Mechanisms Underlying Activity-Dependent Interneuron Development

项目概述：本申请是K99/R00独立之路奖的申请书。我目前是一名 我和纽约大学医学院菲舍尔实验室的高级博士后研究员有广泛的背景 在分子生物学和小鼠遗传学方面。我的职业发展计划旨在获得理论和 在戈德·菲舍尔博士和贝尔纳多·鲁迪博士的指导下进行电生理学实践培训。此外, 发展计划的目的是加强我的陈述、赠款撰写和教学技能。一个 博士后咨询委员会(PAC)将监督我的进展，并评估我是否准备好进入这份工作 并向一个独立的实验室过渡。最后，我将参加课程和研讨会，以 发展神经和神经精神疾病实体的背景，目标是改善我的 能够在与人类病理学的潜在联系中考虑我的研究结果。K99部分 该奖项将在纽约大学医学院的Smilow神经科学项目中进行。这 计划，与纽约大学更大的神经科学社区(神经科学和 Skirball学院)，将提供一个极好的学术环境，在其中完成我的培训和 成功过渡到独立的学术地位。 科学摘要：最近的实验证据表明，内在的遗传程序赋予 具有早期亚型特征的GABA能中间神经元。我们还知道中间神经元参与了 发展过程中相互关联的网络活动。事实上，我之前的工作表明，径向迁徙 Calretinin和Reelin的形态发育，但不包括血管活性肠肽中间神经元。 主要亚型来源于尾神经节隆起(CGE)，是活动依赖的。此外，我们 已经发现谷氨酸能驱动对于调节正常发育所需的活动是必不可少的 轴突和树突接近出生后第一周的末期。然而，通过这些机制 活性对神经元间成熟的调节作用尚不完全清楚。 这一提议旨在揭示为中间神经元提供 板层靶向以及轴突和树突的正常形成所需的神经递质(目标1)。 此外，该项目将探索谷氨酸受体在形态发育中的作用(目标2)。 最后，这项研究计划的一个长期目标是描述发育中的中间神经元的连接模式 当它们整合到大脑皮层回路中，并评估神经元活动如何调节这一过程 生成此模式(目标3)。 在出生后第一周，当活动依赖于大脑皮质时，各种神经元群会聚集在皮质中。 中间神经元亚型成熟。在这段时间内出现的谷氨酸能细胞群包括 Cajal-Retzius细胞、谷氨酸能瞬时细胞、亚板细胞和锥体细胞。由于它们的空间和 时间分布，这些队列非常适合为中间神经元提供谷氨酸能驱动，即 是它们形态发育的基础。从每个个体队列中释放的谷氨酸将是 以评估这些种群对神经元间成熟的影响(Subaim 1a)。 GABA能传递在皮质发育的早期阶段也很突出，可能有助于 层流靶向。为了评估GABA在径向迁移中的作用，将阻断GABA受体 药理(苏巴伊姆1b)。虽然我们之前的实验表明，人体对谷氨酸的需求 形态发育，活性敏感成熟的机制还不清楚。 由于nmda受体的发育作用，我们的实验将集中在这些亲离子的研究上。 中间神经元发育过程中的受体。NMDA受体去除后的细胞自主效应 将对CGE中间神经元进行评估。我们的分析还将包括对信号通路运行的研究 这些受体的下游(目标2)。中间神经元经历迁移和发育后的特征 形态，它们整合到大脑皮层回路中。然而，突触输入到特定子集的身份 CGE中间神经元是未知的。单突触病毒跟踪技术将与IN结合使用 子宫电穿孔以揭示成熟中间神经元的连接模式(Subaim 3a)。此外, 我们的实验将评估扰动神经元活动对中间神经元整合的影响。 新生皮质环路(苏巴伊姆3b)。 这项拨款提案中的实验将在小鼠的体感皮质中进行活体实验。这个 然而，从这些研究中得出的原理预计将适用于大脑皮层的其他区域，如 井。更好地理解潜在的神经元发育和GABA能回路的形成 广泛的皮质区域可能有助于我们理解以下疾病的发病机制 神经元间缺陷被认为起了一定的作用。此外，本文中介绍的实验方法 该提案体现了神经生物学领域内跨学科合作的优势。事实上，它 我坚信，概念方法和实验技术的结合 特定的子领域，发育遗传学和电生理学，将继续推进我们的理解 中枢神经系统功能和病理的关系。

Project Summary: This application is for the K99/R00 Pathway to Independence award. I am currently a senior postdoctoral fellow in the Fishell lab at the NYU-School of Medicine and I have an extensive background in molecular biology and mouse genetics. My career development plan is designed to acquire theoretical and practical training in electrophysiology under the guidance of Drs. Gord Fishell and Bernardo Rudy. In addition, the development plan is aimed at strengthening my presentation, grant-writing and teaching skills. A postdoctoral advisory committee (PAC) will oversee my progress and assess my readiness to enter the job market and make the transition to an independent laboratory. Finally, I will take courses and workshops to develop a background in neurological and neuropsychiatric disease entities with the goal of improving my ability to consider my research findings in the context of potential links to human pathologies. The K99 portion of the award would take place within the Smilow Neuroscience Program at NYU-School of Medicine. This program, in combination with the larger Neuroscience community at NYU (Center for Neural Science and Skirball Institute), will provide a superb academic environment in which to complete my training and successfully transition to an independent academic position. Scientific Abstract: Recent experimental evidence has revealed that intrinsic genetic programs endow GABAergic interneurons with an early subtype identity. It is also known that interneurons participate in correlated network activity during development. Indeed, my previous work indicates that the radial migration and morphological development of calretinin and reelin but not vasoactive intestinal peptide interneurons, the major subtypes derived from the caudal ganglionic eminence (CGE), are activity-dependent. Furthermore we have found that glutamatergic drive is essential for mediating the activity required for the proper development of axons and dendrites towards the end of the first postnatal week. However, the mechanisms by which activity regulates interneuron maturation are not fully understood. This proposal is aimed at revealing the identity of the neuronal types that provide interneurons with the neurotransmitters necessary for laminar targeting, and for the proper formation of axons and dendrites (Aim 1). In addition, this project will explore the role of glutamate receptors in morphological development (Aim 2). Finally, a long-term aim of this research plan is to describe the connectivity pattern of developing interneurons as they integrate into cortical circuits, and to assess how neuronal activity may regulate the process by which this pattern is generated (Aim 3). A variety of neuronal cohorts populate the cortex during the first postnatal week, when activity-dependent maturation of interneuron subtypes takes place. Glutamatergic cell cohorts present during this time include Cajal-Retzius cells, glutamatergic transient cells, subplate cells and pyramidal cells. Due to their spatial and temporal distribution, these cohorts are well suited to provide interneurons with the glutamatergic drive that is fundamental for their morphological development. Glutamate release from each individual cohort will be genetically blocked to assess the impact of these populations on interneuron maturation (Subaim 1a). GABAergic transmission is also prominent at early stages of cortical development and may contribute to laminar targeting. To assess the role of GABA in radial migration, GABA receptors will be blocked pharmacologically (Subaim 1b). While our previous experiments have indicated a requirement for glutamate in morphological development, the mechanism responsible for activity-sensitive maturation is not understood. Due to the developmental role of NMDA receptors, our experiments will focus on the study of these ionotropic receptors during interneuron development. The cell-autonomous consequences of NMDA receptor removal in CGE interneuron will be assessed. Our analysis will also include the study of the signaling pathways operating downstream of these receptors (Aim 2). After interneurons undergo migration and develop characteristic morphologies, they integrate into cortical circuits. However, the identity of synaptic inputs to specific subsets of CGE interneurons are unknown. Monosynaptic viral tracing techniques will be used in combination with in utero electroporation to reveal the pattern of connectivity of maturing interneurons (Subaim 3a). In addition, our experiments will assess the impact of perturbing neuronal activity on the integration of interneurons into nascent cortical circuits (Subaim 3b). The experiments in this grant proposal will be carried out in vivo in the mouse somatosensory cortex. The principles that will emerge from these studies, however, are expected to apply to other regions of the cortex as well. A better understanding of interneuron development and GABAergic circuit formation over a potentially broad set of cortical areas is likely to contribute to our understanding of the pathogenesis of diseases in which interneuron defects are thought to play a role. In addition, the experimental approach presented in this proposal exemplifies the advantage of interdisciplinary collaboration within the field of neurobiology. Indeed, it is my conviction that the integration of both the conceptual approach and experimental techniques from two particular subfields, developmental genetics and electrophysiology, will continue to advance our understanding of CNS function and pathology.