Mult-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosonnal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy
Mult-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosonnal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy
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DOI：
10.3389/fncir.2014.00087
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发表时间：
2014-07-24
影响因子：
3.5
通讯作者：
Becchetti, Andrea
中科院分区：
文献类型：
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作者：
Gullo, Francesca;Manfredi, Irene;Becchetti, Andrea
常染色体显性夜间额叶癫痫（ADNFLE）是一种部分睡眠相关癫痫，可由神经元烟碱乙酰胆碱受体（nAChR）突变引起。我们采用多电极阵列（MEA）记录方法研究了表达或不表达adnfl连接的nAChR亚基（β 2-V287L）的小鼠新皮质培养物的自发放电活性。在实验中，从数千个神经元中取样，记录了超过10万个向上状态。数据分析使用快速滑动窗口程序，计算上状态持续时间的直方图。与WT不同的是，表达β 2-V287L的培养表现出长时间（10-32秒）的突触诱导的上状态放电事件。GABA(A)受体的阴性（gabazine，青霉素G）和阳性（苯二氮卓类）调节剂可防止这种长时间向上状态的发生。卡马西平（CBZ）是ADNFLE患者的首选药物，在微摩尔浓度下也能抑制长上升状态。在表达β 2-V287L的培养物中，无论是没有药物治疗还是有药物治疗，对动作电位波形都没有明显影响。我们的研究结果表明，人类通道病的小鼠模型所显示的自发超兴奋性的某些方面可以在神经元培养中复制。特别地，我们的培养代表了自发癫痫样活动的体外慢性模型，即不需要用惊厥药进行预处理。这为体外研究β 2-V287L在突触形成中的作用开辟了道路。此外，我们在MEA平台上的新皮层培养可以确定长期药物治疗对自发性网络高兴奋性的影响（这在短期大脑切片中是不可能的）。我们在本文中阐述的方法也应该大大促进抗癫痫药物（aed）的初步筛选，从而减少体内实验的数量。
Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (beta 2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing beta 2-V287L displayed long (10-32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABA(A) receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up states at micromolar concentrations. In cultures expressing beta 2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study en vitro of the role of beta 2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.