Intraspecific chemosignals in the main olfactory system

描述(由申请人提供)：种内化学信号的嗅觉提供关于社会和性别地位、基因构成和物种身份的感官信息。在这项建议中，我们描述了研究这些化学信号诱导的活动及其潜在的转导机制的实验。以前我们已经证明，主嗅觉系统在信息素信号传递中同时使用cAMP和磷脂酶C介导的途径。基因敲除了环核苷酸门控通道亚单位2(CNGA2)，它消除了cAMP信号，但并不消除信息素诱导的激活。在我们的初步研究中，我们发现一个新的信号成分，瞬时受体电位通道M5(Trpm5)，存在于小鼠成熟的OSNs中，参与信息素和尿液成分的嗅觉信号传递。CNGA2和Trpm5的敲除都导致了主要的嗅觉上皮和嗅球的严重异常，表明Trpm5在嗅觉信号中的功能重要性。我们推测Trpm5参与了主嗅觉系统种内化学信号的传递。拟议的实验分为三个目标。1)我们将测试功能Trpm5是否对种内信号诱发的肾小球激活模式至关重要。我们将研究种内化学信号诱导的嗅球激活，使用Fos蛋白的免疫标记作为神经元激活标记，并使用肾小球映射程序构建肾小球激活图。然后，我们将确定激活的肾小球是否收到表达Trpm5的OSN的输入，以及Trpm5的敲除是否改变激活模式。2)我们将利用解剖学和免疫细胞化学的方法，测试表达Trpm5的OSN是否提供感觉输入，以维持在单一CNGA2基因敲除小鼠中发现的酪氨酸羟化酶活性，以及CNGA2和Trpm5的双重敲除是否会导致主要嗅觉系统的异常和神经元死亡。3)检测Trpm5是否参与OSNs种内化学信号的信号转导。我们将研究Trpm5-GFP、Trpm5基因敲除-GFP、CNGA2基因敲除-TRPM5GFP小鼠OSN对信息素和尿液成分的电和钙反应，并使用钙成像和电生理记录方法确定Trpm5及其相关通路的功能。综上所述，这些研究将提供有关种内化学线索的信号处理以及Trpm5在主要嗅觉系统中的功能作用的基础知识。 公共卫生相关性：在这项应用中，我们建议研究嗅觉信号机制和由嗅觉上皮离子通道功能障碍引起的异常。众所周知，嗅觉缺陷与衰老和疾病有关。我们的研究将为嗅觉功能障碍的临床治疗提供最终有用的知识。

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.