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

信息素是由犁鼻器(VNO)中独特的受体识别的，进而影响各种动物的内分泌状态和行为。虽然众所周知，VNO介导性和发育不同的行为和神经内分泌反应，但调节这些功能的细胞机制还不是很清楚。信息素效应的差异可能部分是由于信息素受体的不同表达。在这个项目中，我建立了犁鼻感受器神经元(VRN)和副嗅球神经元(AOB)的共培养系统，以评估犁鼻系统中每种神经元在信息素信号处理中的作用。首先，我用钙离子成像技术结合电刺激和药理学分析，展示了培养的VRN和AOB神经元之间的突触形成。其次，分离的AOB神经元与VRN共培养可诱导VRN成熟。…为了更多地表征受体的表达，我研究了来自培养的VRN中信息素受体V2R家族不同亚家族的两个代表(VR1和VR4)。免疫印迹分析显示，VRN中VRN的VR1和VR4的表达被诱导，并且随着培养时间的延长而增加，这种作用在VRN单独培养中没有观察到。此外，我们使用微电极将小鼠尿中的带电化合物离子导入VRN，并在培养AOB细胞或不培养AOB细胞的情况下，用钙成像方法分析VRN的反应。当尿液化合物以-2μA的电流喷射到与AOB细胞共培养的VRN时，VRN的亚群明显显示出持续的钙离子浓度升高。单独注射低于-5μA的电流对VRN无影响。在没有AOB细胞的情况下，没有观察到这样的钙升高。这些结果表明，VRN通过与AOB神经元相互作用而表达信息素受体，进而获得对尿液中化合物的反应性。较少

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