ION CHANNELS IN EPILEPSY AND AS TARGETS FOR ANTIEPILEPTIC DRUGS

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。在本报告期内，我们继续关注谷氨酸受体和谷氨酸受体介导的突触传递。nmda型谷氨酸受体的功能活性既需要谷氨酸结合，也需要一种内源性粘连剂的结合，这种粘连剂被认为是甘氨酸。然而，其他几种氨基酸，包括d -丝氨酸，当与谷氨酸共同作用时，可以激活NMDA受体。虽然d -氨基酸在细菌中很重要，但通常认为它们不会出现在哺乳动物中。然而，最近在哺乳动物大脑中发现了高水平的d -丝氨酸，这表明该氨基酸可能是NMDA受体的内源性凝血剂。我们观察到d -氨基酸氧化酶，一种选择性降解d -丝氨酸的酶，在培养的海马神经元中使用全细胞膜片钳记录来评估，极大地减弱了NMDA受体介导的神经传递。外源性d -丝氨酸完全逆转了该酶的抑制作用，而d -丝氨酸本身并不增强NMDA受体介导的突触反应。这些观察结果表明，d -丝氨酸是NMDA受体甘氨酸位点的内源性调节剂，它可能完全占据某些功能突触的甘氨酸位点。修饰d -丝氨酸代谢酶活性的药物，包括最近克隆的哺乳动物丝氨酸消旋酶，可能有助于减少癫痫中可能发生的NMDA受体的过度激活。一些广泛使用的抗癫痫药物被认为是通过使用依赖性阻断电压激活钠通道起作用的。然而，钠通道调制防止癫痫发作的方式尚不清楚。我们检验了钠通道阻断抗惊厥药的假设，包括苯妥英和唑尼沙胺，通过抑制突触谷氨酸释放起作用。利用全细胞电压钳技术记录了AMPA受体介导的大鼠海马CA1锥体神经元自发和诱发突触电流。临床相关浓度的唑昔亚胺诱导自发性兴奋性突触电流的频率和幅度大幅降低，而不改变其动力学特性。相比之下，小型兴奋性突触后电流的振幅和频率不受药物影响，表明唑尼亚胺在突触前起作用。唑尼亚胺对辐射层低频刺激引起的兴奋性突触电流影响最小。然而，高频率刺激对兴奋性递质的释放有明显的使用依赖性抑制作用。广泛使用的钠通道阻断抗惊厥药苯妥英也有类似的作用。我们的结论是，钠通道阻断抗惊厥药通过影响谷氨酸释放机制来减少谷氨酸介导的兴奋性突触传递。研究还在继续检查谷氨酸受体介导的神经传递和杏仁核中的突触可塑性机制，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。我们之前已经证明，外包膜刺激引起的杏仁核基底外侧神经元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。此外，我们观察到海碱盐受体激活诱导了一种新型的NMDA受体不依赖的持久突触促进。我们现在使用原位杂交组织化学证明GluR5盐酸盐受体mRNA在杏仁核基底外侧高水平表达；其他盐酸盐受体亚基mrna表达水平较低。GluR5盐酸盐受体可能是钙渗透的。为了验证钙通过GluR5盐酸盐受体进入介导GluR5盐酸盐受体依赖的持久突触促进的假设，我们用膜渗透钙螯合剂BAPTA-AM处理杏仁核切片。暴露于BAPTA-AM的切片未显示GluR5盐酸盐受体介导的突触可塑性，尽管突触反应在很大程度上未受影响。Fura-2成像证实BAPTA-AM导致细胞内钙的大量减少。我们得出结论，GluR5含有海因酸盐受体介导一种新型的钙依赖性持久突触促进。我们提出GluR5盐酸盐受体可以作为癫痫高兴奋性和癫痫发生的介质，例如发生在对盐酸盐受体激动剂（包括神经毒素盐酸盐和domoate）的反应中，我们进一步建议阻断GluR5盐酸盐受体的药物可能有助于预防和治疗某些形式的癫痫。

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. In the present reporting period, we continued our focus on glutamate receptors and glutamate receptor-mediated synaptic transmission. Functional activity of NMDA-type glutamate receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine. However, several other amino acids including D-serine can activate NMDA receptors when coapplied with glutamate. Although D-amino acids are prominent in bacteria, they generally are thought not to occur in mammals. However, high levelsof D-serine have recently been found in mammalian brain, suggesting that the amino acid could serve as an endogenous coagonist for NMDA receptors. We observed that D-amino acid oxidase, an enzyme that selectively degrades D-serine, greatly attenuates NMDA receptor-mediated neurotransmission as assessed using whole-cell patch-clamp recordings in cultured hippocampal neurons. The inhibitory effects of the enzyme were fully reversed by exogenously applied D-serine which did not by itself potentiate NMDA receptor-mediated synaptic responses. These observations indicate that D-serine is an endogenous modulator at the glycine site of NMDA receptors and that it may fully occupy this site at some functional synapses. Agents that modify the activity of D-serine metabolic enzymes, including the recently cloned mammalian serine racemase, could be useful to diminish excessive activation of NMDA receptors as may occur in epilepsy. Several widely used antiepileptic drugs are believed to act via use-dependent block of voltage-activated sodium channels. However, the way in which sodium channel modulation protects against seizures is not well understood. We examined the hypothesis that sodium channel blocking anticonvulsants, including phenytoin and zonisamide, act through inhibition of synaptic glutamate release. AMPA receptor-mediated spontaneous and evoked synaptic currents were recorded in CA1 pyramidal neurons of the rat hippocampal slice using whole-cell voltage clamp techniques. Clinically-relevant concentrations of zonisimide induced a large reduction in the frequency and amplitude of spontaneous excitatory synaptic currents without altering their kinetic properties. In contrast, the amplitude and frequency of miniature excitatory postsynaptic currents was unaffected by the drug, indicating that zonisimide acts presynaptically. Zonisimide had minimal effects on excitatory synaptic currents evoked by low frequency stimulation of the stratum radiatum. However, with high frequency stimulation there was a dramatic use-dependent inhibitory action on the release of excitatory transmitter. The widely used sodium channel blocking anticonvulsant phenytoin had similar actions. We conclude that sodium channel blocking anticonvulsants have a common action to reduce glutamate-mediated excitatory synaptic transmission through effects on glutamate release mechanisms.Studies were also continued examining glutamate 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 previously demonstrated that a component of the excitatory synaptic response evoked in basolateral amygdala neurons by stimulation of the external capsule is mediated by kainate receptors containing the GluR5 subunit. Moreover, we observed that kainate receptor activation induces a novel form of NMDA receptor-independent enduring synaptic facilitation. We now demonstrate using in situ hybridization histochemistry that GluR5 kainate receptor mRNA is expressed at high levels in the basolateral amygdala; other kainate receptor subunit mRNAs were expressed at lower levels. GluR5 kainate receptors may be calcium permeable. To examine the hypothesis that calcium entry via GluR5 kainate receptors mediates GluR5 kainate receptor-dependent enduring synaptic facilitation, amygdala slices were treated with the membrane permeant calcium chelator BAPTA-AM. Slices exposed to BAPTA-AM failed to show GluR5 kainate receptor-mediated synaptic plasticity although synaptic responses were largely unaffected. Fura-2 imaging confirmed that BAPTA-AM produced a substantial reduction in intracellular calcium. We conclude that GluR5 containing kainate receptors mediate a novel form of calcium-dependent enduring synaptic facilitation. We propose that GluR5 kainate receptors could serve as mediators of epileptic hyperexcitability and epileptogenesis, such as occurs in response to kainate receptor agonists including the neurotoxins kainate and domoate, and we further suggest that drugs that block GluR5 kainate receptors could be useful in the prevention and treatment of some forms of epilepsy.