Determining the role of dysregulated GABA uptake by reactive astrocytes in thalamic circuit hyperexcitability and seizures

项目总结与摘要 后天性癫痫可发生在中风或创伤性脑损伤等脑损伤之后，并且 尤其影响老年人和儿童。然而，没有有效的治疗或预防策略。 用于创伤后癫痫(PTE)。寻找治疗靶点的关键是了解癫痫的发生， 从最初的损伤到癫痫发展的潜伏期。反应性星形胶质细胞或星形胶质细胞增多症， 形式是对神经侮辱的反应，与癫痫的发生和顽固性密切相关， 抗药性，癫痫。为了了解反应性星形胶质细胞如何在特定疾病中发挥作用 通过癫痫等回路异常，迫切需要了解它们如何影响复杂的 神经元活动。 丘脑与PTE有关-在皮质损伤和中风后，丘脑成为 过度兴奋，并在癫痫发作前发展为慢性星形胶质细胞增生症。使用病毒模型 我之前描述的选择性诱导的星形胶质细胞增生症，我将研究细胞和环路 反应性星形胶质细胞驱动丘脑回路过度兴奋并使癫痫发作的机制。我的 初步的电生理学和转录学研究表明， 在炎症的背景下，星形胶质细胞功能障碍和神经回路过度兴奋。在这份提案中，我将 检验丘脑反应性星形胶质细胞通过减少GABA值下调GABA摄取的工作假说 GABA转运体，最终导致丘脑皮质神经元紧张性GABA电流增强 导致丘脑回路过度兴奋和癫痫发作。 为了验证这一假设，我将描述双向操纵星形细胞GABA的效果 丘脑皮质神经元对GABA能信号的摄取，体外丘脑内环路的节律发生， 以及清醒、行为正常的小鼠对癫痫的易感性。我将使用双腺相关病毒(AAV)CRISPR- CAS9降低野生型小鼠星形细胞GABA转运体表达的方法(目标1)，以及一种AAV- 介导过表达的方法选择性增强野生型星形胶质细胞GABA转运蛋白的表达 丘脑星形胶质细胞增多症和丘脑皮质兴奋性异常的小鼠(目标2)。 拟议的工作将利用病毒性星形胶质细胞增生症方法与切片相结合的选择性。 以及活体神经回路的电生理学评估。这些结果将阐明我们的基本观点 了解星形细胞GABA摄取和神经回路可塑性。此外，通过调查 反应性星形胶质细胞的功能变化，使病理回路活动和介导癫痫的发生， 拟议的工作将确定干预和预防PTE的潜在治疗靶点。

PROJECT SUMMARY & ABSTRACT Acquired epilepsies can occur following brain lesions such as stroke or traumatic brain injury, and particularly affects elderly people children. However, there is no effective treatment or prevention strategy for post-traumatic epilepsy (PTE). Key to finding therapeutic targets is understanding epileptogenesis, the latent period between the initial injury and the development of epilepsy. Reactive astrocytes, or astrogliosis, form in response to neurological insults, and are strongly associated with epileptogenesis and with intractable, drug-resistant, epilepsies. In order to understand how reactive astrocytes contribute to diseases characterized by circuit abnormalities such as seizures, there is an urgent need to understand how they affect complex neuronal activity. The thalamus has been implicated in PTE—following cortical injuries and stroke, the thalamus becomes hyperexcitable, and develops chronic astrogliosis preceding the onset of seizures. Using a viral model of selectively induced astrogliosis that I previously characterized, I will investigate the cellular and circuit mechanisms by which reactive astrocytes drive circuit hyperexcitability in the thalamus and enable seizures. My preliminary electrophysiological and transcriptomic studies have suggested a direct, mechanistic link between astrocyte dysfunction and neural circuit hyperexcitability, in the context of inflammation. In this proposal, I will test the working hypothesis that thalamic reactive astrocytes downregulate GABA uptake via reduction of GABA transporters, which leads to enhanced tonic GABA currents in thalamocortical neurons, ultimately resulting in thalamic circuit hyperexcitability and seizures. To test this hypothesis, I will characterize the effects of bidirectional manipulation of astrocytic GABA uptake on GABAergic signaling in thalamocortical neurons, rhythmogenesis of the intrathalamic circuit in vitro, and seizure susceptibility in awake, behaving mice. I will use a dual adeno-associated virus (AAV) CRISPR- Cas9 approach to decrease astrocytic GABA transporter expression in wild-type mice (Aim 1), and an AAV- mediated overexpression approach to selectively enhance astrocytic GABA transporter expression in wild-type mice that have thalamic astrogliosis and abnormal thalamocortical hyperexcitability (Aim 2). The proposed work will harness the selectivity of the viral astrogliosis approach in combination with slice and in vivo electrophysiological assessments of neuronal circuits. These results will elucidate our basic understanding of astrocytic GABA uptake and neural circuit plasticity. Furthermore, by investigating the functional changes in reactive astrocytes that enable pathological circuit activity and mediate epileptogenesis, the proposed work will identify potential therapeutic targets to intervene and prevent PTE.