Neuronal inhibition of astroglial cell proliferation is membrane mediated

Previously we have used a microwell tissue culture assay to show that early postnatal mouse cerebellar astroglia have a flattened morphology and proliferate rapidly when they are cultured in the absence of neurons, but develop specific cell-cell contacts and undergo morphological differentiation when they are co-cultured with purified granule neurons (Hatten, M. E., 1985, J. Cell Biol., 100:384-396). In these studies of cell binding between neurons and astroglia, measurement with light and fluorescence microscopy or with [35S]methionine-labeled cells indicated that the kinetics of the binding of the neurons to astroglial cells are rapid, occurring within 10 min of the addition of the neurons to the growing glia. 6 h after neuronal attachment, astroglial DNA synthesis decreases, as shown by a two- to fivefold decrease in [3H]thymidine incorporation, and glial growth ceases. No effects on astroglial cell growth were seen after adding medium conditioned by purified cerebellar neurons cultured in the absence of astroglia, by astroglia cultured in the absence of neurons, or by a mixed population of cerebellar cells. This result was unchanged when any of these media were concentrated up to 50-fold, or when neurons and astroglia were cultured in separate chambers with confluent medium. Two groups of experiments suggest that membrane-membrane interactions between granule neurons and astroglia control astroglial cell growth. First, neurons fixed with dilute amounts of paraformaldehyde (0.5%) bound to the astroglia with the same kinetics as did living cells, inhibited DNA synthesis, and arrested glial growth within hours. Second, a cell membrane preparation of highly purified granule neurons also bound rapidly to the glia, decreased [3H]thymidine incorporation two- to fivefold and inhibited astroglial cell growth. The rate of the decrease in glial growth depended on the concentration of the granule neural membrane preparation added. A similar membrane preparation from purified cerebellar astroglial cells, PC12 cells, 3T3 mouse fibroblasts, or PTK rat epithelial cells did not decrease astroglial cell growth rates. Living neurons were the only preparation that both inhibited glial DNA synthesis and induced the astroglial cells to transform from the flat, epithelial shapes they have when they are cultured without neurons to highly differentiated forms that resemble Bergmann glia or astrocytes seen in vivo. These results suggest that membrane-membrane interactions between neurons and astroglia inhibit astroglial proliferation in vitro, and raise the possibility that membrane elements involved in glial growth regulation include neuron-glial interaction molecules.