Animal care and treatment were in accordance with the guidelines of the International Association for the Study of Pain . Adult C57Bl/6, TRPV1-/- and TRPA1-/- as well as TRPV1-/-TRPA1-/- double knockout mice were used. Breeding pairs of TRPV1 and TRPA1 knockout mice were obtained from Dr. John Davis  and Dr. David Corey  and backcrossed to C57BL/6. Double knockout animals were generated in our animal facility by cross-mating knockouts of both strains. All animals were genotyped by the previously reported primers.
Animals were killed in pure CO2 atmosphere. Dorsal root ganglia of all lumbar and the first two thoracic segments of the spinal column were excised and transferred in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, USA) solution containing 50 μg/ml gentamicin (Sigma). The ganglia were treated with 1 mg/ml collagenase and 0.1 mg/ml protease for 30 minutes (both from Sigma) in TNB 100 solution supplemented by TNB 100 lipid-protein-complex, 100 μg/ml streptomycin and penicillin (all from Biochrom, Berlin, Germany) and 200 μg/ml glutamine (Invitrogen, Carlsbad, USA) for 20 minutes at 37°C. After three wash steps, the suspension was triturated in TNB solution using a fire-polished silicone-coated Pasteur pipette. The cells were plated on poly-D-lysine-coated (Sigma) cover slips and cultured in TNB medium overnight at 37°C in a humidified 5% CO2 atmosphere.
Patch Clamp Recordings
HEK293t cells were transfected with plasmids of hTRPV1 (John Davis) or mTRPA1 (Ardem Patapoutian) along with GFP as reporter plasmid using Polyfect (Qiagen, Hilden, Germany) according to the manufacturer's protocol. After incubation for one day, the cells were replated in 35 mm culture dishes and used for experiments within 1–2 days. GFP-expressing cells were identified by fluorescence.
Recording electrodes were pulled from borosilicate glass tubes (GB150T-8P, Science Products, Hofheim, Germany) to give a resistance of 3.5 – 5.0 MΩ (P97, Sutter, Novato, CA). The external solution contained (in mM) 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, 4 glucose, adjusted to pH 7.4. The internal solution contained (in mM) 140 KCl, 1.6 MgCl2, 2 EGTA and 10 HEPES and was adjusted to pH 7.4. Recordings were performed at room temperature and cells were held at -60 mV. Membrane currents were acquired with an Axopatch 200B amplifier and pClamp 10 software (Molecular Devices, Sunnyvale, CA). Solutions were applied with a RSC-100 gravity-driven solution changer (Bio-Logic, Claix, France). All cells were first exposed to morphine, then to capsaicin or mustard oil. TRPA1 was sensitized by PLC activator m-3M3FBS (1 μM, Sigma).
Cells were stained by 5 μM fura-2 AM and 0.02% pluronic F-127 (both from Invitrogen) dissolved in the TNB medium for about 30 min in the incubator, followed by a short wash-out period to allow fura-2 AM ester hydrolysis. The cover slips were placed in a custom-made chamber and mounted on a Zeiss Axiovert inverse microscope with a 40× NeoFluar objective. Cells were continuously superfused throughout the experiment with extracellular fluid (in mM: NaCl 145, KCl 5, CaCl2 1.25, MgCl2 1, Glucose 10, Hepes 10) at a flow rate of approximately 0.3 ml/min at room temperature. Cells were illuminated with a 75W xenon arc lamp and a monochromator alternating between 340 and 380 nm of wavelength (Photon Technology International, New Jersey, USA). Images were acquired at 1 Hz with 200 μs exposure time using a CCD camera, controlled by Image Master software (PTI, Birmingham, USA). Fluorescence ratios were computed for regions of interest adapted to the neurons. All experimental protocols were pre-programmed using a custom-made software that controls the valves of a 7-channel gravity-driven common-outlet superfusion system .
C57/Bl6 mice of either sex with an average weight of 20 g (range 12–27 g) were used. The skin of both hind paws distal to the knee was subcutaneously excised. The skin flaps with an average weight of 105 mg (range 57–135 mg, n = 36) were fixed to acrylic rods by surgical threads with the corium exposed. During this procedure the skin flaps were constantly immersed in synthetic interstitial fluid (SIF, in mM: NaCl 107.8, KCl 3.5, NaHCO3 26.2, NaH2PO4 1.7, Na-gluconate 9.6, sucrose 7.6, glucose 5.6, CaCl2 1.5 and MgSO4 0.7  equilibrated with carbogen (pH 7.4). The fixed skin flaps were placed in test tubes and mounted in a shaking bath of 32°C. After washing for 30 minutes, each experiment was composed of four consecutive 5 min incubation periods in test tubes filled with 0.8 ml SIF. In the first two periods basal CGRP release was determined, during the third period the preparation was chemically or thermally stimulated, the fourth period was for the post-stimulation control. One separate protocol provided two stimulations in incubation periods two and four. The contralateral hind paw skin of each animal was used as a matched-pairs control without conditioning chemicals.
Enzyme-immuno assay for CGRP
Immediately after the incubation, 100 μl of the solution were processed using a commercial CGRP enzyme immune assay kit (SPIbio, France). The details of this method were described previously . The antibodies used are directed against human α/β-CGRP but are 100% cross-reactive against mouse and rat CGRP. The detection level is about 2 pg/ml. Samples of SIF and all stimulation solutions measured on the same enzyme immune assay 96-well plate served as controls.
The calcium influx elicited by a stimulus was quantified by evaluating the area under the curve (AUC) of the application period. Absolute increases in calcium concentrations were calculated based on Rmin and Rmax . Compared to the foregoing reference period a mean increase of the intracellular calcium concentration of at least 15 nM throughout the application period was considered as activation. At the end of all protocols a 10 s stimulus of KCl 60 mM was applied as control and normalization reference. Cell diameter was calculated as the mean of two orthogonal axes. Repeated measurements and independent groups in calcium imaging experiments were compared by ANOVA and HSD post-hoc test. CGRP release experiments were performed using both hindpaw skin flaps of the same animal and compared using the Wilcoxon matched pairs test. Statistical analysis was performed using Statistica 7 (Tulsa, OK, USA), sigmoidal dose-response curves were fitted by Origin 7.5 (Northhampton, MA, USA). Data are presented as mean ± SEM; p < 0.05 was considered significant.