2009-Gao and Cao-JN

Cellular/Molecular Activation of Extracellular Signal-Regulated Kinase in the Anterior Cingulate Cortex Contributes to the Induction and Expression of Affective Pain Hong Cao,1* Yong-Jing Gao,1,2*Wen-Hua Ren,1 Ting-Ting Li,1 Kai-Zheng Duan,1 Yi-Hui Cui,3 Xiao-Hua Cao,3 Zhi-Qi Zhao,1 Ru-Rong Ji,2 and Yu-Qiu Zhang1 1Institute of Neurobiology, Institutes of Brain Science and State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China, 2Department of Anesthesiology, Brigham andWomen’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, and 3Shanghai Institute of Brain Functional Genomics, Key Laboratory of Brain Functional Genomics, Ministry of Education, and Science and Technology Commission of Shanghai Municipality, East China Normal University, Shanghai 200062, China The anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. However, little is known about the molecularmechanisms involved. The present study demonstrated that extracellular signal-regulated kinase (ERK) activation in the ACC plays a crucial role inpain-relatednegative emotion. Intraplantar formalin injectionproduceda transientERKactivation in laminaeV-VI and a persistent ERK activation in laminae II-III of the rostral ACC (rACC) bilaterally. Using formalin-induced conditioned place avoidance (F-CPA) in rats, which is believed to reflect the pain-related negative emotion, we found that blockade of ERK activation in the rACC with MEK inhibitors prevented the induction of F-CPA. Interestingly, this blockade did not affect formalin-induced two-phase spontaneous nociceptive responses and CPA acquisition induced by electric foot-shock or U69,593, an innocuous aversive agent. Up- stream, NMDA receptor, adenylyl cyclase (AC) and phosphokinase A (PKA) activators activated ERK in rACC slices. Consistently, intra-rACC microinjection of AC or PKA inhibitors prevented F-CPA induction. Downstream, phosphorylation of cAMP response ele- ment binding protein (CREB) was induced in the rACC by formalin injection and byNMDA, AC and PKA activators in brain slices, which was suppressed by MEK inhibitors. Furthermore, ERK also contributed to the expression of pain-related negative emotion. Thus, when rats were re-exposed to the conditioning context for retrieval of pain experience, ERK and CREB were reactivated in the rACC, and inhibiting ERK activation blocked the expression of F-CPA. All together, our results demonstrate that ERK activation in the rACC is required for the induction and expression of pain-related negative affect. Introduction Pain is evoked by noxious body stimuli or conditioned stimuli that predict noxious stimulation (Vogt, 2005). Many forms of pain are associated with varying severities and qualities of emo- tion distress. Clinical observations are increasingly indicating that in chronic pain patients, pain-related negative affect is more disabling than pain itself (Crombez et al., 1999). Recently, pain- related negative affect has gained more attention. Accumulating evidence suggests that the anterior cingulate cortex (ACC) is a key structure for pain affect (Price, 2000). Intra- rostral ACC (rACC) injection of kynurenic acid, awide-spectrum excitatory amino acid receptor antagonist, blocked the acquisi- tion of formalin-induced conditioned place avoidance (F-CPA) (Johansen and Fields, 2004). Our previous studies also demon- strated that blockade of NMDA but not AMPA/KA receptors in the rACCeliminated F-CPA, suggesting that activation ofNMDA receptor in the rACC is necessary for the induction of pain- related negative emotion (Lei et al., 2004a; Ren et al., 2006). NMDA receptor is highly expressed in the ACC and plays a crit- ical role in mediating long-lasting synaptic plasticity (Monyer et al., 1994; Wei et al., 2001; Zhao et al., 2005). It is also generally believed that maintenance of long-term synaptic plasticity re- quires gene expression and protein synthesis (Bailey et al., 1999). To understand themechanisms underlying NMDA-mediated af- fective pain, it is important to identify the signaling pathways that couple NMDA receptors to gene expression. Extracellular signal-regulated kinase (ERK), a family member of mitogen-activated protein kinases (MAPKs), has been impli- cated in NMDA-dependent synaptic plasticity, such as learning/ memory and pain hypersensitivity in the hippocampus and spi- nal cord (Ji et al., 1999; Karim et al., 2001, Krapivinsky et al., 2003; Received Sept. 9, 2008; revised Jan. 11, 2009; accepted Feb. 12, 2009. This work was supported by the National Natural Science Fund of China (NSFC Grants 30425022, 30528019, 30870835, 30821002, 30670682, and30500153),National Basic ResearchProgramofChina (Grants 2006CB500807, 2007CB512303, and 2007CB512502), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT), and National Institutes of Health Fogarty International Research Collaboration Award (FIRCA Grant TW7180).We thank Prof. B.M. Li’s group for technical support in theMorris watermaze andM. Han for neuropathic pain experiments. *H.C. and Y.-J.G. contributed equally to this work. Correspondence should be addressed to either of the following: Dr. Yu-Qiu Zhang, Institute of Neurobiology, Institutes of Brain Science, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China, E-mail: yuqiuzhang@fudan.edu.cn; or Ru-Rong Ji, Department of Anesthesiology, BrighamandWomen’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, E-mail: rrji@zeus.bwh.harvard.edu. DOI:10.1523/JNEUROSCI.4300-08.2009 Copyright © 2009 Society for Neuroscience 0270-6474/09/293307-15$15.00/0 The Journal of Neuroscience, March 11, 2009 • 29(10):3307–3321 • 3307 Kawasaki et al., 2004; Wei et al., 2006). In cell lines and cultured neurons, activated ERK translocates from the cytosol to the nu- cleus where it phosphorylates cAMP response ele3ment binding protein (CREB), subsequently activating cAMP response element (CRE)-mediated gene transcription (Treisman, 1996; Impey et al., 1998; Bozon et al., 2003). A series of work have highlighted a critical role of the ERK cascades in triggering gene transcription after synaptic activation (Sweatt, 2001; Ji et al., 2002). Moreover, ERK is known to integrate other signaling pathways, such as cAMP/protein kinase A (PKA) pathway (Hu and Gereau, 2003; Kawasaki et al., 2004). Given the similarities in molecular mechanisms underlying synaptic plasticity in different regions in the CNS (Ji et al., 2003), we hypothesized that molecular mechanism underlying pain- related negative emotion in the rACCmay share common signal- ing pathwayswith nociceptive sensitization in the spinal cord and learning/memory in the hippocampus. In this study, using a ro- dent formalin painmodel that allows simultaneousmeasurement of a learned behavior that directly reflects the pain affect (F-CPA) and “acute” formalin-induced “pain” behavior (paw lifting, lick- ing, and flinching) that reflects the noxious stimulus parameters (intensity, localization) (Johansen et al., 2001), we demonstrated that the NMDA receptor-cAMP-PKA-ERK signaling pathway in the rACC is also involved in pain-related negative affect. In par- ticular, ERK activation in the rACC is required both for the in- duction and expression of affective pain but not for nociceptive pain. Materials andMethods Animals and reagents. Experiments were performed on adult (weighing 220–250 g) and young (4-week-old)male SpragueDawley rats for in vivo studies and brain slice studies, respectively. Animals were obtained from Experimental Animal Center of Fudan University and were on a 12:12 light-dark cycle with a room temperature of 23� 1°C, and received food and water ad libitum. Before experimental manipulations, the animals were given a period of 7 d to adjust to the new surroundings. All experi- ments were performed in according with the guidelines of the Interna- tional Association for the Study of Pain, andwere approved by the Shang- hai Animal Care and Use Committee. All the reagents used in the present study including NMDA, APv (DL- 2-amino-5-phosphonovaleric acid), U69,593 (5�,7�,8�)-(�)-N- methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneecet- amide), PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran- 4-one), U0126 (1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene ethanolate), Sp-cAMP (Sp-Adenosine 3�,5�-cyclicmonophos- phothioate triethyammonium salt hydrate), Rp-cAMP (Rp-Adenosine 3�,5�-cyclic monophosphorothiote triethyammonium salt hydrate), SQ22536 (9-(Tetrahydro-2-furanyl)-9H-purin-6-amine), and forskolin were purchased fromSigma.U69,593 (0.16mg/ml) was dissolved in 20% propylene glycol/saline and stirred overnight. PD98059 (10 mM) and U0126 (2 mM) were dissolved in 10% and 35% dimethylsulfoxide (DMSO), respectively. NMDA, APv, Sp-cAMP, Rp-cAMP, forskolin, and SQ22536 were dissolved in normal saline (NS). The DMSO concen- tration used in slice experiment was 0.1–0.2%. Conditioned place avoidance. Conditioned place avoidance (CPA) was conducted as described previously (Gao et al., 2004), with slight modifi- cations. The place conditioning apparatus consists of three opaque acrylic compartments. Two large ones are conditioning compartments (30 � 30 � 30 cm) and a smaller one is a neutral choice compartment (15 � 20 � 30 cm, length � width � height). The conditioning com- partments are placed in parallel and separated by a wall with a square door (10� 10 cm). The neutral compartment is laid in front of the two conditioning compartments with two doors (10 � 10 cm) to them. A movable transparent ceiling covers each compartment. The two condi- tioning compartments are both painted black, but one is decorated with a transverse white band and contains an odor produced by 1.0% acetic acid; the other is decorated with a white vertical band and has an odor of cinnamon. The floors of the conditioning compartments are also differ- ent: one is made from Plexiglas, and the other is from a polyester board with a metal band on it, which can provide an electric shock. Thus, the two conditioning compartments have distinct visual, olfactory and tac- tile cues. The neutral compartment is white, absent of distinctive odor and has a solid acrylic floor with a slope. The experimental process consists of three distinct sessions: a precon- ditioning session, a conditioning session and a test (postconditioning) session. CPA task processing takes 4 d. Day 1 is a preconditioning day. At the beginning, a rat was placed in the neutral compartment. After habit- uating for 2 min, the entrance to each conditioning compartment was opened. When the rat enters any conditioning compartment, the doors connecting neutral and conditioning compartments were closed. The rat was allowed to explore the two conditioning compartments freely for 15 min. A timer automatically recorded the time spent in each of the com- partments in a blind manner. Rats that spent�80% (720 s) on one side on that daywere eliminated from the subsequent experiments. Day 2 and 3 are conditioning days. For formalin injection and foot-shock experi- ments, the rat received no treatment on day 2, and was randomly con- fined to one of the conditioning compartments for 45 min. On day 3, for producing formalin-inducedCPA (F-CPA), the rat was given a unilateral hindpaw intraplantar injection of 5% formalin (50 �l) or NS (control), and then restrained in the other conditioning compartment for 45 min. For electric foot-shock-induced CPA (S-CPA), the rat received an elec- tric shock (0.5 mA for 2 s) every 8–10 min in the other conditioning compartment during the 45-min training session. For U69,593 experi- ments, rats received no treatment in the morning on day 2, and were randomly confined to one of the conditioning compartments for 1 h. After 5 h, in the afternoon, the ratswere given a subcutaneous injection of U69,593 (0.16mg/kg) and then confined in the other conditioning com- partment for 1 h. On day 3, the same trials as day 2 were repeated. Both the treatments (formalin, shock, U69,593, vehicle, or no treatment) and the compartments were counterbalanced. Day 4 is postconditioning day. The procedure is the same as day 1. The time animals spent in each compartment was measured. F-CPA retrieval. Rats received the same training trials in days 1–3 of conditioning procedure as F-CPA. But onday 4, theywere placed into the formalin-paired compartment for 10 min to retrieve formalin-induced pain experience. Sham CPA retrieval control rats received the same con- ditioning procedure as F-CPA retrieval, but on day 4, they were placed into the nonformalin-paired compartment for 10 min. Nonretrieval rats received the same training trials as F-CPA retrieval, but on day 4, they were allowed to remain in their home cages without 10 min F-CPA retrieval before kill. Morris water maze task.The watermaze consists of a black round tank, which has a diameter of 150 cm and height of 54 cm and is filled with water (24� 2°C) to a depth of 38 cm. The water is made opaque so that the submerged platform (9.0 cm in diameter, 2.0 cm below the water surface) is invisible. The training and testing protocols were essentially as described (Jin et al., 2005). The training procedure consists of two ses- sions with a 30 min interval in between, each session consisting of six consecutive trials. The submerged platform is located at the central po- sition of the southeast quadrant of the tank. The starting position is randomly selected, but counterbalanced among the four positions. A rat was allowed to search for the submerged platform for 60 s. If successful in locating the platform within 60 s, the rat was allowed to stay on the platform for 30 s; if not, it was directed to the platformand allowed to stay there for 30 s. Thereafter, the rat was returned to a holding cage. The next trial began after an intertrial interval of 30 s. A three-trial retention test was conducted 48 h after the training. For each rat at each trial, the submerged platform was fixed at the target quadrant and the starting pointwas at the position opposite to it. Each rat was giving 60 s to locate the submerged platform. If successful in finding the platform within 60 s, the rat was immediately returned to a holding cage for 60 s before next trial began. If unsuccessful in locating the plat- form within 60 s, the rat was directed to the platform and allowed to stay there for 30 s, and then was returned to a holding cage for 30 s before the next trial. 3308 • J. Neurosci., March 11, 2009 • 29(10):3307–3321 Cao et al. • ERK in Affective Pain Immediately after the retention test was completed, the rat was tested in a visible platform version of the Morris water maze. The platform was raised to above the water surface and covered with white gauze tomake it highly visible. Each animal was placed on the visible platform for 30 s before testing. The starting position for any given rat from the groupswas selected randomly, but once selected it was fixed for that rat, whereas the visible platformwas randomly placed among the four quadrants. The rat was allowed to locate the visible platform for 60 s in each trial. If success- ful in finding the platform, the rat was returned immediately to a holding cage; if not, the rat was removed from water and returned to a holding cage. The next trial began after an intertrial interval of 60 s. A total of three trials were conducted for each rat. Navigation of each animal in the water maze was monitored using a video camera, a tracking system and tracking software (SanDiego Instru- ments). The escape latency and swimming traces were recorded for sub- sequent analysis. Formalin test. To minimize an environmental influence on perfor- mance of the F-CPA training task, we developed an automaticmovement recorder by modifying a previous automatic detection system (Jett and Michelson, 1996; Xie et al., 2005), which can simultaneously record the two phases of the formalin-induced nociceptive behavioral response during F-CPA training task (Gao et al., 2004; Ren et al., 2006). Briefly, after intraplantar injection of formalin, the animal was immediately placed into the conditioning compartment on the conditioning day. There was a spring balance under the floor of each conditioning com- partment. Nociceptive behavior (agitation) elicited by formalin injec- tion, such as licking, flinching, shaking, elevating, clutching and favoring the affected paw, induces vibration of the spring balance, which can be converted into electrical signals via an electromagnetic transducer. The electrical signals were amplified, filtered (1.0 kHz) and fed into a com- puter system that allowed quantitative recording of the number of agita- tion events and construction of time histograms. The histograms were recorded automatically for 45 min, and the time course of changes of movement events per 5 min were plotted. To validate the accuracy of this automatic recording, we also per- formed classic manual observation simultaneously. A mirror was posi- tioned below a chamber at a 45°C angle for unobstructed observation of the rat’s paws. The responses to formalin injection were monitored by measuring the time the animal spent on lifting, licking, and shaking the affected paw per 5 min during a 45 min observation period. A weighted pain score for each animal was calculated using the following formula (Tanimoto et al., 2003): formalin pain score � [seconds spent on paw elevation� 2� (seconds spent licking or biting injected paw)]/300. Chronic constriction injury of the sciatic nerve and measurement of ther- mal hyperalgesia andmechanical allodynia.Rats were deeply anesthetized with chloral hydrate (40 mg/kg). The skin of right hind limb was steril- ized with iodine tincture, then 75% alcohol, and the right sciatic nerve exposed at the mid-thigh level by blunt dissection of the biceps femoris. For producing chronic constriction injury (CCI), four chromic gut (4–0) ligatures were tied loosely around the nerve� 1 mm apart, prox- imal to its trifurcation, as described by Bennett and Xie (1988). For sham surgery, the sciatic nerve was isolated but not ligated. After CCI or sham surgery, the overlying muscles and skin were closed respectively in layers with 4–0 silk sutures and dusted with antibiotic power. Thermal hyperalgesia was assessed by measuring the paw withdrawal latency (PWL) in response to a radiant heat source usingHargreaves’ test. Briefly, rats were placed individually into Plexiglas chambers on an ele- vated glass platform, under which a radiant heat source (model 336 combination unit, IITC/Life Science Instruments) was applied to the glabrous surface of the paw through the glass plate. The heat source was automatically turned off when the rat lifted the foot, allowing the mea- surement of paw withdrawal latency (PWL). The heat was maintained at a constant intensity, which produced a stable PWL of�10–12 s in nor- mal animals. A 20 s cutoff was used to prevent tissue damage in the absence of a response. After acclimation to the test chambers, both hind- paws were tested independently with 10 min intervals between trials. Mechanical allodynia was assessed by measuring the paw withdrawal threshold (PWT) in response to a calibrated series of von-Frey hairs (Stoelting) ranging from 0.6 to 26 g. Animals were placed individually into wire mesh-bottom cages, and allowed to acclimatize for�30min. A series of 10 calibrated von Frey hairs were applied to the central region of the plantar surface of one hindpaw in ascending order (0.6, 1, 1.4, 2, 4, 6, 8, 10, 15, and 26 g). Aparticular hairwas applied until buckling of the hair occurred. This was maintained for�2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal re- sponsewas considered valid only if the hindpawwas completely removed from the customi