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bioRxiv · 10.1101/2025.07.23.666263

NG2-Glia Heterogeneity Across Cortical Layers

Barriola, S.·Delgado-Garcia, L. M.·OJALVO-SANZ, A. C.·PERNIA-SOLANILLA, C.·FIGUERES-ONATE, M.·Lopez-Mascaraque, L.
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Abstract

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, 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, principal component analysis, 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 finding 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/666263v1_ufig1.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@1aad0c5org.highwire.dtl.DTLVardef@17049dborg.highwire.dtl.DTLVardef@100e966org.highwire.dtl.DTLVardef@db5b58_HPS_FORMAT_FIGEXP M_FIG C_FIG Main points- Morphological variation of NG2-glial cells by cortical layers - Cells in deep cortical layers are larger and more complex - NG2-glia in upper layers and corpus callosum share similar morphology - NG2-glia vs. astrocytes: distinct structural features

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Barriola, S., Delgado-Garcia, L. M., OJALVO-SANZ, A. C., PERNIA-SOLANILLA, C., FIGUERES-ONATE, M., Lopez-Mascaraque, L.. 2025-07-27. NG2-Glia Heterogeneity Across Cortical Layers. https://doi.org/10.1101/2025.07.23.666263

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