Different developmental potentials of diencephalic and mesencephalic dopaminergic neurons in vitro

Morphological and functional differentiation of dopamine (DA) neurons was compared in dissociated cultures from gestational day 14 rat mesencephalon and diencephalon. Numbers of tyrosine hydroxylase-immunoreactive (TH-IR) neurons relative to all neurons were 4 and 1.7 times higher in mesencephalic than in diencephalic cultures at 6 and 13 days in vitro (DIV), respectively. Morphological maturation of diencephalic DA neurons was retarded in comparison to mesencephalic DA neurons. There were gross differences in soma size and length of processes between the two types of DA neurons, the appearance of which was strongly reminiscent of DA cell types described in vivo. Functional maturation of DA neurons was quantified by measuring uptake and Ca2+-dependent K+-stimulated release of [3H]DA per TH-IR neuron. As early as 6 DIV, DA uptake by mesencephalic DA neurons was saturable, was sensitive to benztropine and reserpine, and could be displaced by unlabeled DA. Twenty to 30% of the radioactivity accumulated could be released upon depolarization within a period of 5 min. At 6 DIV, influx of [3H]DA into diencephalic DA neurons was almost insensitive to benztropine, reserpine and unlabeled DA. Even after 13 DIV, diencephalic DA uptake was characterized by a markedly lower initial velocity, a longer time needed to reach saturation, a lower uptake capacity, and a lower sensitivity to benztropine than mesencephalic DA uptake. The releasable pool was very small and did not increase between DIV 6 and 13. The results demonstrate that mesencephalic DA neurons in vitro differentiate considerably faster than diencephalic DA neurons and gain functional competence very early in brain development. Comparison with data on adult nigrostriatal and hypothalamic DA systems suggests that the in vitro differences reflect a fundamental regional diversity of DA neurons.