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。在本报告期间，我们继续研究GluR5盐酸盐受体介导的杏仁核神经传递和突触可塑性机制，杏仁核是动物模型中癫痫发生的关键脑部位，也是人类癫痫发作的常见主要焦点。我们也开始研究GluR5盐酸盐受体在体内大鼠杏仁核和杏仁核切片制备中癫痫发作和癫痫发生中的作用。利用原位杂交组织化学和RT-PCR，我们之前已经证明了GluR5盐酸盐受体mRNA在杏仁核基底外侧的高密度表达。我们现在已经证实了GluR5盐酸盐受体亚基的存在，使用免疫细胞化学的抗体直接针对GluR5亚基的c端区域。免疫电镜显示杏仁核基底外侧有许多突触的标记。这种标记在很大程度上是突触后的，与我们的观察一致，即GluR5盐酸盐受体的突触激活在基底外侧神经元中诱导了快速的兴奋性突触反应。为了确定GluR5盐酸盐受体的编辑程度，从显微切片中提取mRNA，进行RT-PCR，然后用BbVI31限制性内切酶切割。一部分GluR5 mrna未经编辑，表明它们可以形成钙透性盐酸盐受体。因此，来自杏仁核基底外侧主要神经元的全细胞膜片钳记录显示，GluR5盐酸盐受体介导的突触反应是向内矫正的，如果参与反应的GluR5盐酸盐受体未被编辑并且钙可渗透，则可以预期。我们之前观察到GluR5 kainate受体激活诱导了一种新型的NMDA受体独立的持久突触促进，这需要钙通过钙渗透（未编辑的）GluR5 kainate受体进入。这种新的突触可塑性是由外包膜基底外侧杏仁核神经元兴奋性传入的低频激活引起的。这种新颖的突触可塑性的一个独特特征是它不是输入通路特异性的。外囊刺激诱导GluR5盐酸盐受体介导的突触促进导致基底杏仁核明显的非刺激性兴奋输入的强度增强。相反，我们通过场电位和细胞内记录表明，通过高频刺激外囊输入，可以在杏仁核基底外侧神经元中诱导传统形式的长期增强（LTP）；这个LTP是特定于输入的。GluR5盐酸盐受体介导一种新形式的活动依赖性突触增强的证明，提出了GluR5盐酸盐受体可能在癫痫高兴奋性和癫痫发生中发挥作用的可能性，例如发生在对盐酸盐受体激动剂（包括神经毒素kainate和domoate）的反应中。这项工作进一步表明，GluR5盐酸盐受体的药物阻断可能有助于预防和治疗某些形式的癫痫。为了研究GluR5盐酸盐受体选择性激活介导癫痫发生的可能性，我们使用了GluR5激动剂ATPA。在大鼠杏仁核中微量注射ATPA可引起急性癫痫发作。低浓度的ATPA也会引起杏仁核切片细胞外记录的癫痫样放电。这些放电可以通过GluR5盐酸盐受体的十氢异喹诺酮类拮抗剂消除。因此，我们的研究首次证明了GluR5盐酸盐受体的选择性激活可诱导癫痫活动。为了研究这种癫痫样活动的生理基础，我们探讨了ATPA影响基底外侧杏仁核中GABA-A受体介导的突触抑制强度的可能性。在外胶囊刺激引起的场反应记录中，ATPA被发现可以减少GABA-A受体介导的抑制，这是在成对脉冲协议中评估的。此外，在全细胞电压钳记录中，ATPA抑制了局部刺激引起的抑制性突触电流（IPSCs）。我们的结论是，海碱盐受体激活在基底外侧杏仁核中部分通过抑制GABA介导的抑制诱导癫痫样活动。其他兴奋性氨基酸受体的激活，包括AMPA受体，不会引起同样的效果。这些结果可能解释了盐酸盐受体激动剂（如盐酸盐和多磺酸盐）诱导癫痫样活动的独特能力，并表明在某些情况下，盐酸盐受体的选择性拮抗剂可能具有抗惊厥药的活性。

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 neurons by external capsule stimulation is mediated by kainate receptors containing the GluR5 subunit. In the present reporting period, we continued to examine GluR5 kainate receptor mediated neurotransmission and synaptic plasticity mechanisms in the amygdala, a key brain site for epileptogenesis in animal models and a common primary focus for seizures in human epilepsy. We also began studies examining the role of GluR5 kainate receptors in seizures and epileptogenesis in the rat amygdala in vivo and in the amygdala slice preparation. Using in situ hybridization histochemistry and RT-PCR, we had previously demonstrated a high density of GluR5 kainate receptor mRNA expression in the basolateral amygdala. We now have confirmed the presence of GluR5 kainate receptor subunits using immunocytochemistry with an antibody directed against the C-terminal region of the GluR5 subunit. Immunoelectron microscopy demonstrated labeling of many synapses in the basolateral amygdala. The labeling was largely postsynaptic, compatible with our observation that synaptic activation of GluR5 kainate receptors induces a fast excitatory synaptic response in basolateral neurons. To determine the extent of editing of GluR5 kainate receptors, mRNA was extracted from microdissections and subjected to RT-PCR followed by restriction endonuclease cleavage with BbVI31. A proportion of the GluR5 mRNAs were unedited, indicating that they can form calcium permeable kainate receptors. Accordingly, whole cell patch clamp recordings from principal neurons in the basolateral amygdala revealed that GluR5 kainate receptor mediated synaptic responses are inwardly rectifying, as expected if the GluR5 kainate receptors that contribute to the response are unedited and calcium permeable. We previously observed that GluR5 kainate receptor activation induces a novel form of NMDA receptor-independent enduring synaptic facilitation that requires calcium entry through calcium-permeable (unedited) GluR5 kainate receptors. This novel synaptic plasticity is induced by low frequency activation of excitatory afferents to basolateral amygdala neurons in the external capsule. A unique characteristic of this novel synaptic plasticity is that it is not input pathway specific. Induction of GluR5 kainate receptor-mediated synaptic facilitation by external capsule stimulation results in enhanced strength of a distinct non-stimulated excitatory input from the basal amygdala. In contrast, we have shown through both field potential and intracellular recordings that a conventional form of long-term potentiation (LTP) can be induced in basolateral amygdala neurons by high-frequency stimulation of the external capsule input; this LTP is input-specific. The demonstration that GluR5 kainate receptors mediate a novel form of activity-dependent synaptic enhancement raises the possibility that GluR5 kainate receptors could play a role in epileptic hyperexcitability and epileptogenesis, such as occurs in response to kainate receptor agonists including the neurotoxins kainate and domoate. The work further suggests that pharmacological blockade of GluR5 kainate receptors could be useful in the prevention and treatment of some forms of epilepsy. To investigate the possibility that selective activation of GluR5 kainate receptors mediates epileptogeneis, we used the GluR5 agonist ATPA. Microinjection of ATPA into the rat amygdala resulted in acute seizures. ATPA at low concentrations also induced epileptiform discharges recorded extracellularly in the amygdala slice. These discharges could be eliminated by decahydroisoquinolone antagonists of GluR5 kainate receptors. Our studies therefore demonstrate for the first time that selective activation of GluR5 kainate receptors induces epileptic activity. To examine the physiological basis of this epileptiform activity, we explored the possibility that ATPA affects the strength of GABA-A receptor-mediated synaptic inhibition in the basolateral amygdala. In recordings of field responses evoked by external capsule stimulaion, ATPA was found to diminish GABA-A receptor-mediated inhibition as assessed in a paired-pulse protocol. In addition, in whole cell voltage clamp recordings, inhibitory synaptic currents (IPSCs) evoked by local stimulation were depressed by ATPA. We conclude that kainate receptor activation induces epileptiform activity in the basolateral amygdala, in part, by suppressing GABA mediated inhibition. Activation of other excitatory amino acid receptors, including AMPA receptors, does not cause the same effect. These results likely explain the unique ability of kainate receptor agonists, such as kainate and domoate, to induce epileptiform activity and suggest that selective antagonists of kainate receptors could have activity as anticonvulsants under some circumstances.