Figure 2
BDNF is involved in NR2B phosphorylation by NRG1. (A) NRG1-induced TrkB activation was reduced by pre-treatment with ErbB4 inhibitor, AG1478 (5 μM) for 20 min. The upper panel shows a representative autoradiogram of pTrkB and TrkB, and the lower panel represents normalized pTrkB values. (Bi) Immunoblot analysis shows ErbB4 activation with NRG1, which was blocked by AG1478. The upper panel shows a representative autoradiogram of pErbB4 and ErbB4, and the lower panel represents normalized pErbB4 values. (Bii) NRG1 induced activation of ErbB4 was inhibited by AG1478, but not by K252a. Cortical neurons were treated with 5 μM AG1478, an inhibitor of ErbB4, or K252a, an inhibitor of TrkB, for 20 min prior to the addition of NRG1. Neurons were fixed and stained with phospho-ErbB4 and parvalbumin (PV) antibodies, and visualized with Cy3 (red) and Cy2 (green) coupled secondary antibodies, respectively. Scale bar, 20 μm. (Ci) NRG1-induced increase in BDNF levels in culture medium was blocked by pre-treatment of AG1478 or Picrotoxin (100 μM; 30 min pre-treatment). (Cii) GABA agonist, muscimol (50 μM; 30 min pre-treatment) increased BDNF levels in culture medium. (Di) Immunoblot analysis of NRG1-induced activation of TrkB and NR2B, in presence of BDNF neutralizing antibody (1 μg/ml) or control IgG antibody pre-treated for 30 min. Quantification of (Dii) pTrkB and (Diii) pNR2B showed a significant reduction in their levels with BDNF neutralizing antibody. (E) Inhibition of GABAA receptor activity with Picrotoxin suppresses NRG1-induced activation of TrkB. The upper panel shows a representative autoradiogram of pTrkB and TrkB, and the lower panel represents normalized pTrkB values. (F) Chelating intracellular Ca2+ with BAPTA-AM (50 μM) has no effect on NRG1-induced activation of TrkB. Data represent mean ± SEM. (n = 3). *P < 0.05 vs WT control; $P < 0.05 vs WT NRG1 treatment.