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 shown to be involved in the regulation of LHRH neuronal function, but little is known about the contribution of astroglial-derived factors in the regulation of LHRH neuron development. In order to gain insight into the mechanisms regulating the development of these cells, at morphological and biochemical levels we characterized the neurotrophic effects exerted by young astrocytes (maintained in culture for 8 days in vitro) and old astrocytes (maintained 26 days) on the differentiation, proliferation, and phenotypic expression 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 caused 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 processes/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) neurons 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 of [(3)H]thymidine than GT(1-1) neurons cultured without glia. By contrast, in the presence of old glia [(3)H]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 staining revealed that the incorporation of bromodeoxiuridine (BrdU) was restricted 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 glia. At the functional level, coculture of GT(1-1) neurons with young glia increased the spontaneous release of LHRH as compared to GT(1-1) neurons grown in the absence of glia. By contrast, in the presence of old glia LHRH release in the medium was significantly lower than in controls. Conditioned medium 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 neurotrophic and functional properties, indicating that they involved a peptide factor. Suppression of bFGF activity in ACM-Y reduced its neurotrophic activity by approximately 40%, but did not affect its LHRH release-promoting effects. By contrast, neutralization of endogenous bFGF activity in GT(1-1) neurons cocultured with young glia counteracted both neurotrophic and functional effects of young glia. Treatment of old glia with bFGF rescued its neurotrophic and functional effects on GT(1-1) cells. Moreover, the ACM of aged bFGF-treated old glia was the most powerful neurotrophic stimulus for GT(1-1) neurons. These results suggest that: 1) soluble peptidic factors, including bFGF, and mechanism(s) requiring coculture are responsible for the highly potent neurotrophic and functional effects of young glia; 2) the inhibitory effects of old glia on neurite outgrowth and LHRH release are mediated in part by soluble 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 potent neurotrophic and functional effects on immortalized LHRH neurons. (c) 2000 Wiley-Liss, Inc.
Gallo
F
F
Department of Pharmacology, Medical School, University of Catania, 95125 Catania, Italy.
Morale
M C
MC
Spina-Purrello
V
V
Tirolo
C
C
Testa
N
N
Farinella
Z
Z
Avola
R
R
Beaudet
A
A
Marchetti
B
B
D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D018920:Coculture Techniques; D016222:Fibroblast Growth Factor 2 / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007150:Immunohistochemistry; D009457:Neuroglia / Q000378:metabolism / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D009479:Neuropeptides / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D009479:Neuropeptides; D016222:Fibroblast Growth Factor 2; D007987:Gonadotropin-Releasing Hormone