Ascorbic acid in mesencephalic cultures: Effects on dopaminergic neuron development.
Item
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Title
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Ascorbic acid in mesencephalic cultures: Effects on dopaminergic neuron development.
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Identifier
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AAI9432345
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identifier
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9432345
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Creator
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Kalir, Henry Hanoch.
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Contributor
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Adviser: Catherine Mytilineou
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Neuroscience | Biology, Molecular
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Abstract
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Ascorbic acid (AA) exists in high intracellular concentrations in fetal rat brain. In mesencephalic (MB) cultures prepared from E14 rat embryos, AA drops sharply to undetectable levels when no ascorbic acid is added to the medium, creating a model of scorbutic neuronal tissue. Cultures were treated both chronically and acutely with 0.2 mM AA, and compared to controls (scorbutic cultures) by using morphological and biochemical indices.;In the chronically-treated AA cultures the following were noted on 7 and 14 days in vitro (DIV): (1) A marked increase in glial proliferation demonstrated by glial fibrillary acidic protein staining. (2) Increased neurite growth and number of dopaminergic neurons on tyrosine hydroxylase (TOH) staining. (3) A significantly higher dopamine (DA) uptake ( ({dollar}\sp3{dollar}H) DA uptake). (4) Significantly higher levels of DA and 3,4-dihydroxyphenylacetic acid (DOPAC). (5) A gradual increase in the capacity to accumulate and retain the intracellular AA with culture maturation, reaching the embryonal levels by DIV 14.;The acute effects of AA on the levels of DA in MB cultures were then investigated, and correlated with the results of TOH immunocytochemistry, ({dollar}\sp3{dollar}H) DA uptake and AA levels over a period of 48 hrs following the addition or omission of 0.2 mM AA in DIV10 cultures. The results showed an increase in the number of TOH stained neurons and the extent of staining after 24 hrs exposure to AA, with an increase in the DA content only after 48 hrs. The AA levels reached a maximum value at 24 hrs and declined in a manner similar to the chronically AA-treated cultures. The ({dollar}\sp3{dollar}H) DA uptake remained unchanged.;The effects of AA on TOH activity were investigated by measuring the levels of L-dihydroxyphenylalanine (L-DOPA) in naive, L-AA and D isoascorbic acid (D-AA--the biologically inactive epimer) treated young cultures from DIV 0 through 2. These experiments showed a marked increase in L-DOPA levels at 24 hrs only in the L-AA treated group but not in the D-AA or control groups.;The data presented in this thesis indicate that the cultures not previously treated with AA are capable of AA accumulation and retention in a manner similar to the chronically AA-treated cultures, and that the acute effects on the DA content are not due to an increase in neuronal survival and could be the result of regulation of TOH activity and possibly other enzymes in the DA synthetic and catabolic chain.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
