Neuroanatomy in mouse models of Rett syndrome is related to the severity of Mecp2 mutation and behavioral phenotypes

Background Rett syndrome (RTT) is a neurodevelopmental disorder that predominantly affects girls. The majority of RTT cases are caused by de novo mutations in methyl-CpG-binding protein 2 (MECP2), and several mouse models have been created to further understand the disorder. In the current literature, many studies have focused their analyses on the behavioral abnormalities and cellular and molecular impairments that arise from Mecp2 mutations. However, limited efforts have been placed on understanding how Mecp2 mutations disrupt the neuroanatomy and networks of the brain. Methods In this study, we examined the neuroanatomy of male and female mice from the Mecp2tm1Hzo, Mecp2tm1.1Bird/J, and Mecp2tm2Bird/J mouse lines using high-resolution magnetic resonance imaging (MRI) paired with deformation-based morphometry to determine the brain regions susceptible to Mecp2 disruptions. Results We found that many cortical and subcortical regions were reduced in volume within the brains of mutant mice regardless of mutation type, highlighting regions that are susceptible to Mecp2 disruptions. We also found that the volume within these regions correlated with behavioral metrics. Conversely, regions of the cerebellum were differentially affected by the type of mutation, showing an increase in volume in the mutant Mecp2tm1Hzo brain relative to controls and a decrease in the Mecp2tm1.1Bird/J and Mecp2tm2Bird/J lines. Conclusions Our findings demonstrate that the direction and magnitude of the neuroanatomical differences between control and mutant mice carrying Mecp2 mutations are driven by the severity of the mutation and the stage of behavioral impairments.


Background
Rett syndrome (RTT) is a neurodevelopmental disorder caused, in over 90% of cases, by sporadic mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2) [1]. Although boys carrying MECP2 mutations do not typically survive infancy, girls experience a period of normal development between 6 and 18 months of age, followed by a decline in fine motor coordination skills, speech, autonomic irregularities, cognitive abilities, and stereotypic *Correspondence: rylan.allemang-grand@sickkids.ca 1 Mouse Imaging Centre, 25 Orde Street, M5T 3H7 Toronto, Ontario, Canada 2 Neurosciences and Mental Health, Hospital for Sick Children, 555 University Ave, M5G 1X8 Toronto, Ontario, Canada Full list of author information is available at the end of the article hand movements [2][3][4][5]. These behavioral impairments have been associated with an overall decrease in brain weight along with structural disruptions at the cellular level, such as decreased dendritic length, reduce spine density and cell body size that have been observed posthumous in female RTT patients [6][7][8]. However, the type and location of MECP2 mutation influence the severity of these phenotypes, with disruptions within the functional domains of MECP2 as well as mutations near the N terminus leading to more detrimental outcomes than mutations that occur near the C terminus [9][10][11].
To further understand RTT, mouse models have been engineered to tease apart how Mecp2 disruption affects the brain and behavior. Similarly to humans, Mecp2-null mice experience a period of normal development followed by a loss of motor control, disrupted autonomic regulation and impaired learning and memory [12,13]. Further investigation into the biology of these models has highlighted the importance of Mecp2 within the brain where it mediates transcription through epigenetic modifications to the chromatin structure [14]. Mice that lack functional Mecp2 have fewer and weaker excitatory synaptic connections [15,16] and impaired synaptic plasticity [13,17,18]. Additionally, loss of Mecp2 within the norepinephrine [19], dopaminergic, and serotonergic [20] neurotransmitter systems and within the hypothalamic [21], interneuronal [22,23], and astroglia [24,25] cellular populations leads to circuit-specific impairments in the brain and behavior. Along with the heterogeneity of cellular and molecular phenotypes, region-specific differences in excitability have been found across the brain [16,26]. The culmination of these findings suggest that rather than targeting a specific cell type or neurotransmitter system, Mecp2 plays a global role that is integral to normal brain structure and function.
Continued efforts into understanding the pathophysiology caused by Mecp2 disruption requires emphasis on system-wide metrics in order to identify key nodes and networks of susceptibility. Magnetic resonance imaging (MRI) has the ability to acquire high-resolution, neuroanatomical information across the mouse brain [27][28][29]. Additionally, the high spatial resolution of MRI and the statistical analyses applied to the acquired images has previously been used to phenotype structural differences in mouse models of neurodegenerative and developmental disorders [30][31][32][33][34] and is sensitive to subtle changes in neuroanatomy following training on learning and memory paradigms [35,36]. Thus, MRI is an important tool for localizing and quantifying anatomical differences across the brain and may reveal important insights into RTT when used in mouse models with Mecp2 mutations.
Previous MRI studies of human RTT patients have identified volume loss in frontal gray matter, basal ganglia, substantia nigra, midbrain, cerebellum, and brainstem [37][38][39]. MRI studies of Mecp2-null mice also show volume reductions in many of the same regions as humans, suggesting that these models recapitulate the gross anatomical impairments of the human phenotype [40,41]. However, these studies limited their analyses to a few regions of interest only providing a snapshot of the profile of volumetric changes within the Mecp2 disrupted brain.
To overcome this limitation, we examined male and female mice from three Mecp2 mouse models using whole-brain anatomical MRI sequences paired with deformation-based morphometry to identify the regions of the brain affected by Mecp2 disruption. To determine how Mecp2 mutation severity affects brain structure, we included Mecp2 mouse models with severe phenotypes caused by silencing of the Mecp2 gene by the complete removal of exon 3 / 4 (Mecp2 tm1.1Bird/J [42]) and via a STOP-neomycin cassette within intron 2 (Mecp2 tm2Bird/J [18]) with mice possessing less severe phenotypes driven by a truncation mutation at amino acid 308 which eliminates the C-terminal end of Mecp2 (Mecp2 tm1Hzo [43] (Table 1). In addition to the comparison between mutant and wild-type mice, we also explored the relationship between the progression of RTT-related phenotypic impairments and the neuroanatomy to further understand the link between brain structure and behavior. Our findings demonstrate that the structure of the mouse brain is dependent on Mecp2 in adulthood, with variability in neuroanatomical outcomes driven by the type of the mutation as well as the severity of phenotypic impairments.

Experimental animals
In this study, three different Mecp2 mouse lines were used for experimentation: B6.129P2-Mecp2 tm2Bird/J (Mecp2 tm2Bird/J ) female mice on a C57BL/6J background were purchased from Jackson Laboratory (stock no. 6849) and maintained by breeding to C57BL/6J males. Mecp2 tm2Bird/J females were also crossed with CBA.CaJ males obtained from Jackson laboratories (stock no. 0654) to establish a C57/CBA hybrid colony. 129-Mecp2 tm1.1Bird/Jus mice on a 129S6/SvEv background were acquired from the existing active colony at the Toronto Centre for Phenogenomics. B6.129S-Mecp2 tm1Hzo/J were acquired from James Ellis' laboratory at the Hospital for Sick Children (Toronto, ON) or purchased directly from Jackson Laboratory (stock no. 5439). All mice were housed 2-5 per cage and maintained on a 12-hour light/dark cycle with ab libitum access to food and water. The Toronto Centre for Phenogenomics Animal Care Committee approved all experiments.
Experimental groups included Mecp2 mutant (hemi-, hetero-or homozygotes) and matched WT control mice from both sexes that were either 60 or approximately  Table 1.

Scoring Rett-related phenotypes
As previously outlined [18], experimental mice were scored on six symptoms known to arise from Mecp2 disruption: lack of mobility, abnormal gait, presence of a hindlimb clasp, increased tremors, breathing irregularities, and overall deterioration in general condition. These behaviors were scored as absent (0), present (1), or severe (2), and a total score was generated by adding the six scores together. Body weight and phenotype scores were measured on the laboratory bench in the same location and time of day prior to perfusion and imaging.

Tissue Preparation for MRI
Mice were anesthetized and intracardially perfused as previously described [44]. Briefly, following transcardial perfusion with heparin, 4% paraformaldehyde and 2 mM ProHance (Bracco Diagnostics), heads were decapitated and the skin, lower jaw, ears, and the cartilaginous nose tip were removed. The remaining skull containing the brain was soaked overnight in 4% paraformaldehyde/2 mM Pro-Hance and then transferred to PBS, 0.02% sodium azide and 2 mM Prohance for at least 30 days prior to magnetic resonance imaging (MRI) acquisition [45].

Image acquisition
Over the time course of the study, major hardware and software upgrades were made to our imaging system in an effort to increase throughput and image resolution. Thus, the anatomical MRI scans for many of the experimental groups were acquired using different imaging hardware (e.g., gradient size and strength) and acquisition/sequence parameters (see Table 1 for the imaging parameters used for each experimental groups). Although the base acquisition and sequence parameters differed between some of the experimental groups, all were scanned in the same multi-channel 7.0-T MRI scanner (Agilent Technologies) with a T2-weighted, fast spin echo sequence to optimize signal to noise and gray to white matter contrast.
At the beginning of the study, anatomical images were acquired in three custom-built solenoid coils within an insert gradient (6-cm inner diameter bore, rise time of 150 μs, maximum gradient strength = 100 g/cm) with the following sequence parameters: Cartesian acquisition of k-space, TR = 325 ms, and TEs = 10 ms per echo for 6 echos with the center of k-space acquired on the 4th echo, 4 averages, field-of-view (FOV) = 14 × 14 × 25 mm 3 , matrix size = 432 × 432 × 780, 32 μm isotropic resolution, total imaging time was 11 h. Images were downsampled to 64 μm prior to analysis [46]. Upgrades to our sequence design and coil array allowed for more than 3 brains to be imaged in parallel, increasing throughput using 16 solenoid coils (30 cm inner diameter bore, maximum gradient strength of 12 g/cm; using the following image sequence parameters: Cartesian acquisition of kspace, TR = 2000 ms, echo train length = 6, TEeff = 42 ms, 2 averages, FOV = 14 × 28 × −25 mm 3 , matrix size = 250 × 504 × 450, 56 μm isotropic resolution, for total imaging time of 11.7 h) [35]. By the end of the study, a stronger gradient (2 cm inner diameter bore, maximum gradient strength = 30 g/cm) was installed and a new acquisition scheme was used: cylindrical acquisition of k-space, TR = 350 ms, echo spacing = 12 ms for 6 echoes, TEef f= 30 ms, FOV = 20 × 20 × 25 mm 3 , matrix = 504 × 504 × 630, 40 μm isotropic resolution, for a total imaging time of approximately 14 h.

Image registration
The registration method has previously been outlined in detail [28,35]. In this study, image registration was used to align all images within an experimental group into the same space such that corresponding anatomical features become superimposed, thereby allowing statistical comparison. After a series of iterative linear and nonlinear registration steps, a consensus average representing each individual brain and the deformations of each image to this average is generated [47][48][49]. The Jacobian determinant, a metric of expansion or contraction of a voxel, was then extracted from the deformations fields, converted to a logarithmic scale and blurred with a 200 μm FWHM Gaussian smoothing kernel. The resulting blurred Jacobians are then used as a measure of volume within each voxel of the brain and are fed into our statistical analyses as the dependent variable. To determine the volume of the independent anatomical regions, an atlas with 159 segmented regions [50][51][52] was aligned with each experimental group's consensus average allowing for the labels to be back-propagated to each individual brain. The unblurred Jacobian determinants are then summed within the segmented labels to generate a measure of volume within each anatomical region.
In this study, six separate registration pipelines were used to align brain images of wild-type and mutant mice from the experimental groups.

Analysis and statistics
In order to determine the effect of Mecp2 disruption on the neuroanatomy within each experimental group, a linear model was computed within every voxel of the brain relating the fixed effect of genotype (mutant vs. WT) to the absolute Jacobian determinant. In order to determine the direction and magnitude of the effect, the percent difference in volume was calculated between mutant and WT mice within voxels that survived correction for multiple comparisons. The percent difference in volume was also computed across 159 segmented anatomical regions. For these analyses, the group containing the mutant mice (hemizygous, heterozygous, homozygous) was compared to their corresponding controls, thus normalizing the effect within each experimental group.
Voxelwise correlations between neuroanatomical volume and phenotype score were derived using a linear model relating the normalized Jacobian determinant to total phenotype score. In order to determine whether males and females are differentially affected by Mecp2 disruption, a sex (male, female) by Mecp2 status (silent, functional) interaction was computed using the absolute Jacobian determinants as the dependent variable. The silent group contained the hemizygous Mecp2 308/y[B6,P200] males and the homozygous Mecp2 308/308[B6,P200] females, both of which lack WT copies of Mecp2, while the functional groups were composed of their matched WT controls. Differences in total brain volume and body weight were computed using the Student's t test. The false discovery rate correction was used to control for multiple comparisons [53]. Statistical analysis were conducted using the RMINC package (https://github.com/Mouse-Imaging-Centre/RMINC) in the R statistical environment (http://www.r-project.org).

Neuroanatomical differences in mice possessing the Mecp2 tm1Hzo truncation mutation
To determine the effect of the Mecp2 tm1Hzo truncation mutation on the brain, we scanned P60 hemizygous and P200 hemizygous, heterozygous, homozygous and matched WT controls with high-resolution MRI and used a voxelwise and regional-based analysis within segmented anatomical regions to determine the location, direction, and magnitude of the neuroanatomical differences.
In order to compare any sex differences, a sex by Mecp2 status interaction was run comparing male hemizygous and female homozygous Mecp2 tm1Hzo mice. Although this analysis did not survive correction for multiple comparisons, minor localized differences were found in the inferior and superior colliculi at uncorrected thresholds (uncorrected t = 2.43, p < 0.01).

Neuroanatomical differences in Mecp2-null models: Mecp2 tm1.1Bird and Mecp2 tm2Bird
In order to further characterize the neuroanatomical phenotypes in the Mecp2 mouse brain, we repeated the experiment using two mouse models of Rett syndrome in which the Mecp2 gene is completely silent: Mecp2 tm1.1Bird and Mecp2 tm2Bird . Interestingly, the Mecp2 tm2Bird gene differentially affects the body weight of mice depending on the background strain and sex, significantly increasing weight in C57/CBA hybrid males (Mecp2 STOP (Fig. 3b).
Interestingly, significant volumetric decreases relative to WT were found across the hemizygous male and heterozygous female brain in all experimental groups possessing a fully disrupted Mecp2 gene (Mecp2 NULL/y[129,P60] , Fig. 4a Fig. 4d; t = 2.15, 5% FDR). In all groups, mutants had severe volumetric decreases across the frontal (13-18%), motor (12.9-16.4%), somatosensory a b Fig. 3 Loss of a functional copy of Mecp2 leads to drastic decreases in total brain volume. a Body weight (grams) and b total brain volume (mm 3 (Fig. 4e). Furthermore, linear trends were found between neuroanatomical volume and phenotype score in both Mecp2 NULL/y[129,P60] (uncorrected t = 1.8, p < 0.05, Fig. 5a) and Mecp2 STOP/y[B6,P60] (uncorrected t = 1.83, p < 0.05, Fig. 5b) hemizygous males. In both experimental groups, negative correlations were found within the cingulate cortex area 24b, primary and secondary motor cortices and primary somatosensory cortex (Fig. 5c). Additional negative correlations between phenotype score and volume were found in the globus pallidus and basal forebrain in the Mecp2 NULL/y[129,P60] brain and within the striatum of the Mecp2 STOP/y[B6,P60] group.

Discussion
In this study, we imaged the brains of three different Mecp2 mouse models using anatomical MRI parameters paired with deformation-based morphometry to identify the brain regions affected by Mecp2 mutations. Our primary comparison of interest was between the Mecp2 tm1Hzo truncation model, that possess a STOP sequence downstream of codon 308 which eliminates the C-terminal of the coding sequence [43], with two models that do not express functional Mecp2 protein, either by the complete removal of exon 3 and 4 (Mecp2 tm1.1Bird [42]) or via a STOP-neomycin cassette within intron 2 (Mecp2 tm2Bird [18]).
We reasoned that susceptible brain regions would show a similar neuroanatomical phenotype in mutant mice from all three Mecp2 models. Regardless of mutation type, regional volumes of the frontal, cingulate, sensory, and motor cortices, as well as the striatum, thalamus, and white matter tracts were smaller in mutant mice relative to their WT controls. Interestingly, these neuroanatomical changes recapitulate the findings from human Rett syndrome, highlighting a particular cross-species susceptibility within cortex, caudate putamen, and white matter [37,38,54]. However, the foremost difference between experimental groups was the magnitude of the volumetric differences between genotypes. In the Mecp2 tm1Hzo group, the majority of neuroanatomical differences between mutants and controls were within the range of 5-10% with isolated peaks of volumetric differences greater than 20% localized within specific brain regions. However, mutants carrying an early occurring truncation mutation that disrupts the functional domains of Mecp2 had volumetric decreases ranging from 15 to 30% that spanned across all cortical and subcortical areas. Thus, despite similarities in the direction of the volumetric effects within a subset of regions, mutations that ablate the functional domains of Mecp2 have a more detrimental effect on the brain than the truncation of the C-terminal.
Furthermore, the volume within many of these regions directly related to the severity of phenotypic impairments. P60 Mecp2 NULL/y[129,P60] and Mecp2 STOP/y[B6,P60] males with more severe behavioral phenotypic impairments had correspondingly smaller volume within voxels of the striatum, cingulate cortex area 24b and 32, primary and secondary motor cortices, striatum, and globus pallidus (Fig. 5). While many of these findings were bilateral, correlations between neuroanatomy and phenotype score were found to be localized to the left striatum in the Mecp2 STOP/y[B6,P60] mutant brain. Previous studies have demonstrated that structural volumes within the C57BL/6J mouse brain are asymmetrical within particular cortical and subcortical regions, including the striatum [55]. Although a direct structure/function relationship has not be drawn, the findings suggest that an underlying mechanism exists which may skew the functional capacity within one hemisphere over the other. Furthermore, unilateral structural changes within the striatum have previously been shown to occur following training on a procedure memory task, demonstrating a hemisphericspecific responsiveness to behavioral outcomes [35]. The culmination of these findings provide an explanation for a b c the unilateral correlations within the striatum which may represent a hemispheric-specific susceptibility to increasing phenotype severity in the Mecp2-null brain. At this time, we are unable to fully understand the nature of the relationship between RTT-related phenotypic impairments and the volumetric changes within corresponding neuroanatomical areas. In order to make sense of the relevance of this relationship, additional biochemical and histological assays would need to be added to the imaging and behavioral data to ascertain its diagnostic or therapeutic potential. Interestingly, the volume of frontal cortical regions have also been shown to be correlated with behavioral impairments in girls with Rett syndrome [39], highlighting that the relationship between volume and behavior is particularly important to further investigate.
The behavioral/volume relationship seen here may explain the difference in magnitude of the neuroanatomical effects between the Mecp2 tm1Hzo and Mecp2-null models (Mecp2 tm1.1Bird and Mecp2 tm2Bird ). As seen in both humans and mice, late occurring truncations of Mecp2 lead to a less severe progression and overall behavioral outcome than early occurring mutations that ablate the functional domains [9,10,42,43]. With the established connection between phenotype and volume within particular brain regions, the differences in neuroanatomical effects between these models may be a reflection of the deviation in phenotypic progression and severity. However, this phenomena cannot explain the entire profile of neuroanatomical differences between the models because not all regions are correlated with phenotype score.
Additional neuroanatomical deviations may be driven by differences in the underlying cellular environment. Complete loss of Mecp2 within the brain leads to a reduction in dendritic length, spine density and immunoreactive synaptic markers within the motor cortex and hippocampus [15,56,57]. Despite a slight reduction in postsynaptic density length and impaired LTP, no obvious histological and dendritic irregularities were found in the Mecp2 tm1Hzo brain [13,43]. Thus, the drastic cellular and structural deficits observed at the neuropil in the Mecp2null brain compared to the Mecp2 tm1Hzo may compound throughout the levels of the neuroanatomy leading to greater volumetric effects at the mesoscopic level measured by MRI. Although histological analyses are required to determine how the underlying cellular disruptions are driving the volume within these regions, previous studies have demonstrated that MRI paired with deformationbased morphometry is sensitive to subtle changes at the neuropil [35].
Many studies of Mecp2 mouse models use males as their experimental animals to avoid phenotypic variability caused by mosaic X-chromosome inactivation in females. However, a particular concern in the field is that too many studies are conducted using males to model a neurodevelopmental disorder that predominantly affects females. In our study, we included heterozygous and homozygous female mice to determine any sex-specific interactions that may lead to differential outcomes on the neuroanatomy. Interestingly, we found that the pattern of neuroanatomical changes is preserved between males and females from both the Mecp2 tm1Hzo and Mecp2-null experimental groups when matching for disease progression rather than age. This was seen on a qualitative inspection between Mecp2 tm2Bird males and females and a direct statistical comparison between Mecp2 tm1Hzo hemizygous males and homozygous females, both of which lack a wildtype copy of Mecp2. The lack of an interaction between sex and Mecp2 status suggests that males can be used to model the neuroanatomical phenotypes caused by Mecp2 disruptions.
We also found that despite differences in body weight between P60 Mecp2-null male mice, the magnitude and direction of neuroanatomical changes between the two different models without functional Mecp2 (Mecp2 NULL/y[129,P60] and Mecp2 STOP/y[B6,P60] ), and the two Mecp2 STOP models on different backgrounds (Mecp2 STOP/y[B6,P60] and Mecp2 STOP/y[Hyb,P60] ) was very similar, highlighting that a separate mechanism drives differences in the neuroanatomy and body weight. These findings suggest that disruption of the functional domains of Mecp2 leads to a penetrant effect within the brain that transcends differences in genetic background.
Along with finding regions that were similarly affected across Mecp2 models, we were also able to identify brain regions that were differentially affected by the severity of Mecp2 mutation. Compared to the Mecp2-null groups that show global volumetric reductions, the Mecp2 tm1Hzo mutation increased the volume of the mutant cerebellum compared to controls. Additionally, cerebellar volumes were dependent on the amount of functional Mecp2 alleles, showing a stepwise increase in volume from WT to heterozygous to homozygous females. These findings demonstrate that the severity of Mecp2 disruption leads to differential effects on the cerebellum.
Unlike the P200 Mecp2 308/y[B6,P200] , Mecp2 308/x[B6,P200] , and Mecp2 308/308[B6,P200] experimental groups, the P60 Mecp23 308/y[B6,P60] hemizygotes are larger than WT in total brain volume and locally across many brain regions. These neuroanatomical differences between P60 and P200 hemizygous males may be related to the stage of pathogenesis, with early stages characterized by mild phenotypes and larger volumes, which later transitions into smaller volumes as the phenotype progresses with age. However, a longitudinal study would be needed to understand the spatial and temporal dynamics of this growth trajectory within the Mecp2 308/y brain. Overall, these findings are important because they demonstrate that the type of Mecp2 mutation as well as the stage of pathogenesis can contribute unwanted variance which needs to be controlled when measuring neuroanatomy, particularly in studies that use these outcomes as diagnostic tools or to measure treatment success.
Many of these affected regions are part of neural networks that govern specific behaviors and phenotypes that are impaired by Mecp2 mutations. One of the hallmark behavioral phenotypes of Rett syndrome is an impairment of motor abilities, manifesting as a loss of purposeful hand movements and coordination in humans [2] and impairments on tasks of motor ability, such as beam walking and rotarod in Mecp2 mouse models [12]. In both species, motor control is governed by the conserved dopaminergic networks that originates in the dopamine-synthesizing cells of the substantia nigra and ventral tegmental area and projects through the striatum, globus pallidus, cortex, and thalamic nuclei [58]. Previous work has shown that Mecp2 mutations within the substantia nigra and ventral tegmental area leads to the loss of dopamine and dopaminergic metabolites that contributes to the classical parkinsonian features of RTT [20,59]. Furthermore, selective deletion and reactivation of Mecp2 within the dopamine-synthesizing neurons of the substantia nigra [60] and the dorsal striatum [61] has demonstrated its importance for psychomotor control. It is perhaps not surprising then that mutant mice from all experimental groups had volume reductions within important nodes of this neural network, such as the primary and secondary motor cortices, striatum, and globus pallidus. Moreover, the volume within these regions was correlated with the progression of Rett-related phenotypes. These findings demonstrate that volume within brain regions that govern motor behaviors are sensitive to Mecp2 mutations and are tightly related to the stage of behavioral pathogenesis.
In summary, we identified regions of the brain that are similarly affected regardless of mutation severity, sex, and background which are correlated with changes in phenotypic profile. These findings demonstrate that particular regions of the brain are dependent on functional Mecp2 expression and are thus, most susceptible to Mecp2 disruption.

Conclusions
As our understanding of the etiology of neurodevelopmental disorders digs deeper into the biology, treatment interventions are becoming more specific, targeting cellular mechanisms and pathways. Although treatment strategies are still in development for RTT, many studies conducted with Mecp2 mouse models have tested a variety of pharmacological and genetic interventions that target the cellular imbalance caused by Mecp2 disruption [12,18,56,[62][63][64]. The findings from these studies demonstrate that the behavioral and cellular impairments can be rescued highlighting that the brain retains an innate plasticity that can be recruited to restore function in adulthood [65]. However, in order to accurately assay treatment success, noninvasive biomarkers that provide specific information regarding the neurobiological treatment response across the brain are needed. In our study, we demonstrate that MRI paired with deformation-based morphometry is a sensitive method that can detect global changes in the brain influenced by a variety of genetic disruptions and the progression in behavioral phenotypes. Thus, MRI would serve as an important diagnostic tool for assaying the efficacy of treatment interventions on the neuroanatomy, particularly across important neural networks that govern classical RTT symptoms.