The microtubule-associated protein 6 (MAP6) is involved in the neuromorphogenesis and in brain disorders. Mice without MAP6 (MAP6-KO) are characterized by severe impairments of behavior summarizing some of the clinical features of schizophrenia. In this study, we use 3D DTI tractography to characterize the MAP6 mouse model and results are validated by histology and fluorescent microscopy on cleared brains. In addition to DTI, volumetric imaging analysis was performed to detect brain anatomy alterations.

All experiments were conducted on female wild type (WT) and MAP6-KO fixed brains. For MRI, C57Bl6/129 SvPas-F1 genetic background mice, obtained by crossing pure heterozygote 129SvPas MAP6 mice with pure heterozygote C57BL6 MAP6 mice were used while for histology and cleared brains, Thy1-eYFP MAP6 mice, obtained by the crossing of Th1-eYFP line H mice (Jackson Labs) and MAP6 mice were used.

MRI: The brains were fixed by transcardiac perfusion of a 4% paraformaldehyde solution in phosphate buffered saline containing 6.25mM of Gd-DOTA (Guerbet Laboratories), a paramagnetic MRI contrast agent. After removing surrounding skin and muscles, the skulls containing the intact brains were immersed in the same solution during 4 days and then conserved in Fomblin-oil (FenS chemicals) for MRI acquisitions performed at least 11 days after fixation. Gd-DOTA was used to reduce the T₁ relaxation time permitting short repetition times for MR scan acceleration.T₁ and T₂ reached constant values of 62ms and 16ms and were shortened by a factor of about 30 and 5 respectively. Fomblin-oil was used to avoid magnetic susceptibility artifacts since its chemical composition does not contain hydrogen atoms. Microscopic 3D DTI was performed at 7 T Bruker Biospec Avance III using 3D spin-echo DTI sequence (TE=16ms/TR=90ms, Δ=3.5ms, δ=8 ms, 6 diffusion directions, b=1500 s/mm², spatial resolution: 80 µm³). In addition, in order to characterize the brain anatomy, volumetry analysis was performed using T_1w MRI with isotropic high spatial resolution of 60µm³ obtained with 3D echo gradient sequence (TE=5.5ms/TR=27.5ms) at 9.4 T.

Fluorescent microscopy on cleared brains: Thy1-eYFP MAP6 mice were cleared using the CUBIC method¹. Each middle-hemisphere was incubated in Cubic1 reagent containing 25% urea, 25% N,N,N’,N’-tetrakis (2-hydroxypropyl) ethylenediamine and 15% Triton X-100 for 10 days at 37°C under gently agitation.

Data processing: 3D DTI tractography was performed with MedInria software² and acquisition of the cleared brains was performed with confocal microscope (Zeiss, LSM 710) using x10 objective. The full half-fornix was reconstructed using ImageJ 3D stitching tool. The 3D reconstructions of the fornices obtained from 3D DTI tractography and microscopy on cleared brains were set to the same spatial resolution and registered using rigid transformations with Nyfti Reg software³. Statistical analyses were performed using Welchs’t test, and data were represented as means ± SEM (standard error of means).

In Thy-eYFP-H mouse strain, the eYFP fluorescent protein allows epifluorescence imaging of the full fornix using clearing brain method⁴. 3D DTI tractography and fluorescent microscopy images clearly show that the post-commissural fornix is truncated in MAP6-KO mice (Fig.1) and confirm our previous results obtained from 2D DTI acquisitions⁵ exploiting the fractional anisotropy parameter. The fluorescent microscopy on cleared brain validates the accuracy of our 3D-DTI tractography imaging of mouse brain (Fig.1). 3D DTI tractography highlights new major alterations in white matter (Fig.2) observed for the mammilary tract, anterior commissures, the stria medularis, the corpus callosum and more particularly for the corticospinal tract belonging to the pyramidal tract which is almost truncated (data not shown). Volumetry analysis exhibits strong decrease of the cerebellum and the thalamus volume. Structures as the hippocampal formation and the globus pallidus remain unaffected. Altogether, our result showed that MAP6 deficiency induces not only inhomogeneous decrease in structure volumes and alterations in specific white matter tracts, but a new structural organization of the brain. These biological results are obtained thanks to the development of a high spatial resolution 3D DTI which was validated using microscopy analysis on cleared brains. This work shows the potential of DTI as well as volumetric MRI to better understand the functions of MAP6 in development and maintenance of neuronal connectivities.