Vesicle movement was analyzed using KymographClear and KymographDirect (59). Time-lapse movies were loaded into ImageJ, and the KymographClear toolset was used to create kymographs from sections of axons. Axons chosen for analysis were isolated from other axons and had multiple clearly visible vesicles that move through the region during the image sequence. Representative axons were chosen from each field of view, with six to eight axons analyzed from each culture. At least three independent cultures were analyzed for each condition. Vesicle tracks were manually traced in ImageJ, and the tracks were recorded using KymographClear. The kymograph and manual traces were loaded into KymographDirect, where information on average fluorescence intensities and velocities was obtained for each vesicle track. Data were imported into Origin for statistical analysis.

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