Panel A shows schematic illustrating viral labeling of gastrocnemius and tibialis anterior motor neurons and their spinal cord projections. Panel B shows fluorescence image identifying gastrocnemius and tibialis anterior muscles using TdTomato and GFP labeling. Panel C shows microscopy image and intensity profile defining criteria for inappropriate synapses based on VGlut1, proprioceptive, and dendritic signals. Panel D shows fluorescence microscopy images of motor neuron somata and dendrites in WT, C3 deficient, and CD47 deficient mice. Panel E shows bar graphs and microscopy images quantifying inappropriate synapses on motor neuron somata and dendrites across genotypes.Bottom of Form
Inappropriate synapses on motor neurons formed by proprioceptive fibers originate in antagonistic muscles in C3 −/− and CD47 −/− mice. (A) Schematic revealing the strategy for labelling proprioceptive fibers originating in Gs muscle and labelling of motor neurons innervating the antagonistic TA muscle. TA motor neurons were labelled by injection of AAV6-GFP in TA muscle, and Gs proprioceptors were labelled by injection of CAV2CRE virus in the Gs muscle in PV::FlpO;Ai65 mice. Injections were performed at P0/1, and tissue was harvested at P6/7. (B) Superimposed fluorescent and transmitted-light photograph of the hindlimb injected at P5, showing the Gs muscle in red (TdTomato) and the TA muscle in green (GFP). (C) Confocal image of an inappropriate synapse. Inset shows the line profile across the synapse (white arrow in low-magnification image), which was used as our criterion for synaptic contact. Graph shows the signal intensity profile for each of the three immunoreactive signals. Gs proprioceptive fibers labelled with DsRed (red), TA MN dendrites (green), and VGlut1 synapses (blue). (D) Z-stack projection confocal images of L4 TA motor neurons from PV::FlpO;Ai65 (left), PV::FlpO;Ai65;C3−/− (middle), and PV::FlpO;Ai65;CD47−/− (right) mice. Z-stack distance: 3.5 μm. Top row, scale bar: 100 μm. Magnified image from the yellow dotted box in the soma (center row) and dendrite (bottom row). Yellow arrows point to inappropriate synapses. Scale bar: 20 μm. Higher magnification insets from white dotted boxes showing inappropriate synapses on dendrites (bottom row) in PV::FlpO;Ai65;C3−/− and PV::FlpO;Ai65;CD47−/− mice only. White arrows highlight inappropriate synapses. Scale bar: 2 μm. (E) Percentage of motor neurons with inappropriate synapses (on soma and dendrites). Left graph, WT: N = 3 mice; C3−/−: N = 3; CD47−/−: N = 3 (age: P5–P7). Middle and right graphs: normalized number of inappropriate somatic (center) and dendritic (right) synapses per motor neuron (WT: N = 5; C3−/−: N = 6; CD47−/−: N = 6 sections from three mice per group). Significance: *P < 0.05, **P < 0.001, and ***P < 0.001; one-way ANOVA with multiple comparisons using Bonferroni’s test. MN, motor neuron.