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

项目摘要 这份指导式职业发展奖提案描述了一个综合培训计划， 推进我的职业生涯的目标是运行一个独立的R 01资助的生物医学研究实验室 专注于癫痫的研究。目前，没有办法在癫痫发作之前预防高危人群的癫痫。 癫痫发作的出现，并且对于患有医学难治性癫痫的患者的治疗选择有限。 在我的导师库尔特博士的指导下，我设计了一个培训计划， 协调，强大的一套国家的最先进的技术-包括电生理学，光遗传学，和两个- 光子钙成像-在体外，然后在清醒，行为实验动物体内。拟议 研究测试了一个假设，即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确定亚型，即快速尖峰细胞（“FS细胞”）的异常活性。这种多式联运 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 以及预防和治疗癫痫的新治疗策略的应用。 候选人：我目前是芝加哥儿童医院神经科的助理教授。 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/博士医学科学家，具有强大的背景， 神经科学博士，在生理学和神经科学从纽约大学在伯纳多博士的实验室 鲁迪我在CHOP/UPenn完成了为期五年的儿科神经病学临床培训课程， 儿童癫痫在一般神经病学和神经遗传学诊所在CHOP。这一建议建立在我的 对疾病的神经生物学的长期兴趣和对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K 08奖项将为我提供关键的培训， 支持确保成功过渡到独立和长期成就和生产力， 神经科学家和学术儿科神经学家在癫痫领域。我的目标是成为R 01资助的 研究小鼠模型癫痫的独立研究者， 导向疗法，可以转化为，并改变，病人护理。 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 宾夕法尼亚大学CHOP神经系统功能动态成像中心和转化研究 CHOP的癫痫计划;他有多个研究癫痫的RO 1赠款。库尔特博士也有一个强大的轨道 指导那些已经成为癫痫领域领导者的学员的记录。他的实验室 位于艾布拉姆森研究大楼，其中4楼和5楼是专门用于神经科学 研究，并包括一个非常成功的科学家谁感兴趣，并致力于合作小组 我的职业发展和成功。库尔特博士和我建立了一个优秀的导师团队， 指导建议的研究和我的整体职业发展的执行。我将掌握 临床领域的癫痫神经遗传学的指导下，埃里克马什，医学博士，哲学博士、科科长 神经遗传学，CHOP神经病学分部，他还经营着一个由R 01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， 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的系列研讨会。我的职业发展计划包括 在动态成像，光遗传学和癫痫动物模型的研究，课程在严格的培训 关键学科领域，以及如何正确进行科学和运行一个 研究实验室。这项申请得到了CHOP神经科的热情支持， 宾夕法尼亚大学神经病学系。 研究：我的初步结果表明，在海马中存在异常的GABA能突触抑制 在一个成熟的颞叶癫痫动物模型中，即所谓的“齿状门”失败。 而不是一个普遍的失败的抑制，我已经确定，一个定义的子集的GABA能抑制 癫痫时齿状回中间神经元异常活动。这一建议将建立在我的初步 数据来检验假设：（1）齿状门的机制基础是前馈抑制 特别是由快速尖峰的中间神经元提供，和（2）操纵癫痫脑中的FS细胞活性 利用光遗传学可以重建正常的电路活动。我预测，靶向沉默的快速尖峰细胞， 对照条件将再现癫痫回路病理学并增强癫痫中这些细胞的活性 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 快速发放的中间神经元和突触抑制在齿状回中的作用，以及建立重要的 颞叶癫痫发病机制的贡献。 这个指导职业发展奖将最终定位我翻译的见解收集到的基本 神经科学研究，以告知和激励未来尝试有针对性的治疗癫痫的基础上， GABA能中间神经元的操纵。

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.