Putative pheromones in the main olfactory system

描述（由申请人提供）：哺乳动物嗅觉包括两个并行的信号处理系统。主嗅上皮（莫伊）通过cAMP信号通路检测空气中的气味。犁鼻器（VNO）通过磷脂酶C（PLC）依赖性激活TRP 2通道检测信息素。然而，信息素检测并不完全由VNO介导。我们已经出乎意料地表明，信息素2-庚酮和2，5-二甲基吡嗪（CYP 1A 1）诱发的莫伊的环核苷酸门控通道亚基A2敲除（CNGA 2 KO）小鼠与破坏cAMP途径的场电位，与行为检测一致。当在野生型小鼠中测试时，这些信息素诱导的反应在莫伊中对cAMP途径的抑制剂显著不太敏感，但与其他气味剂相比对PLC抑制剂更敏感，表明对照动物中存在cAMP依赖性和非依赖性机制。重要的是，在CNGA 2 KO和野生型小鼠两者中，2-庚酮和异戊四醇激活了主嗅球（MOB）中的相当的肾小球亚组。活化的肾小球还包括一些项链状肾小球，其被表达鸟苷酸环化酶D（GC-D）通路的嗅觉神经元的轴突靶向。 本研究拟进一步研究信息素在莫伊和莫伊信息素输入激活脑区的转导机制。假设PLC-和/或GC-D-依赖性信号通路介导CNGA 2 KO小鼠对2-庚酮和2-庚酮的反应;对这些信息素的莫伊反应激活了主要的嗅觉皮层和大脑区域，这些区域接收信息素输入并调节社交和性活动。目标1。确定PLC和/或GC-D依赖性信号通路是否介导莫伊中对推定信息素的反应。我将通过使用Ca 2+成像和CNGA 2 KO和对照小鼠中的药理学试剂的组合来检查这些通路的参与。目标二。识别MOB中的2-庚酮和DMP激活的肾小球和高阶脑区中的2-庚酮激活的神经元。使用Fos蛋白表达作为免疫标记的活性标记，我将系统地绘制气味激活的肾小球。我将通过计数Fos阳性神经元的数量并比较暴露于2-庚酮和NH 4的小鼠与CNGA 2 KO和WT小鼠中相同基因型的未刺激对照之间的这些数量来确定激活的脑区域。 从这项研究中产生的数据将相关的信号通路参与莫伊信息素检测到中央活动，并有助于我们的整体理解的嗅觉系统所使用的策略，以区分生物相关的气味和莫伊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.