Cross-terms between imaging and diffusion gradients, unaccounted for during tensor calculations, can lead to erroneous estimation of diffusivity and fractional anisotropy (FA) in regions of isotropic and anisotropic diffusion. Cross-term of magnitude 136.8 ± 1.6 s/mm2, artificially introduced in the slice-encode direction caused an increase in fractional anisotropy (FA) in isotropic phantom from 0.0546 ± 0.0001 to 0.0996 ± 0.0001, while the change in chimpanzee brain depended on the orientation of the white matter (WM). Mean diffusivity (MD), remained unchanged in isotropic phantom, but increased by ~20% in the WM due to cross-terms. A bias was observed in the principal eigenvectors in both phantom and chimpanzee brain, resulting in significant increase in midline crossing fibers along the bias than perpendicular to it in tractography in chimpanzee brain. Post-hoc correction of these artifacts was achieved by estimating the cross-term factors using calibration scans on an isotropic phantom and modifying the b-matrix before tensor calculation. Upon correction, the FA and MD values closely resembled the values obtained from sequence without cross-terms, and the bias in principal eigenvectors was eliminated. Customized sequences involving large b-values, high-resolution imaging, or long diffusion or echo times should therefore be evaluated and any residual cross-terms corrected before implementation.
Background
High throughput, brain-wide analysis of neural circuit connectivity is needed to understand brain function across species. Combining such tractography techniques with small animal models will allow more rapid integration of systems neuroscience with molecular genetic, behavioral, and cellular approaches.
Methods
We collected DTI and T2 scans on 3 series of 6 fixed mouse brains ex vivo in a 9.4 Tesla magnet. The DTI analysis of ten mouse brains focused on comparing prelimbic (PL) and Infralimbic (IL) probabilistic tractography. To validate the DTI results a preliminary set of 24 additional mice were injected with BDA into the IL and PL. The DTI results and preliminary BDA results were also compared to previously published rat connectivity.
Results
We focused our analyses on the connectivity of the mouse prelimbic (PL) vs. infralimbic (IL) cortices. We demonstrated that this DTI analysis is consistent across scanned mice, with prior analyses of rat IL/PL connectivity, and with mouse PL and IL projections using the BDA tracer.
Conclusions
High-throughput ex vivo DTI imaging in the mouse delineated both common and differential connectivity of the IL and PL cortex. The scanning methodology provided a balance of tissue contrast, signal-to-noise ratio, resolution and throughput. Our results are largely consistent with previously published anterograde staining techniques in rats, and the preliminary tracer study of the mouse IL and PL provided here.