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.

项目总结 这份有指导的职业发展奖提案描述了一个旨在 推动我的职业生涯朝着管理一个由R01资助的独立生物医学研究实验室的目标前进 专注于癫痫的研究。目前，没有办法在高危人群发生癫痫之前预防癫痫 对于难治性癫痫患者，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，成功地学习和应用了 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- 光子钙成像-在体外，然后在清醒的，在体内表现的实验动物。建议数 研究验证了一种假设，即癫痫模型中的大脑回路功能障碍是由于 一种特定亚型的抑制性中间神经元，即快速尖峰细胞(“FS细胞”)的异常活动。这种多式联运 对癫痫的电路水平机制的分析将产生新的结果，这将有助于发展 以及应用新的治疗策略来预防和治疗癫痫。 应聘者：我目前是北京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内科和神经科学系 宾夕法尼亚大学(UPenn)。我是医学博士兼内科科学家，有很强的 神经科学，在纽约大学贝尔纳多博士的实验室获得生理学和神经科学博士学位 鲁迪。我在宾夕法尼亚州立大学完成了为期五年的儿科神经学临床培训计划，现在请注意 在CHOP的普通神经病学和神经遗传学诊所的癫痫儿童。这项建议是建立在我的 长期以来一直对疾病的神经生物学感兴趣，并对突触抑制和 大脑皮层内的GABA能抑制中间神经元。这个K08奖项将为我提供批判性的培训和 支持确保向独立和长期成就和生产力的成功过渡 神经学家和癫痫领域的学术儿科神经学家。我的目标是成为R01资助的 独立研究人员在小鼠模型上研究癫痫的发展机制 可以转化为和改变病人护理的定向疗法。 环境：我的导师是道格拉斯·库尔特博士，他是癫痫领域的知名研究员，也是先驱 动态成像方法在癫痫发病机制研究中的应用。库尔特博士是 宾夕法尼亚大学CHOP/UPN神经系统功能动态成像中心及其翻译研究 CHOP的癫痫项目；他有多项研究癫痫的RO1奖学金。库尔特博士也有一个很好的履历 指导那些成为癫痫领域领导者的受训学员的记录。他的实验室 位于艾布拉姆森研究大楼，4楼和5楼致力于神经科学 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 为我的事业发展和成功干杯。库尔特博士和我组建了一支杰出的导师团队 指导所建议的学习和我的整体职业发展。我将在这一领域获得成功。 在癫痫神经遗传学临床领域的指导下 他还经营着一家由R01资助的基础神经科学实验室。 培训将在CHOP/UPenn进行，这是一个学术上丰富的神经科学社区，拥有广泛的 科学互动的资源和机会，包括各种可用的课程和 正在进行的神经科学、神经学和癫痫相关研讨会系列。我的职业发展计划包括 在动态成像、光遗传学和动物模型癫痫研究方面的严格培训， 关键学科领域，以及关于如何正确开展科学和管理 研究实验室。这项申请得到了CHOP和CHOP神经病学部的热情支持 宾夕法尼亚大学神经科。 研究：我的初步结果显示，在海马区存在异常的GABA能突触抑制 在一个已建立的颞叶癫痫动物模型中；即所谓的“齿状门”的失败。 与其说是抑制的普遍失败，不如说我已经确定了GABA能抑制的一个定义的子集 癫痫患者齿状回中间神经元活动异常。这项建议将建立在我初步的基础上 数据来检验假设：(1)齿状门的机制基础是前馈抑制 (2)癫痫脑内FS细胞活动的调控 利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 控制条件会重现癫痫回路病理，增强癫痫患者体内这些细胞的活性 大脑会恢复正常的抑制。这些结果将提供有关正常功能的新信息 和突触抑制在齿状回中的作用，以及建立重要的 在颞叶癫痫发病机制中的作用。 这个有指导的职业发展奖最终将使我能够翻译从基础知识中收集到的见解 神经科学研究为癫痫的靶向治疗提供信息和激励未来的尝试 GABA能中间神经元的操纵。