ASTROCYTE-DEPENDENT AND ASTROCYTE-INDEPENDENT PHASES OF THE DEVELOPMENT AND SURVIVAL OF RAT EMBRYONIC DAY 14 MESENCEPHALIC, DOPAMINERGIC-NEURONS IN CULTURE

A primary neuronal culture was prepared from the ventral mesencephalon , centered on the A8, A9 and A10 dopaminergic nuclei of the embryonic day 14 rat, and studied from 12 h to 28 days. At 12 h after plating, a nd before cell death ensued, 95% of the cells stained positive for neu ron specific enolase; 20% for tyrosine hydroxylase; 5% for vimentin an d <0.1% for glial fibrillary acidic protein. In the presence of the mi totic inhibitor cytosine arabinoside (2.0 mu M), neuronal growth and s urvival were surprisingly normal up to the ninth day in culture, but d eteriorated rapidly thereafter. In the absence of a mitotic inhibitor, and in the presence of proliferating but non-confluent glia, the tyro sine hydroxylase positive neurons that survived to the 10th day, had r etracted neurites and a rounded soma, suggesting an inhibition of cell development. Those tyrosine hydroxylase positive neurons that survive d this adverse phase of development tended to produce elaborate neurit ic profiles after the 11th day, coincident with confluence of the astr ocyte monolayer at the 12th day. By the 21st day in culture, and persi sting up to the 28th day, 60% (61 +/- 10, n = 20) of the surviving neu rons stained positive for tyrosine hydroxylase. When plated on an esta blished, ventral mesencephalic monolayer of astrocytes, at the seventh day in culture, neuritic growth and branching of the tyrosine hydroxy lase positive neurons were greater, compared with similar neurons grow n on poly-D-lysine, and the signs of arrested development (retraction of neurites and rounded soma) seen at the 10th day after plating on po ly-D-lysine, were not observed. We conclude that in the primary cultur e studied, and under the experimental conditions used, the survival of dopaminergic neurons was independent of glia during the first nine da ys, and critically dependent on glia thereafter. The resurgence of gro wth of dopaminergic neurons after 10 days in vitro, and their subseque nt selective survival in culture, suggest that confluent type-1 astroc ytes produce factors that act selectively on the dopaminergic neuronal phenotype. The successful identification of these dopaminergic-specif ic, neurotrophic factors could lead to an increased understanding of t he etiology of Parkinson's disease, and suggest new directions for the rapeutic intervention.