Contribution Of ERK/MAPK Cascade In Anterior Cingulate Cortex To Pain-related Aversion In Rat

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage,which including a sensory-discriminative and an emotional-affective component.The severities and qualities of negative affect of pain is the most characteristic among different feelings.Clinical observations indicate that the patients with chronic pain suffer from as much emotional disturbance as pain sensation per se.Physiological arousal and hypersensitivity to painful stimuli cause unpleasant feelings to patients,such as fear,anxiety,anger,depression and even a suicidal tendency.These negative affective states in turn enhance pain perception.Along with the advances in animal praxiology,cognitive psychology,and human functional brain imaging,experimental studies on pain-related emotion and affection in animals are possible.Accumulating evidence from morphological, electrophysiological,neuroimaging,and behavioral studies as well as early clinical observation indicates that the anterior cingulate cortex(ACC) is involved in pain-related emotion,cognition,memory and anticipation.However,little is known about the molecular and cellular mechanisms underlying pain-related aversion in ACC.Extracellular signal-regulated kinase(ERK) is a member of mitogen-activated protein kinase(MAPK) family.Activation of ERK/MAPK cascade in dorsal horn neurons of the spinal cord following peripheral noxious stimulation contributes to both short-term and long-term pain hypersensitivity.Since evidence has showed that the ACC is involved in pain emotion,how about the relationship between ERK/MAPK cascade in ACC and pain-related aversion?The overall goal of our study is to investigate the activation of ERK/MAPK in the ACC and its contribution to persistent pain-induced negative emotion using behavioral,immunohistochemical,Western Blot and electrophysiological approaches.Firstly,we observed the contribution of ERK activation in ACC to pain-related emotion using immunohistochemistry,Western Blot and behavioral training techniques.Intraplantar(i.pl.) injection of 5%formalin 50μl induced a rapid and sustained ERK activation in bilateral ACC neurons.Intra-ACC microinjection of MEK(ERK kinase) inhibitor PD098059(10,1 nmol) or U0126(1μg) could dose-dependently block the formation of F-CPA,but did not affect neither formalin-induced nociceptive behavior nor S-CPA(electric foot shock-induced conditioned place avoidance),a fear conditioning paradigm.Long-term potentiation could be induced in ACC slice by high frequency stimulation(2-train,100 Hz,1 sec) using field potential recording method.AP-5,the antagonist of NMDA receptor,blocked the induction of cingulate LTP,indicating that LTP in this experiment was NMDA-receptor-dependent.Furthermore,a MEK inhibitor,PD98059(50μM) perfusion could completely blocked the induction of LTP. Consistent with the results above,we found ERK was activated in response to LTP-inducing high frequency stimulation.Our study demonstrated that NMDA receptor and ERK activation was necessary for the induction of LTP in rat anterior cingulate cortex.The upstream modulation of ERK/MAPK activation was further studied. Incubation of ACC slices with NMDA receptor agonist,NMDA(50μM),Adenylyl cyclase(AC) activator,Forskolin(50μM) or cAMP-dependent protein kinase A (PKA) activator,Sp-cAMP(50μM) produced significant activation of ERK,which was suppressed by pre-incubation with corresponding respective antagonist AP-5(50 p-M),SQ22536(50μM),Rp-cAMP(50μM),as well as MEK inhibitor,PD98059(50μM).Similarly,F-CPA was also blocked by pre-microinjection of SQ22536(1,0.1, 0.01 nmol) or Rp-cAMP(10,1 nmol) in bilateral ACC.These findings suggested that NMDA receptors were coupled to ERK through cAMP/PKA pathway,which was also involved in pain-related aversion.Activated ERK can be translocated to the nucleus leading to the phosphorylation of transcription factor such as CREB.Using immunohistochemistry and Western Blot, our results showed that CREB is activated following i.pl.injection of 5%formalin 50μl and the upregulated pCREB could be suppressed by pre-intra-ACC microinjection of PD98059 or Rp-cAMP.Also,incubation of ACC slices with NMDA(50μM), Forskolin(50μM) or Sp-cAMP(50μM) produced robust phosphorylation of CREB, which could be suppressed by pre-incubation with their corresponding antagonist AP-5(50μM),SQ22536(50μM),Rp-cAMP(50μM) and PD98059(50μM).The double immunostaining showed that 97%pERK-positive neurons were co-localized with pCREB.These results demonstrated that NMDA receptor-dependent cAMP/PKA-ERK-CREB signaling transduction existed in ACC and might contribute to pain-related aversion.We also investigated ERK activation in the expression of pain-related aversion. Following F-CPA retrieval,ERK and CREB in the ACC were activated.Bilateral intra-ACC injection of PD098059 also suppressed F-CPA retrieval-induced phosphorylation of ERK and CREB in the ACC.Moreover,pre-delivery of another MEK inhibitor U0126,either 30 min or 6 hrs before testing on the post-conditioning day,blocked the acquisition of F-CPA.We therefore hypothesized that NMDA receptor activation in the ACC might trigger the intracellular ERK/CREB signal transduction pathway and its related protein synthesize,which was necessary for the expression of pain-related emotion.Taken together,our data indicates that activation of ERK/MAPK in the ACC following nociceptive conditioning depends on NMDA receptor-dependent cAMP/PKA pathway and leads to the activation of CREB.We conclude that activation of ERK/MAPK cascade in the ACC is necessary for both acquisition and expression of pain-related aversion.