Figure 7: GABA_BR regulates neuronal development in vivo through cAMP/LKB1 signalling.

(a) Left: confocal image of LKB1 phosphorylation at PKA-site serine S431 (pLKB1-S431) immunostaining (green) from a coronal section of rat somatosensory cortex at E21. Slices were counterstained with Hoechst (blue). Scale bar, 100 μm. Right: high-magnification images of SVZ, IZ and CP. Note the abundance of pLKB1-S431 in SVZ and CP. Scale bar, 10 μm. (b) Left: western blot showing pLKB1-S431, upon bath application of vehicle (control), baclofen, CGP and forskolin to cortices acutely dissected at E17 (see Supplementary Fig. S10). Right: quantification of the fold-increase average of pLKB1-S431 compared with control, in five independent experiments as in left. GABA_BR inhibition by CGP (10 μM) increased pLKB1-S431, mimicking forskolin treatment (Kruskal–Wallis one-way ANOVA on ranks, P<0.001; post-hoc Student–Newman–Keuls method, P<0.05). Conversely, pretreatment with baclofen (10 μM) significantly reduced forskolin-induced pLKB1-S431 (P<0.05). (c) Confocal images of pLKB1-S431 immunostaining (red) and nucleus staining (Hoechst, blue) from a coronal SVZ section of a rat at E21 previously transfected with control siRNAc (green) or siRNA2 (green). Arrowheads point to highly transfected cells, whereas the asterisk marks a low transfected cell for comparison. Scale bar, 10 μm. Right: average cytoplasmic fluorescence measured at the cell body for all cells analysed (diamonds; three animals each per experimental case) as in the example on the left. The average±s.e.m. is reported on the right. The asterisk indicates statistically significant difference (Mann–Whitney test P<0.001). (d) Confocal images of EGFP fluorescence in coronal sections of somatosensory cortices from P16 rats transfected in utero with the indicated constructs. Scale bar, 150 μm. (e) Quantification of the percentage of neurons that did not complete their migration at P16 in slices from animals transfected with the indicated constructs. Asterisks indicate statistically significant difference (Kruskal–Wallis one-way ANOVA, P<0.001; post-hoc Dunn’s method, P<0.05). Numbers in parentheses: number of rats processed (1–2 averaged slices per animal). (f) Quantification of the average EGFP fluorescence intensity (normalized to the number of transfected Tomato-positive cells and field background) of axonal projecting areas, as in Fig. 4c. NS, not significant.