Ascorbic Acid in Mesencephalic Cultures: Effects on Dopaminergic Neuron Development

Abstract

Ascorbic acid exists in high intracellular concentrations in fetal rat brain. In mesencephalic cultures the cellular ascorbic acid content drops sharply to undetectable levels when no ascorbic acid is added to the medium, thus creating a model of scorbutic neuronal tissue and affording the study of ascorbic acid's effects on mesencephalic cell development and function. Cultures treated with 0.2 mM ascorbic acid were compared with controls (scorbutic cultures) by using morphological and biochemical indices. Ascorbic acid cultures at 7 and 14 days in vitro showed a marked increase in glial proliferation on glial fibrillary acidic protein staining and increased neurite growth and number on tyrosine hydroxylase staining. Significantly higher dopamine uptake and levels of dopamine and 3,4-dihydroxyphenylacetic acid were also observed after 7 and 14 days of ascorbic acid treatment. The capacity to accumulate ascorbic acid and the ability to retain the intracellular ascorbic acid developed gradually as the cultures matured. Ascorbic acid reached the embryonal levels by day 14 in vitro. We conclude that although neuronal cultures can survive and grow in the absence of detectable levels of ascorbic acid, its presence exerts a broad effect on dopamine neuron morphology and biochemical functioning either directly or through increased glial proliferation, or possibly both.

Ascorbate compartmentalization in the CNS

“…At one extreme, when media are not supplemented with ascorbate or precursors of GSH, cells are antioxidant deficient. Indeed, ascorbate is completely absent in cells cultured in ascorbate-free media (Raps et al, 1989;Kalir and Mytilineou, 1992;Makar et al, 1994). At the other extreme, ascorbate levels much higher than physiological can be obtained, with up to 7 mM in cultured glia after prolonged exposure to ascorbate (Siushansian and Wilson, 1995).…”

“…Whether ascorbate is synthesized in the liver, as in most mammals, or acquired from the diet, as in humans and a few other species (Chatterjee et al, 1975), it is carried to the brain via plasma. It is then actively transported across the blood-brain barrier (Spector and Lorenzo, 1973;Lam and Daniel, 1986) and subsequently taken up into brain cells from the extracellular fluid (Wilson, 1989;Kalir and Mytilineou, 1992;Rice et al, 1994). Each step in this transport process concentrates ascorbate: it is roughly 0.05 mM in plasma (Spector, 1977) and 0.5mM in CSF (Stamford et al, 1984), with overall brain tissue levels of 2-3mM (Milby et al, 1982;Rice et al, 1995).…”

“…Up to 80% of tissue ascorbate and GSH is lost from brain slices after only brief incubation (McIlwain et ,11., 1956;Schenk et al, 1982;Rice et al, 1994). While GSH can be maintained to a variable extent in cultured cells, ascorbate is usually below detection limits (Raps et al, 1989;Yudkoff et al, 1990;Kalir and Mytilineou, 1992;Sato et al, 1993;Ratan et al, 1994;Makar et al, 1994). Because loss of ascorbate and GSH leaves cells in an antioxidant-deficient state, many in vitro studies are inadvertently conducted under conditions of oxidative stress.…”

Levodopa Is Toxic to Dopamine Neurons in an in Vitro but Not an in Vivo Model of Oxidative Stress

“…Ascorbate levels are very low in mesencephalic cultures (Kalir and Mytilineou, 1991), and ascorbate protects against levodopa toxicity. In contrast, concentrations of ascorbic acid are high TABLE 3 Levels of L-dopa, dopamine, and dopamine metabolites in 5-day-old rat pups injected with levodopa and BSO Rats were injected subcutaneously on postnatal day 5 with 100 mg/kg levodopa and/or 200 mg/kg BSO in a volume of 10 l/10 g body weight.…”

“…The levels of levodopa, dopamine, and dopamine metabolites were assayed by HPLC with amperometric detection as described previously (Kalir and Mytilineou, 1991). In brief, brain tissue was frozen on dry ice and stored at Ϫ80°C.…”