Contribution Of NMDA Receptor In The Rostrol Anterior Cingulate Cortex To Pain-Related Affect

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. It consists of two components, sensory discrimination and affective motivation. Neuroimaging and electrophysiological studies in humans and animals revealed that both noxious stimuli and predictive cues of noxious stimulus activate the anterior cigulate cortex (ACC). Lesion of the ACC, especially the rostral part of ACC abolished the pain-like aversion. A number of studies have demonstrated the pivotal role of excitatory amino acids, especially the glutamate, in excitatory synaptic transmission in the ACC. Our previous study showed that the injection of APV, a glutamate binding site (glu site) antagonist of NMDA receptors, in ACC (rACC) abolished formalin-induced conditioned place avoidance (F-CPA) which reflects the affective component of pain. Taking all listed above into account, it is therefore suggested that activation of glutamate NMDA receptors in the ACC plays important role for the induction of pain-related negative affect.Functional NMDA receptors consist of the essential NR1 subunit and at least one of NR2 subuits, namely NR2A, NR2B, NR2C and NR2D. The function of NMDA receptor is regulated by many factors. This heteromeric ion channel receptor has many binding and modulatory sites, among which is the glycine binding site (gly site) in NR1 subunit. Opening of NMDA ion channel requires the occupation of not only glu site but also gly site by their specific ligands. Thus, gly site is also named as co-agonist site of NMDA receptor. From clinical perspective, both competitive and non-competitive NMDA receptor antagonists produce unacceptable side-effects including psychotomimesis and ataxia. Hence, the development of gly site or subtype-selective NMDA antagonists has currently become one of the most promising strategies.Previous work in our lab has exhibited that, D-serine (D-ser) enhanced the NMDA-induced current in the ACC slice and superfusion of DAAO, a D-ser degradation enzyme, attenuated NMDA current. This suggested that the gly site of NMDA receptor in ACC is not saturated and endogenous D-ser, which participates in the regulation of NMDA receptor, is an endogenous agonist of gly site. Based on the above-mentioned evidence, we first systematically investigated the role of gly site in NMDA receptor of the ACC especially rostral part (rACC) and D-ser in pain related negative affect. Considering NR2A and NR2B subunits are the major NR2 subtypes found in forebrain areas, we performed a primary investigation on the contribution of NR2A and NR2B subunits in the acqusition of pain-related negative affect respectively and explored the possible mechanism of the involvement of NMDA receptor on the acqusition of pain-related negative affect.Formalin induced conditioned place avoidance (F-CPA) behavioral model was employed in present study. When unilateral intraplantar (i.pl.) injection of formalin was paired with an arbitrarily given context, the avoidance behavior to the context can be induced in rats. Combining behavioral (in vivo) methods with immunohistochology and immunoblot (in vitro) technique, the main results we obtained are as following:(1) With slight modification to previously described CPA model, our present training protocol can establish more stable F-CPA in rats. When unilateral intraplantar injection of formalin(5%, 50μl) was paired with a particular compartment in the place conditioning apparatus, 95% rats spent significant less time in the conditioning paired context. This indicated that rat received conditioned training can form strong avoidance to the noxious stimulus-paired context;(2) To test the effect of gly site in NMDA receptor to pain-related negative affect, intra-rACC injection of 7-Cl-KYNA (0．02, 0．2, 2 mM, 0．6μl per side)was performed 20 min prior to F-CPA conditioning. At the doses of 2 and 0．2 mM, 7-Cl-KYNA microinjected into the rACC completely eliminated F-CPA acquisition. As a control, we also observed the effect of 7-Cl-KYNA on another conditioned place avoidance, which is fear (weak plantar electric shock) induced conditioning place avoidance (S-CPA). When electric shock (0．5 mA, 2 s) was paired with particular compartment in the place conditioning apparatus, the rat CPA was observed. Microinjection of 7-Cl-KYNA (2 mM) into the rACC before S-CPA conditioning did not block the acquisition of S-CPA;(3) To investigate the effect of D-ser, an endogenous agonist of gly site in NMDA receptor on pain related negative affect, DAAO (0．01, 0．1 unit, 0．6μl per side) was microinjected into the rACC 20 min prior to F-CPA conditioningRats received 0．1 unit DAAO failed to perform CPA. As a control, we further examined the effect of blockade of gly site of NMDA receptor and exhaustion of the endougenous agonist in the rACC on the acute pain response elicited by unilateral injection of formalin. Bilateral injection of both DAAO (0．1 unit) and 7-Cl-KYNA (2 mM) in rACC did not change the formalin biphasic nociceptive behavioral response;(4) To examine the NMDA subunit function in rACC on the pain-related negative affect acqusition, we first detected the expression of NR1, NR2A and NR2B subunit in rACC. NMDA receptor NRl subunit basicly expressed intensivelyon both sides of the rACC. NR2A and NR2B subunits in the bilateral rACC also exhibited a moderate basal expression. Similar to NRl subunit, NR2A and NR2B immuno-positive (IP) cells mainly distributed in the layersⅡ-Ⅵwith a decreasing gradient from the layerⅡtoⅥ. In rACC, NR1, NR2A-and NR2B-IP cells were predominantly neurons. After unilateral intraplantar injection of formalin (5%, 50μ.l) for 40 minutes or 6 hours, the expression of NR1 in all layers of the ipsilateral and contralateral rACC did not obviously change. In contrast, peripheral formalin significantly up-regulated theNR2A and NR2B expression on both sides of the rACC at 40 minutes and 6 hours after injection. Microinjection of a NR2A selective antagonist NVP-AAM077 (0．012μg/μl, 0．6μl per side), or a NR2B selective antagonist Ifenprodil (0．2μg/μl, 0．6μl per side) into the rACC 20 min before F-CPA conditioning completely blocked F-CPA acquisition.(5) For further investigating the possible mechanism of the involvement of NMDA receptor and the subunit in the acquisition of pain related negative affect, we inspected the effect of activation of NMDA receptor on the phosphorylation level of intracellular signal molecules, in present study, ERK and CREB, in rACC. Exposure of the rACC slice to the NMDA (20μM) perfusate for 10 min, the phosphor-ERK (pERK) and phosphor-CREB (pCREB) expression was significantly increased. Pre-application of 50μM 7-Cl-KYNA/50μM APV/0．2 U/ml DAAO/3μM Ifenprodil for 10 min in perfusion solution, the activation of ERK and CREB was eliminated. Pre-exoposure to PD98059 (50μM) for 40 min, the activation of ERK and CREB induced by NMDA in rACC was diminished.These results indicated that, fuctional activation of gly site of NMDA receptor and NMDA NR2A and NR2B subunit is necessary for the pain related negative affect, and NMDA receptor induced activativation of ERK-CREB signal pathway in rACC is considered to be the cellular and molecular mechanism of the pain-related negetive affect acquisition. In conclusion, selectively inhibition of gly site in NMDA receptor or blockade of NR2A or NR2B subunit might be a preferred target for the prevention of pain-related emotional disturbance.