Axonal structure-function relationships across experimental modalities

The structure-function relationship of myelinated nerve fibers (Waxman & Bennett, 1972) links axon diameter and myelin thickness, commonly expressed as the g-ratio, to conduction velocity via saltatory mechanisms. Here, we investigated this relationship in the transcallosal motor pathway of the rat brain by combining functional and structural metrics in the same animals. Transcallosal conduction times (TCTs) were measured using local field potentials (LFPs) evoked by optogenetic stimulation of excitatory neurons in the motor cortex. Conduction velocity estimates were obtained by combining TCTs with transcallosal tract lengths derived from diffusion MRI (dMRI)-based tractography. Fluorescent labeling of the viral optogenetic construct verified the tractography trajectories. In parallel, axon diameter and g-ratio were quantified using both dMRI and transmission electron microscopy (TEM). To assess dehydration-induced tissue shrinkage associated with conventional TEM preparation ("Epon-TEM"), we also performed cryo-fixation followed by TEM ("Cryo-TEM") in a separate group. This revealed diameter-dependent axonal shrinkage, yielding a correction factor of 37%. Shrinkage correction improved agreement between dMRI and Epon-TEM estimates, although dMRI remained biased toward larger axons. When translated via the structure-function relationship, TCTs corresponded to smaller axons near the mode of the TEM diameter distribution, while dMRI-based diameters predicted TCTs that were too short compared with the recorded LFP latencies. Altogether, our findings show that structural and functional metrics differ in their sensitivity profiles. Accounting for such modality-dependent sensitivities facilitates the investigation of structure-function relationships, advancing our understanding of how microstructure supports neural communication.