A critical importance of polyamine site in NMDA receptors for neurite outgrowth and fasciculation at early stages of P19 neuronal differentiation

We have investigated the role of N-methyl-d-aspartate receptors (NMDARs) and γ-aminobutyric acid receptors type A (GABAARs) at an early stage of P19 neuronal differentiation. The subunit expression was profiled in 24-hour intervals with RT-PCR and functionality of the receptors was verified via fluo-3 imaging of Ca2+ dynamics in the immature P19 neurons showing that both NMDA and GABA excite neuronal bodies, but only polyamine-site sensitive NMDAR stimulation leads to enhanced Ca2+ signaling in the growth cones. Inhibition of NR1/NR2B NMDARs by 1 μM ifenprodil severely impaired P19 neurite extension and fasciculation, and this negative effect was fully reversible by polyamine addition. In contrast, GABAAR antagonism by a high dose of 200 μM bicuculline had no observable effect on P19 neuronal differentiation and fasciculation. Except for the differential NMDAR and GABAAR profiles of Ca2+ signaling within the immature P19 neurons, we have also shown that inhibition of NR1/NR2B NMDARs strongly decreased mRNA level of NCAM-180, which has been previously implicated as a regulator of neuronal growth cone protrusion and neurite extension. Our data thus suggest a critical role of NR1/NR2B NMDARs during the process of neuritogenesis and fasciculation of P19 neurons via differential control of local growth cone Ca2+ surges and NCAM-180 signaling.