Ca2+ dynamics at the frog motor nerve terminal

Abstract

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.

Ca2+from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

“…Therefore, we conclude that the total amount of buffer present in the terminals of NMJs under study (endogenous and added dye) was less than the equivalent of 0.5 mM concentration of our theoretical mobile buffer. This is consistent with the experimentally derived estimate of low buffer capacity in the frog NMJ (Suzuki et al, 2000) and also puts an upper limit on the amount of buffering provided by the addition of the Ca 2ϩ indicator in our experiments.…”

“…Our simulations also indicate that increasing the number of open channels per AP (e.g., by widening APs pharmacologically or by lengthening voltage-clamp steps) should increase the cooperativity. It is believed that the frog NMJ has a relatively low buffer capacity of ϳ50 (Suzuki et al, 2000), and our experimental and simulation results support this conclusion. In our simulations increasing buffer capacity by a factor of five above experimental estimates (using 0.5 mM of buffer) reduces the estimated channel cooperativity by Ͻ20%.…”

“…Measurements of the endogenous Ca 2ϩ buffer capacity in frog NMJ are limited, but there is some evidence that the buffer capacity may be as low as 50 (Suzuki et al, 2000). To test the dependence of our conclusions on endogenous buffer capacity, we assumed the presence of different amounts (0 -0.5 mM) of a buffer with a rather high affinity (dissociation constant, K D ϭ 2 M), fast kinetics (k on ϭ 3 ϫ 10 8 M Ϫ 1 /s), and slow diffusion (27.5 m 2 /s) (Burrone et al, 2002).…”

Augmentation Increases Vesicular Release Probability in the Presence of Masking Depression at the Frog Neuromuscular Junction

“…Figure 6 A shows pseudocolor ratio images before (A1), during (A2), and 1.75 sec (A3) and 186 sec (A4 ) after a conditioning train. The ratio images indicate that the free Ca 2ϩ in the nerve terminal increased during repetitive stimulation and decayed thereafter, as observed previously (Almers and Neher, 1985;Delaney et al, 1989;Swandulla et al, 1991;Delaney and Tank, 1994;Kamiya and Zucker, 1994;Regehr et al, 1994;Lin et al, 1998;Suzuki et al, 2000;Rosenmund et al, 2002;Zucker and Regehr, 2002) The rise and decay of free Ca 2ϩ in the nerve terminal from a single preparation during and after conditioning trains with 2 mM Ca 2ϩ is shown in Figure 6 B (continuous line with some variability). For this experiment, the terminals were loaded with bis-fura-2.…”

“…Fluorescence ratio imaging techniques were used to measure average intracellular free Ca 2ϩ in motor nerve terminals during and after repetitive stimulation (Almers and Neher, 1985;Delaney et al, 1989;Suzuki et al, 2000). Ca 2ϩ indicators were loaded into frog motor nerve terminals by backfilling the nerves (Peng and Zucker, 1993;Wu and Betz, 1996).…”