Figure 8: GABAB2 downregulation affects axon/dendrite growth and polarization of cortical neurons in vitro. | Nature Communications

Figure 8: GABAB2 downregulation affects axon/dendrite growth and polarization of cortical neurons in vitro.

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

Figure 8

(a) Cultured cortical neurons (3 DIV) immunostained for axons (Smi-312, blue) and dendrites (MAP2; red). Cells were treated with vehicle (control), baclofen (10 μM), CGP (10 μM) or forskolin (20 μM) 3 h after plating, or transfected with siRNA2 at plating. All drug-treated neurons were transfected with siRNA (EGFP) vector or siRNAc for visualization. Scale bar, 50 μm. (b) Total neurite length and axonal and dendritic relative lengths of neurons as in a. Asterisks: significant difference compared with controls (one-way ANOVA, P<0.001; post-hoc Dunn’s method, P<0.05). (c) Total branch number and axonal and dendritic relative branch numbers of the data set in b. Asterisks: significant difference compared with controls (Kruskal–Wallis One-way ANOVA, P<0.001; post-hoc Dun’s method, P<0.05. (d) Neurons presenting no axon (baclofen) or multiple axons (CGP, siRNA2), following treatment as in a. Scale bar, 50 μm. (e) Percentage of neurons (four independent experiments, ~250 cells per condition) as in a and d, characterized by one single axon (black), no axon (red) and multiple axons (blue). Asterisks: significant difference compared with controls (one-way ANOVA, P<0.001; post-hoc Holm–Sidak method, P<0.05). (f) Neurons reconstructed for each condition as in a with location of the cells artificially arranged for axon (blue) and dendrite (red) display. SA, single axon; MA, multiple axons; NA, no axon. Scale bar, 100 μm. (g) Neurons cultured on a substrate coated with stripes (dark grey) of baclofen (1 μM) or CGP (1 μM) and immunostained (4 DIV) for specific neuronal (Tuj-1) and axonal (Smi-312) markers. Arrowhead points to an axon turning at a baclofen stripe. Scale bar, 25 μM. (h) Preferential index (defined in the text) for axon initiation on control F-BSA (5 μg ml−1), F-cAMP (20 μM), baclofen or CGP stripes (3–4 independent experiments, ~300 cells per condition). In some experiments, forskolin (20 μM) or SQ22536 (10 μM) were bath applied to cultures. Asterisks: significant difference compared with controls (Kruskal–Wallis one-way ANOVA, P=0.016; post-hoc Student–Newman–Keuls method, P<0.05).

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