Basic fibroblast growth factor (bFGF) acts on both neurons and glia to mediate the neurotrophic effects of astrocytes on LHRH neurons in culture

Luteinizing hormone-releasing hormone (LHRH) neurons play a pivotal role in the neuroendocrine control of mammalian reproduction. Astrocytes were show n to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulati on of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemica l levels we characterized the neurotrophic effects exerted by young astrocy tes (maintained in culture for 8 days in vitro) and old astrocytes (maintai ned 26 days) on the differentiation, proliferation, and phenotypic expressi on of immortalized hypothalamic LHRH (GT(1-1)) neurons in vitro. Culturing GT(1-1) cells in the presence of young glia for different time intervals ca used a marked acceleration in the acquisition of their neuronal phenotype. At all times examined, GT(1-1) cells cocultured with young glia exhibited a significantly greater extension of processes/cell, larger number of proces ses/cell and greater surface area of growth cones than GT(1-1) cells grown over nonglial adhesive substrates (polylysine). By contrast, when GT(1-1) n eurons were cocultured with old glia, the length of neuronal processes and the growth cone surface area were significantly lower than in control GT(1- 1) neurons cultured in the absence of glia. At 3 days in vitro (DIV), GT(1- 1) neurons cocultured with young glia exhibited a 50% lower incorporation o f [H-3]thymidine than GT(1-1) neurons cultured without glia. By contrast, i n the presence of old glia [H-3] thymidine incorporation was significantly higher in cells cocultured with glia than in GT(1-1) neurons cultured alone . Localization of the proliferating cells by dual immunohistochemical stain ing revealed that the incorporation of bromodeoxiuridine (BrdU) was restric ted to nuclei of GT(1-1) neurons when these were cocultured with young glia , but associated with both neurons and astrocytes in the presence of old gl ia, At the functional level, coculture of GT(1-1) neurons with young glia i ncreased the spontaneous release of LHRH as compared to GT(1-1) neurons gro wn in the absence of glia. By contrast, in the presence of old glia LHRH re lease in the medium was significantly lower than in controls. Conditioned m edium of young glia (ACM-Y) induced significant neurotrophic and functional effects on GT(1-1) cells, but these effects were 50% less potent than the coculture itself. Heat denaturation of ACM-Y totally abolished its neurotro phic and functional properties, indicating that they involved a peptide fac tor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activit y by approximately 40%, but did not affect its LHRH release-promoting effec ts. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neur ons cocultured with young glia counteracted both neurotrophic and functiona l effects of young glia. Treatment of old glia with bFGF rescued its neurot rophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged b FGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1 ) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, an d mechanism(s) requiring coculture are responsible for the highly potent ne urotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by s oluble inhibitory molecules and in part by factors requiring coculture with old glia; 3) old glia may revert to a growth-supporting state when treated with bFGF and this functional shift involves a diffusible molecule with po tent neurotrophic and functional effects on immortalized LHRH neurons. Syna pse 36:233-253, 2000. (C) 2000 Wiley-Liss, Inc.