Panel A shows confocal images of L4/5 spinal cord ventral horns from various mouse genotypes at different postnatal days, stained with antibodies against ChAT and VGlut1. Panel B is a bar graph showing the number of VGlut1 synapses per motor neuron soma in different mouse genotypes at P10. The x-axis represents different genotypes, and the y-axis represents the number of synapses. Panel C is a bar graph showing the number of L4 motor neurons per section in different mouse genotypes at P5. The x-axis represents different genotypes, and the y-axis represents the number of motor neurons. Panels D and E are line graphs showing the normalized amplitude change of spinal reflexes challenged at different frequencies in different mouse genotypes at P5 and P10, respectively. The x-axis represents the number of stimuli, and the y-axis represents the normalized amplitude. Panel F is a line graph showing the righting time for different mouse genotypes from P1 to P10. The x-axis represents postnatal age in days, and the y-axis represents righting time in seconds. Panels G and H are line graphs showing the body weight gain in different mouse genotypes over time. The x-axis represents postnatal age in days, and the y-axis represents body weight in grams.
Effects of genetic deletion of C3 alone, CD47 alone, or combined in the sensory-motor circuit. (A) Single optical plane confocal images of L4/5 spinal cord ventral horns from WT, C3−/−, CD47−/−, and C3−/−CD47−/− mice at P1 (left column) and WT, C3−/−, C3+/−, CD47+/−, CD47−/−, and C3+/−CD47+/− at P10 (middle and right columns), with antibodies against ChAT (blue) and VGlut1 (white). Double homozygous knockout mice for C3 and CD47 (C3−/−CD47−/−) were not viable after ∼P3. Scale bar: 50 μm. (B) Number of VGluT1 synapses per motor neuron soma in WT, C3+/−, CD47+/− (hets) and C3+/−CD47+/− (double het) mice at P10 (N = 3 mice/group). Number of motor neuron somata analyzed: WT: n = 17 MNs; C3+/−: n = 16; CD47+/−: n = 13; C3+/−CD47+/−: n = 22 (grey data points), from N = 3 mice/group (colored data points). Significance: *P < 0.05, **P < 0.001, and ***P < 0.001; one-way ANOVA, Tukey’s post hoc test. (C) Number of L4 motor neurons per 75-μm-thick section for three spinal cord sections per animal from WT (N = 3), C3−/− (N = 3), and CD47−/− (N = 3) mice at P5. Quantification was performed from a single side of the spinal cord. (D) Normalized (with respect to first response) amplitude change of spinal reflex challenged at 0.1 and 10 Hz in WT (blue: 0.1 Hz, N = 4 mice; 10 Hz, N = 3), C3−/− (green, 0.1 Hz, N = 4; 10 Hz, N = 4), and CD47−/− mice (purple: 0.1 Hz, N = 4; 10 Hz, N = 4) at P5. Significance: C3−/− versus WT: *P < 0.05; two-way ANOVA multiple comparisons with Bonferroni’s test. (E) Same analysis as in E but at P10. WT (blue, 0.1 Hz, N = 8; 10 Hz, N = 8), C3−/− (green: 0.1 Hz, N = 4; 10 Hz, N = 4), and CD47−/− (purple, 0.1 Hz, N = 3; 10 Hz, N = 3). No significant difference was observed; two-way ANOVA with multiple comparisons using Bonferroni’s test. (F) Righting time for WT (N = 16 mice), C3+/− (N = 4), CD47+/− (N = 7), and C3+/−CD47+/− (N = 6) animals from P1 until P10. (G) Body weight gain in WT (N = 16), CD47+/− (N = 9), C3+/− (N = 4), and C3+/−CD47+/− (N = 6) mice. No significance was observed; one-way ANOVA with multiple comparisons using Bonferroni’s test. (H) Body weight gain in WT (N = 16), CD47−/− (N = 17), C3−/− (N = 16), and C3+/−CD47+/− (N = 6) mice. Significance: C3−/− versus WT: *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with multiple comparisons using Bonferroni’s test. MN, motor neuron.