Altered Dendrites in YFP-MeCP2 Mutant Mice Figure 2. Dendritic branching and length is reduced in YFP+ MeCP2J mutant neurons. A, B) Reconstructions of representative WT and mutant YFP+ L5 pyramidal neurons based on the Sholl analysis branch patterns in C1 and D1. Scale bar (A) = 50 mm. C1) Basal compartment Sholl analysis showing the number of dendrite crossings as a function of distance from the soma. Mutant YFP+ neurons have significantly fewer dendritic branches 40–70 mm from the cell body. D1) Apical compartment Sholl analysis of the summed dendritic length per Sholl radius. Mutant YFP+ neurons have less total dendritic length per Sholl radius in L1–2. Dendrite lengths in radii greater than 300 mm correspond to long, laterally extending dendrites that are near and parallel to the pial surface. C2, D2) Percent dendritic length as a function of branch order in basal and apical compartments. The inset in (D2) shows branch order hierarchy. In basal dendrites only, mutant YFP+ neurons exhibit significantly reduced dendritic complexity with proportionally more dendritic length in lower branch orders. C3) A cumulative total length plot for basal dendrites shows that the main divergence in total dendritic length occurs less than ,120 mm from the soma. The average maximum dendritic length is also reduced in mutant YFP+ neurons. D3) A cumulative total dendritic length plot of apical tuft branches shows no minimal divergence except close to the pial surface, where mutant YFP+ cells extend fewer lateral branches. doi:10.1371/journal.pone.0031896.g002 length of individual apical dendrites. Similarly, no difference was observed when total length analysis was restricted to apical tuft branches within less than 100 mm of the pia (not shown). Although there was an interaction between branch order and genotype (F6,28 = 2.547, P = 0.0428), with mutant neurons tending to have a larger proportion of total dendritic length in primary and secondary branches, post-hoc tests found no significant differences at any specific branch order (Fig. 2D2). The cumulative average dendritic lengths only diverged close to the pial surface to a final difference of 10.3% less total apical length in mutant cells, although this effect was not statistically significant (Fig. 2D3). Overall, these findings suggest that, in contrast to the basal compartment, the MeCP2J mutation reduces higher-order branching in YFP+ L5 apical tuft dendrites in a relatively non-specific manner, with some reductions of lateral dendritic branching in L1. Representative apical tuft dendrites (reconstructed from the Sholl analysis) are shown in Fig. 2A and 2B. Any net losses in total dendritic length will reduce the total synaptic input for a given neuron, but this may be either mitigated or exacerbated by changes in dendritic spine density. To address this question we quantified the number of spines in the dendritic regions that showed the largest differences in the Sholl analyses, i.e. proximal basal dendrites and the apical tuft (Fig. 3A, 3D). Basal spine counts started after the first bifurcation (typically 20–50 mm from the soma) and apical tuft segments were not more than 60 mm from the pial surface. We also counted spines along the primary apical dendrite, 100–200 mm from the soma, and secondary oblique dendrites immediately projecting from the primary apical dendrite over this same region (Fig. 3B, 3C). Mutant neurons exhibited altered spine density, but only in some dendritic compartments (Fig. 3E). Spine density in the apical tuft was reduced by 47.4% (WT: 0.854 mm2160.0774; Mut: 0.449 mm2160.0261; t23 = 4.469, P = 0.0002); in secondary apical dendrites, density was reduced by 54.5% (WT: 1.751 mm2160.123; Mut: 0.797 mm2160.0657; t28 = 6.860, P,0.0001). In mutant primary apical and basal dendrites, however spine density remained unchanged (Primary apical, WT: 1.933 mm2160.167, Mut: 1.648 mm2160.0903; t28 = 1.623, P = 0.1159); (Basal, WT: 0.720 mm2160.0539, Mut: 0.756 mm2160.0733; t23 = 0.3716, P = 0.3716). These compart- ment-specific effects suggest that mutant neurons experience a significant net loss of synaptic input in the apical tuft, due to the compounded effects of both lower spine density and reduced dendritic branching. Thy-1-YFP expression is reduced in MeCP2J mutant mice In addition to the morphological differences in MeCP2J mutant YFP+ neurons, we also observed an unexpected reduction in the number of YFP+ cells in mutant animals relative to WT littermates. To further characterize these differences we performed a qualitative survey of cortical YFP expression patterns (Fig. 4). YFP expression varied in two significant ways (summarized in Fig. 4E). First, despite some inter-litter variability, the density of labeling in both genotypes followed a similar pattern of variation across cortical regions. In some cases these differences had sharp boundaries correspondent with functional divisions of the cortex [53]. The highest densities of YFP expression tended to occur in more rostral and medial regions (e.g. motor, cingulate, and retrosplenial cortex) while the lowest densities tended to occur in caudal and lateral regions (e.g. visual, ectorhinal, entorhinal, and piriform cortex). Second, YFP density appeared to be lower in MeCP2 mutants relative to WT littermates in most cortical regions. Some regions were more dramatically affected than others, such as visual cortex, which was usually marked by an almost complete absence of YFP+ cells (Fig. 4A, 4C). We quantified the percentage of YFP+ cells in three areas (frontal association, motor, and retrosplenial cortex) as a fraction of all fluorescent Nissl-stained L5 neurons (Fig. 4B). In all three regions, mutant mice had fewer YFP+ neurons (one-way ANOVA, F5,17 = 63.36, P,0.0001). The fraction of YFP+ neurons was decreased by 65.6% in frontal cortex, 65.3% in motor cortex, and 71.8% in retrosplenial cortex (t5.6.751, P,0.001 in all pairs) (Fig. 4F). The reductions occurred in spite of higher neuronal density in these same regions (Fig. 4D; one-way ANOVA, F5,12 = 3.689, P,0.0001), with density increases of 46% in frontal cortex, 32% in motor cortex and 24% in retrosplenial cortex. The reduced number of observed YFP+ cells could result from 1) more extensive transgene silencing in the mutant animals, with YFP+ neurons having similar expression levels in both genotypes or 2) overall downregulation of YFP expression levels in MeCP2 mutant animals, so that some neurons expressed YFP below detection limits. Neuronal transgene expression under the thy1.2 cassette begins during postnatal days P6–P10 [54], so we examined the developmental expression patterns of YFP in littermates from both genotypes at early postnatal stages to late maturity (1–18 wks) to compare these two alternatives. Representative examples are shown in Fig. 5. In both genotypes, we found large, bright individual neurons as early as P9. By P12–P14 the number of YFP+ cells increased sharply in WT, but remained low in MeCP2 mutants. YFP+ cells continued to increase in number in both genotypes until reaching stable mature levels at approximately one month of age. MeCP2 mutant mice maintained lower numbers of YFP+ cells at all later developmental stages. Similar confocal fluorescence imaging conditions (exposure, gain, laser intensity) were used in all cases, but increasing any or all of these parameters did not reveal any additional cells in either genotype. Further- more, while the somata of YFP+ L5 pyramidal cells were 24.1% smaller in mutant male mice (WT: 181.1 mm2611.1, Mut: 137.4 mm269.0; t9 = 3.097, P = 0.0128), there was no correlation between soma size and fluorescence intensity level in either genotype (r2,0.06471, d.f..41, both genotypes; data not shown). These results suggest that the reduced number of YFP+ neurons in mutant mice results from transgene silencing rather than as a consequence of a higher proportion of cells expressing YFP below detection limits. PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e31896