Quantification of GABApre Synaptic Proteins in SMA

Images of synaptic terminals were acquired on a Leica SP8 confocal microscope using a 40× objective with 5× digital zoom at a 2048 × 2048 optical resolution. Acquisition settings for excitation and fluorescence detection parameters were identical for each genotype, SMA controls and SMA+AAV9-BDNF. The GAD65 and GAD67 terminals were analyzed within a 10 μm confocal z-stack at 300 nm step intervals. Surface area and staining intensities were determined using Leica LAS AF imaging software. Relative synaptic protein levels were quantified by assessing the mean gray values, defined as the sum of the gray values of all the pixels in a region of interest (pixel sum), divided by the number of pixels in that region (pixel count), as we reported recently^{32 (link)}. Regions of interest were defined as the outline of positively stained terminals. To quantify the relative levels of GABApre synaptic proteins (GAD65 and GAD67), only varicosities directly juxtaposed to proprioceptive afferent terminals (VGluT1+) were considered.