As described previously [40 (link)–42 (link)], neurons on glass coverslips were placed in a 35-mm petridish containing the Hibernate E low-fluorescence medium (BrainBits) on a heated stage of 37°C, and imaged with a 63×/N.A.0.9 water-immersion objective with excitation at 561 nm or 488 nm. Time-lapse movies were obtained continually with 3–5 sec intervals before and after Antimycin A (100 μM, Sigma-Aldrich) was added. Axons longer than 50 μm were selected for recording. Movie length ranged from 120 to 300 min. TMRM (T668, Molecular Probes) was applied at 250 nM for 10 min when needed. For quantification, kymographs were generated from time-lapse movies by ImageJ, representing a 100-sec period either right before or following different time points after addition of Antimycin A. Each kymograph was then imported into a macro written in Labview (NI, TX), and individual mito-dsRed puncta were traced using a mouse-driven cursor at the center of the mito-dsRed object. Using Matlab (The MathWorks, MA), we determined the following parameters: 1) the instantaneous velocity of each mitochondrion, 2) the average velocity of those mitochondria that are in motion, 3) the percent of time each mitochondrion is in motion, 4) stop frequency, and 5) turn back frequency. The intensity of mitochondria is measured using ImageJ.