A Controllable Microfludic Gradient Device for Studying Neuronal Polarization

这个R21提案的总体目标是一个用于研究神经元极化的可控微流控梯度装置，是为了展示和探索一种新型微流控浓度梯度发生器的能力，因为它与发育和再生神经生物学有关。该装置由一系列通过微通道相互连接的交替细胞培养室和试剂通道组成，在用于神经元培养的静态细胞培养室中建立并维持稳定的浓度梯度。小分子（药物制剂和第二信使）和大分子（神经营养素和其他蛋白质）的浓度梯度在该装置中很容易实现和量化。因此，可以完成神经元极化和轴突寻路对微环境信号响应的定量研究。将追求三个具体目标。具体目标1涉及优化所提出的微流体装置的设计，以及它的制造和测量。具体目标2和3侧重于评估神经元极化和轴突寻路的四种假设，这些假设目前无法使用当前的协议进行量化。具体而言，Specific Aim 2涉及评估胚胎大鼠海马神经元在响应设备中建立的微环境线索（BDNF和膜渗透性cAMP类似物）时的生长和引导。特异性目的3包括确定已知引导线索（BDNF和Sema3A）的局部浓度梯度是否可以诱导胚胎非洲爪蟾脊髓神经元的轴突特异性。将定量评估远程轴突制导所需的浓度梯度要求。这项工作如果成功，将通过提供哺乳动物极化和轴突引导的第一个定量描述，对发育神经生物学产生重大影响。由于多种引导信号协同作用来调节体内的这些发育过程，因此未来的研究必须定量地检查这些环境信号的相互作用。由于这是一项涉及微流体、生物工程和神经生物学的高度跨学科合作，因此R21与NINDS和NIBIB的任务相关。提议的R21团队由加州大学伯克利分校机械工程副教授Lydia L. Sohn (PI)，斯坦福大学材料科学与工程助理教授Sarah Heilshorn （co-PI）和加州大学伯克利分校分子与细胞生物学系神经生物学教授兼系主任Mu-ming Poo（顾问）组成。首席研究员/项目主任（最后，第一，中）：孙，李Lydia

The overarching goal of this R21 proposal, A Controllable Microfluidic Gradient Device for Studying Neuronal Polarization, is to demonstrate and explore the capabilities of a novel microfluidic concentration-gradient generator, as it relates to developmental and regenerative neurobiology. The device, consisting of a series of alternating cell-culture chambers and reagent channels that are interconnected via micro-channels, establishes and maintains steady concentration gradients within a static cell-culture chamber for neuronal culture. Concentration gradients of both small molecules (pharmaceutical agents and second messengers) and macromolecules (neurotrophins and other proteins) are easily achieved and quantified in this device. Thus, quantitative studies of neuronal polarization and axon pathfinding of neurons in response to micro- environmental cues can be accomplished. Three Specific Aims will be pursued. Specific Aim 1 involves optimizing the design of the proposed microfluidic device, as well as its fabrication and measurement. Specific Aims 2 and 3 focuses on evaluating four hypotheses of neuronal polarization and axon pathfinding that are not currently quantifiable using current protocols. In particular, Specific Aim 2 involves assessing growth and guidance of embryonic rat hippocampal neurons in response to defined micro-environmental cues (BDNF and a membrane-permeable cAMP analogue) established in the device. Specific Aim 3 involves determining if localized concentration gradients of known guidance cues (BDNF and Sema3A) can induce axon specification in embryonic Xenopus spinal cord neurons. The concentration gradient requirements needed for long-range axon guidance will be quantitatively assessed. The proposed work, if successful, will have significant impact in developmental neurobiology by providing the first quantitative description of mammalian polarization and axon guidance. Because multiple guidance cues work in concert to regulate these developmental processes in vivo, it would be imperative for future studies to examine quantitatively the interplay of these environmental signals. Because this is a highly-interdisciplinary collaboration involving microfluidics, bioengineering, and neurobiology, this R21 is relevant to the missions of both NINDS and NIBIB. The proposed R21 team consists of Lydia L. Sohn (PI), Associate Professor of Mechanical Engineering at University of California, Berkeley, Sarah Heilshorn (co-PI), Assistant Professor of Materials Science & Engineering at Stanford University, and Mu-ming Poo (consultant), Professor and Division Head of Neurobiology in the Dept. of Molecular & Cellular Biology at University of California, Berkeley. Principal Investigator/Program Director (Last, First, Middle): Sohn, Lydia Lee