Search

Our studies have been directed to the identification of local, naturally-occurring molecules that support substantia nigra (SN) dopaminergic (DA) neuron survival. We have previously demonstrated that local Type I astrocytes selectively increase the dopaminergic population [30,31]. However, the mechanism of action remains to be defined. To determine whether survival is elicited through diffusible agents, Type I astrocyte conditioned medium (CM) was tested on SN dissociates. After 7 days of exposure to CM, DA neuronal integrity was monitored immunocytochemically with antibody to tyrosine hydroxylase (TH), the DA biosynthetic enzyme, or by TH catalytic assay. CM increased TH+ cell number greater than 2-fold, suggesting that a soluble factor(s) promoted neuron survival. Neurons cultured in serum free medium (SFM) are known to contain few, but detectable numbers of glia [34]. To examine whether CM affected neurons directly, or indirectly through glia, glial populations were stained with antibody against the glial marker, glial fibrillary acidic protein (GFAP). We employed several approaches to define the potential role of glia. Initially, CM was compared to basic fibroblast growth factor (bFGF), a glial mitogen that reportedly enhances nigral DA neuron survival. bFGF enhanced TH activity in our system, as well, but the effect was blocked by the mitotic inhibitor 5-fluorodeoxyuridine (FDUR), which kills dividing glia. In parallel studies CM increased enzyme activity and TH cell number in cultures exhibiting GFAP+ cells. To define the role of these glial cells in the CM effect, we completely eliminated astrocytes in CM-treated cultures employing alpha-aminoadipic acid (AA; 10-30 microM), a specific gliotoxin. At a concentration of AA that eliminated detectable GFAP+ cells, CM continued to elicit a significant increase in TH cell number. These data suggest that, in contrast to effects of bFGF, the DA neurotrophic activity in CM acts directly on nigral neurons to enhance survival.

In order to develop a reliable source of human astrocytes for in vitro studies, we established a primary explant culture of a human encephalocele. This culture yielded a population of cells which were karyotypically normal, morphologically resembled astrocytes, expressed glial fibrillary acid protein, and responded mitogenically to exogenous growth factors. We conclude that white matter derived from human encephaloceles can be used to generate pure populations of normal astrocytes.

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have trophic effects on rat neocortical neurons in vitro. Concentration-response studies reveal that EGF maximally stimulates neuronal survival and process outgrowth at approximately 10 ng/ml, while the maximal effect of bFGF is seen at 10-30 ng/ml. Treatment with maximal concentrations of bFGF results in cultures containing a greater number of neurons with long processes, as well as greater branching of processes, than does treatment with EGF. When EGF and bFGF are added together to cultures the effects are not additive. In addition, bFGF is capable of supporting the survival of neurons previously treated with EGF. These findings indicate that EGF and bFGF affect a largely overlapping population of neocortical neurons, but that bFGF may be a more effective trophic agent for these cells.

In primary neuronal cultures the activation of protein kinase C (PKC) by tumor-promoting phorbol esters blocked growth factor-induced neuronal survival and neurite extension. Depletion of PKC markedly facilitated both epidermal growth factor and basic fibroblast growth factor-inducible neurite extension. Inhibition of PKC by H-7 also stimulated neurite extension. These results suggest that down-regulation of PKC in neurons may be required for trophic factor action.

Homogeneous cultures of astrocytes and oligodendrocytes provide an excellent model system for studying the regulation of glial structure and function. Recently, a chemically defined (CD) medium was developed for purified cultures of astrocytes, thus eliminating the requirement for serum and providing a controlled system for the study of astroglial properties. Due to the widespread use of astrocyte cultures and the potential benefits to be gained from using a defined medium, astrocyte cultures raised in CD medium were analyzed for purity as well as morphological and biochemical properties. Purity was assessed using immunocytochemical staining for glial fibrillary acidic protein (GFAP) and fibronectin. Astrocytes raised in CD medium are 95% pure using the expression of GFAP as a criterion. Fewer than 1% of the cells in CD medium stained positive for fibronectin eliminating the possibility that CD medium is selective for meningeal or endothelial cells. Astrocytes raised in CD medium exhibit a striking degree of morphological differentiation as seen in scanning electron micrographs. They also exhibit a high degree of biochemical differentiation illustrated by increases in the specific activity of S-100 protein and the induction of glutamine synthetase by glucocorticoids. A defined medium that supports the proliferation of rat astrocytes and enhances numerous morphological and biochemical properties should greatly facilitate the study of factors controlling glial proliferation and differentiation.