All experimental procedures were approved by the Intramural Animal Care and Use Committee at the Kyungpook National University, and followed the guidelines of the National Institute of Health. Twenty male Sprague–Dawley rats (170–190 g) were used for this study: Three rats for light microscopic (LM) immunohistochemistry, three rats for routine electron microscopy, eight rats for a behavioral assay for mechanical allodynia after chronic constriction injury of infraorbital nerve (CCI-ION), followed by electron microscopic (EM) postembedding immunohistochemistry, and six rats for a glutamate release assay.

Rats were anesthetized with intraperitoneal injection of a mixture of ketamine (40 mg/kg) and xylazine (4 mg/kg), and surgery to induce CCI-ION was performed according to the original protocol (Imamura et al., 1997). Briefly, a one-centimeter incision was made along the gingivo-buccal margin, next to the first upper molar. Approximately 5 mm segment of the infraorbital nerve was isolated from the surrounding tissue, and two 5–0 chromic gut (C540, AILEE Co., LTD, Busan, Korea) ligatures were tied loosely around it. The incision was closed with two 4–0 silk sutures. In sham-operated animals, the surgery was identical, except that no ligatures were placed around the infraorbital nerve.

Rats were habituated for 30 min to a cage in a darkened and noise-free room. Withdrawal responses were measured after 10 trials of 4-s duration in 10-s intervals of constant air-puff pressure, as described previously (Ahn et al., 2009). The response threshold was determined as the air-puff pressure at which the rat responded in 5 of the 10 trials. The cut-off pressure was 40 psi.

For immunofluorescence, naïve rats were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused intracardially with 100 mL heparinized 0.9% saline, followed by 500 mL freshly-prepared fixative, containing 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer at pH 7.4 (PB). Trigeminal ganglions (TGs) were dissected out and postfixed in the same fixative for 2 h at 4°C, then cryoprotected in 30% sucrose in PB overnight at 4°C. The next day, 30-μm-thick sections were cut on a freezing microtome. Sections were treated with 50% ethanol in 10% normal donkey serum (NDS, Jackson ImmunoResearch, West Groove, PA) for 30 min, and incubated overnight in a rabbit anti-substance P (SP, 1:2,000; 20064, Immunostar, WI) or a rabbit anti-calcitonin gene-related peptide (CGRP, 1:5000; 24112, Immunostar) antibody alone or in combination of guinea pig anti-SP (1:1,000; AB5892, Chemicon, CA) or goat anti-CGRP antibody (1,500; AB36001, Chemicon) with rabbit anti-Piezo1 antibody (1,300; APC087, Alomone Labs, Jerusalem). On the next day, the sections were incubated with a Cy3-conjugated donkey anti-rabbit antibody alone or a combination of a Cy3-conjugated donkey anti-guinea pig or donkey anti-goat antibody and a FITC-conjugated donkey anti-rabbit antibodies (1,200, in PB, Jackson ImmunoResearch) for 3 h. Sections were mounted on slides, coverslipped with Vectashield (Vector Laboratories, Burlingame, CA), and examined on a Zeiss Axioplan 2 fluorescence microscope. Micrographs were obtained with an Exi digital camera (Q-imaging Inc., Surrey, CA), attached to the microscope. Images were converted to grayscale and brightness and contrast were enhanced using identical adjustment steps for all images. The threshold level for defining neurons as immunopositive was determined at 100–120 gray level in images with 256 gray levels using Image J software (NIH, Bethesda, MD).

The specificity of the immunostaining was evaluated by omission of the primary or secondary antibodies, which completely eliminated the specific staining. Specific immunostaining with SP and Piezo1 was also completely abolished by preadsorption with the respective peptides (SP: S6883, Sigma-Aldrich, 10 μg/mL; Piezo1: BLP-PC087, Alomone Labs, 8 μg/mL).

On day 22 after surgery, when mechanical allodynia is most prominent, rats of the sham-operated and CCI-ION groups, as well as naïve rats, were deeply anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused intracardially with 100 mL heparinized 0.9% saline, followed by 500 mL freshly-prepared fixative, containing 1% PFA and 2.5% glutaraldehyde in 0.1 M PB. TGs were dissected out and fixed in the fixative used for perfusion for an additional 2 h at 4°C. Sections were cut at 50 μm on a Vibratome and osmicated in 1% osmium tetroxide in PB for 30 min. After that, sections were dehydrated in graded ethanol, flat-embedded in Durcupan ACM (Fluka, Buchs, Switzerland) between Aclar plastic films (EMS, Hatfield, PA), and cured for 48 h at 60°C. Small chips containing the ophthalmo-maxillary region of the TG were cut out of the wafers and glued onto blank resin blocks with cyanoacrylate. Thin sections were cut with a diamond knife, mounted on formvar-coated single slot nickel grids, and stained with uranyl acetate and lead citrate. Grids were examined on a Hitachi H7500 electron microscope (Hitachi, Tokyo, Japan) at 80 kV accelerating voltage. Images were captured with Digital Micrograph software driving a cooled CCD camera (SC1000; Gatan, Pleasanton, CA) attached to the microscope, and saved as TIFF files.

Postembedding immunogold labeling for glutamate was performed according to the method described previously by our laboratory (Paik et al., 2012, 2021). Briefly, grids were treated for 6 min in 1% periodic acid, to etch the resin, and for 10 min in 9% sodium periodate, to remove the osmium tetroxide. Grids were then transferred to tris-buffered saline containing 0.1% triton X-100 (TBST; pH 7.4) for 10 min. Grids were further incubated with rabbit polyclonal antiserum against glutamate (1:80,000; G6642, Sigma-Aldrich; RRID: AB_259946) in TBST containing 2% human serum albumin for 4 h at room temperature. To eliminate cross-reactivity, the diluted antiserum was preadsorbed overnight with glutaraldehyde (G)-conjugated amino acids (600 μM glutamine-G, 200 μM β-alanine-G, and 100 μM aspartate-G; Ottersen et al., 1986). After rinsing in TBST, grids were incubated in goat anti-rabbit IgG antibody coupled to 15 nm gold particles (1:25 in TBST containing 0.05% polyethylene glycol; BioCell, Cardiff, United Kingdom; RRID: AB_1769134) for 2 h at room temperature. Grids were counterstained with uranyl acetate and lead citrate, and examined with the electron microscope, images were captured and saved as above.

The specificity of the immunolabeling was evaluated by omission or replacement of the primary antiserum with normal rabbit serum or by preadsorption of the diluted anti-glutamate serum with 300 μM glutamate-G, which abolished the specific immunostaining. It was also confirmed on “sandwiches” of rat brain macromolecule-glutaraldehyde fixation complexes of GABA, glutamate, taurine, glycine, aspartate and glutamine (Ottersen, 1987; Paik et al., 2007), as routinely practiced in our laboratory.

We divided the TG neurons into three groups, based on the size of their somata: small (<400 μm² in cross-sectional area), medium (400–800 μm²), and large (>800 μm²; Bae et al., 2018). Twelve TG neurons of each group, together with their enveloping SGCs, in each of 4 rats with CCI-ION, and 4 sham-operated rats, were used for quantitative analysis. The density of gold particles coding for glutamate (number of gold particles/μm²) was measured in 80–110 electron micrographs from each of 3 small, 3 medium-sized, and 3 large neuronal somata in each TG using a digitizing tablet and Image J software (NIH, Bethesda, MD). The density of gold particles was determined as the number of gold particles in randomly-selected rectangular areas of 4 μm² of the cytoplasm of each neuron (6, 9 and 12 rectangular areas per small, medium-sized, and large neurons, respectively, or a total area of 24–48 μm² per neuron) and in their enveloping SGCs (3–12 randomly-selected areas of the cytoplasm, or a total area of 5–50 μm² per SGC). The gold particles over the nucleus and the mitochondria were excluded from analysis. The density of gold particles over extracellular areas was used to normalize for different background density in different sections and different immunohistochemical runs.

Rats, anesthetized with intraperitoneal injection of ketamine (40 mg/kg) and xylazine (4 mg/kg), were decapitated and the heads were transferred to ice-cold DPBS. The TGs were dissected out, separated from the surrounding dura, and transferred to ice-cold HBSS. TGs were then minced with a sterile razor blade, placed in a collagenase solution (5 mg/mL in HBSS; C9891, Sigma-Aldrich), and incubated for 20 min at 37°C, shaking every 5 min. After centrifugation at 1,300 rpm for 5 min, the pellet was dissociated with 1 mL of 0.125% trypsin (15090–046, Gibco, MA), and incubated for 10 min at 37°C. After another centrifugation at 1,300 rpm for 5 min, the pellet was mechanically dissociated with a Pasteur pipette, placed in DMEM containing 10% FBS, and incubated in 5% CO2 incubator at 37°C. The DMEM medium was changed every 3 days. After 3 weeks, cells were plated into a 12-well culture plate at 5×10⁵ cells per well.

Cultured SGCs were tested using a glutamate assay kit (ab83389, Abcam, Cambridge, United Kingdom) according to the manufacturer’s protocol. Briefly, the cells were treated with an extracellular solution (in mM, 140 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 10 glucose) containing either 0.125% DMSO as a vehicle or 5 μM Ionomycin, 200 μM ATP, and 100 μM Bz-ATP, and 50 μL of the solution was harvested into a 96-well plate at 5 min, 15 min, and 30 min post treatment. The absorbance at 450 nm wavelength was measured with a plate reader (iMark, Biorad, CA).

Data were presented as mean ± SD (extracellular glutamate concentration in Figure 1) or mean ± SEM (air-puff threshold in Figure 2 and normalized value of gold particle density in Figure 3). Statistical analysis was performed using SPSS v.21.0 software (SPSS Inc., Chicago, IL). Difference between groups in extracellular glutamate concentration and in air-puff threshold was examined using two-way ANOVA, followed by Bonferroni post-hoc test. Even though sample size for analysis of extracellular glutamate concentration (in Figure 1) is small (n = 3 per group) power analysis showed 90% power at p < 0.05. Difference between groups in normalized value of gold particle density was examined using unpaired student t-test: Interanimal variability in normalized value of gold particle density within the same group was insignificant (one-way ANOVA), and the data could be pooled per group for analysis. Values of p < 0.05 were considered statistically significant. The correlation of glutamate density in each neuron to that in its enveloping SGC was studied using Pearson correlation analysis.

In the trigeminal SGCs culture, the level of extracellular glutamate increased significantly 15–30 min after treatment with the calcium-ionophore ionomycin (vehicle vs. ionomycin: 3.87 ± 1.22 vs. 14.67 ± 1.97 nmoL/mL, p = 0.001 at 15 min, 5.07 ± 0.46 vs. 22.00 ± 1.44 nmoL/mL, p < 0.001 at 30 min after treatment with ionomycin. Values are mean ± SD, two-way ANOVA), ATP (vehicle vs. ATP: 5.60 ± 0.69 vs. 8.40 ± 0.40 nmoL/mL, p = 0.004 at 30 min after ATP treatment), and Bz-ATP (vehicle vs. Bz-ATP: 4.13 ± 0.92 vs. 7.60 ± 0.69 nmoL/mL, p = 0.007 at 15 min, 3.60 ± 0.40 vs. 10.27 ± 0.46 nmoL/mL, p < 0.001 at 30 min after Bz-ATP treatment), suggesting that SGCs, when stimulated, release glutamate (Figure 1).

Behavioral analysis demonstrated that chronic constriction injury of the infraorbital nerve (CCI-ION) produces prolonged mechanical allodynia in the facial area innervated by the infraorbital nerve (whisker pad). Sham-operated rats rarely showed alteration in air-puff thresholds. However, CCI-ION rats showed significant decrease in air-puff threshold on day 13 (sham vs. CCI-ION: 40.00 ± 0.00 vs. 19.17 ± 6.51 psi, p = 0.015, Values are mean ± SEM, two-way ANOVA), which persisted until day 22 (sham vs. CCI-ION: 40.00 ± 0.00 vs. 5.25 ± 0.31 psi, p < 0.001), when mechanical allodynia was most prominent, and when we sacrificed the animals for analysis of glutamate level in trigeminal neurons and their enveloping SGCs (Figure 2).

Since its introduction more than 30 years ago, the method of assessing the concentration of glutamate in the CNS by determining the density of immunogold particles in cellular and subcellular structures, such as cell bodies and axon terminals, has been applied successfully by many, including our laboratory (Ottersen, 1989a, b; Shupliakov et al., 1992; Paik et al., 2021). Moreover, since the cytoplasm of SGCs is more electron-dense than that of neurons, it is also possible to distinguish the two cell types and compare the concentration of glutamate in them to that in the extracellular matrix.

In SGCs that envelop neurons of all sizes (small, medium-sized, and large), the normalized values of the density of gold particles coding for glutamate was significantly higher in the CCI-ION group than in the control group (sham vs. CCI: 4.80 ± 0.55 vs. 9.32 ± 1.11, p = 0.002 in SGCs enveloping small soma, 4.86 ± 0.34 vs. 8.45 ± 0.75, p < 0.001 in SGCs enveloping medium-sized soma, 4.36 ± 0.49 vs. 7.57 ± 0.70, p = 0.001 in SGCs enveloping large soma, unpaired student t-test. Values are mean ± SEM, Figures 3,4). Also in neurons of all sizes, the normalized values of the gold particle density was significantly higher in the CCI-ION group than in controls (sham vs. CCI: 11.07 ± 1.05 vs. 20.61 ± 1.81, p < 0.001 in small soma, 11.88 ± 0.89 vs. 22.37 ± 1.71, p < 0.001 in medium-sized soma, 13.39 ± 0.85 vs. 22.69 ± 2.14, p = 0.001 in large soma, unpaired student t-test. Values are mean ± SEM, Figures 3,4). The density of gold particles in SGCs was positively correlated with that in the neurons they envelop in both CCI-ION and control groups (correlation coefficient r = 0.7778 in sham group, r = 0.7627 in CCI-ION group, Pearson correlation analysis; Figure 4).

Immunofluorescent staining for SP or CGRP alone or in combination with Piezo1 revealed frequent close apposition between small SP+ or CGRP+ (likely nociceptive), and between large Piezo1+ (likely non-nociceptive, mechanosensitive) neurons and small SP+ or CGRP+ neurons (Figures 5A–I). Electron microscopic examination of serial sections showed that, in some parts of the interface between SGCs of nearby neurons, they were only separated by a ~ 50 nm wide gap, similar to the synaptic cleft of the neuromuscular junction (Gabbiani and Cox, 2010). In addition, closely apposed neurons sharing a single SGC were also frequently observed (Figures 5J–M).