GENETIC DETERMINANTS OF ODORANT RESPONSIVENESS

我们研究的总体目的是了解嗅觉信息在嗅上皮水平上的加工逻辑及其向嗅球的投射。我们测试的假设是嗅觉受体（OR）基因的产物是嗅觉感觉神经元（OSNs）气味反应的关键决定因素。我们的方法包括构建或使用基因靶向小鼠，其中表达给定or基因的活神经元可以通过其绿色荧光蛋白（GFP）的表达来识别和分离。这种遗传策略提供了一个独特的和前所未有的机会来研究从内源性基因表达定义的OR的活神经元。对气味剂面板的反应将用钙成像技术进行分析。这种强大而新颖的小鼠靶向诱变和钙成像的结合将揭示哺乳动物嗅觉编码的一般原理。在具体目标1中，我们将严格检查ORs对表达它们的osn的气味反应的贡献。我们将确定特定OR基因的表达是否与特定气味反应谱紧密相关。同时，我们将确定是否OR基因的表达是必要的和充分的，以赋予气味反应概况。目的是通过功能手段检查单受体/单神经元假说，并提供ORs在气味反应中的作用的关键评估。在具体目标2中，我们将以相反的方式研究嗅觉。从一组确定的气味剂开始，我们将确定在对这些气味剂有反应的osn中表达的ORs的光谱。面对嗅觉系统令人生畏的数字复杂性，我们设计了两种方法来将这个问题简化为可管理的格式。目的是在生理学相关的背景下定义气味剂和ORS的配体/受体关系。在Specific Aim 3中，我们将克隆位于嗅球特定区域的10-20个肾小球的OR基因，并确定表达这些基因的osn亚群的气味反应谱。我们想为球茎的一小部分编制一个综合地图，包括遗传解剖和功能数据。目标是开创嗅觉系统图谱的组装，其分辨率从未达到过。综上所述，提出的研究应该在哺乳动物嗅觉的生理学和分子生物学之间建立期待已久和急需的桥梁。

The overall aim of our studies is to understand the logic of the processing of olfactory information at the level of the olfactory epithelium and its projection to the olfactory bulb. The hypothesis that we test is that the products of the olfactory receptor (OR) genes are critical determinants of the odorant responsiveness of olfactory sensory neurons (OSNs). Our approach involves the construction or use of gene-targeted mice in which live neurons expressing a given OR gene can be identified and isolated by virtue of their expression of the green fluorescent protein (GFP). This genetic strategy provides a unique and unprecedented opportunity to study living neurons that express a defined OR from endogenous genes. Responses to panels of odorants will be assayed with calcium imaging techniques. This powerful and novel combination of targeted mutagenesis in the mouse and calcium imaging will reveal general principles of mammalian olfactory coding. In Specific Aim 1, we will critically examine the contribution of ORs to odorant responses of the OSNs that express them. We will determine if expression of a particular OR gene correlates tightly with a particular odorant response profile. Simultaneously we will determine if expression of an OR gene is both necessary and sufficient to confer an odorant response profile. The goal is to examine the single receptor/single neuron hypothesis by functional means and to provide a critical assessment of the role of ORs in odorant responsiveness. In Specific Aim 2, we will study olfaction in the reverse manner. Starting with a defined set of odorants, we will determine the spectrum of ORs that are expressed in OSNs that respond to these odorants. Faced with the daunting numerical complexity of the olfactory system, we have devised two ways of reducing this question to a manageable format. The goal is to define ligand/receptor relationships of odorants and ORS within a physiologically relevant context. In Specific Aim 3, we will clone OR genes that map to 10-20 glomeruli in a defined area of the olfactory bulb and determine the odorant response profile of the subsets of OSNs that express these genes. We want to compile an integrated map for a small part of the bulb, comprising genetic anatomical and functional data. The goal is to pioneer the assembly of an atlas of the olfactory system at a resolution that has never been achieved before. Taken together, the proposed studies should form the long-awaited and much-needed bridges between the physiology and molecular biology of olfaction in mammals.