Analysis of activity-dependent interactions between microglia and synapses

7.项目摘要/摘要 为了在成熟的神经系统中实现突触电路的精致精确特性， 未成熟的突触形成一个粗略的线路图，在出生后的发育过程中必须重塑。虽然它是 明确神经活动驱动发育性突触重塑，其潜在机制尚不完全 明白了。这份K99/R00计划将支持我的职业发展，因为我正在研究潜在的蜂窝和 在发育中的大脑中驱动活性依赖性突触重构的分子机制。 我已经确定，小胶质细胞，驻留在中枢神经系统的免疫细胞，吞噬突触元素 对神经活动变化作出反应的出生后大脑。这些数据提出了一个耐人寻味的可能性 小胶质细胞是一种驱动活性依赖性突触重构的细胞机制。然而，它是未知的 无论是小胶质细胞主动吞噬完整突触还是被动吞噬突触残留物。在……里面 此外，依赖活性的小胶质细胞-突触相互作用背后的分子机制是 未知。这些问题将在波士顿儿童基金会的指导阶段得到解答 在贝丝·史蒂文斯博士和迈克尔·格林伯格博士的指导下，医院和哈佛医学院进行了合作。目标 1旨在测试小胶质细胞正在主动吞噬完整突触的假说。 神经活动的变化。在这样做的过程中，我将学习病毒介导的基因传递来荧光标记特定的 神经回路和双光子活体成像，实时分析小胶质细胞-突触相互作用。目标2是 旨在研究这些相互作用背后的分子机制。在遗传学和蛋白质组学之后 筛选，我已经确定了一个候选者，白介素12(IL-12)。我将测试IL-12调节的假设 IL-12和IL-12受体KO小鼠体内成像的活性依赖的小胶质细胞-突触相互作用。 具体目标3(将在我自己的实验室完成)将确定活动的功能意义- 依赖的小胶质细胞-突触相互作用通过检验这些相互作用调节 突触回路的功能发育。我将在一段特定的时间内从基因上去除小胶质细胞 出生后发育和评估突触结构和生理。为了更多地操纵小胶质细胞的功能 具体地说，我还将测试IL-12途径的作用。此外，作为替代和/或未来的方向， 我的基因和蛋白质组筛查已经确定了我将测试的其他几个假定的分子途径。 鉴于异常的突触回路和小胶质细胞功能障碍现在已经与几个 对于神经发育和精神障碍，这一提议将具有广泛的影响。我长期的职业生涯 作为一名独立研究员，我的目标是将我的发现转化为了解小胶质细胞和其他胶质细胞如何 类型可能导致与自闭症和其他疾病相关的突触回路异常 精神分裂症。这个奖项将帮助我完成培训，并提供一个学习技术的机会 并为成功的R01申请获得数据，为我未来的工作提供资金。

7. Project Summary/Abstract To achieve the exquisite precision characteristic of synaptic circuits in the mature nervous system, immature synapses form a crude wiring diagram that must remodel during postnatal development. While it is clear that neural activity drives developmental synaptic remodeling, the underlying mechanisms are not fully understood. This K99/R00 proposal will support my career development as I investigate underlying cellular and molecular mechanisms driving activity-dependent synaptic remodeling in the developing brain. I have firmly established that microglia, the resident CNS immune cells, phagocytose synaptic elements in the postnatal brain in response to changes in neural activity. These data raise the intriguing possibility that microglia are a cellular mechanism driving activity-dependent synaptic remodeling. However, it is unknown whether microglia actively phagocytose intact synapses or passively phagocytose synaptic remnants. In addition, the molecular mechanisms underlying activity-dependent microglia-synapse interactions are unknown. These questions will be addressed during the mentored phase of the award at Boston Children's Hospital and Harvard Medical School under the guidance of Dr. Beth Stevens and Dr. Michael Greenberg. Aim 1 is designed to test the hypothesis that microglia are actively phagocytosing intact synapses in response to changes in neural activity. In doing so, I will learn viral-mediated gene delivery to fluorescently label specific neural circuits and 2-photon in vivo live imaging to analyze microglia-synapse interactions in real time. Aim 2 is designed to investigate molecular mechanisms underlying these interactions. After genetic and proteomic screens, I have identified a candidate, interleukin 12 (IL-12). I will test the hypothesis that IL-12 regulates activity-dependent microglia-synapse interactions by in vivo imaging in IL-12 and IL-12 receptor KO mice. Specific Aim 3 (to be completed in my own laboratory) will determine the functional significance of activity- dependent microglia-synapse interactions by testing the hypothesis that these interactions regulate the functional development of synaptic circuits. I will genetically ablate microglia during a specific window of postnatal development and assess synapse structure and physiology. To manipulate microglia function more specifically, I will also test the role of the IL-12 pathway. Furthermore, as an alternative and/or future direction, my genetic and proteomic screens have identified several other putative molecular pathways that I will test. Given that aberrant synaptic circuits and microglial dysfunction have now been linked with several neurodevelopmental and psychiatric disorders, this proposal will have broad implications. My long term career goal as an independent investigator is to translate my findings to understand how microglia and other glial cell types may contribute to synaptic circuit abnormalities associated with disorders such as autism and schizophrenia. This award will help me to complete my training and provide an opportunity to learn techniques and obtain data for a successful R01 application to fund my future work.