CaV2.1 R192Q knock-in (KI) and wild-type (WT) mouse littermates were used . All experimental procedures were in strict accordance with the Italian and EU regulation on animal welfare and were previously approved by the SISSA ethics committee. Mouse genotyping was performed as previously reported .
Culture procedure and protocol for macrophage-trigeminal ganglion co-cultures
Trigeminal ganglion cultures were obtained from P12-14 mice as described before  and, after 48 h from plating, employed for patch clamping, macrophage functional tests or molecular/cell biology experiments. From each mouse two ganglia were excised and used to prepare one petri dish. Control and test cultures were always run in parallel on the same day.
To explore the potential interaction between macrophages and trigeminal ganglion cells, in a series of experiments, we supplemented standard trigeminal ganglion cultures with the addition of host macrophages. To this end, mouse host macrophages (MФ) were extracted from the peritoneal cavity of WT adult mice 72 h after a single intraperitoneal injection of Brewer thioglycollate medium (0.4 g/kg, 3% wt/vol, Sigma, Milan, Italy) . Primary macrophages were plated in DMEM/10% FBS medium and kept for 14 d in culture (with change of medium every 48 h). Thereafter, macrophages were collected, counted in a Burker chamber, and transferred (as a batch of 120,000 cells), for co-cultures experiments, to Petri dishes containing trigeminal ganglia (from WT or R192Q KI mice) dissociated on the same day. Experiments were performed after 2 d of co-culturing. For the ATP assay and Fluoresbrite® YG latex Microspheres experiments, we used multiwall plates in which an equivalent number of macrophages (30,000 cells/cm2) were plated together with trigeminal neurons (from WT or R192Q KI mice) for co-cultures experiments. We estimated that this addition increased the percent of macrophages from about 3–5 to 10–15% of the total DAPI-positive cell population in a region of interest (ROI) of 640 × 480 μm. It should be noted that, under the present experimental conditions, it was not possible to distinguish host macrophages from resident ganglion macrophages.
Immunocytochemistry of trigeminal ganglia in culture from WT or R192Q KI mice was performed as already described . For immunocytochemistry of trigeminal ganglia in situ, a series of 14 μm-thick longitudinal sections of trigeminal ganglia was collected. The following antibodies were used: anti-β-tubulin III (1:1000; Sigma) for neuronal staining, and anti-Iba1 (1:300, Wako, Osaka, Japan), a microglia/macrophage-specific marker of macrophage lineage in central and peripheral neuronal tissue [19, 20]. For secondary immunostaining AlexaFluor goat-anti-mouse 488- or goat-anti-rabbit 594-conjugated antibodies (1:300 Invitrogen; S.Giuliano Milanese, Italy) were used; nuclei were counterstained with DAPI (1:1000, Sigma). Images in a 640 × 480 μm ROI were visualized with a Zeiss Axioskop fluorescence microscope (Zeiss, Zurich, Switzerland), and analyzed with either MetaMorph software (Molecular Devices, Downingtown, PA, USA) or ImageJ software (NIH, USA) with ITCN plugin. 3D reconstructions (Z-stack; 0.5 μm steps) of high magnification confocal images (Leica TCS SP2, Wetzlar, Germany) of intact ganglia were obtained with ImageJ software and quantified with ImageJ Voxel counter (voxel, μm3).
Macrophage phagocytosis tests were performed by incubating cultures with FITC-conjugated Zymosan A (Zy-FITC; 1 mg/ml, Sigma) for 10 min at 37°C, fixed in 4% paraformaldehyde and processed for immunofluorescence . Active macrophages were considered when taking up ≥ 1 granule of Zy-FITC. After counting the average number of granules/active macrophage, the phagocytosis index was calculated as the percentage of Zy-FITC-positive macrophages multiplied by the number of Zy-FITC granules per single cell . Experiments were also performed with Fluoresbrite® YG latex Microspheres (1.00 μm, PolySciences, Warrington, PA, USA) on trigeminal neuronal cultures and macrophages co-cultures (incubation was 15 min at 37°C). Fluorescence signals were detected with a Perkin Elmer fluorimeter at 488 nm wavelength. Cumulative probability plots were constructed as shown before .
Real time - PCR and protein analysis
Total mRNA was extracted from cultures of WT or KI mouse peritoneal macrophages as described before . Real-time PCR reactions were run in duplicate in an iQ5 thermocycler using IQ SyBr Green Supermix Reactions (Bio-Rad Hercules, CA, USA), with specific primers for P2X3  or Cav2.1 (Fw: 5′-GAAGTCCATCATAAGTCTGTTGTT-3′ and Rw: 5′- GCCACCGAACAGCTGCAT-3′) . All primer sequences were designed using Beacon designer (PREMIER Biosoft International, Palo Alto, CA, USA) and were previously validated [8, 17]. Calculations for relative mRNA transcript levels were performed using the comparative method between cycle thresholds of different reactions [8, 17]. Quantitative PCR was performed in duplicate following the MIQE guidelines .
Western blotting was performed as described earlier [8, 16], using antibodies against anti-P2X3 (1:300; Alomone, Jerusalem, Israel), anti-β-tubulin III (1:2.000; Sigma) or anti-actin (1:3.000; Sigma). Grey values were quantified with Scion Image software (Scion, Frederick, Maryland, USA) or Uviband (Uvitec, Cambridge, UK). Total protein content of ganglia was measured with the BCA kit purchased from Sigma.
ATP release assay
Basal ATP concentrations in the extracellular medium collected from 24 h trigeminal ganglion (WT or KI) cultures were measured with ENLITEN ATP Assay (Promega, Italy), according to the manufacturer’s instructions. Extracellular ATP was also measured in the medium of macrophage-neuronal co-cultures following the addition of 30,000 host macrophages /cm2 (5 h).
P2X3 receptor-mediated currents were recorded, under patch clamping conditions in whole-cell configuration, from trigeminal neurons obtained from WT or R192Q KI mice, using the specific agonist α,β-methylene-ATP (α,β-meATP) applied with a fast superfusion system (Rapid Solution Changer RSC-200; BioLogic Science Instruments, Claix, France). Full details of the electrophysiological methods have been previously reported [8, 17]. Trigeminal neurons were superfused continuously (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). Patch pipettes (3–5 MΩ resistance) were filled with the following solution (in mM): 140 KCl, 0.5 CaCl2, 2 MgCl2, 2 Mg2ATP3, 2 GTP, 10 HEPES, and 10 EGTA (pH adjusted to 7.2 with KOH). Cells were held at −60 mV. Data were filtered at 1 KHz and acquired by means of a DigiData 132XInterface and pClamp 8.2 software (Molecular Devices, Sunnyvale, CA, USA). We measured current peak amplitude, current rise-time (10–90% of peak amplitude), onset of desensitization (estimated by calculating the first-time constant of current decay, τfast), and recovery from desensitization (with paired-pulse experiments in which α,β-meATP applications were spaced at 30-s interval). Recovery was expressed as % of the first response in each pair [9, 17].
To find out how WT P2X3 receptor currents might have been affected by the number of host macrophages, we compared current amplitudes after adding 120,000 or 300,000 or 1 million MФ for 48 h to the primary ganglion cultures. Under these conditions, while control WT currents had an average amplitude of −290 ± 30 pA (n = 15), co-culturing with 120,000 host macrophages raised the average amplitude of P2X3 currents to −500 ± 60 pA (n = 24; p < 0.05). Larger macrophage numbers (300,000 or 1 million) led to current amplitudes of −660 ± 90 (n = 6) or −540 ± 70 pA (n = 7), values that were not significantly different from the one observed with 120,000 macrophages. Hence, further experimental tests were routinely carried out with 120,000 host macrophages.
Data are expressed as mean ± standard error of the mean (SEM), where n indicates the number of independent experiments or the number of investigated cells. Statistical analysis was performed using the Student’s t-test, or the Mann–Whitney rank sum test after the software-directed choice of parametric or non-parametric data, respectively (Sigma Stat and Sigma Plot, Systat Software Inc., San Jose, CA, USA). A p value of ≤ 0.05 was accepted as indicative of a statistically significant difference.