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

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.

Acetylcholine-Induced Inhibition of Presynaptic Calcium Signals and Transmitter Release in the Frog Neuromuscular Junction

“…At the end of loading protocol, all terminals in the proximal part of the nerve trunk had sufficient levels of fluorescence to allow [Ca 2+ ] i recordings. We (in this paper), as well as others have shown that the loading of the fluorescent dye in the nerve ending does not significantly alter the physiological parameters of secretion, such as quantal content and frequency of mEPC (Wu and Betz, 1996; Samigullin et al, 2015). …”

“…Our own observations (Samigullin et al, 2015) as well as observations of others (Wu and Betz, 1996) failed to detect any appreciable influence of loaded Ca 2+ probe on the amplitude of the postsynaptic response or on the frequency of the miniature end-plate potentials. Nonetheless we additionally performed control experiments to compare spontaneous endplate currents (mEPC) and quantal release before and after Ca 2+ probe loading.…”

“…Each nerve-muscle preparation was loaded with Ca 2+ -sensitive dye by soaking nerve stump in 50 mM solution of fluorescent Ca 2+ -indicator Oregon Green 488 BAPTA-1 Hexapotassium Salt (Molecular Probes, Eugene, Oregon, USA); for details of loading technique see (Peng and Zucker, 1993; Wu and Betz, 1996; Tsang et al, 2000; Samigullin et al, 2015). At the end of loading protocol, all terminals in the proximal part of the nerve trunk had sufficient levels of fluorescence.…”

“…Fluorescent signal was recorded using photometric setup on the base of Olympus BX-51 microscope with x60 water-immersion objective connected to photodiode S1087 (Hamamatsu, Japan) as described in Sinha and Saggau (1999) and Samigullin et al (2010, 2015). The region for recording was selected by optical viewfinder (Till Photonics, Munich, Germany).…”

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

“…The rate of entry of Ca 2+ into the nerve ending, interaction with the dye, and diffusion in the cytoplasm all affect the rise time of the Ca 2+ transient. The decay time of the fluorescent signal depends on the affinity of the dye, the speed of the Ca 2+ interaction with intracellular buffers, and the removal by ion pumps 35 . The amplitude analysis of Ca 2+ transients can be used to study the influence of various substances on the calcium entry that participates in neurotransmitter release 33 .…”