A role of periaqueductal grey NR2B-containing NMDA receptor in mediating persistent inflammatory pain

Abstract

The midbrain periaqueductal grey (PAG) is a structure known for its roles in pain transmission and modulation. Noxious stimuli potentiate the glutamate synaptic transmission and enhance glutamate NMDA receptor expression in the PAG. However, little is known about roles of NMDA receptor subunits in the PAG in processing the persistent inflammatory pain. The present study was undertaken to investigate NR2A- and NR2B-containing NMDA receptors in the PAG and their modulation to the peripheral painful inflammation. Noxious stimuli induced by hind-paw injection of complete Freund's adjuvant (CFA) caused up-regulation of NR2B-containing NMDA receptors in the PAG, while NR2A-containing NMDA receptors were not altered. Whole-cell patch-clamp recordings revealed that NMDA receptor mediated mEPSCs were increased significantly in the PAG synapse during the chronic phases of inflammatory pain in mice. PAG local infusion of Ro 25-6981, an NR2B antagonist, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in rats. Hyperoside (Hyp), one of the flavonoids compound isolated from Rhododendron ponticum L., significantly reversed up-regulation of NR2B-containing NMDA receptors in the PAG and exhibited analgesic activities against persistent inflammatory stimuli in mice. Our findings provide strong evidence that up-regulation of NR2B-containing NMDA receptors in the PAG involves in the modulation to the peripheral persistent inflammatory pain.

Figure 1

Up-regulation of NR2B receptors in PAG after injury. (A) Western blot results showed that the expression level of GluR1 and NR2B receptors, but not NR2A receptor, was increased in the PAG after CFA injection. Hyp reversed expression level of GluR1 and NR2B receptors. (B) Summary of expression of NR2A, NR2B, GluR1 receptors in the PAG after CFA-injection. Values were significantly different from saline control ** p < 0.01; from CFA-injected alone ^# p < 0.05.

Figure 2

Immunostaining of NMDA receptors in PAG. Immunostaining was performed in the coronal midbrain slices containing the PAG. (A) Expression of NR2A in saline control, CFA, and CFA + Hyp treated mice. (B) Expression of NR2B in saline control, CFA, and CFA + Hyp treated mice. Scalebar (in B): A, B, 500 μM. DMPAG: Dorsomed periaqueductal gray; LPAG: Lateral periaqueductal gray.

Figure 3

Enhanced NMDA receptor-mediated transmission after injury. (A) NMDA receptor-mediated mEPSCs recorded in PAG neurons at a holding potential of -30 mV. Representative traces show NMDA receptor-mediated mEPSCs in the saline, Hyp, CFA, and CFA + Hyp treated mouse. (B) Cumulative frequency (left) and amplitude (right) histogram of the mEPSCs from the cells in (A). Filled circles, from a saline control mouse; open circles, Hyp treated mouse; filled trangles, from a CFA-injected mouse; open trangles, from a CFA + Hyp treated mouse. (C) Summary of mEPSCs frequency (left) and amplitude (right) in neurons from the saline, Hyp, CFA, and CFA + Hyp treated mice. Values were significantly different from saline control * p < 0.05; from CFA-injected alone ^# p < 0.05.

Figure 4

Basal glutamatergic synaptic transmission in the PAG. (A) AMPA receptor-mediated mEPSCs recorded in PAG neurons at a holding potential of -70 mV.Representative traces show AMPA receptor-mediated mEPSCs in the saline, Hyp, CFA, and CFA + Hyp treated mouse. (B) Cumulative frequency (left) and amplitude (right) histogram of the mEPSCs from the cells in (A). Filled circles, from a saline control mouse; open circles, Hyp treated mouse; filled trangles, from a CFA-injected mouse; open trangles, from a CFA + Hyp treated mouse. (C) Summary of mEPSCs frequency (left) and amplitude (right) in neurons from the saline, Hyp, CFA, and CFA + Hyp treated mice.

Figure 5

Changes in the PWL by AP-5 and Ro 25-6981. (A) Representative coronal section of rat midbrain showing PAG injection sites. Scale bar, 500 μm. Aq: aqueduct; DMPAG: Dorsomed periaqueductal gray; LPAG: Lateral periaqueductal gray. (B) PWL in the CFA-treated paws (ipsilateral) by AP-5 and Ro25-6981 infusion. (C) PWL in the right hind paws (contralateral) by AP-5 and Ro25-6981 infusion. Values were significantly different from saline control * p < 0.05, ** p < 0.01; from CFA-injected alone ^# p < 0.05.

Figure 6

Changes in the PWL by Hyperoside. (A) PWL in the CFA-treated paws (ipsilateral) from the control, Hyp, CFA + saline, and CFA + Hyp treated mice. (B) PWL in the right hind paws (contralateral) from the control, Hyp, CFA + saline, and CFA + Hyp treated mice. Values (in A and B) were significantly different from saline control * p < 0.05, ** p < 0.01; from CFA-injected alone, # p < 0.05. (C) Paw volume in the CFA-treated and contralateral paws from control and Hyp treated mice. Values (in C) were significantly different from contralateral paws ** p < 0.01.