Confocal image stacks were flattened into maximum intensity projections (MIPs) prior to cell counts using the ImageJ Cell Counter plugin (Kurt De Vos, University of Sheffield). Cells occluded by the image boundary or lacking a nucleus clearly delineated by the Nissl stain were not counted, to eliminate partially imaged cells from above and below the planes included in the MIP. For YFP cell counts, YFP+ cells whose Nissl stains did not fit these criteria were similarly excluded. Qualitative analysis of YFP expression patterns in the cortex used five WT/mutant littermate pairs (11–13 wks). Entire brains were cut in 200 mm coronal sections (excluding the olfactory bulbs and cerebellum) and serially mounted. Due to the marked volume reduction in the mutant mouse brains relative to WT, rostrocaudal position was established with reference to distinctive anatomical features visible in the coronal plane instead of Bregma, with reference to coronal plates in the Paxinos Mouse Brain Atlas [53]. These included: fusion of the medial orbital cortex to the anterior olfactory bulb; disappearance of the rhinal fissure; fusion of the genu of the corpus callosum; emergence of the anterior commissure and third ventricle; appearance of the dentate gyrus; and appearance and separation of the medial mammillary nucleus. After establishing the range of tissue sections over which a cortical region occurred each region was assigned a score from 0 to 4 as a qualitative estimate of the number of large YFP+ pyramidal cells. A score of 0 reflected very few or no cells and 4 indicated very dense labeling. A median value of the score for each cortical region was taken from all mice within each genotype and plotted using a heat map, where white represents few to no cells and dark orange represents the highest observed density of YFP-expressing neurons. Soma traces and dendritic analyses were performed on five F1 WT/mutant littermate pairs (8–13 wks). Neuron reconstructions and soma traces were performed by a single observer blind to genotype. All 3D confocal image stacks of motor cortex were obtained from coronal tissue sections rostrally bounded by the first appearance of the corpus callosum and caudally by appearance of the anterior commissure, corresponding to a rostrocaudal depth of ,2 mm. Soma traces were performed using ImageJ. YFP+ somata were outlined in the focal plane where each cell had its maximum in- focus area. Partially occluded cell bodies at the image boundaries were not used. Prior to outlining each cell, the image was zoomed 126. Post-hoc analysis of the mean signal:background ratio defining the cell boundary was determined by examining 20 neurons randomly selected from previous traces in two YHM littermates. The average YFP fluorescence intensity at the cell boundary was 7.86 background intensity and not less than 36. Apical and basal dendrite tracings were performed on confocal image stacks using Igor Pro 6.05 software (Wavemetrics) with the Migor 4 custom analysis plugin developed by Jamie Boyd (UBC). 3D traces of apical or basal compartments were generated in Migor using a Cintiq 21 UX interactive pen display (Wacom). Apical and basal compartments were separately imaged to allow adequate resolution of fine dendritic branches in YFP+ neurons. For basal compartments, image stacks were oriented such that somata of YFP+ Layer 5 pyramidal neurons were centered along the y- axis, to permit capture of the maximum length of dendritic branching possible, as well as a minimum of 100 mm of the apical dendrite. Somata were selected from a sub-volume of the z-stack defined by a zone of exclusion comprising ,100 mm from the outer edges of the x and y axes and the central 100 mm within the z-axis. At 406 magnification the field of view in the x-y axes was 2 318 mm . Due to variable shrinkage in the z-axis during the mounting process the excluded z-axis guard zones were of variable length but typically ,25 mm. Traces of the first 100 mm of the Altered Dendrites in YFP-MeCP2 Mutant Mice primary apical dendrite (and any secondary branches) were included as part of the basal compartment. For apical compart- ment traces, the pial surface of the brain was aligned parallel and contiguous with the upper boundary of the x-axis in the image plane, permitting capture of apical dendrite into deep Layer 3. A standard reference point for comparing dendritic structures was established by initiating all traces 300 mm from the pial surface. Traces were not included if obvious truncations of lateral branches occurred. Statistical analysis of the 3D Sholl data from individual neuron reconstructions used a repeated measures ANOVA with genotype and distance from origin as predictor variables and number of dendrite crossings per Sholl radius (basal) or summed dendrite length per Sholl radius (apical) as the response variables. Differences at specific radii were analysed using Bonferroni post- hoc t-tests. In the basal compartment, 3D Sholl cross analysis was performed by counting the number of dendrites intersecting concentric spherical radii at 10 mm intervals. In the apical compartment, Sholl lengths were measured by summing all of the segments of dendrite length occurring within each 20 mm Sholl radius. Maximum dendritic length was defined as the largest Sholl radius crossed by at least one dendritic segment. Total cumulative dendrite length measurements were the summed length of all dendrites in each of the basal and apical compartments. Percent dendritic length was calculated as a function of branch order in each compartment. Branch order was defined by the number of branch nodes occurring following a given primary node, such that dendrites with fewer branches would have proportionally more branch length in lower branch orders. Dendritic spine counts were performed using confocal fluores- cence image stacks up to 10 mm deep in the z-axis. Image stacks were smoothed using a 2.0 pixel median filter prior to analysis. Spine counts were obtained from 20–30 mm dendritic segments of randomly selected neurons using the ImageJ Cell Counter plugin. Spines projecting directly above and below the dendrite in the z- axis were not counted. In regions of high spine density (e.g. WT primary apical) potentially overlapping spines were identified by vertically scrolling through confocal z-stacks. Due to the difficulty of unequivocally distinguishing filopodia from long thin spines, spine counts included all types of dendritic protrusions. Tissue sections were obtained from three WT/mutant littermate pairs (11–13 wks). 15–20 cells were analyzed per animal, with total spine counts of 1641 (WT) and 1440 (mutant). Spine density was calculated by dividing the total spine count by the length of dendrite analyzed. Representative images in Fig. 3 were rotated, cropped and adjusted for brightness and contrast. Statistical Analysis Statistics were performed using the Migor 4 plugin (Jamie Boyd, UBC) for Igor Pro (Wavemetrics), Prism 5 v.5.0b (Graphpad Software) and R v.2.9.2 (WU). Unless otherwise stated, all parameters were analyzed using the two-tailed unpaired student’s t-test and results considered significant at P,0.05, with measure- ments reported as mean 6 SEM. Bonferroni’s Multiple Compar- ison Test was used for pairwise comparisons for all ANOVAs. Dataset distributions were tested for normality using D’Agostino & Pearson omnibus normality test. Figures were prepared using Prism 5, ImageJ, and Photoshop CS3 v.10.0 (Adobe; San Jose, CA). Author Contributions Conceived and designed the experiments: DPS JDB DBL KRD. Performed the experiments: DPS. Analyzed the data: DPS KRD. Contributed reagents/materials/analysis tools: JDB DBL KRD. Wrote the paper: DPS. PLoS ONE | www.plosone.org 9 March 2012 | Volume 7 | Issue 3 | e31896