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

Abstract

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 o...

Immortalized hypothalamic luteinizing hormone‐releasing hormone (LHRH) neurons induce a functional switch in the growth factor responsiveness of astroglia: involvement of basic fibroblast growth factor

“…Previous studies from our laboratory have resorted to immortalized hypothalamic LHRH neurons as a model to analyze the early development of GT 1-1 cells in culture, and demonstrated potent neurotrophic and functional effects of glia and astroglial-derived factors on those cells ( [20 -22,11,24,9]). Neutralization of endogenous bFGF activity in the co-culture counteracted the glial-induced morphological and functional effects, suggesting that it played a critical role in the acquisition of the LHRH phenotype by GT 1-1 cells ( [22]). To test the hypothesis that LHRH neurons may exert a reciprocal modulation of the astroglial cell compartment, we assessed the influence of GT 1-1 neuron co-culture on the proliferation and morphologic appearance of hypothalamic astrocytes in response to a number of GFs normally expressed during brain development (i.e.…”

“…6 and 7). The incorporation of [ 3 H]thymidine into DNA was increased and the release of the decapeptide, LHRH, in the culture medium was sharply stimulated approximately four-to fivefold [20,22]. Localization of the proliferating cells by dual GFAP or LHRH and BrdU immunolabeling indicated that under these culture conditions, GFAP-immunoreactive cells had almost stopped to proliferate since only LHRH neuron nuclei showed BrdU incorporation.…”

“…Application of the different GFs, but especially bFGF, to the co-culture induced potent morphological effects, as revealed by GFAP-immunohistochemistry, characterized by extensive glial process extension and branching. The present findings clearly support the notion that a bidirectional functional interaction exists between the two cellular compartments [20][21][22]11,24,9].…”

“…The capacity of this antibody to neutralize bFGF activity had previously been tested in a mouse fibroblast line 3T3 cell growth assay [42] and demonstrated to inhibit dose-dependently bFGF-but not EGF-induced increase in fibroblast cell number. This antibody was also previously shown to counteract glial neurotrophic effects on GT 1-1 neurons [22]. Neutralization of bFGF activity reduced sharply LHRH-immunoreactive neuron survival as well as proliferation (not shown).…”

Basic Fibroblast Growth Factor Priming Increases the Responsiveness of Immortalized Hypothalamic Luteinizing Hormone Releasing Hormone Neurones to Neurotrophic Factors

“…Basic FGF is known to be associated with extracellular matrix and cell membranes and activation of bFGF receptors has been demonstrated to underlie neurite outgrowth through the involvement of extracellular cell matrix molecules ( 39). Taken together with recent observations showing a decrease in LHRH neuronal number and LHRH content in the median eminence of transgenic mice expressing an FGFR‐1 dominant negative in LHRH neurones ( 40) , the present data add support for a role of bFGF in promoting LHRH neuronal differentiation ( 15, 19, 20).…”