Riluzole induces LTD of spinal nociceptive signaling via postsynaptic GluR2 receptors

Abstract

Purpose: Riluzole - a major therapeutic medicine for patients with amyotrophic lateral sclerosis - reportedly has anti-nociceptive and anti-allodynic efficacies in neuropathic pain models. However, little is known about its effect on neurotransmission in the spinal superficial dorsal horn (SDH). The present study aims to investigate the effects of riluzole on the synaptic transmission of SDH nociceptive pathways in both physiological and pathological conditions.

Materials and methods: Spinal nerve ligation was used to produce a neuropathic pain model. Mechanical allodynia behavior was assessed with Von Frey filaments. Riluzole's effects on nociceptive synaptic transmission under both physiological and pathological conditions were examined by patch-clamp recordings in rat SDH neurons.

Results: The principal findings of the present study are three-fold. First, we affirm that riluzole has a remarkable long-lasting analgesic effect on both in vitro and in vivo pathological pain models. Second, the prolonged inhibitory effects of riluzole on spinal nociceptive signaling are mediated by both presynaptic and postsynaptic mechanisms. Finally, endocytosis of post-synaptic GluR2 contributes to the riluzole-induced long-term depression (LTD) of the spinal nociceptive pathway.

Conclusion: The present study finds that riluzole induces LTD of nociceptive signaling in the SDH and produces long-lasting anti-allodynia effects in nerve injury-induced neuropathic pain conditions via postsynaptic AMPA receptors associated with the endocytosis of GluR2.

Keywords: AMPA receptor endocytosis; LTD; SSDH; long-term depression; neuropathic pain; riluzole; superficial dorsal horn.

Figure 1

Riluzole inhibits excitatory synaptic transmission in a concentration-dependent manner. Notes: (A) Traces of eEPSCs in SDH neurons in the presence of different concentrations of riluzole (20, 50, 100, and 500 µM). (B) The histogram indicates the monosynaptic eEPSC peak amplitude in different concentrations of riluzole compared with control (mean ± SEM ^*P<0.05, ^***P<0.001, paired t-test). HPs were clamped at −70 mV. (C) Dose–response curves for IC₅₀ of riluzole. Abbreviations: eEPSC, evoked excitatory postsynaptic currents; IC₅₀, half maximal inhibitory concentration; SDH, superficial dorsal horn.

Figure 2

Blocking of GluR2 endocytosis prevents riluzole-induced LTD of spinal nociceptive inputs. Notes: Riluzole (50 µM)-induced LTD on C fiber (A) and A fiber (C) drive to neurons in SDH. The LTD was prevented by Tat-GluR2 (10 pmol/L). (B, D) Bar graphs showing that averaged peak values of eEPSCs are significantly reduced by 50 µM riluzole compared to those averaged from the control and Tat-GluR2 group. ^***P<0.001, n=6, paired t-test, one-way ANOVA. ^*P<0.05. Abbreviations: GluR2, glutamate receptor 2; LTD, long-term depression; eEPSC, evoked excitatory postsynaptic currents; ANOVA, analysis of variance; SDH, superficial dorsal horn.

Figure 3

Riluzole decreases the frequency and amplitude of mEPSC. Notes: (A) Ten successive episodes of mEPSCs recorded in ACSF. (B) Ten successive episodes of mEPSCs were recorded for riluzole (50 µM). All recordings were made during the application of TTX. (C) Comparison of cumulative probability of the inter-event interval of mEPSCs recorded in ACSF and riluzole. (D) Comparison of cumulative probability of amplitudes. HP = −70 mV. P<0.01, n=8, K–S test. Abbreviations: mEPSC, miniature excitatory postsynaptic current; ACSF, artificial cerebrospinal fluid; TTX, tetrodotoxin.

Figure 4

Riluzole reduces the hyperexcitability of SDH neurons in a slice model of disinhibition. Notes: (A) DR stimulation at C-fiber intensity evoked a biphasic response in a lamina II neuron. (B) The application of bicuculline (2 µM) and strychnine (20 µM) successfully blocked the IPSPs and generated long-lasting repetitive action potentials. (C) Riluzole (50 µM) totally suppressed bicuculline- and strychnine-induced action potentials. HP was clamped at −50 mV in current clamp mode. Abbreviations: SDH, superficial dorsal horn; DR, dorsal root; IPSP, inhibitory postsynaptic potential; HP, hold potential.

Figure 5

A single intraperitoneal (i.p.) injection of riluzole produces long-lasting anti-allodynia effects. Notes: (A) The lowered threshold of mechanical withdrawal indicated that nerve injury induces mechanical allodynia. The nerve injury-produced mechanical allodynia was partially reversed by i.p. injection of riluzole. (B) Bar graph shows the analgesic effect persisted for at least 14 days. ^***P<0.01, one-way ANOVA with Bonferroni post hoc test, n=6. Abbreviations: SNL, spinal nerve ligation; ANOVA, analysis of variance.

Figure 6

Riluzole decreases Glu- and AMPA-induced currents. Notes: (A) Consecutive traces showing the membrane currents induced by Glu (100 µM), AMPA (100 µM), GABA (3 µM), and Gly (30 µM). Riluzole suppressed the peak of inward currents induced by Glu and AMPA, but had little effect on GABA- or Gly-induced currents (P>0.05, paired t-test, n=5). (B) The histogram indicates the comparison of membrane current peaks before and after dosing (mean ± SEM); ^*P<0.05, ^***P<0.001, paired t-test, n=5). Abbreviations: Glu, glutamic acid; AMPA, aminomethylphosphonic Acid; GABA, gamma-aminobutyric acid; Gly, glycine.

Figure 7

The expression of GluR2 receptor in membrane fraction was reduced by riluzole. Notes: (A) Representative images of Western blotting bands. (B) Western blotting analysis demonstrated that riluzole (12 mg/kg intraperitoneal injection) in lumbar spinal cord reduced the expression of the GluR2 receptor in membrane fractions compared with control and vehicle groups (^***P<0.001, one-way ANOVA, n=4, for each group). Abbreviation: GluR2, glutamate receptor 2.