At the nerve terminal, neurotransmitter is released by the fusion of synaptic vesicles with presynaptic plasma membrane. After exocytosis, vesicles are retrieved by endocytosis and recycled for reuse. The coupling of exo- and endocytosis of synaptic vesicles is essential for the maintenance of synaptic transmission. The exo- and endocytosis have been measured by capacitance measurement, or by imaging (e.g. FM dyes, pHluorin). However, there are only few studies that apply both techniques sumultaneously, mainly because of the technical difficulty caused by the small size of the nerve terminl (usually around 1 μm).
The calyx of Held is a large synapse in the auditory brainstem. Because of its large terminal size (-20 μm), it allows us to apply electrophysiological and optical methods and analyze presynaptic mechanisms reliably. In this study, we simultaneously measured the turnover of fusion related synaptic vesicle proteins and plasma membrane by applying imaging method and capacitance measurement, respectively.
We labeled synaptotagmin-2 (a putative calcium censor for exocytosis) with an antibody coupled to pH-sensitive fluorophore cypHer5E to monitor the dynamics during exo- and endocytosis. Fluorescent changes of cypHer were measured simultaneously with membrane capacitance to compare the cycling of synaptic vesicle proteins and that of plasma membrane. By varying stimulus conditions or by applying pharmacological agents to manipulate the time course of endocytosis, we tested how the time courses of both measurements were correlated or not correlated. We also investigated how the amount of stranded (i.e. surface-expressed) synaptotagmin-2 changes with time after stimulation.
In addition, exocytosis was also investigated by labeling synaptic vesicles by FM-dye, and observed the discharge of the dye upon the stimulation of the cell.