White matter fiber bundles enable robust conduction and time-critical signaling despite axon caliber variation

Axon caliber determines signal conduction velocity and varies within and across the bundles that form the brain's wiring. Our knowledge of this organization derives largely from 2D histology, assuming cylindrical axons whose calibers are described by their radius. However, recent 3D histology reveals substantial along-axon variability in radius, calling into question 2D-derived insights into axon radius and conduction velocity. We show in 450,000 3D axon reconstructions from male rats that-despite this individual variation-axon bundles possess stable radius distributions at the ensemble level. These distributions are faithfully captured by 2D cross-sections and yield robust conduction velocity predictions, as along-axon variability has only a modest effect. Conduction in large axons is particularly stable, supporting 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 show how axonal organization gives rise to stable ensemble statistics and conduction, explaining and reinforcing decades of 2D histology-based research.