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（ORN）抑制性嗅觉反应的机制。 具体目标是：（1）确定Ca 2+激活的K+通道是否介导气味诱导的抑制性反应;（2）确定庚醛在转基因小鼠（POMP-I7-IRES-RFP）的ORN中是否仅引起兴奋性或抑制性反应，其中气味受体I7在成熟的ORN中表达。 将利用Ca 2+成像和穿孔膜片钳记录的组合来检查气味诱导的抑制反应及其对K+和细胞内Ca 2+的依赖性。 将通过将POMP-I7-IRES-RFP的cDNA构建体注射到受精的小鼠卵母细胞中来产生转基因小鼠。嗅觉标记蛋白（OMP）启动子的近端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）将用于产生编码I7气味受体和红色荧光蛋白的双顺反子mRNA（RFP; Clontech）。 由于I7对庚醛有特异性反应，这将导致小鼠有很大比例的神经元对特定的气味（庚醛）有反应。 这种小鼠将被用于更严格地研究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。 从这项研究中获得的结果将扩大我们的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.