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.

Effect of the putative cognitive enhancer, linopirdine (DuP 996), on quantal parameters of acetylcholine release at the frog neuromuscular junction

“…Unbiased estimates of quantal release parameters The present study uses a new procedure which avoids the problem of nonuniformity and nonstationarity (Brown et al, 1976) to obtain unbiased estimates of n and p (Provan & Miyamoto, 1993a). Important features of the method are the use of direct quantal counts (m.e.p.ps) and elevated K+ to evoke transmitter release.…”

“…An illustration of the response is presented in Figure 1, in which sample records of m.e.p.ps are shown for control (Figure la (Figure 2a) was, according to the method of Provan & Miyamoto (1993a), equivalent to an increase in m. This increase in m could in turn be due to an increase in n, p, or both n and p (since m = np). To dissociate the effect on m into its quantal components and examine the temporal relationships of the changes, data were recorded for 15 min in Values for m and n are in quanta, whereas p and varp are dimensionless.…”

“…frequency, and the quantal content of the endplate potential (e.p.p.) in Mg2'-depressed preparations (Tsai et al, 1992 We have examined the effect of DuP 996 using a new method for obtaining the quantal -release parameters, m (number of quanta released), n (number of functional release sites), p (probability of release), and varsp (spatial variance in p) (Provan & Miyamoto, 1993a). This approach circumvents the problem of nonuniformity and nonstationarity in the quantal estimates (Brown et al, 1976), as well as the problem of inaccuracies in the nerve-evoked synaptic potential (Zucker, 1977).…”

“…The aims of this study were (a) to examine whether DuP 996 produces a significant increase in quantal ACh release, as monitored by the release parameters, and (b) to elucidate the cellular or subcellular mechanism by which DuP 996 enhances neurosecretion. A preliminary account of these results was presented at the 1992 Meeting of the American Society for Pharmacology and Experimental Therapeutics in Orlando, FL (Provan & Miyamoto, 1992 (Provan & Miyamoto, 1993a). The control solution thus contained (in mM): NaCl 102.5, KCl 10.0, CaCl2 1.8, Tris 2.0 (adjusted to pH 7.2), and glucose 5.6.…”

“…The control solution thus contained (in mM): NaCl 102.5, KCl 10.0, CaCl2 1.8, Tris 2.0 (adjusted to pH 7.2), and glucose 5.6. Muscles were equilibrated for at least 30 min before use, to allow re-establishment of the Donnan equilibrium and ensure that results were obtained during the longterm, steady-state effect of K+ (Cooke & Quastel, 1973 (Hille, 1984 Data collection, analysis and statistics Specific details of the overall procedure-are described elsewhere (Provan & Miyamoto, 1993a). Briefly, the number of quanta released by one nerve impulse (m) was replaced by the number of m.e.p.ps occurring in a 50 ms time interval (bin), and 500 sequential bins were used for each estimate.…”