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Coculture of Rat Melanotrophs with Hypothalamic Cells Enhances Differentiation of Dopaminergic Neurons

The interaction between a glandular target and hypothalamic dopaminergic neurons during development was studied by cocultivating dispersed fetal hypothalamic and adult intermediate lobe rat cells in serum-free medium. In such conditions, hypothalamic neurons aggregated around intermediate lobe cells and were interconnected by well-developed neurites. They could be grown in coculture at lower density than alone. In regard to dopaminergic activity, both tyrosine hydroxylase content and accumulation of [(3)H]DA in the cells were significantly increased in coculture after 6 days in vitro (DIV). These effects persisted until 18 DIV, but were progressively reduced with time. Other neuronal activities (choline acetyltransferase activity or thyroliberin content) were not modified by coculture. Furthermore, dopaminergic activity was not stimulated when hypothalamic neurons were grown in the presence of anterior pituitary cells, nor mesencephalic neurons together with intermediate lobe cells, suggesting selectivity of intermediate lobe cells on hypothalamic DA neurons. After radioautographic labeling of DA neurons at 6 DIV, morphometric analysis revealed that the presence of intermediate lobe cells increased the number of branching points and the total neuritic length, without changing the number of DA neurons, the size of cell bodies, nor the number of neurites emerging from the soma. Cocultured DA neurons at 6 DIV exhibited morphological features of more differentiated neurons, as estimated by morphological analysis of 12 DIV control neurons. Thus, intermediate lobe cells induce in vitro clustering of fetal hypothalamic neuronal somata and accelerate specifically hypothalamic dopaminergic neuron maturation.