Postmitotic neurons were generated from the human
NT-2 teratocarcinoma cell line in a novel rapid differentiation procedure. These neurons were used to establish an in vitro assay system that allows the investigation of hypoxic/ischaemic cell damage and the development of neuroprotective strategies. In experiments of simulated ischaemia, the neurons were subjected to
anoxia and hypoglycaemia. The viability of
NT-2 neuronal cells was significantly reduced by
anoxia especially in the presence of
glutamate, reflecting the cellular vulnerability to excitotoxic conditions. The addition of the
N-methyl-D-aspartate (
NMDA) receptor antagonist
MK-801 reduced
glutamate-induced neuronal damage.
Calcium imaging showed that
NT-2 neurons increased cytosolic
calcium levels in response to stimulation with
glutamate or
NMDA, an effect that was abolished in
calcium free medium and at low pH values. The
NMDA receptor antagonists
MK-801, AP 5 and
ketamine reduced the
NMDA-induced response, suggesting the presence of functional
NMDA receptors in the human neuronal cells. The mitochondrial potential of neurons was estimated using the
fluorescent dye rhodamine 123 (R123). The fluorescence imaging experiments indicated an energetic collapse of mitochondrial functions during
anoxia, suggesting that the human
NT-2 neurons can be used to investigate subcellular processes during the excitotoxic cascade.