Astrocytes protect neurons from neurotoxic injury by serum glutamate.
Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30-100 microM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from approximately 90 microM to less than 1 microM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.
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Department of Neurobiology, The University of Alabama at Birmingham, 25294, USA.
Sontheimer
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D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D001798:Blood Proteins / Q000494:pharmacology; D002450:Cell Communication / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D003470:Culture Media; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006624:Hippocampus / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009498:Neurotoxins / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000494:pharmacology
D001798:Blood Proteins; D003470:Culture Media; D018691:Excitatory Amino Acid Antagonists; D009498:Neurotoxins; D012710:Serum Albumin, Bovine; D018698:Glutamic Acid; D016291:Dizocilpine Maleate