High Resolution Analysis of miR125b in Dendrites via Microfluidic Devices

描述(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(S)也就不足为奇了。由单个神经元的树突在其空间环境中延伸的无数丝状足细胞的相互作用产生了人脑的一系列显着的功能。即使是相邻的丝状足纲，也会遇到不同的局部微环境，并发展出各自的功能。只有通过克服总体平均和用单丝状基分辨率测量分子特征，我们才能理解不同的内在和外在调节因子的相互作用，并解释包括健康和疾病状态的神经功能谱。特别是，在树状乔木的出现和雕刻过程中，对丝状孢子的当地调节器的探测方法的需求尚未得到满足。这一创新方案通过将我们在设计和制造用于超低密度神经元培养的纳升微流控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，满足了这一需求。我们建议使用微流控装置(FD)环境和高分辨率图像分析来探索特定的microRNAs(MiRNAs)在发育中的海马树突起的定位、活性和功能的变化。MiRNAs是一种短的、非编码的RNA，它作为局部蛋白质合成的调节者，特别是在树突形成和神经系统的局部连接过程中。我们的目标是在微通道内控制单个树突的结构和功能。 制造的FDs用于分离单个树枝晶。我们将使用这个系统来定位和影响miR125b在丝状足细胞发育过程中的功能以及对谷氨酸能刺激的反应。这组新颖的研究将解决高分辨率理解miR125b在海马体连接过程中的定位、激活和功能的需要。这一方法将为这一假定的调控因子提供新的见解，为研究miRNA控制单个神经元树突状生成的特性提供新的工具，并有助于修复情感功能障碍、慢性应激、阿尔茨海默病和自闭症模型中的缺陷。

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.