Dendritic spines were three-dimensionally reconstructed (Figures 1F and 2) with the aid of Reconstruct software (Reconstruct 1.0.5.7; available from http://synapses.mcg.edu/; (Fiala, 2005 (link))). Independent traces were drawn for the neck and head of each spine, and three-dimensional distances were measured for the spine length, neck length and neck diameter of spines located at a distance of 13–127 μm from the cell body. Since the neck diameter was not constant, an average diameter was calculated from three measurements obtained proximal, intermediate and distal to the insertion of the spine to the dendrite. Depending on the angle of visualization of the spine, sometimes the head was difficult to distinguish from the neck. In these cases, the border between the head and the neck was traced after rotating the spine in order to view the spine at different angles (Figure 2B). In 14 spines, it was not possible to distinguish a clear head (Figure 2). Since the head usually presented an irregular shape (Figures 1F and 2), an average head diameter was estimated from the measured head volume and length, considering the head as a cylinder (head diameter = 2× square root (head volume/head length/π)). Volumes were measured directly from the three-dimensional reconstructions.
Synapses were identified by the presence of a PSD facing an axon terminal with synaptic vesicles (Figure 1E). Synapses were most frequently cut transversally and were traced as lines that followed the length of the PSD, while those cut tangentially were traced as closed contours to obtain a consistent estimation of synaptic surface area. Tangentially cut postsynaptic densities frequently appeared in adjacent sections with overlapping distribution. In these cases, redundant regions were traced only in one section to avoid overestimation of the surface area. In addition, the average diameter of the PSD was calculated from the values of PSD surface area, assuming a circular shape of the PSD.
The presence of perforations and segregations in the PSD (Figure 1E) allowed the classification of synapses as simple or complex. PSDs resembling disks were considered simple and will be referred as macular, while complex PSDs were considered those with a single or multiple perforations in the PSD, or a segregation of the PSD associated to a single axon terminal.