3D Histology Validates 2D Histology for Axon Radius Distributions and Conduction Velocities

Abstract Axons are the brain’s wiring, organized into bundles that connect nearby and distant regions. Axon caliber determines signal conduction velocity and varies both within and across bundles, reflecting the brain’s diverse functional demands. Much of what we know about this organization derives from 2D histology, assuming cylindrical axons whose calibers are described by their radius. Yet, recent 3D histology reveals that the radius varies along an individual axon—with implications for both characterizing axon caliber and potentially conduction velocity predictions. We show in 450,000 3D rat axon reconstructions that—despite this individual variation—axon bundles possess stable radius distributions at the ensemble level, which 2D cross-sections faithfully represent. This representativeness extends to conduction velocity predictions, as along-axon variation has only modest impact. In particular, large axons exhibit especially stable conduction, emphasizing their key role in time-critical signaling. With 2D sampling validated, we leverage 46 million human corpus callosum axons from 2D histology to determine sample size requirements across neuroscience applications. Our findings reinforce decades of 2D histology-based research on axon organization and its functional implications, while guiding future study design.