Cell culture preparation of TG neurons
Primary cultures of TG ganglion sensory neurons were obtained from C57-Black/6Jico mice or Wistar rats (P10–14; an age reported to show a quasi-adult phenotype for P2X receptors ). Animals were anesthetized by diethyl ether and decapitated (in accordance with the Italian Animal Welfare Act and approved by the Local Authority Veterinary Service). TG were rapidly excised and enzymatically dissociated in F12 medium (Invitrogen Corp, S.Giuliano Milanese, Italy) containing 0.25 mg/ml trypsin, 1 mg/ml collagenase and 0.2 mg/ml DNAse (Sigma) at 37°C. Cells were plated on poly-L-lysine-coated petri dishes in F12 medium with 10% fetal calf serum. For molecular biology or Ca2+ imaging experiments, 2 × 106 cells were plated. For patch clamp experiments, mouse and rat cells were diluted twice. Three week-old TG cultures, that lacked neurons (i.e., negative for the neuron specific marker β-tubulinIII), served as negative control for molecular biology experiments.
Real time RT-PCR
For PCR experiments, total RNA was extracted from TG ganglia or from culture using Trizol reagent (Invitrogen). After DNAse treatment (Ambion, Austin, TX, USA), cDNA synthesis and amplification were obtained using SuperScript III Two-step qRT-PCR kit (Invitrogen). Thirty ng cDNA were amplified with specific oligonucleotides and fluorogenic probes (TaqMan gene expression assays, Applied Biosystems, Applera, Norwalk, CT, USA) in ABI PRISM 7000 Sequence Detection System (Applied Biosystems) in the presence of ROX (Invitrogen) as internal reference dye. mRNA samples from fresh ganglia or cultures at different times were calibrated to obtain similar amplification of the GAPDH housekeeping mRNA. In preliminary experiments, analogous amplification of the samples was obtained also with 18S RNA probes. Nevertheless, to normalize the real time PCR results only with respect to neuronal mRNA, amplification of the neuronal specific β-tubulinIII housekeeping gene was chosen.
Specific TaqMan assays for mouse and rat target mRNA encoding P2X3, P2X2 and TRPV1, neuronal specific β-tubulinIII, GAPDH mRNA and 18S RNA (respective Applied Biosystems catalogue numbers: Mm00523699_m1, Mm00462952_m1, Mm01246282_m1, Rn01460299_m1, Mm00727586_s1, 4352339E FG, 4308329; www.appliedbiosystems.com) were chosen. All assays were validated for linearity of amplification efficiency and quantitative standard curves were obtained using serial dilutions of ganglia rat or mouse TG cDNA. To ensure absence of amplification artifacts, end point PCR products were initially assessed on ethidium bromide-stained agarose gels that gave a single band of the expected size for each assay. Negative controls containing no template cDNA were run in each condition and gave no results. The reactions were quantified when the PCR product of interest was first detected (cycle threshold). Calculations for relative mRNA transcript levels were performed using the comparative CT method (ΔΔCT) between cycle thresholds of different reactions . In particular, the parameter CT (threshold cycle) is defined as the cycle number at which the fluorescence emission exceeds the fixed threshold. The calculation is based on the difference (ΔCT) between the CT values of the target receptor and the neuron-specific housekeeping gene (β-tubulinIII) at each time-point in culture, and then normalized with respect to the ΔCT value of the ganglion.
Rat or mouse TG ganglia or cultures were homogenized in ice-cold lysis buffer containing 10 mM TrisHCl (pH 7.5), 150 mM NaCl, 20 mM EDTA, 1% Triton X-100, 8 M urea and protease inhibitors (Roche, Basel, Switzerland). The procedure was essentially the same as described by Fabbretti et al.. The following polyclonal antibodies were used: P2X3 (1:2000 Neuromics, Bloomington, MN, USA), P2X2 or TRPV1 (1:400 Alomone, Jerusalem, Israel), β-tubulinIII (1:400, Chemicon, Temecula, CA, USA). To ensure correct equal loading reflecting the neuronal cell content in different lysates, protein extracts were quantified with bicinchonic acid (Sigma) and calibrated for the neuronal specific β-tubulinIII. The amount of loaded proteins was in the 20–50 μg/ml range.
TG ganglion tissue was used with a free-floating immunostaining procedure. TG culture cells were fixed in 4% paraformaldehyde for 20 min at room temperature. The following rabbit polyclonal antibodies were used: P2X3 (1:500 from Chemicon), P2X2 and TRPV1 (1:200 from Alomone), and anti-cleaved caspase3 (1:100, Cell Signaling Technology, Beverly, MA, USA). The mouse monoclonal antibodies against the neuron specific β-tubulinIII (1:100, from Chemicon), GFAP (1:200, Sigma) and JNK (1:100, Santa Cruz) were used. Immunofluorescence reactions were visualized using the secondary antibodies AlexaFluor 488 or AlexaFluor 594 (1:500 dilution; Molecular Probes, Invitrogen). The P2X3 antibody used for double immunofluorescence experiments was obtained by immunizing a guinea-pig with the peptide CVEKQSTDSGAYSIGH. The specificity of the guinea-pig anti-P2X3 antibody was evaluated by western immunoblotting and immunofluorescence experiments (dilution 1:500 and 1:200, respectively) of HEK-293 cells transfected with pCDNA-P2X3 . Tissue or cells stained with the secondary antibody only showed no immunostaining. To minimize tissue autofluorescence, TG ganglia were treated with Sudan Black. All images were captured under the same brightness and contrast settings. Control experiments using pre-immune guinea-pig serum gave no signal. In each experiment the number of positive neurons for a given antibody was normalized by dividing the number of positive cells by the number of β-tubulinIII-stained cells (equal to 100 %). For double immunofluorescence experiments the number of neurons stained with a certain antibody was referred as a percent of the total number of cells stained with the other antibody. An average of 500 cells in culture or 1,000 cells in the tissue were counted for each condition. Each data is the mean of at least 3 independent experiments. Results were analyzed with the ImagePro Express software (Media Cybernetics, L.P., Silver Spring, MD, USA).
Scanning electron microscopy
For scanning electron microscopy, 24 h rat or mouse TG cultures were fixed in 2.5% glutaraldehyde (Sigma; in 0.1 buffered phosphate; pH 7.3) for 30 min at 4°C and postfixed with 1% OsO4 (Sigma), dehydrated in ethanol and dried by the critical-point method . Ganglia or cultures were sputter-coated with gold (Electron microscopy sciences, Hatfield, PA, USA) as described . Specimens were observed under a Stereoscan 430i microscope (Leica, Houston, TX, USA). Three hundred cells obtained from 4 different rats and 6 mice were observed. Freshly dissected TGs from two rats were slit open after de-sheathing without enzymatic treatment. Ganglia were fixed for 3 h at 4°C and treated as above.
The level of NGF present in the supernatant or in the cell lysates of TG culture was assessed with using the Emax NGF immunoassay system (Promega, Madison, WI, USA). Rat and mouse TG culture medium was collected after 24 h from plating and concentrated 4-fold for analysis (n = 4). Furthermore, mouse or rat ganglia or cell cultures were homogenated in 200 μl of a buffer containing 137 mM NaCl, 20 mM TrisHCl (pH 8), 1% NP40, 10% glycerol and protease inhibitors (Roche). Samples were diluted (1:6) in the same buffer and processed for ELISA assay. Results were corrected for blank and normalized. In the case of ganglia or cells, quantification was normalized with respect to the genomic DNA content (purified using GenElute mammalian genomic DNA kit, Sigma). The recovery of exogenously added NGF (100 ng/ml) was 63 ± 11 % (n = 3); data were not corrected for recovery. No NGF was detected in the fetal calf serum before adding it to the cultures.
Cells were continuously superfused (2 ml/min) with physiological solution containing (in mM): 152 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH adjusted to 7.4 with NaOH, osmolarity adjusted to 320 mOsm with glucose). Single cells were patch clamped in the whole-cell configuration using pipettes with a resistance of 3–4 MΩ when filled (in mM) with 140 KCl, 0.5 CaCl2, 2 MgCl2, 2 Mg2ATP3, 2 GTP, 10 HEPES and 10 EGTA (pH adjusted to 7.2 with KOH; osmolarity 285 mOsm). Currents were recorded from medium-sized (15–25 μm) nociceptive TG neurons . TG cells were voltage clamped at membrane potential ranging from -70 to -60 mV. Series resistance was compensated by 70 %. α, β-meATP concentration-response curves were obtained by applying the same dose range to each tested cell; results were fitted with a sigmoidal curve (Origin 6.0, Microcal, Northampton, MA, USA) in order to express agonist potency in terms of EC50 values (concentration producing 50 % of the maximum response). Each concentration of α, β-meATP was applied for 2 s every 7 min to obtain full response recovery from desensitization. Capsaicin induces responses with strong tachyphylaxis during repetitive applications  and it can kill primary afferent nociceptors : to circumvent these problems, a single dose of capsaicin was applied to each TG neuron. On mouse neurons, 1 μM capsaicin was used as standard test dose to yield reproducible inward currents, because even a small increment in concentration (10 μM) produced very slowly reversible inward currents as previously reported for sensory neurons . On rat neurons, reproducible responses were evoked with a test (1–10 μM) concentration of capsaicin. This concentration is in excess of the EC50 value for TG neurons . In order to minimize any possible difference in responses between TG neuron preparations, sister dishes were used on each occasion to compare control neurons and neurons treated with 5-HT or NGF (acute or chronic treatment).
Cells were incubated for 40 min at 20–22°C in physiological solution containing Fluo3 (AM ester cell-permeable compound; 5 μM; Molecular Probes), followed by a 30 min washout period. Fluorescence emission was detected with a fast CCD camera (Coolsnap HQ; Roper Scientific, Duluth, GA, USA). Images were acquired with 150 ms exposure time and single cell responses were analysed with the Metafluor software (Metafluor Imaging Series 6.0, Universal Imaging Corporation, Downingtown, PA, USA). Intracellular Ca2+ transients were expressed as percent amplitude increase (ΔF/F0, where F0 is the baseline fluorescence level and ΔF is the increment over baseline). Each event was also visually inspected to exclude artifactual components.
Drug delivery in functional experiments
α, β-meATP, NGF and capsaicin (all from Sigma) were diluted with physiological solution to final concentration and applied by a rapid superfusion system (Rapid Solution Changer RSC-200, BioLogic Science Instruments, Grenoble, France). The time for solution exchange was about 30 ms ). Chronic application of NGF (50 ng/ml) was done by applying this substance to TG cultures for 24 h; cells were patch clamped immediately after washing out this dose of NGF. Twenty-four hour long treatment with 5-HT (10 μM) was carried out in the continuous presence of the monoaminoxidase inhibitor pargyline (100 μM; Sigma) to prevent enzymatic breakdown of this monoamine. Parallel controls were treated with the same concentration of pargyline alone.
Data are presented as the means ± standard error of the mean (n = number of cells, unless otherwise indicated). The statistical significance was assessed with Mann-Whitney rank-sum test and the Wilcoxon test for non parametric data, and with Student's t-test for parametric data (KyPlot, version 2.0, Qualest Co., www.qualest.co.jp). For RT-PCR and Western blot, the films were scanned and band density was measured using CorelDraw Photopaint software (Corel, Berkshire, UK), normalized with β-tubulinIII control band and compared to the tissue value. For real time PCR, the relative mRNA expression of P2X3, P2X2 and TRPV1 in the different samples was normalized to the neuronal β-tubulinIII mRNA content in each condition and correlated with the one of the TG tissue. These experiments were performed in duplicate and repeated thrice for mouse and rat samples. Differences between groups were compared using ANOVA. A P value of < 0.05 was accepted as indicative of a statistically significant difference.