Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord

Abstract

Central sensitization, increased sensitivity in spinal cord dorsal horn neurons after injuries, plays an essential role in the induction and maintenance of chronic pain. However, synaptic mechanisms underlying central sensitization are incompletely known. Growing evidence suggests that proinflammatory cytokines (PICs), such as interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha), are induced in the spinal cord under various injury conditions and contribute to pain hypersensitivity. Using patch-clamp recordings in lamina II neurons of isolated spinal cord slices, we compared the effects of IL-1beta, IL-6, and TNFalpha on excitatory and inhibitory synaptic transmission. Whereas TNFalpha enhanced the frequency of spontaneous EPSCs (sEPSCs), IL-6 reduced the frequency of spontaneous IPSCs (sIPSCs). Notably, IL-1beta both enhanced the frequency and amplitude of sEPSCs and reduced the frequency and amplitude of sIPSCs. Consistently, TNFalpha and IL-1beta enhanced AMPA- or NMDA-induced currents, and IL-1beta and IL-6 suppressed GABA- and glycine-induced currents. Furthermore, all the PICs increased cAMP response element-binding protein (CREB) phosphorylation in superficial dorsal horn neurons and produced heat hyperalgesia after spinal injection. Surprisingly, soluble IL-6 receptor (sIL-6R) produced initial decrease of sEPSCs, followed by increase of sEPSCs and CREB phosphorylation. Spinal injection of sIL-6R also induced heat hyperalgesia that was potentiated by coadministration with IL-6. Together, our data have demonstrated that PICs induce central sensitization and hyperalgesia via distinct and overlapping synaptic mechanisms in superficial dorsal horn neurons either by increasing excitatory synaptic transmission or by decreasing inhibitory synaptic transmission. PICs may further induce long-term synaptic plasticity through CREB-mediated gene transcription. Blockade of PIC signaling could be an effective way to suppress central sensitization and alleviate chronic pain.

Figure 1.

a–e, Potentiation of excitatory synaptic transmission by PICs. a, Patch-clamp recording of sEPSCs in lamina II neurons shows increase in the frequency and amplitude of sEPSCs after perfusion of IL-1β (10 ng/ml, 2 min). a1 and a2 are enlargements of the recording before and after IL-1β treatment, respectively. b, c, Ratio of the frequency (b) and amplitude (c) of sEPSCs after the treatment of IL-1β, TNFα, and IL-6 (10 ng/ml, 2 min). d, Effects of PICs on AMPA-induced currents recorded at holding potential of −70 mV. Top, A mild potentiation of AMPA (10 μm)-induced current by IL-1β. Bottom, Ratio of the amplitude of the AMPA-induced currents after treatment of IL-1β, TNFα, and IL-6 (10 ng/ml, 2 min). e, Effects of PICs on NMDA-induced currents recorded at holding potential of −50 mV. Top, Potentiation of NMDA (50 μm)-induced current by IL-1β. Bottom, Ratio of the amplitude of the NMDA-induced currents after treatment of IL-1β, TNFα, and IL-6 (10 ng/ml, 2 min). *p < 0.05 compared with pretreatment baseline (t test). Inside each column, the number of total recorded neurons and number of responding neurons are indicated.

Figure 2.

a–e, Suppression of inhibitory synaptic transmission by PICs. a, Patch-clamp recording of sIPSCs in lamina II neurons shows decrease in the frequency and amplitude of sIPSCs after perfusion of IL-1β (10 ng/ml, 2 min). The holding voltage is 0 mV. a1 and a2 are enlargements of the recording before and after IL-1β treatment. b, c, Ratio of the frequency (b) and amplitude (c) of sIPSCs after the treatment of IL-1β, TNFα, and IL-6 (10 ng/ml, 2 min). *p < 0.05 compared with pretreatment baseline (t test). Inside each column, number of total recorded neurons and number of responding neurons are indicated. d, A typical lamina II neuron shows inhibition of sIPSCs to both IL-6 and IL-1β treatment (10 ng/ml). Right, Enlarged traces of recordings that are underlined.

Figure 3.

a–d, Suppression of GABA- and glycine-induced currents by PICs. a, b, Inhibition of GABA- (1 mm; a) and glycine- (1 mm; b) induced currents by IL-1β perfusion (10 ng/ml, 2 min). Both currents were recorded at holding potential of 0 mV. c, d, Ratio of the amplitude of the GABA- (c) and glycine- (d) induced currents after the treatment of IL-1β, IL-6, and TNFα (10 ng/ml, 2 min). *p < 0.05 compared with pretreatment baseline (t test). Number of total recorded neurons and number of responding neurons are shown inside each column. For TNFα treatment, only number of recorded neurons is shown, because all neurons failed to respond to TNFα.

Figure 4.

a–f, Induction of CREB phosphorylation and heat hyperalgesia by PICs. a–d, pCREB immunostaining in the superficial dorsal horn of nonstimulated control slice (a) and stimulated slices with IL-1β (b), TNFα (c), and IL-6 (d). Spinal cord slices were treated with the PICs for 30 min (10 ng/ml) and then fixed with 4% paraformaldehyde. Scale bar, 100 μm. e, Colocalization of pCREB (red) with neuronal marker NeuN (green) in the superficial dorsal horn after IL-1β treatment. Scale bar, 20 μm. f, Number of pCREB-positive nuclei of neurons in the superficial dorsal horn (laminas I–II) 30 min after PIC (10 ng/ml) treatment. *p < 0.05 compared with control (ANOVA); n = 4. g, Spinal injection of IL-1β, TNFα, and IL-6 (10 ng) induces heat hyperalgesia. *p < 0.05 compared with corresponding saline control.