Implementation of Microfluidic Automation for Large-Scale Searches of Olfactory N

描述（由申请人提供）：我们最近开发了一种微流体灌注和成像平台，用于大规模检测解离的小鼠嗅觉感觉神经元（OSN）对各种气味剂的响应;我们将该平台称为“芯片上嗅觉平台”。该项目的总体目标是进一步开发现有的芯片嗅觉平台，以提高自动化程度，从而提高数据质量并增加整体实验吞吐量。我们的目标是表征的动态和特异性的OSNs的反应，以各种各样的气味，复杂的气味，和信息素。信息素是一种挥发性的有机化合物，可以引起或调节先天行为，如交配，养育后代，攻击和领土标记。在哺乳动物中，信息素主要由犁鼻器（VNO）检测，但嗅上皮（OE）似乎也在信息素检测中发挥作用，因为（在啮齿动物中）信息素相关的行为和嗅球（OB）的激活仍然存在，即使VNO被移除。虽然人类的VNO与大脑没有可检测的联系，但信息素敏感的OSN在小鼠OE中的作用强烈表明人类的信息素信号传导是通过OE发生的。OB激活显然证明OE中存在参与信息素检测的嗅觉感觉神经元（OSN），但通过传统方法检测这些OSN已被证明是难以捉摸的，这可能是因为它们的数量非常少。因此，在OE中识别这些“信息素专家”OSN将需要高通量检测方法。在这个提议中，我们将利用我们的芯片上的嗅觉平台来检测解离的小鼠OSN已知的气味，气味和信息素的反应。通过同时对数千个OSN进行成像，我们将能够发现罕见的OSN反应。信息素专家小鼠OSN的检测和分离将为小鼠单细胞基因表达研究开辟道路，以更深入地了解人类信息素诱导的行为。在细胞和分子水平上表征信息素反应对于理解大量的先天行为（例如性吸引、母子结合和月经周期同步，仅举几例）至关重要，并且对香水和食品工业至关重要。该项目的成功完成还将为测量大量单细胞行为的各种领域提供普遍适用的平台，例如毒性研究，药物测试和小分子筛选（例如，对于癌症生物标志物），以及在寻找具有罕见，来自大量正常行为细胞的病理行为（例如，血流中存在致瘤细胞）。获得丰富的单细胞统计数据允许辨别细胞的敏感性反应亚群并确定内在细胞行为变异性。在拟议的研究中，我们将应用芯片上的气味平台来筛选OSN，但该平台具有广泛的适用性，可以对大量单个解离细胞进行任何基于成像的高通量筛选。此外，稀有细胞（在> 28，000个微孔的阵列中，每孔仅一个细胞）可以被挑选出来并在表征它们对已知化合物的响应之后手动取回用于进一步分析（例如PCR扩增）。公共卫生相关性：在这项提案中，我们将进一步开发我们的芯片上的气味平台（在以前的R21支持下成功原型化），以检测和表征解离的小鼠OSN对气味剂，复杂气味和信息素的反应的动态，特异性和适应性。通过同时对数千个OSN进行成像，我们将能够发现罕见的OSN反应。信息素专家小鼠OSN的检测和分离将为小鼠单细胞基因表达研究开辟道路，以更深入地了解人类信息素诱导的行为。

DESCRIPTION (provided by applicant): We have recently developed a microfluidic perfusion and imaging platform for large-scale detection of the response of dissociated mouse olfactory sensory neurons (OSNs) to various odorants; we refer to this platform as "smell-on-a-chip platform". The overall goal of this project is to further develop the existing smell-on-a-chip platform to increase automation so as to improve data quality and increase overall experimental throughput. Our goal is to characterize the dynamics and the specificity of the responses of OSNs to a large variety of odorants, complex odors, and pheromones. Pheromones are volatile organic compounds that elicit or modulate innate behaviors such as mating, rearing of young, aggression and territory marking. In mammals, pheromones are primarily detected by the vomeronasal organ (VNO), but the olfactory epithelium (OE) also appears to play a role in pheromone detection because (in rodents) pheromone-associated behaviors and activation of the olfactory bulb (OB) are still present even if the VNO is removed. Although the VNO in humans has no detectable connection to the brain, the role of pheromone-sensitive OSNs in the mouse OE strongly suggests that pheromone signaling in humans occurs through the OE. OB activation is clearly a proof that there are olfactory sensory neurons (OSNs) in the OE that participate in pheromonal detection, yet the detection of these OSNs by traditional methods has proven elusive, likely because they exist in very low numbers. Hence, identification of these "pheromone-specialist" OSNs in the OE will require high-throughput detection methods. In this proposal we will utilize our smell-on-a-chip platform to detect the responses of dissociated mouse OSNs to known odorants, odors and pheromones. By imaging thousands of OSNs simultaneously we will be able to find rarely-occurring OSN responses. The detection and isolation of pheromone-specialist mouse OSNs would open the way for single-cell gene expression studies in mice towards a deeper, molecular-level understanding of pheromonal-induced behaviors in humans. The characterization of pheromonal responses at the cellular and molecular level is of paramount importance for understanding a large number of innate behaviors (such as sexual attraction, mother-child bonding, and menstrual cycle synchronization, to name only a few), and are of vital interest to the perfume and food industries. The successful completion of this project would also provide a platform of general applicability in a variety of fields for measuring the behavior of a large number of single cells, such as in toxicity studies, drug testing, and small-molecule screening (e.g. for cancer biomarkers), and in finding cells with rare, pathological behaviors from a large population of normally-behaving cells (e.g. the presence of tumorigenic cells in the bloodstream). Obtaining rich, single-cell statistics allows for discerning uniquely-responsive sub-populations of cells and for determining intrinsic cell behavior variability. In the proposed study, we will apply the smell-on-a-chip platform to screen OSNs, but the platform has broad applicability to any imaging-based, high-throughput screen of single dissociated cells in large numbers. Furthermore, rare cells (amongst an array of >28,000 microwells, only one cell per well) can be singled out and manually retrieved for further analysis (e.g. PCR amplification) after characterizing their response to known compounds. PUBLIC HEALTH RELEVANCE: In this proposal we will further develop our smell-on-a-chip platform (successfully prototyped under previous R21 support) to detect and characterize the dynamics, the specificity and the adaptation of the responses of dissociated mouse OSNs to odorants, complex odors, and pheromones. By imaging thousands of OSNs simultaneously we will be able to find rarely-occurring OSN responses. The detection and isolation of pheromone-specialist mouse OSNs would open the way for single-cell gene expression studies in mice towards a deeper, molecular-level understanding of pheromonal-induced behaviors in humans.