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bioRxiv · 10.64898/2026.05.15.725074

An organotypic neocortical slice culture for studying neuroglial interactions

Higgins, K. P.·Al Naqib, V. A. B.·Mayo, P.·Lodder, B.·Masuda, T.·Amann, L.·Prinz, M.·Kole, M. H. P.
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Abstract

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

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Higgins, K. P., Al Naqib, V. A. B., Mayo, P., Lodder, B., Masuda, T., Amann, L., Prinz, M., Kole, M. H. P.. 2026-05-15. An organotypic neocortical slice culture for studying neuroglial interactions. https://doi.org/10.64898/2026.05.15.725074

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