Dopamine synthesis, uptake and metabolism in embryonic rat mesencephalic cultures

We have developed an HPLC method using electrochemical detection (ED) to study the synthesis, uptake and metabolism of dopamine (DA) in primary cell cultures dissociated from rat embryonic mesencephalon. The method is rapid and simple and is also able to detect, after 7 days in vitro incubation (DIV), intracellular levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3,4-dihydroxyphenylacetic acid (DOPAC). The amount of DA synthesized and taken up from the cells is directly proportional to in vitro development time; the contents of endogenous DA is related to the number of mesencephalic neurons originally plated. When the dopa decarboxylase inhibitor alpha-methyldopa is added to the incubation medium, it reduces DA levels and conversely increases the amount of L-DOPA in a dose-dependent manner. In mesencephalic-striatal cocultures a statistically significant increase in the amount of DA is observed. This is not observed when either cerebellar or cortical cells are used in the cocultures which confirms the importance of target striatal cells in the maturation of dopaminergic mesencephalic neurons.