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Brain fiber pathways are presumed to follow smooth curves but recent high angular resolution diffusion MRI (dMRI) suggests that instead they follow 3 primary axes often nearly orthogonal. To investigate this, we analyzed axon pathways under monkey primary motor cortex with (1) dMRI tractography, (2) axon tract tracing, and (3) axon immunohistochemistry. dMRI tractography shows the predicted crossings of axons in mediolateral and dorsoventral orientations and does not show axon turns in this region. Axons labeled with tract tracer in the motor cortex dispersed in the centrum semiovale by microscopically sharp axonal turns and/or branches (radii ≤15 µm) into 2 sharply defined orientations, mediolateral and dorsoventral. Nearby sections processed with SMI-32 antibody to label projection axons and SMI-312 antibody to label all axons revealed axon distributions parallel to the tracer axons. All 3 histological methods confirmed preponderant axon distributions parallel with dMRI axes with few axons (&lt;20%) following smooth curves or diagonal orientations. These findings indicate that axons navigate deep white matter via microscopic sharp turns and branches between primary axes. They support dMRI observations of primary fiber axes, as well as the prediction that fiber crossings include navigational events not yet directly resolved by dMRI. New methods will be needed to incorporate coherent microscopic navigation into dMRI of connectivity.

Geometric Navigation of Axons in a Cerebral Pathway: Comparing dMRI with Tract Tracing and Immunohistochemistry