Panel A shows microscopy images comparing VGlut1 and ChAT signals in P5 WT, C3 deficient, and CD47 deficient samples. Panel B shows bar graph quantifying synapse numbers in L4 motor neuron somata at P1, P5, and P10. Panel C shows bar graph measuring synaptic density in the L4 motor neuron dendritic compartment across developmental ages. Panel D shows electrophysiological traces illustrating L4 spinal reflex responses in WT, C3 deficient, and CD47 deficient samples. Panel E shows scatter plot comparing reflex response amplitudes among WT, C3 deficient, and CD47 deficient samples. Panel F shows scatter plot comparing reflex response latencies among WT, C3 deficient, and CD47 deficient samples. Panel G shows line graph comparing righting behavior across ages for WT, CD47 deficient, and C3 deficient groups.
Genetic deletion of C3 or CD47 results in a higher incidence of proprioceptive (VGlut1+) synapses on spinal motor neurons. (A) Confocal images of L4 motor neurons (ChAT, blue) and VGluT1 synapses (VGluT1, white) in WT, C3−/−, and CD47−/− mice at P5. Insets show higher magnification images of a single motor neuron. Scale bar: 50 μm. Images are Z-stack projection 3.5 μm in total distance (at 0.35 μm intervals). (B) Number of VGluT1 synapses per motor neuron soma at P1, P5, and P10 (N = 3 mice/group; colored data points). Number of MN somata analyzed: (WT: P1, n = 18; P5, n = 14; P10, n = 16) (C3−/−: P1, n = 18; P5, n = 17; P10 n = 16) (CD47−/−: P1, n = 15; P5, n = 10; P10, n = 12) are shown in grey data points and bar graphs from all mice per group. (C) Synaptic density of proximal dendrites (0–50 μm from the soma, N = 3 mice/group). Number of MN dendrites analyzed: (WT: P1, n = 30; P5, n = 24; P10, n = 30) (C3−/−: P1, n = 30; P5, n = 30; P10, n = 30) (CD47−/−: P1, n = 30; P5, n = 20; P10, n = 30). Significance: *P < 0.05, **P < 0.001, ***P < 0.001; one-way ANOVA with multiple comparisons using Bonferroni’s test. Statistical comparison was performed for average values in mice (colored data points). (D) Simplified schematic of the ex vivo spinal cord preparation to assess the dorsal-ventral reflex at P5. Representative recordings of L4 ventral root responses following L4 dorsal root stimulation in WT, C3−/−, and CD47−/− mice. The red trace is the average of the first five trials, which are shown in grey. Arrowheads indicate stimulus artifacts. Black arrows indicate peak amplitude in the averaged response. (E) Maximum amplitude of spinal reflexes at P5 (WT: N = 6 mice; C3−/−: N = 8; CD47−/−: N = 4). Significance: *P < 0.05, one-way ANOVA with multiple comparisons using Bonferroni’s test. (F) Latency of responses for the same group as in E. (G) Righting times across the first 10 postnatal days for WT, C3−/−, and CD47−/− mice. Significance: (*) (#) P < 0.05, (**) (##) P < 0.01, and (***) P < 0.001 (compared with WT: CD47 significance #; C3 significance *), one-way ANOVA with multiple comparisons using Bonferroni’s test. MN, motor neuron.