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

本研究主要围绕以下两个方面展开：1)膜片钳记录与荧光记录同时进行的新型记录系统的研制；2)笼状化合物在急性分离的中枢神经系统神经元上的应用。使用改进的试验系统研究M3毒碱样乙酰胆碱、代谢性谷氨酸和5-羟色胺_3受体的反应细节。结果表明，每种受体均通过以下途径诱导细胞超极化：G-蛋白*PLC*IP_3*Ca~(2+)&gt；从细胞内钙离子释放*PLC*IP_3*Ca~(2+)&gt；另一方面，为了得到一种有效的笼状化合物，我们新合成了12种笼状甘氨酸，并寻找了一种方便的笼状化合物，因为从初步的实验中发现，商业上可用的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产物。甘氨酸在紫外光下以S形方式释放能量，在10^~(-5)~10^~(-3)M浓度范围内，释放效率为60%~80%。如上所述，成功地完成了60%以上的初始平面，但荧光测量系统及其与膜片钳技术的结合也存在许多问题。我们的部分荧光测量结果已投入到后续系统的开发中。由于系统的改进被耽搁了很久，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负载对细胞的损伤造成了膜片钳实验的困难，因此开发新的膜透过性和低细胞毒性的荧光芯片也是必要的。

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.