Biochemical characterization of the uptake and release of [ 3H]dopamine by dopaminergic hypothalamic neurons: A developmental study using serum-free medium cultures

Abstract

The development of the biochemical properties of mouse hypothalamic dopaminergic neurons has been analyzed in vivo and in cultures of cells taken on the 16th day of gestation and grown in serum-free medium for up to 3 weeks. In the course of in vivo development, the dopamine (DA) content remains low during fetal life (10% of the adult value), beginning to increase on the 19th fetal day. In contrast, the specific accumulation of [ 3H]DA increased markedly during the last days of gestation from 20% of the adult value on the 16th fetal day to 70–80% of the adult value on Postnatal Day 3. Hypothalamic DA neurons in culture accumulate endogenous DA although at a lower level than in vivo. They take up [ 3H]DA by an active transport system which is specific for DA, and which shows time, temperature, and sodium dependency ( K m = 1 μM). HPLC analysis showed that the newly taken up [ 3H]DA was not metabolized in the short run under the conditions used. It was stored in a form that could be released when neurons were depolarized in a high K + (60 m M) medium. The K +-evoked [ 3H]DA release was found to be strictly dependent on extracellular Ca 2+. Moreover the release of [ 3H]DA was also stimulated by veratridine in a Ca 2+-dependent manner. Similar data have been obtained with the release of endogenous dopamine. No specific uptake and no K +-evoked dopamine release occurred in 2-day-old cultures. The specific [ 3H]DA uptake and the K +-evoked release appeared in 5-day-old cultures and increased with time in culture at least until Day 15. We examined the effects on [ 3H]DA release of polyunsaturated fatty acid, triiodothyronine, and corticosterone, all of which have been shown to play an important role in synaptogenesis in culture. These components, either separately or together, did not modify the percentage of the basal or the stimulated [ 3H]DA release. These results showed that hypothalamic DA neurons grown in serum-free medium progressively acquired the functional properties of adult DA neurons as concerns DA synthesis, DA uptake, and release. From a development point of view, this study suggests that the capacity to specifically take up [ 3H]DA and to respond to high K + concentration is not expressed at early stages of neuronal development. Moreover, the differences in the time course between the development of these biochemical markers on the one hand and the development of synapses studied in parallel cultures on the other hand, suggest that [ 3H]DA uptake and release precedes the appearance of mature synapses and may occur in other structures than synapses, at least at early stages of development.