Synaptic vesicle acidification and exocytosis studied with acridine orangefluorescence in rat brain synaptosomes

The acidification of synaptic vesicles (SV) in rat brain synaptosomes was s tudied using acridine orange (AO) as a fluorescent probe. In synaptosomal s uspensions the AO fluorescence was partially quenched, indicating the prese nce of an acidic compartment. In permeabilized synaptosomes, the quenching was augmented by MgATP and was sensitive to concanamycin A, a specific inhi bitor of the V-type H+-ATPase known to be present in synaptic vesicles. Som e ATP-dependent acidification was also observed without permeabilization, s uggesting that a fraction of synaptosomes (ca. 15%) was unsealed, irrespect ive of the method used to prepare the synaptosomes (sucrose or Ficoll densi ty gradient, sedimentation or flotation). Depolarization of synaptosomes wi th 30 mM KCI resulted in an immediate, albeit small, rise in AO fluorescenc e that was prevented by the removal of Ca2+ or by substituting NaCl for KCI . This response is consistent with depolarization-evoked release of the aci dic contents of an exocytosis-competent pool of synaptic vesicles, represen ting ca. 5% of the total. No further AO release subsequent to the immediate phase was observed in depolarized synaptosomes, which indicates an extreme ly rapid reacidification. The results demonstrate that AO fluorescence is s uitable for monitoring SV acidification within synaptosomes, and may be use d to derive an independent estimate of the relative size of the immediately releasable SV pool. In addition, the use of AO might be advantageous for t he assessment of synaptosomal integrity by comparing the ATP-dependent acid ification in intact and permeabilized synaptosomes.