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

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）对各种气味的反应;我们参考了这个平台 作为“芯片嗅觉平台”该项目的总体目标是进一步发展现有的芯片嗅觉 平台，以提高自动化，从而提高数据质量和增加整体实验吞吐量。 我们的目标是表征OSN对各种各样的 气味、复合气味和信息素。信息素是挥发性有机化合物， 调节与生俱来的行为，如交配、养育后代、攻击性和领土标记。在哺乳动物中， 信息素主要由犁鼻器（VNO）检测，但嗅上皮（OE）也 似乎在信息素检测中起作用，因为（在啮齿动物中）信息素相关的行为和 即使VNO被移除，嗅球（OB）的激活仍然存在。虽然人类的VNO 与大脑没有可检测的联系，信息素敏感的OSN在小鼠OE中的作用强烈 表明，信息素信号在人类发生通过OE. OB激活显然是一个证据， 在OE中有嗅觉感觉神经元（OSN）参与信息素检测，然而， 通过传统方法检测这些OSN已被证明是难以实现的，可能是因为它们存在于非常低的环境中。 号码因此，在OE中识别这些“信息素专家”OSN将需要高通量 检测方法在这项提案中，我们将利用我们的芯片上的气味平台来检测的反应， 解离小鼠OSN已知的气味，气味和信息素。通过对数千个OSN进行成像 同时，我们将能够找到罕见的OSN响应。检测和分离 信息素专家小鼠OSN将为小鼠单细胞基因表达研究开辟道路 对人类信息素诱导的行为有更深入的分子水平的理解。的 在细胞和分子水平上表征信息素反应对于 了解大量的先天行为（如性吸引，母子关系， 月经周期同步，仅举几例），并对香水和食品至关重要 行业 这一项目的成功完成还将提供一个在各种领域普遍适用的平台， 用于测量大量单细胞行为的领域，如毒性研究、药物测试和 小分子筛选（例如癌症生物标志物），以及发现具有罕见病理行为的细胞 来自大量行为正常的细胞（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敏感的细胞亚群， 决定内在的细胞行为变异性。在建议的研究中，我们将应用芯片嗅觉平台 筛选OSN，但该平台具有广泛的适用性，任何基于成像的，高通量的屏幕， 大量的单个分离的细胞。此外，稀有细胞（在> 28，000个微孔的阵列中， 每孔仅一个细胞）可以被挑选出来并手动取回用于进一步分析（例如PCR扩增） 在描述了它们对已知化合物的反应之后。

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.