Pool-independent labelling of synaptic vesicle exocytosis with single vesicle resolution in rat hippocampal neurons

FM dyes are an established tool to analyze synaptic vesicle pools. However, quantitative measurements using FM dyes are typically based on the re-release properties of previously labelled vesicles, which might vary depending on the experimental setup. An FM dye protocol independent of the previous labelling of vesicle membrane has not been applied for quantitative measurements of individual synaptic vesicles before. We therefore analyzed the direct staining of newly exocytosed vesicle membrane with FM dyes in cultured rat hippocampal neurons. In the presence of FM 1-43, stimulation-induced synaptic activity led to a stable fluorescence increase. The quantal release of synaptic vesicles was preserved and its amplitude correlated highly with the exocytic dye loss induced by a subsequent stimulation. Thus, the method presented here provides a tool for the pool-independent measurement of synaptic vesicle exocytosis.

► Staining of newly exocytosed membrane with FM 1-43 with single vesicle resolution. ► Pool-independent labeling of synaptic vesicles independent of vesicle reuse. ► Comparison of various direct and indirect FM dye staining protocols.