Tonic Inhibition in Thalamocortical Network Function and Absence Epilepsy

描述(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y2亚基(Y2R43Q)的精氨酸到谷氨酰胺的替代，这会导致杂合子人类患者的儿童期失神癫痫。此外，杂合表达y2R43Q的敲入小鼠(即RQ小鼠)表现出失神样行为抑制，同时伴有广泛的脑电棘波放电，这两者都被抗失神药物阻断。在异源表达系统中，y2R43Q严重干扰受体的组装或运输，导致预测在受影响的个体中突触抑制应该受到影响。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)中只显示出细微的变化。有趣的是，我们最近发现，由内源性环境中的GABA激活的非突触“紧张性”抑制电流在RQ小鼠的皮层和丘脑的兴奋细胞中完全消失，这导致我们假设这种丧失与它们的癫痫表型缺失有关。然而，这一预测与Crunelli和他的同事的结论相反，他们的结论是“失神发作的产生需要加强的紧张性抑制”。综上所述，这些看似不同的发现导致了一种混合假说，即紧张性抑制必须保持在最佳设定点附近，以允许正确调节丘脑皮质功能。因此，我们的总体假设是，失神发作是由紧张性抑制的失调引起的，无论是过多还是过少，都会使回路偏向失神发作。这项研究的长期目标是a)了解紧张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工具。我们将在丘脑脑片上使用膜片钳和多电极记录，并在体内进行连续的视频/脑电/肌电监测，以确定野生型和突变型细胞、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，使用针对丘脑和皮质中的紧张性抑制素的药物，将突变的特性恢复到它们的野生型水平。我们的具体目标是：目标1-紧张性抑制如何调节固有的和突触驱动的兴奋性？目的2-紧张性抑制如何调节丘脑皮质网络的功能连通性？目标3-紧张性抑制如何调节行为状态，包括失神、癫痫和睡眠？

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?