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（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性疾病，通常在中枢神经系统的初始损伤后发展。为了更好地了解癫痫发生的过程，并为TLE的管理开发创新的治疗方法，已经开发了动物模型，显示出这种癫痫发作障碍的一些特征：癫痫持续状态（SE）时期，这是对中枢神经系统的初始损害，癫痫发作不发生的可变潜伏期，以及最终发展为颞叶起源的复发性自发癫痫发作。最近，我们使用TLE的kainic酸（KA）模型来研究海马（HC）中的“反应性”星形胶质细胞，这是一个已知参与癫痫发作的大脑区域。在ka诱导的癫痫持续状态（SE）后，星形胶质细胞之间的间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发生改变。我们还发现星形胶质细胞在SE后表达kainate受体（KAR）亚基，并假设KARs的激活可以导致钙（Ca2+）瞬态，从而诱导释放调节HC中神经元活动的信号分子。目前的应用将使用靶向路径扫描双光子显微镜（TPS）来同时评估大的星形胶质细胞网络中的快速Ca2+瞬态，这些星形胶质细胞是在KA诱导SE的动物脑切片中获得的。我们雇佣在子宫内电穿孔基因编码的Ca2 +指示目标蛋白质(Lck - GCaMP3)老鼠HC,这样我们可以用大脑切片从成年动物获得确定1)如果激活卡尔斯诱发体细胞Ca2 +的反应性星形胶质细胞信号网络HC和2)如果凹地,诱导和/或其他agonist-induced Ca2 +信号反应性星形胶质细胞的精细过程诱发信号分子的释放,直接影响网络活动在大脑HCKA治疗大鼠潜伏期和慢性癫痫的切片。最后，我们将利用电子显微镜来确定ka诱导SE后HC CA1和CA3区三方突触星形细胞间室的KAR表达、间隙连接偶联和树突鞘是否有超微结构变化。将TPS与HC细胞中Lck-GCaMP3的稳定表达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）后星形细胞中KAR表达的功能作用，无论是在潜伏期还是慢性癫痫中。提出的实验也将确定病理胶质/神经元的相互作用，结构和功能，如何影响电路活动在发育和