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. 2016 May 26;15(10):2251–2265. doi: 10.1016/j.celrep.2016.04.090

Figure 6.

Figure 6

The Presence of DENR C37Y and DENR P121L Disrupts the Long-Term Positioning, Dendritic Branching, and Dendritic Spine Properties of Cortical Projection Neurons

(A) In utero electroporation to study the effects of forced expression of DENR C37Y and DENR P121L on the long-term positioning of E14.5-born, GFP-labeled cells within the P17 postnatal cortex, illustrated as representative coronal sections from each.

(B) There is a significant interaction between treatment groups and the positioning of GFP+ cells within the P17 cortex (F(8,75) = 3.93; p = 0.0006). Compared to control, there is a significant reduction in the proportion of GFP+ cells in layers II–IV following treatment with DENR C37Y (control 0.984 ± 0.008; DENR C37Y 0.947 ± 0.0103; Dunnett’s multiple comparisons ∗∗∗p < 0.005) as well as P121L (control 0.984 ± 0.008; DENR P121L 0.9532 ± 0.0148; Dunnett’s multiple comparisons ∗∗p < 0.01), along with a concomitant increase in the proportion of GFP+ cells in layer V in both DENR C37Y- and DENR P121L-treated cells (control 0.010 ± 0.005; DENR C37Y 0.033 ± 0.0077; DENR P121L 0.0348 ± 0.0110; Dunnett’s multiple comparisons ∗p < 0.05).

(C) Representative images of GFP-labeled, layer II/III cortical projection neurons from each condition.

(D) Sholl analysis of dendritic branching complexity reveals alterations to regional dendritic branching complexity in DENR C37Y- and DENR P121L-treated neurons (C37Y: 40–65 μm; P121L: 30–50 μm; as well as Figures S6F and S6G).

(E and F) Forced expression of DENR C37Y and DENR P121L did not significantly affect the numbers of primary neurites (two-way ANOVA F(2,52) = 1.041; p = 0.3602; n = 17–20 neurons per condition) or their branch points (two-way ANOVA F(2,52) = 1.022; p = 0.3668; n = 17–20 neurons per condition) when compared with control (GFP-only treatment).

(G) Confocal microscopy and image reconstruction of dendritic spines from apical and basal dendrites of GFP-labeled neurons.

(H) Following treatment with DENR C37Y or DENR P121L, there is a significant decrease in the density of dendritic spines along apical dendrites (control 8.417 ± 0.4771; DENR C37Y 6.844 ± 0.2217; DENR P121L 6.295 ± 0.2217; one-way ANOVA F(2,49) = 11.62; p < 0.0001; Bonferroni multiple comparisons test ∗∗p < 0.01; ∗∗∗p < 0.005) as well as basal dendrites (control 9.122 ± 0.3651; DENR C37Y 7.664 ± 0.3635; DENR P121L 7.664 ± 0.3635; one-way ANOVA F(2,52) = 4.759; p = 0.0126; Bonferroni multiple comparisons test ∗p < 0.05).

Graphs plot mean ± SEM. The scale bars represent (A) 200 μm, (C) 25 μm, (G) 20 μm, and (G insets) 5 μm.