Inhibition of PS-dependent spine pruning by MFG-E8 ^D89E disrupts the synaptic maturation of adult-born neurons in the hippocampal DG. (A) Representative image of the DG in the hippocampus of adult WT mice injected with PSVue480 (white; n = 4 mice). White arrows and red arrowheads indicate PS⁺ dots and a pyknotic cell, respectively. Nuclei are stained by Hoechst 33342 (blue). (B) Representative images of the ML of adult WT mice injected with PSVue480 (white), stained for PSD95 (red). The boxes in B are enlarged and shown by orthogonal view (B¹ and B²). Arrows (B, B¹, and B²) indicate PS⁺PSD95⁺ signals (36.3 ± 3.0% [n = 3 mice] of PS⁺ dots are colocalized with PSD95; pooled from two independent experiments). (C) Effect of MFG-E8^D89E on microglial contacts to spines of adult-born neurons in the DG. The density of microglial contacts to tdTomato⁺ spines in the adult Dcx-CreER^T2; R26-EPT; R26-tdTomato (EPT) and Dcx-CreER^T2; R26-D89E; R26-tdTomato (D89E) mice (n = 3 mice each) is shown (spreading, t₍₄₎ = 3.7, P = 0.022, unpaired t test; wrapping, t₍₄₎ = 4.0, P = 0.016, unpaired t test; pooled from three independent experiments). (D) Effect of MFG-E8^D89E on microglial phagocytosis of spines of adult-born neurons in the DG. Representative single z-plane (D), orthogonal (D¹), and surface-rendered 3D (D²) images of the ML of adult EPT mice (n = 4 mice) stained for PSD95 (yellow), CD68 (cyan), and Iba1 (green) are shown. tdTomato⁺ signals (red) are directly observed without staining. Arrowheads indicate PSD95⁺tdTomato⁺ spines incorporated in CD68⁺ lysosomes in Iba1⁺ microglial processes (D and D¹). The percentages of PSD95⁺tdTomato⁺ spines incorporated in CD68⁺ lysosomes in Iba1⁺ microglial processes in adult EPT (n = 4 mice) and D89E (n = 3 mice) mice at 28 dpi are shown (D³; t₍₅₎ = 4.7, P = 0.0054, unpaired t test; pooled from three independent experiments). Also, see Video 6. (E and F) Effect of MFG-E8^D89E on the survival of granule cells in the DG. Representative images of the DG in the hippocampus of adult EPT and D89E mice (n = 3 mice each) stained for DsRed (red) and NeuN (blue) are shown in E. Density of tdTomato⁺NeuN⁺ granule cells in EPT and D89E mice (n = 3 each) at 56 dpi is shown in F (pooled from three independent experiments). (G and H) Effect of MFG-E8^D89E on the spine density of granule cells in the DG. Representative images (G) and densities of protrusions (H, P = 0.0025, Welch’s t test) and mushroom spines (H, t₍₃₆₎ = 2.7, P = 0.0098, unpaired t test) of dentate granule cells in EPT and D89E mice at 56 dpi (n = 3 mice each) are shown. Asterisks and arrows indicate mushroom spines and other protrusions, respectively (G). Parentheses in H indicate the number of analyzed cells. Data are pooled from two independent experiments. (I) Typical recordings of miniature EPSCs from tdTomato⁺ granule cells at 65–74 dpi in EPT (top) and D89E (bottom) mice. (J and K) Cumulative probability distributions of miniature EPSC amplitudes (J, bins: 0.5 pA; P = 0.013, two-sample Kolmogorov–Smirnov test) and interevent intervals (K, bins: 50 ms; P = 0.0014, two-sample Kolmogorov–Smirnov test) in EPT (n = 8 cells from four mice, pooled from four independent experiments) and D89E mice (n = 6 cells from three mice, pooled from three independent experiments). SGZ, subgranular zone; GCL, granule cell layer; ML, molecular layer. Scale bars: A, 5 µm; B and D, 1 µm; G, 2 µm; E, 10 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Data shown are mean ± SEM.