Tonic Inhibition in Thalamocortical Network Function and Absence Epilepsy

描述（由申请人提供）：GABAA受体是中枢神经系统中主要的抑制性神经递质受体。这种受体的突变与遗传性癫痫有关。其中研究得最好的是在y2亚基（y2R43Q）上的精氨酸-谷氨酰胺替代，它会导致杂合人类患者的儿童期缺失癫痫。此外，杂合表达y2R43Q的敲入小鼠（即RQ小鼠）表现出缺失样的行为逮捕，同时伴有广泛性脑电图峰波放电，这两种情况都被抗缺失药物阻断。在异源表达系统中，y2R43Q严重干扰受体组装或运输，导致预测受影响个体的突触抑制应该受到损害。然而，与这些预测形成鲜明对比的是，RQ小鼠在抑制性突触后电流（IPSCs）中仅表现出细微的变化。有趣的是，我们最近发现，由内源性环境GABA激活的非突触性“强直性”抑制电流在RQ小鼠皮层和丘脑的兴奋性细胞中完全丢失，这使我们假设这种丢失是导致它们没有癫痫表型的原因。然而，这一预测与Crunelli及其同事的结论相反，后者认为“癫痫发作的产生需要增强强直性抑制”。综合起来，这些看似不同的发现导致了一种混合假说，即强直性抑制必须保持在最佳设定点附近，以允许丘脑皮质功能的正确调节。因此，我们的总体假设是，失神发作是由强直性抑制失调引起的，因此，过多或过少都会使神经回路偏向于失神发作。本研究的长期目标是a)了解强直性抑制在调节丘脑皮质功能中的确切作用，b)确定导致RQ小鼠癫痫发作的丘脑皮质功能变化，以及c)确定可以挽救这些变化并恢复正常功能的药理学工具。我们将使用膜片钳和多电极记录丘脑皮质切片，以及连续的视频/脑电图/肌电图监测，以确定野生型和突变型之间的细胞，网络和行为特性的具体差异。然后，我们将开发药物治疗方法，使用专门针对丘脑和皮层强直性抑制的药物，将突变特性恢复到野生型水平。我们的具体目标是：AIM 1 -强直抑制如何调节内在和突触驱动的兴奋性？强直抑制如何调节丘脑皮质网络的功能连接？AIM 3 -强直抑制如何调节行为状态，包括癫痫发作和睡眠？

DESCRIPTION (provided by applicant): The GABAA receptor is the main inhibitory neurotransmitter receptor in the CNS. Mutations in this receptor are associated with heritable epilepsy. The best studied of these is an arginine-to-glutamine substitution in the y2 subunit (y2R43Q) that confers Childhood Absence Epilepsy in heterozygous human patients. Furthermore, knock-in mice heterozygously expressing y2R43Q (i.e., RQ mice) display absence-like behavioral arrests concurrent with generalized EEG spike-wave discharges, both of which are blocked by anti-absence drugs. In heterologous expression systems, y2R43Q severely interferes with receptor assembly or trafficking, leading to the prediction that synaptic inhibition should be compromised in affected individuals. In sharp contrast to these predictions, however, RQ mice show only subtle changes in Inhibitory Postsynaptic Currents (IPSCs). Interestingly, we have recently discovered that nonsynaptic "tonic" inhibitory currents, activated by endogenous ambient GABA, are completely lost in excitatory cells of the cortex and thalamus of RQ mice, leading us to hypothesize that such loss is responsible for their absence epilepsy phenotype. This prediction, however, is the opposite of the conclusion, by Crunelli and colleagues, that "enhanced tonic inhibition is required for absence seizure generation". Taken together, these seemingly disparate findings lead to the hybrid hypothesis that tonic inhibition must be maintained near an optimal set point to allow correct regulation of thalamocortical function. Our overall hypothesis is therefore that absence seizures arise from dysregulation of tonic inhibition, such that either too much or too little biases the circuit towards absence seizures. The long term goals of this research are a) to understand the precise role of tonic inhibition in regulating thalamocortical function, b) to identify the changes in thalamocortical function that lead to seizures in RQ mice, and c) to identify pharmacological tools that can rescue these changes and restore normal function. We will employ patch clamp and multielectrode recordings in thalamocortical slices, and continuous video/ EEG/EMG monitoring in vivo to determine the specific differences between wild type and mutant cellular, network and behavioral properties. We will then develop pharmacotherapeutic approaches to rescue the mutant properties back to their wild type levels using drugs that specifically target tonic inhibiton in thalamus and cortex. Our Specific Aims are: AIM 1 - How does tonic inhibition regulate intrinsic and synaptically driven excitability? AIM 2 - How does tonic inhibition regulate the functional connectivity of the thalamocortical network? AIM 3 - How does tonic inhibition regulate behavioral states, including absence seizures and sleep?