ImageJ version 1.43 software (NIH, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/) was used to analyze parameters of mitochondrial morphology and motility as described previously [28, 54]. In detail, the length of single mitochondria (aspect ratio), mitochondrial network formation (form factor) and mitochondrial trafficking parameters (including the number of moving mitochondria and their direction) were evaluated. Fluorescence microscopy images were optimized by adjusting the contrast and subsequently binarized by conversion to 8 bit images. After reduction of unspecific noise of the fluorescence signal, a threshold was applied to the images to define mitochondrial structures. The threshold was kept equal within one experiment. Analysis of the aspect ratio as well as the form factor included the use of the ‘analyze particle’ function in ImageJ program to preclude from inclusion of unspecific background. At least 3 fields of view were used per coverslip, and several thousand mitochondrial structures were analyzed per condition (= neurons at day 40 or day 100). For quantification, all samples within one experiment were stained simultaneously and imaged with identical settings. For analysis of mitochondrial trafficking studies, kymographs were created using
