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

Abstract

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate 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 counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach 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 micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cell…