无镁诱导培养的大鼠皮层神经元癫痫样放电

Magnesium-free medium induces spontaneous recurrent seizure activity in cultured cortical neurons of rats

目的 :观察无镁细胞外液短暂处理培养的胚胎鼠大脑皮层神经元能否诱导自发、反复的惊厥样放电。以期提供一种较好的研究发育中脑癫活动的细胞模型。方法 :采用全细胞电流钳记录技术 ,记录体外培养 12～ 18d的大脑皮层神经元在正常细胞外液、无镁细胞外液、无镁细胞外液处理 3h继而恢复正常细胞外液后 1～ 2h、2 4h、72h神经元电活动的变化。结果 :(1)神经元在正常细胞外液中自发放电形式表现为兴奋性突触后电位 (EPSP)及约 16次 /min左右的动作电位 (n =2 9)。 (2 )无镁细胞外液孵育 3h ,孵育期间 10 0 %的细胞 (n =2 5 )放电形式发生改变 ,表现为两种形式 :持续强直的高频爆发及“楔形”去极化。 (3)经无镁细胞外液孵育 3h恢复正常细胞外液后 2h 10 0 %的细胞发生异常放电 (n =12 ) ,72h仍有近 80 %的细胞发生异常放电 ,主要表现为阵发性持续棘波样爆发、“楔形”去极化及PDSs样发作 (paroxysmaldepolarizingshifts)。 结论 :培养的胚胎鼠皮层神经元予短暂的无镁处理后可诱导自发、反复的惊厥样活动 ,且惊厥样活动可持续 72h 。

SUMMARY Objective: To investigate if cortical cultures treated with Magnesium free media could be induced to develop spontaneously recurring epileptiform discharges. Methods: Experiment was carried out using whole cell current clamp recording techniques on cultured cortical neurons.Cultures were exposed to Mg 2+ free media for 3 h,returned to regular media containing normal level magnesium,and maintained for 72 h.During and after the treatment,we detect the epileptiform discharges. Results: (1) whole cell recordings from control cultured cortical neurons( n =29)manifested spontaneous excitatory postsynaptic potentials(EPSPs) and individual action potentials with frequence of about 16/min. (2) During the Mg 2+ free media exposure, all cultures developed epileptiform discharges( n =25), including continuous tonic high frequency burst discharges (9 of 25 neurons) and 'wedge shaped' depolarization (16 of 25 neurons). (3) Following the restoration of the normal Mg 2+ concentration, continuous tonic high frequency burst discharges attenuated and the neurons manifested recurrent seizure activity with paroxysmal depolarizing shifts (PDSs) and sustained spike discharges from 12 to 57 Hz. At 2 h after Mg 2+ free media treatment all cultures ( n =12) displayed epileptiform discharges: PDSs (8 of 12 neurons), sustained spike discharges (2 of 12 neurons) and 'wedge shaped' depolarization (2 of 12 neurons). At 24 h after Mg 2+ free media treatment in 22 of 26 neurons displayed epileptiform discharges: PDSs (15 of 26 neurons), sustained spike discharges (1 of 26 neurons) and 'wedge shaped' depolarization (6 of 26 neurons). At 72 h after Mg 2+ free media treatment in 21of 27 neurons displayed epileptiform discharges: PDSs (17 of 27 neurons) and sustained spike discharges (4 of 27 neurons). Conclution: Brief treatment with Mg 2+ free media of cultured cortical neurons could induce recurrent spontaneous seizure activity and these discharges could last 72 h. This neuronal culture model of spontaneous seizure discharge may provide a useful tool to epilepsy study.