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），这是一种毁灭性的癫痫发作障碍，很难用抗惊厥药控制，通常在对中枢神经系统的初始侮辱后会出现。为了更好地了解癫痫发生的过程并开发了用于管理TLE的创新治疗方法，已经开发出了动物模型，这些模型表现出了这种癫痫发作障碍的某些标志：癫痫持续状态（SE）的时期，是对CNS的最初侮辱，在癫痫发作中最初的侮辱，在癫痫发作期间不再发生可变的潜伏期。最近，我们使用了TLE的Kainic Acid（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）之后，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中，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之后表达海藻酸盐受体（KAR）亚基，并假设KAR的激活会导致钙（Ca2+）瞬变，从而诱导调节HC中神经元活性的信号分子的释放。本应用将使用靶向路径扫描2光子显微镜（TPS）同时评估从用KA处理的动物获得的大脑切片中大型星形胶质细胞网络中的快速Ca2+瞬变，以诱导SE。我们使用子宫电穿孔来靶向遗传编码的Ca2+，表明蛋白质（LCK-GCAMP3）向大鼠HC靶向大鼠HC，以便我们可以使用从成年动物获得的大脑切片来确定1）KARS的激活是否会在HC中引起反应性星形胶质细胞的网络中的Somatic Ca2+信号，并在HC中+////2），以及/2）。反应性星形胶质细胞诱导信号分子的释放，该信号分子直接影响潜在时期和慢性癫痫的HC脑切片中的网络活性。最后，我们将使用电子显微镜来确定KAR表达，间隙连接耦合和树突状宿舍是否存在超构型变化。在HC细胞中，TPS与LCK-GCAMP3的稳定表达的综合使用是技术成就，这将有助于我们在潜在时期和慢性癫痫病中对KAR表达在状态癫痫持续状态（SE）之后的星形胶质细胞中的功能作用。提出的实验还将确定结构性和功能的病理神经胶质/神经元相互作用如何影响发育过程中的电路活性 癫痫的持久性。最后，预计拟议的实验将导致对这种毁灭性癫痫发作疾病的治疗，预防和/或治疗的创新治疗方法的新分子靶标识别。