Characterizing neuronal cell bodies in white matter tracts of the human brain

Until the discovery of white matter neurons (WMN) in the 19th century, white matter (WM) was considered to be completely devoid of neuronal cell bodies. Despite evidence consistently showing neuronal soma within cortical WM and their purported implication in neuropsychiatric disorders, these neurons are understudied and have not been characterized in human long-range WM tracts. Using postmortem human brain tissue, we investigated the presence, densities and proportions of excitatory/inhibitory neurons in the uncinate fasciculus (UF) and corpus callosum (CC). We also investigated the ventromedial prefrontal cortex (vmPFC) to validate our methods by comparing our results with previously reported densities of neurons in cortical WM. To identify WMN, we employed fluorescence in situ hybridization with excitatory (SLC17A7) and inhibitory (GAD1) neuronal markers and subsequently validated these neurons at the protein level with NeuN immunohistochemistry. We found that the density of WMN in the vmPFC corresponded with previous independent estimates. The UF displayed a similar, though slightly lower density of WMN compared to the vmPFC, while the CC had a far lower density of WMN than both of these regions. Due to the higher-than-expected density of WMN in the UF, we validated the findings at a second location along the UF temporal segment and confirmed the presence of substantial numbers of WMN in this tract. This research constitutes the first ever validated observation of WMN in human long-range WM tracts, laying the foundation for future research on the phenotype and function of these neurons, and how they may be affected in brain disorders.