Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation

Ca2+ influx through voltage-gated Ca2+ channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca2+ signals may also influence the neuronal differentiation of neural stem / progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca2+ transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca2+ signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+) / MAP2(+)) displayed time-dependent increases in intracellular Ca2+ transients, with Delta F/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba2+ current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 mu m nifedipine. To determine the influence of Ca(v)1-mediated Ca2+ influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine ( 5 mu m) or the L-channel activator Bay K 8644 ( 10 mu m). The latter treatment significantly increased the percentage of beta-III-tubulin(+) / MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca2+ signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca2+ transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca2+ channels, especially the Cav1, and that Ca2+ influx through these channels plays a key role in promoting neuronal differentiation.