Image processing and quantification

Maximum intensity projections of confocal z-stacks and in general all downstream processing and image analysis steps were instead performed in Fiji (^{Schindelin et al., 2012}). ImageJ custom macros enabled semi-automated identification, drawing and ID assignment of MN somata according to VAChT signal. Regions of interest (ROIs) were then extracted from ISH channels (upon a background subtraction step, radius 5) and their size was measured retaining information on the receptor probe, the experimental group and the MN ID in their name tag. A similar approach was adopted to measure cytoplasmic PKA phospho-epitopes and whole-cell misfSOD1 accumulation as mean fluorescence intensity per MN cross section area. ISH signal was quantified as spot density per μm² by means of custom Jython and ImageJ macro scripts exploiting the difference of Gaussian detector shipped with Trackmate plugin (^{Tinevez et al., 2017}); peak size and intensity threshold were set upon visual inspection of the experimental group given the slight variability and efficiency of probe penetration in different batches.