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，刺激外囊刺激杏仁基底外侧核(BLA)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，可能介导杏仁核某些类型的癫痫发生。GluR5海氨酸受体在BLA神经元中产生的突触反应是内向整流性和钙通透性的。在BLA神经元的脑片记录中，我们证明了广泛使用的抗癫痫药物托吡酯选择性和有效地抑制GluR5海人酸受体介导的突触反应。托吡酯拮抗红藻氨酸受体的能力很耐人寻味，因为没有其他临床使用的抗癫痫药物以治疗浓度针对这些受体。在本报告期间，我们试图从内向整流和假定的钙通透性两个方面来描述BLA主神经元中AMPA受体的特性。缺乏GluR2亚单位的AMPA受体是向内整流的，是钙离子通透性的。我们使用免疫电子显微镜来确定大鼠BLA中突触与GluR2亚单位的AMPA受体的程度；为了进行比较，我们在海马区进行了平行检查。我们还记录了杏仁核脑片，以检查AMPA受体介导的BLA主神经元中诱发的突触电流的电压依赖性特性。在光镜下，GluR2免疫反应定位于BLA神经元的核周和近端树突，在海马CA1和CA3亚区的锥体细胞层也有密集的标记。在BLA的电子显微镜下，大多数突触是不对称的，突触后密度(PSD)明显。它们含有与PSD相对的透明的球形小泡，并且主要分布在脊椎上(86%)，表明它们主要与BLA主神经元一起。在BLA中，只有11%的形态突触与GluR2免疫反应阳性的突触后元件相连，而在海马区CA1和CA3区，76%和71%的突触后元件被标记。当突触发生时，BLA和海马区的突触染色完全是突触后的，尤其是在PSD上。在全细胞电压钳记录中，72%的BLA主神经元表现出AMPA受体介导的内向整流性突触电流。尽管BLA主神经元表达核周膜和近端树突状GluR2免疫反应，但与这些神经元相连的突触很少表达GluR2，而且大多数主神经元具有内向整流AMPA介导的突触电流，这表明GluR2对突触的靶向受到限制。与主神经元上的AMPA受体不同，BLA神经元的AMPA受体在推测的钙渗透性方面与GluR5海人酸受体相似。因此，BLA主神经元上的AMPA受体，就像这些神经元上的GluR5海氨酸受体一样，可能在突触可塑性、癫痫发生和兴奋性方面发挥作用。杏仁核主要神经元突触的AMPA受体的不同寻常的特性可能有助于该脑区独特的癫痫易感性。

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 (BLA) 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. Synaptic responses generated by GluR5 kainate receptors in BLA neurons are inwardly rectifying and calcium permeable. In brain slice recordings from BLA neurons, we demonstrated that topiramate, a widely used antiepileptic agent, selectively and potently inhibits GluR5 kainate receptor mediated synaptic responses. The ability of topiramate to antagonize kainate receptors is intriguing inasmuch as no other clinically used antiseizure medication targets these receptors at therapeutic concentrations. In the present reporting period, we sought to characterize the properties of AMPA receptors in BLA principal neurons with respect inward rectification and presumed calcium permeability. AMPA receptors that lack the GluR2 subunit are inwardly rectifying and are calcium permeable. We used immunoelectron microscopy to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor-mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled. Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Unlike the hippocampus where AMPA receptors onto principal neurons are calcium impermeable, BLA neuron AMPA receptors are similar to GluR5 kainate receptors in their presumed calcium permeability. Thus, AMPA receptors on BLA principal neurons, like the GluR5 kainate receptors on these neurons, could play roles in synaptic plasticity, epileptogenesis and excitoxicity. The unusual properties of AMPA receptors at principal neuron synapses in the amygdala may contribute to the unique epileptic susceptibility of this brain region.