J. Phy&iol. (1976), 262, pp. 553-581 With 6 text-ftgure8 Printed in Great Britain

553

SPONTANEOUS SUBMINIATURE END-PLATE POTENTIALS IN MOUSE DIAPHRAGM MUSCLE: EVIDENCE FOR SYNCHRONOUS RELEASE

By M. E. KRIEBEL, F. LLADOS AND D. R. MATTESON From the Department of Physiology, Upstate Medical Center, State University of New York, 766 Irving Avenue, Syracuse, New York 13210, U.S.A.

(Received 9 February 1976) SUMMARY

1. Miniature end-plate potentials (min.e.p.p.s) were recorded from small muscle cells of mouse diaphragms. Min.e.p.p. amplitude histograms showed successive peaks which were integral multiples of the smallest peak. The smallest potentials (submin.e.p.p.s) averaged 0-3-0-6 mV and the mean of the larger min.e.p.p.s averaged 3-7 mV, depending on the muscle cell diameter. There was a positive correlation between time-topeak and min.e.p.p. amplitude. Time-to-peak of the submin.e.p.p.s fell slightly below the regression line through the larger min.e.p.p.s. 2. Sometimes min.e.p.p. amplitude distributions changed spontaneously such that the mean of the major mode min.e.p.p.s decreased twofold during which time the mean of the submin.e.p.p.s did not change. Spontaneous decreases were most pronounced during low frequencies of release (10/min) achieved at 320 C. 3. Small changes in temperature (2° C steps in the range 32-40° C) greatly altered the number of peaks of min.e.p.p. amplitude histograms without noticeably changing the position of the submin.e.p.p. peak. At 320 C submin.e.p.p.s composed 5-20 % of the histograms and the amplitude of the major mode peak was twelve to fifteen-times that of the submin. e.p.p.s. Over-all bell-shaped distributions were obtained at 370 C which showed up to eight peaks with the major peak at the fourth to sixth peak. Temperatures slightly above 37° C gave a flat distribution with the mean amplitude at the third peak. Min.e.p.p. amplitude histograms were initially skewed (mostly small min.e.p.p.s) after a 40° C heat challenge. 4. Two to eight-times the normal concentration of Ca2+ in the saline reversibly increased the min.e.p.p. frequency and also decreased the mean of the major mode min.e.p.p.s (two to nine-times) without noticeably changing the mean of the submin.e.p.p.s.

MAHLON E. KRIEBEL AND OTHERS 554 5. Botulinum toxin A, 105 x intraperitoneal median lethal dose (1 05 i..LD,)/ml., almost abolished. min.e.p.p.s in 30-90 min. The relative proportion of submin.e.p.p.s increased and the mean of the major mode min.e.p.p.s usually did not change during the initial decrease in frequency. Major mode min.e.p.p.s essentially ceased after 200-1000 were generated and remaining min.e.p.p.s of some cells showed skewed distributions with three small peaks that were integral multiples of the submin.e.p.p. peak. Smaller min.e.p.p.s were more resistant to block than the larger min.e.p.p.s and, although frequencies were low, small min.e.p.p.s were recorded after 4 hr of botulinum toxin incubation. 6. Colchicine (5 x 104 M) within minutes reduced the major mode min. e.p.p.s by half (mean of major peak reduced to sixth or seventh peak). Additional colchicine (10-3M) reduced the major mode min.e.p.p. amplitude to a fifth of that of control (mean of major mode min.e.p.p.s at the third peak) with no change in position of the submin.e.p.p. peak. Min.e.p.p. amplitudes slowly recovered to half control values after washing. 7. The multiple peak distributions and the changes in the number of peaks that were integral multiples of the smallest peak indicated that most spontaneous min.e.p.p.s were composed of subunits and that the release of a single subunit generated the submin.e.p.p. INTRODUCTION

Cooke & Quastel (1973) noticed a class of small miniature end-plate potentials (min.e.p.p.s) from the rat diaphragm following high min.e.p.p, frequencies induced by nerve terminal depolarization. They proposed that these small min.e.p.p.s may be partially filled quanta resulting from either a secondary release or a premature release of newly formed quanta. Kriebel & Gross (1974) working with the frog sartorius also reported a class of small min.e.p.p.s (subminiature end-plate potentials, submin.e.p.p.s) that composed 2-5% of the min.e.p.p.s in the normal preparation. They were able to shift most min.e.p.p.s into the small class with successive heat challenges or periods of tetanic nerve stimulation. These observations, along with the fact that many min.e.p.p. amplitude histograms showed multiple peaks, led them to propose that the larger min.e.p.p.s resulted from synchronously released subunits. There are further indications of synchronous release in that Dennis & Miledi (1974) found that the unitary evoked response in the frog regenerating neuromuscular junction was larger than the min.e.p.p. They suggested along with several other possibilities that the larger unitary evoked response resulted from the synchronous release of subunits. In mouse diaphragms we have observed a class of small min.e.p.p.s

SUBMINIATURE END-PLATE POTENTIALS

555 (5-20% of total) which have the same shape and time characteristics as the larger min.e.p.p.s. These were particularly evident in small muscle cells with high input resistance. In such cells the min.e.p.p.s were relatively large (cf. Katz & Thesleff, 1957) with means of 4-8 mV and amplitude histograms showed peaks which were integral multiples of the submin. e.p.p.s. The submin.e.p.p.s were not due to contamination of the records by min.e.p.p.s from other sites of innervation remote from the recording electrode, as their time-to-peak was at least as short as that of the larger min.e.p.p.s (cf. Bennett & Pettigrew, 1974a, 1975). We report here that the percentage of submin.e.p.p.s and the number of multiple peaks of min.e.p.p. amplitude histograms were readily altered with botulinum toxin type A, colchicine and changes in temperature and in calcium concentration. We conclude that the smallest peak in min.e.p.p. amplitude histograms represents the subunit of spontaneous transmitter release and that larger min.e.p.p.s are generated by the synchronous release of several subunits. Results have been briefly reported elsewhere (Matteson & Kriebel, 1975; Kriebel & Matteson, 1975a, b). METIRODS We have greatly increased the resolution of min. e.p.p. amplitude histograms by working with small muscle cells with high input impedance which are common in 9-28 day-old mice. Mice were killed by decapitation. Hemidiaphragms were removed from the animal and placed in a controlled temperature bath which was mounted to the stage of a compound microscope (200 x magnification). Bath temperature was maintained by perfusing water of the appropriate temperature through a chamber located between the bath floor and microscope condenser. Changes in bath temperature were usually achieved in a few minutes, which was relatively rapid compared to the time required to collect enough min e.p.p.s for a histogram. The bathing medium contained (in mwm): NaCl, 125; KCl, 5; CaCl2, 2; NaHCO3, 24; NaH2PO4, 1; and glucose, 11; and was re-circulated with a bubble lift (95% 02- 5% C02, bubbled through water). Conventional 3 M-KCl micropipettes (30-50 MQ) were positioned orthogonally against muscle fibres with a manipulator and penetration was accomplished by ringing the amplifier with the negative capacitance control. Double the calcium concentration was used in many preparations to promote electrode sealing, thus greatly improving the chance of maintaining muscle cell resting potentials for several hours. Neostigmine methylsulphate (10-6-10-7 g/ml.) (Roche Laboratories, Nutley, N.J.) was used in order to make the submin. e.p.p.s as large as possible (cf. Liley, 1956). Since min.e.p.p.s generated in small cells were occasionally large enough to initiate muscle fibre action potentials, we usually added tetrodotoxin (2 x 10-6 g/ml.) (Sankyo, Tokyo, Japan), which does not have an apparent effect on spontaneous transmitter release (Elmqvist & Feldman, 1965; Miyamoto & Volle, 1974). Colchicine (City Chemical Corp., N.Y.) was dissolved in saline and added to the bath to achieve final concentrations of 10-4-10-3 M. Botulinum toxin type A (supplied by Dr E. J. Schantz, University of Wisconsin) stored in 0-05 M acetate buffer at pH 4-2 was diluted in saline and then added to the bath to achieve a final concentration of 105-104 intraperitoneal median lethal dose (i.P. LD'0)/ml. Min.e.p.p.s were recorded on 35 mm film either directly or from magnetic tape.

5.56

5MAHLON E. KRIEBEL AND OTHERS

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Film was projected on to graph paper and min.e.p.p. amplitudes were determined to the nearest 50 or 100 #iV depending on the noise level (50-100 1sV). (Min.e.p.p.s were measured by one person and recorded by a second.) Final amplitude histograms were constructed by adding successive histograms and sometimes combining pairs of histobars. In order to determine the exact start of min.e.p.p.s for time-to-peak measurements, signals were recorded on moving film (Fig. 1 A) with the oscilloscope sweep in the free run mode (2 msec/cm). Negrete, del Castillo, Escobar & Yankelevich (1972b) found that the relationship between amplitudes and rise times was not changed when they recorded without negative capacitance in the pre-amplifler, although the time-to-peak was doubled and the amplitude lowered by 30%. We did not use negative capacitance in the pre-amplifler in order to keep the noise level as low as possible. Distortion of min.e.p.p.s was held to a minimum by performing most experiments at 32-34o C, which slowed the min.e.p.p. time course. A time constant of 400 Mtec was used for most experiments, which increased the min.e.p.p. time-to-peak by 40 % and lowered the xnin.e.p.p. amplitude by 8 % when compared to min.e.p.p.s recorded with negative capacitance (time constant, 125 ,ssec). Slightly lower than normal temperatures also promoted preparation stability so that longer

recording periods were possible. RESULTS

Time-to-peak analysis of minxe.p.p.s We recorded a class of small min.e.p.p.s (submin.e.p.p.s) in small muscle fibres (0.25-0. 6 mV, depending on cell size (Fig. 1 A)) that formed a bellshaped distribution (first peak at 0-5 mY in Fig. 2A-C, J). The percentage of submin.e.p.p.s was usually greatest at slightly lower than normal body temperatures (32-34O C). At body temperatures the percentage of submin. e-p-p.s in young mice was occasionally as low as 3 % and was comparable to that in the adult frog sartorius preparation (Kriebel & Gross, 1974). In five different preparations the mean rise time of min.e.p.p.s composing the submin.e.p.p. population fell on or below the regression line of the larger min.e.p.p.s. Fig. lB shows four time-to-peak vs. min.e.p.p. amplitude Fig. 1. A, selected oscilloscope film strips (not continuous) showing subminiature end-plate potentials (submin.e.p.p.s, encircled) and slow timeto-peak intermediate sized min.e.p.p.s. Offsets on rising phase of slow time-to-peak min.e.p.p.s, suggesting two-events, are indicated with arrows. Intermediate sized min.e.p.p. with normal time-to-peak is indicated by an asterisk. Calibration: 1-4 mV between traces; each trace is 20 msec long. B, min.e.p.p. amplitude v8. time-to-peak plots. The min.e.p.p.s used for these curves are from the same series as those for histograms in Fig. 2A-D. Regression lines were calculated from the range of min.e.p.p.s indicated by the continuous part of the line. Many min.e.p.p.s of intermediate amplitudes at the start of the experiment (Fig. 2 A and B) had a long time -to -peak and were probably composed of two or more subunits. Many of these appeared to have offsets on the rising phase (indicated by arrows on the oscilloscope records). Each point on the regression line indicates mean rise time of all rnine.p.p.s in a 400,uV interval. Bars indicate mean + 1 S.D. of observation.

Submin.e.p~p.s are represented by the first two points on the plots.

SUBMINIATURE END-PLATE POTENTIALS

559 plots. The min.e.p.p.s. of the four plots of Fig. 1 B were used to construct the first four successive min.e.p.p. amplitude histograms of Fig. 2 (i.e. Fig. 2A-D). The submin.e.p.p.s of Fig. 2A-D are plotted as the first two points in the amplitude vs. time-to-peak curves of Fig. 1B. This analysis shows that submin.e.p.p.s were generated at the same junction as the larger min.e.p.p.s and not at a remote junction (cf. Katz & Kuffler, 1941; Fatt & Katz, 1951; del Castillo & Katz, 1954; Bennett & Pettigrew, 1974a). Quite unexpectedly, during the early periods of some experiments the time-to-peak of min.e.p.p.s two to four times greater than submin.e.p.p.s was longer than predicted by the regression line and more variable than the time-to-peak of the larger min.e.p.p.s (Fig. 1B, a and b). These large variances were not seen at normal temperatures, in which case the major mode was only four to seven-times the smallest mode. However, the mean and variance were found to decrease during the course of some experiments (Fig. 1 B, c and d) and peaks of intermediate-sized min.e.p.p.s became apparent in amplitude profiles (Fig. 2E-G, K). Many min.e.p.p.s which had a long time-to-peak showed inflexions on the rising phase, suggesting that two min.e.p.p.s were almost sychronously generated (Fig. 1A). These min.e.p.p.s. were analysed as single events. Min.e.p.p.s which showed clear breaks or offsets on the rising phase ('doublet min.e.p.p.s') were analysed as two events (see Kriebel & Stolper, 1975). Fig. 2. Successive amplitude histograms showing a spontaneous decrease in mean min.e.p.p. amplitude (A-D) and the effect of excess Ca2+ ions (E-I) on min.e.p.p. frequency and amplitude. These histograms show all recorded min.e.p.p.s during 120 min of continuous monitoring at 340 C. Min.e.p.p.s (8 %) that occurred during fly-back of the oscilloscope beam were missed. The muscle cell resting potential was -65 mV at the start and -64 mV at the end. Neostigmine, 106 g/ml.; 9-day-old mouse. A-D, show a spontaneous decrease in mean min.e.p.p. amplitude with no detectable change in the mean of submin.e.p.p.s. In D, the mean min.e.p.p. amplitude was stationary. J, shows histograms A-D together. E-G, calcium was added which doubled the min.e.p.p. frequency, reduced the mean min.e.p.p. amplitude but did not change the mean of submin.e.p.p.s (cf. the first peak in J to that in L). G, the initial frequency increase with eight-times Ca2+ concentration was large since a few minutes were required for Ca2+ to equilibrate (L) in the total re-circulating volume of saline. K, shows histograms E-G together and L, H and I. A, 269 min.e.p.p.s/20 min, 13 min.e.p.p.s/min; 20 min elapsed betv. een the start of dissection and the start of recording; B, 329 min.e.p.p.s/20 min, 16 min.e.p.p.s/min; C, 398 min.e.p.p.s/20 min, 20min.e.p.p.s/min;D, 293min.e.p.p.s/l5min, 20min.e.p.p.s/min; E,4 x Ca2+ saline, 225 min.e.p.p.s/5 min, 45 min.e.p.p.s/min; F, 6 x Ca2+ saline, 341 min.e.p.p.s/10 min, 34 min.e.p.p.s/min; G, 8 x Ca2+ saline, 634 min. e.p.p.s/10 min, 63 min.e.p.p.s/min; H, 8 x Ca2+ saline, 383 min.e.p.p.s/ 10 min, 38 min.e.p.p.s/min; I, 8 x Ca2+ saline, 408 min.e.p.p.s/10 min, 40

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Spontaneous change in min.e.p.p. amplitude profiles In low-frequency cells (achieved at 32-34° C) the major mode min.e.p.p.s were initially twelve to fifteen-times the smallest mode min.e.p.p.s (submin.e.p.p.s) but usually decreased within the first hour of recording and stabilized at about ten to twelve times the smallest mode (Fig. 2D). This change was not apparent in preparations initially placed in a 370 C bath. During spontaneous decreases in mean major mode min.e.p.p. amplitudes, the amplitude of the submin.e.p.p.s did not noticeably decrease. Boyd & Martin (1956 a) reported a progressive decrease in mean min.e.p.p. amplitude but ascribed this to a curare-like effect of neostigmine. Nevertheless, we observed decreases in the mean min.e.p.p. amplitude with either low (10-7 g/ml.) or no neostigmine salines. In Fig. 2, histograms A-D are bell-shaped but when added together (Fig. 2J) the larger min. e.p.p.s form a skewed distribution. In many skewed amplitude histograms the intermediate-sized min.e.p.p.s showed multiple peaks which were integral multiples of the smallest mode, providing there were enough min.e.p.p.s in the histograms (note first two peaks in Fig. 2J).

Effect of excess Ca2+ on min.e.p.p. amplitude distributions Addition of Ca2+ ions to make the Ca2+ concentration in saline two to eight-times normal increased the min.e.p.p. frequency (Boyd & Martin, 1956a; Hubbard, 1961; Hubbard, Jones & Landau, 1968) and also decreased the mean of the major mode min.e.p.p.s without noticeably changing the mean of the submin.e.p.p. peak (Kriebel & Gross, 1974) (Fig. 2E-G). This decrease in mean min.e.p.p. amplitude was most obvious when control frequencies were very low and the major mode min.e.p.p. relatively large compared to that of the submin.e.p.p. mode. In one preparation an eight-times calcium concentration in the solution decreased the min.e.p.p. amplitude to 15% of the control such that the min.e.p.p. histogram showed only two peaks, a submin.e.p.p. peak and a second peak that was twice as large as the submin.e.p.p.s. Initial low min.e.p.p. frequencies were usually attained at 320 C (see below); and, at normal temperatures and thus greater min.e.p.p. frequencies, excess calcium did not have as dramatic an effect on min.e.p.p. amplitudes and frequencies (see Kriebel & Gross, 1974, for similar findings on the frog).

Effect of temperature on min.e.p.p. amplitude distributions The min.e.p.p. frequency of very small muscle cells at temperatures of 32-34° C was relatively low (5-10/min) (cf. Kuno, Turkanis & Weakly, 1971) and the percentage of submin.e.p.p.s was relatively high (10-20 %).

SUBMINIATURE END-PLATE POTENTIALS 563 Consequently, we equilibrated many preparations at this temperature to clearly define the submin.e.p.p. peak in amplitude histograms (Fig. 3Aa, Bf). An increase in temperature from 320 C to normal body temperature increased min.e.p.p. frequency (Liley, 1956, found the Q28-38 to be 3-4 for the rat) and also reduced the major mode min.e.p.p. to a value of four to seven-times the submin.e.p.p. (Fig. 3A, B) (cf. Boyd & Martin, 1956a). The occurrence of multiple peaks that were integral multiples of the submin.e.p.p. peak was most pronounced when the mean min.e.p.p. amplitude was only four-times the submin.e.p.p. (Fig. 3Ac, Be). Temperatures above 380 C decreased the submin.e.p.p. amplitudes so that peak intervals also decreased with an increase in temperature. However, the two selected examples (Fig. 3A, B) show relatively large changes in the position of the major mode min.e.p.p.s with small changes in temperature to minimize post-synaptic effects. At temperatures of 34-36' C the major mode was usually about seven to Fig. 3. Successive amplitude histograms showing effect of small temperature challenges on the mean min.e.p.p. amplitude and the distribution of amplitudes. These plots show all recorded min.e.p.p.s. Temperatures were changed in about 1 min, which was relatively rapid compared to the duration of each challenge. A, this preparation (10-day-old mouse) was equilibrated for 1 hr at 340 C and frequency and mean min.e.p.p. amplitude were stationary. Muscle cell resting potential was -72 mV at the start and -70 mV at the end of 268 min of continuous recording. Note that the first peak (submin.e.p.p.s) inhistogram (a) has the same mean as the first peak in (g). Neostigmine, 10-7 g/ml.; normal Ca2+ concentration in saline. (a) 34° C, 824 min.e.p.p.s/60min, 14 min.e.p.p.s/min; (b) 370 C, 659 min.e.p.p.s/16 min, 41 min.e.p.p.s/min; (c) 340 C, 591 min.e.p.p.s/40 min, 15 min.e.p.p.s/min; (d) 320 C, 558 min. e.p.p.s/56 min, 10 min.e.p.p.s/min; (e) 29° C, 1005 min.e.p.p.s/80 min, 12 min.e.p.p.s/min; (f) 38° C, 500 min.e.p.p.s/12 min, 41 min.e.p.p.s/min; (g) 390 C, 686 min.e.p.p.s/4 min, 71 min.e.p.p.s/min. B, this preparation (14-day-old mouse) was equilibrated at 340 C for 30 min and the mean min.e.p.p. amplitude and frequency were stationary. Neostigmine, 106g/ml.; double Ca2+ concentration in saline. (a-c) are histograms from one muscle cell; resting potential was -73 mV at start and -70 mV at end of 28 min of continuous recording. (d-f ) are histograms from an adjacent cell; resting potential was -78 mV during 36 min of continuous recording. Note that the means of the submin.e.p.p.s in (d) and (e) at 380 C are the same as the submin.e.p.p. mean in (f ) at 340 C. (d) and (e) show the mean stationary and multiple peaks that are integral multiples of the submin. e.p.p. peak. The peak intervals in (d) and (e) are the same as in (f ). (a) 340 C, 482 min.e.p.p.s/16 min, 30 min.e.p.p.s/min; (b) 350 C, 437 min.e.p.p.s/8 min, 54-5 min.e.p.p.s/min; (c) 370 C, 199 min.e.p.p.s/4 min, 50 min.e.p.p.s/min; (d) 38° C, 484 min.e.p.p.s/2 min, 242 min.e.p.p.s/min; (e) 380 C, 497 min. e.p.p.s/2 min, 248 min.e.p.p.s/min; (f) 340 C, 1325 min.e.p.p.s/32 min, 41 min.e.p.p.slmin.

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MAHLON E. KRIEBEL AND OPHERS nine-times the submin.e.p.p. mode, which still contained relatively too many submin.e.p.p.s to give a bell-shaped appearance of the histogram profile. At 370 C, the major mode was four to seven times the smallest mode and, ignoring the multimodality of these plots, the over-all histogram profile was usually bell-shaped (Fig. 3A b, Bc and d). Larger cells at 370 C, which probably generated submin.e.p.p.s below the recording resolution, produced smooth bell-shaped amplitude profiles and were similar to those reported by others (Fatt & Katz, 1952; Boyd & Martin, 1956 b; Miledi, 1960; Wernig, 1975; Bennett, Florin & Woog, 1974; Bennett & Florin, 1974; Tonge, 1974 b). When the bath temperature was increased from 37 to 400 C, min.e.p.p. frequencies increased ten to forty-times (cf. Liley, 1956; Boyd & Martin, 1956a) with major mode min.e.p.p.s reduced to three or four times the submmn.e.p.p. mode (Fig. 3Ag). The over-all profile was either a flat distribution, i.e. the number of min.e.p.p.s in each of the first three or four peaks was about the same (Fig. 3 Ag) or the distribution was skewed to the submin.e.p.p.s. During above normal heat challenges, there were bursts of min.e.p.p.s of only the small mode size and extended periods of heating produced min.e.p.p. histograms which showed mainly submin.e.p.p.s. Cooke & Quastel (1973) also found an increase in small min.e.p.p.s after high frequencies of release with focal depolarization. Repeated heat challenges on frog preparations shifted all min.e.p.p.s into the small mode class (Kriebel & Gross, 1974) but this was not as easily done in the mouse preparation. After heat challenges the min.e.p.p. amplitude profiles returned towards normal within an hour or two. 566 566

Effect of botulinum toxin type A on min.e.p.p. amplitude distributions We were able to record all min.e.p.p.s from single end-plates after the addition of botulinum toxin and found that min.e.p.p. frequencies progressively decreased (control 10-60/min) during 30 mmn-i hr 30 mmn and then precipitously fell to only a few per minute (Spitzer, 1972; Tonge, 1974a) (Figs. 4, 5A). The number of min.e.p.p.s generated during botulinum toxin poisoning varied from 225 to 1000, depending on the muscle size, min.e.p.p. frequency and botulinum toxin dose. We did not do an extensive dose-response study but we did find that a 105 X intraperitoneal median lethal dose (i.r. LD50)fml. blocked the release of quanta about twice as fast as a 104fm1. dose. The time required to reach the final reduction in min.e.p.p. frequency was also temperature dependent in that we did not observe a significant decrease in frequency at 300 C for 3 hr with a 5 x 104 I.r. LD5wlml. dose (cf. Burgen, Dickens & Zatman, 1949) but when the bath was warmed to 340 C min.e.p.p.s essentially ceased within a few minutes.

SUBMINIATURE END-PLATE POTENTIALS 567 In agreement with Spitzer (1972), who recorded min.e.p.p.s after 10-5 hr, min.e.p.p.s were not completely blocked even 3 hr after the initial drastic reduction in frequency since a few small min.e.p.p.s were always recorded (1/min) (Fig. 5B) and occasionally a 'burst' of small min.e.p.p.s was observed. We did not succeed in recording from a single cell for 4 hr but the small min.e.p.p.s recorded from botulinum toxin incubated cells were comparable to control submin.e.p.p.s in adjacent cells. The most notable observations were: that the mean of the major mode usually did not decrease during the decrease in min.e.p.p. frequency, although the relative percentage of major mode min.e.p.p.s decreased; and that the mean of the submin.e.p.p.s did not decrease (Figs. 4 and 5A). Some min.e.p.p. amplitude histograms which were recorded either after the initial reduction in min.e.p.p. frequency (Fig. 5Ad) or several hours after the initial reduction (Fig. 5Bd) showed three or four multiple peaks. After min.e.p.p.s essentially ceased, we were sometimes able to stimulate the release of quanta by probing the junctional region with the microelectrode (Thesleff, 1960). In addition, two to four-times Ca2+ concentration seemed to retard the blocking action of botulinum toxin (Thesleff, 1960). Repeated washing did not restore min.e.p.p. activity.

Effect of colchicine Colchicine reduced the mean min.e.p.p. amplitude to a stable value within a few minutes with little change in frequency (Turkanis, 1973) and with the generation of as few as fifty to 200 min.e.p.p.s (Fig. 6). We found that a colchicine dose of 5 x 10-4 M usually reduced the mean min.e.p.p. by half with no reduction in the submin.e.p.p. mode. A further dose increase to 10-3 M sometimes reduced the major mode min.e.p.p. to a fifth that of the control. The most striking observation in these experiments was that the smaller and intermediate peaks of control histograms became the dominant peaks with a colchicine challenge. Moreover, colchicine neither changed peak intervals nor time-to-peak of most min.e.p.p.s. Even with high colchicine doses a few min.e.p.p.s the size of the control major mode min.e.p.p.s were generated (Fig. 6G, L). We also noted an increase in the number of offsets on the rising phases of some min.e.p.p.s and the occurrence of min.e.p.p.s, with flat tops. There were too many of these to be accounted for by chance coincidence of two or more min.e.p.p.s. When colchicine was washed from the bath there was 50% recovery in min.e.p.p. amplitude (there was little or no recovery at the frog neuromuscular junction (Turkanis, 1973)). During recovery, the peak intervals and peak positions in successive min.e.p.p. amplitude histograms did not change As min.e.p.p.s became larger, peaks were added to the right side of the distribution and subtracted from the left side (histograms G to L of Fig. 6).

MAHLON E. KRIEBEL AND OTHERS

568

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SUBMINIATURE END-PLATE POTENTIALS A (b)

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Fig. 4. Effect of botulinum toxin type A, 3 x 104 intraperitoneal median lethal dose (i.P. LD50)/ml., on min.e.p.p. amplitude distributions and selected oscilloscope records. These are successive histograms showing all recorded min.e.p.p.s during 96 min (4 min of control) of continuous recording. 330 C; 23-day-old mouse; neostigmine, 10-7 g/ml.; normal Ca2+ concentration in saline. Resting potential at start, -64 mV; at end, -60 mV. A, (a) control histogram. The mean min.e.p.p. amplitude and frequency were stationary; 266 min.e.p.p.s/4 min, 66-5 min.e.p.p.s/min. (b) film strip: submin.e.p.p.s are indicated with an arrow. Calibration: 3-5 mV between traces; each trace is 0 5 sec long. B, botulinum toxin added to bath; 666 min. e.p.p.s/12 min, 55-5 min.e.p.p.s/min. C, 647 min.e.p.p.s/12 min, 54 min. e.p.p.s/min. D, 378 min.e.p.p.s/12 min, 31-5 min.e.p.p.s/min. Note that the means of major mode min.e.p.p.s and submin.e.p.p.s are the same as in the control, A. E, (a) 112 min.e.p.p.s/24 min, 4-6 min.e.p.p.s/min. (b) film strip: (not continuous) submin.e.p.p.s are indicated with arrows. Calibration same as in A. F, 27 min.e.p.p.s/32 min, 0-9 min.e.p.p.s/min. The frequency was dropping through the sequence of histograms B-F.

569

MAHLON E. KRIEBEL AND OTHERS

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1I2 Millivolts Fig. 5. A, effect of botulinum toxin (104 i.P. LD50/ml.) on min.e.p.p. amplitude distributions: 17-day-old mouse, neostigmine, 10-7 g/ml. Three-times normal Ca2+concentration in saline. (a) control cell: resting potential -74 mV; 270 C; 1018 min.e.p.p.s/24 min, 42 min.e.p.p.s/min. (b-d) adjacent experimental cell. The preparation had been incubated with botulinum toxin for 20 min before the start of (b). (b), (c) and (d) are successive and show all recorded min.e.p.p.s during 60 min of continuous recording. Resting potential, -67 mV at start; -76 mV at end. (b) 551 min.e.p.p.s/8 min, 69 min.e.p.p.s/min; 27° C. (c) 527 min.e.p.p.s/16 min, 33 min.e.p.p.s/min; 27° C. (d) 165 min.e.p.p.s/36 min. Min.e.p.p. frequency dropped to less than 1/min towards end of recording period. In an attempt to generate as many min.e.p.p.s as possible, we raised the bath temperature to 350 C for most of this recording period. Additional Ca2+ ions (final concentration five-times normal) were added near the end but they increased the frequency little. B, min.e.p.p. amplitude histograms from a control cell and three experimental cells which had been incubated with botulinumtoxin (5 x 10 i.P. LD50 /ml.) for 4 hr; 18-day-old mouse; neostigmine, 10-7 g/ml. doublenormal Ca2+ concentration in saline. (a) control cell: resting potential, -77 mV at start; -66 mV, at end; 280 C; 455 min.e.p.p.s/16 min, 28 min.e.p.p.s/min. (b) first experimental cell. Min.e.p.p.s recorded for 20 min after 4 hr of botulinum toxin incubation. Resting potential, -65 mV at start; -68 mV, at end. The temperature was raised to 35' C in an attempt to increase the min.e.p,.p. frequency. (c) second experimental cell. Min.e.p.p.s recorded for 20 min after 4 hr 20 min incubation in botulirxum toxin. Resting potential, -70 mV at start; -73 mV, at end; 350 C. (d) third experimental cell. Min.e.p.p.s recorded for 20 min after 4 hr 40 min incubation with botulinum toxin. Resting potential -78 mV; 35° C. Most of these min.e.p.p.s occurred during a 2 min burst of min.e.p.p.s.

572

MAHLON E. KRIEBEL AND OTHERS

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573

MAHLON E. KRIEBEL AND OTHERS

574

DISCUSSION

Relationship of submin.e.p.p.s to major mode min.e.p.p.s By working with small muscle cells (cf. Katz & Thesleff, 1957) we have increased the resolution of submin.e.p.p.s to show that they form a bellshaped distribution. Moreover, the mean of the submin.e.p.p. mode did not progressively change or move nearer to the noise level with high frequencies of release induced with temperature or calcium challenges, which decreased the major mode min.e.p.p. mean to 15-35 % of the control, or during spontaneous decreases in the mean amplitude. Neither colchicine nor botulinum toxin changed the submin.e.p.p. mean, although these drugs greatly reduced the major mode min.e.p.p. mean during the generation of only a few hundred min.e.p.p.s (cf. Katz, 1962). Small quanta released from Schwann cells have not been reported in the mammalian preparation (Miledi & Slater, 1970) as were found by Birks, Katz & Miledi (1960) at the frog denervated junction. Kriebel & Gross (1974) presented evidence that submin.e.p.p.s in the frog were not the same as Schwann cell min.e.p.p.s. On the other hand, the time-to-peak plots (Fig. 1) show that the submin. Fig. 6. Effect of colchicine and wash-out on min.e.p.p. amplitude distributions. These are successive histograms showing all recorded min.e.p.p.s for 44 min of continuous recording. Neostigmine, 10-7 g/ml. Twice normal Ca2+, 260 C. Resting potential, - 75 mV at start; -72 mV at end; 21 -day-old mouse.

A, control: 1754 min.e.p.p.s/12 min, 146 min.e.p.p.s/min. The means of submin.e.p.p.s and major mode min.e.p.p.s and of frequency were stationary. B, min.e.p.p.s during 0- 5 min after the addition of 5 x 10-4 M colchicine. Note that the mean min.e.p.p. amplitude had already begun to decrease. C, 482 min.e.p.p.s recorded during the following 3 min; 160 min.e.p.p.s/ min. The mean of major mode min.e.p.p.s were stationary during this period. D-F, these are 1 min sections after the addition of a second dose of colchicine (final concentration, 10-3 M) showing progressive transition states (each histogram is 1 min): 132 min.e.p.p.s/min, 396 total. G, (a) 1107 min.e.p.p.s recorded in the next 9 min after stationary state was reached; 123 min.e.p.p.s/min. Note that the mean amplitude has been reduced four-times, while the submnin.e.p .p. amplitude has remained the same as in the control, A. (b) and (c) are the last two 1 min sections of G showing that the mean min.e.p.p. amplitude and peaks were stationary before washout; 140 min.e.p.p.s/min. H-K, these are 2 min sections after the first wash-out. Note that while peaks were emerging to the right they were being subtracted from the left during recovery. L, 803 min.e.p.p.s recorded in 8 min after a second wash-out with saline; 100 min.e.p.p.s/min. The mean min.e.p.p. amplitude was half that of the control, while the mean of the submin.e.p.p.s remained unchanged.

575 SUTBMINIATURE END-PLATE POTENTIALS e.p.p.s were not generated at a remote or distributed junction, since they fell on, or below, the regression line through the larger min.e.p.p.s (cf. Bennett & Pettigrew, 1974a; Katz & Kuffler, 1941; Hartzell, Kuffler & Yoshikami, 1975; Fatt & Katz, 1951). However, it is possible that some junctions were multiply innervated since Bennett & Pettigrew (1974a) found that 20 % of the junctions of diaphragm end-plates of a 2-weekpost-natal rat had multiple innervations. Nerve endings of different axons at the same end-plate would generate min.e.p.p.s showing similar time characteristics but could conceivably have different quantal sizes. Thus, most of our experiments were done on mice that were older than 21 days since Redfern (1970) found single innervation by 16-18 days and Bennett & Pettigrew (1974a) found single axon innervation by 3-4 weeks in the rat. Some of the increase in time-to-peak of the larger min.e.p.p.s (Fig. 1) may be accounted for by spreading activation (Negrete, del Castillo, Escobar & Yankelevich, 1972a, b); i.e. relatively larger amounts of ACh must spread tangentially in the synaptic cleft to reach available receptors. However, Hartzell et al. (1975) found no correlation between time-to-peak and magnitude of miniature end-plate currents. The transformation from current to voltage would account for the positive correlation reported here. The multiple peaks of amplitude histograms indicate that there are either many classes of quanta that are differentially affected by the challenges used here, that filling of vesicles is quantal, or that subunits are synchronously released to generate the larger min.e.p.p.s (Kriebel & Gross, 1974). We propose that submin.e.p.p.s do not represent partially filled quanta for several reasons (cf. Cooke & Quastel, 1973; Highstein & Bennett, 1975): firstly, min.e.p.p. amplitude histograms showed multiple peaks that were integral multiples of the submin.e.p.p.s; secondly, the over-all profiles of amplitude histograms were altered after the generation of only a few hundred min.e.p.p.s although peak intervals did not change; and thirdly, the amplitude of the major peak was greatly changed with various challenges that did not alter the mean of the submin.e.p.p.s (temperature, Ca2+ ions, botulinum toxin and colchicine). If one assumes that all min.e.p.p.s result from the release of a single quantum, it would be difficult to explain a decrease in mean min.e.p.p. amplitude by a decrease in quantal size for three reasons: the mean of the submin.e.p.p. peak was not shifted towards the noise; the over-all profile of the major mode min.e.p.p.s, changed from a bell-shaped distribution (370 C) to a distribution with a flat top and a steep profile to the left side of the min.e.p.p. amplitude distribution (Fig. 3A g); and the multiple peaks of control histograms and flat top histograms had the same position and peak interval.

MAHLON E. KRIEBEL AND OTHERS 576 The dramatic decrease in mean min.e.p.p. amplitude with incubation with colchicine or botulinum toxin is difficult to reconcile with quantized vesicle filling. This hypothesis would require that the available store of vesicles be as low as 125 and that filling of vesicles occur just before release (cf. Highstein & Bennett, 1975). Min.e.p.p. amplitude histogram profiles Disregarding multiple peaks, the over-all profiles of some distributions were neither bell-shaped nor skewed to the larger min.e.p.p.s (Liley, 1957), butshowed two distinct parts. The larger min.e.p.p.s usually formed a bell-shaped part (Figs. 3Ae, 4B, 5Aa) or a slightly skewed part (Fig. 2J). The second part of the histograms consisted of smaller min.e.p.p.s which were generally too numerous to form a simple skew distribution (all control histograms but 3Ba) and many times formed multiple peaks that were integral multiples of the first peak; there are two obvious peaks in Fig. 2J and several small peaks in Fig. 6A. The over-all bell-shaped part of many distributions (Fig. 3Bd) appears similar to the amplitude distributions reported by others (Fatt & Katz, 1952; Bennett et al. 1974; Boyd & Martin 1956 b; Bennett & Florin, 1974; Tonge, 1974 b). Skewed distributions (Fig. 2J) towards larger min.e.p.p.s (Liley, 1956, 1957) indicate either interaction or a brief drag effect between quanta (cf. Martin & Pilar, 1964; Cohen, Kita & Van der Kloot, 1974a, b; Blackman, Ginsborg & Ray, 1963) or simply a larger quantal class (Heuser, 1974). Dennis, Harris & Kuffler (1971) also reported synchronous quantal release at the parasympathetic synapse in the frog heart. Martin & Pilar (1964) found a drag effect in the ciliary ganglion and proposed that a primary event may be capable of releasing one or several quanta although the number of failures would be unknown. Kriebel & Stolper (1975) have shown in very low frequency cells in the frog sartorius that the temporal occurrence of min.e.p.p.s was often not described by a Poisson distribution since there were too many doublet min.e.p.p.s. The activation of a primary release mechanism may be a branching Poisson, i.e. activation of a release mechanism may follow Poisson statistics but each activation may generate several quanta (Cohen et al. 1974b). However, Hubbard & Jones (1973) failed to find significant interaction between mammalian min.e.p.p.s but did show that each release site may be more ordered than a Poisson process. We propose that a release mechanism synchronizes several subunits to generate most spontaneous min.e.p.p.s. Long time-to-peak min.e.p.p.s In some preparations min.e.p.p.s of intermediate size had long and highly variable time-to-peaks (Fig. 1). Some of these min.e.p.p.s had a

577 SUBMINIATURE END-PLATE POTENTIALS flat top and appeared similar to those produced by colchicine. Fig. 1 is of interest since the large variance in time-to-peak of intermediate min. e.p.p.s decreased when the flat top min.e.p.p.s ceased. We suggest that the long time-to-peak intermediate sized min.e.p.p.s (Fig. 1 A) resulted from the asynchronous release of subunits. Katz & Miledi (1973) reported a different class of flat-top min.e.p.p.s from the frog nerve-muscle junction that were

10-20 % smaller than the mean, which were found when an external electrode was pressed against the nerve-muscle junction. They concluded that the external electrode compressed the junctional element so as to impede the diffusion of transmitter from the synaptic cleft. Yet additional possible classes of long time-to-peak min.e.p.p.s have been reported at the rat regenerating junction and at the developing junction (Bennett et al. 1974; Bennett, McLachlan & Taylor, 1973; Bennett & Pettigrew, 1974b). Gage & McBurney (1975) also determined that miniature end-plate currents at the toad junction fell either into 50-500 msec or 0 5-5 msec classes.

Effect of botulinum toxin and colchicine on min.e.p.p. amplitude distributions We found that botulinum toxin did not completely block submin.e.p.p.s, although the percentage of submin.e.p.p.s increased. As the frequency of major mode min.e.p.p.s decreased to zero during the generation of a few hundred min.e.p.p.s, the mean of the major mode min.e.p.p.s did not change. Spitzer (1972) reported a broadening of min.e.p.p. amplitude histograms and an increase in smaller min.e.p.p.s near the noise level in the rat diaphragm with botulinum toxin poisoning. Spitzer (1972) and Bray & Harris (1975) found that min.e.p.p. amplitudes were increased during nerve stimulation and Harris & Miledi (1971) reported that the evoked unitary events recorded in a botulinum toxin blocked frog preparation were larger than min.e.p.p.s. Harris & Miledi (1971) proposed that there were either different classes of quanta or that a small class could be synchronously released. Duchen & Tonge (1973) and Tonge (1974a) noted an increase in the percentage of small min.e.p.p.s and great variations in min.e.p.p. amplitude profiles in mouse muscle with tetanus toxin; and Duchen & Tonge (1973) observed that nerve stimulation increased the amplitude of min.e.p.p.s to the normal size. However, Mellanby & Tbompson (1972) found that tetanus toxin only decreased min.e.p.p. frequency. Boroff, del Castillo, Evoy & Steinhardt (1974) observed that botulinum toxin greatly reduced the amplitude of frog min.e.p.p.s such that most min.e.p.p.s were lost to the noise. They found that depolarization increased the frequency of min.e.p.p.s without altering the min.e.p.p. amplitude whereas hyperpolarization greatly increased min.e.p.p. amplitudes, which

578

MAHLON E. KRIEBEL AND OTHERS they interpreted to demonstrate two classes of quanta. Boroff et al. (1974) also found that the larger min.e.p.p.s progressively became smaller and they suggested that botulinum toxin blocked filling of vesicles. We found that the number of min.e.p.p.s generated during botulinum toxin incubation was of the same order of magnitude as the immediately available store (300-1000 quanta suggested by Ginsborg, 1970). The effects of colchicine on transmitter release have been matters of controversy. Perisic & Cuenod (1972) demonstrated that intra-ocular injections of colchicine in the pigeon greatly reduced the evoked response recorded in the optic tectum after stimulating the optic nerve. It has also been reported that colchicine decreases the mean quantal content of the end-plate potential (Katz, 1972) and the min.e.p.p. amplitude (Turkanis, 1973) in the frog neuromuscular junction. On the other hand, when colchicine was applied to motor nerves in doses which inhibit axoplasmic transport, thus inducing denervation changes in muscles, neither the spontaneous nor the evoked release of transmitter was modified (Albuquerque, Warnick, Tasse & Sansone, 1972; Hofmann & Thesleff, 1972). We have found that colchicine reduces the min.e.p.p. amplitude in the mouse diaphragm. Although the major mode min.e.p.p. mean was reduced to a fifth of that of the control, the mean of the submin.e.p.p.s did not change, demonstrating little or no anticholinergic action. However, a post-synaptic action on the frog has been reported (Turkanis, 1973; Spoor & Ferguson, 1965; M. E. Kriebel, unpublished). With a four- or fivefold reduction in min.e.p.p. amplitude, the multimodality of the first four peaks was obvious, although the over-all min.e.p.p. amplitude distribution was bell-shaped. These distributions were different from the three or four peaks obtained near the end of botulinum toxin incubation (Fig. 5Ad and Bd) which exhibited an over-all skew distribution towards the submin.e.p.p.s. We suggest that the multimodal distribution obtained after incubation with either botulinum toxin or colchicine can be explained by postulating that the submin.e.p.p.s represented the release of single subunits (first peak) and that synchronous release of subunits gave rise to the integral peaks. Botulinum toxin may first block the synchronizing mechanism since the mean of the major mode min.e.p.p.s was not reduced and, secondly, block single subunit releases (submin.e.p.p.s) since the submin. e.p.p. frequency eventually decreased. Colchicine may interfere with mobilization of subunits since the number of subunits composing a min. e.p.p. was reduced but not the min.e.p.p. frequency. This research was supported by N.I.H. Grant 5RO1NS11996-02. We thank Mrs Kathleen Hamer for her excellent technical assistance and Dr Cordell Gross for helpful discussions. We thank Dr E. J. Schantz for supplying botulinum toxin.

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