TNFα Enhances Calcium Influx by Interacting with AMPA Receptors in the Spinal Dorsal Horn Neurons.

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine and has been implicated in pain regulation. Neuronal activity in the spinal dorsal horn contributes to nociceptive transmission. However, it is not fully understood how TNFα affects the activity of spinal dorsal horn neurons. In the present study, we used calcium imaging to characterize the mechanism by which TNFα regulates calcium influx in the cultured spinal dorsal horn neurons. We observed that TNFα incubation caused an increase in fluorescent intensity of Fura-2, a specific intracellular calcium indicator, in the cultured spinal dorsal horn neurons, and such effect was significantly inhibited by co-incubation with R7050, a selective TNFα receptor antagonist, which suggests that TNFα can enhance calcium influx and increase neuronal activity via activating TNFα receptors in the spinal dorsal horn. Using double immunofluorescence staining, we showed that TNFα receptors were co-expressed with a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor subunit GluA1 in the spinal dorsal horn neurons. We further observed that treatment with 1-naphthyl acetyl spermine (NASPM), a specific calcium-permeable AMPA receptor blocker, completely blocked the effect of TNFα incubation on calcium influx in the cultured neurons. Moreover, lipopolysaccharide (LPS)-induced calcium influx was inhibited by co-incubation with R7050. Together, our results suggest that TNFα in the spinal dorsal horn can increase calcium-indicated neuronal activity through the interaction between its receptor and calcium-permeable AMPA receptors.