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

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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