Three dimensional analysis of dendrites of the CNS neurons.

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We have developed a method of extracting three dimensional information quantitatively from high voltage electron microscope stereo images. To improve the accuracy of the result, we introduced into Hitachi HVEM H-1250 a rotation free zoom optical system and a side entry rotation tilt stage with a feed back control mechanism for the tilt angle. The microscope is also equipped with an image intensifier in combination with a real time image processor to reduce the beam damage to the specimen.Number, length and diameter of dendritic spines of the granule cell in the rat dentate gyrus were measured with the aid of this system. 5 m thick Golgi preparations were used for the measurement.Spine densities of 3.4, 2.3, and 2.0 per 1mu m at proximal (P), middle (m), and distal (d) portion of the dendrite respectively were obtained, which were 1.6-2.0 fold of the previous light microscopical report. Since the diameter of the dendrite decreases from proximal to distal portion, the spine densities per unit surface area of the dendrite was about equal, 0.8/mu m^2, throughout the dendritic tree.Spines of various shapes , calyx shape,mushroom shape, club shape , thin thread-like etc. were found , however the large stubby spines which were reported to be 20-40 % by light microscopy were rare, less than 2%. Mean three dimensional spine length were 1.25mu m, 1.26mu m, and 1.24 mu m at p, m, and d portions respectively, which were about 1.4 times longer than those measured in two dimensions.The surface area of the spines were measured using morphological parameters , and it was found that the total surface areas of the dendrite was doubled by the addition of the spines at each dendritic portion.It became evident that the resolution of the light microscope was not sufficient for the precise morphometry of the dendritic spine.