Ion Channels In Epilepsy And As Targets For Antiepilepti

本项目的目的是基于抗癫痫药物与神经元离子通道系统的相互作用，探索合理开发抗癫痫药物的新策略。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。最近的研究主要集中在海藻酸型谷氨酸受体上。我们已经证明，刺激外囊刺激杏仁核基底外侧神经元所引起的兴奋性突触反应的一个组成部分是由含有GluR5亚单位的海人酸受体介导的，我们已经证明这些受体诱导了一种新的突触可塑性，可以介导杏仁核某些类型的癫痫发生。在本报告期间，我们继续研究GluR5海人酸受体作为杏仁核癫痫活动的媒介的作用，杏仁核是人类癫痫发作的常见主要病灶。我们还研究了GluR5海人酸受体作为抗癫痫药物(AED)开发的新靶点的可能性。已有研究表明，低浓度的海人藻酸可在体外培养的大鼠海马区和大脑皮层脑片上诱发自发癫痫样活动。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受体而发生尚不确定。利用杏仁核片的胞外场记录，我们进行了药理学研究，证明选择性激活GluR5海人酸受体可以诱导自发的癫痫样放电。结果还表明，红藻氨酸的体外致痫作用可能与其对红藻氨酸受体的特异性作用有关，而与AMPA受体无关。由于海人藻酸受体的激活引起癫痫样活动(至少在BLA中)，可以想象，阻断海人藻酸受体可以抑制癫痫活动，在这种情况下，海人藻酸受体将是潜在的AED靶点。事实上，我们发现托吡酯是一种广谱的AED，其作用机制尚不清楚，它选择性地抑制BLA中GluR5红藻氨酸受体介导的突触电流，但对AMPA受体的反应效果要差得多。对自发和微小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是通过突触后作用实现的。托吡酯阻断GluR5红藻氨酸受体的反应很慢，这表明它可能通过影响调节红藻氨酸受体活性的第二信使机制而间接发挥作用。托吡酯是一种高效的AED，能够在低浓度下阻断红藻氨酸受体，这一证明支持了红藻氨酸受体可能是AED开发的一个有价值的靶点的概念。为了探讨选择性激活GluR5海人酸受体介导癫痫发生的可能性，我们在大鼠体内进行了GluR5激动剂ATPA的研究。杏仁核内微量注射ATPA可导致急性癫痫发作。此外，一些动物追踪了几个月后出现了自发性癫痫发作。这些研究表明，杏仁核中GluR5海氨酸受体的激活不仅会导致癫痫发作，还可能导致突触生理上的持久变化，从而易于自发癫痫发作。因此，海人藻酸受体拮抗剂除了对癫痫有强烈的保护作用外，还可能具有长期的抗癫痫作用。

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.