Photographs

were taken at 16,000 of the first 10–12 frames containing synapses found on dendrites within the white matter located ventro-laterally to the L3/L4 area used for MN synapse evaluation. These synapses were then blindly scored and the Selleckchem ROCK inhibitor lengths of their dendrites measured using Image J. Statistical analysis was performed by Inhibitors,research,lifescience,medical the Design and Analysis Unit at WFSM. A mixed models approach was initially used to identify differences between WT and SOD1 while accounting for the repeated measures within each mouse and correlation between measures within each mouse. Statistical difference was determined using a least square means table. To confirm changes in C-terminals, immunohistochemistry was performed. For the immunohistochemical quantification of C-terminals in the VH,

cholinergic terminals surrounding MNs were identified using an antibody to vesicular acetylcholine transporter (VAChT; Table ​Table1)1) and biotinylated secondary antibody and DAB Inhibitors,research,lifescience,medical to visualize reaction product (Vector Laboratories). The number of VAChT+ terminals surrounding at least 10 large α-MNs per animal, three animals per group, were then counted on an Olympus BX-50 microscope using 100× oil immersion and through focus. Statistical differences between WT and SOD1 groups were determined Inhibitors,research,lifescience,medical using unpaired t-tests. Characterization of VH white matter For VH white matter measurements, axon number and size and glia number were determined in two 100× fields, one immediately ventral and one immediately lateral to the L3–L4 VH. Using Image J thresholding software under constant conditions, the number and mean size of axons was determined. In these same images the number of glia were also counted. For white Inhibitors,research,lifescience,medical matter width, five measures

of width were made in a spoke-like fashion around the VH, then averaged and compared. Statistical differences between WT and SOD1 groups or between P14 and P30 were determined using unpaired Inhibitors,research,lifescience,medical t-tests. Identification and evaluation of ultrastructure of NMJs Specific muscles were dissected from the mice used for ultrastructural analysis of the spinal cord described above and placed in fixative overnight at 4°C. Muscles were cut at 600 μm on a tissue chopper, then reacted for cholinesterase and to reveal NJMs using Karnovsky’s method with the following changes: sodium cacodylate buffer was used instead of PBS and the tissue was incubated for 1 h or until the endplates were visible under the dissecting microscope (Karnovsky, 1964). Specimens were then embedded in Araldite 502 using a Lynx processor. One micron sections and subsequent 700 Å thin sections were cut using an LKB ultramicrotome, then counterstained with either toluidine blue for 1 μm sections, or uranyl acetate in 100% methanol and subsequently lead citrate for thin sections, which were viewed with a Zeiss EM 10 electron microscope.