Figure 4.
PS is involved in microglial phagocytosis of spines of adult-born neurons in the OB. (A) Representative image of the EPL in the OB of adult Dcx-CreERT2; R26-tdTomato (control) mice at 28 dpi (n = 4 mice) stained for DsRed (red) and Iba1 (green). The boxed areas in A are enlarged in A1–A3. Contacts between microglia and spines are classified into three types: touching (A1, asterisks), spreading (A2, arrowheads), and wrapping (A3, arrows). (B and C) Effect of MFG-E8D89E on spine contacts by microglia in adult-born neurons in the OB. Representative images of the EPL in the OB of adult Dcx-CreERT2; R26-EPT; R26-tdTomato (B, EPT, n = 5 mice), and Dcx-CreERT2; R26-D89E; R26-tdTomato (C, D89E, n = 3 mice) mice at 28 dpi stained for DsRed (red) and Iba1 (green) are shown. The boxed areas (B and C) are displayed on the right-hand side (0 µm). Asterisks, arrowheads, and arrows in the three consecutive images (B, C, right) indicate microglial touching to, spreading on, and wrapping of spines, respectively. (D–F) Density of microglial contacts (touching [D], spreading [E], and wrapping [F]) to spines in the adult control (n = 4 mice), naris-opened or occluded EPT (n = 5 mice each) or D89E (n = 3 mice each) mice (E, for comparison among control, EPT, and D89E mice, F(2,9) = 10.8, P = 0.0041, one-way ANOVA; control versus D89E, P = 0.0045, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,12) = 72.6, P = 2.0 × 10−6, Focclusion (1,12) = 8.87, P = 0.012, two-way ANOVA; EPT versus D89E in opened, t(6) = 4.4, P = 0.018, opened versus occluded in EPT, t(8) = −3.8, P = 0.021, EPT versus D89E in occluded, t(6) = 8.0, P = 0.00082, unpaired t test; F, for comparison among control, EPT, and D89E mice, F(2,9) = 93.6, P = 9.5 × 10−7, one-way ANOVA; control versus D89E, P = 1.6 × 10−6, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,12) = 128, P = 9.1 × 10−8, Focclusion (1,12) = 18.3, P = 0.0011, two-way ANOVA; EPT versus D89E in opened, t(6) = 15.4, P = 1.9 × 10−5, opened versus occluded in EPT, t(8) = −5.2, P = 0.018, EPT versus D89E in occluded, t(6) = 7.6, P = 0.0011, unpaired t test). Pink bars indicate data from controlmice. Data are pooled from 10 independent experiments. (G) Representative images of EPL in the OB of adult EPT mice (n = 4 mice) stained for PSD95 (yellow), CD68 (cyan), and Iba1 (green). tdTomato+ signals (red) are directly observed without staining. The boxed area in G is enlarged (G1) and is shown by orthogonal (G2) and a surface-rendered 3D view (G3). White arrowheads indicate tdTomato+PSD95+ spine incorporated in CD68+ lysosomes within Iba1+ processes (G1). Also, see Video 3. (H) Density of tdTomato+PSD95+ spines incorporated in CD68+ lysosomes within Iba1+ processes in the adult control (n = 3 mice), naris-opened or occluded EPT (n = 4 [opened] or 3 [occluded] mice) or D89E (n = 3 mice each) mice (for comparison among control, EPT, and D89E mice, F(2,7) = 41.0, P = 0.00014, one-way ANOVA; control versus D89E, P = 0.00018, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,9) = 110.7, P = 2.3 × 10−6, Focclusion (1,9) = 16.8, P = 0.0027, Fmouse×occlusion (1,9) = 8.8, P = 0.016, two-way ANOVA; EPT versus D89E in opened, t(5) = 7.6, P = 0.0025, opened versus occluded in EPT, t(5) = −4.2, P = 0.035, EPT versus D89E in occluded, t(4) = 7.3, P = 0.0074, unpaired t test). Pink bar indicates data from control mice. Data are pooled from six independent experiments. EPL, external plexiform layer. Scale bars: A–C, 10 µm; G, 5 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.005; adjusted with Bonferroni correction. Data shown are mean ± SEM.

PS is involved in microglial phagocytosis of spines of adult-born neurons in the OB. (A) Representative image of the EPL in the OB of adult Dcx-CreERT2; R26-tdTomato (control) mice at 28 dpi (n = 4 mice) stained for DsRed (red) and Iba1 (green). The boxed areas in A are enlarged in A1–A3. Contacts between microglia and spines are classified into three types: touching (A1, asterisks), spreading (A2, arrowheads), and wrapping (A3, arrows). (B and C) Effect of MFG-E8D89E on spine contacts by microglia in adult-born neurons in the OB. Representative images of the EPL in the OB of adult Dcx-CreERT2; R26-EPT; R26-tdTomato (B, EPT, n = 5 mice), and Dcx-CreERT2; R26-D89E; R26-tdTomato (C, D89E, n = 3 mice) mice at 28 dpi stained for DsRed (red) and Iba1 (green) are shown. The boxed areas (B and C) are displayed on the right-hand side (0 µm). Asterisks, arrowheads, and arrows in the three consecutive images (B, C, right) indicate microglial touching to, spreading on, and wrapping of spines, respectively. (D–F) Density of microglial contacts (touching [D], spreading [E], and wrapping [F]) to spines in the adult control (n = 4 mice), naris-opened or occluded EPT (n = 5 mice each) or D89E (n = 3 mice each) mice (E, for comparison among control, EPT, and D89E mice, F(2,9) = 10.8, P = 0.0041, one-way ANOVA; control versus D89E, P = 0.0045, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,12) = 72.6, P = 2.0 × 10−6, Focclusion (1,12) = 8.87, P = 0.012, two-way ANOVA; EPT versus D89E in opened, t(6) = 4.4, P = 0.018, opened versus occluded in EPT, t(8) = −3.8, P = 0.021, EPT versus D89E in occluded, t(6) = 8.0, P = 0.00082, unpaired t test; F, for comparison among control, EPT, and D89E mice, F(2,9) = 93.6, P = 9.5 × 10−7, one-way ANOVA; control versus D89E, P = 1.6 × 10−6, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,12) = 128, P = 9.1 × 10−8, Focclusion (1,12) = 18.3, P = 0.0011, two-way ANOVA; EPT versus D89E in opened, t(6) = 15.4, P = 1.9 × 10−5, opened versus occluded in EPT, t(8) = −5.2, P = 0.018, EPT versus D89E in occluded, t(6) = 7.6, P = 0.0011, unpaired t test). Pink bars indicate data from controlmice. Data are pooled from 10 independent experiments. (G) Representative images of EPL in the OB of adult EPT mice (n = 4 mice) stained for PSD95 (yellow), CD68 (cyan), and Iba1 (green). tdTomato+ signals (red) are directly observed without staining. The boxed area in G is enlarged (G1) and is shown by orthogonal (G2) and a surface-rendered 3D view (G3). White arrowheads indicate tdTomato+PSD95+ spine incorporated in CD68+ lysosomes within Iba1+ processes (G1). Also, see Video 3. (H) Density of tdTomato+PSD95+ spines incorporated in CD68+ lysosomes within Iba1+ processes in the adult control (n = 3 mice), naris-opened or occluded EPT (n = 4 [opened] or 3 [occluded] mice) or D89E (n = 3 mice each) mice (for comparison among control, EPT, and D89E mice, F(2,7) = 41.0, P = 0.00014, one-way ANOVA; control versus D89E, P = 0.00018, Dunnett’s test; for comparison of EPT versus D89E and opened versus occluded, Fmouse (1,9) = 110.7, P = 2.3 × 10−6, Focclusion (1,9) = 16.8, P = 0.0027, Fmouse×occlusion (1,9) = 8.8, P = 0.016, two-way ANOVA; EPT versus D89E in opened, t(5) = 7.6, P = 0.0025, opened versus occluded in EPT, t(5) = −4.2, P = 0.035, EPT versus D89E in occluded, t(4) = 7.3, P = 0.0074, unpaired t test). Pink bar indicates data from control mice. Data are pooled from six independent experiments. EPL, external plexiform layer. Scale bars: A–C, 10 µm; G, 5 µm. *, P < 0.05; **, P < 0.01; ***, P < 0.005; adjusted with Bonferroni correction. Data shown are mean ± SEM.

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