Axonal transport Retrograde axonal transport was determined

using previously published protocols (Ferri et al. 2003) with the exception that Alexa-Fluor-CTB was injected into the muscles rather than fluorogold. SOD1G93A and WT mice were injected at P20 with Alexa-Fluor-CTB to label different MN pools. Because of the small size of muscles in preweaned mice P20 was the earliest time point when we were confident that injections were within individual muscles. Anesthesia induction using isofluorane took place using a vaporizer set at 5.0%, and mice were kept under anesthesia for the entire surgical procedure with the vaporizer between 1.5% and 2.0%. Once the TA and soleus Inhibitors,research,lifescience,medical muscle were exposed and identified, a volume of 4 μL Alexa-Fluor-555 Inhibitors,research,lifescience,medical CTB (5 μg/μL) was injected in the TA, and 2 μL Alexa-Fluor-488 CTB(5 μg/μL) was injected in the soleus muscle to identify both pools of fast-type and slow-type

MNs, respectively. Muscle injection was carefully and slowly performed to avoid diffusion of the dye to adjacent muscles. A delay of several seconds was used prior to removing the needle from the muscle in order to minimize any dye reflux. Following the injection, surgical incisions were closed using a 4.0 silk-suture, and the mice were sacrificed between 0 and 96 h after injection. Mice were perfused with 2% paraformaldehyde Inhibitors,research,lifescience,medical and the spinal cords removed. Spinal cords were cryoprotected in 20% sucrose, frozen in OCT medium, and sectioned at 20 μm using a cryostat (Leica Microsystems, Wetzlar, Germany). All spinal cord sections Inhibitors,research,lifescience,medical were collected, counterstained with DAPI, and MNs were examined under a regular

epi-fluorescence microscope at 20×. The number of labeled MNs was determined. MNs were analyzed if they possessed a FK228 nmr nucleus and exhibited Alexa-Fluor CTB label. MNs were not distinguished on the basis of intensity of CTB label. Statistical differences were determined Inhibitors,research,lifescience,medical using ANOVA followed by Tukey–Kramer post hoc test. To determine MN size, P30 mice were injected as described above. Four days following injection, mice were perfused and tissue processed as described above. MNs were analyzed if they possessed a nucleus and Non-specific serine/threonine protein kinase exhibited Alexa-Fluor CTB label. MN area was determined using NIH Image J software. Statistical significance was determined using one-way ANOVA followed by Bonferrroni’s multiple comparison test. Motor function Gait dynamics Gait dynamics were recorded using ventral plane videography, as previously described in detail (Hampton et al. 2004; Kale et al. 2004; Hampton and Amende 2010). Briefly, we used a motor-driven treadmill with a transparent treadmill belt (DigiGait Imaging System, Mouse Specifics, Inc., Quincy, MA). A high-speed digital video camera was positioned below the transparent belt to focus on the ventral view of subjects walking on the belt. An example of an SOD1 mouse being tested is shown (insert video).