Regulation By Noradrenergic α2Receptors Of Spinal NMDA Receptor-Mediated Synaptic Nociceptive Transmission

Objective:The α2adrenoceptors are highly enriched in spinal dorsal horn. Following peripheral tissue injury, intrathecal application of α2adrenoceptor agonists effectively alleviates the pathological pain hypersensitivity, although the precise mechanisms are not fully understood.The NMDA (N-methyl-D-aspartic acid)-subtype glutamate receptor hyperfunction is considered to be a key event in the etiology of pathological pain. The present study aimed to investigate the regulatory effect of α2adrenoceptors on spinal NMDA receptor-mediated synaptic nociceptive transmission and reveal the possible related molecular mechanisms.Method:This study utilized behavior tests, patch clamp recording and immunoblotting analysis to explore the signaling pathway evoked by α2adrenoceptors to regulate spinal NMDA receptor function in spinal dorsal horn of mice with inflammatory pain.Results:(1) The inflammatory pain was induced by intraplantar injection of Complete Freund’s Adjuvant (CFA) in mice, and the spinal cord slices were prepared at24h post-CFA. Whole-cell patch clamp recordings in lamina Ⅱ neurons illustrated that clonidine significantly decreased the amplitudes of NMDAR-mediated monosynaptic responses in inflamed mice through activation of α2A-subtype adrenoceptor.(2) No significant alteration in the paired-pulse ratio (PPR;=2nd response/1st response) of NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) before and after clonidine application in inflamed mice indicated the postsynaptic origin.(3) Intracellular loading of nonhydrolyzable GDP analog GDP-β-S through the recording pipettes blocked, whereas direct inhibition of cAMP-dependent protein kinase (PKA) mimicked, the inhibitory effect of clonidine on NMDAR currents, implicating that Gi protein/PKA signaling was involved in clonidine action.(4) Biochemical analysis in vivo revealed that intrathecal clonidine administration specifically decreased the content of GluN2B subunit-containing NMDAR (GluN2B receptor) at synaptosomal membrane fraction of spinal dorsal horn in inflamed mice.(5) Electrophysiological recordings in vitro further demonstrated that GluN2B receptor-selective inhibitor ifenprodil dramatically reduced NMDAR synaptic responses in inflamed mice and more importantly, occluded the synaptic inhibition produced by clonidine. These data suggested that the noradrenergic suppression of inflammatory pain might involve the blockade of GluN2B receptor-mediated nociceptive transmission in spinal dorsal horn.Conclusion:α2adrenoceptors inhibited NMDA receptor-mediated nociceptive transmission in spinal dorsal horn of mice with inflammatory pain through the Gi protein/PKA/GluN2B receptor signaling pathway, which might contribute to the alleviation of chronic inflammatory pain.