High Resolution Analysis of miR125b in Dendrites via Microfluidic Devices

描述（由申请人提供）：空间定义的亚细胞异质性决定神经元功能。因此，疾病的起源可以追溯到连接大脑的树突丝状足的异常行为就不足为奇了。由单个神经元树突延伸的无数丝状足在其空间环境中的相互作用产生了人类大脑的显著功能范围。即使是相邻的丝状足也会遇到不同的局部微环境，并发展出独特的功能。只有克服种群的总体平均和测量分子特征与单丝足分辨率，我们才能理解各种内在和外在调节的相互作用，并解释神经功能的频谱，包括健康和疾病状态。特别是，在树突乔木的出现和雕刻过程中，对丝状伪足的局部调节进行探测的方法尚未得到满足。这项创新提案通过将我们在设计和制造超低密度神经元培养的纳米级微流体环境方面的专业知识与我们在神经元细胞生物学方面的专业知识相结合，解决了这一需求。我们建议使用微流体装置（FD）环境和高分辨率图像分析来探测海马树突发育过程中特定microRNAs （miRNAs）的定位、活性和功能变化。mirna是短的非编码rna，作为局部蛋白质合成的调节剂，特别是在树突发生和神经系统的局部布线过程中。我们的目标是控制微通道内单个树突的结构和功能

DESCRIPTION (provided by applicant): Spatially defined sub-cellular heterogeneity determines neuron function. Thus, it is not surprising that disease origins can be traced back to the aberrant behavior(s) of dendritic filopodia that wire the brain. Interactions of the myriad filopodia extended by dendrites of individual neurons in their spatial contexts generate the remarkable range of functionalities of the human brain. Even adjacent filopodia encounter distinct local micro-environments and develop individual functionalities. Only by overcoming ensemble averaging of populations and measuring molecular signatures with single- filopodium resolution can we understand the interplay of the diverse intrinsic and extrinsic regulators, and explain the spectrum of neurological functions encompassing healthy and disease states. In particular, there is an unmet need for ways of probing of local regulators of filopodia during the emergence and sculpting of the dendritic arbor. This innovation proposal addresses this need by integrating our expertise in designing and fabricating nanoliter microfluidic environments for ultra-low density neuronal cultures with our expertise in the cell biology of neurons. We propose to use microfluidic device (¿FD) environments and high resolution image analysis to probe changes in localization, activity, and function of specific microRNAs (miRNAs) in developing hippocampal dendrites. miRNAs are short, non-coding RNAs that act as regulators of local protein synthesis, especially during dendrogenesis and local wiring of the nervous system. Our objective is to control the structure and function of individual dendrites within micro-channels of fabricated ¿FDs to isolate individual dendrites. We will use this system to map and influence miR125b functioning in filopodia during their development and in response to glutamatergic stimulation. This novel set of studies will address the need for understanding with high resolution the localization, activation, and function of miR125b during wiring of the hippocampus. This approach will provide new insights on this putative regulator, new tools for studying properties of miRNA control of dendrogenesis in single neurons, and contribute to effective strategies for restoring defects in models of affective dysfunctions, chronic stress, Alzheimer's disease, and autism.