Development and characterisation of a glutamate-sensitive motor neurone cell line

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glu tamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell countin g techniques demonstrated a mild but significant cell death (similar to 30% , p < 0.01) following a 24-h exposure to 1 mM glutamate that could be preve nted by the presence of the glutamate receptor antagonists (+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 mu M) and 2,3- dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 mu M) . AS an indication of glutamate receptor functional activity a novel approa ch was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a microelectrochemical sensor following addition of glut amate receptor agonists to the cell bathing solution. Alterations in intrac ellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in in tracellular calcium concentrations that is prevented by the presence of glu tamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamater gic toxicity in motor neurone degeneration.