Ion Channels in Epilepsy and as Targets for Antiepileptic Drugs

细胞电生理记录技术被用来研究药物调制的神经递质门控和电压激活的离子通道在脑切片，培养的神经元和异源细胞转染克隆的离子通道亚基基因。在动物模型中进行了相关研究。 在一系列的实验中，我们揭示了红藻氨酸受体（KAR），含有GluR 5亚基（KAR-G5）在癫痫发作诱导和癫痫发生的作用。这些受体是离子型谷氨酸受体的一个亚组，与NMDA和AMPA受体不同。我们使用KAR-G5受体敲除小鼠和KAR-G5激动剂ATPA来确定KAR-G5受体激活的行为、临床癫痫发作和电生理效应。 通过在视频EEG记录期间进行选择性GluR 5激动剂ATPA的缓慢尾静脉输注，我们证明了KAR-G5的选择性激活介导行为停滞，随后是肌阵挛痉挛，其显示与EEG相关的尖锐波。在较高剂量的ATPA下发生的阵挛性和强直性癫痫发作不是由KAR-G5介导的，并且最有可能是由于AMPA受体激活，因为ATPA在较高浓度下失去其选择性。这些数据在体外切片记录和自发活动的行为测试中得到证实。 在另一种方法中，我们通过腹腔注射红藻氨酸诱导SE来触发大鼠癫痫。SE后7至13天，我们进行了24小时视频EEG监测，显示所有动物的癫痫发生体征（EEG癫痫发作、临床癫痫发作、尖波）。第二天，处死大鼠进行体外切片记录（全细胞膜片钳，细胞外记录），以表征KAR-G5活化对GABA传递的功能影响的潜在变化。GABA是人类和啮齿动物成年大脑中的主要抑制性神经递质。 我们的合作者Maria Braga发表的实验表明，健康大鼠杏仁核切片制备中KAR-G5的激活具有双向效应。低浓度的ATPA（1 μ M）增加GABA能传递，但高剂量（10 μ M）反而减少GABA能传递。我们发现，癫痫杏仁核对低浓度ATPA的反应降低，反映GABA能传递（去抑制）增强较少。然而，10 μ M的ATPA在癫痫动物的脑片中仍然诱导爆发活动。这表明KARG 5受体的功能可能以兴奋超过抑制的方式改变。进一步的研究正在调查癫痫发生过程中可能发生变化的细胞位置和详细的亚基组成。 此外，我们使用AMPA受体（也是离子型谷氨酸受体）的竞争性拮抗剂进行了治疗研究。AMPA受体携带绝大多数由谷氨酸（主要兴奋性神经递质）诱导的快兴奋性电流。这使得它们成为治疗癫痫持续状态的有希望的靶点。该研究是在小鼠中进行视频脑电图记录。我们选择了两个任意的时间点（早期和晚期）与AMPA拮抗剂治疗，并将其与车辆和地西泮进行比较。 在早期（连续EEG癫痫发作活动的前5分钟后）和晚期（25分钟后）的两个时间点，AMPA拮抗剂在成功中断正在进行的癫痫发作活动方面上级溶剂和地西泮，随后不会复发癫痫发作。AMPA拮抗剂治疗后未发现任何死亡。地西泮组的死亡率（57%）甚至超过溶剂组，这很可能是由于对呼吸和心血管系统的不良反应。 AMPA拮抗剂治疗的潜在抗癫痫（SE后癫痫保护）作用目前正在通过SE后长达20周的24小时视频EEG监测筛查自发性癫痫发作的发生率进行评价。

Cellular electrophysiological recording techniques are used to study drug modulation of neurotransmitter-gated and voltage-activated ion channels in brain slices, cultured neurons and heterologous cells transfected with cloned ion channel subunit genes. Correlative studies are carried out in animal models. In a series of experiments we revealed the roles of kainate receptors (KAR) that contain the GluR5 subunit (KAR-G5) in seizure induction and epileptogenesis. These receptors are a subgroup of ionotropic glutamate receptors which are distinguishable from NMDA and AMPA receptors. We used KAR-G5 receptor knockout mice and the KAR-G5 agonist ATPA to determine the behavioral, clinical seizure and electrophysiological effects of KAR-G5 receptor activation. By performing slow tail vain infusion of the selective GluR 5 agonist ATPA during Video-EEG recordings we demonstrated that the selective activation of KAR-G5 mediates a behavioral arrest followed by myoclonic jerks which show a sharp wave as EEG correlate. Clonic and tonic seizures occurring at higher doses of ATPA are not mediated by KAR-G5 and are most likely due to AMPA receptor activation as ATPA loses its selectivity at higher concentrations. These data were confirmed in in vitro slice recordings and behavioral testing for locomotor activity. In a different approach we triggered epilepsy in rats by induction of SE with intraperitoneal injections of kainic acid. Seven to thirteen days after SE we performed 24h Video-EEG monitoring, which revealed signs of epileptogenesis in all animals (EEG seizures, clinical seizures, sharp waves). The following day the rats were sacrificed for in vitro slice recordings (whole cell patch clamp, extracellular recordings) to characterize potential changes in the functional effects of KAR-G5 activation on GABA transmission. GABA is the main inhibitory neurotransmitter in the adult brain of humans as well as rodents. Experiments published by our collaborator Maria Braga showed that activation of KAR-G5 in the amygdala slice preparation of healthy rats has a bidirectional effect. Low concentrations of ATPA (1uM) increase GABAergic transmission but higher doses (10uM) rather decrease GABAergic transmission. We found that in the epileptic amygdala the response to low concentrations of ATPA is reduced, reflecting less enhancement of GABAergic transmission ( disinhibition). However, 10 uM ATPA did still induce bursting activity in the slices of epileptic animals. This suggests that the function of KARG5 receptors may be changed in a way that excitation outweighs inhibition. Further studies investigating the cellular location and detailed subunit composition that may have changed during epileptogenesis are underway. Furthermore we performed a therapeutic study using a competitive antagonist at the AMPA receptor, also an ionotropic glutamate receptor. AMPA receptors carry the vast majority of the fast excitatory currents induced by glutamate (main excitatory neurotransmitter). This makes them a promising target as treatment for status epilepticus. The study was performed in mice under Video-EEG recordings. We chose two arbitrary time points (early and late) for treatment with the AMPA antagonists and compared it with vehicle and diazepam. At both time points early (after the first 5 min of continuous EEG seizure activity) and late (25 min later) the AMPA antagonist was superior to both vehicle and diazepam in successfully interrupting the ongoing seizure activity without later reoccurrence of seizures. We did not see any mortality after the AMPA antagonist treatment. The mortality rate in the diazepam group (57%) exceeded even the vehicle group what is most likely due to the depressant effect on the respiratory and cardiovascular system. The potential antiepileptogenic (protection against epilepsy after SE) effect of the AMPA antagonist treatment is currently being evaluated by screening for the occurrence of spontaneous seizures with 24h Video-EEG- monitoring sessions up to 20 weeks after SE.