Anatomical insights into the superior longitudinal system from integrative in- vivo and ex-vivo mapping

Charting the organization of white matter (WM) pathways is essential for understanding the functioning of the human brain. This study provides a comprehensive, anatomically enhanced characterization of the superior longitudinal system (SLS) by integrating in-vivo tractography with ex-vivo dissection within the same radiological space. Using a data-driven cortex-to-cortex pairing approach leveraging gyral-sulcal macroanatomical landmarks, we reconstructed the dorsal associative connectivity of the frontal cortex in 39 healthy participants. We identified 45 SLS components, of which (i) 22 were validated and refined through ex-vivo dissection, (ii) 17 were deemed anatomically plausible despite lacking ex-vivo confirmation, and (iii) 6 were classified as anatomically implausible. The anatomical description of plausible sub-SLS templates revealed fundamental organizational principles of the system: (i) a medio-lateral and dorso-ventral hierarchy, where dorsal regions connect dorsally and ventral regions ventrally, and (ii) a depth-dependent organization, with shorter, superficial fibers linking proximal areas and longer, deeper fibers connecting distal regions. Pearson's correlation confirmed a significant positive relationship between streamline length and distance from the cortex (r = 0.689, p < 0.001). This study emphasizes the need for a distributed and integrated understanding of brain connectivity beyond classical bundle definition, and provides a robust anatomical foundation for future population-based WM atlases using bundle-specific tractography. By integrating in-vivo tractography with ex-vivo dissection, this study maps the subcomponents of the Superior Longitudinal System and classifies them based on their anatomical plausibility, revealing the hierarchical and depth-dependent organization of association fibers of the brain.