Probing vesicle dynamics within small synapses

Sustained synaptic transmission at the hippocampal synapse (O∼1 μm) requires continuous exocytosis and subsequent endocytosis of synaptic vesicles (O∼35 nm). The kinetics of this process have been widely studied, however, due to a lack of appropriate techniques, little is known about the mobility of small vesicles within the synapse. Fluorescence fluctuation spectroscopy (FFS) was introduced by Jordan (2000) to study vesicle dynamics in resting synapses. This study is supplemented here with Monte-Carlo simulations to propose a quantitative model of vesicle mobility. Vesicle movement was found to be slow with a diffusion coefficient of 5 •10-5 μm2/s and restricted to a cage of ∼50 nm. The effects of disabling motor proteins and treatment with a phosphatase blocker on vesicle dynamics were also analyzed using simulations.Furthermore, the development of a single particle technique used to monitor single fluorescently labelled vesicles both, at rest and during stimulation of the synapse, is described. An automated algorithm identified single vesicles and tracked their movement in real-time. Vesicle mobility was studied in specific vesicle populations that differed in their mode of endocytosis from the plasma membrane. Results from the particle tracking and the FFS study were found to be in good agreement. Vesicle mobility was observed to increase, but was still slow during stimulation.Additionally, a theoretical investigation of the performance of single photon counting devices with dead-time for the task of single particle tracking was performed. A Maximum Likelihood based algorithm is presented which allows to determine the position of single subresolution fluorescent particles, and yields a precision higher than expected from the total number of photons collected with these detectors.