1. Motor axons were injected ionophoretically with one of five Ca2+-sensitive dyes (fluo-3, Calcium Green-2, Calcium Green-5N, flu0-3FF and Oregon Green BAPTA-5N). Changes in fluorescence (ΔF/F(rest)) 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/F(rest) were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in ΔF/F(rest) began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. ΔF/F(rest) was graded with bath [Ca2+]. Both ΔF/F(rest) 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, K,≃ 60 μM) stimulation-induced increases in ΔFΔ F/F(rest) were measured in the presence of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP, 100 μM). During brief stimulus trains (4 at 50 Hz) in 3,4-DAP, the EPP exhibited profound depression, but tile 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, K(d) ≃ 0.5-1 μM) stimulation-induced increases in ΔF/F(rest) could also be measured in normal physiological saline. Increases in ΔF/F(rest) 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/F(rest) 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.
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