Spine density measurements consisted of marking all of the spines on two randomly selected Order 2 branches (due to their proximity to the soma) as well as two randomly selected Order 4 branches (due to their distal nature from the soma) for each cell.. Only branches over 20μm in length were included in the study. Spine density was calculated per 10μm of dendritic length. Finally, spine phenotyping was done on the first 7–13 spines of each order 2 and order 4 branches for a total of between one hundred to 110 phenotyped spines per animal. Spines were classified, based on the principles outlined in Irwin et al, 2002, as either immature or mature according to their shapes (Figure 2).The total density of mature versus immature spines was then calculated for each animal. In addition, at the site of each spine included in analysis, the dendritic width was measured to ensure that the thickness of dendritic branches did not differ between any of the three animal groups. No significant differences existed between groups in the width of dendrites used for spine phenotypes.
