Anatomical Gradients in Proliferation and Differentiation of Embryonic Rat CNS Accessed by Buoyant Density Fractionation: α3, β3 and γ2 GABAA Receptor Subunit Co-expression by Post-mitotic Neocortical Neurons Correlates Directly with Cell Buoyancy

Authors

  • Dragan Maric,

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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  • Irina Maric,

    Corresponding author
    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
    • D. Maric, as above

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  • Wu Ma,

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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  • Fatiha Lahojuji,

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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  • Roland Somogyi,

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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  • Xiling Wen,

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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  • Werner Sieghart,

    1. Department of Biochemical Psychiatry, University Clinic for Psychiatry, Vienna, Austria.
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  • Jean-Marc Fritschy,

    1. Institute of Pharmacology, University of Zurich, Zurich, Switzerland
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  • Jeffery L. Barker

    1. Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 2C02, Bethesda, Maryland 20892, USA.
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Abstract

Development of the CNS occurs as complex cascade of pre-programmed events involving distinct phases of cell proliferation and differentiation. Here we show these phases correlate with cells of specific buoyant densities which can be readily accessed by density gradient fractionation. Sprague-Dawley dams were pulse-labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at El1–22 dissociated with papain into single-cell suspensions. Proliferative cell populations were assessed by anti-BrdU and propidium iodide staining using flow cytometry. Cell differentiation was evaluated using molecular and immunocytochemical probes against mRNAs and antigens differentiating the neuroepithelial, neuronal and glial cell lineages. The results show the emergence of distinctive spatiotemporal changes in BrdU+ populations throughout the CNS during embryonic development, which were followed by corresponding changes in the cellular distributions of antigens distinguishing specific cell types. Fractionation of neocortical cells using discontinuous Percoll gradients revealed that an increasing number of cells increase their buoyancy during corticogenesis. Immunocytochemical and molecular characterization showed that the proliferative and progenitor cell populations are for the most part associated with lower buoyancy or higher specific buoyant densities (> 1.056 g/ml) whereas the post-mitotic, differentiated neurons generally separated into fractions of higher buoyancy or lower specific buoyant densities (<1.043 g/ml). Immunostaining with antibodies against several GABAA receptor subunits (α3, β3, γ2) revealed that the highest percent (70–90%) of immunopositive cells could be identified in the most buoyant, differentiating neurons found in the cortical plate/subplate regions, with the lowest percent of the immunopositive cells found in the least buoyant, proliferative and progenitor cell populations originating from the ventricular/subventricular zones. Taken together, these results indicate that buoyant density is distinguishing characteristic of embryonic CNS cells transforming from primarily proliferative to mainly differentiating, and that fractionation of these cells according to their buoyant densities provides rapid access to the properties of specific cell lineages during the prenatal period of CNS development.

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