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Aberrant striatal anatomy and gray matter connectivity networks in mice lacking autism-associated gene CNTNAP2.

Marco Pagani^1,2, Alberto Galbusera¹, and Alessandro Gozzi¹

¹Functional Neuroimaging Laboratory, Center for Neuroscience and Cognitive Systems, Istituto Italiano di Tecnologia, Rovereto, Italy, ²CIMeC, Center for Mind/Brain Sciences, University of Trento, Rovereto, Italy

Synopsis

Mice lacking CNTNAP2 exhibit robust autism-like behavioral traits, including stereotyped behaviors and excessive self-grooming. By using high resolution morpho-anatomical imaging in CNTNAP2 mutant mice, we identified marked volumetric alterations subcortical substrates implicated in ASD motor stereotypy, with a prominent involvement of the dorsal striatum. Importantly, we also show that in mutant mice, striatal but not cortical regions, exhibit dramatically expanded gray matter network extension, encompassing aberrant trophic interaction between limbic, subcortical and prefrontal regions. The observed striatal volumetric and gray matter network abnormalities serve as a plausible morpho-anatomical substrate for some of the stereotypy exhibited by CNTNAP2 mutant mice.

INTRODUCTION

CNTNAP2 is an autism-associated gene^1-4 that encodes CASPR2, a trans-membrane cell-adhesion protein involved in the clustering of K+ channels^5,6. CNTNAP2 deficient mice exhibit decrease in dendritic arborization and spine development⁷, as well as autistic-like motor stereotypy and hyperactivity⁸, and reduced prefrontal connectivity⁹. The broad spectrum of phenotypic alteration observed in carriers of homozygous CNTNAP2 mutations¹⁰ suggests the involvement of distributed brain substrates in the phenotypic expression of this condition. However, attempts to identify the brainwide substrates affected by CNTANP2 via imaging of human carriers of CNTNAP2 polymorphisms, have produced inconsistent result, possibly reflecting clinical heterogeneity in the mutations mapped^11,12. As a result of this, the brainwide anatomical substrates affected by homozygous loss of CNTNAP2, a condition with high autism penetrance, remain undetermined. To probe a mechanistic involvement of CNTNAP2 in shaping gray matter (GM) volume and trophic dynamics occurring between connected brain regions - known as structural covariance MRI (scMRI) networks - we used high resolution structural MRI in Cntnap2-/- and control mice^13,14. The approach revealed aberrant striatal anatomy and GM connectivity networks in CNTNAP2 mice, which serve as a plausible morpho-anatomical substrate for some of the stereotypy produced by this mutation.

METHODS

High-resolution morpho-anatomical T2-weighted MR imaging of ex-vivo Cntnap+/+ (n=10) and Cntnap-/- (n=14) mouse brains was performed at 7 Tesla, using a FLASH sequence with an isotropic voxel size of 70 µm s recently described¹⁴. Inter-group differences in local volumes were mapped with VBM and volumetric anatomical labelling¹³. To confirm regional location of morphometric alterations in the striatum, a vertex-wise shape analysis based on spherical harmonics was employed¹⁵. We also used seed-based mapping (t>3.5, pc=0.01) to probe growth dynamics between brain regions (structural covariance network mapping - scMRI). scMRI networks between eighteen representative neuroanatomical volumes were clustered using the k-means method¹⁴.

RESULTS

High-resolution VBM mapping revealed focal bilateral areas of increased GM volume in dorsal striatum and hippocampal formation, as well as a decrease GM volume in the cerebellum and ventral tegmental area in Cntnap2-/- mice compared to wild-type littermates (t> |2|, pc<0.01, Fig.1). Importantly, striatal shape analysis revealed the presence of aberrant striatal enlargement in Cntnap2-/- mice, with an outward bilateral displacements in the dorsomedial surface of this structure, a key site for habitual and repetitive behaviour in human and rodents^16,17 (Fig.2). Analogous effects were observed using automated anatomical labelling (Fig.3).

GM connectivity network matrices for Cntnap2-/- and control mice are shown in Fig. 4. scMRI mapping of homotopic GM connectivity revealed increased inter-hemispheric connectivity across all the regions examined in Cntnap2-/- mice with respect to control littermates (Fig.4). However, when regions were clustered based on their heterotopic connectivity, two distinct sets of regions were identified. Of particular relevance was a set of subcortical regions including the striatum, amygdala, thalamus, which appeared to exhibit large heterotopic connectivity than all the rest of cortical regions. This effect was apparent when scMRI network of the striatum was mapped using seed-based approach (Fig. 5), revealing a dramatic reorganization of the striatal GM covariance in mutants with respect to control mice, with an aberrant involvement of subcortical and prefrontal substrates that was not present in control mice. This results suggest that the striatal hypertrophism observed in CNTNAP2 mutants may affect the coordinated growth of a set of connected regions, resulting in brainwide aberrancies in GM networks organization.

CONCLUSIONS

Here, we show that homozygous mice lacking CNTNAP2 exhibit macroscale volumetric alterations in the striatum and other subcortical regions previously associated to ASD. Our data also provide a mechanistic link between CNTNAP2 deficiency and scMRI dynamics, supporting the view that hyperactivity and motor stereotypy exhibited by these mice could be generated by network-level interactions between striatum and neighbouring subcortical regions, which serve as a plausible morpho-anatomical substrate for some of the stereotypy produced by this mutation. We expect this observation to spur analogous multivariate GM analyses in human carriers of CNTANP2 mutations.

Acknowledgements

The study was funded by a grant from the Simons Foundation (SFARI 314688, A.G.)

References

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12.Uddén, J., et al., A common variant of the CNTNAP2 gene is associated with structural variation in the left superior occipital gyrus. Brain and language, 2016.

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Figures

VBM revealed morphoanatomical alterations in regional GM volume in dorsal striatum in Cntnap-2-/- mice. Representative coronal slice reconstructions of the areas showing a statistically significant differences in GM volume in mutants compared to control littermates. Increase of GM volume was evident in the dCPu, dHPC, PrS and DS bilaterally. Decrease of gray-matter volume was detected in the Cb and VTA. Cb, cerebellum; dCPu, dorsal striatum; dHPC, dorsal hippocampus; DS, dorsal subiculum; PrS, presubiculum; VTA, ventral tegmental area.

Shape analysis highlighted outward displacement of the dorsal striatum in Cntnap2-/- mice. Medial view of the right and left striatum. A vector pointing from each vertex of the average shape of the WT towards the corresponding vertex of the striatum of Cntnap2-/- mice was calculated, where the positive value indicates a local measure of outward displacement up to 100um both of the right (a) and left (c) dorsal striatum of mutant mice. Statistical maps (b and d) confirmed that significant outward deformations in mutant mice are mainly located in the dorso-medial portion of the striatum.

Automated anatomical labelling. Consistent with VBM findings, volumetric anatomical labelling showed increased volume in the striatum, dorsal subiculum, presubiculum and hippocampus, as well as a reduction in the volume of the cerebellum and ventral tegmental area in Cntnap2-/- compared to WT littermates. Asterisks indicate the significance levels of comparisons between groups (∗p ≤ 0.05).

CNTNAP deficiency results in aberrant subcortical scMRI network connectivity. (a) Gray matter correlations between eighteen representative homotopic cortical and extra-cortical neuroanatomical volumes of interest (VOIs) was calculated for control and mutant mice. (b) ScMRI between homotopic regions was found to be uniformly increased in Cntnap2-/- mice (p=0.002), suggesting a role of CNTNAP in modulating the growth dynamics of contralateral homotopic regions. (c) k-means cluster analysis showing that scMRI networks involving the striatum be hyper-correlated with neighbouring heterotopic areas in Cntnap2-/- mice. Cortical networks showed comparable heterotopic connectivity networks across the two groups.

Striatal covariance GM networks in Cntnap2-/- mice exhibit aberrant anatomical organization. Correlation maps for unilateral gray matter volume seeds located in right and left striatum of control (a-b) and mutant (c-d) mice. In control mice, both seeds exhibited the involvement of homotopic regions in the contralateral hemisphere as previously shown in wild type mice [18]. In mutant mice, scMRI networks showed aberrant neuroanatomical distribution, involving the nucleus accumbens, amygdala and thalamus plus prefrontal areas (t>3.5, pc=0.01).

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)

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