Development of in vitro models of NMDA excitotoxicity and assessment of excitotoxicity modulation by neurosteroids

Mouse and rat primary hippocampal cultures, along with mouse fibroblast cultures transfected with N-methyl- scD-aspartate (NMDA) receptor subunits, were developed as models of NMDA receptor-mediated excitotoxicity. These systems were subsequently used to evaluate the modulation of excitotoxicity by the neurosteroids pregnenolone sulfate and pregnanolone sulfate.;Excitotoxicity was not elicited reproducibly in the rat hippocampal culture model. Furthermore, immunohistochemical characterization of neurons and glial cells in these cultures was not accomplished. However, measurement of excitotoxicity in the mouse hippocampal culture model was characterized, and thus, pregnenolone sulfate and pregnanolone sulfate modulation of NMDA excitotoxicity was examined. While several concentrations of pregnenolone sulfate appeared to positively modulate NMDA excitotoxicity, vehicle toxicity was marked. Consequently, the modulation of NMDA toxicity by pregnenolone sulfate, at concentrations ranging from 0.001 mM to 0.1 mM, was difficult to establish. In contrast, it was determined with greater confidence that pregnanolone sulfate, at a concentration of 0.05 mM, negatively modulates NMDA toxicity.;A novel model system, consisting of mouse fibroblasts expressing either human recombinant NMDA receptor subunits (NR) 1a/2A or NR1a/2B was successfully developed as a further model of NMDA receptor dependent toxicity. The system was subsequently employed to examine pregnenolone sulfate and pregnanolone sulfate modulation of excitotoxicity. Pregnenolone sulfate at a concentration of 0.01 mM failed to modulate NMDA receptor mediated toxicity, while 0.10 mM pregnanolone sulfate negatively modulated toxicity in both the NR2A and NR2B expressing cell lines.;In summary, pregnenolone sulfate possibly potentiated NMDA excitotoxicity while pregnanolone sulfate reduced NMDA excitotoxicity in the mouse hippocampal culture model of toxicity. In the mouse fibroblast model of NMDA excitotoxicity, pregnenolone sulfate had no modulatory effect on NMDA receptor mediated excitotoxicity, at a single dose. Pregnanolone sulfate, however, negatively modulated the toxicity, independent of NMDA receptor subunit composition.