Ion Channels In Epilepsy And Targets For Antiepileptics

本项目的目的是探索基于与神经元离子通道系统相互作用的抗癫痫药物的合理开发新策略。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。最近的研究集中在红藻氨酸型谷氨酸受体。我们已经证明，兴奋性突触反应的一个组成部分引起的基底外侧杏仁核神经元的外囊刺激是介导的红藻氨酸受体含有GluR 5亚基，我们已经表明，这些受体引起一种新形式的突触可塑性，可以介导某些类型的癫痫发生在杏仁核。在本报告期间，我们研究了托吡酯与GluR 5红藻氨酸受体介导的基底外侧杏仁核神经元突触反应的相互作用。 托吡酯是一种广泛使用的抗癫痫药物，其作用机制尚不清楚。据报道，该药物与各种离子通道类型相互作用，包括AMPA/红藻氨酸受体。托吡酯对AMPA/红藻氨酸受体的潜在作用是有趣的，因为阻断这些受体的药物在用于筛选抗癫痫药物的动物模型中是高度有效的，但没有其他临床使用的抗癫痫药物以治疗浓度靶向这些受体。在全细胞电压钳记录大鼠基底外侧杏仁核的主要神经元在体外脑片，托吡酯在低浓度选择性抑制cardiac孤立兴奋性突触电流介导的红藻氨酸受体含有GluR 5亚基。托吡酯也部分抑制AMPA受体介导的EPSC，但疗效较低。托吡酯没有改变成对脉冲实验中的易化程度，并且它降低了微型EPSC的振幅而不影响其频率，表明突触反应的阻断发生在突触后。GluR 5红藻氨酸受体的抑制可能是托吡酯抗惊厥活性的关键机制。此外，这些结果支持的概念，GluR 5红藻氨酸受体代表一种新的抗癫痫药物开发的目标。 为了确定托吡酯对GluR 5红藻氨酸受体的抑制作用是否与药物在体内的抗惊厥作用有关，我们测定了托吡酯对小鼠静脉输注各种离子型谷氨酸受体激动剂诱导的癫痫发作的保护活性。托吡酯产生剂量依赖性升高的阈值由ATPA，GluR 5红藻氨酸受体的选择性激动剂输注诱导的阵挛发作。托吡酯对红藻氨酸（AMPA和红藻氨酸受体的混合激动剂）诱导的阵挛性癫痫发作的保护效果较差。托吡酯不影响AMPA或NMDA诱发的阵挛性发作。与此相反，托吡酯升高了高剂量这些不同谷氨酸受体激动剂诱导的强直性癫痫发作的阈值。我们的研究结果与托吡酯对阵挛性发作活动的影响是由于其特异性阻断GluR 5红藻氨酸受体的可能性一致。对强直性癫痫发作的保护作用可能由药物的其他作用介导。连同我们的体外细胞电生理结果，目前的观察结果强烈支持托吡酯的独特作用机制，其中包括选择性阻断GluR 5红藻氨酸受体。

The objective of this project is to explore new strategies for the rational development of antiepileptic drugs based upon their interaction with neuronal ion channel systems. 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. Recent studies have focused on kainate-type glutamate receptors. We have demonstrated that a component of the excitatory synaptic response evoked in basolateral amygdala neurons by external capsule stimulation is mediated by kainate receptors containing the GluR5 subunit and we have shown that these receptors elicit a novel form of synaptic plasticity that could mediate some types of epileptogenesis in the amygdala. In the present reporting period, we investigated the interaction of topiramate with GluR5 kainate receptor mediated synaptic responses in basolateral amygdala neurons. Topiramate is a widely used antiepileptic agent whose mechanism of action is poorly understood. The drug has been reported to interact with various ion channel types, including AMPA/kainate receptors. The potential action of topiramate on AMPA/kainate receptors is intriguing inasmuch as drugs that block these receptors are highly effective in animal models used in the screening of antiepileptic drugs, but no other clinically used antiseizure medication targets these receptors at therapeutic concentrations. In whole-cell voltage-clamp recordings from principal neurons of the rat basolateral amygdala in the in vitro brain slice, topiramate at low concentrations selectively inhibited pharmacologically isolated excitatory synaptic currents mediated by kainate receptors containing the GluR5 subunit. Topiramate also partially depressed predominantly AMPA-receptor-mediated EPSCs, but with lower efficacy. Topiramate did not alter the degree of facilitation in paired-pulse experiments, and it reduced the amplitude of miniature EPSCs without affecting their frequency, demonstrating that the block of synaptic responses occurs postsynaptically. Inhibition of GluR5 kainate receptors could represent a key mechanism underlying the anticonvulsant activity of topiramate. Moreover, these results support the concept that GluR5 kainate receptors represent a novel target for antiepileptic drug development. To determine if the inhibitory action of topiramate on GluR5 kainate receptors as shown in brain slice recordings is relevant to the anticonvulsant effects of the drug in vivo, we determined the protective activity of topiramate against seizures induced by intravenous infusion of various ionotropic glutamate receptor agonists in mice. Topiramate produced a dose-dependent elevation in the threshold for clonic seizures induced by infusion of ATPA, a selective agonist of GluR5 kainate receptors. Topiramate was less effective in protecting against clonic seizures induced by kainate, a mixed agonist of AMPA and kainate receptors. Topiramate did not affect clonic seizures induced by AMPA or NMDA. In contrast, the thresholds for tonic seizures induced by higher doses of these various glutamate receptor agonists were all elevated by topiramate. Our results are consistent with the possibility that the effects of topiramate on clonic seizure activity are due to its specific blockade of GluR5 kainate receptors. Protection from tonic seizures may be mediated by other actions of the drug. Together with our in vitro cellular electrophysiological results, the present observations strongly support a unique mechanism of action of topiramate, which involves selective blockade of GluR5 kainate receptors.