DEVELOPMENT OF NEW SYSTEM USED CAGED-COMPOUND BY WHICH THE PHYSIOLOGICAL FUNCTION OF CNS NEURONS CAN BE CLARIFIED.

开发使用笼状复合物的新系统，通过该系统可以阐明中枢神经系统神经元的生理功能。

基本信息

批准号：
02557005
负责人：
AKAIKE Norio
金额：
$ 8.9万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

In this study, the theme was focused on the following two points ; 1) development of the new recording system by which the patch-clamp and fluorometrical recordings can be performed simultaneously, 2) application of caged compound to CNS neurons acutely dissociated from mammalian brain. Improved trial system was used to investigate the details of the responses via M3 muscarinic acetylcholine, metabotropic glutamate and serotonin_3 receptors. As results, it was revealed that each receptor induces hyperpolarization via following pathway ; G-protein* PLC * IP_3 * Ca^<2+> release from intracellular Ca^<2+> store * K^+ channel open. On the other hand, to obtain an effective caged compound, we newly synthesized twelve caged glycines and searched the convenient one, because it was found from the preliminary experiment that the liberation efficiency of the commercially available compounds was quietly low. Quantitative analysis revealed N-[1-(2-Nitrophenyl) ethyloxycarbonyl] glycine as the most effectively photolyzed one. Glycine was liberalized UV energy dependently in as sigmoidal manner, and the liberation efficiency was 60 to 80 % between the concentration range from 10^<-5> to 10^<-3> M.As described above, more than 60 % of initial plane was successfully finished, but many problems were also found in the fluorometrical measurement system and the combination of it with patch-clamp technique by the detailed biological experiment used trial system. A part of our results for fluorometrical measurement has been putted into the development of the succeeding system. Since the improvement of system was delayed so much, we could not put the system into practical use. However, the points to carry out the experiment was realized. The development of new membrane-permeable and low cell toxic fluorescent die is also necessary, because cell damage by loading of fura 2/AM caused the difficult patch-clamp experiment.

本次研究的主题主要集中在以下两点； 1）开发新的记录系统，通过该系统可以同时进行膜片钳和荧光记录，2）将笼状化合物应用于与哺乳动物大脑急剧分离的中枢神经系统神经元。改进的试验系统用于研究 M3 毒蕈碱乙酰胆碱、代谢型谷氨酸和 5-羟色胺_3 受体的反应细节。结果表明，每种受体通过以下途径诱导超极化； G-蛋白* PLC * IP_3 * Ca^<2+> 从细胞内 Ca^<2+> 储存中释放 * K^+ 通道打开。另一方面，为了获得有效的笼状化合物，我们新合成了12种笼状甘氨酸，并寻找方便的笼状甘氨酸，因为从初步实验发现，市售化合物的释放效率相当低。定量分析表明 N-[1-(2-硝基苯基)乙氧基羰基]甘氨酸是最有效的光解甘氨酸。甘氨酸以S形方式依赖于UV能量释放，并且在10^-5至10^-3M的浓度范围内释放效率为60至80％。如上所述，初始的60％以上飞机顺利完成，但通过详细的生物实验用试验系统，也发现荧光测量系统及其与膜片钳技术的结合存在许多问题。我们的荧光测量结果的一部分已用于后续系统的开发。由于系统的改进被拖延了很多，所以我们无法将系统投入实际使用。然而，进行实验的要点已经实现。开发新的膜渗透性和低细胞毒性荧光芯片也是必要的，因为加载 fura 2/AM 造成的细胞损伤导致膜片钳实验变得困难。