Method for the determination of trajectory angles of directional secretory vesicles in cultured astrocytes

Astrocytes provide a principal pathway for glutamate uptake in the mammalian brain, a task accomplished by the powerful action of excitatory amino acid transporters (EAAT) 1 and 2. These transporters are synthesized within the endoplasmic reticulum and are then trafficked to the plasma membrane. The characteristics of their intracellular traffic within astrocytes have not been investigated. We monitored the trafficking of secretory vesicles laden with the recombinant fluorescent protein chimera of EAAT2 in cultured astrocytes. Such vesicles appeared as fluorescent puncta, and their trafficking parameters were obtained using original algorithms, which we describe here in detail. We determined the maximal displacement, average instantaneous speed, and trajectory angle of individual puncta/vesicles, with angles near 0° indicating radial movement directly away from or toward the nucleus and angles near 90° indicating tangential movement. Analysis of these trafficking parameters demonstrated that trafficking of EAAT2-laden vesicles has typical characteristics expected of the trafficking of secretory vesicles in cultured astrocytes. The distribution of trajectory angles for directional vesicles, i.e. those with a maximal displacement greater than 1 μm within the 40-s time-lapse imaging, was found to be unimodal, with angles near 0° being the most prominent (mode 7°). These measurements are in good agreement with previous measurements of trajectory angles of similar trafficking vesicles carrying cannabinoid receptor 1, evidencing the validity and robustness of our analytical approach and algorithms.