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. 2020 Jan 9;21(3):e48512. doi: 10.15252/embr.201948512

Figure EV5. Related to Fig 6. Additional dendritic spine defects revealed in vitro and in vivo .

Figure EV5

  • A
    Analysis of 3D‐reconstructed spine head diameters in DG revealed increased proportion of spines with enlarged heads at P30, but no changes in adults. Scale bars: 5 μm, ***P < 0.001, Kolmogorov–Smirnov test.
  • B, C
    Analysis of dendritic spine remodeling in vitro. E18.5 hippocampal neurons were transfected with GFP (at DIV 14), and the same dendrite segments were scanned at DIV 21 and DIV 25. Spine density decreased in WT neurons (DIV21 vs. DIV 25: 9.6 ± 2.2 spines/10 μm vs. 8.4 ± 2.4 spines/10 μm, P = 0.003, n = 21 neurons) but not in knockout neurons (DIV21 vs. DIV 25: 10.3 ± 2.3 spines/10 μm vs. 9.9 ± 2.6 spines/10 μm, P = 0.25, n = 37 neurons). Scale bars: 5 μm, mean ± SD, *P < 0.05, paired t‐test.
  • D, E
    At DIV 25, neurons were photographed, treated with 5 nM Sema3F or control Fc for 3 h, and photographed again. CRMP2 deficiency prevents Sema3F‐induced spine elimination. WT neurons: Fc treatment (before vs. after) 7.8 ± 2 vs. 8.6 ± 1.8 spines/10 μm (P = 0.07, n = 10 neurons), Sema3F treatment (before vs. after) 9.2 ± 3.2 vs. 8.4 ± 2 spines/10 μm (P = 0.01, n = 15 neurons). Crmp2 −/− neurons: Fc treatment (before vs. after) 9 ± 2.5 vs. 9.4 ± 2.9 spines/10 μm (P = 0.04, 16 neurons), Sema3F treatment (before vs. after) 10.9 ± 2.2 vs. 11.2 ± 2.4 spines/10 μm (P = 0.2, 41 neurons). Scale bars: 5 μm, mean ± SD, *P < 0.05, paired t‐test.
  • F, G
    Analysis of DiOlistically labeled dendritic spine density in hippocampal CA1 region, secondary apical dendrites (n = 3 mice per genotype, 36 dendrites) (WT 15.2 ± 2.8 spines/10 μm, crmp2 −/− 13.3 ± 3.4 spines/10 μm, P = 0.02). Scale bars: 5 μm, *P < 0.05, t‐test.