movements, impaired balance, and loss of ambulation [2,35]. Early Golgi impregnation studies of neuron morphology identified dendritic branching losses in Layer 5 (L5) pyramidal cells of the motor cortex [11,15]. L5 pyramidal neurons are not a unitary class, however, and can be grouped into subtypes based on phylogeny, gene expression profiles, morphology, electrophysiol- ogy, and axonal projection targets [36–41]. Since MeCP2 mutations could potentially affect any of these features, the selection of a cell population for phenotypic analysis must take these properties into account. Transgenic labeling provides a convenient method for identifying some of these populations, as in the widely used YFP-H line (B6.Cg- Tg(Thy1-YFPH)2Jrs/J ([42] also [38,43–48]). YFP-H mice express yellow fluorescent protein under the Thy-1 promoter in a restricted set of L5 cortical neurons. In the motor-frontal cortex of YFP-H mice, YFP-expressing (YFP+) pyramidal neurons have electrophys- iological properties and patterns of synaptic connectivity that are distinct from both neighbouring non-YFP+ cells and from YFP+ cells in other cortical regions [38,40,49]. We crossed YFP-H mice with the ‘‘Jaenisch’’ (MeCP2J) mouse line, Mecp2tm1.1Jae/Mmcd, which has an in-frame N-terminal deletion of Mecp2 exon 3 [20]. A similar cross-breeding strategy was used for a different mutation, the protein-null Mecp2tm1.1Bird or ‘‘Bird’’ line (MeCP2B), in which Thy- 1-GFP-labeled L5 neurons revealed significant spine losses throughout the dendritic arbor [32]. The rationale for the current study was motivated in part by the erroneous initial classification of both MeCP2B and MeCP2J mice as harboring protein-null mutations [20,21]. In addition to our own immunohistochemical findings (unpublished data), multiple lines of evidence have emerged demonstrating that the MeCP2J line expresses a partly functional truncated MeCP2. These include the presence of stable MeCP2 mRNA transcripts, divergent gene expression profiles, and a milder phenotype in terms of brain weight, brain region volumes, and dendritic spine morphology [30,31,50–52]. In the context of these reports and previous analyses focused on L2/3 neurons in MeCP2J mice [28,29,31], we examined dendrite architecture and spine density in YFP+ L5 cells in the motor cortex of wildtype (WT) and MeCP2J mutant animals. Results YFP+ mutant neurons have selective reductions in dendrite length, branching, and spine density The large size of L5 pyramidal neurons and the density of YFP labeling in YFP-H mice precluded the imaging of entire cells, so 3D confocal fluorescence image stacks were obtained indepen- dently for dendrites in both the apical and basal compartments. Basal dendrite image stacks were centered on L5 YFP+ somata, while apical stacks were bounded by the pial surface, allowing visualization of the most distal branches of the apical tuft (Fig. 1A). Manual 3D reconstructions were made of the complete dendritic arbor in each compartment for all neurons passing exclusion criteria (Fig. 1B; see Methods). The largest differences we observed in YFP+ mutant cell morphology were in the basal arbor (Fig. 2C1–3). First, although very long individual dendrites were often observed in mutant neurons, the average maximum dendritic length was reduced by 9.1% (128.1 mm63.463 in mutant vs. 140.9 mm63.782 in WT, t8 = 2.490, P = 0.0375). 3D Sholl analysis revealed no difference in the number of primary dendrites in mutant neurons, but fewer bifurcations in a zone 40–70 mm from the soma (Fig. 2C1; one-way ANOVA, F14,112 = 3.908, P,0.001; t8.2.999, P,0.05). Two-way repeated measures ANOVA also revealed a significant interaction between branch order and genotype (Fig. 2C2; F6,28 = 6.849, Altered Dendrites in YFP-MeCP2 Mutant Mice Figure 1. 3D Sholl analysis of apical and basal arbors in YFP+ L5 motor cortical neurons. A) Confocal microscopic image stacks from 200 mm tissue sections showing YFP+ dendrites in apical (upper) and basal (lower) compartments (WT male mouse). Scale bar = 50 mm. B) Representative 3D traces from a WT neuron. 10 mm Sholl radii were used in the basal compartment and 20 mm radii in the apical compartment. The pial surface is shown as a dotted line with the apical Sholl origin defined as a point 300 mm from the pial surface. Depth in the z-axis is color-coded. doi:10.1371/journal.pone.0031896.g001 P = 0.0001). Mutant neurons showed a corresponding increase in the proportion of dendrite length within the first branch order (+5.93%; t6 = 5.053, P,0.01), and significantly less in the third branch order (23.5%; t6 = 2.987, P,0.05). Plotting the cumulative increase in total basal dendritic length vs. distance from the soma (Fig. 2C3) showed that the loss of higher-order branching close to the soma reduced the total dendritic length for MeCP2 mutant neurons by 18.6% (WT: 1192.0 mm661.54; Mut 969.8 mm665.54; t8 = 2.472, P = 0.0386). The largest divergence in cumulative dendritic length occured between approximately 40–130 mm from the soma. WT YFP+ neurons therefore maintain a larger number of dendritic branches in proximal portions of the basal arbor, as shown in reconstructions of representative ‘‘average’’ neurons from the Sholl analysis for each genotype (Fig. 2A–B). In the apical compartment the Sholl analysis was modified to accommodate the fact that secondary apical dendrites can extend over long distances roughly parallel to the pial surface without crossing a Sholl radius. We therefore measured the summed dendritic lengths per 20 mm radius. The Sholl origin was arbitrarily defined as a point on the apical dendrite 300 mm from the pial surface (Fig. 1B). In motor cortex this point is situated in cortical layer 3 in both mutant and WT mice, and corresponds to a distance of ,100–200 mm from L5A somata in the WT and slightly less in the mutant. Differences in the apical compartment were more subtle than those seen in basal dendrites (Fig. 2D1–3). Mutant neurons had up to 26.6% less summed dendritic length in Sholl radii 60 mm from the pial surface, corresponding to losses in the neuropil-dense cortical Layer 1 (Fig. 2D1; one-way ANOVA, F16,128 = 2.143, P = 0.01; Bonferroni post-hoc tests t8.3.174, P,0.05). This did not significantly change the total dendritic length in tuft branches (WT: 526.1 mm628.86; Mut: 472.9 mm622.07; t8 = 2.472, P = 0.1809), and we found no difference in the average maximum PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e31896