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gdavid@newssun.med.miami.edu

1. Motor axons were injected ionophoretically with one of five Ca(2+)-sensitive dyes (fluo-3, Calcium Green-2, Calcium Green-5N, fluo-3FF and Oregon Green BAPTA-5N). Changes in fluorescence (delta F/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 delta F/Frest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in delta F/Frest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. delta F/Frest was graded with bath [Ca2+]. Both delta F/Frest and the EPP were reduced, but not eliminated, by omega-conotoxin GVIA (5-10 microM). 3. For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA-5N, Kd approximately 60 microM) stimulation-induced increases in delta F/Frest were measured in the presence of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP, 100 microM). 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 approximately 0.5-1 microM) stimulation-induced increases in delta F/Frest could also be measured in normal physiological saline. Increases in delta F/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 delta F/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.

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