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

Tumor necrosis factor-alpha (TNF alpha) affects several CNS functions such as regulation of sleep, body temperature, and feeding during pathology. There is also evidence for TNF alpha involvement in physiological sleep regulation, e.g., TNF alpha induces sleep and brain levels of TNF alpha increase during prolonged wakefulness. The immediate cause of enhanced TNFa production in brain is unknown. We investigated whether glutamate could signal TNF alpha 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 TNF alpha. Immunostaining for neuron specific enolase revealed that the cultures were 50-60% neuronal and 40-50% non-neuronal cells. TNFa 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 TNF alpha, 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 TNF alpha was expressed by similar to 25% of the neurons and similar to 75% of the glial cell in the culture. Stimulation of the cultures with glutamate did not increase the percentage of cells expressing TNFa. We conclude that TNF alpha 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 TNF alpha production. These findings support the hypothesis that TNF alpha can participate in normal physiological regulation of sleep and feeding. (c) 2005 Elsevier B.V. All rights reserved.