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

描述（由申请人提供）：我们最近开发了一个微流控灌注和成像平台，用于大规模检测游离小鼠嗅觉感觉神经元（OSNs）对各种气味的反应；我们把这个平台称为“芯片上的气味平台”。该项目的总体目标是进一步发展现有的芯片嗅觉平台，提高自动化程度，从而提高数据质量，提高整体实验吞吐量。我们的目标是表征osn对各种各样的气味、复杂气味和信息素的反应的动态和特异性。信息素是一种挥发性有机化合物，它可以引发或调节诸如交配、抚养后代、侵略和领土标记等先天行为。在哺乳动物中，信息素主要是由嗅鼻器官（VNO）检测到的，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与大脑没有可检测到的联系，但信息素敏感的OSNs在小鼠OE中的作用强烈表明，人类的信息素信号是通过OE发生的。OB的激活显然证明了OE中存在参与信息素检测的嗅觉感觉神经元（OSNs），但通过传统方法检测这些嗅觉感觉神经元被证明是难以捉摸的，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，我们将利用我们的嗅觉芯片平台来检测解离的小鼠osn对已知气味、气味和信息素的反应。通过同时对数千个OSN进行成像，我们将能够发现很少发生的OSN响应。信息素特异性小鼠OSNs的检测和分离将为小鼠单细胞基因表达研究开辟道路，从而在分子水平上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先天行为（如性吸引、母子关系和月经周期同步，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提供一个广泛适用于各种领域的平台，用于测量大量单细胞的行为，例如毒性研究，药物测试和小分子筛选（例如癌症生物标志物），以及从大量正常行为的细胞中发现具有罕见病理行为的细胞（例如血液中存在致瘤细胞）。获得丰富的单细胞统计数据可以识别细胞的独特响应亚群，并确定固有的细胞行为可变性。在本研究中，我们将应用芯片上的气味平台来筛选osn，但该平台广泛适用于任何基于成像的、高通量的大量单个解离细胞的筛选。此外，稀有细胞（在28000个微孔中，每孔只有一个细胞）可以被挑选出来，并在确定它们对已知化合物的反应后进行人工检索，以进行进一步分析（例如PCR扩增）。公共卫生相关性：在本提案中，我们将进一步开发我们的芯片嗅觉平台（在以前的R21支持下成功原型），以检测和表征解离小鼠OSNs对气味剂，复杂气味和信息素的反应的动力学，特异性和适应性。通过同时对数千个OSN进行成像，我们将能够发现很少发生的OSN响应。信息素特异性小鼠OSNs的检测和分离将为小鼠单细胞基因表达研究开辟道路，从而在分子水平上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.