Figure 2: GABAB2 downregulation affects neuronal radial migration in vivo. | Nature Communications

Figure 2: GABAB2 downregulation affects neuronal radial migration in vivo.

From: Non-hyperpolarizing GABAB receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1

Figure 2

(a) Confocal images of tdTomato fluorescence in coronal sections of rat somatosensory cortices at different ages after in utero transfection (at E17) with pRNAT-U6.3 (EGFP) siRNA empty vector together with pCAG-IRES-tdTomato (Tomato, Control), or functional GABAB2-siRNA (siRNA2) together with Tomato construct (n=16–64 animals/experimental age). Arrowheads point to example neurons that did not complete radial migration. Scale bars, 200 μm. (b) Quantification of the number of neurons transfected with either siRNA vector/Tomato or control siRNAc/Tomato (controls, black) or neuron expressing either siRNA1 (light grey) or siRNA2 (dark grey), and residing at the VZ, IZ, or CP (marked by the dotted lines in a) at E21. Numbers are expressed as a percentage of the total number of fluorescent cells in the same section. Asterisks indicate statistically significant difference compared with controls (one-way ANOVA, P=0.004; post-hoc Holm–Sidak method, P<0.05). n=number of rats processed (1–3 averaged slices per animal). (c) Quantification of the number of control neurons (black) or neurons expressing either siRNA1 (light grey) or siRNA2 (dark grey) that did not complete their migration at different postnatal ages. Asterisks indicate statistically significant difference compared with controls (Kruskal–Wallis one-way ANOVA, P<0.001; post-hoc Dunn’s method, P<0.05). Numbers in parentheses: number of rats processed (1–3 averaged slices/ per animal). (d) Confocal images of tdTomato fluorescence (green) from empty vector/Tomato- (control) or siRNA2/Tomato-transfected slices stained (at P16) for upper-layer marker Cux1 (red) and nuclear marker Hoechst (blue), showing no defects in general layering of siRNA2-transfected cortices. Dotted white lines delimitate the boundaries of the cortical layers. Scale bar, 150 μm. LII–LIV: layer II–IV; WM: white matter. (e) Cumulative distribution of the average tdTomato-fluorescence intensity at the soma in neurons transfected with control vectors (EGFP/Tomato or siRNA2/Tomato) in the CP (black and light grey, respectively) or with siRNA2/Tomato in ectopic cells (gray) was quantified as a measure of the GABAB2 downregulation extent at P16. Ectopic cells exhibited higher tdTomato fluorescence intensity, consistent with severe developmental defects associated with higher levels of siRNA2 expression (Kolmogorov–Smirnov test).

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