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.

项目摘要 该修订的职业发展奖提案描述了一项旨在的综合培训计划 将我的职业发展朝着运行独立R01资助的生物医学研究实验室的目标 专注于情节的研究。目前，没有办法在危险中预防危险的人 癫痫发作的出现，对于医学上顽固性癫痫患者的治疗选择有限。 在我的心理指导下，库尔特博士，我设计了一个培训计划，以成功学习和应用 协调的，强大的最先进技术集，包括电生理学，光遗传学和两种技术 光子钙成像 - 体外，然后在醒着中，在体内表现出实验动物。提议 研究检验了以下假设：癫痫模型中脑电路功能障碍是由于 抑制性中间神经元（快速刺激性细胞）定义的亚型的异常活性（“ FS细胞”）。这个多模式 分析癫痫的电路级机制将产生新的结果，这将有助于发展 并应用新颖的治疗策略来预防和治疗癫痫。 候选人：我目前是儿童医院神经病学系的助理教授 费城（CHOP）和神经病学和神经科学系 宾夕法尼亚大学（Upenn）。我是M.D./ph.d。具有强大背景的身体科学家 神经科学，已获得博士学位。 Bernardo博士实验室的纽约大学生理学和神经科学博士学位 鲁迪。我在Chop/Upenn完成了一项为期五年的儿科神经病学临床培训计划，现在要注意 Chop的一般神经病学和神经遗传学诊所的癫痫儿童这个建议建立在我的 对疾病神经生物学的长期兴趣，并在突触抑制和 大脑皮层中的GABA能抑制性中间神经元。该K08奖将为我提供重要的培训和 支持确保成功过渡到独立性以及长期成就和生产力的支持 癫痫领域的神经科学家和学术儿科医生。我的目标是成为R01资助 独立研究者研究小鼠模型中的发作，以告知机械的发展 可以转化并转化患者护理的定向疗法。 环境：我的心理是道格拉斯·库尔特（Douglas Coulter） 在将动态成像方法应用于癫痫机制的研究中。库尔特博士是 Chop/Upenn和翻译研究的神经系统功能动态成像中心 CHOP的癫痫计划；他有多次研究癫痫的RO1补助金。库尔特博士也有一条强大的曲目 在癫痫领域成为领导者的心理学员的记录。他的实验室 位于艾布拉姆森研究大楼，四楼专门用于神经科学 研究并包括一个非常成功的科学家的合作小组，他们对此感兴趣并承诺 我的职业发展和成功。库尔特博士和我已经建立了一个杰出的心态团队 指导拟议研究和我的整体职业发展的执行。我将在 在埃里克·马什（Eric Marsh）的指导下，癫痫神经遗传学的临床领域 神经遗传学，神经病学部门，在CHOP上，他还经营了R01资助的基本神经科学实验室。 培训将在Chop/Upenn进行，这是一个学术丰富的神经科学社区的培训 科学互动的资源和机会，包括广泛的可用课程和多个 持续的神经科学，神经病学和与癫痫相关的精液系列。我的职业发展计划涉及 在动态成像，光遗传学和动物模型中癫痫的研究中进行了严格的培训 关键主题领域以及如何正确地进行科学并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的神经病学的支持， Upenn神经病学系。 研究：我的初步结果表明，海马中有异常的GABA能突触抑制作用 在临时叶癫痫的良好动物模型中；也就是说，随着所谓的“牙齿门”的失败。 我已经确定了GABA能的定义子集，而不是成为一般的抑制失败 齿状回中的神经元在癫痫中表现出异常活性。该建议将基于我的初步 数据以测试以下假设：（1）齿状门的机械基础是进料向前抑制 特别由快速刺激的中间神经元提供，（2）操纵癫痫大脑中FS细胞活性 使用光遗传学可以重建正常的电路活动。我预测将快速加速细胞的靶向沉默 控制条件将重现癫痫电路病理学并增强这些细胞在癫痫病中的活性 大脑将恢复正常的抑制作用。这些结果将提供有关正常功能的新信息 快速刺激的中间神经元和突触抑制在齿状回中的作用，并确定重要的 临时叶癫痫发病机理的机械贡献。 这个修改的职业发展奖最终将使我翻译从基本的见解中。 神经科学研究为基于癫痫的有针对性治疗的未来动机告知和动机尝试 操纵GABA能中间神经元。