10.1371/journal.pone.0026089.g004 Figure 4  The BDNF-evoked neuronal IEG response is counteracted by ephrin-A5 co-application.
Wild-type (black bars) and Srf mutant (white bars) cortical neurons were treated for 20 minutes (A, B, D, F, H) or 4h (C, E, G, I) with guidance cues, followed by quantification of mRNA levels of indicated genes. (A) Srf mRNA levels were not altered by any treatment. In conditional Srf mutant cultures, Srf mRNA levels are reduced approximately two-fold compared to wild-type. (B, D, F, H) Expression of the IEGs c-fos (B), Egr1 (D), Egr2 (F) and Arc (H) was up-regulated in wild-type neurons by BDNF, but not ephrin-A5 alone. Ephrin-A5 inhibited BDNF-evoked IEG up-regulation in wild-type neurons. As revealed by Srf mutant neurons, BDNF-induced gene regulation of Egr1, Egr2, Arc and weaker c-fos required SRF. (C, E, G, I) Long-term exposure to BDNF, but not ephrin-A5 alone, elevated expression of the IEGs c-fos (C), Egr1 (E), Egr2 (G) and Arc (I) in wild-type neurons. At this extended exposure (4h) to both guidance cues, co-application of ephrin-A5 failed to decrease BDNF-stimulated gene expression. This is inline with the activation profile of Eph receptors reaching their maximum at 10-60 mins after ephrin-A5 addition (see Fig. 2).