Abstract
1
Motor axons were injected ionophoretically with one of five Ca2+‐sensitive dyes (fluo‐3, Calcium Green‐2, Calcium Green‐5N, fluo‐3FF and Oregon Green BAPTA‐5N). Changes in fluorescence (ΔF/Frest) within motor terminal boutons following a single action potential and brief stimulus trains were monitored with high temporal resolution using a confocal microscope.
2
Stimulation‐induced increases in ΔF/Frest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in ΔF/Frest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. ΔF/Frest was graded with bath [Ca2+]. Both ΔF/Frest and the EPP were reduced, but not eliminated, by ω‐conotoxin GVIA (5–10 μm).
3
For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA‐5N, Kd≈ mm) stimulation‐induced increases in ΔF/Frest were measured in the presence of the K+ channel blocker 3,4‐diaminopyridine (3,4‐DAP, 100 mm). During brief stimulus trains (4 at 50 Hz) in 3,4‐DAP, the EPP exhibited profound depression, but the fluorescence increase associated with each stimulus showed little decrement, suggesting that depression was not mediated by a reduction in Ca2+ entry.
4
For dyes with a higher affinity for Ca2+ (e.g. fluo‐3, Kd≈ 0.5–1 μm) stimulation‐induced increases in ΔF/Frest could also be measured in normal physiological saline. Increases in ΔF/Frest were much greater with 3,4‐DAP present, but the amplitude decreased with successive stimuli due to partial dye saturation.
5
Calculations suggested that following a single action potential the average [Ca2+] within a bouton increased by up to 150 nm in normal saline and 940 nM in 3,4‐DAP. With low affinity dyes the ΔF/Frest measured near the membrane had a higher peak amplitude and a faster early decay than that measured in the centre of the bouton, suggesting that substantial spatial [Ca2+] gradients exist within boutons for at least 15 ms following stimulation.
