TRANSDUCTION OF INHIBITORY ODORANT RESPONSES IN MOUSE

这项建议的重点是研究小鼠嗅觉感受器神经元(ONs)抑制嗅觉反应的机制。具体目的是：(1)确定钙激活的Kplus通道是否介导气味诱导的抑制反应；(2)确定在气味受体I7在成熟Orn中表达的转基因小鼠(pomp-i7-IRES-RFP)中，庚醛是否能引起特异性的兴奋性或抑制性反应。气味诱导的抑制反应及其对K+和细胞内钙的依赖性将利用钙离子成像和穿孔膜片钳记录相结合的方法进行研究。转基因小鼠将通过将pomp-I7-IRES-RFP的cDNA构建注入受精的小鼠卵母细胞中而产生。嗅觉标记蛋白(OMP)启动子近端0.9kb将驱动小鼠I7气味受体的表达。一个内部核糖体进入位点(IRES)将被用来创建编码I7气味受体和红色荧光蛋白(RFP；Clontech)的双顺反子mRNA。由于I7对庚醛的反应是特异的，这将导致小鼠有很大比例的神经元对一种特殊的气味--庚醛做出反应。这只小鼠将被用来更严格地研究参与抑制反应的机制，以及同一细胞中兴奋和抑制通路之间的相互作用。这项研究的结果将扩大我们对气味检测和嗅觉编码的知识。

The focus of this proposal is to study the mechanisms underlying inhibitory olfactory responses in mouse olfactory receptor neurons (ORNs). Specific aims are: (1) to determine whether Ca2+-activated Kplus channels mediate odor-induced inhibitory responses; (2) to determine if heptaldehyde elicits exclusively excitatory or inhibitory responses in ORNs of transgenic mice (POMP-I7-IRES-RFP) where odor receptor I7 is expressed in mature ORNs. Odor- induced inhibitory responses and their dependence on K+ and intracellular Ca2+ will be examined utilizing a combination of Ca2+ imaging and perforated patch-clamp recording. The transgenic mice will be generated by injection of a cDNA construct of POMP-I7-IRES-RFP into fertilized mouse oocytes. The proximal 0.9 Kb of the olfactory marker protein (OMP) promoter will drive expression of the mouse I7 odor receptor. An internal ribosomal entry site (IRES) will be used to create a bicistronic mRNA encoding for I7 odor receptor and red fluorescent protein (RFP; Clontech). Since I7 responds to heptaldehyde specifically, this will result in a mouse with a large percentage of neurons responding to a specific odor, heptaldehyde. This mouse will be used to more stringently study mechanisms involved in inhibitory responses and the interaction between excitatory and inhibitory pathways in the same cells. Results gained from this study will expand our knowledge of odor detection and olfactory coding.