Activation of Extracellular Signal-Regulated Kinase in the Anterior Cingulate Cortex Contributes to the Induction and Expression of Affective Pain

Animals and reagents.

Experiments were performed on adult (weighing 220–250 g) and young (4-week-old) male Sprague Dawley 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 experiments were performed in according with the guidelines of the International Association for the Study of Pain, and were approved by the Shanghai Animal Care and Use Committee.

All the reagents used in the present study including NMDA, APv (DL-2-amino-5-phosphonovaleric acid), U69,593 (5α,7α,8β)-(+)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneecetamide), 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′-cyclic monophosphothioate 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 from Sigma. U69,593 (0.16 mg/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 concentration 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 modifications. 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 compartments 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 conditioning 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 different: 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 tactile 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 preconditioning 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 habituating 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 compartments in a blind manner. Rats that spent >80% (720 s) on one side on that day were eliminated from the subsequent experiments. Day 2 and 3 are conditioning days. For formalin injection and foot-shock experiments, the rat received no treatment on day 2, and was randomly confined to one of the conditioning compartments for 45 min. On day 3, for producing formalin-induced CPA (F-CPA), the rat was given a unilateral hindpaw intraplantar injection of 5% formalin (50 μl) 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 electric 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 experiments, 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 rats were given a subcutaneous injection of U69,593 (0.16 mg/kg) and then confined in the other conditioning compartment 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 on day 4, they were placed into the formalin-paired compartment for 10 min to retrieve formalin-induced pain experience. Sham CPA retrieval control rats received the same conditioning 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 water maze 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 sessions with a 30 min interval in between, each session consisting of six consecutive trials. The submerged platform is located at the central position 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 platform and 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 point was 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 platform 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.

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 to make 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 groups was selected randomly, but once selected it was fixed for that rat, whereas the visible platform was randomly placed among the four quadrants. The rat was allowed to locate the visible platform for 60 s in each trial. If successful 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 (San Diego Instruments). The escape latency and swimming traces were recorded for subsequent analysis.

Formalin test.

To minimize an environmental influence on performance of the F-CPA training task, we developed an automatic movement 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 compartment. Nociceptive behavior (agitation) elicited by formalin injection, 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 computer system that allowed quantitative recording of the number of agitation 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 performed classic manual observation simultaneously. A mirror was positioned 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 thermal hyperalgesia and mechanical allodynia.

Rats were deeply anesthetized with chloral hydrate (40 mg/kg). The skin of right hind limb was sterilized 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, proximal 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 using Hargreaves' test. Briefly, rats were placed individually into Plexiglas chambers on an elevated 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 measurement of paw withdrawal latency (PWL). The heat was maintained at a constant intensity, which produced a stable PWL of ∼10–12 s in normal animals. A 20 s cutoff was used to prevent tissue damage in the absence of a response. After acclimation to the test chambers, both hindpaws 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 ∼30 min. 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). A particular hair was 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 response was considered valid only if the hindpaw was completely removed from the customized platform. Each hair was applied 5 times at 5 s intervals. If withdrawal responses did not occur more than twice during five applications of a particular hair, the next ascending hair in the series was applied in a similar manner. Once the hindpaw was withdrawal from a particular hair three out of the five consecutive applications, the rat was considered responsive to that hair. The PWT was defined as the lowest hair force in grams that produced at least three withdrawal responses in five tests. After the threshold was determined for one hindpaw, the same testing procedure was repeated on the other hindpaw at 5-min interval.

Intra-rACC drug infusions.

Rats were anesthetized with intraperitoneal chloral hydrate (40 mg/kg), and securely placed into a sterotaxic device with bregma and lambda at a horizontal level. A 30 gauge stainless steel cannula with a 33 gauge stainless steel stylet plug was bilaterally implanted 0.5 mm above the rACC injection site [anteroposterior (AP) + 2.6 from bregma, mediolateral (ML) ± 0.6, dorsoventral (DV) −2.5] according to the atlas of Paxinos and Watson (1998). In some animals, double cannulae were implanted and two injections (one injection per cannula site) were applied for each side of the rACC (four injections in total) with the following coordinates: AP +3.0 from bregma, ML ±0.6, DV −2.5, and AP +1.6 from bregma, ML ±0.6, DV −2.2, respectively. The cannula was anchored to the cranium with three stainless steel screws and dental acrylic. Animals were allowed to recover for 5–6 d, and began to gain weight before next experimental procedure. At the end of the experiment, brains were sectioned for cresyl violet staining to verify cannula position and injection site.

Microinjection was performed through a 33 gauge stainless-steel injection cannula that extended 0.5 mm beyond the tip of the guide cannula. The injection cannula was connected to a 1 μl Hamilton syringe with PE-10 tubing. A total volume of 0.5 μl per hemisphere of either vehicle or drug was injected over a 5 min period. The injection cannula was left in place for an additional 5 min to minimize spread of the drug along the injection track.

To examine the effect of drugs on the induction (acquisition) of F-CPA, S-CPA, or U69,593-induced CPA, intra-rACC microinjection of drugs or vehicle was performed 30 min before intraplantar formalin (or foot-shock, s.c. U69,593) on conditioning day. To examine the effects of drugs on F-CPA expression, the drugs or vehicle were administered into the bilateral rACC 30 min or 6 h before F-CPA test on postconditioning day. To examine whether drugs per se produced place preference or avoidance in rats without formalin treatment, drugs or vehicle were injected into the bilateral rACC 30 min before conditioning on conditioning day. Training procedures were performed as F-CPA (described above) except that formalin was replaced by NS. To examine the effects of drugs on Morris water maze task, the drug or vehicle was administered into the bilateral rACC 30 min before training in water maze. To examine the effects of drugs on formalin-induced acute spontaneous nociceptive response, the drug or vehicle was administered into the bilateral rACC 30 min before intraplantar injection of formalin. To examine the effects of drugs on CCI-induced behavioral hypersensitivity, drug or vehicle was administered on day 3 after CCI operation when the thermal hyperalgesia and mechanical allodynia reliably developed, and behavioral testing was performed at 1, 2, 3, 4, and 5 h after intra-rACC injection. This behavioral testing protocol was based on our previous study showing that PD98059 produces the best anti-neuropathic pain effect at 3 h after injection (Zhuang et al., 2005). All behavioral tests were performed blind with respect to drugs injected.

Western blots.

After defined survival times, rats were killed by overdose of Chloral hydrate (80 mg/kg) and the brain was quickly removed. The rACC was dissected on ice using a Rat Brain Matrix (Stoelting Company). Briefly, three coronal brain slices (1 mm thick) containing rACC (AP 3.7–0.7 from bregma) were cut. Then the brain slices were separated into left and right half from the sagittal suture. Finally, the rACC tissues were dissected using a surgical blade according to the atlas of Paxinos and Waston, and rapidly frozen in liquid nitrogen. Frozen samples were homogenized in a lysis buffer (12.5 μl/mg tissue) containing a mixture of protease inhibitors (Roche) and PMSF (Sigma). After incubating in ice for 30 min, samples were centrifuged at 10,000 rpm for 15 min at 4°C. The supernatants were used for Western blotting.

Equal amount of protein (∼20 μg) was loaded and separated in 10% Tris-Tricine SDS-PAGE gel. The resolved proteins were transferred onto polyvinilidene difluoride (PVDF) membranes (Amersham Bioscience). The membranes were blocked in 10% non-fat milk for 2 h at room temperature (RT), and incubated overnight at 4°C with mouse anti-phospho-ERK (pERK1 and pERK2; 1:3000, Sigma), mouse anti-ERK (total ERK1 and ERK2; 1:100,000, Sigma), rabbit anti-pCREB (1:3000, Upstate Biotechnology), rabbit anti-CREB (total CREB; 1:1000, Sigma), or rabbit anti-pPKA RII (1:1000, Upstate Biotechnology) primary antibody. The blots were then incubated with the secondary antibody, goat anti-mouse or goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (1:1000, Pierce), for 2 h at 4°C. Signals were finally visualized using enhanced chemiluminescence (ECL, Pierce) and the blots were exposed onto X-films for 1–10 min. All Western blot analysis was performed at least three times, and consistent results were obtained.

Immunohistochemistry.

After defined survival times, rats were killed by overdose of chloral hydrate (80 mg/kg) and perfused transcardially with NS followed by 4% paraformaldehyde in 0.1 m phosphate buffer (PB, pH 7.4). Brains were then removed, postfixed in the same fixative for 6 h at 4°C, and immersed in 10–30% gradient sucrose in PB for 24–48 h at 4°C for cryoprotection. Coronal sections (30 μm) were cut in a cryostat (Leica 1900) and processed for immunofluorescence. Briefly, the sections were blocked with 10% donkey serum in 0.01 m PB saline (PBS, pH 7.4) with 0.3% Triton X-100 for 1 h at RT and incubated overnight at 4°C with mouse anti-pERK (1:2000; Sigma) or rabbit anti-pCREB (1:3000; Upstate Biotechnology) primary antibody in PBS with 1% normal donkey serum and 0.3% Triton X-100. After three 15 min rinses in PBS, the sections were incubated in fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG (1:200; Jackson ImmunoResearch) or rhodamine-conjugated donkey anti-rabbit IgG (1:200, Jackson ImmunoResearch) for 2 h at 4°C, then washed in PBS. For pERK/NeuN, pERK/MAP-2, pERK/OX-42, pERK/GFAP, pCREB/NeuN, pERK/pCREB, pERK/NR1, or pCREB/Fos double immunofluorescence, the sections were incubated with a mixture of rabbit anti-pERK (1:200, Cell Signaling) and mouse anti-NeuN (neuronal marker, 1:5000, Millipore), mouse anti-MAP-2 (pyramidal cell marker, 1:1000, Millipore), mouse anti-OX-42 (microglia marker, 1:5000, Serotec), or mouse anti-GFAP (astrocyte marker, 1:3000, Sigma), or mouse anti-pERK (1:2000) and rabbit anti-pCREB (1:3000), or rabbit anti-pCREB (1:3000) and mouse anti-NeuN (1:5000), or mouse anti-pERK (1:2000) and rabbit anti-NR1 (1:200, Millipore), or rabbit anti-pCREB (1:3000) and goat anti-Fos (1:200, Santa Cruz Biotechnology) overnight at 4°C. All sections were coverslipped with a mixture of 50% glycerin in PBS, and then observed with a Leica SP2 confocal laser-scanning microscope. The specificity of immunostaining was verified by omitting the primary antibodies, and immunostaining signal disappeared after omitting primary antibodies. The specificity of primary antibodies was verified by the preabsorption experiment. Brain sections were first incubated with a mixture of pERK or pCREB primary antibody and the corresponding blocking peptide for phospho-p44/42 (10 μg/ml; Cell signaling) or phospho-CREB (20 μg/ml; Cell Signaling) overnight, followed by secondary antibody incubation. pERK and pCREB immunostaining signal was abolished after absorption (supplemental Fig. 1, available at www.jneurosci.org as supplemental material).

Brain slice preparation.

Coronal brain slices containing the rACC were obtained from young rats (4-week old). After anesthetizing with isoflurane, rats were decapitated. The brain was quickly removed and submerged in preoxygenated (95% O₂ and 5% CO₂) cold artificial CSF (ACSF) containing (mm): 126 NaCl, 4.0 KCl, 1.25 MgCl₂, 26 NaHCO₃, 1.25 NaH₂PO₄, 2.5 CaCl₂, and 10 glucose. The osmolarity was adjusted to 300 mosmol/L and the pH to 7.35. Slices (380 μm thick) were cut with a vibratome (Leica VT1000S) and transferred to an oxygenated chamber at RT (22 ± 1°C) for at least 1 h before further processing.

For immunohistochemical experiments, the rACC slices were treated with different agonist (or activators) at RT: NMDA (20, 50, and 100 μm), forskolin (50 μm), or Sp-cAMP (50 μm) for 10 min. NMDA receptors antagonist APv (50 μm), adenylyl cyclase (AC) inhibitor SQ22536 (50 μm), PKA inhibitor Rp-cAMP (50 μm), or MAP kinase kinase (MEK) inhibitor PD98059 (50 μm) was added to ACSF 40 min before and during stimulation. Untreated or 0.2% DMSO-treated slices served as controls. Subsequently, the slices were rapidly immersed in cold 4% paraformaldehyde and fixed for 60 min, and then processed for immunostaining.

Quantification and statistics.

For the quantification of Western signals, x-ray films with blotting bands were scanned. Bio-Rad Image Analysis System (quantity one, Bio-Rad) was then used to measure the integrated optic density of the bands. For the quantification of immunoreactive signals, six non-adjacent sections (30 μm) through the rACC were randomly selected. The numbers of pERK- and pCREB-labeled cells were counted in laminae II-VI of the rACC that was captured inside the optic field using a computerized image analysis system (Leica Qwin 500). The areas sampled within the rACC were: 520 × 480 μm in the laminae II-III and 480 × 520 μm in the laminae V-VI. Four to five rats were included in each group for quantification of Western blot and immunohistochemistry results. Differences between groups were compared using one-way ANOVA followed by post hoc Dunnett's test or using Student's t test if only two groups were applied.

In the case of CPA experiments, the CPA scores represent the time spent in the treatment-paired compartment on the preconditioning day (day 1) minus the time spent in the same conditioning compartment on the postconditioning day (day 4). The differences in CPA scores among drug-treated groups were compared using Student's t test for a two-group comparison or one-way ANOVA followed by post hoc Dunnett's test for multiple group comparison. In addition, the absolute time spent in the treatment-paired compartment on the preconditioning day versus the postconditioning day was compared in training (F-CPA, S-CPA), sham training, vehicle or drug-treated animals using paired t test. For formalin-induced nociceptive behaviors, the agitation-induced vibration events (using automatic movement recorder) and formalin pain scores (using manual detection) per 5 min were analyzed using a two-way (drug treatment × time) ANOVA with a post hoc comparison for analysis of the differences at each 5-min period. For CCI-induced behavioral hypersensitivity, Pre-CCI baseline measures were analyzed by one-way ANOVA. Pre-and postdrug time course measures for both hyperalgesia and allodynia were analyzed by two-way (treatment × time) ANOVA followed by a post hoc test. For Morris water maze experiments, the differences in the escape latencies to find both the submerged and visible platform between drug- and vehicle-treated groups were compared using Student's t test. All data were expressed as mean ± SEM, and the accepted level of statistical significance for all experiments was p < 0.05.

All the behavioral testing and quantification of Western blot and immunohistochemical experiments were performed blind with respect to treatments.