Development of fluorometric microscope system and its application to intracellular Ca^<2+> measurements in neurons

钙离子作为细胞内的第二信使参与细胞的各种活动。它调节神经系统中神经递质的释放，被认为是突触长时程增强中最重要的因素。因此，研究细胞内Ca ~（2+）的动态变化对了解细胞功能具有重要意义。这项研究的重点是细胞内钙离子的动力学，并试图同时研究其行为与electrophysicological behaviors.In 1987年，我们已经发明了一种荧光显微镜系统，使我们能够测量intracelluar钙动力学与足够的时间分辨率。其应用之一是分析由缓激肽产生的神经母细胞瘤细胞系的Ca反应。另一部分是对大鼠海马原代培养细胞内钙释放机制的研究。在这两种制备中，我们都成功地记录了细胞内Ca反应。在1988年，我们改进了我们的系统，使我们能够测量钙反应从多个点连续。通过这种方法，我们分析了由毒蕈碱激动剂对鸡毛细胞产生的钙反应。传出神经支配在内耳毛细胞上已知已有40年，但其确切的作用模式尚不清楚。我们在毛细胞水平上阐明了传出抑制性神经支配的潜在机制，但该系统的缺点是用于多点测量时时间分辨率相对较低。它具有10毫秒的时间分辨率，并且当从单个点测量时足以用于大多数Ca响应。我们现在正在改进这种时间分辨率，特别是当从多个点测量荧光时。整个系统的开发都是与奥林巴斯显微镜公司合作完成的。

Ca ion is known as an intracellular second messenger in a various cell activities. It regulates a release of neurotransmitter in the nervous system, and is considered as the most important factor in the long term potentiation of a synapse. It is therefore innevitable to study the dynamics of intracellular Ca ions in understanding cellular functions. This research is focusing on the dynamics of intracellular Ca ions and is trying to study its behaviour simultaneously with electrophysiclogical behaviour of the cell.In 1987, we have invented a fluorescence photomicroscope system which enabled us to measure intracelluar Ca dynamics with sufficient time resolution. One of its aplication was to analyze the Ca response of a neuroblastoma cell line which was generated by bradykinine. The other is the analysis of intracellular Ca release mechanism studied in the hypocampus primary cell culture of a rat. In both preparation we have succeeded in recording intracellular Ca response. In 1988, we have improved our system and enabled us to measure Ca responses from multiple points simultanously. By this method, we have analyzed the Ca responses generated by a muscarinic agonist on the chick hair cell. Efferent innervation is known for 40 years on the inner ear hair cell, but its exact mode of action is not yet known. We have made it clear the underlying mechanism in the efferent inhibitory innervation on the level of hair cells.A drawback of this system is a relatively slow time resolution when used for multiple spots measurements. It has 10 msec time resolution and is sufficient for most Ca response when measured from a single spot. We are now improving this time resolution particularly when fluorescence are measured from multiple spots. All the development of system was under cooperation with Olympus Microscope Company.