Glutamate induces the expression and release of tumor necrosis factor-α in cultured hypothalamic cells

Tumor necrosis factor-α (TNFα) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNFα involvement in physiological sleep regulation, e.g., TNFα induces sleep and brain levels of TNFα increase during prolonged wakefulness. The immediate cause of enhanced TNFα production in brain is unknown. We investigated whether glutamate could signal TNFα 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 TNFα. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFα 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 TNFα, 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 TNFα was expressed by ∼25% of the neurons and ∼75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFα. We conclude that TNFα 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 TNFα production. These findings support the hypothesis that TNFα can participate in normal physiological regulation of sleep and feeding.