NG2-Glia Heterogeneity Across Cortical Layers.

NG2-glia are a unique and heterogeneous glial cell population with diverse roles in the central nervous system. However, their morphological diversity across brain regions and cortical layers remains poorly understood. Here, we use StarTrack labeling and in utero electroporation at embryonic day 14 (E14) to reconstruct individual NG2-glial cells in the adult mouse cortex and corpus callosum. Through detailed two- and three-dimensional morphometric analyses, including Sholl analysis, principal component analysis (PCA), and hierarchical clustering, we uncover striking layer-specific patterns. NG2-glia in deep cortical layers (L5-6) exhibit significantly larger somatic areas, more elaborate arborizations, and higher process complexity compared to those in superficial layers (L1-4) and the corpus callosum. In contrast, NG2-glia in layer 1 and the corpus callosum share a compact morphology characterized by smaller somata and simplified processes, suggesting common microenvironmental constraints. Moreover, Sholl analysis, PCA, and hierarchical clustering reveal distinct morphological subpopulations within the NG2-glial population and highlight heterogeneity in upper cortical layers. Comparative analyses with astrocytes reveal fundamental structural differences: NG2-glia have thinner, longer processes and larger enclosing radii but occupy smaller volumes, whereas astrocytes form denser, more compact arbors with higher branch numbers. Together, our findings establish the first comprehensive morphological atlas of cortical adult NG2-glia, highlighting region- and layer-specific adaptations that likely underlie their diverse roles in CNS physiology and repair.