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Department of Physiology, UCLA School of Medicine 90095, USA.

1. Action potential (AP)-induced fluorescence transients were measured, using Ca2+ indicators and a spot-detection method, at single nerve terminals of a cultured Xenopus neuromuscular junction preparation with simultaneous measurement of neurotransmitter release. 2. Transients obtained using the low affinity Ca2+ indicator Oregon Green 488 BAPTA-5N (OGB-5N) exhibited rapid rising (t1/2 (time at which one-half of the peak fluorescence was attained) = 0.54 ms) and decaying (tau fast = 1.9 ms) phases. The higher affinity indicator Oregon Green 488 BAPTA-2 (OGB-2) produced transients with significantly slower kinetics (t1/2 = 2 ms; tau slow = 73 ms). 3. Tetanic stimulation elicited distinct increases in fluorescence in response to each AP. Each OGB-5N fluorescence increase was more rapid than those observed using OGB-2. Furthermore, a smaller proportion of residual fluorescence at the end of the train was observed using OGB-5N. 4. When OGB-5N was used, a significant [Ca2+] increase was observed prior to the release of neurotransmitter. This was not observed when OGB-2 was used. 5. We conclude that the use of localized optical detection coupled with low affinity Ca2+ indicators can help elucidate rapid changes in presynaptic [Ca2+] dynamics underlying evoked neurotransmitter release.