Stimulation-induced changes in [Ca2+] in lizard motor nerve terminals

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 (Delta F/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 Delta F/F-rest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in Delta F/F-rest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. Delta F/F-rest was graded with bath [Ca2+]. Both Delta F/F-rest and the EPP were reduced, but not eliminated, by omega-conotoxin GVIA (5-10 mu M). 3. For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA-5N, K-d approximate to 60 mu M) stimulation-induced increases in Delta F/F-rest were measured in the presence of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP, 100 mu M). 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, K-d approximate to 0.5-1 mu M) stimulation-induced increases in Delta F/F-rest could also be measured in normal physiological saline. Increases in Delta F/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 Delta F/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.