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Spatially localized Ca2+ domains are thought to play a key role in action potential (AP)-evoked neurotransmitter release at fast synapses. We used a stage-scan confocal spot-detection method and the low-affinity Ca2+ indicator Oregon Green 488 BAPTA-5N to study the spatiotemporal profile of presynaptic AP-induced Ca2+ domains. Families of scanned AP-induced fluorescence transients were detected from spot locations separated by 200–300 nm, within the vicinity of Ca2+ entry sites. Typically, the largest transient in a particular scan peaked within ∼1 msec and decayed with rapid (τ1 of 1.7 msec) and slow components (τ2 of 16 msec, τ3 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. Three-dimensional graphs of fluorescence versus time and spot displacement revealed the presence of AP-induced fluorescence domains that dissipated within ∼7 msec. The size of fluorescence domains were estimated from the full-width at half-maximum of gaussian fits to isochronal ΔF/F plots and ranged from 0.6 to 3.0 μm, with a mean ± SD of 1.6 ± 0.6 μm. Model simulations of a localized Ca2+ entry site predicted the major features of the fluorescence transients and suggested that, within ∼1 msec of the initiation of the Ca2+ current, both the fluorescence domain and the underlying Ca2+ domain do not extend significantly beyond the site of entry. Consistent with this prediction, the intracellular addition of EGTA (up to 2 mm) accelerated the decay of the measured transients but did not affect the domain size.

Measurement of Action Potential-Induced Presynaptic Calcium Domains at a Cultured Neuromuscular Junction