AB10529 Sigma-AldrichAnti-GluR-2 Antibody, CT

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are critically involved in the excitatory synaptic transmission, and blocking AMPARs at the spinal level reverses neuropathic pain. However, little is known about changes in the composition of synaptic AMPARs in the spinal dorsal horn after peripheral nerve injury. AMPARs lacking the GluA2 subunit are permeable to Ca(2+), and their currents show unique inward rectification. We found that AMPAR-mediated excitatory postsynaptic currents (AMPAR-EPSCs) of spinal dorsal horn neurons exhibited a linear current-voltage relationship in control rats, whereas AMPAR-EPSCs of dorsal horn neurons displayed inward rectification in rats with spinal nerve injury. In nerve-injured rats, compared with control rats, the GluA2 protein level was significantly less in the plasma membrane but was greater in the cytosolic vesicle fraction in the dorsal spinal cord. However, the GluA1 protein levels in these fractions did not differ significantly between nerve-injured and control rats. Blocking N-methyl-d-aspartate receptors (NMDARs) abolished inward rectification of AMPAR-EPSCs of dorsal horn neurons in nerve-injured rats. Furthermore, inhibition of calpain or calcineurin, but not protein kinase C, completely blocked nerve injury-induced inward rectification of AMPAR-EPSCs of dorsal horn neurons. In addition, blocking GluA2-lacking AMPARs at the spinal cord level reduced nerve injury-induced pain hypersensitivity. Our study suggests that nerve injury increases GluA2 internalization and the prevalence of GluA2-lacking AMPARs in the spinal dorsal horn to maintain chronic neuropathic pain. Increased prevalence of spinal GluA2-lacking AMPARs in neuropathic pain is mediated by NMDARs and subsequent stimulation of calpain and calcineurin signaling.

Sensitization of dorsal horn neurons (DHNs) in the spinal cord is dependent on pain-related synaptic plasticity and causes persistent pain. The DHN sensitization is mediated by a signal transduction pathway initiated by the activation of N-methyl-d-aspartate receptors (NMDA-Rs). Recent studies have shown that elevated levels of reactive oxygen species (ROS) and phosphorylation-dependent trafficking of GluA2 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPA-Rs) are a part of the signaling pathway for DHN sensitization. However, the relationship between ROS and AMPA-R phosphorylation and trafficking is not known. Thus, this study investigated the effects of ROS scavengers on the phosphorylation and cell-surface localization of GluA1 and GluA2. Intrathecal NMDA- and intradermal capsaicin-induced hyperalgesic mice were used for this study since both pain models share the NMDA-R activation-dependent DHN sensitization in the spinal cord. Our behavioral, biochemical, and immunohistochemical analyses demonstrated that: 1) NMDA-R activation in vivo increased the phosphorylation of AMPA-Rs at GluA1 (S818, S831, and S845) and GluA2 (S880) subunits; 2) NMDA-R activation in vivo increased cell-surface localization of GluA1 but decreased that of GluA2; and 3) reduction of ROS levels by ROS scavengers PBN (N-tert-butyl-α-phenylnitrone) or TEMPOL (4-hydroxy-2, 2, 6, 6-tetramethylpiperidin-1-oxyl) reversed these changes in AMPA-Rs, as well as pain-related behavior. Given that AMPA-R trafficking to the cell surface and synapse is regulated by NMDA-R activation-dependent phosphorylation of GluA1 and GluA2, our study suggests that the ROS-dependent changes in the phosphorylation and cell-surface localization of AMPA-Rs are necessary for DHN sensitization and thus, pain-related behavior. We further suggest that ROS reduction will ameliorate these molecular changes and pain.