Effect of recurrent epileptiform discharges induced by magnesium-free treatment on developing cortical neurons in vitro.
As seizures in infants and children often originate from the neocortex, neocortical epilepsy models may be appropriate for studying epileptiform activity and seizure-induced injury in the developing nervous system. However, the characterization of epileptiform activity or seizure-induced injury in cultured developing cortical neurons has seldom been reported. Therefore, We attempted to establish a cultured developing cortical neuronal epilepsy model, and to study the subsequent effect on neurons. Cultures were exposed to Mg(2+)-free media for 3 h, and then returned to regular media. Using whole-cell patch-clamp intracellular recording techniques, we found that spontaneously recurrent epileptiform discharges for at least 72 h could be induced after transient Mg(2+)-free treatment. Neuron morphology following Mg(2+)-free treatment demonstrated no prominent alterations. At different time points (6, 24 and 72 h) after Mg(2+)-free treatment, neuronal viability, identified by trypan blue staining and LDH activity, and apoptosis, measured by flow cytometry, showed modest but non-significant (P>0.05) changes compared with the age-matched control group after various culture periods (6 and 17 days) in vitro. Mitochondrial metabolic activity, measured by MTT assay, significantly decreased by 15% at 6 h after Mg(2+)-free treatment (P<0.05) in neurons cultured for 6 days, and at 24 h showed a 29% decrease in neurons cultured for 17 days (P<0.05). In conclusion, brief Mg(2+)-free treatment constitutes a cultured developing cortical neuron 'seizure' model, and can induce transient mitochondrial dysfunction without cell loss.
Cao
Hai-Yan
HY
Division of Child Neurology, Department of Pediatrics, Peking University First Hospital, No. 1, Xi'anmen Dajie, Beijing 100034, PR China.
Jiang
Yu-Wu
YW
Liu
Zhen-Wei
ZW
Wu
Xi-Ru
XR
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000196:embryology / Q000503:physiopathology; D003470:Culture Media; D004594:Electrophysiology; D004622:Embryo, Mammalian; D004827:Epilepsy / Q000503:physiopathology; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology*; D008928:Mitochondria / Q000378:metabolism; D023421:Models, Animal; D009474:Neurons / Q000473:pathology* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D012640:Seizures / Q000503:physiopathology*
D003470:Culture Media; D008274:Magnesium