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

钙离子是细胞内的第二信使，参与各种细胞活动。它调节神经系统中神经递质的释放，被认为是突触长期增强的最重要因素。因此，研究细胞内钙离子的动力学是理解细胞功能的必然要求。本研究的重点是细胞内钙离子的动力学，并试图同时研究其与细胞的电物理行为。1987年，我们发明了一种荧光显微系统，使我们能够以足够的时间分辨率测量细胞内钙动力学。它的一个应用是分析由缓激碱产生的神经母细胞瘤细胞系的钙反应。二是分析大鼠垂体下原代细胞培养过程中钙离子在细胞内的释放机制。在这两种制备中，我们都成功地记录了细胞内钙的反应。1988年，我们改进了我们的系统，使我们能够同时从多个点测量Ca响应。用这种方法，我们分析了一种毒蕈碱激动剂对鸡毛细胞产生的钙反应。传出神经支配内耳毛细胞已有40年的历史，但其确切的作用方式尚不清楚。我们在毛细胞水平上明确了传出抑制性神经支配的潜在机制。该系统的一个缺点是，当用于多点测量时，时间分辨率相对较慢。它具有10毫秒的时间分辨率，从单个点测量时足以满足大多数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。所有系统的开发都是与奥林巴斯显微镜公司合作进行的。

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.