Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

Dmitry Samigullin,Dmitry Samigullin,Dmitry Samigullin,Eugeny Nikolsky,Nijaz Fatikhov,Andrey Skorinkin,Ellya Bukharaeva,Eduard Khaziev,Eduard Khaziev

doi:10.3389/fnsyn.2014.00029

Abstract

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

Highlights

Calcium ions (Ca2+) play a leading role in the initiation, maintenance, and plasticity of neurotransmitter release at chemical synapses in both the central and peripheral nervous systems (Llinás et al, 1992; Chow et al, 1994; Augustine, 2001; Burnashev and Rozov, 2005; Schneggenburger and Neher, 2005; Pang and Südhof, 2010)
ACTION POTENTIAL INDUCED RESPONSES OF Ca2+ DYES WITH DIFFERING AFFINITIES FOR Ca2+ The peak of the fluorescence signal in nerve terminals loaded with the low-affinity dye Magnesium Green (MG) was about 2.6 times smaller than in terminals loaded with the high-affinity dye Oregon Green BAPTA-1 (OGB-1)
Identical inward calcium currents evoked in nerve terminals loaded with dyes with different properties are accompanied by fluorescence signals with different amplitude and temporal properties

Summary

Introduction

Calcium ions (Ca2+) play a leading role in the initiation, maintenance, and plasticity of neurotransmitter release at chemical synapses in both the central and peripheral nervous systems (Llinás et al, 1992; Chow et al, 1994; Augustine, 2001; Burnashev and Rozov, 2005; Schneggenburger and Neher, 2005; Pang and Südhof, 2010). The influx of Ca2+ into the nerve terminal following the action potential initiates the release of neurotransmitter and the subsequent postsynaptic potential. Optical methods may be employed to estimate the action-potential-induced Ca2+ influx and the subsequent distribution of Ca2+ ions in the axoplasm. The basis of these methods is the recordings of fluorescence transients which arise as a result of changes in the fluorescence intensity of specific calcium-sensitive dyes that interact with Ca2+ ions (DiGregorio and Vergara, 1997; Sabatini and Regehr, 1998; Suzuki et al, 2000; Sabatini et al, 2002; Luo et al, 2011). The profile of intracellular Ca2+ distribution, and the parameters of the fluorescence signal, is defined by the calcium influx and by the concentration of endogenous calciumbinding proteins

Methods

Results

Conclusion