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

细胞内Ca^<2>浓度监测实验方法的建立

基本信息

项目摘要

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
近年来的研究表明,细胞内Ca^2+在哺乳动物中枢神经系统的各种神经功能和神经细胞死亡中起着重要作用。因此,了解细胞内Ca^2+浓度在不同条件下的变化([Ca^2+]i),不仅在基础神经科学领域,而且在临床上对于阐明神经元损伤的机制和预防都是有意义的。本研究的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,从成熟的胎鼠中取出海马或纹状体,并进行分离的细胞培养。在体外培养约14天后,使用细胞测量[Ca^<2+>]i。将钙离子指示剂fura-2加载到神经元中。将细胞固定在荧光倒置显微镜上。使用氙灯作为光源,并使用340 nm和360 nm的干涉滤光片。显微镜图像是通过 ...更多信息 一台硅增强靶摄像机。将光纤垂直固定在细胞图像上,光纤的另一端连接到光电二极管,光电二极管的输出被放大、积分并用笔式记录器记录。此外,还结合ARGUS Ⅱ图像分析仪,以图像形式检测细胞内[Ca^<2+>]i的变化,并进一步将其应用于神经药理学研究,即检测细胞内[Ca^<2+>]i对各种神经递质的反应变化及缺氧后这些反应的异常。生长抑素被认为是一种遍及神经和内分泌系统的抑制性神经肽。然而,我们获得的数据表明,生长抑素诱导培养的大鼠海马神经元[Ca^<2+>]i增加。生长抑素作用的Ca^<2+>来源是细胞外液的内流.用α-芋螺毒素GVIA进行的实验表明,在大鼠海马中生长抑素受体与N型Ca^<2+>通道偶联。此外,胆囊收缩素也以同样的机制使培养的大鼠纹状体内[Ca^<2+>]i增加,这种方法有助于观察神经元内[Ca^<2+>]i的变化和了解细胞功能,因为细胞内[Ca^<2+]i的调节是信号转导的重要组成部分之一。少

项目成果

期刊论文数量(37)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Kito,S.: "Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography" The Journal of Histochemistry and Cytochemistry. 38. 1725-1731 (1990)
Kito,S.:“通过体外放射自显影研究年龄对大鼠脑中 NMDA 受体复合物的影响”《组织化学和细胞化学杂志》。
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S.Murakami: "The colocalization of substance P-and somatostatin-like peptides in neurons of the entopeducular nucleus of rats" Peptides. 10. 973-977 (1989)
S.Murakami:“P 物质和生长抑素样肽在大鼠内髓核神经元中的共定位”肽。
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Miyoshi, R., et al.,: "Age-related changes of strychnine-insensitive glycine receptors in rat brain studied by in vitro autoradiography" Synapse. 6. 338-343 (1990)
Miyoshi, R. 等人:“通过体外放射自显影研究大鼠大脑中士的宁不敏感甘氨酸受体的年龄相关变化”突触。
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R.Miyoshi: "Basal Ganglia III" Plenum Press, (1990)
R.Miyoshi:“Basal Ganglia III”Plenum Press,(1990)
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Miyoshi, R., et al.,: "Quantitative autoradiographic localization of the M_1 and M_2 subtypes of muscarinic acetylcholine receptors in the monkey brain" Japanese Journal of Pharmacology. 51. 247-255 (1989)
Miyoshi, R. 等人:“猴脑中毒蕈碱乙酰胆碱受体 M_1 和 M_2 亚型的定量放射自显影定位”,《日本药理学杂志》。
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