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: 8439602
• 负责人: John A. White
• 金额: $ 36.88万
• 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/8439602
• 关键词: 3-Dimensional; Achievement; Adult; Afferent Pathways; Agonist; Animal Model; Animals; Anticonvulsants; Astrocytes; Brain; Brain region; Calcium; Calcium Signaling; Cells; Chronic; Computer Architectures; 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; Photons; 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; neurotransmission; novel; patch clamp; receptor expression; reconstruction; research study; response; transmission process

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. PUBLIC HEALTH RELEVANCE: Temporal lobe epilepsy is a seizure disorder with devastating effects, particularly in the large number of patients for whom current treatments are ineffective. The purpose of the proposed work is to use ground- breaking imaging technology coupled with genetically encoded calcium indicating proteins to study this disease in interacting networks of both nerve cells and glial metabolic support cells. A likely outcome of the proposed work will be entirely new ways to assess potential pharmacological therapies for epilepsy.

描述(申请人提供)：颞叶癫痫(TLE)是一种破坏性的癫痫，很难用抗惊厥药物控制，通常发生在中枢神经系统的初始损伤之后。为了更好地了解癫痫的发生过程和开发治疗TLE的创新方法，已经建立了显示这种癫痫障碍的一些特征的动物模型：作为对中枢神经系统的初始侮辱的癫痫持续状态(SE)期，在此期间不发生癫痫发作的可变潜伏期，以及最终发展为源于颞叶的反复自发性癫痫发作。最近，我们使用红藻氨酸(KA)的TLE模型来研究海马区(HC)的“反应性”星形胶质细胞，海马区是已知参与癫痫发生的大脑区域。在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，在与癫痫发作相关的脑区，星形细胞网络结构发生了变化。我们还发现，星形胶质细胞在SE后表达海人酸受体(KAR)亚单位，并假设KAR的激活可以导致钙(Ca~(2+))瞬变，从而诱导调节HC神经元活动的信号分子的释放。目前的应用将使用定向路径扫描双光子显微镜(TPS)来同时评估从KA诱导SE的动物获得的脑片中大型星形胶质细胞网络中的快速钙瞬变。我们利用宫内电穿孔技术将基因编码的钙指示蛋白(LCK-GCaMP3)靶向大鼠HC，这样我们就可以使用从成年动物获得的脑片来确定1)KAR的激活是否诱导HC反应性星形胶质细胞网络的体细胞钙信号转导；2)KAR诱导和/或其他激动剂诱导的反应性星形胶质细胞精细过程中的钙信号是否诱导信号分子的释放，从而直接影响KA治疗大鼠的HC脑片在潜伏期和慢性癫痫期间的网络活动。最后，我们将使用电子显微镜来确定在KA诱导的SE后，在HC的CA1和CA3区的三部分突触的星形胶质细胞间是否存在KAR表达、缝隙连接偶联和树突被膜的超微结构变化。TPS与LCK-GCaMP3在HC细胞中的稳定表达相结合是一项技术成就，将有助于我们理解KAR在癫痫持续状态(SE)潜伏期和慢性癫痫中的功能作用。拟议的实验还将确定病理性神经胶质/神经元的相互作用，包括结构和功能，如何影响发育和 顽固性癫痫。最后，预计拟议的实验将导致为治疗、预防和/或治愈这种毁灭性癫痫障碍的创新治疗方法确定新的分子靶点。 公共卫生相关性：颞叶癫痫是一种癫痫发作障碍，具有毁灭性的影响，特别是在目前治疗无效的大量患者中。这项拟议工作的目的是使用突破性的成像技术，结合遗传编码的钙指示蛋白，在神经细胞和神经胶质代谢支持细胞的相互作用网络中研究这种疾病。这项拟议工作的一个可能结果将是评估癫痫潜在药物疗法的全新方法。