Calcium Signaling in a Model of Temporal Lobe Epilepsy

描述（由申请人提供）：颞叶癫痫（TLE）是一种难以用抗惊厥药物控制的破坏性癫痫发作疾病，通常在CNS初始损伤后发生。为了更好地了解癫痫发生的过程并开发用于管理TLE的创新治疗方法，已经开发了表现出这种癫痫发作障碍的一些特征的动物模型：癫痫持续状态（SE）期，其作为对CNS的初始损伤，可变的潜伏期，在此期间不发生癫痫发作，以及复发的最终发展，源于颞叶的自发性癫痫最近，我们使用红藻氨酸（KA）模型的TLE研究“反应性”星形胶质细胞在海马（HC），一个已知的大脑区域参与癫痫发作的产生。KA诱导的癫痫持续状态（SE）后星形胶质细胞之间的缝隙连接偶联显著增加。因此，星形胶质细胞网络结构在与癫痫发作相关的大脑区域中发生改变。我们还发现，星形胶质细胞表达红藻氨酸受体（KAR）亚基SE后，并假设激活KAR可以导致钙（Ca 2+）瞬变，诱导释放信号分子，调节HC中的神经元活动。本申请将使用靶向路径扫描双光子显微镜（TPS）来同时评估从用KA处理以诱导SE的动物获得的脑切片中的星形胶质细胞的大网络中的快速Ca 2+瞬变。我们采用子宫内电穿孔靶向遗传编码的钙指示蛋白因此，我们可以使用从成年动物获得的脑切片来确定1）KAR的激活是否诱导HC中反应性星形胶质细胞网络中的体细胞Ca 2+信号传导，和2）KAR诱导的和/或其他激动剂诱导的Ca 2+信号传导是否与大鼠HC中的细胞因子（Lck-GCaMP 3）的激活相关。在潜伏期和慢性癫痫期间，反应性星形胶质细胞的精细过程中的信号传导诱导直接影响从KA处理的大鼠获得的HC脑切片中的网络活性的信号传导分子的释放。最后，我们将使用电子显微镜，以确定是否有超微结构的变化，KAR的表达，间隙连接耦合，和树突ensheathment在星形胶质细胞室的三角突触的CA 1和CA 3地区的HC KA诱导SE后。TPS与HC细胞中Lck-GCaMP 3的稳定表达的组合使用是一项技术成就，这将有助于我们理解癫痫持续状态（SE）后星形胶质细胞中KAR表达的功能作用，无论是在潜伏期还是在慢性癫痫中。拟议的实验还将确定病理性神经胶质/神经元相互作用，无论是结构上的还是功能上的，如何影响发育过程中的回路活动， 癫痫的持续性最后，预计所提出的实验将导致识别用于治疗、预防和/或治愈这种破坏性癫痫发作障碍的创新治疗方法的新型分子靶点。

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE), a devastating seizure disorder that is difficult to control with anticonvulsant drugs, often develops following an initia insult to the CNS. In order to better understand the process of epileptogenesis and to develop innovative therapeutic approaches for the management of TLE, animal models have been developed that exhibit some of the hallmarks of this seizure disorder: a period of status epilepticus (SE) which serves as the initial insult to the CNS, a variable latent period during which seizures do not occur, and the eventual development of recurrent, spontaneous seizures of temporal lobe origin. Recently we used the kainic acid (KA) model of TLE to investigate 'reactive' astrocytes in the hippocampus (HC), a brain region known to be involved in seizure generation. There is a significant increase in gap junction coupling between astrocytes following KA-induced status epilepticus (SE). Therefore, the astrocytic network architecture is altered in brain regions associated with seizure generation. We also discovered that astrocytes express kainate receptor (KAR) subunits following SE and hypothesize that activation of KARs can result in calcium (Ca2+) transients that induce the release of signaling molecules that modulate neuronal activity in the HC. The present application will use targeted path scanning 2-photon microscopy (TPS) to simultaneously evaluate rapid Ca2+ transients in large networks of astrocytes in brain slices obtained from animals treated with KA to induce SE. We employ in utero electroporation to target a genetically encoded Ca2+ indicating protein (Lck- GCaMP3) to the rat HC so that we can use brain slices obtained from adult animals to determine 1) if activation of KARs induces somatic Ca2+ signaling in networks of reactive astrocytes in the HC and 2) if KAR- induced and/or other agonist-induced Ca2+ signaling in the fine processes of reactive astrocytes induces the release of signaling molecules that directly influence network activity in HC brain slices obtained from KA- treated rats during both the latent period and chronic epilepsy. Finally, we will use electron microscopy to determine if there are ultrastructura changes in KAR expression, gap junction coupling, and dendritic ensheathment in the astrocyte compartment of the tripartite synapse of the CA1 and CA3 regions of the HC following KA-induced SE. The combined use of TPS with the stable expression of Lck-GCaMP3 in cells of the HC is a technical achievement that will contribute to our understanding of the functional role of KAR expression in astrocytes following status epilepticus (SE), both in the latent period and in chronic epilepsy. The proposed experiments will also determine how pathologic glial/neuronal interactions, both structural and functional, influence circuit activity during the development and persistence of epilepsy. Finally it is anticipated that the proposed experiments will lead to the identification of novel molecular targets for innovative therapeutic approaches for the treatment, prevention, and/or cure of this devastating seizure disorder.