Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia

Abstract

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.