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

Lookup NU author(s): Dr Christopher Eggett, Dr Stephen Crosier, Dr Philip Manning, Fiona Menzies, Emeritus Professor Calum McNeil, Professor Pamela Shaw

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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 (~30%, ρ < 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 μM) and 2,3-dioxo-6-nitro- 1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 μM). 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 microelectrochemical 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 cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.


Publication metadata

Author(s): Eggett CJ, Crosier S, Manning P, Cookson MR, Menzies FM, McNeil CJ, Shaw PJ

Publication type: Article

Publication status: Published

Journal: Journal of Neurochemistry

Year: 2000

Volume: 74

Issue: 5

Pages: 1895-1902

ISSN (print): 0022-3042

ISSN (electronic): 1471-4159

Publisher: Wiley-Blackwell Publishing Ltd.

URL: http://dx.doi.org/10.1046/j.1471-4159.2000.0741895.x

DOI: 10.1046/j.1471-4159.2000.0741895.x

PubMed id: 10800932


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