Sections were imaged using an SP5 Leica confocal microscope and analyzed using LASAF software (Leica). For all immunohistochemical analysis, at least three animals were used for each genotype. For SMN quantification, the number of motor and proprioceptive neurons with either present or absent nuclear Gems from L1 – L3 spinal segments were counted using a ×40 objective from z-stacks (0.5 μm steps) scans. At least 30 motor and proprioceptive neurons were included from each animal for each genotype. For motor neuron counts, we analyzed z-stacks images (at 3 μm intervals) collected for each section that contained a fluorescent signal from L2 retrogradely labeled motor neurons as previously described for L1 motor neurons13 (link). Sections were scanned using a ×20 objective. Only motor neurons (ChAT+) that contained the nucleus were counted in order to avoid double counting of adjoining sections.
Quantitative analysis of VGluT1 immunoreactive synaptic densities on motor neurons at P4 and P11 were performed on stacks of optical sections scanned using an ×40 objective throughout the whole section thickness at 0.35 μm z-steps to include the whole cell body and dendrites of retrogradely labeled and ChAT+ motor neurons. To obtain density estimates, we measured all VGluT1+ contacts on dendritic segments at 50 μm sequential distances (0–50, 50–100, 100–150) from the cell body and divided this number by the total linear length of all dendritic segments in each compartment as described previously13 (link). For VGluT1 motor neuron soma counts, only motor neurons with a whole cell body present within the z-stack were included.
To determine the extent of NMJ innervation, NMJ synapses were acquired using an ×20 objective and z-stack images were scanned at 2 μm intervals. Images were analyzed off-line using LASAF software. NMJs were only considered innervated if the presynaptic nerve terminal completely co-localized with the postsynaptic endplate.
Analysis of Kv2.1 and Kv2.2 channels were performed from single optical plane images acquired with an ×63 oil objective at 4096×4096 dpi resolution using an SP5 Leica confocal microscope. Only motor neuron somata (identified by ChAT immunoreactivity) in which the nucleus was present were included in the analysis. To calculate the coverage by Kv channel on motor neuron soma, a line was drawn along the soma perimeter to acquire the fluorescence intensity (expressed in arbitrary units), avoiding the area in which primary dendrites were present using LAS X software (Leica). A baseline fluorescence intensity measurement was achieved by drawing a straight line within the cytoplasm. The fluorescence intensity measurements were exported into Excel as x–y coordinates (x: distance in μm; y: fluorescence intensity in arbitrary units). The fluorescence signal above 3 Standard Deviations (SD) of the baseline intensity measurement was considered expression of Kv immunoreactivity along the soma perimeter (Supplementary Fig. 14C ), whereas the signal below was considered as background. The distance with fluorescence intensity above 3 standard deviations was calculated for each motor neuron and Kv channel coverage was expressed as a percentage of the total perimeter of the motor neuron soma.
Quantitative analysis of VGluT1 immunoreactive synaptic densities on motor neurons at P4 and P11 were performed on stacks of optical sections scanned using an ×40 objective throughout the whole section thickness at 0.35 μm z-steps to include the whole cell body and dendrites of retrogradely labeled and ChAT+ motor neurons. To obtain density estimates, we measured all VGluT1+ contacts on dendritic segments at 50 μm sequential distances (0–50, 50–100, 100–150) from the cell body and divided this number by the total linear length of all dendritic segments in each compartment as described previously13 (link). For VGluT1 motor neuron soma counts, only motor neurons with a whole cell body present within the z-stack were included.
To determine the extent of NMJ innervation, NMJ synapses were acquired using an ×20 objective and z-stack images were scanned at 2 μm intervals. Images were analyzed off-line using LASAF software. NMJs were only considered innervated if the presynaptic nerve terminal completely co-localized with the postsynaptic endplate.
Analysis of Kv2.1 and Kv2.2 channels were performed from single optical plane images acquired with an ×63 oil objective at 4096×4096 dpi resolution using an SP5 Leica confocal microscope. Only motor neuron somata (identified by ChAT immunoreactivity) in which the nucleus was present were included in the analysis. To calculate the coverage by Kv channel on motor neuron soma, a line was drawn along the soma perimeter to acquire the fluorescence intensity (expressed in arbitrary units), avoiding the area in which primary dendrites were present using LAS X software (Leica). A baseline fluorescence intensity measurement was achieved by drawing a straight line within the cytoplasm. The fluorescence intensity measurements were exported into Excel as x–y coordinates (x: distance in μm; y: fluorescence intensity in arbitrary units). The fluorescence signal above 3 Standard Deviations (SD) of the baseline intensity measurement was considered expression of Kv immunoreactivity along the soma perimeter (
