Mesencephalic type 1 astrocytes rescue dopaminergic neurons from death induced by serum deprivation

Abstract

We have established a primary neuronal culture of the embryonic day 14 rat, ventral mesencephalic region, centered on the A8, A9, and A10 dopaminergic nuclei (≃1.0 mm3 of tissue). At 16 hr after plating on a substrate of poly-D-lysine, in a serum-free or serum-supplemented growth medium, using a microisland culturing method, 95% of the cells stained positive for neuron-specific enolase, 20% for tyrosine hydroxylase, and <5% for vimentin. When the growth medium was supplemented with 10% fetal calf serum, the percentage of tyrosine hydroxylase-positive neurons increased significantly (p < 0.05) at the 7th and 10th days in culture, compared with the percentage present at 16 hr after plating. When cultured in a serum-free growth medium, the percentage of tyrosine hydroxylase-positive neurons decreased to <5% and to 0% by the 5th and 7th days, respectively, while the percentage of GABA-IR neurons increased. The addition of serum to the serum- free culture rescued dopaminergic neurons from death induced by serum deprivation. The effect of serum was dependent both on the time of addition after plating, and on the percentage added. When the cells were plated in a serum-free medium, on a confluent, type 1 astrocyte monolayer, prepared from the ventral mesencephalon of the embryonic day 16 rat, the survival of dopaminergic neurons increased significantly (p < 0.01) at DIV5, versus survival after plating on poly-D-lysine. Conditioned medium prepared from the same mesencephalic type 1 astrocyte monolayer also rescued dopaminergic neurons from death. The rescue mediated by the astrocyte monolayer or the conditioned medium was not inhibited by the mitotic inhibitor cytosine arabino furanoside (1.0 μM). Type 1 astrocyte monolayers and conditioned media prepared from the striatum and cerebral cortex of the embryonic day 16 rat had weaker trophic effects than those mediated by mesencephalic glia. We conclude that serum deprivation causes the selective death of dopaminergic neurons in a primary culture of the rat E14 ventral mesencephalon. Type 1 astrocytes or the conditioned medium from type 1 astrocytes can rescue dopaminergic neurons from death induced by serum deprivation. The dissection technique used, which yields a high percentage (20%) of tyrosine hydroxylase- positive neurons, the low percentage of glia in the initial culture (<5.0% were vimentin positive), the use of a microisland culturing method, and the use of serum deprivation as the stimulus to induce cell death will be of value in future experiments designed to isolate dopaminergic neurotrophic factors from type 1 astrocytes.