Cortical neurons culture to study C-Jun N-Terminal kinase signaling pathway

Excitotoxicity via N-methyl-D-aspartate receptors (NMDA-r) is a key mechanism of neurodegeneration following ischemia and other brain pathologies. We here describe the use of a fast, reliable and easily reproducible in vitro model of excitotoxicity induced with NMDA stimulation of cortical neurons. Such a protocol can be used to study the intracellular pathways involved in NMDA-induced death. Here wefocused on JNK signaling, a pathway that plays a determinant role in excitotoxic death of cortical neurons. Our proposed model can additionally be used to test different drugs designed to prevent excitotoxic neuronal death. As an example we describe the use of the cell permeable specific JNK inhibitor peptide as a neuroprotective agent. This chapter will give methodological suggestions to help researchers prepare cortical neurons, to develop an in vitro model of neuronal death by stimulating neurons with NMDA, to quantify neuronal death in vitro using LDH assay and propidium staining, to analyze the intracellular pathways involved (JNK pathway) in neuronal death, and to perform protocols of neuroprotection.