Development and Characterisation of a Glutamate‐Sensitive Motor Neurone Cell Line

Abstract

Modification of the growth conditions of NSC-34 mouse neuroblastoma ϫ 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 (ϳ30%, p Ͻ 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (ϩ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d ]cyclohepten-5,10-imine maleate (10 M ) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f ]quinoxaline-7-sulphonamide (1 M). 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. Key Words: Excitotoxicity-NSC-34 cells-Glutamate -Free radicalsMotor neurone disease.

Atorvastatin Protects NSC-34 Motor Neurons Against Oxidative Stress by Activating PI3K, ERK and Free Radical Scavenging

“…To slow cell proliferation and enhance differentiation, the medium was exchanged for 1:1 DMEM and Ham's 12 with 1 % FBS, 1 % PS, and 1 % non-essential amino acids [13]. The medium was changed every 2 days and the cells were grown for up to 7 days to allow them to differentiate into motor neuron-like cells with increased neurite formation [13].…”

“…These cells have been widely used as in vitro models of ALS and other disorders affecting motor neurons. They are used especially after inducing differentiation by serum depletion [13], because primary motor neurons have a limited proliferative capacity and are short-lived in culture. In addition, they are potentially useful for studying motor neuron dysfunction in response to toxins [14].…”

Nuclear transport impairment of amyotrophic lateral sclerosis‐linked mutations in FUS/TLS

“…Transfection was performed using Lipofectamine Plus reagent or Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. To transfect differentiated NSC-34 (NSC-34D), cells were grown to 80% confluence on cover glass, and the growth medium was exchanged for a medium comprising 1:1 DMEM plus Ham F12, 1% fetal calf serum, and 1% modified Eagle medium nonessential amino acids 18 following transfection using Lipofectamine 2000 (Invitrogen). After 48 hours in this medium, these differentiated cells (NSC-34D) were fixed for immunofluorescence staining as described below.…”

Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice

“…NSC-34 cells were cultured until they differentiated to a neuronal phenotype (NSC-34 D ) using medium comprising: 1:1 DMEM/Ham's F12, 1% FBS and 1% non-essential amino acids (NEAA) as previously described (Eggett et al, 2000).…”