Unbiased estimates of quantal release parameters and spatial variation in the probability of neurosecretion

Abstract

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n) and probability of release (p), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 microM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La(3+)-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n and p and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.