In vitro formation of type 2 astrocytes derived from postnatal rat hypothalamus or cerebral cortex

The origin and function of type 2 astrocytes in the optic nerve are now well described, but there are few and controversial observations concerning their origin and functional significance in other regions of the mammalian brain. We here describe primary and highly enriched secondary glial cultures obtained from postnatal (P0-P6) rat hypothalami and cerebral cortices that included glial cells with morphological and immunocytochemical characteristics of type 2 astrocytes. The somata of such astrocytes were characteristically small and polygonal; they bore several processes with few branches. They were highly immunoreactive for glial fibrillary acidic protein and the surface antigens, A2B5 and NSP4; they were immunonegative for myelin basic protein and galactocerebroside. They grew on top of a continuous monolayer of much larger, flattened cells, that were glial fibrillary acidic protein-positive but A2B5- and NSP4-negative. In cultures derived from tissues younger than postnatal day 4, their appearance required the addition of adult (horse) serum to the culture milieu; they appeared spontaneously in cultures from older animals. Analysis of the origin of these cells, including experiments using tritiated thymidine incorporation, indicated that these astrocytes resulted from asymmetric divisions of the flat glial cells in the basal layer of the cultures. After their first appearance which varied according to the age of the source tissue, they were continuously generated, with a generation time no longer than 48 h; the life-span of individual cells was found to not exceed one week in neuron-free primary glial cultures. They displayed important process motility but did not show any significant migratory activity. The ready inducibility of glial cells showing many characteristics of type 2 astrocytes, in cultures derived from different brain areas, suggests that type 2-like astrocytes or their committed precursors are not restricted to particular neural structures, but are probably widely distributed within the mammalian brain. Their functional significance within the different brain areas remains to be determined.