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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.

本研究主要围绕以下两个方面展开：1）开发一种新的膜片钳记录系统，使膜片钳和荧光记录同时进行; 2）笼状化合物在急性分离的哺乳动物中枢神经系统神经元中的应用。用改良的试验系统研究M3胆碱能受体、代谢型谷氨酸受体和胆碱酯酶3受体的反应。结果表明，每个受体都通过以下途径诱导超极化：G蛋白 * PLC * IP_3 * 细胞内Ca^<2 +>库释放Ca^<2+* K^+通道开放。另一方面，为了获得有效的笼状化合物，我们新合成了12个笼状甘氨酸并寻找方便的笼状甘氨酸，因为从初步实验中发现，市售化合物的释放效率很低。定量分析表明N-[1-（2-硝基苯基）乙氧羰基]甘氨酸是最有效的光解之一。甘氨酸以S形方式释放紫外能量依赖性，并且在10 μ M至10 μ M的浓度范围内释放效率为60%至80%<-5><-3>。如上所述，成功地完成了超过60%的初始平面，但是通过使用试验系统的详细生物实验，在荧光测量系统及其与膜片钳技术的结合中也发现了许多问题。我们的荧光测量结果的一部分已被纳入后续系统的开发。由于系统的改进滞后，我们无法将该系统投入实际使用。然而，进行实验的要点是实现的。由于Fura 2/AM负载对细胞的损伤导致膜片钳实验困难，因此开发新型的膜透性和低细胞毒性的荧光染料也是必要的。