Panel A shows fluorescence microscopy images at postnatal day 0 stained for ChAT, VGlut1, C1q, and CD47, with enlarged examples identifying CD47 and C1q tagged synapses, CD47 only synapses, and C1q only synapses. Panel B shows corresponding fluorescence microscopy images at postnatal day 5 with enlarged examples of the different synapse labeling patterns. Panel C shows a bar graph of the number of double tagged C1q and VGlut1 synapses per cell at postnatal days 0 and 5. Panel D shows fluorescence microscopy images at postnatal days 0 and 5 stained for Iba1, C1q, CD47, and VGlut1, with enlarged regions showing labeled structures. Panel E shows three dimensional surface renderings of microglia from wild type, C3 deficient, and CD47 deficient mice stained for Iba1 and CD68. Panel F shows a bar graph of the percentage of microglia containing VGlut1 and CD47 positive residues at postnatal days 0, 5, and 10. Panel G shows a bar graph of the percentage of CD68 volume in microglia across wild type, C3 deficient, and CD47 deficient mice. Panel H shows a bar graph of microglial volume for the three genotypes. Panel I shows three dimensional microglial renderings from C3 deficient and CD47 deficient mice, with Iba1, CD68, and VGlut1 labeling and an enlarged inset of labeled structures. Panel J shows a bar graph comparing the percentage of microglial volume occupied by CD68 and VGlut1 in C3 deficient and CD47 deficient mice.
Microglia engulf synapses tagged by C1q, CD47, or combined. (A and B) Orthogonal view of images immunoreactive against C1q (green), CD47 (red), VGlut1 (white), and ChAT (blue) at P0 (A) and P5 (B). Circles indicate instances where a VGlut1 synapse is tagged by CD47 only (red circle), C1q only (green circle), or combined by both CD47 and C1q (yellow circle). Inserts at the bottom represent higher magnifications. Yellow arrows indicate double tagging of VGluT1 synapses by CD47 and C1q. Red arrows indicate tagging by CD47 only. Green arrows indicate tagging by C1q only. Scale bar in A and B: 10 μm. (C) Quantification of VGluT1 synapses double-tagged at P0 (grey bar) and P5 (blue bar). Grey data points are the number of synapses, and colored data points are averages from individual mice (N = 4 at P0 and N = 3 at P5). Significance: **P < 0.01; Unpaired t test. (D) Single optical plane confocal images of immunoreactivity against Iba1 (blue), C1q (green), CD47 (red), and VGluT1 (white) at P0 and P5. Inserts, higher magnification of the area indicated by dotted box. Yellow arrows indicate VGlut1 residues within microglia tagged by C1q and CD47. Red arrows indicate VGluT1 residues within microglia tagged by CD47 only. Scale bar: 5 μm. (E) 3D reconstructions of microglia (Iba1; yellow) and CD68 (red), showing the presence of CD68 within microglia. (F) Percentage of microglia in the LMC L4 motor neuron region containing VGlut1 residues tagged with CD47. Each data point represents a mouse (P0 and P10: N = 3; P5: N = 4). ***P < 0.001, one-way ANOVA, Tukey’s post hoc test. (G) Percentage of CD68 volume within microglia. Grey data points represent the number of microglia analyzed. Colored data points are the average from individual mice (N = 3 mice/group). (H) Microglia volume in WT, C3−/−, and CD47−/− mice. Grey points are the number of microglia analyzed, and colored data points are the average from individual mice (N = 3 mice/group). Significance: **P < 0.001, ***P < 0.001; ns: no significance; one-way ANOVA with multiple comparisons using Bonferroni’s test. (I) 3D reconstruction of microglia obtained from z-stack confocal images (inset) from C3−/− and CD47−/− mice. The 3D reconstruction depicts Iba1 (cyan), CD68 (yellow), and VGluT1 (red); inset shows immunostaining for Iba1 (green), CD68 (red), and VGluT1 (blue). Scale bars: 5 μm. (J) Percentage of microglia volume filled with either CD68 or VGluT1 in C3−/− and CD47−/− mice. ns: no significance. Unpaired t test.