Miniature endplate potential frequency and amplitude determined by an extension of Campbell's theorem

Abstract

A method based upon an extension of Campbell's theorem is used to measure the amplitude, waveform, and frequency of occurrence of miniature endplate potentials (mepps) at rapidly secreting neuromuscular junctions of frog cutaneous pectoris muscles. Measurements of the variance, skew, and power spectrum of the fluctuations in membrane potential are used to deduce the mepp parameters. These estimates of mepp amplitude and frequency are insensitive to slow drifts in membrane potential that preclude the conventional application of Campbell's theorem, which uses the mean and variance. The new method becomes unreliable at high mepp frequencies because the distribution of the values of membrane potential approaches a Gaussian thereby reducing the accuracy of skew measurements. Frequencies approaching 10(4) s-1 can be measured, however, if the data are high-pass filtered. The method has been tested with computer simulated data and applied to junctions exposed to La3+; the effects of Ca2+ on the La3+-induced secretion have been explored. Some muscles were fixed after treatment with La3+, and changes in nerve terminal ultrastructure were assessed by morphometric analysis of electron micrographs. Horseradish peroxidase was used to obtain information about vesicle recycling.

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

“…This method selectively measures quantal secretion and is insensitive to non-quantal secretion. Its results are valid in spite of slow, spurious changes in the average membrane potential, non-stationary MEPP rates and non-linear summation of MEPPs (Segal et al 1985;Fesce et al 1986a). However, these values of <h> overestimate, and the values of <r> underestimate, the true ones, because the MEPPs recorded at a single junction are not equal in amplitude.…”

“…The high-gain AC record was put through the low-pass Butterworth filter (1250 Hz), then through a high-pass RC filter, and the output was sampled by the computer at a rate of 2500 Hz. The RC filter suppresses the lowfrequency spectral components which arise from non-stationarities in the rate and reduces the random errors of the measurements (Segal et al 1985;Fesce et al 1986 a). A10 s sample of data was collected (25000 digitized points), and the variance, V (2nd central moment or cumulant), the skew, S (3rd central moment or cumulant), and the 4th cumulant (or semi-invariant), C4, were calculated (5 s computation time).…”

“…the cumulants had to double before their net values were used to estimate MEPP rate and amplitude). The net values of the cumulants were corrected for non-linear summation (the correction was significant only for rates > 100/s) and the mean MEPP rate, <r>, and amplitude factor, <h>, were calculated from the equations (Rice, 1944;Segal et al 1985;Fesce etal. 1986a…”

Making quantal analysis more convenient, fast, and accurate: User-friendly software QUANTAN

“…This situation may be further complicated by a non-stationarity of asynchronous release. Solving these problems requires fluctuation analysis of postsynaptic currents (Segal et al, 1985;Fesce et al, 1986;Rossi et al, 1994) and taking into account non-stationary synaptic activity Sakaba, 2001a, 2003), which is beyond the capabilities of the QUANTAN package.…”

“…This situation may be further complicated by a nonstationarity of asynchronous release. Thus, fluctuation analysis of postsynaptic currents has been developed (Segal et al, 1985;Fesce et al, 1986;Rossi et al, 1994) to estimate the release rate and mEPSC parameters under conditions when the rate of asynchronous release is high and individual quantal events are not detectable. This approach has been adapted and further developed by Sakaba (2001a,b, 2003) who took into account non-stationary synaptic activity, which could result from postsynaptic receptor desensitization or residual glutamate in the synaptic cleft and residual postsynaptic current.…”