Ion Channels in Epilepsy and as Targets for Antiepilepti

本项目旨在探索基于抗癫痫药物与神经元离子通道系统相互作用的合理开发新策略。利用细胞电生理记录技术研究了药物对脑切片、培养神经元和转染克隆离子通道亚基基因的异源细胞中神经递质门控和电压激活离子通道的调节作用。在动物模型上进行了相关研究。最近的研究主要集中在盐酸盐型谷氨酸受体上。我们已经证明，外部胶囊刺激在基底外侧杏仁核（BLA）神经元中引起的兴奋性突触反应的一个组成部分是由含有GluR5亚基的海碱盐受体介导的，我们已经证明，这些受体引发了一种新的突触可塑性形式，可以介导杏仁核中某些类型的癫痫发生。GluR5盐酸盐受体在BLA神经元中产生的突触反应是向内矫正和钙渗透的。在BLA神经元的脑切片记录中，我们证明了托吡酯，一种广泛使用的抗癫痫药物，选择性地和有效地抑制GluR5盐酸盐受体介导的突触反应。托吡酯拮抗海因酸盐受体的能力令人感兴趣，因为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。在本报告期间，我们试图表征BLA主神经元中AMPA受体的特性，包括向内矫正和假定的钙通透性。缺乏GluR2亚基的AMPA受体是向内整流的，并且是钙渗透的。我们使用免疫电镜来确定大鼠BLA突触具有GluR2亚基的AMPA受体的程度；为了进行比较，在海马体中进行了平行检查。我们还记录了杏仁核脑切片，以检查AMPA受体介导的BLA主神经元诱发突触电流的电压依赖性特性。光镜下GluR2免疫反应仅限于BLA神经元核周和近端树突；海马CA1和CA3亚区锥体细胞层也存在密集标记。在BLA的电子显微镜下，大多数突触不对称，突触后密度（PSD）明显。它们含有透明的球形囊泡，与PSD相对，主要位于棘上（86%），表明它们主要与BLA主神经元有关。BLA中只有11%的形态学突触连接到显示GluR2免疫反应性的突触后元件上，而海马亚区CA1和CA3中分别有76%和71%的突触后元件被标记。BLA和海马的突触染色，当它发生时，只发生在突触后，在PSD上特别重。在全细胞电压钳记录中，72%的BLA主神经元表现出AMPA受体介导的突触电流，这些突触电流是由外囊刺激引起的，并向内整流。尽管BLA主神经元表达核周和近端树突GluR2免疫反应性，但这些神经元上很少有突触表达GluR2，而且多数主神经元具有向内校正ampa介导的突触电流，这表明GluR2靶向突触受到限制。与海马不同的是，在海马中，主要神经元上的AMPA受体是钙不渗透性的，而BLA神经元的AMPA受体在其假定的钙渗透性方面与GluR5盐酸盐受体相似。因此，BLA主神经元上的AMPA受体与这些神经元上的GluR5盐酸盐受体一样，可能在突触可塑性、癫痫发生和兴奋性中发挥作用。杏仁核主要神经元突触AMPA受体的不寻常特性可能导致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.