Analysis of dendritic spines in rat CA1 pyramidal cells intracellularly filled with a fluorescent dye

The dendritic branching pattern and the distribution of dendritic spines were studied in hippocampal neurones with an improved technique. In slices taken from adult Wistar rats, CA1 pyramidal cells were filled with Lucifer yellow and examined under a laser-scanning confocal microscope. The basal dendrites were found evenly distributed inside a regular cupola-shaped volume. Their total length was about 4,500 microns. The branches divided between one and three times, with the initial segments comprising less than 2%, and the long terminal segments (mean length, 119 microns) including more than 80% of the total length of the basal dendrites. The apical dendritic branches emerged obliquely from the main shaft, ran for a distance of 50 to 250 microns, and made up a total length of about 5,100 microns in stratum radiatum and between 1,100 and 3,200 microns in stratum lacunosum-moleculare. The mean total length of the dendritic tree was 11,900 microns. All values were corrected for shrinkage. Shrinkage was measured in three dimensions and was 20.2% in the horizontal (x/y) plane and 40.9% in the vertical (z) plane. Both the basal and the apical dendritic branches were covered by regularly spaced spines. When corrected for dehydration-induced shrinkage and for hidden spines, the density was 1.80 and 2.00 spines/microns dendritic length for the basal and apical dendritic branches, respectively. Apart from the initial parts of the branches, which had few or no spines, the spines were remarkably evenly spaced. In particular, the distance between spine heads was significantly different from a random distribution, suggesting a regulatory process for the spacing of spines.