Chloride currents in peripheral nociceptive neurons have been implicated in the generation of afferent nociceptive signals, as Cl⁻ accumulation in sensory endings establishes the driving force for depolarizing, and even excitatory, Cl⁻ currents. The intracellular Cl⁻ concentration can, however, vary considerably between individual DRG neurons. This raises the question, whether the contribution of Cl⁻ currents to signal generation differs between individual afferent neurons, and whether the specific Cl⁻ levels in these neurons are subject to modulation. Based on the hypothesis that modulation of the peripheral Cl⁻ homeostasis is involved in the generation of inflammatory hyperalgesia, we examined the effects of inflammatory mediators on intracellular Cl⁻ concentrations and on the expression levels of Cl⁻ transporters in rat DRG neurons.

We developed an in vitro assay for testing how inflammatory mediators influence Cl⁻ concentration and the expression of Cl⁻ transporters. Intact DRGs were treated with 100 ng/ml NGF, 1.8 μM ATP, 0.9 μM bradykinin, and 1.4 μM PGE₂ for 1–3 hours. Two-photon fluorescence lifetime imaging with the Cl⁻-sensitive dye MQAE revealed an increase of the intracellular Cl⁻ concentration within 2 hours of treatment. This effect coincided with enhanced phosphorylation of the Na⁺-K⁺−2Cl⁻ cotransporter NKCC1, suggesting that an increased activity of that transporter caused the early rise of intracellular Cl⁻ levels. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl⁻ importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In contrast, the mRNA levels of the two transporters did not change markedly during this time. These data demonstrate a fundamental transition in Cl⁻ homeostasis toward a state of augmented Cl⁻ accumulation, which is induced by a 1–3 hour treatment with inflammatory mediators.

Our findings indicate that inflammatory mediators impact on Cl⁻ homeostasis in DRG neurons. Inflammatory mediators raise intracellular Cl⁻ levels and, hence, the driving force for depolarizing Cl⁻ efflux. These findings corroborate current concepts for the role of Cl⁻ regulation in the generation of inflammatory hyperalgesia and allodynia. As the intracellular Cl⁻ concentration rises in DRG neurons, afferent signals can be boosted by excitatory Cl⁻ currents in the presynaptic terminals. Moreover, excitatory Cl⁻ currents in peripheral sensory endings may also contribute to the generation or modulation of afferent signals, especially in inflamed tissue.