Selective culture of neurons from rat cerebral cortex: morphological characterization, glutamate uptake and related enzymes during maturation in various culture media.
Dissociated cerebral cortex of fetal rat was grown in a serum-free, chemically defined medium (CDM) (containing insulin, progesterone, estradiol, transferrin, putrescine, selenium and 15 mM KCl) and compared with cultures grown in a medium containing 20% fetal calf serum (SCM). Neurons survived well using either medium, but in the serum-free medium the cellular population was exclusively neuronal (at 96%), while glial cells began to proliferate after one week in the SCM. The various cellular morphologies are described in the present report and the presence of immunological markers characteristic of neurons was investigated. Autoradiographic experiments have been performed after incubation with various putative neurotransmitters and we have shown the presence of a strikingly high proportion of glutamatergic neurons in these cultures. Glutamate high affinity uptake was also greatly increased in neuronal cultures maintained in a CDM compared to a SCM, especially in young cultures. The development of different enzymes involved in the metabolism of glutamate was also studied; the specific activity of glutaminase increased in culture and was found to be higher in a CDM than in a SCM, while the inverse was true for glutamine synthetase. The relative proportion of both enzymes in neurons compared to glial cells was opposite, as neuronal cultures had higher levels of glutaminase and glial cultures were enriched in glutamine synthetase activity. It seems that the proportion of glutamate-neurons increases when cultured in a CDM compared to a SCM and we suggest that this culture procedure may provide a purely neuronal population enriched in mature glutamatergic neurons. It may thus be useful for future in vitro studies on glutamate and GABA metabolism in neurons.
Borg
J
J
Spitz
B
B
Hamel
G
G
Mark
J
J
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism; D005333:Fetus; D005968:Glutamate Decarboxylase / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D005972:Glutaminase / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D051381:Rats
D005971:Glutamates; D018698:Glutamic Acid; D005972:Glutaminase; D005968:Glutamate Decarboxylase; D005974:Glutamate-Ammonia Ligase