Cell Body

We measured intracellular pH in single cells by live-cell ratiometric imaging experiments using the fluorescent pH- sensitive BCECF dye^{48 (link)}. DRG cultures were incubated with 1 µM BCECF-AM (Life Technologies, Italy) for 20 min at room temperature in Tyrode standard solution (TS) of the following composition in mM: NaCl 154; KCl 4; CaCl₂ 2; MgCl₂ 1; 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES) 5; glucose 5.5; NaOH to pH 7.4. After washing in TS, cells were placed under a Leica DMI6000 epifluorescent microscope equipped with S Fluor × 40/1.3 objective and BCECF was alternatively excited at 490 nm and 450 nm (monochromator Polychrome IV, Till Photonics, Germany) while recording at emission wavelengths > 525 nm. Data was acquired every 3 s (Hamamatsu, Japan) with MetaFluor software (Molecular Devices, Sunny-vale, CA, USA). Image time series were analysed with ImageJ (Rasband W.S., NIH, Bethesda MD) and OriginPro 9.1 (OriginLab, USA) softwares to obtain the background subtracted ratio of the mean pixel intensity within a region of interest encompassing a neuronal cell body. The ratios were converted into the pH_i value by the high K⁺/nigericin technique. At the end of the experiment we obtained for each neuron three calibration points at pH values of 5.5, 6.5 and 7.5 (Calibration Buffer Kit, Life Technologies) that in almost all cells were best fitted by a straight line, whose parameters were used to convert ratios to pH values.

Adult mice expressing GCaMP3 (6–12 weeks, male and female) were anesthetized using ketamine (KETAVET; Zoetis; 100 mg/kg), xylazine (Rompun; Bayer; 15 mg/kg), and acepromazine (Elanco; 2.5 mg/kg). Depth of anesthesia was confirmed by pedal reflex and breathing rate. Animals were maintained at a constant body temperature of 37°C using a heated mat (VetTech). Lateral laminectomy was performed at spinal level L3–5. In brief, the skin was incised longitudinally, and the paravertebral muscles were cut to expose the vertebral column. Transverse and superior articular processes of the vertebrae were removed using OmniDrill 35 (WPI) and microdissection scissors. To obtain a clear image of the sensory neuron cell bodies in the ipsilateral dorsal root ganglion (DRG), the dura mater and the arachnoid membranes were carefully opened using microdissection forceps. Artificial spinal fluid (values are in mm: 120 NaCl, 3 KCl, 1.1 CaCl₂, 10 glucose, 0.6 NaH₂PO₄, 0.8 MgSO₄, and 18 NaHCO₃, pH 7.4 with NaOH) was constantly perfused over the exposed DRG during the procedure to maintain tissue integrity. The animal was mounted onto a custom-made clamp attached to the vertebral column, rostral to the laminectomy. The trunk of the animal was slightly elevated to minimize interference caused by respiration. The DRG was isolated by coating with silicone elastomer.
Images were acquired using a Leica SP8 confocal microscope. A 10× dry, 0.4-N.A. objective with 2.2 mm working distanced was used, with image magnification of 0.75–3× optical zoom. GCaMP3 was excited using a 488 nm laser line (3–10% laser power). GCaMP was detected using a hybrid detector (60% gain). 512 × 512-pixel images were captured at a frame rate of 1.55 Hz, bidirectional scan speed of 800 Hz, and pixel dwell time of 2.44 μs.
Noxious and innocuous stimuli were applied to the left hindpaw, ipsilateral to the exposed DRG. For thermal stimuli, the ventral side of the paw was immersed with ice-water (nominally 0°C), acetone (100%) or water heated to 55°C using a Pasteur pipette. For delivery of precise temperature stimuli, a Peltier-controlled thermode (Medoc) was used. For mechanical stimuli, a pinch with serrated forceps was used. An interval of at least 30 s separated each stimulus application.