Panel A shows a fluorescence microscopy image of the spinal cord from a C1q deficient mouse, stained for ChAT, CD47, and VGlut1. Panel B shows bar graphs of the percentage of synapses on motor neuron somata tagged by CD47 at postnatal days 5 and 10, comparing wild type and C1q deficient mice. Panel C shows bar graphs of CD47 tagged synapses on motor neuron dendrites within 0 to 50 micrometers at postnatal days 5 and 10. Panel D shows fluorescence microscopy images of C1q deficient L4 spinal cord at postnatal days 1, 5, and 10, stained for ChAT and VGlut1. Panel E shows bar graphs of the number of synapses on L4 motor neuron somata at postnatal days 1, 5, and 10. Panel F shows bar graphs of synaptic density in the L4 motor neuron dendritic compartment within 0 to 50 micrometers. Panel G shows electrophysiological trace recordings of the L4/L5 spinal reflex in wild type and C1q deficient mice, with stimulus artifacts and evoked responses indicated. Panel H shows scatter plots comparing spinal reflex amplitude and latency between wild type and C1q deficient mice, with a significant increase in amplitude but no significant difference in latency.
Effects of C1q genetic deletion on the sensory-motor circuit. (A) Single-plane confocal image of VGluT1 (red), CD47 (green) and ChAT (blue) immunoreactivity in a C1q−/− mouse at P5. Scale bar: 50 μm. (B) Percentage of somatic synapses tagged by CD47 in WT (blue) and C1q−/− (red) mice at P5 and P10 (N = 3 mice/group; colored points in the graph). Grey data points (and bars) represent the number of synapses analyzed in: WT: n = 14 at P5, n = 16 at P10; C1q−/−: n = 17 at P5, n = 13 at P10 for N = 3 mice/group. Colored points represent average values per mouse. (C) Percentage of dendritic synapses tagged by CD47. Number of primary dendrites (0–50 μm from the soma); WT: n = 24 at P5, n = 30 at P10; C1q−/−: n = 24 at P5, n = 30 at P10 (grey points) for N = 3 mice/group (colored points represent average values per mouse). Significance: *P < 0.05; One-way ANOVA multiple comparisons with Bonferroni’s test. (D) Confocal images of L4/5 spinal cord ventral horns from C1q−/− mice at P1, P5, and P10 showing immunoreactivity against ChAT (blue) and VGlut1 (white). Scale bar: 50 μm. Image shown in A is the same image as shown in P5 (middle image) with the exception that VGluT1 synapses appear in white for clarity and comparison with images at P1 and P10. (E) Number of VGluT1 synapses per motor neuron soma at P1, P5, and P10 mice (N = 3 mice/group; colored points in the graph) in WT and C1q−/− mice. Number of motor neuron somata analyzed, WT: n = 18 at P1, n = 14 at P5, n = 16 at P10; C1q−/−: n = 12 at P1, n = 17 at P5, n = 13 at P10. *P < 0.05, **P < 0.01, One-way ANOVA, Tukey’s post hoc test. (F) Synaptic density (# synapses/50 μm dendrite) for proximal dendrites of motor neurons (N = 3 mice/group; colored points in the graph). Number of dendrites analyzed, WT: n = 30 at P1, n = 24 at P5, n = 30 at P10; C1q−/−: n = 30 at P1, n = 24 at P5, n = 30 at P10. ns: no significance. (G) Electrical responses from ventral roots following stimulation of the L4 (or L5) dorsal root in WT and C1q−/− mice. Red traces show the average of first five responses (grey) acquired at 0.1 Hz. Arrowhead indicates stimulus artifact. The dotted line indicates baseline. Horizontal blue arrows indicate peak amplitude of the averaged response. (H) Amplitude and latency of spinal reflexes at P5 in WT (blue, N = 6 mice) and C1q−/− (red, N = 5) mice. Significance: *P < 0.05, Unpaired t test.