Supplemental information

2. 2. Intra-dorsal striatum injection

2.2.1. Surgery for microinjection

Prior to surgery, mice were anesthetized with pentobarbital 50 mg/kg, i.p. The mice were placed in a stereotaxic apparatus, and a guide cannula (9 mm, 24 gauge) was implanted laterally into the DSt after a hole was drilled through the skull. Each cannula was cemented in place by affixing dental acrylic to stainless steel screws that had been secured in the skull. The ventral end of the guide cannula was placed relative to the bregma according to the atlas of Paxions and Franklin (2003). The coordinates were as follows: + 0.86 mm A/P, + 1.5 mm M/L, and -2.7 mm D/V. In order to prevent occlusion, an obstructer (9 mm, 26 gauge) was inserted into each cannula guide following surgery. Both the guide cannula and obstructer were trimmed.

2.2.2. Microinjection

The obstructer was removed from the guide cannula and 26 gauge stainless steel microinjector-linked PE tubing was inserted. This microinjector was cut to a length that extended 1 mm below the ventral end of the guide cannula and into the DSt. Infusions were performed over a 60 second time period to introduce a total volume of 1 μl per site. Following the microinjection, the injector was left in place for 60 seconds, in order to allow the administered solution to diffuse away from the tip of the cannula. The obstructor was then replaced into the guide cannula.

2.3. Quantitative Receptor Autoradiography

2.3.1. Tissue preparation

After the CPP test, mice were sacrificed by decapitation. Mouse brains were carefully removed, frozen in dry ice for 5 minutes, and then stored at -80°C. For autoradiographic studies, mouse brains were cut into 20-µm coronal sections at -19°C using a cryostat (Leica, Wetzlar, Germany).

2.3.2. TRPV1 receptor binding

Brain sections were incubated in assay buffer consisting of 10 mM HEPES, 5 mM KCl, 5.8 mM NaCl, 0.75 mM MgCl2, 320 mM sucrose, and 1 mg/ml bovine serum albumin (BSA, pH 7.4) containing 1 nM [3H] Resiniferatoxin (38.31Ci/mmol, Perkin-Elmer ) for 60 min at room temperature. Nonspecific binding was controlled by adding 1 μM unlabelled Resiniferatoxin to a parallel series of sections. Following incubation, sections were washed in 20 mM Tris-HCl (+ 0.1 % BSA) for 4 x 10 min at 4°C and dipped into distilled water at 4°C to remove buffer salts. The sections were allowed to air dry and were exposed to Kodak BioMax MR Film (Eastman Kodak Co., Rochester, NY, USA) along with [3H] autoradiographic microscalesTM (Amersham Bio-Science, NJ, USA) at 4°C for 6 weeks.

2.3.3. µ-Opioid receptor binding

Brain sections were pre-incubated in 50 mM Tris-HCl, pH 7.4, plus 0.9% NaCl for 30 min to remove endogenous opioids. Binding was carried out in 50 mM Tris-HCl, pH 7.4 containing 4 nM [3H]DAMGO (56.8 Ci/mmol, Perkin-Elmer) at room temperature for 60 min. Slides were washed three times with ice-cold Tris-HCl buffer (5 min), rapidly dried with cool air, and exposed to Kodak BioMax MR film (Eastman Kodak) along with [3H]autoradiographic microscalesTM (Amersham Bio-Science) at 4°C for 3 weeks.

Autoradiography films were developed at room temperature for 1 minute and fixed for 2 minutes. The films were quantified using Molecular Dynamic Image Quant software version 3.3. Specific regions of interest were defined using the mouse brain atlas (Paxinos and Franklin, 2004). Standard curves constructed using [3H]MicroscalesTM were used to convert the optical density of each region into fmol/mg wet brain tissue. Mean densities were determined from at least two sections per region per mouse by blind observer.

2.4. Real-time RT- PCR

2.4.1. Tissue preparation

After the behavioral test, mice were sacrificed by decapitation. Mouse brains were quickly removed and frozen in dry ice. A coronal section (Bregma +1.7 to +0.74 mm) was cut according to the mouse brain atlas (Paxinos et al, 2004). The DSt (dorsal striatum) regions were punched bilaterally using a Harris Uni-Core 1.20 (Ted Pella, Inc., Redding, CA, USA). These brain tissues were used for real-time RT-PCR and Western blot assays.

2.4.2. Real-time RT-PCR

Total RNA was isolated from brain tissues using the RNeasy microkit (Qiagen, Valencia, CA, USA) and quantified spectrophotometrically. Total RNA (0.5 µg) was reverse transcribed using the SyperScriptTM III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using oligo dT. Equal amounts of cDNA were subjected to PCR with TRPV1 primers (forward: 5’-AGCCATGCTCAATCTGCAC-3’ and reverse: 5’-TGCTGTCTGGCCCTTGTAG-3’) and using the SensiMixPlus SYBR Kit (Quantace, Alexandria, NSW, Australia) in a Rotor-Gene 6000 real-time amplification system (Corbett Research, Mortlake, NSW, Australia). Reactions (total volume, 20 µl) were incubated at 95°C for 10 min, followed by 45 cycles of PCR amplification with denaturation (95°C for 10 s), primer annealing (58°C for 15 s), and extension (72°C for 20 s). Non-template controls were included to ensure specificity. The amount of TRPV1 mRNA was normalized to β-actin mRNA (forward: 5’-AGAGGGAAATCGTGCGTGAC-3’ and reverse: 5’-CAATAGTGATGACCTGGCCT-3’). The comparative critical threshold, ∆∆Ct, was calculated using Corbett Research Software version 1.7.75. The relative TRPV1 mRNA expression of each sample was a mean of triplicate measurements.

2.5. Western blot

Tissues were homogenized in lysis buffer containing inhibitor cocktail (Sigma). Homogenates were centrifuged for 20 min at 15,000 rpm at 4°C. The supernatant was recovered and protein concentrations were determined using a Bio-Rad protein assay. Samples (50 µg) resuspended in 4X Laemmli’s sample buffer (Sigma) were separated by 6% SDS-PAGE and transferred to polyvinylidene fluoride membranes. Nonspecific binding sites were blocked by incubation overnight with 5% BSA in Tris-buffered saline (TBS). Membranes were incubated with TRPV1 antibody (1:1000, Abcam), anti-adenyl cyclase 1 antibody (1:1000, Littleton), anti-phospho-p38 MAPK (Thr180/Tyr182, 1:1000, Cell Signaling), rabbit anti-p38 MAPK antibody (Thr180/Tyr182, 1:1000, Cell Singnaling), anti-phospho-NF-κB p65 antibody (1:1000, Cell Signaling) overnight or with β-actin antibody (1:10,000, Sigma) for 15 minutes at 4°C. The PVDF membranes were incubated with horseradish peroxidase-coupled goat anti-rabbit antibody (1: 2500; Cell Signaling) or peroxidase-coupled goat anti-mouse antibody (1: 3000; Promega) for 2 h at room temperature. Antibody binding sites were visualized on Kodak BioMax MR Film (Eastman Kodak Co., Rochester, NY, USA) using a Western Lightning Detection Kit (Perkin Elmer Life Science). Specific bands were quantified by densitometric analysis using Image Gauge software version 4.0. Data were calculated as relative ratio to the control.

2.6. Immunohistochemistry

2.6.1. Tissue preparation

After CPP, mice were cardially perfused with a perfusion solution containing 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 4 h then put in 30% sucrose for 48 h at 4°C. The brains were frozen and sectioned coronally at 40 μm using a cryostat (Leica, Wetzlar, Germany).

2.6.2. Immunohistochemistry

Brain sections of the drug-treated and control groups were selected from the same bregma levels and processed in parallel. Sections were incubated in 0.3% H2O2 in TBS for 15 min, then in a blocking solution containing 10% goat or horse serum and 1% BSA in TBS for 1 h at room temperature, then incubated with the primary antibody for 12-24 h at 4°C. Primary antibodies were anti-p-p38 MAPK (Thr180/Tyr182) rabbit monoclonal antibody (1:50; Cell Signaling), rabbit anti-adenylate cyclase 1 polyclonal antibody (1:200; Novus Biologicals), goat anti-adenylate cyclase 8 polyclonal antibody (1:50, Santa Cruz Biotechnology), or rabbit anti-pNF-κB/p65 (S536) polyclonal antibody (1:100, Abcam). For immunoperoxidase staining, biotinylated goat anti-rabbit, goat anti-guinea pig, or horse anti-goat IgG antibody (7 μg/ml; Vector Laboratories) was applied for 60 min at room temperature, followed by incubation with an avidin-biotin horseradish peroxidase complex (Vector Laboratories) for 60 min at room temperature. The peroxidase label was detected by incubating in 3,3’-4,4’-diaminobenzidine (Sigma). Images were taken using a DP digital microscope camera (Olympus Optical Co., Ltd., Tokyo, Japan) connected to a microscope (BX 51, Olympus, Tokyo, Japan).

Fig. S1

Fig. S1. The signaling cascade that TRPV1 modulates behavioral effects of morphine. Morphine binds to opioid receptors to activate G protein. G protein in turn activates cascades including AC1, MAPK, and NF-κB to synthesize new molecules related to addictive behavior. TRPV1, one of these new molecules, is activated by morphine through the PKA or PKC pathways, allowing Ca2+ influx into the cell to facilitate the phosphorylation of MAPK and NF-κB, which accelerates addictive behavioral response. G, G protein; TRPV1, transient receptor potential vanilloid type 1; AC1, adenylate cyclase 1; p38 MAPK, p38 mitogen-activated protein kinase; NF-κB, nuclear-kappa B.