Mechanisms For Peripheral Tissue Injury To Dynamically Regulate The Synaptic Expression Of AMPA Receptors In Spinal Dorsal Horn

Objective:AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-subtype glutamate receptors mediate the majority of fast excitatory synaptic transmission in central nervous system (CNS). The synaptic trafficking of AMPA receptors plays a critical role in a myriad of pathophysiological processes, such as synaptic plasticity, learning and memory. The conveyance of nociceptive information from periphery to pain-relative brain region also requires the involvement of AMPA receptors in spinal dorsal horn. The present study was designed to investigate the dynamic regulation by peripheral tissue injury of the synaptic expression of spinal AMPA receptors during the initiation and development of chronic inflammatory pain.Methods:Complete Freund’s Adjuvant (CFA) was injected into the plantar surface of mouse hindpaws to establish the animal model of chronic inflammatory pain. Nociceptive behavioral tests, biotinylation assay of surface protein expression and immunoblotting were performed to probe the molecular mechanisms underlying the plastic change of synaptic AMPA receptors in spinal dorsal horn during inflammatory pain.Results:(1) Intrathecal application of AMPA receptor-selective non-competitive antagonist GYKI52466prior to CFA injection blocked the reduction of paw withdrawal thresholds (PWT) of mice in response to innocuous Von Frey filament stimulation, suggesting that AMPA receptors in spinal dorsal horn was involved in the induction of inflammatory allodynia.(2) Intrathecal GYKI52466application at0.5h post-CFA injection alleviated the inflammatory pain in a dose-dependent manner. When intrathecal administration was delayed at12h post-CFA injection, the analgesic potency of GYKI52466was markedly impaired. When intrathecal GYKI52466injection was delayed at24h post-CFA, it totally lost the ability to relieve the mechanical allodynia, suggesting that spinal AMPA receptors only participated in the early maintenance of chronic inflammatory pain.(3) In agreement with these behavioral data, immunoblotting analysis illustrated that the expression of AMPA receptor GluRl subunit in synaptosomal membrane fraction of spinal dorsal horn noticeably increased at0.5h after CFA injection, which peaked at1h and lasted for6h. At12～24h post-CFA injection, the synaptic contents of GluR1subunits in allodynic mice no longer differed from those in control mice. These data implicated that peripheral tissue lesion time-dependently recruited AMPA receptors at synapses to strengthen the synaptic conveyance of nociceptive signals, which conferred to AMPA receptors an important role in the initiation and early maintenance of inflammatory pain.(4) Although CFA transiently enhanced the synaptic clustering of AMPA receptors, it generated little effects on the total protein level, surface expression and cytosolic distribution of AMPA receptors, suggesting that the synaptic accumulation of AMPA receptors might be attributable to the lateral diffusion of the receptors following CFA injection.(5) The present study further demonstrated that the activation of phosphatidylinositol3-kinase (PI3K.) shared high similarity in the temporal pattern with AMPA receptor synaptic expression. We found that PI3K activity noticeably increased at0.5h after CFA injection, which peaked at1h and lasted for6h. At12h and24h post-CFA injection, P13K activity in allodynic mice no longer differed from that in saline-injected control littermates.(6) Animal behavioral tests showed that the intrathecal application of PI3K-selective inhibitor wortmannin prior to CFA injection dose-dependently blocked the reduction of PWT values. Intrathecal wortmannin application at0.5h post-CFA injection also effectively alleviated the inflammatory pain. However, when intrathecal administration was delayed at12h and24h post-CFA injection, the analgesic action of wortmannin was completely abrogated, suggesting that PI3K was also involved in the induction and early maintenance of inflammatory pain, as was observed with AMPA receptors.(7) Western blot experiments provided direct evidence that spinal wortmannin (0.5μg) application at0.5h post-CFA injection reversed the synaptic accumulation of AMPA receptors, suggesting the importance of PI3K activity in the regulation of AMPA receptor trafficking in spinal dorsal horn.(8) PI3K might take effect by promoting the synaptic distribution of postsynaptic density-95(PSD-95).Conclusion:Peripheral tissue injury time-dependently stimulated PI3K activity in spinal dorsal horn to facilitate the synaptic localization of PSD-95, through which AMPA receptors were recruited at synapses to mediate the inducation and early maintenance of chronic inflammatory pain.