1 Institutes of Neurobiology, Institute of Brain Science and State Key Laboratory of Medical Neurobiology, Shanghai Medical College, Fudan University, Shanghai 200032, China 
2 Department of Life Science, Fudan University, Shanghai 200433, China

Abstract 

Objective
The rostral anterior cingulate cortex (rACC) is implicated in processing the emotional component of pain. N-methyl-D-aspartate receptors (NMDARs) are highly expressed in the rACC and mediate pain-related affect by activating a signaling pathway that involves cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and/or extracellular regulated kinase (ERK)/cAMP-response element-binding protein (CREB). The present study investigated the contributions of the NMDAR glycine site and GluN2B subunit to the activation of ERK and CREB both in vitro and in vivo in rat rACC.
Methods
Immunohistochemistry and Western blot analysis were used to separately assess the expression of phospho-ERK (pERK) and phospho-CREB (pCREB) in vitro and in vivo. Double immunostaining was also used to determine the colocalization of pERK and pCREB.
Results
Both bath application of NMDA in brain slices in vitro and intraplantar injection of formalin into the rat hindpaw in vivo induced significant up-regulation of pERK and pCREB in the rACC, which was inhibited by the NMDAR antagonist DL-2-amino-5-phospho-novaleric acid. Selective blockade of the NMDAR GluN2B subunit and the glycine-binding site, or degradation of endogenous D-serine, a co-agonist for the glycine site, significantly decreased the up-regulation of pERK and pCREB expression in the rACC. Further, the activated ERK predominantly colocalized with CREB.
Conclusion
Either the glycine site or the GluN2B subunit of NMDARs participates in the phosphorylation of ERK and CREB induced by bath application of NMDA in brain slices or hindpaw injection of 5% formalin in rats, and these might be fundamental molecular mechanisms underlying pain affect.

Keywords

N-methyl-D-aspartate receptor; glycine site; GluN2B; D-serine; extracellular regulated kinase/cAMP-response element-binding protein signaling pathway; rostral anterior cingulate cortex

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