神経系における機能解明を志向したNOのバイオイメージング

Imaging for revealing the real function of NO in the central nervous system

神経系におけるNOの役割は，未だ混沌としている。その原因の一つとして，生理的に機能しているNOを直接リアルタイムで測定できる簡便な方法がなく，詳細な議論ができなかったことが挙げられる。そこで，われわれはバイオイメージングに用いるためのNO感受性蛍光色素DAF-2 DAを開発した。DAF-2 DAは培地に加えるだけで，細胞膜を透過し，加水分解を受け，細胞内にとどめることができる。この色素をラット海馬スライスに適用したところ，NMDA刺激により，CA1領域の細胞内蛍光強度が有意に上昇し，NOの可視化に成功した。このようにDAFを用いることで，NOの時空間的な量的変化を調べることが可能となり，今後，NOの作用解析に寄与するものと考えている。

　The biological functions of nitric oxide (NO) in the neuronal system remain controversial. One reason for this is the difficulty of direct, real-time detection of NO, which hinders the discussion based on the quantity and the distribution. In order to monitor NO directly, which is produced at low concentration and has a short half-life, a new technique is necessary to follow its level changes rapidly and accurately. We developed novel fluorescent indicators, diaminofluoresceins (DAFs), for the direct detection of NO production from living cells. These indicators enabled the specific real-time detection of NO with high sensitivity.The detection limit was 5 nM. A membrane-permeable derivative, DAF-2 DA, was also synthesized to load the dye into cells.

　Using a novel fluorescence indicator, DAF-2 DA, for direct detection of NO, we examined both acute rat brain slices and organotypic culture of brain slices to ascertain the NO production sites. The brain slices including hippocampus were loaded with 10μM of the dye for 30 min. The fluorescence intensity in the CAl region of the hippocampus was augmented significantly by the addition of NMDA (1 mM), comparing to the controls. This NO production in the CAl region was also confirmed in cultured hippocampus. This is the first direct evidence of NO production in the CA1 region. There were also fluorescent cells in the cerebral cortex after stimulation with NMDA.

　Imaging techniques using DAF-2 DA should be very useful for the clarification of neuronal NO functions.