Development of the basic system for the stereological analysis in the brain

大脑体视分析基本系统的开发

基本信息

批准号：
05558093
负责人：
KOSAKA Toshio
金额：
$ 9.73万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Detailed structural analyzes, especially quantitative analyzes, are essential to understanding the neuronal organization of the central nervous system and its function. In the present research project we have tried 1) to establish the bases for the stereological analyzes, particularly optical disector analysis, in the central nervous system and 2) to establish the methods of image analyzes for evaluating the immunoreaction intensities of individual somata and synaptic terminals in brain samples at the light microscopic level.For the optical disector analysis, we stained 50 mm thick sections fluorescent immunocytochemically, and examined by means of a confocal laser scanning light microscope (CLSM). The penetration of immunostaining from the section surfaces was carefully evaluated and the conditions of samples which can be used for the optical disector analysis were established. Then the established methods allowed us to analyze various cell groups defined chemically by means of conventional light microscope as well as CLSM.The methods for evaluating the intensities of immunoreactivities in individual elements such as neuronal somata and synaptic boutons were developed and applied on determining the distributions of two isozymes of GABA synthesizing enzymes, GAD 67 and GAD 65, in hippocampal GABAergic neurons. Images were acquired by TV camera attached to conventional light microscope for the analyzes of somata and by CLSM for the analyzes of synaptic boutons. Relative reaction intensities on individual elements were analyzed by personal computer using an image analysis software NIH Image.

详细的结构分析，尤其是定量分析，对于了解中枢神经系统的神经元组织及其功能至关重要。在目前的研究项目中，我们尝试：1）建立中枢神经系统体视学分析，特别是光学解剖分析的基础，2）建立评估个体体细胞和突触末梢免疫反应强度的图像分析方法对于光学解剖分析，我们对 50 毫米厚的切片进行荧光免疫细胞化学染色，并通过共焦激光扫描光学显微镜 (CLSM) 进行检查。仔细评估了免疫染色从切片表面的渗透，并建立了可用于光学解剖分析的样品条件。然后，所建立的方法使我们能够通过传统光学显微镜和 CLSM 分析化学定义的各种细胞群。开发了评估神经元体和突触布顿等单个元素的免疫反应强度的方法，并将其应用于确定分布海马 GABA 能神经元中两种 GABA 合成酶 GAD 67 和 GAD 65 的同工酶。通过连接到传统光学显微镜的电视摄像机获取图像以分析体细胞，并通过 CLSM 获取图像以分析突触按钮。使用图像分析软件NIH Image通过个人计算机分析各个元素的相对反应强度。