Dendritic branching and length is reduced in YFP⁺ MeCP2J mutant neurons.

<p>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 µm. 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 µm 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 µm 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 µm 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.</p

3D Sholl analysis of apical and basal arbors in YFP⁺ L5 motor cortical neurons.

<p>A) Confocal microscopic image stacks from 200 µm tissue sections showing YFP⁺ dendrites in apical (upper) and basal (lower) compartments (WT male mouse). Scale bar = 50 µm. B) Representative 3D traces from a WT neuron. 10 µm Sholl radii were used in the basal compartment and 20 µm radii in the apical compartment. The pial surface is shown as a dotted line with the apical Sholl origin defined as a point 300 µm from the pial surface. Depth in the z-axis is color-coded.</p

MeCP2J mutant mice have reduced numbers of YFP-expressing cortical neurons.

<p>A, C) Fewer YFP⁺ cells are observed across the entire cortex in mutant males (200 µm coronal sections, ∼ Bregma −2.2 mm in WT). Some cortical regions are more dramatically affected than others, such as visual cortex (red arrow) and auditory cortex (blue arrow). Sharp boundaries correspond to functional regions (Au, auditory; S, somatosensory; V, visual; RS, retrosplenial). Scale bar = 0.5 mm. Overall cortical YFP expression patterns are shown as a qualitative heat map in E), where white indicates few to no neurons and dark orange indicates the most dense cortical labeling. The number of YFP⁺ cells is generally higher in rostral and medial region in both genotypes. B) Fluorescent Nissl staining (red) of L5 motor cortex. A limited subset of pyramidal neurons express YFP. Scale bar = 100 µm. D) Neuronal density is increased in frontal (Fr), motor (M) and retrosplenial (RS) cortex in mutant mice. F) The percentage of YFP⁺ pyramidal neurons is reduced in three cortical regions (F, frontal; M, motor; RS, retrosplenial).</p

Developmental progression of cortical YFP expression.

<p>Left column: 200 µm coronal sections from YFP-H MeCP2J littermate pairs (∼ Bregma 0.14 mm in WT). The anterior commissure was used as a reference anatomical marker. Right column: maximum intensity projections of confocal fluorescence imaging z-stacks from cingulate cortex. The red box in P9 mutant (rotated 90° clockwise) corresponds to the region imaged in each brain. Single bright YFP⁺ L5 pyramidal neurons are observed in both genotypes as early as P9. By P12 YFP expression has been strongly upregulated in the WT, after which the number of YFP⁺ cells continues to increase more slowly until stabilizing to mature levels sometime between P21 and P40. The number of YFP⁺ cells in mutant males also continues to increase, but at a slower rate, and final levels remain below those seen in the WT. All confocal fluorescence images shown were obtained from the brain section shown except P9 WT, which was obtained from a tissue section 400 µm anterior due to sparse YFP labeling at this age. Left column scale bar = 0.5 mm. Right column scale bar = 100 µm.</p

Spine density is variably affected in different dendritic compartments in YFP⁺ MeCP2J mutant L5 pyramidal neurons.

<p>A–D) Spines were quantified in 20–30 µm segments in four dendritic compartments for WT and mutant YFP⁺ neurons. Scale bar (A) = 5 µm (all images). A) Apical tuft dendrites were sampled <60 µm from the pial surface. B) Primary apical dendrites were imaged between 100–200 µm from the soma. C) Secondary apical dendrites originating in the same region as (B) were sampled beginning at the branch point from the primary apical dendrite. D) Basal dendrite spine counts were initiated following the first dendritic bifurcation. E) Spine density is reduced by 47.4% in the apical tuft and by 54.5% in secondary apical dendrites.</p