β-Bungarotoxin preferentially blocks one class of miniature endplate potentials

598

Brain Research, 192 (19801 598 602 ~t: Elsevier/North-Holland Biomedical Press

fl-Bungarotoxin preferentially blocks one class of miniature endplate potentials

F. L L A D O S , D. R. M A T T E S O N and M. E. K R I E B E L

Department of Physiology, Upstate Medical Center, State University of New York, Syracuse, N.Y. 13210 (U.S.A.) (Accepted February 21st, 1980)

Key words: fl-bungarotoxin - - neuromuscular junction - - M E P P s

Miniature endplate potential (MEPP) amplitudes of both the frog and mouse neuromuscular junctions do not form a simple bell-shaped distribution. In the frog neuromuscular preparation, there was a distinct class of sub-MEPPs (s-MEPPs) with an amplitude about 1/5 to 1/7 that of the classical MEPP 4,11,12. Moreover, the histograms showed multiple peaks that were integral multiples of the s-MEPP 11,1z,22. Direct evidence for preferred MEPP amplitudes that were integral multiples of the sMEPP was demonstrated with multiple exposure oscilloscope pictures in which the MEPPs triggered the trace 11. These observations and results of various challenges suggested that MEPPs are composed of subunits the same size as s-MEPPs. Thus, a subunit hypothesis was proposed to explain the following data: (1) that amplitude histograms showed multiple peaks; (2) that the peaks were integral multiples of the smallest peak; (3) that there was stationarity of peaks with large sample sizes; and (4) that the number of peaks was changed with various challenges that altered the MEPP frequencyla,12,13, 23. In the mouse diaphragm preparation the amplitude of s-MEPPs was usually 1/10 to 1/15 that of the classical MEPPs 7A3 and with a high signal to noise ratio, Kriebel et al. a3 found amplitude histograms with multiple peaks that were integral multiples of the s-MEPP peak. In addition to multiple peaks, MEPP amplitudes fell into one of two general classes. The larger MEPPs composed an overall bellshaped distribution ('beI1-MEPPs') and represented those studied by previous investigators. The smaller MEPPs formed an overall skew distribution which sometimes showed several peaks 1~. Histograms that showed integral peaks have been fit with a model based on the variance of the s-MEPP and the assumption that all MEPPs are multiples of the s-MEPP. The actual s-MEPP variance was determined by subtracting the noise and measurement error components from the observed apparent variance (see Refs. 15, 16, figure legend). These data and predictive model answer the objections raised by Miller et al. 17 and Katz and Miledi 1° against the subunit hypothesis. We report here that the MEPPs composing the 'bell-shaped' distribution (i.e. the classical MEPPs) were preferentially blocked by fl-bungarotoxin (fl-BuTX) and that

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Fig. 1. Effect of fl-BuTX on MEPP amplitude distribution. A: control histogram, 20 min of recording. After the addition of 20/~g/ml fl-BuTX there was a period of depression lasting approximately 5 min followed by a 4-fold increase in frequency that reached its peak 40 min after the drug was added. B, C and D: successive 12 min periods 1 h after the addition of fl-BuTX. E: B, C and D combined. Temperature was kept at 26 °C during the experiment. Resting potential 70 mV at the start and 66 mV at the end. 21-day-old mouse. The curves are predicted from a model derived under the following assumptions : (1) s-MEPPs result from the release of a subunit of transmitter. The subunit amplitude is assumed to be normally distributed with mean ~u) and variance (~r2 + trot2), where (or2) is the actual variance of the subunit and (aM ~) represents measurement error (with noise); (2) larger amplitude MEPPs result from the synchronous release of two or more subunits. Subunits sum linearly; therefore, the release ofj subunits would produce a normally distributed population of MEPP amplitudes with mean (j~) and variance (jcrz + crM2); (3) the overall amplitude distribution is, therefore, composed of the sum of these subpopulations. Each subpopulation must be multiplied by a weighting factor (Wj) which represents the conditional probability that a MEPP belongs to the jth population. The probability density function for k subpopulations is, therefore, as follows: f(x) = W1 • N1 (x) + W2 N2 (x) + . . . + Wk • Nk (x). Where f (x) represents the probability of observing a MEPP of amplitude x, and Nj (x) is the normal probability density function with mean (j~) and variance (ja 2 + ~rM2). For any observed amplitude histogram, estimates of the parameters in this function (the Ws, and or) are obtained as maximum likelihood estimates is. The probability density function was used to produce a fit to the observed histograms (the smooth lines). The data were also fit with a bimodal model and the fit for the skew-MEPPs was not as significant as that of the summed release model (see ref. 15 for details on the Model).

600 the smaller MEPPs composing the ~skewed' distribution were much more resistant to fl-BuTX (Fig. i). The isolated mouse diaphragm was bathed in a small volume of recirculated buffered saline and electrophysiological techniques were conventional ~3. MEPPs were recorded on tape and subsequently filmed with an oscilloscope camera. The noise was usually 50-100 #V and the s-MEPP mean 250-400 #V. The film was projected onto graph paper such that the trace was confined between two lines of the graph paper. MEPP amplitudes were read to the nearest half line (25-50 /~V). A concentrated solution of purified fl-BuTX (Miami Serpentarium Labs., Miami, Florida) was added to give a final concentration of 20/~g/ml. The effect of fl-BuTX on the frequency was found to be like that reported by previous investigators2,~,~4, '~0. First, there was a depression in MEPP frequency (both bell- and skew-MEPPs) which persisted for 5-10 rain. The depression effect was greatest on the belI-MEPPs. The initial depression phase was followed by a 3-4 fold increase in frequency of bellMEPPs which persisted for about an hour with this dose. The MEPPs also appeared in bursts as reported by Abe et al. 1. The third phase was expressed as a fall in the frequency of the belI-MEPPs until only the skew-MEPPs remained, and finally, the frequency of the skew-M EPPs also fell after 2 or 3 h (we have not followed the effect longer). The preferential block has not been reported by previous investigators2,,~,~4, 20 working with fl-BuTX, presumably because they did not resolve the skew-MEPPs from the noise in the recording system. Fig. IB, C and D shows that essentially skewMEPPs remain after the high frequency phase. The beI1-MEPP mean did not decrease during the high frequency phase but belI-MEPPs simply ceased as with botulinum toxin poisoning 13. Although Chang and Lee 6 have demonstrated that fl-BuTX had no post-synaptic effect, it is important to point out that we followed the time course of/~BuTX poisoning at single junctions and found that the position of the s-MEPP mean and small peaks (Fig. 1) did not decrease as would be expected with a postsynaptic effect. Note that 3 successive histograms which show all MEPPs after the high frequency phase demonstrate stationarity of peaks and that the second and third peaks are integral multiples of the smallest. Thus, we also conclude that fl-BuTX has no postsynaptic effect and that fl-BuTX preferentially blocks the mechanism that generates the bell-MEPPs. We believe that the skew-MEPPs generated after the high frequency phase (Fig. 1E) are uncontaminated by the primary releasing mechanism which generates the bellMEPPs. Two different release mechanisms are also indicated with botulinum toxin (BTX) poisoning which also preferentially blocks the beI1-MEPPs 13. The BTX blocking action differs in that it does not induce the transient increase in MEPP frequency that/%BuTX induces. It has been shown that in early periods of reinnervation and in postnatal muscle of the mouse diaphragm that the skew-MEPPs initially dominate the MEPP histogram distributions 19. Also, at chick 3 and frog junctions s,9 the percentage of belI-MEPPs increases as reinnervation develops. It may be that the mechanism of release giving rise to the belI-MEPPs is a more complex event and thus more likely to be compromised with drug challenges. The overall profiles of MEPP amplitude control histograms (Fig. I A) are composed of two parts, the small MEPPs composing the 'skewed' profile and the

601 larger MEPPs composing the 'bell-shaped' distribution. The skewed MEPPs of Fig. 1A show multiple integral peaks. However, the sample size of the beU-MEPPs is too small to analyze for significant integral peaks. Kriebel et a113 have demonstrated that the intervals between the peaks are the same for both classes of MEPPs and thus proposed that MEPPs result from the summed release of a single class of subunits and the release of a subunit generates the s-MEPP. The morphological correlate to a sMEPP (or sub-unit) or the skew-class of MEPPs is not known 21. We conclude from the fl-BuTX experiments presented here that the mechanism that generates the bell-MEPPs is different than that which generates the skew-MEPPs, that skew-MEPP frequencies are not appreciably altered with fl-BuTX after bellMEPP generation has ceased, and that skew-MEPPs are composed of integral multiples of subunits.

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