Single synaptic vesicle tracking in individual hippocampal boutons at rest and during synaptic activity

How synaptic vesicles move within central nervous synapses to their docking sites at the plasma membrane is widely discussed in synaptic physiology. This question is especially difficult to investigate in the small hippocampal boutons, which themselves can slowly move during observation in primary cell culture. Here, we describe a single particle tracking method using dual fluorescent dye labels that enabled us to visualize the movements of a single vesicle and the respective synaptic bouton simultaneously during resting conditions and stimulation. We found vesicle mobility to be very low in the absence of stimulation, in line with previous studies. Interestingly, mobility was also found to be low during synaptic activity. We found that vesicles labeled preferentially via early, late, and spontaneous endocytotic mechanisms behaved similarly at rest and during stimulation.