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.