Establishment of Experimental Method for Monitoring Intracellular Ca^<2+> Concentration

最近的研究表明，细胞内钙离子在哺乳动物中枢神经系统的各种神经元功能和神经细胞死亡中起着重要作用。因此，了解细胞内钙离子浓度在不同条件下的变化，不仅在基础神经科学领域，而且在临床上对阐明神经元损伤的机制及其预防具有重要意义。在本研究项目中，主要目的是建立一种在单细胞基础上测定[Ca~(2+)]i的方法。首先，取成熟胎鼠的海马体或纹状体，进行细胞分离培养。体外培养约14天后，用细胞测定[Ca^&lt；2+&gt；]i。将钙指示剂Fura-2载入神经元。将细胞安装在荧光倒置显微镜上。光源为氙灯，干涉滤光片波长为340 nm和360 nm。通过…获得了显微图像更粗糙的是硅强化的目标摄像机。将一根光纤垂直固定在细胞图像上，光纤的另一端与光电二极管相连，光电二极管的输出被放大、积分并用笔式记录器记录。此外，结合自动图像分析仪Argus II，以图像的形式检测细胞内[Ca^&lt；2+&gt；]i的变化，并将该方法应用于神经药理学研究，即检测细胞在低氧暴露后对各种神经递质的[Ca^&lt；2+&gt；]i的变化以及这些反应的异常。生长抑素被认为是一种贯穿神经和内分泌系统的抑制性神经肽。然而，我们获得了生长抑素诱导培养的大鼠海马神经元[Ca~(2+)&Gt；]i升高的数据。生长抑素作用的钙来源是细胞外液的流入。用刀豆蛋白GVIA进行的实验表明，生长抑素受体与大鼠海马区的N型钙通道偶联。此外，还证实了CCK以同样的机制使培养的大鼠纹状体[Ca^&lt；2+&gt；]i升高。由于细胞内[Ca^&lt；2+&gt；]i的调节是信号转导的重要部分之一，因此该方法有助于观察神经元内[Ca^&lt；2+]i的变化，了解细胞的功能。较少

Recent studies have demonstrated that the intracellular Ca^<2+> plays an important role in various neuronal functions and neuronal cell death in the mammalian central nervous system. Therefore, it is significant to understand how intracellular Ca^<2+> concentration changes under various conditions ([Ca^<2+>]i) not only in the field of basic neuroscience but also clinically to elucidate mechanisms of neuronal impairments and their prevention. In this research project, the primary purpose was to establish a method for measuring [Ca^<2+>]i on the single cell basis. First, either the hippocampus or striatum was removed from matured fetal rats and dissociated cell cultures were done. After about 14 days in vitro, cells were used for measurement of [Ca^<2+>]i. A Ca^<2+> indicator, fura-2, was loaded into neurons. Cells were mounted on a fluorescence inverted microscope. A xenon lamp as a light source and interference filters for 340 nm and 360 nm were used. Microscope images were obtained th … More rough a silicon-intensified target video camera. An optic fiber was vertically fixed on the cell image, and the other end of the fiber was connected to a photodiode, the output of which was amplified, integrated and recorded with a pen recorder. In addition, an automatic image analyzer, ARGUS II, was combined and changes of [Ca^<2+>]i were detected as image.At the next step, we applied the method to neuropharmacological studies, that is, changes of [Ca^<2+>]i in response to various neurotransmitters and abnormalities of these responses after hypoxic exposure of cells were detected. Somatostatin has been considered to be an inhibitory neuropeptide throughout the nervous and endocrine systems. Nevertheless, we obtained the data that somatostatin induced an increase of [Ca^<2+>]i in cultured rat hippocampal neurons. The Ca^<2+> source for the effect of somatostatin was an influx from extracellular fluids. From experiments using - conooxin GVIA, it was indicated that somatostatin receptors couple with N-type Ca^<2+> channels in the rat hippocampus. In addition, it was demonstrated that cholecystokinin increased [Ca^<2+>]i in cultured rat striatum in the same mechanism.This method is useful for observing changes of [Ca^<2+>]i in neurons and understanding the cellular functions, since regulation of intracellar [Ca^<2+>]i is one of the most important parts of signal transduction. Less