Buffered Saline (HBS) with 2 mM Pluronic-F127 (Invitrogen) for 15 minutes at 37uC/5% CO2. Cells were rinsed 2 times with HBS and left to rest at 37uC/5% CO2 for 30 minutes before use. During imaging, cells were perfused with HBS containing 2 mM tetrodotoxin (TTX, Tocris), 10 mM bicuculline (Tocris), 50 mM D- (2)-2amino-5-phosphono-valeric acid (D-APV, Tocris) and 20 mM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris) at a rate of 0.5 ml/minute. Images were captured every 3 seconds using a spinning disc confocal microscope (DSU; Olympus) equipped with a 406 oil Plan ApoN objective (1.4 NA), electron-multiplying charge-coupled device camera (Hamamatsu), and Slidebook software (Version 5; Olympus). After acquiring images for 1 min, 1 mM of capsaicin was focally applied by pressure ejection (Picospritzer II, General Valve Corporation) for 20 seconds. Depending on the experiment, capsaicin was applied between 1 and 4 additional times. At the end of each experiment, the cultures were depolarized by application of 56 mM KCl for 20 seconds. Regions (2.5 by 2.5 mm) were drawn along axons that responded to both capsaicin and KCl and the percent change in fluorescence intensity from baseline ([(F2F0)/F0]*100) was calculated within these regions using MetaMorph software. All regions were averaged by axon; all axons were averaged by animal and animal averages were compared by genotype and treatment. For analysis of somal responses, circular regions were drawn just inside the boundaries of the cell body and [(F2F0)/F0]*100 was calculated within these regions as described above. Immunoblotting For TRPV1, DRG. P21 WT and Type III Nrg1+/2 DRG cultures were grown in a 6-well plate, rinsed 2 times on ice with PBS, lysed into a modified RIPA buffer (95 mM NaCl, 25 mM Tris pH 7.4, 10 mM EDTA, 1 mM EGTA, 10 mM ammonium molybdate, 1 mM sodium orthovanadate, 1% SDS and protease inhibitors) and homogenized using a pipette tip. Lysate was left on ice for 10 minutes followed by centrifugation at 10,000 rpm for 10 minutes at 4uC. Lysate was mixed with Laemmli buffer, heated at 37uC, separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane (Whatman). The membrane was then blocked for 1 hour in 5% milk (Carnation)/Tris Buffered Saline (TBS) and antibodies directed against TRPV1 (rabbit, 1:1000, RA14113 Neuromics) and GAPDH (mouse, 1:5000, CB1001 Calbiochem) were applied in 5% milk/Tris Buffered Saline with 0.1% Tween-20 (TBS-T) overnight at 4uC. After three 10-minute rinses in TBS-T at room temperature, secondary antibodies conjugated to Alexa680 or Alexa800 (Molecular Probes) or IRDye-700 or 800 (Rockland) were applied at a concentration of 1:5000 diluted in 5% milk/TBS-T for 1 hour at room temperature. Membranes were scanned with an Odyssey Infrared Imaging System (Li-Cor Biosciences) and band intensities were analyzed using Odyssey Image Analysis software. The intensity of the TRPV1 band was normalized to that of GAPDH to control for variable protein loading. TRPV1-GAPDH ratios were compared by genotype. For pAKT and pERK, DRG. P21 WT and Type III Nrg1+/2 cultures grown in a 12-well culture plate for 36–48 hours were serum starved for 4 hours before being treated with sErbB4-ECD (300 ng/ml for 15 minutes) or NGF (100 ng/ml for 5 minutes) at 37uC, rinsed 2 times on ice with TBS and homogenized with a pipette tip in lysis buffer (1% Ipegal, 30 mM HEPES, 100 mM NaCl, 25 mM NaF, 10 mM EDTA, 4 mM EGTA, 15 mM sodium pyrophosphate, 10% glycerol, protease and phosphatase inhibitors, 1 mM PMSF and 5 mM sodium orthovanadate). The lysate was left on ice for 10 minutes followed by centrifugation at 10,000 rpm for 10 minutes at 4uC. Lysate was mixed with Laemmli buffer, heated Type III Nrg1, TRPV1 and Pain Sensation to 90uC for 10 minutes, separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was then blocked for 1 hour in 5% milk/TBS and antibodies directed against pAKT (Ser473, rabbit, 1:1000, 9271 Cell Signaling Technology), pERK (Thr202/Tyr204, mouse, 9106 Cell Signaling Technology) and GAPDH were applied in 5% milk/TBS-T overnight for 2 nights at 4uC. Secondary antibodies conjugated to Alexa680 or IRDye-700 were applied at a concentration of 1:5000 in 5% milk/ TBS-T for 1 hour at room temperature and bands were detected by scanning with the Odyssey Infrared Imaging System. Subsequently, membranes were re-blocked in 5% milk/TBS-T and incubated in antibodies directed against AKT (rabbit, 1:1000, 9272 Cell Signaling Technology) and ERK (rabbit, 1:1000, 9102 Cell Signaling Technology) diluted in 5% milk/TBS-T overnight at 4uC followed by detection with secondary antibodies conjugated to Alexa800 or IRDye-800 for one hour at room temperature. Membranes were rescanned using the Odyssey system and band intensities were analyzed using Odyssey Image Analysis software. Ratios of pAKT-AKT and pERK-ERK were determined for each sample and compared by treatment and/or genotype. For TRPV1, TrkA and Ret, Hindpaw. P21 WT and Type III Nrg1+/2 hindpaw galabrous skin samples were lysed into a modified RIPA buffer (95 mM NaCl, 25 mM Tris pH 7.4, 10 mM EDTA, 1 mM EGTA, 10 mM ammonium molybdate, 1 mM sodium orthovanadate, 1% SDS and protease inhibitors) and homogenized using a dounce homogenizer. Lysate was left on ice for 10 minutes followed by centrifugation at 10,000 rpm for 10 minutes at 4uC. Lysate was mixed with Laemmli buffer, heated at 95uC, separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose membrane (Whatman). The membrane was then blocked for 1 hour in 5% milk (Carnation)/Tris Buffered Saline (TBS) and antibodies directed against TRPV1 (rabbit, 1:500, RA14113 Neuromics) and GAPDH (mouse, 1:5000, CB1001 Calbiochem) or TrkA (rabbit, 1:500, gift of L.F. Reichardt), Ret (goat, 1:500, GT15002) and GAPDH (mouse, 1:5000, CB1001 Calbiochem) were applied in 5% milk/Tris Buffered Saline with 0.1% Tween-20 (TBS-T) overnight at 4uC. After three 10-minute rinses in TBS-T at room temperature, secondary antibodies conjugated to Alexa680 or Alexa800 (Molecular Probes) or IRDye-700 or 800 (Rockland) were applied at a concentration of 1:5000 diluted in 5% milk/TBS-T for 1 hour at room temperature. Membranes were scanned with an Odyssey Infrared Imaging System (Li-Cor Biosciences) and band intensities were analyzed using Odyssey Image Analysis software. The intensity of the TRPV1, TrkA and Ret bands were normalized to that of GAPDH to control for variable protein loading. TRPV1, TrkA or Ret-GAPDH ratios were compared by genotype. RT-PCR Analysis of Type III Nrg1 Splice Variants Relative levels of Type III Nrg1 transcripts containing the TMc exon versus those containing the b3 exon were measured by semi- quantitative RT-PCR. Total RNA from thoracic, lumbar and sacral level DRG from P21 male mice was purified using Trizol and 2 mg was converted to cDNA using Superscript III (Invitrogen). 0.5 mg of each cDNA was amplified using either a CRD 59 and TMc 39 primer pair, a CRD 59 and b3 39 primer pair, or all three primers together (primer concentrations in each instance were 200 ng). The resulting products were resolved on an agarose gel and relative ethidium fluorescence was quantified. The predicted product sizes for the CRD – TMc product was 359 bp (assuming the b1 splice form was amplified) and 309 bp for the CRD-b3 Primer sequences used were: CRD59CAGGAACT- PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25108