Ion Channels In Epilepsy And As Targets For Antiepilepti

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由红藻氨酸受体介导的含有GluR5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。在本报告所述期间，我们继续研究GluR5红藻氨酸受体作为杏仁核癫痫活动介质的作用，杏仁核是人类癫痫发作的常见主要焦点。我们还研究了GluR5红藻氨酸受体可能成为抗癫痫药物（AED）开发的新靶点的可能性。众所周知，低浓度的红藻氨酸可在海马和新皮质的体外脑切片制备物中诱导自发性癫痫样活动。然而，由于红藻氨酸是一种非选择性激动剂，其促惊厥活性是否通过激活红藻氨酸或AMPA受体发生尚不确定。使用杏仁核切片的胞外场记录，我们进行了药理学研究，证明选择性激活GluR5红藻氨酸受体可以诱导自发性癫痫样放电。结果还表明，红藻氨酸的体外致癫痫活性可能与红藻氨酸受体而不是AMPA受体的特异性作用有关。由于红藻氨酸受体的激活会诱发癫痫样活动（至少在BLA中），因此可以想象，红藻氨酸受体的阻断可以抑制癫痫发作活动，在这种情况下，红藻氨酸受体将代表潜在的AED靶点。事实上，我们发现，托吡酯，一种广谱AED，其作用机制知之甚少，选择性抑制GluR5红藻氨酸受体介导的突触电流在BLA，但对AMPA受体反应的有效性低得多。自发和微型突触电流的研究证实，托吡酯对GluR5红藻氨酸受体介导的突触反应的影响至少部分通过突触后作用发生。托吡酯阻断GluR5红藻氨酸受体反应是缓慢的，这表明它可能间接起作用，可能是通过影响调节红藻氨酸受体活性的第二信使机制。托吡酯，一种高效的AED，能够在低浓度下阻断红藻氨酸受体的证明支持了红藻氨酸受体可能代表AED开发的有价值的靶点的概念。为了研究选择性激活GluR5红藻氨酸受体介导癫痫发生的可能性，我们在大鼠体内用GluR5激动剂ATPA进行了研究。杏仁核内微量注射ATPA可引起急性癫痫发作。此外，一些动物在几个月后出现自发性癫痫发作。这些研究表明，杏仁核中GluR5红藻氨酸受体的激活不仅诱导癫痫发作，而且还可能导致突触生理学的持久改变，从而易于自发性癫痫发作。因此，红藻氨酸受体拮抗剂可能有长期的抗癫痫作用，除了他们的能力，以防止急性发作。

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 have continued studies examining the role of GluR5 kainate receptors as mediators of epileptic activity in the amygdala, a common primary focus for seizures in human epilepsy. We have also examined the possibility that GluR5 kainate receptors could represent novel targets for antiepileptic drug (AED) development. It is well recognized that low concentrations of kainate can induce spontaneous epileptiform activity in in vitro brain slice preparations of hippocampus and neocortex. However, because kainate is a nonselective agonist, whether its proconvulsant activity occurs through activation of kainate or AMPA receptors is uncertain. Using extracellular field recordings in the amygdala slice, we carried out pharmacological studies demonstrating that selective activation of GluR5 kainate receptors can induce spontaneous epileptiform discharges. The results also indicated that the in vitro proepileptic activity of kainate may relate to effects specifically on kainate receptors and not AMPA receptors. Since activation of kainate receptors elicits epileptiform activity (at least in the BLA), it is conceivable that blockade of kainate receptors could suppress seizure activity, in which case kainate receptors would represent potential AED targets. In fact, we found that topiramate, a broad spectrum AED whose mechanism of action is poorly understood, selectively inhibits GluR5 kainate receptor-mediated synaptic currents in the BLA, but is much less effective against AMPA receptor responses. Studies of spontaneous and miniature synaptic currents confirmed that the effects of topiramate on GluR5 kainate receptor-mediated synaptic responses occur at least in part via a postsynaptic action. Topiramate block of GluR5 kainate receptor responses was slow, suggesting that it could act indirectly, perhaps by affecting second messenger mechanisms that modulate the activity of kainate receptors. The demonstration that topiramate, a highly effective AED, is able to block kainate receptors at low concentrations supports the concept that kainate receptors may represent a worthwhile target for AED development. To investigate the possibility that selective activation of GluR5 kainate receptors mediates epileptogenesis, we conducted studies with the GluR5 agonist ATPA in the rat in vivo. Microinjection of ATPA into the amygdala resulted in acute seizures. In addition, some animals followed for several months developed spontaneous seizures. These studies demonstrate that activation of GluR5 kainate receptors in the amygdala not only induces seizures, but may also lead to enduring alterations in synaptic physiology that predisposes to spontaneous seizures. Therefore, kainate receptor antagonists may have long-term antiepileptogenic actions in addition to their ability to protect acutely against seizures.