Fractone-associated N-sulfated heparan sulfate shows reduced quantity in BTBR T+tf/J mice: a strong model of autism

Abstract

BTBR T+tf/J (BTBR) mice show abnormal social, communicatory, and repetitive/stereotyped behaviors paralleling many of the symptoms of autism spectrum disorders. BTBR also show agenesis of the corpus callosum (CC) suggesting major perturbations of growth or guidance factors in the dorsal forebrain [1]. Heparan sulfate (HS) is a polysaccaride found in the brain and other animal tissues. It binds to a wide variety of ligands and through these ligands modulates a number of biological processes, including cell proliferation and differentiation, migration and guidance. It is aggregated on fractal-like structures (fractones) in the subventricular zone (SVZ), that may be visualized by laminin immunoreactivity (LAM-ir), as well as by HS immunoreactivity (HS-ir). We report that the lateral ventricles of BTBR mice were drastically reduced in area compared to C57BL/6J (B6) mice while the BTBR SVZ was significantly shorter than that of B6. In addition to much smaller fractones for BTBR, both HS and LAM-ir associated with fractones were significantly reduced in BTBR, and their anterior-posterior distributions were also altered. Finally, the ratio of HS to LAM in individual fractones was significantly higher in BTBR than in B6 mice. These data, in agreement with other findings linking HS to callosal development, suggest that variations in the quantity and distribution of HS in the SVZ of the lateral ventricles may be important modulators of the brain structural abnormalities of BTBR mice, and, potentially, contribute to the behavioral pathologies of these animals.

Fig. 1

Representative cresyl violet stained sections of B6 and BTBR mouse brains at AP points +1.0 through −1.0. Scale bars are 1mm.

Fig. 2

Quantification of B6/BTBR differences in lateral ventricles: A. area (in pixels, p<0.001 between strain differences indicated with ###, differences between AP points in B6 indicated with * and **, for p<0.05 and p<0.01 respectively); B. subventricular zone length (in pixels).

Fig. 3

Characteristics of individual fractones (B6 n=1083; BTBR n=345): A. total average size (in pixels, p<0.01 indicated with **); B. Size with respect to AP distribution (in pixels); C. total average intensity of LAM-ir (in grayscale 1–256, p<0.001 indicated with ***); D. Intensity of LAM-ir with respect to AP distribution (in grayscale 1–256, p<0.05 and p<0.001 indicated with * and ***); C. total average intensity of HS-ir (in grayscale 1–256, p<0.001 indicated with ***); D. Intensity of HS-ir with respect to AP distribution (in grayscale 1–256); E. total average ratio of HS to LAM-ir (p<0.001 indicated with ***); F. ratio of HS to LAM-ir with respect to AP distribution.

Fig. 4

B6 and BTBR lateral ventricles: A–C. B6 lateral ventricle at AP+0.0 as revealed by A. LAM-ir; B. HS-ir; C. both LAM and HS-ir; D–F. BTBR lateral ventricle at AP+0.0 as revealed by D. LAM-ir; E. HS-ir; F. both LAM and HS-ir; G. magnification of the B6 subventricular zone showing individual fractones; H. magnification of the BTBR subventricular zone showing individual fractones. Scale bar for A–C and D–F is 250µm; for G–H, 50 µm