Quantitatve analysis of GABAergic neurons in the mouse limbic system using confocal laser scanning microscopy.

GABA能神经元可以分为几个化学定义的亚群。特别是，一些钙结合蛋白，如小清蛋白（PV），钙结合蛋白（CaBP），钙视网膜蛋白（CR）已被证明是位于特定的神经元亚群。本研究采用激光共聚焦扫描显微镜（CLSM）和无偏立体计数法（unbiased stereopogical counting method）对小鼠海马结构神经元亚群的分布进行定量研究。将切片进行荧光三重染色，即针对不同Ca结合蛋白进行双重免疫染色，并用碘化丙啶（PI）染色。在配备XY载物台的CLSM（MRC 1000，Bio-Rad）中，使用荧光染色切片的随机选择区域中获得三个系列的光学图像。 ...更多信息 三道激光束用宏程序设计语言（MPL）编写程序，自动控制XY载物台的步进运动和CLSM图像的采集。采用PI标记的光学切片，体视学方法测定海马各亚区神经元的数密度。将PI标记的光学切片与无偏计数框叠加，并使用个人计算机（Macintosh 8100，Apple）根据光学剥离器原理进行分析。无偏取样的神经元的化学性质可通过免疫标记的光学切片来确定，含PV的神经元的数密度极低，每个淋巴管中小于4，000个/mm^3。CaBP阳性神经元主要分布于齿状回颗粒细胞层（GL）和海马CA 1区皮层（SP）。CA 1的GL和SP中CaBP神经元的密度分别为550，000个/mm^3和100，000个/mm^3，分别约占所有神经元的60%和30%。齿状回门区CR神经元数量较多。CR神经元的密度为180，000个/mm^3，约占门区所有神经元的20%。少

GABAergic neurons can be classified into several chemically defined subpopulations. In particular, some calcium-binding proteins such as parvalbumin (PV), calbindin (CaBP), and calretinin (CR) have been shown to be located in specific subpopulations of neurons. The object of this study was to establish the efficient method for quantification of the distribution of these neuronal subpopulations using optical imeges obtained with confocal laser scanning microscope (CLSM) and unbiased stereopogical counting method.The mouse hippocampal formation was cut into 50 mum thick sections on a vibratome at the plane randomly selected along the septo-temporal axis of the hippocampus. The sections were fluorescently triple-stained, that is, double-immunostained for different Ca-binding proteins and stained with propidium iodide (PI). Three series of optical images in the randomly selected fields of the fluoro-stained sections were obtained in CLSM (MRC 1000, Bio-Rad) equipped with a XY-stage using t … More hree channels of laser beams. The stepwise movement of the XY-stage and collection of CLSM images was controlled automatically using the program writen with Macro Programming Language (MPL). The numerical density of the neurons in the each layrs of the hippocampal subdivisions was estimated stereologically using series of PI-labeled optical sections. The PI-labeled optical sections were superimposed with unbiased counting frame and analyzed in accordance with optical disector principle using a personal computer (Macintosh 8100, Apple). The chemical nature of the neurons unbiasedly sampled was determined by examinning the immuno-labeled optical sections.The numerical density of the PV-containing neurons was extremely low, and less than 4,000/mm^3 in every lyer. CaBP-containing neurons were pariculary numerous in the granule cell layr (GL) of the dentate gyrus and stratum pyramidale (SP) of the CA1 region. The density of CaBP neurons in the GL and SP of the CA1 were 550,000/mm^3 and 100,000/mm^3, respectively, which were about 60% and 30% of all neurons, respectively. CR neurons were numerous in the hilus of the dentate gyrus. The density of CR neurons was 180,000/mm^3, which was about 20% of all neurons in the hilus. Less