Calcium Signaling in a Model of Temporal Lobe Epilepsy

颞叶癫痫模型中的钙信号传导

• 批准号: 8990193
• 负责人: John A. White
• 金额: $ 2.39万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990193
• 关键词: 3-Dimensional; Achievement; Adult; Afferent Pathways; Agonist; Animal Model; Animals; Anticonvulsants; Astrocytes; Brain; Brain region; Calcium; Calcium Signaling; Cells; Chronic; Coupled; Coupling; Data; Dendrites; Development; Disease; Dyes; Electron Microscopy; Electroporation; Epilepsy; Epileptogenesis; Evaluation; Exhibits; Frequencies; Gap Junctions; Generations; Genome; Glutamates; Hippocampus (Brain); Image; Imaging technology; Kainic Acid; Kainic Acid Receptors; Knowledge; Label; Lead; Mediating; Metabolic; Microscopy; Modeling; Molecular Target; Monitor; Neurons; Outcome; Pathologic; Patients; Population; Prevention; Process; Proteins; Pyramidal Cells; Rattus; Recurrence; Research; Role; Scanning; Seizures; Signal Transduction; Signaling Molecule; Slice; Status Epilepticus; Supporting Cell; Synapses; Temporal Lobe; Temporal Lobe Epilepsy; Testing; Therapeutic; Tissues; Transfection; Transposase; Viral; Work; cell type; in utero; innovation; mature animal; network architecture; neurotransmission; novel; patch clamp; receptor expression; reconstruction; research study; response; transmission process; two-photon

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.

描述（由申请人提供）：颞叶癫痫 (TLE) 是一种破坏性癫痫病，很难用抗惊厥药物控制，通常在中枢神经系统受到初始损伤后发生。为了更好地了解癫痫发生的过程并开发治疗 TLE 的创新治疗方法，我们开发了动物模型，这些模型表现出这种癫痫疾病的一些特征：一段癫痫持续状态 (SE) 时期，这是癫痫发作的初始阶段。对中枢神经系统的损害，不发生癫痫发作的可变潜伏期，以及最终发展为颞叶起源的复发性自发性癫痫发作。最近，我们使用 TLE 的红藻氨酸 (KA) 模型来研究海马体 (HC) 中的“反应性”星形胶质细胞，海马体是已知参与癫痫发作的大脑区域。 KA 诱导的癫痫持续状态 (SE) 后星形胶质细胞之间的间隙连接耦合显着增加。因此，与癫痫发作相关的大脑区域的星形胶质细胞网络结构发生了改变。我们还发现星形胶质细胞在 SE 后表达红藻氨酸受体 (KAR) 亚基，并假设 KAR 的激活可导致钙 (Ca2+) 瞬变，从而诱导信号分子释放，从而调节 HC 中的神经元活动。本申请将使用靶向路径扫描 2 光子显微镜 (TPS) 同时评估从用 KA 治疗诱导 SE 的动物脑切片中获得的大型星形胶质细胞网络中的快速 Ca2+ 瞬变。我们采用子宫内电穿孔将基因编码的 Ca2+ 指示蛋白 (Lck-GCaMP3) 靶向大鼠 HC，以便我们可以使用从成年动物获得的脑切片来确定 1) KAR 的激活是否会在反应性星形胶质细胞网络中诱导体细胞 Ca2+ 信号传导HC 中和 2) 如果 KAR 诱导和/或其他激动剂诱导的 Ca2+ 信号在反应性星形胶质细胞的精细过程中诱导直接影响信号分子的释放从 KA 治疗的大鼠在潜伏期和慢性癫痫期间获得的 HC 脑切片中的网络活动。最后，我们将使用电子显微镜来确定 KA 诱导 SE 后 HC CA1 和 CA3 区三联突触的星形胶质细胞室中 KAR 表达、间隙连接耦合和树突鞘的超微结构是否发生变化。 TPS 与 HC 细胞中 Lck-GCaMP3 稳定表达的结合使用是一项技术成就，将有助于我们了解癫痫持续状态 (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在慢性癫痫中。拟议的实验还将确定病理性胶质细胞/神经元相互作用（结构和功能）如何影响发育和发育过程中的电路活动。 癫痫持续存在。最后，预计所提出的实验将导致识别新的分子靶点，用于治疗、预防和/或治愈这种破坏性癫痫病的创新治疗方法。