Modulation of the kinetics of evoked quantal release at mouse neuromuscular junctions by calcium and strontium

Abstract

The effects of calcium and strontium on the quantal content of nerve-evoked endplate currents and on the kinetic parameters of quantal release (minimal synaptic delay, value of main mode of synaptic delay histogram, and variability of synaptic delay) were studied at the mouse neuromuscular synapse. At low calcium ion concentrations (0.2-0.6 mmol/L), evoked signals with long synaptic delays (several times longer than the value of main mode) were observed. Their number decreased substantially when [Ca 2+ ] o was increased (i.e. the release of transmitter became more synchronous). By contrast, the early phase of secretion, characterized by minimal synaptic delay and accounting for the main peak of the synaptic delay histogram, did not change significantly with increasing [Ca 2+ ] o . Hence, extracellular calcium affected mainly the late, 'asynchronous', portion of phasic release. The average quantal content grew exponentially from 0.09 ± 0.01 to 1.04 ± 0.07 with the increase in [Ca 2+ ] o without reaching saturation. Similar results were obtained when calcium was replaced by strontium, but the asynchronous portion of phasic release was more pronounced and higher strontium concentrations (to 1.2-1.4 mmol/L) were required to abolish responses with long delays. Treatment of preparations with 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) (25 lmol/L), but not with ethylene glycol-bis(2-aminoethylether)-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester (EGTA-AM) (25 lmol/L), abolished the responses with long delays. The dependence of quantal content and synchrony of quantal release on calcium and strontium concentrations have quite different slopes, suggesting that they are governed by different mechanisms. Keywords: BAPTA-AM, calcium, EGTA-AM, quantal release, strontium, time course of the evoked quanta release.

Modeling of quantal neurotransmitter release kinetics in the presence of fixed and mobile calcium buffers

“…This agrees with experimental data obtained from Purkinje cells, where it was shown that the mobile endogenous buffer calbindin decreased the amplitude of spatially distributed Ca 2+ concentration (Schmidt et al 2003). In experiments at neuromuscular junctions, we also observed a decrease in quantal content in the presence of a calcium buffer-BAPTA-AM (Bukharaeva et al 2007), which indicates a lowering of intracellular calcium concentration.…”

“…The results of mathematical modeling investigations in the presence of endogenous fixed and mobile calcium buffers fit with the changes in TCS that we observed in experiments at the neuromuscular junction of mice by varying the extracellular Ca 2+ content and applying exogenous buffers with rapid kinetics, such as BAPTA-AM (Samigullin et al 2005;Bukharaeva et al 2007). During the recording of uni-quantal endplate currents under increased concentrations of Mg 2+ , it was found that a decrease in Ca 2+ concentration from 0.4 to 0.2 mM produces, together with a decrease in quantal content, an increase in the fluctuation of synaptic delays, i.e., a desynchronization [ Fig.…”

“…The importance of assessing the role of calcium buffers is supported by the fact that only 1-5% of the entering Ca 2+ can be considered to be "free" calcium (Ahmed and Connor 1988), whereas the vast majority of the entering calcium is practically immediately bound to calcium-binding sites, the mobility of which determines the nature of Ca 2+ diffusion from their entry region into nerve endings and the sites where vesicles are docked (Zhou and Neher 1993;Gabso et al 1997;Muller et al 2005). The model-in agreement with previous experiments (Samigullin et al 2005;Bukharaeva et al 2007)-showed that the presence of an endogenous fixed calcium buffer, the diffusion coefficient of which is very much smaller than the diffusion coefficient of free Ca 2+ , brings about the desynchronization of secretion under conditions of decreased extracellular Ca 2+ and synchronization upon adding a mobile buffer.…”

“…Despite this, at an increased Ca 2+ flux, the early phase of the TCS grew more rapidly at the expense of a higher number of transmitter quanta released with a shorter synaptic delay; that is, this early phase was accelerated in the model situation, when no buffers are assumed. Still, in the experimental work performed by ourselves (Samigullin et al 2005;Bukharaeva et al 2007) as well as by other authors (Barrett and Stevens 1972;Van der Kloot 1988;Parnas et al 1986), no changes in the kinetics of the early phase of neurotransmitter secretion were found with a higher concentration of Ca 2+ , According to the Kolmogorov-Smirnov criteria, the difference between the curves is statistically significant if the confidence limits of the two curves do not overlap and we can, therefore, assume that some retarding calcium buffers must be present in nerve terminals.…”

“…In our recent experiments with the mouse neuromuscular synapse, we showed that Ca 2+ ions modify the time course of release of acetylcholine (Samigullin et al 2005;Bukharaeva et al 2007). The decrease in extracellular Ca 2+ lowered the quantal content as expected and caused an increase in the fluctuations of synaptic delays; that is, it desynchronized secretion.…”