Expression and Localization of NMDA Receptor GluN2 Subunits in Dorsal Horn Pain Circuits across Sex, Species, and Late Postnatal Development

Abstract

Despite being essential mediators of pain processing, the molecular identity of N -methyl- d -aspartate receptor (NMDAR) subtypes in nociceptive dorsal horn circuits is poorly understood, especially between sexes and in humans. Given the importance of GluN2 subunits in shaping NMDAR function and plasticity, we investigated the expression and localization of specific GluN2 NMDAR variants in the dorsal horn of viable spinal cord tissue from male and female rodents and human organ donors. Analysis of single-cell/nuclei sequencing datasets and quantitative reverse transcriptase polymerase chain reactions revealed that the GluN2A ( GRIN2A ) and GluN2B ( GRIN2B ) subunits are robustly expressed in dorsal horn neurons of mice, rats, and humans, with moderate expression of GluN2D ( GRIN2D ). Immunohistochemistry with antigen retrieval demonstrated that GluN2A, GluN2B, and GluN2D proteins are all preferentially localized to the superficial dorsal horn (SDH) of both adult rats and humans, which is conserved between males and females. Surprisingly, we found that these GluN2 NMDAR subunits are enriched in the lateral SDH in rats but not in humans, while presynaptic and neuronal markers are symmetrically distributed across the rat mediolateral axis. A dramatic shift in localization of GluN2A to the lateral SDH was observed across later postnatal development (PD21–PD90) in both male and female rats, with a corresponding change in synaptic NMDAR currents. This discovery of changes in NMDAR subunit distribution during maturation and between species will shed light on the physiological roles of NMDARs and their potential as therapeutic targets for pain.