Cells were harvested in 100 l immunoprecipitation buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 10% glycerol, 0.1% NP-40). in the ventricular zone and subventricular zone (SVZ) gives rise to most of the neurons and glial cells that form the brain in higher vertebrates1C3. These cells in the Anisodamine beginning migrate toward the pial surface, known as the preplate. The preplate consists of two unique cell types, the Cajal-Retzius cells, and a deeper zone of cells called the subplate cells. From this region, neuronal cell body migrate back to form the cortical plate where the cortical layers IICVI develop4. In rodents, neurogenesis starts early, around embryonic day 9 Anisodamine (E9), and most neurons that form the different layers in the mature cortex reach their positions by migrating radially toward the cortical plate such that those neurons that finally reside in the deeper layers of the cortex migrate first, and later arriving neurons give rise to the more superficial layers, thus generating an inside-out pattern5C9. Both intrinsic cues and growth factors influence mitosis, cell cycle exit, migration and synaptic differentiation of neurons in the developing cortex10. TrkB and TrkC, the receptors for BDNF and NT-3, are expressed early in developing neurons in the cortical ventricular zone and SVZ and during migration toward their final position in the cortex11,12. Inhibition of TrkB and TrkC via overexpression of dominant-negative constructs results in reduced numbers of proliferating neural precursor cells in the ventricular zone and SVZ, delayed migration and, ultimately, Anisodamine disturbed localization of neurons in the cortical layers13. Similar defects in migration of early cortical Rabbit Polyclonal to CNGB1 neurons were observed in mice in which the cytoplasmic tyrosines in the TrkB receptor that mediate docking of Src homology 2 domain-containing-transforming protein C (SHC) and fibroblast growth factor receptor substrate 2 (FRS2) adaptors and Anisodamine phospholipase C (PLC) were inactivated, thereby abolishing activation of downstream signaling pathways once the tyrosine kinase activity of the TrkB receptors has been activated. However, it is unclear from these findings whether BDNF or NT-3, the ligands for TrkB and TrkC receptors, activate tyrosine kinases in early neurons to mediate these effects13. We examined the activation of TrkB and TrkC in the developing cortex of embryonic mice. We found that these receptors were activated at early stages of cortical development. Although TrkB and TrkC could be stimulated by BDNF and NT-3, the activation of these receptors was not affected in knockout mice lacking BDNF14 and/or NT-3 (ref. 15). Unexpectedly, activation of EGFR by EGF in isolated cortical precursor cells resulted in a strong transactivation of TrkB and also TrkC. This transactivation is responsible for the effects of EGF around the migration of early cortical neurons from your VZ/SVZ toward the cortical layers. Our results suggest that the functions of TrkB and TrkC in newborn neurons of the developing cortex go beyond serving as specific receptors for BDNF and NT-3, and include the mediation and integration of additional signals, particularly those from your EGFR. Thus, TrkB and TrkC transactivation appears to be an essential mechanism for coordination of cortical differentiation when early neuronal cells migrate and integrate into the layers that form the cerebral cortex in higher vertebrates. RESULTS Ligand-independent activation of TrkB in cortical neurons During development, high levels of TrkB are.