Intraspecific chemosignals in the main olfactory system

描述（由申请人提供）：种内化学信号的嗅觉感知提供有关社会和性别地位，基因组成和物种身份的感官信息。在这个建议中，我们描述了实验来研究这些化学信号诱导的活动及其潜在的转导机制。以前我们已经表明，主要的嗅觉系统使用cAMP-和磷脂酶C-介导的信息素信号传导途径。基因敲除环核苷酸门控通道亚基2 (CNGA2)，可以消除cAMP信号，但不会消除信息素诱导的激活。在我们的初步研究中，我们发现一种新的信号成分，瞬时受体电位通道M5 (TRPM5)，存在于参与信息素和尿液成分嗅觉信号传导的小鼠成熟osn亚群中。敲除CNGA2和TRPM5会导致主嗅上皮和嗅球发生严重畸变，这表明TRPM5在嗅觉信号传导中的功能重要性。我们假设TRPM5参与了主要嗅觉系统中种内化学信号的信号传递。提出的实验分为三个目的。1)我们将测试功能性TRPM5是否对种内信号诱发的肾小球激活模式至关重要。我们将使用Fos蛋白的免疫标记作为神经元激活标记，并使用肾小球定位程序构建肾小球激活图，研究种内化学信号诱导的嗅球激活。然后，我们将确定激活的肾小球是否接受来自表达TRPM5的osn的输入，以及敲除TRPM5是否会改变激活模式。2)我们将通过解剖和免疫细胞化学方法检测表达TRPM5的osn是否提供感觉输入以维持CNGA2单敲除小鼠中发现的酪氨酸羟化酶活性，以及CNGA2和TRPM5双敲除是否会导致主嗅觉系统异常和神经元死亡。3)我们将测试TRPM5是否参与osn种内化学信号的信号转导。我们将表征TRPM5- gfp、TRPM5敲除gfp、CNGA2敲除trpm5gfp小鼠的osn对信息素和尿液成分的电和Ca2+反应，并利用Ca2+成像和电生理记录方法确定TRPM5和相关途径的功能。综上所述，这些研究将为了解种内化学信号的信号处理和TRPM5在主要嗅觉系统中的功能作用提供基础知识。

DESCRIPTION (provided by applicant): Olfactory perception of intraspecific chemosignals provides sensory information about social and sexual status, genetic makeup, and species identity. In this proposal we describe experiments to investigate activities induced by these chemosignals and their underlying transduction mechanisms. Previously we have shown that the main olfactory system employs both cAMP- and phospholipase C- mediated pathways in pheromone signaling. Genetic knockout of the cyclic nucleotide-gated channel subunit 2 (CNGA2), which eliminates the cAMP signaling, does not eliminate pheromone-induced activation. In our preliminary study, we find that a novel signaling component, Transient Receptor Potential channel M5 (TRPM5), is present in a subset of mouse mature OSNs involved in olfactory signaling of pheromones and urinary components. Knockout of both CNGA2 and TRPM5 results in profound aberration in the main olfactory epithelium and bulbs, indicating the functional importance of TRPM5 in olfactory signaling. We hypothesize that TRPM5 is involved in signaling of intraspecific chemical cues in the main olfactory system. The proposed experiments fall into 3 aims. 1) We will test whether functional TRPM5 is crucial for intraspecific signal-evoked glomerular activation patterns. We will examine intraspecific chemosignals-induced activation in olfactory bulbs use immunolabeling of Fos protein as a neuronal activation marker and construct glomerular activation maps using a glomerular mapping program. We will then determine whether activated glomeruli receive input from the TRPM5-expressing OSNs, and whether knockout of TRPM5 alters the activation patterns. 2) We will test whether TRPM5-expressing OSNs provide sensory input to sustain tyrosine hydroxylase activity found in single CNGA2 knockout mice and whether double- knockouts of both CNGA2 and TRPM5 result in abnormality and neuronal death in the main olfactory system using anatomical and immunocytochemical approaches. 3) We will test whether TRPM5 is involved in signal transduction of intraspecific chemosignals in OSNs. We will characterize electrical and Ca2+ responses to pheromones and urine components in OSNs from TRPM5-GFP, TRPM5 knockout-GFP, CNGA2 knockout-TRPM5GFP mice and determine the functions of TRPM5 and associated pathways using Ca2+ imaging and electrophysiological recording methods. Taken together, the proposed studies will provide fundamental knowledge on signaling processing of intraspecific chemical cues and functional roles of TRPM5 in the main olfactory system. PUBLIC HEALTH RELEVANCE: In this application, we propose to investigate olfactory signaling mechanisms and the abnormality resulted from dysfunction of ion channels in the olfactory epithelium. Olfactory deficits are known to associate with aging and diseases. Our studies will provide knowledge ultimately useful for clinical treatment of olfactory dysfunctions.