| Co-Investigator(Kenkyū-buntansha) |
YASUI Shouzo KYUSHU INST.TECHNOL.CONTROL.ENGIN.SCI.PROF., 情報工学, 教授 (50132741)
KOIDE Ikuo INTERDEC CHIEF, 課長
KUDO Yoshihisa MITSUBISHI KASEI INST.LIFE SCI.DIRECT., 主任研究員
NABEKURA Junich TOHOKU UNIV.NEUROPHYSIOL.ASSIST.PROF., 医学部, 助手 (50237583)
KAWA Kazuyoshi TOHOKU UNIV.NEUROPHYSIOL.ASSOCI.PROF., 医学部, 助教授 (70125839)
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| Research Abstract |
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.
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