High-resolution measurement of the time course of calcium-concentration microdomains at squid presynaptic terminals

Transmitter release is considered to be a secretory event triggered by localized calcium influx which, by binding to a low-affinity Ca2+ site at the presynaptic active zone, initiates vesicular exocytosis (1-7). In previous experiments with aequorin-loaded presynaptic terminals we visualized, upon tetanic presynaptic stimulation, small points of light produced by calcium concentration microdomains of about 300 microM (5). These microdomains had a diameter of about 0.5 microns (5) and covered 5-10% of the total presynaptic membrane with an average density of 8.4 microns2 per 100 microns2, corresponding closely to the size and distribution of the active zones in that junction (6, 7). To understand in more detail the nature of these concentration microdomains, we obtained rapid video images (400/s) after injecting the photoprotein n-aequorin-J into the presynaptic terminals of squid giant synapses. Using that experimental approach, we determined that microdomains evoked by presynaptic spike activation had a duration of about 800 microseconds. Spontaneous quantum emission domains (QEDs) observed at about the same locations as the microdomains were smaller in amplitude, shorter in duration, and less frequent. These results illustrate the time course of the calcium concentration profiles responsible for transmitter release. Their extremely short duration compares closely with that of calcium current flow during a presynaptic action potential and indicates that, as theorized in the past (6-8), intracellular calcium concentration at the active zone remains high only for the duration of transmembrane calcium flow.