title: Glutamate induces the expression and release of tumor necrosis factor-alpha in cultured hypothalamic cells.
abstract: Tumor necrosis factor-alpha (TNFalpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFalpha involvement in physiological sleep regulation, e.g., TNFalpha induces sleep and brain levels of TNFalpha increase during prolonged wakefulness. The immediate cause of enhanced TNFalpha production in brain is unknown. We investigated whether glutamate could signal TNFalpha production because glutamate is a neurotransmitter associated with cell activation and wakefulness. We used primary cultures of fetal rat hypothalamic cells to examine the expression and release of TNFalpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFalpha was detected in both the media and cells under basal conditions. Stimulation of the cells with 1 mM glutamate for 2 h produced an increase in media content of TNFalpha, whereas cell content was elevated at earlier time points. Using trypan blue exclusion and MTT assays, there was no evidence of cell toxicity with this stimulation protocol. Immunocytochemical staining revealed that TNFalpha was expressed by approximately 25% of the neurons and approximately 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFalpha. We conclude that TNFalpha is constitutively expressed and released by healthy cultures of hypothalamic cells and that activation of the cells with a non-toxic challenge of glutamate increases TNFalpha production. These findings support the hypothesis that TNFalpha can participate in normal physiological regulation of sleep and feeding.
authors.affiliation: Program in Neuroscience, Department of VCAPP, College of Veterinary Medicine, Washington State University, 205 Wegner Hall, Pullman, WA 99164-6520, USA.
authors.forename: Alok
authors.initials: A
authors.lastname: De
authors.forename: James M
authors.initials: JM
authors.lastname: Krueger
authors.forename: Steven M
authors.initials: SM
authors.lastname: Simasko
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002452:Cell Count / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects*; D018698:Glutamic Acid / Q000494:pharmacology*; D007031:Hypothalamus / Q000166:cytology* / Q000187:drug effects; D007150:Immunohistochemistry / Q000379:methods; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D014409:Tumor Necrosis Factor-alpha; D018698:Glutamic Acid; D010751:Phosphopyruvate Hydratase