Animals

Pirt-GCaMP3 mice were generated as described previously [20, 21]. In Pirt-GCaMP3 mice, GCaMP3 expression is driven by the Pirt promoter, which is a pan-DRG and TG-selective promoter, resulting in almost all primary sensory neurons expressing GCaMP3. MrgprB2 knock-out (KO) mice were purchased from The Centre for Phenogenomics (Ontario, Canada) and were backcrossed to C57BL/6 or Pirt-GCaMP3 mice. To generate PAC1 conditional KO mice, we first crossed C57BL/6N-Atm1BrdAdcyap1r1tm1a(KOMP)Wtsi/MbpMmucd mice (MMRRC #46,500) with B6N(B6J)-Tg(CAG-Flpo)1Afst/Mmucd mice (MMRRC #36,512). The IRES: lacZ trapping cassette was excised using methods described in previous studies [22, 23]. The resultant PAC1 floxed/floxed mice were subsequently bred with SM2-cre-ERT2 (JAX# 019079) or Pirt-cre mice. Mice were housed three to five per cage with a 12-hour light/dark cycle and fed water and mouse chow ad libitum. Male mice used for experiments were 6–12 weeks old. All animal procedures were approved by the University of Texas Health at San Antonio (UTHSA) Animal Care and Use Committee (IACUC). All experiments were performed following the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Peptides and drugs

PACAP38 was purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). QWF, interleukin 6 (IL-6), R-7050, SB366791, and PA-8 were purchased from Tocris Bioscience/Bio-Techne Corporation (Minneapolis, MN, USA). Compound 48/80 was obtained from MilliporeSigma (St. Louis, MO, USA), and a stock solution was prepared by dissolving in distilled water. PACAP38 (1 ng/5 µL) and IL-6 (0.1 ng/5 µL) were prepared in synthetic interstitial fluid (SIF) [24] and administered to dura. R-7050 (10 mg/kg), SB366791 (1 mg/kg), and PA-8 (10 mg/kg) were prepared in 5% DMSO and 5% Tween-80 in 0.9% NaCl and administered intraperitoneally (i.p.).

Repetitive restraint stress

Restraint stress was performed as previously described [25, 26] with modification. Animals were placed in tubular type restrainers (RWD, China) and were restrained for 1 h per day for 3 consecutive days. The restraint was carefully adjusted to permit sufficient respiration, and precautions were taken to prevent injury from the confinement restrainer. Control mice were isolated in a different room where they were subjected to a 1 h deprivation of food and water over three consecutive days. To prevent the potential transmission of stress characteristics, mice exposed to stress were housed distinctly apart from the control group.

Behavioural tests

The facial mechanical von Frey test was performed according to previously published methods with some modifications [27]. Mice were habituated to the experimenter’s smell and hand touch for 2 days and acclimated in a plexiglass chamber with 4 oz paper cups for 2 h/d for 3 days. Baseline testing of cutaneous facial (periorbital region) sensitivity touched by von Frey filament was conducted for 3 days. The baseline of the facial von Frey test was set when a withdrawal threshold of approximately 0.5 to 0.7 g was reached. The thresholds were determined using the Dixon “up-and-down” method [28]. Grimace pain behaviour was measured in five characterized facial areas (orbital, nose, cheek, ears, and whiskers) on a scale of 0 to 2 (0 = not present, 1 = somewhat present, 2 = clearly present) as previously published [29]. We assessed orbital tightening, nose bulge, cheek bulge, ear position, and whisker change. The experimenter handling the mice and the data analyst were blinded to experimental conditions.

TG exposure surgery for in vivo intact Pirt-GCaMP3 Ca2+ imaging

Mice were anesthetized by i.p. injection of Ketamine/Xylazine (approximately 80/10 mg/kg) (MilliporeSigma, St Louis, MO, USA), and ophthalmic ointment (Lacri-lube; Allergen Pharmaceuticals) was applied to the eyes. The right-side dorsolateral skull was exposed by removing skin and muscle. A patch of dorsolateral skull (parietal bone between the right eye and ear) was removed using a dental drill (Buffalo Dental Manufacturing, Syosset, NY, USA) to make a cranial window (∼ 10 × 10 mm). The TG was then exposed by aspirating overlying cortical tissue. During the surgery, the mouse’s body temperature was maintained on a heating pad at 37 °C ± 0.5 °C and monitored by rectal probe. We performed in vivo imaging one day after the cessation of repetitive stress for stressed mice, while PACAP38-injected mice were used 3 h after injection.

In vivo intact TG Pirt-GCaMP3 Ca2+ imaging

In vivo intact TG Pirt-GCaMP3 Ca2+ imaging in live mice was performed for 2–5 h immediately after exposure surgery as previously described [20]. After the exposure surgery, mice were laid abdomen-down on a custom-designed platform under the microscope. For in vivo intact TG Pirt-GCaMP3 Ca2+ imaging, the animal’s head was fixed by a head holder to minimize movements from breathing and heartbeats. During the imaging session, body temperature was maintained at 37 °C ± 0.5 °C on a heating pad and monitored by rectal probe. Anaesthesia was maintained with 1–2% isoflurane using a gas vaporizer with pure oxygen. Live images were acquired at ten frames per cycle in frame-scan mode at ∼ 4.5 to 8.79 s/frame, ranging from 0 to 100 μm, using a 5 × 0.25 NA dry objective at 512 × 512 pixels or higher resolution with solid diode lasers tuned at 488 nm and emission at 500–550 nm. An average of 2,893 ± 59 neurons per TG (∼ 10% of total TG neurons [30, 31]) were imaged (∼ 2 Hz), and small regions of TG neurons were imaged at greater speed (> 40 Hz). von Frey filaments (0.4 g) and noxious water (4 °C and 50 °C) were applied to the different divisions of the animal’s face divided by TG branches for one imaging cycle after four baseline imaging cycles. Noxious hot water (2 mL) was gently applied with a pipette to the fur skin of the mouse’s face. Capsaicin (500 µM, 10 µl) was injected intracutaneously into the different TG branches using a syringe after four baseline imaging cycles. To prevent mechanical stimulation while injecting capsaicin into the skin, the syringe needle was inserted into the skin before imaging.

For imaging data analysis, raw image stacks were collected, deconvoluted, and imported into ImageJ (NIH). Optical planes from sequential time points were re-aligned and corrected using the stackreg rigid-body cross-correlation-based image alignment plugin. Ca2+ signal amplitudes were expressed as Ft (fluorescence intensity in each frame)/F0 (average fluorescence intensity during the first one to four frames). All responding cells were verified by visual examination of the raw imaging data. Neurons displaying changes in amplitude originating from Ca2+ signals without any external stimulation were categorized as spontaneous neurons. In contrast, neurons exhibiting changes in amplitude following specific stimuli were classified as activated neurons. We employed ImageJ software equipped with Feret’s diameter analysis tool to measure the diameter of the cell bodies of the collected cells. Based on this analysis, cells with a diameter of less than 20 μm were classified as small, those with a diameter between 20 and 25 μm were classified as medium, and those exceeding 25 μm were categorized as large diameter neurons.

Dura injection

Dura injection was performed as previously described [27]. Drugs or compounds were injected into the junction of the sagittal and lambdoid sutures of dura mater in a volume of 5 µl via a modified internal cannula (P1 Technologies, Roanoke, VA, USA) under brief anaesthesia by isoflurane. The length of the injection needle was adjusted to 0.5 to 0.8 mm.

In vivo imaging in dura mater

Mice were anesthetized by i.p. injection of Ketamine/Xylazine (80/10 mg/kg) (MilliporeSigma, St Louis, MO, USA), and ophthalmic ointment was applied to the eyes. The scalp was shaved and sterilized using 70% EtOH. To form a cranial window, a ∼ 3 × 3 mm round area of the right parietal skull was carefully removed. Using dental cement (Lang Dental Manufacturing, Wheeling, IL, USA), a crown was made around the cranial window, and the brain was covered with SIF. Texas-Red conjugated 2k Dalton dextran (Nanocs) was injected into the tail vein (100 µl of 1 mg/ml in saline) to visualize blood vessels in dura mater. Anesthetized mice were imaged with a single photon confocal microscope (Carl Zeiss) using a 40× water-immersion objective (1.0 NA, Carl Zeiss). The vessel diameter and Ca2+ transient of trigeminal afferent nerve fibres were measured using ImageJ software.

Immunofluorescence imaging in dura mater

Animals were anesthetized with Ketamine/Xylazine (80/10 mg/kg) 3 h after PACAP38 injection (MilliporeSigma, St Louis, MO, USA), and cardiac perfusion fixation was conducted using 4% paraformaldehyde. After fixation, the dura mater was dissected and postfixed for 24 h in 4% paraformaldehyde. The collected tissues were incubated with primary antibodies: PACAP (Santa Cruz), βIII-tubulin (Abcam), and Rhodamine Avidin D (Vector Laboratories) for 24 h. Tissues were then incubated with Alexa-Fluor 488 or 647 (1:500; Invitrogen)-labelled polyclonal secondary antibodies, and slides were cover-slipped using a mounting medium (Invitrogen). Degranulated mast cells were identified by the presence of scattered granules adjacent to the cell.

Mast cell culture and imaging

Peritoneal mast cells were isolated from adult male and female mice as previously described [32]. Briefly, 5 mL of ice-cold mast cell dissociation medium (MCDM; HBSS with 3% FBS and 10 mM HEPES, pH7.2) was injected into the peritoneal cavity, and the abdomen was massaged for 60s; peritoneal fluid was collected and centrifuged at 200 g for 5 min at room temperature. The pellets were resuspended in 2 mL MCDM, layered over 4 mL of isotonic 70% Percoll solution (MilliporeSigma, St Louis, MO, USA), and centrifuged at 500 g for 20 min at 4 °C. The purity of isolated mast cells was > 95%, as determined by avidin staining. The mast cells were resuspended in DMEM with 10% FBS, 100 U/mL penicillin, 50 mg/mL streptomycin, and 25 ng/mL recombinant mouse stem cell factor (mSCF; Peprotech, Cranbury, NJ, USA) and plated onto glass coverslips coated with 30 mg/mL fibronectin (MilliporeSigma, St Louis, MO, USA). LAD2 human mast cells (Laboratory of Allergic Diseases 2), provided by Professor Dr. Xinzhong Dong from Johns Hopkins University (MD, USA), were cultured in StemPro-34 SFM medium (Life Technologies, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human stem cell factor (Peprotech, Cranbury, NJ, USA) at 37 °C, 5% CO2. Cell culture medium was hemi-depleted every week and replaced with fresh medium. For Ca2+ imaging, the cell suspensions were seeded onto glass coverslips coated with fibronectin and incubated for 2 h (37 °C, 5% CO2). The cells were loaded with Fluo-8 Ca2+ dye (5 µM) for 30 min and imaged in Ca2+ imaging buffer (CIB; 125 mM NaCl, 3 mM KCl, 2.5 mM CaCl2, 0.6 mM MgCl2, 10 mM HEPES, 20 mM glucose, 1.2 mM NaHCO3, 20 mM sucrose, adjusted to pH 7.4 with NaOH) using a confocal (Carl Zeiss, Oberkochen, Germany) or widefield fluorescence microscope (Carl Zeiss) system.

Ca2+ imaging in HEK293 cells

HEK293 cells were transiently transfected with lipofectamine 2000 (Invitrogen) using a total of 2 µg cDNA per dish (35 mm). Plasmid DNAs encoding the human MrgprX2 (pcDNA3.1) and mouse MrgprB2 (pcDNA3.1), provided by Professor Dr. Xinzhong Dong from Johns Hopkins University (MD, USA), were co-transfected with Gα15 (pcDNA3.1) at a ratio of 9:1. A surrogate expression marker, tdTomato (60 ng), was co-transfected along with other plasmids. Forty-eight hours following transfection, cells were loaded with AM esters of the Ca2+ indicators, Fluo-8 (1 µM; Abcam, Cambridge, MA, USA) along with Pluronic F-127 (0.04%; Life Technologies, Carlsbad, CA, USA) for 30–45 min at 37 °C in the dark, in standard extracellular solution (SES) containing 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM D-glucose, pH 7.40. Cells were viewed on an upright Zeiss Examiner/A1 microscope fitted with a 40× water-immersion objective (0.75 NA, 2.1 mm free working distance, Carl Zeiss) and with an Axiocam 705 color camera (Carl Zeiss). Fluorescence images were taken every 5 s using the Zeiss Zen Blue software module. Cells were imaged in SES at room temperature; drugs were bath applied into the chamber at 400 µL/min following 10 cycles of baseline imaging, and responses were monitored for an additional 50 cycles. The percentage of cells responding to PACAP38 among total tdTomato-expressing cells was calculated to quantify the PACAP38 response.

Mast cell degranulation assay

Mast cell degranulation was measured by β-hexosaminidase release assay as previously described for peritoneal mast cells [33] or LAD2 [34]. Isolated mouse mast cells or LAD2 were cultivated and then sensitized overnight with mouse monoclonal anti-DNP-IgE (MilliporeSigma) or human IgE (MilliporeSigma). Cells were washed three times and resuspended in HEPES buffer. The cells were incubated with PACAP38 (1 µM, Phoenix Pharmaceuticals) and Compound 48/80 (1 µg/ml, MilliporeSigma) for 45 min at 37 C°. β-hexosaminidase released into the supernatants and cell lysates was quantified by hydrolysis of p-nitrophenyl N-acetyl-β-D-glucosamide (MilliporeSigma) in 0.1 M sodium citrate buffer (pH 4.5) for 90 min at 37 °C. The percentage of β-hexosaminidase release was calculated as a percent of total content.

ELISA

Dura mater and blood were collected between 8:00 and 10:00 a.m. after restraint stress. The collected dura maters were washed with PBS to remove blood. Blood was collected in vacutainers containing K3EDTA and centrifuged at 1000 g for 15 min at 4 °C. PACAP38 levels were determined using the PACAP38 ELISA kit (Antibodies.com, Cambridge, UK).

Statistical analysis

All statistical analyses were performed in Prism (GraphPad). Error bars are defined as the mean ± S.E.M. Differences were considered significant at p < 0.05. Use of statistical tests are noted in the figure legends.