Ion Channels in Epilepsy and as Targets for Antiepilepti

癫痫中的离子通道和抗癫痫靶标

• 批准号: 7143853
• 负责人: MICHAEL A. ROGAWSKI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7143853
• 关键词: AMPA receptors; amygdala; anticonvulsants; calcium flux; disease /disorder model; drug screening /evaluation; electrophysiology; epilepsy; glutamate receptor; hippocampus; immunoelectron microscopy; kainate; laboratory rat; membrane channels; membrane permeability; molecular /cellular imaging; molecular pathology; neural plasticity; neural transmission; neuropharmacology; neuroregulation; pharmacokinetics; synapses; synaptic vesicles; tissue /cell culture; transfection; voltage /patch clamp

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.

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（BLA）神经元的外囊刺激是由红藻氨酸受体介导的含有GluR 5亚基，我们已经表明，这些受体引起一种新的形式的突触可塑性，可以介导某些类型的癫痫发生在杏仁核。BLA神经元中GluR 5红藻氨酸受体产生的突触反应是向内整流和钙渗透的。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GluR 5红藻氨酸受体介导的突触反应。托吡酯拮抗红藻氨酸受体的能力是令人感兴趣的，因为没有其他临床使用的抗癫痫药物在治疗浓度下靶向这些受体。在本报告所述期间，我们试图表征BLA主要神经元中AMPA受体的内向整流和假定的钙渗透性特性。缺乏GluR 2亚基的AMPA受体是内向整流的，并且是钙可渗透的。我们用免疫电镜来确定在何种程度上在大鼠BLA的突触有AMPA受体与GluR 2亚基;为了比较，在海马进行了平行检查。我们还记录了杏仁核脑片，以检查在BLA主神经元的AMPA受体介导的诱发突触电流的电压依赖性。在光镜水平上，GluR 2免疫反应定位于BLA神经元的胞体和近端树突;密集的标记也存在于海马子区CA 1和CA 3的锥体细胞层。在BLA的电子显微照片中，大多数突触是不对称的，具有明显的突触后密度（PSD）。它们含有透明的球形囊泡，与PSD并列，主要位于棘上（86%），表明它们主要与BLA主神经元在一起。只有11%的形态突触在BLA上的突触后元素，显示GluR 2免疫反应，在海马子字段CA 1和CA 3，其中76%和71%的突触后元素被标记。在BLA和海马的突触染色，当它发生时，是专门的突触后，特别是在PSD重。在全细胞电压钳记录，72%的BLA主神经元表现出AMPA受体介导的突触电流引起的外囊刺激，向内整流。虽然BLA主要神经元表达核周和近端树突状GluR 2免疫反应，这些神经元上的突触很少表达GluR 2，和主要神经元的优势有内向整流AMPA介导的突触电流，这表明GluR 2的突触靶向是有限的。与海马体不同，在海马体中，主神经元上的AMPA受体是钙不可渗透的，BLA神经元AMPA受体在其假定的钙渗透性方面与GluR 5红藻氨酸受体相似。因此，AMPA受体的BLA主要神经元上，这些神经元上的GluR 5红藻氨酸受体一样，可以发挥作用的突触可塑性，癫痫和兴奋性。杏仁核中主要神经元突触AMPA受体的不寻常特性可能有助于该脑区独特的癫痫易感性。