A Controllable Microfludic Gradient Device for Studying Neuronal Polarization

这项R21提案的总体目标是研究神经元极化的可控微流体梯度装置，旨在展示和探索新型微流体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。该装置由一系列交替的细胞培养室和通过微通道互连的试剂通道组成，在用于神经元培养的静态细胞培养室内建立并维持稳定的浓度梯度。小分子（药剂和第二信使）和大分子（神经营养因子和其他蛋白质）的浓度梯度在该装置中容易实现和定量。因此，可以完成对微环境线索响应的神经元极化和神经元轴突寻路的定量研究。将实现三个具体目标。具体目标1涉及优化所提出的微流体装置的设计，以及其制造和测量。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方案进行量化。具体而言，特定目标2涉及评估胚胎大鼠海马神经元响应于装置中建立的定义的微环境线索（BDNF和膜渗透性cAMP类似物）的生长和指导。具体目标3涉及确定是否局部浓度梯度的已知的指导线索（BDNF和Sema 3A）可以诱导胚胎非洲爪蟾脊髓神经元轴突规格。将定量评估长程轴突引导所需的浓度梯度要求。这项工作如果成功，将通过首次定量描述哺乳动物的极化和轴突导向，对发育神经生物学产生重大影响。由于多个指导线索协同工作，以调节这些发展过程中的体内，这将是必要的，为未来的研究定量研究这些环境信号的相互作用。由于这是一个涉及微流体、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的任务都相关。拟议的R21团队由Lydia L. Sohn（PI），加州大学伯克利分校机械工程系副教授，Sarah Heilshorn（co-PI），斯坦福大学材料科学与工程系助理教授，以及Mu-ming Poo（顾问），神经生物学系教授兼系主任。他是加州大学伯克利分校的分子与细胞生物学教授。主要研究者/项目负责人（最后，第一，中间）：Sohn，Lydia Lee

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