Fig. 3. Complement inhibition rescues synapse loss in Nptx2^KO brains.

(A) Shown are representative confocal images and 3D surface reconstructions of Homer1 (blue), Vglut1 (magenta), and CD68 (green) in mouse cortical layers II to IV. Scale bars, 5 μm. (B and C) The relative amount of Homer1 (B) and Vglut1 (C) puncta within CD68⁺ microglial lysosomes was quantified. P values were determined by one-way ANOVA followed by Šidák post hoc test. n = 5 for WT; n = 7 for Nptx2^KO; n = 7 for C1q^KO; n = 6 for Nptx2^KO;C1q^KO. (D and E) Shown are representative confocal images (D) and quantification (E) of excitatory synapse density around somas of PV⁺ cells in the cortical layers II to V. White arrows indicate synapses, which were identified by colocalization of Homer1 and Vglut1 puncta. P values were determined by one-way ANOVA followed by Šidák post hoc test. n = 5 for WT; n = 7 for Nptx2^KO; n = 7 for C1q^KO; n = 6 for Nptx2^KO;C1q^KO. Scale bars, 5 μm and 2 μm (inset). (F and G) Shown are representative confocal images (F) and quantification (G) of excitatory synapse density around somas of NeuN⁺ cells in cortical layers II to V. White arrows indicate synapses, which were identified by colocalization of Homer1 and Vglut1 puncta. P values were determined by one-way ANOVA followed by Šidák post hoc test. n = 5 for WT; n = 7 for Nptx2^KO; n = 7 for C1q^KO; n = 6 for Nptx2^KO;C1q^KO. Scale bars, 5 μm and 2 μm (inset). (H) Shown are representative confocal images and 3D surface reconstructions of CD68 (green), Vglut1 (magenta), and Homer1 (blue) in control IgG and C1q-blocking antibody-injected Nptx2^KO cortices. Scale bars, 5 μm and 1 μm (inset). (I) Volume of CD68⁺ microglial lysosomes in the cortices of Nptx2^KO mice injected with control IgG and C1q-blocking antibody was quantified. P value was determined by paired t test. n = 7. (J and K) The relative amount of Homer1 (J) and Vglut1 (K) puncta within CD68⁺ microglial lysosomes was quantified in the cortices of Nptx2^KO mice injected with isotype IgG antibody or C1q-blocking antibody. P values were determined by paired t test. n = 7. (L) Shown are representative confocal images of immunostained NeuN (gray), Homer1 (blue), and Vglut1 (magenta) in the cortical layers II-V of Nptx2^KO mice injected with control IgG or C1q-blocking antibody. White arrows indicate synapses, which were identified by colocalization of Homer1 and Vglut1. P value was determined by paired t test. n = 7; Scale bars, 5 μm and 2 μm (inset). (M) Shown are representative confocal images of immunostained PV (gray), Homer1 (blue), and Vglut1 (magenta) in the cortical layers II to V of Nptx2^KO mice injected with control IgG or C1q-blocking antibody. White arrows indicate synapses, which were identified by colocalized Homer1 and Vglut1 puncta. Scale bars, 5 μm and 2 μm (inset). (N) Excitatory synapse density around somas of NeuN⁺ cells was quantified. P value was determined by paired t test. n = 7. (O) Excitatory synapse density around somas of PV⁺ cells was quantified. P value was determined by paired t test. n = 7. Data are presented as means ± SEM. Each dot represents data from one mouse.