Experimental animals and pain model
Both male and female C57BL/6 mice and male Fos-tTA-eGFP transgenic mice (Jackson Laboratory, stock #: 018306) weighing 20-28 g were used for the experiment. The mice were housed 4-6 per cage in a temperature-controlled room (23 ± 1 ℃, 12 h/12 h light/dark cycle) and maintained with pellet diet and tap water ad libitum except for fasting periods. All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC, SNU-191001-9-1) at Seoul National University. The experiments were also designed and performed in accordance with the International Association for the Study of Pain (IASP) guidelines. All efforts were made to reduce animal suffering and decrease the number of animals used. For 24 hr fasting, food was removed from the cage but free access to water. For refeeding, mice were given normal chow for 2 hr following 24 hr fasting. We used a modified CFA-induced chronic inflammatory pain model to prolong pain behaviors for more than 20 days, as previously described [24]. 20 μL of undiluted CFA (Sigma-Aldrich) was injected subcutaneously into the plantar surface of the left hind with a 0.3 mL insulin syringe. A booster injection was given 7 days after the first injection. For electrophysiological recordings, the mice were injected with the same amount of CFA into both hind paws. All experiments were performed by a researcher blinded to treatment condition.
Pain behavior tests
The behavior tests were conducted as described previously [1, 24]. For spontaneous pain measurement, mice were acclimated in the cage at least for a week and then adapted in an acrylic observation chamber (size ranges 12×12×12 cm) before the experiment at least three times for three hours. A mirror was located at 45° angle below the chamber to observe the paws. Mice were video recorded using a video camera for 30 min. The time spent licking or biting was measured by an observer who was blinded to the treatment.
To assess mechanical allodynia, 50% paw withdrawal threshold was measured using von Frey filaments (North Coast Medical). Mice were acclimated in the cage at least for a week and then adapted in an acrylic cylinder (6.5 cm diameter, 17 cm height) on the metal mesh floor before the experiment at least three times. All animals were left to an acrylic cylinder on the metal mesh floor for 30 min prior to the mechanical test. The 50% paw withdrawal threshold was determined based on the up-down method with an ascending series of von Frey filaments (0.16 g, 0.4 g, 0.6 g, 1 g, and 2 g).
Administration of drugs
Sulpiride and SR 141716 were purchased from Tocris. For systemic administration, sulpiride 100 mg/kg was diluted in 10% DMSO (Sigma-Aldrich), and 2 μL of acetic acid glacial (Duksan) was added, then pH was adjusted with NaOH (pH 7.4). For direct brain infusion, sulpiride (5 μg/μL) was dissolved in 0.9% normal saline and unilaterally microinjected (0.5 μL over 5 min) into the contralateral NAcS. Injectors were left in place for an additional 2 min before removal. SR 141716 (10 mg/kg) was diluted in 0.9% normal saline with 10% DMSO and 1% tween 80.
Clozapine-N-oxide (CNO) was purchased from Tocris. For systemic injection, CNO 10 mg/kg was dissolved in 0.9% normal saline and intraperitoneally injected 30 min prior to the pain behavior test. For microinjection, CNO (3 μM) was dissolved in the 0.9% saline and administrated into the NAcS 5 min prior to the pain behavior test. For electrophysiology, CNO (3 μM) was dissolved in the bath solution.
Stereotaxic surgeries
C57BL/6 mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and implanted with a guide cannula (26-gauge, 4.6 mm of length, Plastics One) aimed at the NAcS (coordinates: anteroposterior (AP) 1.4 from bregma, mediolateral (ML) 0.5 from the midline, dorsoventral (DV) −4.6 from skull surface). When inserted, microinjector tips extended 0.5 mm beyond the guide. The aIC (coordinates: anteroposterior (AP) 1.8 from bregma, mediolateral (ML) 2.5 from the midline, dorsoventral (DV) −3.5 from skull surface) was injected with virus.
The following vectors were used for single-site Designer receptors exclusively activated by designer drugs (DREADD) and cre-EGFP expression: pAAV-hSyn-DIO-hM4D(Gi)-mCherry, retrograde pAAV-CaMKIIa-hM3D(Gq)-mCherry, pENN-AAV-CaMKIIa-HI.GFP-Cre-WPRE-SV40 (Addgene). Viral vectors were injected at the rate of 0.2 μL/min, with a 10-min diffusion time. The injection volume for the single-site injections was 0.5 μL. The misplaced cannula or virus injections were excluded from the analysis. DREADD expression was allowed to accumulate for 2 weeks before systemic/local vehicle and CNO injections.
Immunohistochemistry
Mice were administrated with pentobarbital (50 mg/kg), then transcardially perfused, first with PBS and then with a 4% paraformaldehyde solution. Brains were post-fixed at 4°C in paraformaldehyde for 1 d following the extraction and cryoprotected in 30% sucrose for 3 days. The brains were then sectioned into 40 μm slices and preserved in PBS. For the DAB staining, free-floating sections were rinsed in PBS, incubated for 30 min in 0.3% hydrogen peroxide PBS solution to quench endogenous peroxidase activity, rinsed several times in PBS, and incubated in a blocking solution that contained 5% normal goat serum diluted in 0.3% Triton X-100 in PBS for 60 min. The sections were incubated in rabbit anti-c-Fos polyclonal antibody (1:1000 dilution, ab11137 Abcam) diluted in blocking serum for 48 hr at 4°C. After incubation in the primary antibody, the sections were rinsed three times for 10 min in PBS and incubated for 2 hr in biotinylated secondary antiserum made from goat anti-rabbit antibody (1:200 dilution; Vector Laboratories) in PBS. Then the sections were processed with ABC kit (PK-6100, Vectastain ABC kit, Vector laboratories) for an hour and visualized with DAB kit (DAB substrate kit for peroxidase, Vector laboratories). After several rinses in PBS, the sections were mounted on coated glass slides, air dried, dehydrated through a series of graded ethanol, and clearing agents, then permounted (Sigma-Aldrich).
To detect D1R, D2R, and CaMKII expression, sections were incubated overnight in blocking buffer (0.3% Triton X-100, 2% bovine serum in PBS) at room temperature. The primary antibodies rabbit anti-D1R (1:200 dilution, ab20066, Abcam), mouse anti-D2R (1:50 dilution, b-10, Santa Cruz), and mouse anti-CaMKII (1:100 dilution, sc-5306, Santa Cruz) were diluted in the blocking solution, and the sections were incubated for 48 hr at 4°C. The slices were washed three times for 10 min each in 0.1 M PBS. To visualize D2R expression, sections were incubated for 2 hr in biotinylated anti-mouse IgG (Vector, PB-9200). Then washed with 0.1 M PBS three times for 10 min each. The secondary antibody, FITC donkey anti-rabbit (1:200 dilution, 706-095-148, Jackson ImmunoResearch Laboratories), Alexa 488 donkey anti-mouse (1:200 dilution, 715-545-150 Jackson ImmunoResearch Laboratories), Streptavidin (1:200 dilution, s11223, Invitrogen), and DAPI (1:5000 dilution, D9542, Sigma Aldrich) were diluted in the secondary antibody buffer (0.1% Triton X-100, 2% BSA) and incubated 2 hr at room temperature. The sections were then washed three times with 1×PBS for 10 min each and mounted using Vectashield Mounting media (Vector Laboratories).
Image analysis
Quantification of c-Fos was performed on slices from bregma +2 to +1.6 mm (4-6 sections per mouse, n=3 each group) for aIC and bregma +1.4 to +1.0 mm (4-6 sections per mouse, n=3 each group) for NAc. Mounted slides were examined under the bright-field microscope (DM5000B, Leica), and images were all taken at 10X magnification at one time to maintain identical lighting intensity and color balance. Averaged number of c-Fos positive neurons was determined using inverted color images in ImageJ software (National Institutes of Health).
All immunofluorescent-stained sections were imaged on a confocal microscope (LSM 700, Carl Zeiss). We collected 4 sections per mouse (n=3 mice each group), and images were all taken at 200X magnification. Image analysis was performed manually by identifying and counting D1R+, D2R+, CaMKII+ in the same area.
Electrophysiology
Mice were sacrificed by cervical dislocation and decapitated. The brain was immediately transferred to ice-cold sucrose cutting solution (189 mM sucrose, 10 mM D-glucose, 26 mM NaHCO3, 3 mM KCl, 10 mM MgSO4.7H2O, 1.25 mM NaH2PO4, and 0.1 mM CaCl2), which was bubbled continuously with 95% O2 and 5% CO2. The brain was sagittally dissected in a Petri dish containing an ice-cold cutting solution. The brain was glued onto a brain holder, which was placed in a buffer tray containing ice-cold cutting solution. Subsequently, 250-μm sagittal sections were obtained using a vibrating microtome (Leica, VT-1200). The sections (slices containing anterior commissure) were then transferred to a chamber on a nylon mesh containing external solution (in mM: 124 NaCl, 3 KCl, 1 MgSO4.7H2O, 1.25 NaH2PO4, 10 D-glucose, 24 NaHCO3, and 2 CaCl2, pH 7.4, osmolality 320-330 mOsm/kg H2O) bubbled with 95% O2 and 5% CO2 at 33°C. It was incubated for 1 hr. The slices could be maintained in a healthy state for up to 8 h and were transferred to the recording chamber as required.
The slices were continuously perfused with external solution (the same as above) at a rate of 1.0–2.0 ml/min. The recordings were made at room temperature. The neurons were visualized using infra-red differential interference contrast (IR-DIC) microscopy with a 40x water immersion objective and video imaging camera (BX51WI, Olympus). Electrophysiological recordings were obtained with a Multiclamp 700B amplifier, Digidata 1330A converter, and pClamp 10.3 software (Molecular Devices), sampled at 20 kHz, and filtered at 2 kHz.
Patch pipettes were pulled from borosilicate glass capillaries (OD 1.5 mm, I.D. 0.86 mm; Harvard Apparatus, Edenbridge, United Kingdom) on a horizontal puller (P-97, Sutter Instruments). The pipette tip resistance was 6–8 MΩ. The pipette offset potential was adjusted with the amplifier. The pipettes were filled with an internal solution containing the following components (in mM: 105 potassium gluconate, 20 KCl, 10 HEPES-Na+, and 0.1 EGTA, pH 7.25 adjusted with KOH, osmolality 280 mOsm/kg H2O). Recordings usually began >5 min after obtaining access to the cell. Only one neuron in each slice was exposed to a tested compound a single time, and the slice was replaced after one test on a single cell was performed. The recordings were analyzed with Clampfit 10.7 (Molecular Devices).
Statistical Analysis
Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, USA). Comparison between two groups was made using the unpaired Student's t-test. For multiple comparisons, data were analyzed using two-way ANOVA followed by the post hoc Bonferroni test. Detailed statistics for each experiment were shown in the figure legend. Data are presented as mean ± SEM. Differences with p < 0.05 were considered significant.