Establishment of in vitro model for the vomeronasal system and analysis of the synapse formation between the vomeronasal and accessory olfactory neurons

信息素是通过犁鼻器（VNO）上的特异性受体检测到的，进而影响各种动物的内分泌状态和行为。尽管VNO介导性和发育不同的行为和神经内分泌反应是众所周知的，但调节这些功能的细胞机制还不太清楚。信息素效应的差异可能部分是由于信息素受体的差异表达。本研究建立了犁鼻受体神经元（vomeronasal receptor neurons，VRN）和副嗅球（accessory olfactory bulb，AOB）神经元共培养系统，探讨犁鼻系统中各神经元在信息素信号处理中的作用。首先，我使用钙成像技术结合电刺激和分析钙离子，证明了培养的VRNs和AOB神经元之间的突触形成。第二，与分离的AOB神经元共培养可诱导VRNs成熟。 ...更多信息 为了表征受体的表达，我研究了两个代表（VR 1和VR 4）从不同的亚家族的V2 R家族的信息素受体在培养VRNs。蛋白质印迹分析显示VRNs中VR 1和VR 4的表达被诱导，并且随着共培养的天数而增加，在VRNs单独培养中没有观察到这种效果。此外，我们使用微电极将小鼠尿液中的带电化合物离子电渗作用于VRN，并通过Ca^2+成像方法分析有或无培养的AOB细胞的VRN反应。当尿液中的化合物被排出到与AOB细胞共培养的VRN中，电流为-2的UNFA时，VRN亚群明显显示出持续的Ca^<2+>增加。单独注入低于-5UNFA的电流对VRN没有影响。在没有AOB细胞的情况下，没有观察到这种Ca^<2+>的增加。这些结果表明，VRNs通过与AOB神经元相互作用，表达信息素受体，然后获得对尿液中化合物的反应性。少

Pheromones are detected by unique receptors in the vomeronasal organ (VNO), and then affect endocrinal status and behavior in various kinds of animals. Although it is well known that the VNO mediates sexually and developmentally different behavioral and neuroendocrine responses, the cellular mechanisms regulating these functions are not, much understood. The differences in pheromonal effects might be in part due to differential expression of pheromone receptors. In this project, I established the co-culture system of vomeronasal receptor neurons (VRN) and accessory olfactory bulb (AOB) neurons to assess roles of each neuronal species in the vomeronasal system on the pheromonal signal processing. Firstly, I demonstrated the synapse formation between cultured VRNs and AOB neurons using a calcium imaging technique combined with electrical stimulation and analyzing pharmacologically. Secondly, it was showed that the maturation of VRNs was induced by co-culture with dissociated AOB neurons. … More To characterize the receptor expression, I examined two representatives (VR1 and VR4) from different subfamilies of the V2R family of pheromone receptors in cultured VRNs. A western blotting analysis showed the expression of VR1 and VR4 in VRNs was induced and increased with days in co-culture, an effect which was not observed in the VRN-alone culture. Moreover, we applied charged compounds in mouse urine iontophoretically to the VRNs using a microelectrode and analyzed VRN response by a Ca^<2+> imaging method with or without cultured AOB cells. When urine compounds were ejected to the VRNs co-cultured with AOB cells with a current of -2μA, subpopulation of VRNs clearly showed long-lasting Ca^<2+> increases. Injections of a current below -5μA alone had no effect to the VRNs. Such Ca^<2+> increases were not observed without AOB cells. These results indicate that VRNs result in expressing pheromone receptors by interacting with AOB neurons, and then acquiring responsiveness to compounds in urine. Less