Fig. 8. Activation of PI3-kinase by neurotrophins and its role in tangential migration. (A) AKT phosphorylation in GE explants induced by BDNF or NT4 and visualized by western blotting with anti-phospho-AKT antibodies. Note that both BDNF and NT4 induce AKT phosphorylation (top panel), which is suppressed by preincubation with LY294002 (50 µM), a specific PI3-kinase inhibitor (lanes 1, 4 and 6). Lower panel indicates western analysis of the same blot with anti-AKT antibodies as a loading control. (B-E) Inhibition of PI3-kinase suppresses tangential migration of MGE-GFP cells. E15 MGE-GFP explants were co-cultured with P2-3 cortex for 24 hours in presence of control (B, DMSO), DMSO plus recombinant NT4 (50 ng/ml, C), or recombinant NT4 (50 ng/ml) plus 50 µM LY294002 (a PI3-kinase inhibitor, D). (E) Quantification of the results shown in B-D using the migration index as described in Fig. 6E (n=8 explants in each condition from four independent experiments). Note that inhibition of PI3-kinase with LY294002 treatment suppresses the NT4 effect, indicating that the effect of NT4 on migration requires PI3-kinase activation. Also indicated in green (full squares) is the quantification for treatment with LY294002 alone (ANOVA; F=0.623; not significantly different from LY294002+NT-4, see text). Comparison with Fig. 7F indicates that in LY294002-treated slices, migration is reduced to levels seen with K252a, consistent with the interpretation that endogenous neurotrophins regulate tangential migration via TrkB and PI3-kinase activation. (F) Quantification of the effect of inhibiting MEK1/2 activity (U0126, 10 µM) and inhibiting phospholipase C (U73122, 1 µM) on tangential migration of MGE-derived cells. Conventions are as in Fig. 8E. The migration index curves obtained for U0126 and U73122 do not differ significantly for the control (DMSO) curves [ANOVA test; F=1.021 and F=0.875, respectively]. Scale bar: 500 µm in B-D.
