Effect of NMDA on staurosporine-induced activation of caspase-3 and LDH release in mouse neocortical and hippocampal cells

To achieve a better understanding of developmentally regulated NMDA- and staurosporine-induced apoptotic processes, we investigated the concerted action of these agents on caspase-3 activity and LDH release in neocortical and hippocampal cell cultures at different stages in vitro (DIV). Hoechst 33342 and MAP-2 stainings were additionally employed to visualize apoptotic changes and cell damage. The vulnerability of neocortical cells to NMDA was more prominent at later culture stages, whereas hippocampal neurons were more susceptible to NMDA treatment at earlier stages. A persistent activation of caspase-3 by staurosporine was found at all experimental stages. Despite of certain differences in susceptibility to NMDA and staurosporine, both tissues responded to regulatory action of NMDA towards staurosporine-activated caspase-3 in a similar way. Combined treatment with NMDA and staurosporine resulted in a substantial increase in caspase-3 activity in neocortical and hippocampal neurons on 2 DIV. Additive effects were also observed in neocortical cultures on 12 DIV. In contrast, NMDA substantially inhibited staurosporine-induced caspase-3 activity on 7 DIV in neocortical and hippocampal cultures. Additionally, pro-apoptotic effects of 17β-estradiol were attenuated by NMDA on 7 DIV. Changes in vulnerability to NMDA- and staurosporine-mediated activation of caspase-3 were not strictly related to LDH release. Our data revealed that NMDA can both enhance and inhibit the staurosporine-induced neuronal cell apoptosis. The pro-apoptotic effect of NMDA was exhibited at early and late culture stages, whereas the anti-apoptotic effect was transient occurring on 7 DIV only.