Putative phermones in the main olfactory system

描述(申请人提供)：哺乳动物的嗅觉包括两个并行的信号处理系统。主嗅觉上皮(MOE)通过cAMP信号通路检测空气中的气味。犁鼻器(VNO)通过依赖磷脂酶C(PLC)激活Trp2通道来检测信息素。然而，信息素的检测并不完全由VNO介导。我们意外地表明，信息素2-庚酮和2，5-二甲基吡嗪(DMP)在cAMP途径中断的环核苷酸门控通道亚单位A2基因敲除(CNGA2KO)小鼠的MOE中诱发了场电位，这与行为检测一致。当在野生型小鼠身上测试时，MOE中的这些信息素诱导的反应对cAMP途径的抑制剂明显不敏感，但与其他气味物质相比对PLC抑制剂更敏感，这表明对照动物中同时存在cAMP依赖和非依赖的机制。重要的是，2-庚酮和DMP在CNGA2KO和野生型小鼠的主要嗅球(MOB)中激活了类似的肾小球亚群。激活的肾小球还包括一些项链肾小球，这些肾小球是表达鸟苷环化酶D(GC-D)途径的嗅觉神经元轴突的靶标。 这一建议旨在进一步研究通过MOE信息素输入在MOE和激活的脑区中推测的信息素的转导机制。假设依赖PLC和/或GC-D的信号通路都介导了CNGA2KO小鼠对2-庚酮和DMP的反应；MOE对这些信息素的反应激活了主要的嗅觉皮质和大脑区域，这些区域接受信息素输入，调节社会和性活动。目的1.确定依赖于PLC和/或GC-D的信号通路是否介导了对MOE中可能的信息素的反应。我将通过在CNGA2KO和对照组小鼠身上使用钙离子成像和药物的组合来检验这些通路的参与。目的2.在MOB和高级脑区的激活神经元中鉴定2-庚酮和DMP激活的肾小球。使用Fos蛋白表达作为免疫标记的活性标记，我将系统地定位气味激活的肾小球。我将通过计算Fos阳性神经元的数量，并将暴露于2-庚酮和DMP的小鼠与CNGA2KO和WT小鼠中相同基因类型的非刺激对照组小鼠之间的这些数字进行比较，来确定激活的大脑区域。 这项研究产生的数据将把参与MOE信息素检测的信号通路与中枢活动联系起来，并有助于我们全面了解嗅觉系统用来区分生物相关气味的策略，以及MOE对生殖和社会互动的影响。

DESCRIPTION (provided by applicant): Mammalian olfaction encompasses two parallel signal-processing systems. The main olfactory epithelium (MOE) detects airborne odorants via the cAMP-signaling pathway. The vomeronasal organ (VNO) detects pheromones via phospholipase C (PLC)-dependent activation of TRP2 channels. However, pheromone detection is not exclusively mediated by the VNO. We have shown unexpectedly that pheromone 2-heptanone and 2,5-dimethylpyrazine (DMP) evoked field potentials in the MOE of cyclic nucleotide-gated channel subunit A2 knockout (CNGA2 KO) mice with a disrupted cAMP pathway, consistent with behavioral detection. When tested in wild type mice, these pheromone-induced responses in MOE were significantly less sensitive to inhibitors of the cAMP pathway, but more sensitive to a PLC inhibitor as compared to other odorants, indicating the presence of both cAMP-dependent and -independent mechanisms in control animals. Importantly, 2-heptanone and DMP activated a comparable subset of glomeruli in the main olfactory bulbs (MOB) in both CNGA2 KO and wild type mice. Activated glomeruli also included some necklace glomeruli, which are targeted by axons of olfactory neurons expressing the guanylyl cyclase D (GC-D) pathway. This proposal intends to further study transduction mechanisms of putative pheromones in the MOE and activated brain areas by MOE pheromonal inputs. Hypotheses are that both PLC- and/or GC-D-dependent signaling pathways mediate responses to 2-heptanone and DMP in CNGA2 KO mice; and the MOE responses to these pheromones activate the main olfactory cortex and brain areas that receive pheromonal inputs and regulate social and sexual activities. Aim 1. Determine whether PLC- and/or GC-D-dependent signaling pathways mediate responses to putative pheromones in the MOE. I will examine the involvement of these pathways by using Ca2+ imaging and a combination of pharmacological agents in both CNGA2 KO and control mice. Aim 2. Identify 2-heptanone and DMP-activated glomeruli in the MOB and -activated neurons in higher-order brain areas. Using Fos protein expression as an activity marker in immunolabeling, I will map systematically odor-activated glomeruli. I will determine activated brain areas by counting the number of Fos-positive neurons and comparing these numbers between mice exposed to 2-heptaone and DMP and the non-stimulated controls of the same genotypes in both CNGA2 KO and WT mice. Data generated from this study will correlate signaling pathways involved in MOE pheromone detection to central activity, and contribute to our overall understanding of the strategies used by the olfactory system to discriminate biologically relevant odorants and the influence of MOE on reproduction and social interaction.