Figure 10. Increasing the intracellular concentration of the calcium buffer EGTA further delays both the calcium signal in boutons and the onset of asynchronous release.
(A) Sample traces comparing the time-dependent Fluo-5F fluorescence increases during stimulation for selected boutons of CaP motor neurons dialyzed with the indicated EGTA concentrations. (B) Cumulative data comparing the effects of 0.5 mM (blue; 61 boutons from 4 fish) and 5 mM (red; 44 boutons from 3 fish) EGTA on the time required to reach 20% maximal fluorescence change in boutons of ω-conotoxin GVIA-treated fish. The distances from the reference point were determined using Imaris filament reconstruction and binned (30 μm bin size). Control neuron dialyzed with 0.5 mM EGTA (black; 47 boutons from 3 fish) is shown for comparison. (C) Sample patch clamp recordings of muscle EPCs performed at three different EGTA concentrations in ω-conotoxin GVIA-treated fish. The action potentials are not shown but the stimulus lasted 35 s for the bottom trace and 20 s for the other traces. (D) The associated integrated synaptic currents as a function of time with 100 Hz stimulation beginning at time 0. Only a single example is shown for 25 mM EGTA because this concentration blocked most transmission in other trials. (E) Comparisons of time to reach peak release for all recordings made using 0.5 mM (4.8 ± 0.4 s, n = 8) and 5 mM (10.5 ± 2.4 s, n = 13) EGTA in ω-conotoxin GVIA-treated fish. Data set for 5 mM EGTA was duplicated from Figure 2 for comparison.