Figure 2. Sites of action potential-evoked Ca²⁺ entry and phalloidin puncta are colocalized at the presynaptic terminal.

Aa, action-potential-evoked Ca²⁺ responses recorded using Ca²⁺-sensitive dye, Oregon Green 488 BAPTA-1. Evoked Ca²⁺ entry occurs at discrete and localized ‘hotpots’ along the length of the axon, as shown here. Ab, injection of fluorescently labelled phalloidin (Alexa Fluor 568 phalloidin) into this axon marks phalloidin puncta (red) only at the same locations as evoked Ca²⁺ responses (n = 9 puncta). Every identifiable phalloidin punctum, conversely, demonstrates a resolvable Ca²⁺ signal (n = 18 phalloidin puncta, in 8 axons), both at the same region as previously measured (shown in Ac) and at other regions of same axon as in B. Ba, phallodin puncta at other regions of axon also show action-potential-evoked Ca²⁺ hotspots, as seen in Bb.