Axonal Transport Measurements

Prior to the measurements, the cultured DRG neurons were loaded with MitoTracker Red FM (50 nM; Invitrogen, Carlsbad, CA, USA). To visualize the mitochondria, the MitoTracker was excited at 580 nm by a TILL Poly V light source (Gräfelfing, Germany). During the measurements, the DRG neurons were perfused in HEPES solution (containing 148 mM NaCl, 5 mM KCl, 0.1 mM MgCl₂•1.6H₂O, 10 mM glucose, 10 mM HEPES, and 2 mM CaCl₂•2.2H₂O) at 37°C by a heated gravity-fed perfusion system (Multichannel systems, Reutlingen, Germany), and 200 images were recorded at 1 Hz by a cooled charge-coupled device camera (Sensicam QE, PCO, Kelheim, Germany) using TILL VisION. To analyze different parameters of mitochondrial movement, kymographs, or spatiotemporal maps were obtained using Igor Pro (Wavemetrics, Portland, USA) using custom-written routines based on a previous described analysis algorithm [27].