PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B

Abstract

Studies suggest that the scaffolding protein, postsynaptic density protein-95 (PSD-95), is involved in multiple neurological dysfunctions. However, the role of PSD-95 in the anterior cingulate cortex (ACC) in neuropathic pain (NP) has not been investigated. The current study addressed the role of PSD-95 in the ACC in NP and its modulating profile with NMDA receptor subunit 2B (NR2B). The NP model was established by chronic constriction injury (CCI) of the sciatic nerve, and mechanical and thermal tests were used to evaluate behavioral hyperalgesia. Protein expression and distribution were evaluated using immunohistochemistry and western blotting. The results showed that PSD-95 and NR2B were co-localized in neurons in the ACC. After CCI, both PSD-95 and NR2B were upregulated in the ACC. Inhibiting NR2B with Ro 25-6981 attenuated pain hypersensitivity and decreased the over-expression of PSD-95 induced by CCI. Furthermore, intra-ACC administration of PSD-95 antisense oligonucleotide not only attenuated pain hypersensitivity but also downregulated the NR2B level and the phosphorylation of cyclic AMP response element-binding protein. These results demonstrated that PSD-95 in the ACC contributes to NP by interdependent activation of NR2B.

Figure 1

Cellular distribution of NR2B and PSD-95 in the anterior cingulate cortex. Double immunofluorescence staining shows colocalization of NR2B with NeuN and PSD-95, but not with GFAP or Iba1. Scale bar = 100 μm. NR2B: NMDA receptor subunit 2B; NeuN: neuron-specific nuclear protein; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adaptor molecule-1; PSD-95: postsynaptic density protein-95.

Figure 2

CCI-induced behavioral hypersensitivity, NR2B/PSD-95 upregulation, and CREB activation in the ACC. (A) Schematic diagram of the experiment. (B) The anatomical boundaries used for ACC tissue harvest. In comparison with the sham group, the CCI group showed significantly lower thermal latency (C) and mechanical threshold (D) on the 7th day after surgery (***P < 0.001, independent t-test, n = 6). Western blot showing that compared to that in the sham group, the expression of NR2B (E) and PSD-95 (F) in the ACC was bilaterally increased in the CCI group (***P < 0.001, **P < 0.01, vs. Sham, one-way ANOVA, n = 4); while the total CREB (G) did not change, the level of pCREB (H) was bilaterally increased in the CCI group (***P < 0.001, vs. Sham, one-way ANOVA, n = 4). GAPDH was loading control and error bars represent standard error of the mean (SEM). Original images of blots are available in supplementary Fig. S2. ACC: anterior cingulate cortex; CCI: chronic constriction injury; Ipsi: Ipsilateral; Contr: Contralateral; NR2B: NMDA receptor subunit 2B; CREB: Cyclic AMP response element-binding protein; pCREB: phosphorylated CREB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Ro 25-6981 attenuated behavioral hypersensitivity and inhibited the PSD-95 upregulation induced by CCI. (A, B) Schematic diagram of the experiments. In comparison with DMSO, Ro 25-6981 increased the thermal latency (C) and mechanical threshold (D) (*P < 0.05, **P < 0.01, ***P < 0.001, vs. CCI + DMSO, two-way ANOVA, n = 6). (E, F) Ro 25-6981 suppressed PSD-95 over-expression induced by CCI (**P < 0.01, vs. sham; ^##P < 0.01, vs. CCI + DMSO, one-way ANOVA, n = 3). GAPDH was loading control and error bars represent standard error of the mean (SEM). Original images of blots are available in supplementary Fig. S4. CCI: chronic constriction injury; DMSO: dimethyl sulfoxide; PSD-95: postsynaptic density protein-95; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 4

PSD-95 AS-ODN attenuated behavioral hypersensitivity, inhibited the PSD-95/NR2B over-expression and CREB activation induced by CCI. (A) Schematic diagram of the experiment. (B, C) The location of ACC catheterization and microinjection. (D, E) In comparison with the PEI, the thermal latency and mechanical threshold were significantly higher in the AS-ODN group (*P < 0.05, ***P < 0.001, vs. CCI + PEI, two-way ANOVA, n = 6). (F) Western blot showed that the expression of PSD-95 in the AS-ODN group was significantly lower than that in the PEI group (*P < 0.05, vs. CCI + PEI, independent t-test, n = 3). (G) The expression of NR2B in the AS-ODN group was significantly lower than that in the PEI group (**P < 0.01, vs. CCI + PEI, independent t-test, n = 3). (H, I) The expression level of CREB was not altered significantly. The phosphorylated CREB was significantly lower than that in the PEI control group (***P < 0.001, vs. CCI + PEI, independent t-test, n = 3). GAPDH was loading control, and error bars represent standard error of the mean (SEM). Original images of blots are available in supplementary Fig. S3. ACC: anterior cingulate cortex; CCI: chronic constriction injury; PEI: polyethyleneimine; AS-ODN: antisense oligonucleotide; PSD-95: postsynaptic density protein-95; NR2B: NMDA receptor subunit 2B; CREB: cyclic AMP response element-binding protein; pCREB: phosphorylated CREB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 5

The effect of ACC catheterization and manipulation on locomotive abilities of the front paws. (A, B) The strength of grasp of the front paws showed no significant differences, compared to the control groups (n = 6). AS-ODN: antisense oligonucleotide; ACC: anterior cingulate cortex.

Figure 6

A schematic presentation of PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B. The material of the schematic diagram is provided by Figdraw.