Measurement of action potential-induced presynaptic calcium domains at a cultured neuromuscular junction

Spatially localized Ca2+ domains are thought to play a key role in action p otential (AP)-evoked neurotransmitter release at fast synapses. We used a s tage-scan confocal spot-detection method and the low-affinity Ca2+ indicato r Oregon Green 488 BAPTA-5N to study the spatiotemporal profile of presynap tic AP-induced Ca2+ domains. Families of scanned AP-induced fluorescence tr ansients were detected from spot locations separated by 200-300 nm, within the vicinity of Ca2+ entry sites. Typically, the largest transient in a par ticular scan peaked within similar to 1 msec and decayed with rapid (tau(1) of 1.7 msec) and slow components(tau(2) of 16 msec, tau(3) of 78 msec). As the spot was incrementally displaced, transients progressively exhibited a slowing in their time-to-peak and a loss of the fast decay component. Thre e-dimensional graphs of fluorescence versus time and spot displacement reve aled the presence of AP-induced fluorescence domains that dissipated within similar to 7 msec. The size of fluorescence domains were estimated from th e full-width at half-maximum of gaussian fits to isochronal Delta F/F plots and ranged from 0.6 to 3.0 mu m, with a mean +/- SD of 1.6 +/- 0.6 mu m. M odel simulations of a localized Ca2+ entry site predicted the major feature s of the fluorescence transients and suggested that, within similar to 1 ms ec of the initiation of the Ca2+ current, both the fluorescence domain and the underlying Ca2+ domain do not extend significantly beyond the site of e ntry. Consistent with this prediction, the intracellular addition of EGTA ( up to 2 mM) accelerated the decay of the measured transients but did not af fect the domain size.