Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre− littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre− mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre− mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre− littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

Research Highlights▶Peripherin-promoter driven Cre expression drives a selective 75% knockdown of NMDA receptors in sensory neurons. ▶Knockdown mice respond normally to acute thermal and mechanical pain tests. ▶Knockdown mice have a 50% decrease in pain behaviors during phase 2 of the formalin test. ▶NMDA receptors on sensory nerves are important in the development of sensitized pain states.