ASTROGLIAL DIFFERENTIATION IS REQUIRED FOR SUPPORT OF NEURITE OUTGROWTH

Models of astrocyte differentiation stress a lineage program that invo lves a progressive loss of astroglial support of neuronal differentiat ion. These models predict that astroglial promotion of neurite extensi on declines with the ''age'' of the astrocyte. An alternative view is that astroglial support of neurite growth is regulated by epigenetic f actors that induce the cells either to differentiate and support neuro nal functions or to undergo cell proliferation and fail to support neu rons. To compare the contribution of astroglial cell ''age'' to astrog lial support of neurite extension, mouse cerebellar astroglia were mai ntained in vitro for 3-90 d, and assayed for their ability to support neurite formation. When cultured in isolation, astroglial support of n eurite extension declined with time in vitro, as assayed by quantifyin g outgrowth from explants of pontine nuclei, falling from a robust lev el just after the astroglia were harvested to negligible levels 21-90 d later. Since previous studies have shown that neurons can change the state of astroglial cells (Hatten, 1985), we tested the neurite promo ting activity of astroglia that were cultured for 21-90 d in vitro and subsequently induced to differentiate by the addition of neurons. Whe n granule neurons were added to aged astroglia and pontine explants pl ated 2 d later, neurite growth from the explants was exuberant, regard less of the time astroglia spent in vitro prior to the addition of neu rons. The state of astroglia that were growth promoting or growth inhi biting was examined by bromodeoxyuridine staining and with antisera to glial filament protein. Aged astroglia cultured alone and thus inhibi tory to axon growth, proliferated at high rates and had polygonal shap es. In contrast, aged astroglia to which neurons had been added, proli ferated at low rates and developed process-bearing stellate shapes. To test further whether proliferation levels related to the growth-suppo rting properties of astroglia, astroglia were plated alone in medium w ithout serum, or with the addition of transforming growth factor-beta 1, each treatment known to arrest proliferation. In both cases, promot ion of neurite growth was restored in aged astroglia, but the morpholo gy of astroglia did not correlate with the ability to support neurite growth. Finally, the growth-inhibiting properties of aged astroglia do not appear to be mediated by diffusible factors, and require close ap position with living astroglial cells. We conclude that astroglial sup port of neurite extension depends on the state of differentiation of a stroglial cells, and that these properties can be modified by cocultur e with neurons or conditions that arrest of astroglial proliferation, irrespective of astroglial ''age.''