TRANSDUCTION OF INHIBITORY ODORANT RESPONSES IN MOUSE

本研究的重点是研究小鼠嗅觉受体神经元抑制性嗅觉反应的机制。具体目的是：(1)确定Ca2+激活的Kplus通道是否介导气味诱导的抑制反应；(2)确定七醛是否在成熟的气味受体I7表达的转基因小鼠（POMP-I7-IRES-RFP）的orn中只引起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Ca2+的依赖将利用Ca2+成像和穿孔膜片钳记录的组合进行检查。将POMP-I7-IRES-RFP cDNA构建体注射到受精卵细胞中产生转基因小鼠。嗅觉标记蛋白（OMP）启动子的近0.9 Kb将驱动小鼠I7气味受体的表达。内部核糖体进入位点（IRES）将用于创建编码I7气味受体和红色荧光蛋白（RFP; Clontech）的双链mRNA。由于I7对七乙醛有特殊的反应，这将导致老鼠有很大比例的神经元对一种特定的气味——七乙醛做出反应。这只小鼠将被用于更严格地研究涉及抑制反应的机制以及同一细胞中兴奋和抑制途径之间的相互作用。本研究的结果将扩展我们在气味检测和嗅觉编码方面的知识。

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.