Dynamic two-photon calcium imaging and optogenetic manipulation of epileptic brain circuits in an experimental model of temporal lobe epilepsy

颞叶癫痫实验模型中癫痫脑回路的动态双光子钙成像和光遗传学操作

• 批准号: 9295077
• 负责人: ETHAN M GOLDBERG
• 金额: $ 18.42万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9295077
• 关键词: Achievement; Acute; Affect; American; Animal Model; Animals; Appearance; Area; Award; Biomedical Research; Brain; Calcium; Cell Therapy; Cell physiology; Cells; Cerebral cortex; Cerebrum; Characteristics; Child Care; Childhood; Chronic; Chronic Disease; Clinic; Clinical; Communities; Data; Development; Development Plans; Disease; Doctor of Medicine; Doctor of Philosophy; Electrophysiology (science); Ensure; Environment; Epilepsy; Exhibits; Experimental Models; Failure; Floor; Functional disorder; Funding; Future; Gene Deletion; Glean; Goals; Grant; Head; Hippocampus (Brain); Human; Image; In Vitro; Individual; Interneurons; Intractable Epilepsy; Investigation; K-Series Research Career Programs; Knowledge; Label; Laboratories; Laboratory Research; Learning; Mediating; Medical; Mentors; Mentorship; Methods; Modeling; Mus; Mutation; Nervous System Physiology; Neurobiology; Neurologist; Neurology; Neurons; Neurosciences; Neurosciences Research; Outcome; Pathogenesis; Pathologic; Pathology; Patient Care; Patients; Pediatric Hospitals; Pediatric Neurology; Pennsylvania; Perforant Pathway; Pharmacologic Substance; Philadelphia; Physicians; Physiology; Picrotoxin; Population; Positioning Attribute; Preparation; Preventive Intervention; Productivity; Pyramidal Cells; Recruitment Activity; Research; Research Personnel; Resistance; Resources; Risk; Role; Running; Science; Scientist; Seizures; Series; Slice; Somatostatin; Specificity; Stem cells; Syndrome; Techniques; Temporal Lobe Epilepsy; Testing; Training; Training Activity; Training Programs; Training Support; Translating; Translational Research; United States; Universities; awake; base; calcium indicator; career; career development; cell type; clinical care; cognitive function; dentate gyrus; design; entorhinal cortex; experience; feeding; granule cell; imaging modality; in vivo; insight; interest; medical schools; microendoscope; mouse model; multimodality; neurogenetics; novel; novel therapeutics; optogenetics; pre-clinical; prevent; professor; programs; receptor; reconstitution; response; success; synaptic inhibition; targeted treatment; tool; two-photon

PROJECT SUMMARY This mentored career development award proposal describes an integrated training program designed to advance my career towards the goal of running an independent R01-funded biomedical research laboratory focused on the study of epilepsy. Currently, there is no way to prevent epilepsy in at-risk individuals prior to the appearance of seizures, and there are limited treatment options for patients with medically intractable epilepsy. With the guidance of my mentor, Dr. Coulter, I have designed a training plan to successfully learn and apply a coordinated, powerful set of state-of-the-art techniques – including electrophysiology, optogenetics, and two- photon calcium imaging – in vitro and then in awake, behaving experimental animals in vivo. The proposed research tests the hypothesis that brain circuit dysfunction in a well-established model of epilepsy is due to abnormal activity of a defined subtype of inhibitory interneuron, the fast-spiking cells (“FS cells”). This multimodal analysis of circuit-level mechanisms of epilepsy will yield novel results that will contribute to the development and application of novel therapeutic strategies to prevent and treat epilepsy. Candidate: I am currently Assistant Professor in the Division of Neurology at The Children's Hospital of Philadelphia (CHOP) and Departments of Neurology and Neuroscience at The Perelman School of Medicine at the University of Pennsylvania (UPenn). I am an M.D./Ph.D. physician-scientist with a strong background in neuroscience, having received a Ph.D. in Physiology & Neuroscience from NYU in the laboratory of Dr. Bernardo Rudy. I completed a five-year clinical training program in pediatric neurology at CHOP/UPenn and now take care of children with epilepsy in General Neurology and Neurogenetics Clinic at CHOP. This proposal builds on my long-standing interest in the neurobiology of disease and established interests in synaptic inhibition and GABAergic inhibitory interneurons in the cerebral cortex. This K08 award will provide me with critical training and support to insure a successful transition to independence and long-term achievement and productivity as a neuroscientist and academic pediatric neurologist in the field of epilepsy. My goal is to become an R01-funded independent investigator studying epilepsy in mouse models to inform the development of mechanistically oriented therapies that could be translated to, and transform, patient care. Environment: My mentor is Dr. Douglas Coulter, an established investigator in the field of epilepsy and a pioneer in the application of dynamic imaging methods to the study of epilepsy mechanisms. Dr. Coulter is Director of the Center for Dynamic Imaging of Nervous System Function at CHOP/UPenn and the Translational Research Epilepsy Program at CHOP; he has multiple RO1 grants studying epilepsy. Dr. Coulter also has a robust track record of mentoring trainees who have gone on themselves to be leaders in the field of epilepsy. His laboratory is located in the Abramson Research Building, where the 4th and 5th floors are dedicated to neuroscience research and include a collaborative group of highly successful scientists who are interested in and committed to my career development and success. Dr. Coulter and I have constructed an outstanding mentorship team to guide the execution of the proposed studies and my overall career development. I will attain mastery in the clinical field of epilepsy neurogenetics under the guidance of Eric Marsh, M.D., Ph.D., Head of the Section on Neurogenetics, Division of Neurology, at CHOP, who also runs an R01-funded basic neuroscience laboratory. Training will occur at CHOP/UPenn, an academically enriching neuroscience community with extensive resources and opportunities for scientific interaction, including a wide range of available coursework and multiple ongoing neuroscience-, neurology-, and epilepsy-related seminar series. My career development plan involves rigorous training in dynamic imaging, optogenetics, and the study of epilepsy in animal models, coursework in crucial subject areas, as well as formal and informal training in how to properly conduct science and run a research laboratory. This application is supported enthusiastically by the Division of Neurology at CHOP and Department of Neurology at UPenn. Research: My preliminary results show that there is abnormal GABAergic synaptic inhibition in the hippocampus in a well-established animal model of temporal lobe epilepsy; namely, with failure of the so-called “dentate gate.” Rather than being a general failure of inhibition, I have determined that a defined subset of GABAergic inhibition interneuron in dentate gyrus exhibits abnormal activity in epilepsy. This proposal will build on my preliminary data to test the hypotheses that: (1) the mechanistic basis of the dentate gate is feed-forward inhibition specifically provided by fast-spiking interneurons, and (2) manipulation of FS cell activity in the epileptic brain using optogenetics can reconstitute normal circuit activity. I predict that targeted silencing of fast-spiking cells in control conditions will reproduce epileptic circuit pathology and augmenting the activity of these cells in epileptic brain will recover normal inhibition. These outcomes will provide novel information regarding the normal function of fast-spiking interneurons and role of synaptic inhibition in dentate gyrus, as well as establishing important mechanistic contributions to the pathogenesis of temporal lobe epilepsy. This mentored career development award will ultimately position me to translate the insights gleaned from basic neuroscience research to inform and motivate future attempts at the targeted treatment of epilepsy based on manipulation of GABAergic interneurons.

项目概要 该指导性职业发展奖提案描述了一项综合培训计划，旨在 推动我的职业生涯朝着运行独立的 R01 资助的生物医学研究实验室的目标发展 专注于癫痫研究。目前，尚无任何方法可以在治疗前预防高危人群的癫痫病。 癫痫发作，并且对于医学上难治性癫痫患者来说，治疗选择有限。 在我的导师库尔特博士的指导下，我设计了一个培训计划来成功学习和应用 协调、强大的一套最先进的技术——包括电生理学、光遗传学和两种技术 光子钙成像——在体外进行，然后在清醒、有行为的实验动物体内进行。拟议的 研究测试了这样的假设：在一个完善的癫痫模型中，脑回路功能障碍是由于 抑制性中间神经元的特定亚型，即快速尖峰细胞（“FS 细胞”）的异常活动。这种多式联运 对癫痫的回路水平机制的分析将产生新的结果，这将有助于发展 以及预防和治疗癫痫的新治疗策略的应用。 候选人：我目前是儿童医院神经内科助理教授 费城 (CHOP) 和佩雷尔曼医学院神经病学和神经科学系 宾夕法尼亚大学（UPenn）。我是医学博士/博士。具有深厚医学背景的医师科学家 神经科学，获得博士学位。纽约大学贝尔纳多博士实验室生理学和神经科学博士学位 鲁迪.我在 CHOP/UPenn 完成了为期五年的儿科神经病学临床培训计划，现在正在照顾 CHOP 普通神经病学和神经遗传学诊所的癫痫儿童治疗。这个建议建立在我的 对疾病神经生物学的长期兴趣以及对突触抑制和突触抑制的既定兴趣 大脑皮层中的 GABA 能抑制性中间神经元。这个 K08 奖项将为我提供重要的培训和 支持确保成功过渡到独立以及长期成就和生产力 癫痫领域的神经科学家和学术儿科神经学家。我的目标是成为 R01 资助的 独立研究者研究小鼠模型中的癫痫，以从机制上了解癫痫的发展 可以转化为并改变患者护理的定向疗法。 环境：我的导师是 Douglas Coulter 博士，他是癫痫领域的知名研究者和先驱 动态成像方法在癫痫机制研究中的应用。库尔特博士是 CHOP/宾夕法尼亚大学神经系统功能动态成像中心及转化研究 CHOP 癫痫项目；他拥有多项研究癫痫的 RO1 资助。库尔特博士也有强大的履历 指导学员成为癫痫领域领导者的记录。他的实验室 位于艾布拉姆森研究大楼内，四楼和五楼专门用于神经科学 研究并包括一个由非常成功的科学家组成的合作小组，他们对此感兴趣并致力于 为了我的职业发展和成功。库尔特博士和我组建了一支优秀的导师团队 指导拟议研究的执行和我的整体职业发展。我将精通 在癫痫神经遗传学科主任 Eric Marsh 医学博士、哲学博士的指导下开展癫痫神经遗传学临床领域的研究 CHOP 神经病学部门神经遗传学，他还管理着一个由 R01 资助的基础神经科学实验室。 培训将在 CHOP/UPenn 进行，这是一个学术丰富的神经科学社区，拥有广泛的 科学互动的资源和机会，包括广泛的可用课程和多种 正在进行的神经科学、神经病学和癫痫相关研讨会系列。我的职业发展计划包括 动态成像、光遗传学和动物模型癫痫研究方面的严格培训，课程作业 关键学科领域，以及如何正确进行科学研究和运行的正式和非正式培训 研究实验室。该应用程序得到了 CHOP 神经病学部门的热情支持 宾夕法尼亚大学神经病学系。 研究：我的初步结果显示海马区存在异常的GABA能突触抑制 在成熟的颞叶癫痫动物模型中；即，所谓的“齿状门”失效。 我已经确定 GABA 能抑制的一个确定子集，而不是普遍的抑制失败 齿状回的中间神经元在癫痫中表现出异常活动。该提案将建立在我的初步 数据检验假设：（1）齿状门的机制基础是前馈抑制 由快速尖峰中间神经元专门提供，以及（2）控制癫痫脑中的 FS 细胞活动 使用光遗传学可以重建正常的电路活动。我预测快速尖峰细胞的靶向沉默 控制条件将重现癫痫回路病理学并增强癫痫中这些细胞的活性 大脑将恢复正常的抑制。这些结果将提供有关正常功能的新信息 快速尖峰中间神经元的作用和齿状回突触抑制的作用，以及建立重要的 颞叶癫痫发病机制的机制贡献。 这个受指导的职业发展奖最终将使我能够转化从基本知识中收集到的见解 神经科学研究，以告知和激励未来基于以下基础的癫痫靶向治疗尝试 GABA能中间神经元的操纵。