(272) - Agmatine reduction of neuropathic pain requires spinal NMDA receptors containing GluN2B subunits

Abstract

N-methyl-D-aspartate (NMDA) receptors are expressed in the dorsal horn of the spinal cord and are an important target for inhibiting pain signals from reaching the brain. Agmatine, decarboxylated L-arginine, is known to antagonize NMDA receptors, but little is known about the subunit requirements of this antagonism. Here we used a conditional GluN2B KO line of mice to assess the role of GluN2B on agmatine electrophysiological responses as well as on the development and maintenance of neuropathic pain. Spinal GluN2B Selective Deletion: At time of weaning, GluN2B-floxed mice were intrathecally injected with either AAV9-Cre (AAV9.hSyn.HI.eGFP-Cre.WPRE.SV40 Penn Vector Core) to delete spinal GluN2B or with saline as a control. Using rtPCR we observed an average 50% reduction of spinal GluN2B mRNA AAV9-Cre-treated mice relative to control. Electrophysiology: Whole cell patch clamp recordings were taken from spinal cord slices from GluN2B knockdown or control mice. Neurons receiving monosynaptic input from C-fiber afferent terminals in the dorsal horn of the spinal cord were identified. The amplitude and recovery rate of evoked excitatory post-synaptic currents (eEPSCs) was measured following electrical stimulation of the dorsal root and application of .3, 1 or 3 mM agmatine. In control mice, agmatine inhibited NMDA-mediated EPSCs but lacked efficacy in GluN2B-deficient mice. Neuropathic pain: Four weeks after AAV9-Cre injection, mice were intrathecally injected with 10 nmol agmatine or saline control and immediately underwent spared nerve injury (SNI) surgery. Mice were given 3 additional 10 nmol agmatine or saline control injections at 48 hour intervals, and von Frey behavioral thresholds were assessed on alternating days, and 25 days following surgery. The GluN2B-selective NMDA antagonists, agmatine and ifenprodil, lacked efficacy at inhibiting pain behaviors in GluN2B KO mice but not control mice. These data support the proposal that agmatine selectively targets GluN2B and requires GluN2B for therapeutic effectiveness in reversing chronic pain.