All analyses of ultrastructural features were performed on unaltered images that were collected directly from the electron microscope camera. Images

shown in the figures were adjusted only for contrast to Tasocitinib enhance visibility for the reader. Degenerative inclusions (vacuoles, tubulovesicles, autophagic vesicles, and dense core vesicles) have been reported to occur in aging or following ischemia in presynaptic NMJ terminals (Boaro et al. 1998; Tömböl et al. 2002). The criteria previously published in those papers were used when identifying these structures in our material. Inhibitors,research,lifescience,medical Numerous measurements of structures of the NMJ including area of the axon terminal, junctional fold number, length and diameter, length of active zone, area and number of mitochondria were made using Image J software directly from the images acquired with the Orius EM high-resolution camera. The number of vesicles and of docked vesicles was also counted, and the number of vesicles/μm2 axon terminal and the number Inhibitors,research,lifescience,medical of docked vesicles/μm2 of active zone were derived. Presynaptic vesicles were characterized as “docked” vesicles if they were within one vesicle diameter (~50 nm) of the presynaptic active zone (Schikorski Inhibitors,research,lifescience,medical and Stevens 1997, 2001; Tyler and Pozzo-Miller 2001; Rizzoli and Betz 2004). We defined active zone as the length of the presynaptic membrane that directly opposed the postsynaptic

junctional folds. These Inhibitors,research,lifescience,medical criteria were applied for all material analyzed. A total of four WT and four SOD1 animals were analyzed with 5–10 NMJs evaluated for each muscle area (soleus, TA inside, and TA outside subcompartments) in each animal. Statistical analysis was performed by the Design Analysis Core at WFUSM using the restricted maximum likelihood, mixed effect (REML) model. Evaluation of spinal cord mitochondria function Mitochondria were Inhibitors,research,lifescience,medical isolated from fresh livers and spinal cords of age-matched wild-type and SOD1G93A mice by mechanical disruption followed by differential centrifugation as described previously (Del Gaizo et al. 2003; Pedrini et al. 2010). Mitochondria protein content

was then determined by Lowry Assay (Biorad, Hercules, CA). For membrane potential assessment, mitochondria were diluted to 0.5 mg/mL in experimental MRIP buffer (125 mmol/L KCl, 5 mmol/L malate, 1 mmol/L potassium phosphate buffer, pH 7.4, 20 μmol/L EGTA/Tris, and 10 mmol/L Tris/MOPS, pH 7.4) and then incubated with 2 μmol/L tetramethylrhodamine, ethyl ester (Invitrogen) with and without 4 μmol/L CCCP (Sigma-Aldrich St. Louis, MO) and 0.2 μmol/L valinomycin (Sigma) for 10 min at room temperature. Each reaction was then spun at 10,000g, 4°C, for 5 min to pellet mitochondria and 50 μL of the supernatant added to separate wells of a 96 well plate (Corning) and fluorescence measured using excitation filter 485 nm (15 nm band pass) and emission filter of 590 nm (15 nm band pass).