Initial Distance‐Dependent Mean Force Drives Synaptic Vesicle Motion Toward Fusion Sites in Stimulated Hippocampal Neurons

Abstract Neuronal communication occurs through transport and exocytosis of synaptic vesicles (SVs). However, their dynamics during neuronal stimulation remains poorly understood. Here, real‐time, 3D motion of individual SVs undergoing exocytosis in presynaptic terminals is quantitatively investigated. SVs are categorized into two types: Type I showing confined motion near fusion sites until exocytosis and Type II SVs exhibiting unconfined motion before tethering and exocytosis. Type II SVs have a broader fusion time distribution with a higher mean value than Type I SVs. Electrical stimulation increases the straightness of the Type II trajectories toward their fusion sites approximately tenfold. To quantify the straightness of the SV trajectories, a straightness parameter is introduced and its relationship to the mean force exerted on SVs is established. Interestingly, the straightness parameter, and hence mean velocity, increase in a sigmoidal manner with the initial distances of Type II SVs from their fusion sites upon stimulation, which results in a counterintuitive non‐monotonic dependence of their fusion time on the initial distances. A quantitative model is presented that simultaneously explains various experimental results regarding SV transport and fusion dynamics. This work offers new insights into mysterious SV motion at presynaptic terminals and its consequences on synaptic transmission of stimulated neurons.