritation was our inability to pre-set the MN interval precisely. This was due tothe fact that the time of arrival of the M spike at the junction varied from one record to the next,usually becoming earlier as the resting potential diminished. Two alternative methods of directstimulation were available: byinserting another microelectrode into the fibre, it could be stimulatedselectively. This has some advantage because the single-fibre contraction following the M spikeis very weak, but this is outweighed by the necessity of manipulating two internal electrodes andthereby increasing the risk of mechanical damage during the strong MN response (of the wholemuscle). We preferred therefore to use only one microelectrode, for internal recording, andstimulate the muscle with surface electrodes at the pelvic end. The bath was divided into twocompartments with the nerve-free end of the muscle passing through a communicating channel.A short pulse was applied to platinum electrodes on either side of the channel. Before insertingthe recording electrode, its tip was brought close to the surface of the selected junction. Externalaction potentials were observed on the oscilloscope, and the timing of M and N stimuli wasadjusted to give approximately the required interval. This was repeated sufficiently often andin a large enough number of experiments to provide an adequate assortment of MN records.

(b) Resistance changes during end-plate activity. These experiments were based on the methodused by Fatt & Katz (1951, p. 353). It consists in measuring the 'extrinsic' potential changewhich is produced inside the muscle fibre when a known direct current passes through the mem-brane. The current was applied through a second intracellular electrode inserted in the same fibreat a small distance, 50p or less, from the recording electrode.

In the steady-state condition (and disregarding the special case of the end-plate), the 'extrinsic'p.d. V, at the point of application, is related to the applied current I as V =Ilg, whereg = 2/A/ (rmri), rm being transverse resistance of fibre membrane x unit length and ri internallongitudinal resistance per unit length of fibre. In previous experiments (Fatt & Katz, 1951),V was measured, either in the resting muscle fibre, or as the additional p.d. produced by a currentduring the peak and falling phase of the action potential. It was shown that V falls during themuscle spike to a small fraction of the resting value, an effect which is due to a large increase ofmembrane conductance. In the present experiments, the added potential V is measured whena given current I passes through the junctional region of the fibre membrane, and a spike has beenset up by nerve or by direct stimulation. If the nerve-muscle transmitter produces an extrashort-circuit, of leakage resistance R, then the extrinsic p.d. should be further reduced, forV'=I/g', where g'=2/V (rmri) + IIR. The argument will only be used in a qualitative way, becauseduring the action potential the values of rm and R are changing with time. A steady-state condi-tion is therefore not attained, though it is approached fairly closely because of the short timeconstant of the active membrane (Fatt & Katz, 1951, p. 356).The technical difficulties in obtaining an adequate set of records were even greater for this

type of experiment than for the previous one. It was necessary to record at least four actionpotentials from the same end-plate: a pair of M spikes with and without superimposed extrinsicpotential and a corresponding pair of N spikes. After various trials, the following procedure wasadopted. The microelectrodes were inserted into the fibre and two M spikes were recorded, thesecond internal electrode being used to stimulate as well as to produce the extrinsic potential(see Fatt & Katz, 1951, p. 353). Record (i) was obtained with a short outward pulse of current,terminating during the rising phase of the spike. For record (ii) the current pulse was increasedin duration, and often also in strength, so as to build up a conveniently large extrinsic p.d. duringand after the spike. These two records could generally be made without serious damage, for theselected fibre alone contracted and its movement was reduced by fixation of the ends of themuscle. Record (iii) was an N spike which gave rise to the full twitch tension. Before this

END-PLATE ACTIVITY 549record was taken the current-delivering electrode was withdrawn from the fibre, for in our ex-perience the risk of damage during the twitch was much reduced when only one microelectrodewas inside the muscle fibre. If the fibre had survived all this and its resting potential was stillgood, record (iv) was made after re-insertion ofthe second electrode, and now an extrinsic potentialwas superimposed on the N spike. The time interval between nerve stimulus and direct currentpulse had been adjusted during the preliminary exploration of the curarized muscle so that thed.c. pulse started at about the same moment as, or during the rising phase of, the e.p.p. Havingobtained this set of four records, the experiment was continued by repeating as many of them aspossible. But the hazards of the experiment were such that only a very small proportion of thefibres survived the 'first round'.The current I was registered on the second beam of the oscilloscope by recording the p.d. across

a monitor resistance placed between bath and earth. Checks were made with the 'I' electrodeoutside and the ' V' electrode inside the fibre to reveal any recording artifacts due to a potentialdrop in the bath or an unbalance in the ' V' amplifier.Apart from the main difficulty, viz occurrence of mechanical damage before the necessary

records had been taken, there was a source of error which seriously affected a small proportion ofthe experiments. Occasionally, large inconsistencies were found in successive records in the valueof V/I, e.g. in one case after a preceding twitch the extrinsic p.d. was much reduced, although thecurrent intensity was a little larger, and the size of resting and action potentials had remainedunaltered. It is possible that this was due to imperfect retention of the current-supplying electrodein the fibre, with the result that part of the current by-passed the membrane during the secondrecord. It is easy to reject records which show such obvious inconsistencies; the difficulty,however, was to decide whether less conspicuous changes may not have vitiated other experiments.It was particularly important to make certain that the comparison of the two extrinsic potentialsadded, respectively, to M and N spikes was valid and unaffected by such a 'leakage-error'. Aswill be pointed out on p. 659 below, the only safe way of overcoming this difficulty was to assuMethat the membrane resistance reaches the same value during the late parts of the M and Npotentials, so that any observed residual disparity of V/I may be taken as a measure of the'leakage-error' for which the earlier parts of the records should be corrected. In principle, thiscould have been checked by measuring the resting potential with the current-passing electrode,but in practice the switching of the internal electrode is apt to cause stimulation of the fibreby a.c. pick-up.

RESULTSA. Interaction between transmitter and muscle spike

In these experiments an action potential was set up in the muscle fibre ata distant point and allowed to travel to the junction; in addition a nerveimpulse was timed to arrive at the junction during the passage of the musclespike. Normally, the transmitter released by the nerve impulse gives rise toan e.p.p. from which a reduced muscle spike takes off. What happens whenthe transmitter is released during the peak of the electric response of the fibremembrane? Does it still produce its characteristic effect and cut down theamplitude of the action potential, or are the end-plate receptors preoccupiedand made refractory by the electrical activity which has just reached thejunctional region?The answer will be evident from Fig. 1: the immediate effect of the trans-

mitter is to reduce the action potential which falls to a transient plateau some10 mV below the zero line. The initial deflexion which the nerve impulse hasproduced in this record is the opposite of the normal rise of the e.p.p. and itappears that the underlying ionic currents have reversed sign.

J. DEL CASTILLO AND B. KATZIn Fig. 2 a series of responses are shown, all from the same end-plate, but

at different MN intervals. Tracings of these records have been superimposedin Fig. 3 (of. Castillo & Katz, 1954 a). It is instructive to compare records 3, 4and 7 of Fig. 2. Record 3 shows M and N spikes side by side. It shows thecharacteristic differences between the two action potentials (large e.p.p. stepduring the rise and a 'hump' after the peak of the N-response) and confirmsthat the N response fails to reach the peak amplitude of the M spike (Fatt &Katz, 1951; Nastuk, 1953). In record 4, M and N stimuli were combined,keeping the time interval the same as in 3, so that the e.p.p. would startshortly after the peak of the M spike. As in Fig. 1, the immediate effect ofthe transmitter is to produce a reversed deflexion, a fall instead of a rise ofthe internal potential. If we now delay the nerve impulse a little so that theresponse starts about half-way down on the falling phase of the M-spike(record 7), we obtain an e.p.p. of diminished size but normal direction.

Fig. 1. Effect of the transmitter released during a directly excited muscle action potential.Intracellular recording from end-plate of frog's sartorius. Temp. 190 0. The larger of the tworecordls is a simple M spike. Arrow indicates commencement of N response, just before thecrest of the M sjpike.

It appears that there is a reversal point of the e.p.p. som'ewhere betweenthe two levels of membrane potential marked by the arrow in records 4 and 7.Intermediate MN intervals were examined in several other experiments, andone may summarize them by saying that there is a critical range between 0 andabout 25 mVY, negatilve inside, below which the 'e.p.p.' adds to, and abovewhich it subtracts from the muscle spike. This range can be narrowed if weexamine, not only the initial rate of the N response, but the maximu or thetransient plateau which is attained some 0-5-l msec later. Records 4 (or 9)and 7 in Fig. 2, and the superimposed tracings serve to illustrate this point.In records 4 and, 9, the release of the transmitter causes the action potentialto fall to a nearly horizontal level which it reaches after about 0-75 msec. Thislevel is, approximately, at -10 mV in 9, and -17 mV in 4. In record 7, thee.p.p. rises and attains a maximum after about 0-7 msec, at a level of -19 mV.This result was confirmed in several other experiments: we were never able to

550

END-PLATE ACTIVITY 551

make the e.p.p. rise above a level of 10 to 20 mV below the zero line, and whenit started at higher levels it produced a rapid fall of potential to a plateau some10 mV below the zero line. Presumably the 'equilibrium level' of the e.p.p.,i.e. the final leveltowards which the transmitter moves the membrane potential,is somewhere between the levels which the e.p.p. attains from above or below.

Some comments are required on the procedure which we used in measuring and superimposingthe tracings of M and MN responses (Fig. 3). In our best records when a MN potential was

Fig. 2. A series of records from a single end-plate illustrating MN interaction at different intervals.M spikes in 1, 3 and 5; N spikes in 2 and 3. In all MN records, the commencement of theN response is shown by an arrow. Zero-potential lines are shown in all records except 8, inwhich the ele'ctrode resistancewas excessive and the level uncertain. Time marks: milliseconds.

compared with an immediately preceding or following M spike, there was no appreciable differencein resting or action potentials up to the moment of intervention of the N response. There was

usually a small change in latency of the.M spike which was compensated by shifting one recordalong the time base. If resting and action potentials had declined between the records, it wasoften still possible to use them, for the active-membrane potential is not immediately affected bya small depolarization of the fibre. When such records are superimposed it is often found that,in spite of a base-line discrepancy of 10-20 mV, the top halves of the spike potentials with whichwe were concerned still fit satisfactorily (see Fatt & Katz, 1951, p. 354).

A limitation of the present analysis was that little can be said about theeffect of the MN interaction during the steep part of the ascent of the spike.It was impossible to determine with any degree of assurance at what point of

552 J. DEL CASTILLO AND B. KATZ

the rising phase the e.p.p. began to reduce, instead of increase, the rate of thespike potential. It is clear, e.g. from record 6, Fig. 2 and from Fig. 3 B, thatthe transmitter does not prevent the increase of the spike potential above

M +40A +20

0

/ -120IMN -4

~~~~~ ~~~-60-80

1001 msec mV

B M

MN

C M

MN

E

MN

Fig. 3. Superimposed tracings of the experiment of Fig. 2, showing the effect of the transmitter atvarious moments of the muscle spike. Arrows indicate beginning of N response. Record D issomewhat distorted and its resting potential uncertain, because of a faulty electrode insertion.

the end-plate equilibrium level, though within a fraction of a millisecondit reduces and reverses the rate of ascent. A more precise analysis during therising phase was prevented by inaccuracies of our recording technique.

END-PLATE ACTIVITY

The results of this section are summarized schematically in Fig. 4 in whicha typical propagated action potential of the muscle fibre is shown exceedingthe zero by about 35 mV. The equilibrium potential of the active end-plateis indicated by a horizontal bar cutting the M spike at -10 to -20 mV. Thearrows indicate the direction of the potential change which follows the releaseof the nerve-muscle transmitter at different moments. During the early partof the rising phase, the e.p.p. adds to, and accelerates the rise of the spike(e.g. record 1, Fig. 2; see also Eccles, Katz & Kuffler, 1941; Kuffler, 1942 b).Similarly, during the later part of the falling phase the e.p.p. adds to theremainder of the action potential and, with increasing MN intervals, graduallygrows in amplitude (cf. Eccles et al. 1941, p. 376; Kuffler, 1942a). During the

mV+40

+20 -

-20 - / 4 // i

-40 -i ; \ f

-

8

msec

Fig. 4. Diagram illustrating the direction of the e.p.p. which the neuromuscular transmitterproduces if it is released at different phases of the muscle spike. The arrows indicate thedirection and relative magnitude of the potential change due to the release of the transmitter.The shaded band shows the approximate level at which a reversal of sign of the e.p.p. occurs.

peak portion and throughout the phase of reversed membrane potential, thetransmitter reduces the action potential, an effect not previously describedthough it was to be expected from the observations of Fatt & Katz (1951) andNastuk (1953).The interpretation presented in Fig. 4 is tentative and can be challenged on

several grounds. Although it appears that the reversal of the e.p.p., duringthe rise and fall of the action potential, depends simply on the position of themembrane potential, and is related to a unique, time-independent, level, thismay be fortuitous. It is possible that the true relations are more complicated,but for the present our scheme can be put forward as a simple and adequateway of explaining the observed phenomena.

It might be queried why the equilibrium level indicated in Fig. 4 is lowerthan the plateau or 'hump' in the N response itself which quite often occurs

553

J. DEL CASTILLO AND B. KATZseveral millivolts above the zero level (Fatt & Katz, 1951). This 'hump',however, is not a reliable index of the end-plate equilibrium potential, for itsexact position is the resultant of two different modes of electric activity, vize.p.p. and spike, and depends on the relative intensity of the two processes atthat particular moment. In our experience, the position of the 'hump' varieswith the intensity of the transmitter action; it is higher the lower the initialrate of the e.p.p. (i.e. the weaker the transmitter effect). This can be seen notonly by comparing records from different junctions, but also quite clearly ina single fibre when the recording electrode is moved a fraction of a millimetreaway from the end-plate (see Fatt & Katz, 1951, fig. 21). We consider there-fore that the end-plate equilibrium potential is not reached during the 'hump'of the N response, and that the lowest position of the hump which can beregistered (about -10 mV) represents the nearest approach to the equilibriumlevel.

B. The membrane resistance during spike and e.p.p.The results of §A indicate that the end-plate potential reaches an equilibrium

level at -10 to -20 mV. This is clearly compatible with the short-circuithypothesis but does not exclude alternative suggestions such as that putforward by Nastuk (1953). Nastuk supposes that the end-plate receptors arespecific 'sodium-carrier' molecules which may be activated either by directelectric stimulation or by combination with ACh. The sodium-carrying powerof the receptors would depend on the level of the membrane potential and bemodified by ACh in such a way that the receptors become effective at thenormal resting potential but relatively ineffective when the membranepotential reverses. The reduction of theN spike is explained by inefficacy oftheACh-combined receptors to maintain a high Na-permeability of the membranewhen the action potential crosses the 'zero-line'. Against this hypothesis onemight argue that mere failure of the end-plate receptors to contribute to thespike is unlikely to diminish its amplitude, for the myoneural junction occupiesonly a small part on the fibre surface, and a low conductivity of this spot,surrounded by active fibre membrane, would have little effect on the mem-brane potential. This kind of argument was taken further by Eccles (1953,pp. 78, 86) who pointed out that the ionic current observed during the e.p.p.requires a very large increase of conductivity at the end-plate, much greaterthan could be accounted for by the rise of sodium permeability found in theactive fibre membrane. Eccles suggests that anything but a short-circuit ofthe kind postulated by Fatt & Katz (1951) would not provide an adequateexplanation of the e.p.p.

This general argument, however plausible, is not decisive because it is basedon the implicit assumption that the membrane area on which the chemicaltransmitter acts corresponds, at least approximately, to what we can see underthe microscope. Although this is a reasonable assumption, it may yet be found

554

END-PLATE ACTIVITY

that the true contact area is much greater than the histological picture reveals,and the whole argument would then lose its force.A crucial test between 'short-circuit' and 'modified sodium-carrier' hypo-

thesis requires a measurement of the changes in membrane resistance duringM and N spikes, as has been pointed out by Nastuk (1953, p. 270).The method which we used for this purpose was described on p. 548. It

depends on a comparison of four records obtained successively from a singleend-plate and is illustrated in Fig. 5. Record (a) is a muscle spike (M) startedby a brief direct-current pulse. Record (b) (M+D.C.) was obtained by main-taining the current and superimposing a 'catelectrotonic' potential on thespike. Records (c) and (e) are N responses showing the usual properties (end-plate step, reduced peak and 'hump'). Record (d) (N+ D.C.) is anN spike withsuperimposed 'catelectrotonic' potential. The applied current strength wasregistered in the lower traces of records (a), (b) and (d).

There are certain obvious differences between the electrotonic potentialsadded toM andN spikes respectively. It is clear, for instance, that during thepeak portion of the action potential an applied current produces less additionto the N, than to theM spike. During the falling phase, the difference becomesless noticeable. In Figs. 6 and 7 other experiments of this type are illustratedshowing similar results.The change in the added 'extrinsic potential' can be appreciated better

when tracings of spikes, with and without current, are superimposed. In Fig. 8experiments on two end-plates are illustrated in the upper and lower sets oftracings; on the left are M spikes, with and without electrotonic potential; onthe right are corresponding N spikes. In superimposing these action potentials,a common time must be chosen at some point of the rising phase. We took,somewhat arbitrarily, the moment at which the spike crossed the zero line(which is nearly synchronous with the point of its maximum slope). Althoughthis may not be strictly justified on theoretical grounds, we can see no im-portant practical objection to this procedure. The latitude of adjustment in thesuperposition of the two tracings is evidently very small (see also Fatt & Katz,1951, p. 354), and a slight temporal displacement of the rising phases would notseriously affect measurements of the added potentials during the peak andfalling phase. The only region in which our method of superposition gives novalid answer is the steep part of the ascending phase, and this would in anycase be difficult to analyse with the present technique.To evaluate the results, two methods were used.(a) The added potential was measured-with both, M and N, responses-

at a fixed interval after the common zero-potential point. The most suitableinterval was near the peak ofthe 'M+ D.C.' potential, when the added potentialhad reached a relatively large and easily measurable amplitude (cf. Fatt &Katz, 1951). In different experiments, this interval was between 0x6 and

555

J. DEL CASTILLO AND B. KATZ

1-2 msec after the 'zero-point'. The same interval was chosen for the N and'N +D.C.' potentials where it generally coincided with the beginning of theflat portion following the peak of the N response. All measurements were

1M~~

Fig. 5. 'End-plate resistance' during M and N spikes. Five succesive records, from the sameend-plate, of responses to direct (M) and nerve (N) stimuli. For further explanation see text.A zero-potential line is shown with each record. The square pulses under records a, b and dindicate intensity and duration of the applied outward currents.

Fig. 6. Fig. 7.Figs. 6 and 7. End-plate resistance during M and N spikes. Two further examples of the

same type of experiment as in Fig. 5.

made within the region of reversed membrane potential, that is, well withinthe region in which the transmitter gives rise to a reversed end-plate current(section A). Method (a) was simple and fairly accurate because the action

556

END-PLATE ACTIVITY 557potentials were compared at a time when their rates of change were small,and their differences in amplitude relatively large.

(b) In order to avoid errors which might arise from the arbitrary choice ofa single time interval, the mean addition was determined in several experi-ments during the phase of reversed membrane potential. The same period was

M N

Ao

1 msec

. ~~~I 9

8 ~~~~~~~~~~18mV

--Jo I 1 msecOSuAI I

Fig. 8. Superimposed tracings illustrating potential and resistance changes of the active end-plate during direct (M) and nerve (N) stimulation. A and B: records from different end-plates. The superimposed tracings show the additional potential due to an applied currentpulse, registered in the lower part. Ordinary N spikes were recorded before and after addinga direct current pulse.

chosen for both M and N records: it started at the common 'zero-point' andextended over about 1-2 msec, the exact length being determined in eachexperiment by the duration of reversed potential of the N spike.

Results obtained by the first method are shown in Table 1A. The size ofthe 'extrinsic potential' (mV) divided by the strength of the applied current(,uA) gives an approximate measure of the momentary resistance (kQ) acrossthe end-plate membrane. The mean value for the M spike was 2*2 times

J. DEL CASTILLO AND B. KATZ

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558

END-PLATE ACTIVITY 559

greater than for the corresponding instant of the N spike. This ratio remainedunaltered when four suspect experiments were rejected (because of errors dueto one of the following causes: leakage of polarizing electrode; uncertaintyabout level of zero potential; faulty localization of end-plate).The absolute values of the 'end-plate resistance', approximately 55,000 Q

for M, and 30,000 Q for N, are not accurate and, therefore, of little interest.Their inaccuracy is due to the fact that they were measured during anunsteady state before the extrinsic potential had reached its full value. Thisdoes not, however, reduce the significance of the observed difference betweenthe two values: one might expect the true difference, between the' equilibrated'values, to be even greater, for the full development of the electrotonic potentialwould take somewhat longer during the less leaky state associated with theM spike.

The value of 55,000 found here is high compared with the average of 21,000 Q)observed duringthe falling phase oftheM spike by Fatt & Katz (1951). The difference is largely due to the differentmoments of the spike at which the resistance was measured. The value obtained by Fatt & Katzwas a minimu&m to which the resistance fell during the decline of the spike. In the present experi.ments an earlier interval was chosen, close to the transient maximum of the membrane resistancewhich occurs shortly after the peak of the M spike (cf. Fatt & Katz, 1951, fig. 27).

Certain sources of error had to be considered which might influence or scatter the results. Anerror mayarise from extracellular leakage ofcurrent at the tip ofthe current-passingmicroelectrode,as discussed on p. 549 above. A small leakage would be very difficult to check; it would result inan underestimate of the end-plate resistance which may be different each time the electrode isreinserted. Although this source of error could not be completely eliminated, it was important toremove any influence which it might have on the comparison of M and N. A fair way of doingthis is to ignore any residual action of the transmitter at the end of the applied current pulse(about 5 msec after the start of the spike). We assume, in other words, that the membraneresistance has, by this time, reached the same value, independently of the initial differencesoccurring during the peak ofM and N spikes. Any difference observed at the end of the pulse wasascribed to an error in the measurement of the applied membrane current, owing to the suspectedextracellular leakage of the electrode. We have used the 'terminal ratio' of N and M resistances,at the end of the applied current, as a correction factor and recalculated the M/N ratio during theearlier phases of the response: e.g. if the observed N resistance at the end of the 5 msec pulse was10% less than the M resistance, we attributed this to an error and multiplied the observedM/N ratio by a factor of 0 9. The effect of this correction is shown in the last column of Table 1.We obtain a slightly lower mean ratio (2{0-2-1, instead of 2.2) and a more markedly reducedstandard deviation. It is possible, therefore, that an error of this kind may have contributed tothe random scatter of the results without, however, significantly biasing them.Another possible criticism concerns the sequence in which the records were usually made (i.e.

M, M + D.C., N, N + D.C., etc., see p. 548). During a series of stimuli, deterioration occurscausing resting and action potentials to decline. In our best experiments the decaywas negligible(Fig. 8), but the more usual effect can be appreciated from the average resting potentials in Table 1(M: 82 mV; M +D.C.: 79 mV; N: 78 mV; N +D.C.: 74 mV). What probably happens is thatwith each twitch the leakage of the fibre membrane around the impaled electrodes becomes a littleworse and shunts the resting e.m.f. more effectively. Does this progressive change invalidate thecomparison ofM and N in our average results?

Suppose the e.m.f. of the resting membrane is 90 mV, the resistance across it 200,000 QL(Fatt &Katz, 1951; see also the circuit diagrams in Fatt & Katz, 1952a, fig. 10 and Eccles, 1953, fig. 26),

J. DEL CASTILLO AND B. KATZand the diffusion potential across a 'short-circuited' membrane 15 mV. Then a reduction of theresting potential to 79 mV (M + D.C.) would be equivalent to a shunting of the membrane witha local leak of 200,000 x (79 - 15)/(90-79) = 1*16 MO. A reduction to 74 mV (N + D.C.) could beattributed to a shunt of 0 74 MQ. Our measured values (55,000 and 31,000 Q- during M and Nspikes respectively) include these leakage artifacts (viz. 1-17 and 0 74 MQ respectively). In theabsence of such shunting, the end-plate resistances would increase to 57,700 and 32,400 Q2, forM and N respectively, retaining the same ratio. This is a simplified argument, but it suggeststhat little importance need be attached to the decline of the resting potential in these experiments.

In eleven experiments method (b) was used, the resistance across the end-plate being averaged over a period of about 1-2 msec during the peak of thespike. The results are listed in Table 1 B which shows the observed ratio ofMIN resistances, also the effect of the correction for suspected leakage currentdiscussed on p. 559. The four most satisfactory experiments in which therewas little or no decline of the resting potential are marked with asterisks, andtheir mean ratio is shown separately at the end of the table. It was reassuringto find that the MIN ratio was well above the average in these four selectedexperiments.Among the other experiments there were a few which gave inconclusive

results, the ratio of M/N resistances being near unity. The validity of theseexperiments was questionable but their rejection made little difference to themean result (see p. 559). A point of interest was that, while some experimentswere inconclusive, none of them supported the alternative theory according towhich the MIN ratio should be less than unity above, and greater than unitybelow the zero-potential line. In those cases where the ratio did not signi-ficantly differ from unity at the peak of the spike, it either remained at thatlevel or diminished during the falling phase.The results of these experiments show that the release of the transmitter

causes not only a reduction in the peak amplitude of the spike, but a simul-taneous increase of membrane conductance, a conclusion which agrees withthe short-circuit hypothesis, but not with the alternative mechanism proposedby Nastuk (1953).

C. Other experimentsIn order to check the conclusions arrived at in §A, various attempts were

made to discover whether, in the absence of a spike, an e.p.p. can be drivenbeyond the zero-potential line. We tried to superimpose the e.p.p. on a mem-brane which was kept depolarized by the passage of a strong and prolongedoutward current, but the experiment was abandoned because such currentsproduced contracture and rapid injury.We then tried to raise the level of the 'pure' e.p.p. by pharmacological

means and by repetitive stimulation. One way of doing this is to block trans-mission by an excess of both calcium and magnesium ions which lowers theexcitability of the muscle fibre and so reveals large e.p.p.'s without interven-tion of spikes. With a solution containing 20 mM-Mg and 7-2 mM-Ca (replacing

560

END-PLATE ACTIVITYabout 30% of the normally used NaCl concentration), 'pure' e.p.p.'s of up to60 mV amplitude were recorded in fibres whose resting potential was 90 mV.Two nerve impulses at an interval of 3-4 msec set up a muscle spike in somefibres when the amplitude of the e.p.p. exceeded 60 mV. The second e.p.p. wassmaller than the first and added relatively little (Fig. 9). This contrasts withthe well-known facilitation observed at lower peak levels of the e.p.p. (Eccleset al. 1941; Castillo & Katz, 1954b) and indicates that the e.p.p. in thepresent experiments is approaching a 'ceiling'.

a ~~~~~~~~

Fig. 9. End-plate potentials from muscle fibres treated with excess calcium and magnesium.Double nerve impulses in all records except in C2. A: e.p.p.'s at varying short intervals.B and C: e.p.p.'s and spikes. Zero-potential lines are shown in B and C1. C2 shows singlee.p.p. followed by a delayed spike (propagated from a remote junction).

Results of greater significance were obtained when the response to two nerveimpulses was recorded in normal muscle (Fig. 10). The earliest change in theaction potential, brought about by N2, was a second hump at a level slightlybelow the zero line (mean level in seven experiments: -14 mV, S.E. + 1'4 mV).When the experiment was repeated on a prostigmine-treated muscle, thehump lengthened to a plateau whose highest observed level was at -18 mV(Fig. 11). Additional repetitive nerve impulses did not raise the level of thee.p.p. further (cf. Fig. 11), but the records are difficult to interpret after thefirst 5-10 msec because of complication by movement artifacts.To summarize, our attempts to 'drive' the e.p.p. beyond the equilibrium

level indicated by the experiments of §A failed, and the present results stillremain well within the limits set by the short-circuit theory.

DISCUSSION

The main result of this paper is that the neuromuscular transmitter reducesthe resistance as well as the potential of the active fibre membrane. The effectof the transmitter is to lower the membrane potential towards a level of10-20 mV below zero, and this occurs even when the transmitter is releasedduring the peak of a directly initiated muscle spike.

36 PHYSIO. CXXV

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562 J. DEL CASTILLO AND B. KATZ

Fig. 10.

Fig. 11.

Fig. 10. Effect of two nerve impulses. A: upper record shows response to a single impulse, lowerrecord to 2 impulses at short interval. The response to the second impulse is seen as a secondhump beginning at the arrow. B: examples of N1N2 responses from other end-plates, thestart of the N2 potential being indicated by arrows.

Fig. 11. Response to repetitive nerve impulses at prostigmine-treated end-plates. A and B: upperrecords show single, lower records double responses. The second impulse merely raises thelevel of the residual depolarization after the spike. C: response to single and five repetitivenerve impulses (successive stimuli marked by dashes) at another end-plate. Slower timebase. There is evident interference by movement artifacts, but the level of depolarizationdoes not appreciably exceed that observed in A and B.

* - - -Z Z

END-PLATE ACTIVITYThe results are in satisfactory agreement with the predictions of the short-

circuit hypothesis (Fatt & Katz, 1951), but they do not prove it. In the firstplace, neither the 'equilibrium potential' of the active end-plate nor thediffusion potential between myoplasm and surroundings are known accuratelyenough to be certain of their identity. Secondly, there are various possibilitiesbeside a simple short-circuit which may equally well account for the observedresistance and potential changes. For example, a simultaneous increase ofsodium and chloride, or sodium and potassium permeability at the end-platewould produce indistinguishable results. What seems certain is that a selectiveincrease of permeability to sodium alone cannot explain the transmitter effect.The suggestion of a localized short-circuit reaction at the motor end-plate isa simple, but provisional, substitute for that hypothesis.Once we are forced to invoke a simultaneous increase of permeability to more than one species

of ions, it is perhaps simpler to envisage a reaction which leads to the breakdown of an ion barrierrather than one which involves a multiplicity of specific permeability channels. There remainsalso the factthat in the absence of sodium appliedACh can still produce a measurabledepolarization(Fatt, 1950; Nastuk, 1953). This effect would be difficult to explain by increased permeability tosodium, chloride or potassium, and seems to require the transport of yet another ion: e.g. entryof ACh+ itself or, more probably, outflow of internal anions. The observation of this effect is,therefore, another point in favour of a general rather than selective increase of end-plate per-meability. A further study of the action of ACh in the absence of external sodium salts may helpto clear up this problem. The results so far reported have been variable, and it has been difficultto make quantitative predictions, for the theoretical 'short-circuit potential' between myoplasmand outside solution would depend critically on the unknown behaviour of intracellular anionswhen NaCl has been replaced by a non-electrolyte. A method has recently been described byCoombs, Eccles & Fatt (1954) by which the internal anion composition may be changed. It ishoped that an application of this method to the end-plate will throw further light on this matter.

The results described in §A show that the transmitter changes the membranepotential even vhen the release coincides with the peak of electric activity inthe muscle fibre. It appears, therefore, that the receptor molecules of theend-plate are either not engaged in the production of the muscle spike or,if they are, not thereby rendered refractory to a subsequent action of thetransmitter.

It has previously been suggested by Fatt & Katz (1951) that the moleculeswith which ACh combines are specific chemo-receptors which differ from thepostulated 'sodium-carriers' in the surrounding muscle membrane and do notparticipate in the process of electric excitation. The present experiments showthat the excitatory process during the muscle spike fails to preclude the reactionbetween end-plate and transmitter and, therefore, support the view that thetwo mechanisms are independent.

There remains the alternative that the end-plate receptors can respond bothto ACh and to electric stimulation. To maintain this view, one would have toassume that (i) the receptor molecules can participate in two membranereactions leading to different equilibrium potentials and (ii) their affinity to

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J. DEL CASTILLO AND B. KATZACh is so overwhelming that in its presence electric excitation of the receptorscannot occur or proceed. It seems to us that the assumption of independent' chemo-' and 'electro-receptor' molecules is simpler, but there is at present nocritical evidence to decide this matter.

SUMMARY

1. The electrical changes at the motor end-plate have been further investi-gated with the intracellular recording technique.

2. When a nerve impulse arrives at the junction during the passage ofa directly initiated muscle spike (M) it causes the active membrane potentialto move towards a level of about 10-20 mV below the line of zero p.d. If thetransmitter is released at the peak of the M spike, it causes ion current to flow'outward' and the action potential to fall; if the transmitter is released belowthat critical level, it gives rise to 'inward' ion current and to an e.p.p. whichadds to the M spike. The reversal occurs at a level which is close to the esti-mated free diffusion potential between fibre contents and Ringer solution.

3. The resistance across the active end-plate was measured by passinga current pulse through the membrane and observing the added p.d. Themembrane resistance is greatly reduced by the action of the neuromusculartransmitter. The indirect response to a nerve impulse (N spike) is associatedwith a more severe breakdown of the membrane resistance than the responseto a direct stimulus (M spike), in spite of the fact that the voltage amplitudeof the M spike is larger.

4. During repetitive nerve stimulation of normal or prostigmine-treatedmuscle, the end-plate potential could not be made to rise above a level of10-20 mV below the 'zero line'.

5. The evidence presented in this paper is incompatible with the hypothesisthat the neuromuscular transmitter may act on sodium permeability alone.It requires an additional, and simultaneous, increase of permeability to otherions and, to this extent, supports the 'short-circuit' hypothesis previouslyput forward. There is, however, at present no evidence to decide betweena selective increase of permeability to several species of ions and a short-circuitproper, i.e. complete breakdown of a local ion barrier.

We are indebted to Mr J. L. Parkinson for his invaluable assistance. This work was supportedby a research grant from the Nuffield Foundation.

REFERENCESDEL CASTILLO, J. & KATZ, B. (1954a). Potential and resistance changes at the motor end-plate.

J. Phy8iol. 123, 70-71 P.DEL CASTILLO, J. & KATZ, B. (1954b). Statistical factors involved in neuromuscular facilitation

and depression. J. Phy8iol. 124, 574-585.DEL CASTILLO, J. & STARK, L. (1952). The effect of calcium ions on the motor end-plate potential.

J. Physiol. 116, 507-515.

564

END-PLATE ACTIVITY 565COOMBS, J. S., ECCLES, J. C. & FATT, P. (1954). The action of the inhibitory synaptic transmitter.

Au8t. J. Sci. 16, 1-5.ECCLES, J. C. (1953). The Neurophysiological Basis of Mind, chapter m. Oxford: Clarendon Press.ECCLES, J. C., KATZ, B. & KUFFLER, S. W. (1941). Nature of the 'endplate potential' in curarized

muscle. J. Neurophy8iol. 5, 362-387.FATT, P. (1950). The electromotive action of acetylcholine at the motor end-plate. J. Physiol.

111, 408-422.FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intra-cellular

electrode. J. Physiol. 115, 320-370.FATT, P. & KATZ, B. (1952a). The electric activity of the motor end-plate. In: Discussion on

excitation and inhibition. Proc. Roy. Soc. B, 140, 183-186.FATT, P. & KATZ, B. (1952b). Some problems of neuro-muscular transmission. Cold Spr. Harb.

Symp. quant. Biol. 17, 275-280.FATT, P. & KATZ, B. (1952 c). The effect ofsodium ions on neuromuscular transmission. J. Physiol.

118, 73-87.GOLDMAN, D. E. (1943). Potential, impedance, and rectification in membranes. J. gen. Physiol.

27, 37-60.HENDERSON, P. (1907). Zur Thermodynamik der Fliissigkeitsketten. Z. phys. Chem. 59, 118-127.HODGKIN, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26,

339-409.KUFFLER, S. W. (1942a). Responses during refractory period at myoneural junction in isolated

nerve-muscle fibre preparation. J. Neurophysiol. 5, 199-209.KuFFLER, S. W. (1942b). Further study on transmission in an isolated nerve-muscle fibre

preparation. J. Neurophysiol. 5. 309-322.NASTUK, W. L. (1953). The electrical activity of the muscle cell membrane at the neuro-muscular

junction. J. cell comp. Phy8iol. 42, 249-272.NASTUK, W. L. & HODGKIN, A. L. (1950). The electrical activity of single muscle fibers. J. cell.

comp. Physiol. 35, 39-73.