Mechanisms Underlying Activity-Dependent Interneuron Development

描述(申请人提供)：此申请表是K99/R00独立之路奖的申请表。我目前是纽约大学医学院菲舍尔实验室的高级博士后研究员，在分子生物学和老鼠遗传学方面有广泛的背景。我的职业发展计划是为了在戈德·菲舍尔博士和贝尔纳多·鲁迪博士的指导下获得电生理学的理论和实践培训。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就业市场并过渡到独立实验室。最后，我将参加课程和研讨会，以发展神经学和神经精神疾病实体的背景知识，目标是提高我在与人类病理学潜在联系的背景下考虑我的研究结果的能力。该奖项的K99部分将在纽约大学医学院的Smilow神经科学项目中进行。这个项目与纽约大学更大的神经科学社区(神经科学中心和Skirball研究所)相结合，将提供一个极好的学术环境，在其中完成我的培训并成功过渡到一个独立的学术职位。科学摘要：最近的实验证据表明，内在遗传程序赋予GABA能中间神经元一个早期的亚型认同。我们还知道，在发育过程中，中间神经元参与相关的网络活动。事实上，我以前的工作表明，Calretinin和Reelin而不是血管活性肠肽中间神经元的径向迁移和形态发育是活动依赖的，这两种主要亚型来自尾侧神经节隆起(CGE)。此外，我们还发现，谷氨酸能驱动对于调节轴突和树突在出生后第一周末的正常发育所需的活动是必不可少的。然而，活动调节神经元间成熟的机制还不完全清楚。这一建议旨在揭示为中间神经元提供板层靶向以及轴突和树突正确形成所需的神经递质的神经元类型的身份(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，这项研究计划的长期目标是描述发育中的中间神经元在整合到皮质回路时的连接模式，并评估神经元活动如何调节这种模式的产生过程(目标3)。在出生后第一周，当中间神经元亚型发生活动依赖的成熟时，皮质中填充着各种神经元群组。在此期间存在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，这是它们形态发育的基础。每个个体队列的谷氨酸释放将被基因阻断，以评估这些群体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显著，可能有助于板层靶向。为了评估GABA在径向迁移中的作用，将从药理上阻断GABA受体(Subaim 1b)。虽然我们以前的实验表明在形态发育过程中需要谷氨酸，但对活性敏感成熟的机制尚不清楚。由于NMDA受体的发育作用，我们的实验将重点研究这些离子亲电受体在中间神经元发育过程中的作用。将评估移除CGE中间神经元中NMDA受体的细胞自主后果。我们的分析还将包括对这些受体下游的信号通路的研究(目标2)。中间神经元经历迁移并形成特殊的形态后，它们整合到皮质回路中。然而，CGE中间神经元特定亚群的突触输入的同一性 都是未知的。单突触病毒示踪技术将与宫内电穿孔技术相结合，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到新生皮质回路的影响(Subaim 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。然而，从这些研究中得出的原则预计也适用于大脑皮层的其他区域。更好地了解神经元间发育和GABA能神经回路在一系列潜在的广泛皮质区域的形成可能有助于我们理解神经元间缺陷被认为在其中发挥作用的疾病的发病机制。此外，本提案中提出的实验方法体现了神经生物学领域内跨学科合作的优势。事实上，我坚信， 来自发育遗传学和电生理学这两个特殊领域的概念方法和实验技术将继续促进我们对中枢神经系统功能和病理学的理解。

DESCRIPTION (provided by applicant): 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 activiy 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 thei 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.