Ion Channels in Epilepsy and as Targets for Antiepileptic Drugs

应用细胞电生理记录技术，研究了神经递质门控和电压激活离子通道在脑片、培养神经元和异种细胞中对离子通道亚单位基因的药物调节作用。相关研究在动物模型中进行。 在一系列实验中，我们揭示了含有GluR5亚单位(KAR-G5)的红藻氨酸受体(KAR)在癫痫诱导和癫痫发生中的作用。这些受体是离子型谷氨酸受体的一个亚类，与NMDA和AMPA受体不同。我们利用KAR-G5受体基因敲除小鼠和KAR-G5激动剂ATPA来研究KAR-G5受体激活对小鼠的行为、临床发作和电生理的影响。在视频-脑电记录过程中，通过缓慢尾静脉注射选择性GluR 5激动剂ATPA，我们证明了选择性激活KAR-G5介导了行为停止，随后是肌肉阵挛抽动，随着脑电的相关显示出尖锐的波形。在较高剂量的ATPA中发生的阵挛和强直性发作不是由KAR-G5介导的，很可能是由于AMPA受体的激活，因为ATPA在较高浓度时失去了它的选择性。这些数据在体外脑片记录和运动活动的行为测试中得到了证实。 在另一种方法中，我们通过腹腔注射红藻氨酸诱导SE来引发大鼠癫痫。SE后7~13天进行24小时Video-EEG监测，发现所有动物均有癫痫发生的征象(脑电发作、临床发作、尖波)。第二天，大鼠被处死用于体外切片记录(全细胞膜片钳、细胞外记录)，以表征KAR-G5激活对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，在健康大鼠的杏仁核片制备中激活KAR-G5具有双向效应。低浓度的ATPA(1uM)增加GABA能传递，而高剂量(10uM)反而降低GABA能传递。我们发现，在癫痫杏仁核中，对低浓度ATPA的反应减弱，反映出GABA能传递的增强(去抑制)较少。然而，10微米ATPA仍能诱导癫痫动物脑片的爆发活动。这表明KARG5受体的功能可能发生了兴奋大于抑制的变化。进一步的研究正在研究癫痫发生过程中可能改变的细胞位置和详细的亚基组成。 此外，我们使用AMPA受体的竞争性拮抗剂进行了治疗研究，AMPA受体也是一种离子型谷氨酸受体。AMPA受体携带谷氨酸(主要的兴奋性神经递质)诱导的绝大多数快速兴奋电流。这使它们成为治疗癫痫持续状态的有希望的靶点。这项研究是在视频-脑电记录下在老鼠身上进行的。我们选择了两个任意的时间点(早期和晚期)使用AMPA拮抗剂进行治疗，并与赋形剂和安定进行了比较。 AMPA拮抗剂在早期(持续EEG持续发作5min后)和晚期(25min后)均优于赋形剂和安定，在成功阻断持续EEG发作活动而不再发生癫痫发作方面优于安慰剂和安定。在AMPA拮抗剂治疗后，我们没有看到任何死亡。安定组的死亡率(57%)甚至超过了赋形剂组，这很可能是由于对呼吸系统和心血管系统的抑制作用。 AMPA拮抗剂治疗的潜在抗癫痫作用(SE后防止癫痫)目前正在通过对SE后20周内24小时视频-脑电监测会议的自发性癫痫发作的筛查来评估。

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.