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Impaired Nociception and Pain Sensation in Mice Lacking the Capsaicin Receptor
M. J. Caterina, A. Leffler, A. B. Malmberg, W. J. Martin, J. Trafton, K. R. Petersen-Zeitz, M. Koltzenburg, A. I. Basbaum, and D. Julius

Supplementary Material

References and Notes
26. Generation of mice lacking VR1. A 129/SVJ mouse genomic DNA clone (~100 kb) containing part of the VR1 gene was isolated from a bacterial artificial chromosome library by PCR screening with mouse-specific VR1 primers (5�-CAAAGACCCAGAGACGG-3� and 5�-CTGGCCCTTGTAGTAGC-3�) (Genome Systems). A 10-kb Hind III subfragment was used to construct the pBluescript-based (Stratagene) targeting vector shown in Fig. 1. JM1 embryonic stem (ES) cells [M. Qiu et al., Genes Dev. 9, 2523 (1995)] were electroporated with linearized targeting vector DNA and placed under ganclyclovir/geneticin (Life Technologies) double selection. Southern blotting with 3� and 5� probes (Fig. 1) revealed that approximately 1/30 surviving ES clones had undergone homologous recombination at the VR1 locus. C57/Bl6 mouse blastocysts were injected with one of these clones and implanted into pseudopregnant Black/Swiss female mice. Male offspring with chimeric coats were mated with C57/Bl6 females. Resulting VR1 heterozygous offspring were intercrossed to generate the VR1+/+ and VR1-/- mice used in many experiments. For other experiments, VR1+/- males were backcrossed at least four generations to C57Bl/6 females to place the VR1 deficiency on a more nearly isogenetic background. No significant differences were observed between backcrossed and non-backcrossed animals in several assays (hot plate, tail immersion, patch clamp, skin-nerve preparation) carried out under identical conditions. Northern blot analysis was performed as described [M. J. Caterina et al., Nature389, 816 (1997)] using a rat VR1 (nucleotides 94 to 1101) cDNA probe.
28. Immunohistochemistry and statistical analyses. Immunohistochemistry was performed as described [A. B. Malmberg, C. Chen, S. Tonegawa, A. I. Basbaum, Science278, 279 (1997); M. Tominaga et al., Neuron21, 1 (1998); M. J. Caterina, T. A. Rosen, M. Tominaga, A. J. Brake, D. Julius, Nature398, 436 (1999)]. Substance P was detected using rabbit anti-substance P (1:20,000, Peninsula). All other antisera were used at the previously indicated titers.
Throughout the manuscript, unless otherwise indicated, experiments were carried out blind as to the genotype of the animals and all values are given as mean and SEM or median and interquartile range of the 25th to 75th percentile. Statistical analysis consisted of two-sided unpaired T-test, Mann-Whitney U-test or Chi-squared test, as appropriate.
30. Calcium imaging of cultured dorsal root ganglion neurons. For calcium imaging studies, dorsal root ganglia were dissected from adult mice and cultured as previously described [M. J. Caterina, T. A. Rosen, M. Tominaga, A. J. Brake, D. Julius, Nature398, 436 (1999)], except that the culture medium was supplemented with 10% horse serum (Gibco) instead of fetal calf serum and the collagenase used was Type IV (Sigma) [C. L. Stucky and G. R. Lewin, J. Neurosci.19, 6497 (1999)]. Cells were plated on polyornithine/laminin-coated cover slips, loaded with Fura 2-acetomethoxy ester (Molecular Probes) and imaged for cytoplasmic free-calcium, as described [M. J. Caterina et al., Nature389, 816 (1997)]. For IB4 staining, cells were preincubated with fluoresceinated IB4 (16 μg/ml, 20 min, Sigma). In contrast to other analyses, calcium imaging experiments were performed in an unblinded manner.
31. Whole-cell voltage-clamp analysis of cultured dorsal root ganglion neurons. For patch clamp studies, adult mice were killed via CO2 asphyxiation. Dorsal root ganglia from all spinal levels were collected in Dulbecco's modified eagle medium (D-MEM, Gibco) and treated with collagenase (80 min, 0.28 U/ml in D-MEM, Biochrom, Germany) and trypsin (10 min, 25,000 U/ml in PBS without Ca2+ and Mg2+, Sigma). Following trituration and centrifugation, cells were resuspended in Ham's F12 medium (Gibco) containing L-glutamine (3.0 mM), penicillin (100 UI/ml), streptomycin (100 μg/ml), glucose (40 mM), horse serum (10%, Life Technologies) and nerve growth factor (50 ng/mL, Almone labs, Israel), plated on poly-L-lysine (Sigma) coated glass cover slips, cultured at 37°C/5% CO2 and used within 48 hours. In some experiments cells were vitally stained with IB4-FITC (10 μg/ml). For patch clamp recording, cover slips were continously superfused with standard extracellular solution (ECS) containing (in mM) NaCl 145, KCl 5, CaCl2 2, MgCl2 1, Glucose 10, HEPES 10 (adjusted to pH 7.3). HEPES was replaced with 10 mM MES in pH 5 solution. Small- to medium-sized neurons were recorded in the whole-cell voltage-clamp configuration with fire-polished borosilicate electrodes with 3 to 4 MΟ resistance (pipette solution in mM: KCl 140, CaCl2 1, MgCl2 2, EGTA 11, HEPES 10, Mg2ATP 2, pH adjusted to 7.4) using an Axopatch 200B amplifier and pCLAMP6 software on a PC. Data were filtered at 5 kHz and sampled at 10 kHz. There was no significant difference (P always > 0.2 t-test) between the proportion of IB4 positive and negative cells, cell diameter (mean � SD, capsaicin: 22.5 � 3.3 μm VR1+/+, 23.7. � 3.2 μm VR1-/-; protons: 22.8 � 3.2 μm VR1+/+, 23.4 � 3.3 μm VR1-/-; heat: 22.7 � 3.4 μm VR1+/+, 23.6 � 2.7 μm VR1-/-) or capacitance (capsaicin: 23.5 � 7.0 pF VR1+/+, 22.7. � 8.0 pF VR1-/-; protons: 23.4 � 7.2 pF VR1+/+, 21.0 � 6.0 pF VR1-/-; heat: 22.5 � 6.3 pF VR1+/+, 20.9 � 6.1 pF VR1-/-) between the different experimental groups. Capacitance transient and series resistance were compensated before each recording. Holding potential was -80 mV. One cell per cover slip was analyzed, but only if initial seal resistance was > 2 GΟ, leakage current at -80 mV was < 1 nA throughout the recording and resting membrane potential was < -40 mV. Capsaicin and resiniferatoxin (stored as stock solutions in ethanol) were diluted to 1 μM and 300 nM with ECS. Drugs and heat stimuli were delivered using a multichannel, gravity-driven system incorporating rapid-feedback temperature control. In this system, a platinum-covered glass capillary, positioned < 100 μM from the cell under study, is used as a common outlet [I. Dittert et al., J. Neurosci. Meth.82, 195 (1998)]. ECS was heated from 28°C to 60°C within 11 s. Capsaicin, RTX and acidic solution (pH 5) were all applied for 10 s (time for solution change ~100 ms). ECS was applied to the cell before and after drug- and heat-application. At the end of the recordings cells were depolarized to +50 mV (in 5 mV increments of 50 ms duration) to study voltage-gated channels. A total of 7 wild-type and 6 VR1-null mice were used for the patch clamp studies.
32. Skin nerve preparation and single unit recordings. Single cutaneous primary afferent neurons were recorded as described [M. Koltzenburg, C. L. Stucky, G. R. Lewin, J. Neurophysiol.78, 1841 (1997); M. Kress, M. Koltzenburg, P. W. Reeh, H. O. Handwerker, J. Neurophysiol. 68, 581 (1992)]. Adult mice were killed, the hairy skin of the hindlimb removed together with the saphenous nerve, placed corium-side up in an organ bath and superfused with oxygen-saturated, synthetic interstitial fluid (SIF) containing (in mM) 123 NaCl, 3.5 KCl, 0.7 MgSO4, 1.7 NaH2PO4, 2.0 CaCl2, 9.5 sodium gluconate, 5.5 glucose, 7.5 sucrose and 10 HEPES (pH 7.45 � 0.05, temperature 32 � 0.5°C). The nerve was desheathed and single afferent fibers recorded extracellularly using gold wire electrodes. Action potentials were acquired using a low-noise differential amplifier, stored on a PC and analyzed with custom software. Units were initially identified by probing with a blunt glass rod, which activates more than 90% of unmyelinated nociceptive units in this preparation [M. Koltzenburg, C. L. Stucky, G. R. Lewin, J. Neurophysiol.78, 1841 (1997)]. The conduction velocity of each unit was determined by electrically stimulating the receptive field with supramaximal square wave pulses (1.0 ms) using a Teflon-coated steel electrode. Only nociceptors were studied in detail. Units conducting 1.2 to 10 m/s were considered to be thin myelinated (Aδ) fibers and those conducting less than 1.2 m/s as unmyelinated. Then each unit was studied by a standard protocol consisting of a series of mechanical stimuli, followed in that order by cold, heat, pH and capsaicin stimuli. Mechanical sensitivity was determined with calibrated von Frey filaments (tip diameter 0.8 mm, range of force 0.3 to 362 mN) and sustained force stimuli (200 ms rise time, 10 s duration of force plateau, ascending stimuli of 5, 10, 20, 40, 100 and 150 mN separated by an interstimulus interval of 60 s) applied by a computer-driven, feedback-controlled stimulator. The receptive field was then isolated with a small, self-sealing metal ring (5 mm diameter). After removal of solution from the ring, we injected a 10 ml bolus of ice-cold SIF to produce a temperature nadir of 4° to 6°C, measured by a thermocouple on the epidermal side of the receptive field. After passive return to baseline temperature, a heat stimulus (linear ramp from 32° to 47°C in 15 s) was delivered to the epidermal side of the preparation through the translucent bottom of the organ bath with a feedback-controlled halogen bulb. Heat threshold was defined as the temperature that evoked an instantaneous frequency >1 impulse/s [M. Koltzenburg, C. L. Stucky, G. R. Lewin, J. Neurophysiol. 78, 1841 (1997)]. Following thermal testing, the receptive field was exposed for 1 min to SIF at pH 5 (with HEPES replaced by MES), and subsequently with 1 μM capsaicin. Given the low prevalence of heat-sensitive Ad nociceptors in our experiments, we applied an additional, more intense heat stimulus (peak 57°C in 25 s) to 20 thin myelinated units. No units classified as unresponsive at 47°C could be activated by this more intense stimulus.
34. Behavioral assays of nociception. Animals were acclimated for 30 min in individual plexiglass chambers prior to all behavioral experiments. Rotarod, von Frey hair, hot plate, tail pressure, formalin injection, and radiant heating assays were performed as previously described [A. B. Malmberg, C. Chen, S. Tonegawa, A. I. Basbaum, Science278, 279 (1997); Y. Q. Cao et al., Nature392, 390 (1998)]. For vanilloid sensitivity studies, capsaicin (1 to 5 μg in 10 μl saline/10% ethanol/0.5% Tween 80) or resiniferatoxin (5 μl of a 10 μM solution in saline/1% ethanol) was injected intraplantar using a 30G needle, after which the animals were placed on a 30°C metal surface and paw licking and flinching behavior quantified for 10 minutes. Paw diameter was measured with spring-loaded calipers (Mitutoyo). For the tail immersion assay, the animal was wrapped in a towel and gently restrained in the investigator's hand. The distal one third of the tail was then immersed into a preheated water bath and the latency to tail flick recorded.
41. Recording from spinal cord wide dynamic range neurons. Mice (25 to 35 g) were anesthetized with 1.5 mg/kg (i.p.) of 10% urethane and administered dexamethasone (0.2 mg, s.c.) and atropine (0.3 mg, s.c.). Heart rate and core temperature were monitored and maintained within normal range. A laminectomy was performed, the dura retracted and the spinal cord covered with warm saline. Mice breathed spontaneously throughout the experiment. Fine tip (<1.0 μm) tungsten microelectrodes (4 to 5 MΟ at 1 kHz FHC, Brunswick, ME) were used to record extracellular potentials. Unit activity was acquired, digitized and discriminated by computer using Experimenter's Workbench (Datawave Technologies, Thornton, CO). One second bins were used to generate peristimulus time histograms for subsequent data analyses. Neurons were identified by continuously brushing the ipsilateral dorsal hindpaw and characterized as WDR if they responded to brush, pressure, and noxious pinch. These neurons were located at a mean depth of 491 � 13 μm from the surface of spinal cord. Thermal stimuli were delivered with a 3 X 3 mm copper probe heated by a 9W Peltier device. The probe was positioned firmly onto the center of the receptive fields and maintained at 35.5°C between periods of stimulation. Mustard oil (3-isothiocynato-prop-1-ene; Sigma), diluted to 10% with mineral oil, was painted onto the skin around the probe. Recordings were done on 5 mice per genotype.
42. Spinal cord Fos immunoreactivity in heat-treated mice. One hindpaw of male mice, anesthetized with urethane (1.5 mg/kg i.p.), was immersed in a 50°C water bath 10 times, once per minute for 3 s each time. After 90 min, the mice were perfused transcardially with 4% paraformaldehyde. Frozen spinal cord sections (40 μm) were prepared from lumbar level L4/L5 and immunostained for Fos as described [W. J. Martin, C. M. Loo, A. I. Basbaum, Pain82, 199 (1999)] and immunoreactive nuclei counted.
48. Hypersensitivity produced by complete Freund's adjuvant and nerve injury. Complete Freund's Adjuvant (5 μg in 10 ml of a 1:1 emulsion of saline and mineral oil) was injected i.pl. into one hindpaw and mice assayed the following day as described [Y. Q. Cao et al., Nature392, 390 (1998)]. Nerve injury was performed by ligating approximately one-third of the sciatic nerve as described [A. B. Malmberg, C. Chen, S. Tonegawa, A. I. Basbaum, Science278, 279 (1997)].