ANATOMICAL GRADIENTS IN PROLIFERATION AND DIFFERENTIATION OF EMBRYONIC RAT CNS ACCESSED BY BUOYANT DENSITY FRACTIONATION - ALPHA-3, BETA-3 AND GAMMA-2 GABA(A) RECEPTOR SUBUNIT COEXPRESSION BY POSTMITOTIC NEOCORTICAL NEURONS CORRELATES DIRECTLY WITH CELL BUOYANCY

Development of the CNS occurs as a complex cascade of pre-programmed e vents involving distinct phases of cell proliferation and differentiat ion. Here we show these phases correlate with cells of specific buoyan t densities which can be readily accessed by density gradient fraction ation. Sprague-Dawley dams were pulse-labelled with bromodeoxyuridine (BrdU) and selected regions of embryonic (E) CNS tissues at E11-22 dis sociated with papain into single-cell suspensions, Proliferative cell populations were assessed by anti-BrdU and propidium iodide staining u sing flow cytometry. Cell differentiation was evaluated using molecula r and immunocytochemical probes against mRNAs and antigens differentia ting the neuroepithelial, neuronal and glial cell lineages. The result s show the emergence of distinctive spatiotemporal changes in BrdU(+) populations throughout the CNS during embryonic development, which wer e followed by corresponding changes in the cellular distributions of a ntigens distinguishing specific cell types, Fractionation of neocortic al cells using discontinuous Percoll gradients revealed that an increa sing number of cells increase their buoyancy during corticogenesis. Im munocytochemical and molecular characterization showed that the prolif erative and progenitor cell populations are for the most part associat ed with lower buoyancy or higher specific buoyant densities (>1.056 g/ ml) whereas the post-mitotic, differentiated neurons generally separat ed into fractions of higher buoyancy or lower specific buoyant densiti es (<1.043 g/ml). Immunostaining with antibodies against several GABA( A) receptor subunits (alpha 3, beta 3, gamma 2) revealed that the high est percent (70-90%) of immunopositive cells could be identified in th e most buoyant, differentiating neurons found in the cortical plate/su bplate regions, with the lowest percent of the immunopositive cells fo und in the least buoyant, proliferative and progenitor cell population s originating from the ventricular/subventricular zones. Taken togethe r, these results indicate that buoyant density is a distinguishing cha racteristic of embryonic CNS cells transforming from primarily prolife rative to mainly differentiating, and that fractionation of these cell s according to their buoyant densities provides rapid access to the pr operties of specific cell lineages during the prenatal period of CNS d evelopment.