Nitric oxide produced by non-motoneuron cells enhances rat embryonic motoneuron sensitivity to excitotoxins: comparison in mixed neuron/glia or purified cultures

The present study compares the sensitivity to chronic exposure to glutamate agonists of SMI-32-positive rat-derived embryonic motoneurons under both m ixed neuron/glia and purified cultures. We found that in spite of a trophic role of glia on cultured motoneurons, SMI-32-positive cells are more sensi tive to excitotoxicity in the presence of glia than in purified culture, ve ry likely through nitric oxide released by non-neuronal cells. The rank ord er of potency for inducing toxicity after 48 h incubation was AMPA > kainat e > NMDA, with EC50: 0.43, 4.9 and 49 muM, respectively, in mixed neuron/gl ia culture and 14, 32 and 135 muM in purified cultures. The effect of NMDA was dose-dependently potentiated by glycine, with similar potency in the tw o culture conditions. The effect of agonists was completely antagonized by the specific antagonists CNQX, BNQX and MK801 in both culture conditions. Motoneurons were similarly immunoreactive to NR1 and GluR2 antibodies under both mixed neuron/glia and purified cultures, thus confirming the presence of the calcium-impermeant AMPA receptor subtypes and of the obligatory sub unit for NMDA receptors. The effect of kainate in mixed neuron/glia culture was reduced by the addit ion of 40 muM N-nitro-L-arginine Or L-NAME, which shifted the EC50 to 9 muM . By contrast, L-NAME did not modify the effect of kainic acid in purified cultures. These results suggest that the release of nitric oxide by non-neu ronal cells in culture enhances glutamate excitotoxicity in SMI-32-positive cells, and that direct activation of ionotropic glutamate receptors is not enough to explain the mechanism of chronic motoneuron degeneration occurri ng in vivo in amyotrophic lateral sclerosis (ALS). (C) 2001 Elsevier Scienc e B.V. All rights reserved.