Intracellular calcium mobilization and neurite outgrowth in mammalian neurons

Cultured adult rat dorsal root ganglion (DRG) neurons were used to study depolarization-induced Ca2+ mobilization and the effects of intracellular Ca2+ depletion on neurite outgrowth. Cytoplasmic and nuclear Ca2+ signals were visualized in dissociated DRG neurons using confocal scanning laser microscopy and the Ca2+ indicator dye fluo-3. The depolarization-induced Ca2+ signals were highest in neurons during the first few days in culture, prior to neurite extension; during this time nuclear signals exceeded those of the cytoplasm severalfold. After several days in culture, neurons began to arborize, depolarization-induced Ca2+ signals became attenuated, and nuclear signals no longer exceeded those of the cytoplasm. Elevated Ca2+ signals were dependent upon both Ca2+ influx and intact intracellular Ca2+ stores, indicating that the signals are generated by calcium-induced calcium release (CICR). Thapsigargin, an endoplasmic reticulum Ca2+ ATPase inhibitor, depleted intracellular Ca2+ stores and blocked the induction of the large nuclear Ca2+ signals. Treating DRG neurons briefly with thapsigargin (200 nM for 20 min) shortly after plating reduced subsequent neuritogenesis, implying that intact Ca2+ stores are necessary for initiating neurite outgrowth. Immunostaining of DRG neurons with antibodies to Ca2+/calmodulin-dependent kinase II (CaM kinase II) demonstrated that this enzyme is present in the nucleus at early times in culture. These observations are consistent with the idea that CICR triggered by Ca2+ entry subsequent to depolarization may elicit neurite outgrowth by activating nuclear enzymes appropriate for such outgrowth.