Identification of sensory fiber types in mouse temporomandibular joint tissues

Alfaro, Jessie J.; Wang, Exing; Akopian, Armen N.

doi:10.1038/s41598-025-17508-x

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Published: 01 September 2025

Identification of sensory fiber types in mouse temporomandibular joint tissues

Jessie J. Alfaro¹,
Exing Wang²,
R. E.-JOIN Consortium Investigators &
…
Armen N. Akopian^1,3,4

Scientific Reports volume 15, Article number: 32210 (2025) Cite this article

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Abstract

Temporomandibular joint (TMJ) disorders (TMJDs) are linked to heightened nerve sensitivity in TMJ tissues. To set the groundwork for investigating the mechanisms governing this increased responsiveness, this study aimed to identify the types of nerves in the retrodiscal tissue (retrodisc), anterior disc, and joint capsule of mouse TMJ using immunohistochemistry (IHC) and reporter mice. The pan-sensory neuronal marker pgp9.5 revealed no nerves in the articular disc but identified approximately 70% unmyelinated and 30% myelinated fibers in other TMJ tissues. Nearly all sensory fibers in the joint capsule and anterior disc were CGRP⁺ peptidergic fibers, while the retrodisc contained about 80% peptidergic fibers. Notably, CGRP^-/NFH⁺ myelinated non-peptidergic nerves were absent, indicating the absence of non-nociceptive fibers (A-LTMRs) in TMJ tissues. Almost all sensory fibers in the joint capsule and anterior disc were Htr3a⁺, with the retrodisc containing 60–70% Htr3a⁺ fibers. Additionally, TMJ tissues had minimal to no (< 5%) MrgprD⁺, MrgprA3⁺, MrgprC11⁺, somatostatin⁺, or parvalbumin⁺ fibers, except for the retrodisc, which had about 20% Mrgpr⁺ fibers. Excluding articular discs, TMJ tissues were highly vascularized, with blood vessels surrounded by both sensory and sympathetic (TH⁺) nerves. Overall, TMJ tissues were predominantly innervated by peptidergic fibers, with a minor presence of other non-peptidergic fibers.

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Introduction

Temporomandibular joint disorders (TMJDs) represent a diverse group of conditions that disrupt the normal function of the temporomandibular joint (TMJ)^1,2. These disorders can present in both painful and non-painful forms, with pain often being the most debilitating and difficult symptom to manage². Painful TMJDs can be further classified into subtypes such as osteoarthritis, rheumatoid arthritis, and idiopathic disorders^3,4. Each subtype is characterized by distinct pathophysiological mechanisms, which contribute to alterations in the sensory processing within the joint^2,3. This understanding highlights the critical importance of characterizing the sensory innervation of the TMJ. These nerves transmit nociceptive signals that may be altered or amplified in TMJDs, contributing to persistent orofacial pain.

The joint receives its innervation primarily from branches of the mandibular division (V3) of the trigeminal nerve. Specifically, the auriculotemporal nerve (ATN), the masseteric nerve (MN), and the anterior deep temporal nerve are the principal contributors to TMJ sensory input⁵. Prior studies indicate a predominance of C- and A-fiber nociceptors in TMJ tissues, with TMJ afferents expressing neuropeptides such as calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY), and nitric oxide synthase (NOS)^6,7,8,9. CGRP-positive nerves were found in the capsule and the synovial membrane, but not in the articular disc of sheep^10,11. In vivo extracellular recordings from TMJ structures, ATN single nerve preparations, demonstrated that TMJ nerves were classified into the following four subtypes: Adelta-high-threshold mechanonociceptor (Aδ-HTMR) (12.1%), Adelta-polymodal nociceptor (Aδ-POLY) (36.4%), C-HTMR (12.1%), and C-POLY (39.4%)^12,13. Overall, despite accumulated data on sensory neurons innervating TMJ, there is limited information regarding the precise make-up of neuronal subtypes innervating the TMJ tissues, such as the joint capsule and associated synovium, articular disc, anterior disc, and retrodisc.

The development of single-cell RNA sequencing technology opened avenues for precise identifications of sensory nerve subtypes in tissues using IHC with well-defined antibody markers and reporter mouse lines highlighting expressions of sensory neuronal markers²⁰. Accordingly, the main aim of this study was to identify nerve subtypes in each TMJ tissue using IHC and reporter mouse lines.

Results

TMJ tissues examined in this study

TMJ tissues exhibit distinct functions and pathophysiological profiles, and are believed to undergo unique nerve plasticity, gene expression changes and non-neuronal cellular plasticity during TMJD. Therefore, this study focused on characterizing the innervation and vascularization of individual TMJ structures^8,14,15. We generated serial cryosections that sequentially passed through the mandible, temporal bone, and a thin layer of the joint capsule, which contains synovial tissue in its deeper layers (as shown on the schematic; Fig. 1A). As the sections progressed, they encompassed the lateral pterygoid muscle (LPM), condyle, anterior disc, articular disc, and retrodiscal tissue (aka retrodisc) (as shown on the schematic; Fig. 1B). The morphology of sections passing through the joint capsule was illustrated on a H&E labeled paraffin section (Fig. 1C). The morphology of sections containing condyle, articular disc, anterior disc, and retrodiscal tissue was illustrated on a deeper H&E labeled paraffin section (Fig. 1D). Innervation and vascularization were visualized and quantitatively analyzed in cryo sections containing the joint capsule (Fig. 1E), and in the deeper TMJ sections containing condyle, articular disc, anterior disc and retrodisc (Fig. 1F). After visual analysis of many sections, it remains unclear whether the neurovascular elements observed in the anterior disc and retrodisc extend into and are functionally present within the articular disc itself. Thus, some portions of retrodiscal tissue can overlay the articular disc (Fig. 1D). Therefore, innervation and vascularization were not associated to the articular disc due to inconclusive evidence of nerves or blood vessels in this TMJ tissue.

Myelinated and unmyelinated sensory afferents in TMJ tissues

All sensory nerves were identified using the pan-neuronal marker pgp9.5, while myelinated fibers were specifically labeled with NFH antibodies^16,17. We did not reliably detect pgp9.5 + nerves in the articular disc (Suppl. Figure 1). Myelinated nerves (pgp9.5⁺/NFH⁺) were 28.1 ± 7.6% (n = 5) of pgp9.5 in the joint capsule; 30.3 ± 6.1% (n = 6) of pgp9.5 in the anterior disc and 30.2 ± 12% (n = 6) of pgp9.5 in the retrodisc (Figs. 2A, 2B). Remaining pgp9.5⁺ fibers, around 70%, in these TMJ tissues were un-myelinated (pgp9.5⁺/NFH⁻) (Figs. 2A, 2B). This pattern indicates significantly higher innervation by unmyelinated fibers across these TMJ tissues (2-way ANOVA; F (1, 28) = 29.72; P < 0.0001; n = 5–6; Fig. 2B). Overall, in contrast to masticatory muscles, which typically exhibit a higher proportion of myelinated fibers^16,17, TMJ structures were predominantly innervated by unmyelinated sensory nerves. The relative proportions of myelinated and unmyelinated fibers were consistent across the joint capsule, anterior disc, and retrodisc.

Peptidergic and Htr3a-positive sensory afferents in TMJ tissues

We next assessed the presence of CGRP⁺ peptidergic nerves in TMJ tissues. The joint capsule (95.9 ± 16.6%; n = 5) and anterior disc (98.9 ± 12.4%; n = 5) were almost entirely populated by peptidergic sensory fibers (Figs. 3A, 3B), while the retrodiscal tissue contained ≈75% CGRP⁺/pgp9.5⁺ nerves (75.4 ± 9.9%; n = 5; Figs. 3A, 3B). Further analysis using CGRP reporter mice and co-labeling with CGRP and NFH antibodies confirmed that roughly 70% of peptidergic fibers were unmyelinated (73.4 ± 2.3%; n = 5 for joint capsule; 70.3 ± 2.4%; n = 5 for anterior disc; 71.6 ± 2.6%; n = 5 for retrodisc), with the remaining ≈30% myelinated (Figs. 4A, 4B). Notably, TMJ tissues showed minimal-to-no (< 5%) CGRP⁻/NFH⁺ fibers (0.8 ± 0.6%; n = 5 for joint capsule; 6.9 ± 3.4%; n = 5 for anterior disc; 2.9 ± 2.9%; n = 5 for retrodisc), non-peptidergic myelinated sensory nerves typically classified as non-nociceptive Aβ low-threshold mechanoreceptors (A-LTMRs)^11,18,19.

Given that several peptidergic fiber subtypes express Htr3a¹⁷, we examined this further using Htr3a/tdTomato reporter mice. In the joint capsule (99.9 ± 13.6%; n = 6) and anterior disc (90.3 ± 14.1%; n = 5), Htr3a⁺ fibers almost completely overlapped with pgp9.5⁺ nerves, whereas the retrodisc (46.6 ± 5.5%; n = 4) showed only ≈45% overlap (Figs. 5A, 5B). As expected, approximately 70% of Htr3a⁺ fibers were unmyelinated (69.7 ± 7.7%; n = 6 for the joint capsule; 68.1 ± 3.0%; n = 5 for the anterior disc; 75.9 ± 0.9%; n = 4 for the retrodisc), with the remainder being myelinated; and with almost no Htr3a⁻/NFH⁺ fibers were detected (Fig. 5C)¹⁷. In summary, nearly all sensory nerves in the joint capsule and anterior disc are peptidergic and co-express Htr3a. In contrast, the retrodisc contains a distinct subset (≈20%) of non-peptidergic, Htr3a⁻ fibers¹⁷, along with some peptidergic fibers that are likely Htr3a⁻.

Presence of non-peptidergic—MrgprD, MrgprA3, MrgprC11, somatostatin and parvalbumin—sensory afferents in TMJ tissues

Our findings suggest that TMJ tissues are predominantly innervated by peptidergic sensory fibers, with widespread expression of Htr3a. To further validate this, we examined the presence of non-peptidergic nerve subtypes, focusing on MrgprD⁺ fibers, which represent the major class of non-peptidergic neurons (NP-1 group) in the dorsal root ganglia (DRG) and trigeminal ganglia (TG)²⁰. Using MrgprD/tdTomato reporter mice, we found that MrgprD⁺ fibers were scarce in the joint capsule (4.1 ± 1.9%; n = 4 and anterior disc (3.7 ± 2.2%; n = 4), and many sections did not contain MrgprD⁺ nerves. In contrast, the retrodisc showed a modestly higher presence of MrgprD⁺ fibers (10.8 ± 3.2%; n = 4; Figs. 6A, 6B). As a positive control, facial skin displayed dense MrgprD⁺ innervation, confirming the sensitivity of our detection method (Suppl. Figure 2). These results reinforce the conclusion that TMJ tissues are mainly innervated by peptidergic neurons, while non-peptidergic MrgprD⁺ fibers are limited, particularly outside the retrodisc.

Another subset of non-peptidergic fibers, known as the NP-2 group, is marked by MrgprA3⁺ expression^18,20. However, some studies have shown that MrgprA3⁺ neurons are heterogeneous, comprising both peptidergic and non-peptidergic subtypes^21,22. In MrgprA3/tdTomato reporter mice, MrgprA3⁺ nerves were detected only sporadically on few sections of the joint capsule (4.4 ± 3.2%; n = 5) and retrodiscal tissues (6.4 ± 3.2%; n = 4), with none observed in the anterior disc (0.6 ± 0.6%; n = 4; Figs. 6B, 7). A broader subset, marked by MrgprC11⁺, includes certain TrpV1⁺ peptidergic neurons²³. In MrgprC11/tdTomato reporter mice, TMJ tissues displayed only a small number of MrgprC11⁺ nerves on few Sects. (3.7 ± 3.7%; n = 4 for the joint capsule; 2.9 ± 3.0%; n = 4 for the anterior disc; 6.8 ± 3.5%; n = 6 for the retrodisc; Figs. 6B, 8). As with MrgprD⁺ fibers, facial skin, used as a positive control, showed significantly greater innervation by both MrgprA3⁺ and MrgprC11⁺ fibers (Suppl. Figure 2). These results further support the conclusion that TMJ tissues contain very few non-peptidergic sensory fibers, with only limited contributions from MrgprA3⁺ and MrgprC11⁺ subtypes.

The final group of non-peptidergic nociceptors in the DRG and TG is marked by somatostatin (Sst⁺), also referred to as the NP-3 cluster^11,20. In Sst/tdTomato reporter mice, Sst⁺ fibers were not detected in the anterior disc (0 ± 0%; n = 5) or retrodisc (0 ± 0%; n = 6), while the joint capsule contained a small proportion of Sst⁺ nerves observed on several Sects. (5.7 ± 3.4%; n = 6; Figs. 6A, 9). The dura mater was used as a positive control and showed robust Sst⁺ innervation (Suppl. Figure 2), validating the specificity of our detection. Overall, TMJ tissues exhibited very low levels of non-peptidergic innervation. Among the TMJ regions analyzed, the retrodisc consistently contained the highest proportion of non-peptidergic sensory fibers.

Non-peptidergic, non-nociceptive nerves are typically classified as low-threshold mechanoreceptors (LTMRs), which include C-, Aδ-, and Aβ-LTMR subtypes^18,20. These subtypes are commonly marked by expression of vGlut3 or tyrosine hydroxylase (TH) for C-LTMRs, TrkB for Aδ-LTMRs, and TrkC or parvalbumin (PV) for Aβ-LTMRs²⁰. While both mouse and primate TG contain few TH⁺ neurons, they show a high prevalence of PV⁺ neurons^16,17. Similarly, facial skin, used here as a positive control, exhibited abundant PV⁺ nerve fibers (Suppl. Figure 2). To assess the presence of LTMRs in TMJ tissues, we analyzed PV/tdTomato reporter mice. No PV⁺ nerve fibers were detected in the joint capsule, anterior disc, or retrodisc, despite 6–12 sections were scanned for each tissue (Figs. 6B, 10). In summary, TMJ tissues contain very few, if any, non-peptidergic nociceptors or non-nociceptive mechanoreceptors. Among the TMJ regions examined, the retrodisc consistently exhibited the highest proportion of non-peptidergic nociceptors, accounting for approximately 15–20% of the total innervation.

Innervation of vasculature in TMJ tissues

Using α-SMA as a marker for vascular smooth muscle, we clearly identified blood vessels in all TMJ tissues except the articular disc (Figs. 11A, B, 12) , Suppl. Figure 3). To determine whether these vessels were innervated by sympathetic or sensory nerves, we used tyrosine hydroxylase (TH) to label sympathetic fibers and pgp9.5 as a pan-sensory nerve marker. Both sensory and sympathetic nerves were detected in the joint capsule, anterior disc, and retrodisc, but not in the articular disc (Figs. 11A, 12, Suppl. Figure 1, Suppl. Figure 3). Nerves were considered to innervate blood vessels if they overlapped or were located within 50 µm of α-SMA⁺ vessels. Approximately 40% of all TH⁺ sympathetic nerves of the entire TMJ were found in close association with blood vessels (Figs. 11A, B), while sensory pgp9.5⁺ fibers showed a slightly lower association at around 35% (Figs. 11B, 12). Overall, these findings indicate that TMJ tissues, excluding the articular disc, are well vascularized and receive comparable innervation from both sensory and sympathetic nerves. The joint capsule, anterior disc, and retrodisc displayed similar patterns of neurovascular pattern.

Discussion

Studies of painful joint pathologies in the knee have demonstrated that gene expression and cellular plasticity within the synovium play a critical role in the development of hypersensitivity and the persistence of chronic pain^24,25,26. Both neuronal and non-neuronal cell plasticity in the knee synovium contribute significantly to the mechanisms underlying chronic pain²⁷. In the TMJ, the synovium is distributed across three major anatomical components: the joint capsule, the anterior disc, and the retrodiscal tissue²⁸. However, it remains unclear whether these distinct synovial regions of the TMJ possess different cellular compositions or patterns of innervation. Therefore, the rationale for this study was to characterize the types and distribution of nerves innervating specific regions of the TMJ; namely, the joint capsule, the articular disc, the anterior disc, and the retrodiscal tissue. A detailed understanding of the regional innervation patterns may provide insights into the mechanisms of TMJ pain and guide the development of targeted therapies for painful TMJ disorders.

We found that TMJ tissues are primarily innervated by peptidergic unmyelinated sensory fibers, with the remaining fibers consisting largely of peptidergic myelinated afferents. These results are consistent with previous anatomical and electrophysiological studies identifying various neuropeptides in TMJ-innervating nerves^7,8,9,10 and classifying them as C- and A-fiber nociceptors^12,13. While prior research mainly focused on the joint capsule^7,8,9,10, our study expanded the anatomical mapping to include the anterior disc and retrodisc. Although the overall innervation pattern was similar across regions, key differences were observed. The retrodisc contained approximately 20% non-peptidergic fibers, including MrgprD⁺, MrgprA3⁺, and MrgprC11⁺ subtypes, fiber types largely absent in other TMJ regions. In contrast, the joint capsule uniquely harbored Sst⁺ fibers. A further distinguishing feature was the expression of Htr3a: nearly all fibers in the joint capsule and anterior disc expressed Htr3a, whereas only about 50% of retrodisc fibers did. These differences suggest that the retrodisc is innervated by a more diverse and molecularly distinct population of sensory neurons.

Electrophysiological studies revealed that TMJ is innervated by C- and A-fiber nociceptors, which could be classified into two main functional subtypes: polymodal nociceptors and high-threshold mechanoreceptors (HTMRs)^12,13. Building on this, recent integrative analyses of single-cell RNA sequencing data from mouse and human trigeminal ganglia (TG) have identified several molecularly distinct subtypes of peptidergic nociceptors²⁰. Among C-fiber nociceptors, these subtypes include Oprk1⁺, Adra2a⁺, Sstr2⁺, and Dcn⁺ neurons. A-fiber nociceptors are categorized primarily as Smr2⁺ and Bmpr1b⁺ neurons²⁰. Both Smr2⁺ and Bmpr1b⁺ A-nociceptors express Htr3a, while only the Oprk1⁺ and Dcn⁺ subtypes among C-fiber nociceptors express this receptor²⁰. In light of our findings, the retrodisc, where only ~ 50% of sensory fibers are Htr3a⁺, may contain the full range of peptidergic C- and A-nociceptor subtypes. Conversely, the joint capsule and anterior disc, which show near-complete Htr3a expression among their sensory fibers, are likely innervated mainly by Oprk1⁺ and Dcn⁺ C-fiber nociceptors. These differences point to region-specific specialization in nociceptive innervation within TMJ tissues. A proposed summary of nociceptor subtype distribution across TMJ structures is illustrated in Fig. 13.

One potential limitation of this study is utilized solely female mice for MrgprA3-tdTomato and solely male mice for Htr3a/tdTom, CGRP/tdTom, MrgprD/tdTom, MrgC11/tdTom, and α-SMA. Sst/tdTom consisted of male and female mice. Prior studies utilizing bulk and single-cell RNA sequencing have identified some transcriptomic differences between male and female DRG and TG neurons^11,20,29,30. Although these differences were generally modest within neuronal populations, notable sex-specific distinctions were observed, particularly in Aβ-rapid adapting (Aβ-RA) and Aδ-low threshold mechanoreceptors (Aδ-LTMRs) in the naïve state³¹. Thus, we do not anticipate sex differences in innervations of TMJ tissues, since they do not contain Aβ-RA and Aδ-LTMRs. Another limitation lies in the manual counting of nerve fibers from IHC images. This method does not distinguish between individual nerve branches and closely adjacent fibers, potentially leading to over- or underestimation. Additionally, we did not use specific markers for neuronal terminals, raising the possibility that some counted fibers were merely passing through the tissue rather than terminating within it. Another potential limitation of this study is the exclusive use of conventional two-dimensional IHC for analyzing TMJ innervation. While this method is well-established and effective, it does not capture the full complexity of the 3D neural architecture. Advanced imaging techniques such as light sheet fluorescence microscopy could provide additional spatial information and more comprehensive quantification of nerve distribution within the TMJ. However, the primary objective of this study was to identify and characterize the types of sensory neurons innervating the TMJ. For this purpose, two-dimensional IHC imaging represents a practical and cost-effective approach, particularly suitable for high-throughput analysis across multiple tissue samples. It allows for reliable detection of nerve fiber subtypes while maintaining efficiency in data collection and analysis. Nonetheless, light sheet microscopy offers distinct advantages in visualizing 3D nerve architecture and would be especially valuable in follow-up studies aimed at exploring nerve remodeling and plasticity in various mouse models of TMJ disorders.

In conclusion, despite certain limitations, the data presented in this study reliably reveal key patterns of innervation and establish a foundational map of sensory nerve subtypes within the TMJ. The identification of both predominant and less common nerve fiber types across distinct TMJ tissues holds important implications, as different nerve types possess unique transcriptional signatures, biochemical characteristics, and functional responses to environmental cues. These differences underscore the diverse roles that specific nerve populations play in both normal joint physiology and pathological conditions. For example, nociceptive and non-nociceptive neurons may exhibit differential responses to tissue injury, while peptidergic and non-peptidergic fibers are likely to contribute to pain signaling through separate mechanisms. Understanding these distinctions is a central goal in the field of pain research and is essential for the development of precise, targeted therapeutic strategies for managing TMJ-related disorders.

Materials and methods

Ethical approval

This study was conducted in accordance with the ARRIVE 2.0 guidelines³². All animal care and experimental procedures complied with the U.S. Public Health Service Policy on the Use of Laboratory Animals, as well as ethical standards set by the National Institutes of Health (NIH) and the Society for Neuroscience (SfN), with a commitment to minimizing both animal use and suffering. Mice were euthanized via transcardiac perfusion following intramuscular injection of 100 μL of a 1:1 Ketamine/Dexdormitor cocktail. This method, recommended by the AVMA Guidelines for the Euthanasia of Animals, was chosen to ensure minimal distress. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas Health Science Center at San Antonio (UTHSCSA) under protocol number 20220069AR.

Mouse lines

Mice were housed under controlled conditions at approximately 22 °C, with 40–60% relative humidity, and maintained on a 12-h light/dark cycle (lights on at 7:00 AM). Food and water were provided ad libitum in standard home cages. Experiments were performed on adult wild-type C57Bl/6 mice (10–23 weeks old) obtained from The Jackson Laboratory (Bar Harbor, ME). The following transgenic mouse lines were purchased from The Jackson Laboratory: B6.Cg-Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze/J (Ai9; tdTomato; strain 007,909); B6.129P2-Pvalbtm1(cre)Arbr/J (PV^cre; strain 008,069); Mrgprd^{tm1.1(cre/ERT2)Wql}/J (MrgprD^cre/ER; strain 031,286), and B6N.Cg-Sst^{tm2.1(cre)Zjh}/J (Sst^Cre; stock 013,044). The CGRP^cre/ER mouse line was generously provided by Dr. Pao-Tien Chuang (University of California, San Francisco), and the MrgprA3^cre and MrgprC11^cre/ERT2 lines by Dr. Liang Han (Georgia Institute of Technology). The Htr3a^cre line was developed by the Mutant Mouse Resource & Research Centers (MMRRC) at UC Davis. In inducible Cre-carrying lines, Cre recombination was induced in 6–8-week-old mice via three intraperitoneal injections of tamoxifen (100 mg/kg in corn oil), administered every other day. Recombination was expected to occur within two to three weeks post-injection.

Tissue collection and processing

All mice were deeply anesthetized via intramuscular injection of 100 μL of a ketamine (75 mg/kg) and dexdomitor (1 mg/kg) cocktail, then transcardially perfused with 25 mL of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). Following perfusion, mice were decapitated, and the masseter and temporalis muscles were carefully removed. Bilateral TMJs, along with surrounding bone structures, were then dissected. Facial skin overlying the masseter and TMJ, as well as the TG and dura mater, were collected as positive controls for tdTomato fluorescence in afferent fibers. Dissected TMJs were post-fixed overnight in 4% PFA at 4 °C. The next day, tissues were rinsed twice in 1X PB and transferred to 15 mL of 10% ethylenediaminetetraacetic acid (EDTA; ThermoFisher, Cat# 17,892) in ddH₂O, adjusted to pH 7.2–7.4. Decalcification in EDTA was carried out over two weeks, with solution changes every three days. After decalcification, tissues were cryoprotected in 10% sucrose for 18 h, followed by 30% sucrose for another 18 h. TMJs were then embedded in Neg-50 embedding medium (FisherScientific, Cat# 22–110-617) and cryo-sectioned at a thickness of 20 μm.

Immunohistochemistry (IHC)

Immunostaining was performed as previously described^16,33. Briefly, tissue sections were blocked for 90 min at room temperature in a solution containing 4% normal donkey serum (Sigma, St. Louis, MO), 2% bovine gamma-globulin (Sigma-Aldrich, St. Louis, MO), and 0.3% Triton X-100 (Fisher Scientific) in 0.1 M phosphate-buffered saline (PBS). Following blocking, sections were incubated overnight (18 h) at room temperature with primary antibodies. After incubation, sections were washed with 0.1 M PBS to remove unbound primary antibodies, re-blocked, and then incubated for 90 min at room temperature with species-appropriate fluorophore-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA). Sections were then washed three times for five minutes each with 0.1 M PBS, air-dried, and mounted with Vectashield Antifade Mounting Medium (Vector Laboratories, SKU H-1000–10). The following well-characterized primary antibodies were used on mouse tissue sections: anti-neurofilament heavy chain (NFH) chicken polyclonal antibodies (BioLegend, catalog #PCK-592P, 1:300)³⁴; anti-pgp9.5 (Millipore-Sigma, catalog #AB1761-I, 1:400)³⁵; anti-CGRP rabbit polyclonal (Sigma, C8198, 1:300)^36,37,38; anti-tyrosine hydroxylase (TH) rabbit polyclonal (Pel-Freez; Rogers, AR; P40101; 1:400)^39,40; and anti-smooth muscle actin (α-SMA) Cy3-conjugated mouse monoclonal antibody (Sigma, C6198, 1:200)^16,41. Secondary antibodies were generated in donkey (Jackson Immuno-Research).

Counting and imaging of fibers

Images were acquired using a Keyence BZ-X810 all-in-one microscope (Itasca, IL, USA) or a Nikon AX confocal microscope with Z-stack (“sectioning”) functionality. For nerve fiber counting, all images were captured using a 10 × objective. Representative images in figures are cropped versions of 10 × images to increase visibility and resolution. Control IHC was conducted on tissue sections processed identically, but either lacking primary antibodies or both primary and secondary antibodies. Imaging settings were calibrated to ensure that these negative controls produced no detectable nerve fiber signal. Z-stack images for fiber quantification were obtained. For each condition, at least 3 independent tissue sections were analyzed. Actual numbers are presented in the legends for figures. Sensory fiber quantification was performed manually, following previously established protocols¹⁶, to estimate the distribution and density of nerve subtypes innervating TMJ tissues. The rationale and validation for manual fiber counting as a reliable method for detecting and quantifying peripheral nerve fibers have been previously described^16,41. Each quantification analysis had a reference point, which was either pgp9.5 (all sensory nerves) or NFH (all myelinated A-fibers). This reference points (defined as 100%) were based on the mean number of pgp9.5⁺ or NFH⁺ fibers, which were obtained by counting pgp9.5⁺ and NFH⁺ fibers from all sections for each specific TMJ tissue type. This normalization approach was intentionally designed to illustrate inter-animal variability and heterogeneity in nerve innervation.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA). Data are presented as mean ± standard error of the mean (SEM), with “n” indicating the number of mice analyzed for IHC. For each mouse, fibers were counted from at least 5 randomly selected sections and then the counting was averaged. Group differences were evaluated using appropriate statistical tests, including chi-square analysis with Fisher’s exact test, unpaired t-tests, and one-way or two-way ANOVA followed by Bonferroni’s post hoc test, as applicable. A p-value of < 0.05 was considered statistically significant. Where relevant, interaction F-ratios and corresponding p-values are reported.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

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Acknowledgements

We would like to thank Miss. Anahit Hovhannisyan for guidance on IHC. We are grateful to Dr. Pao-Tien Chuang (UC San Francisco, San Francisco, CA) for kindly providing the CGRPcre-ER mouse line; Dr. Liang Han (College of Sciences, Georgia Tech, Atalanta, GA) for kindly providing MrgprA3cre and MrgprC11cre/ER mouse lines generated in Dr. Xinzhong Dong’s laboratory (Johns Hopkins University, Baltimore, MD) and Dr. Liang Han’s laboratory, respectively; and MMRRC at UC Davis for preparing Htr3acre mouse line.

Funding

This research work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health (NIH/NIAMS) through the NIH HEAL (https://heal.nih.gov/) Initiative the Restoring Joint Health and Function to Reduce Pain (RE-JOIN) Consortium UC2 AR082195 (to A.N.A.) and by the National Institute of Dental and Craniofacial Research (NIH/NIDCR) training CO-STAR grant T32 DE014318 (to J.J.A.).

Author information

A list of authors and their affiliations appears at the end of the paper.

Authors and Affiliations

Integrated Biomedical Sciences (IBMS) Program, The School of Medicine, The University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, 78229, USA
Jessie J. Alfaro & Armen N. Akopian
Department of Cell Systems and Anatomy, The School of Medicine, The University of Texas Health Science Center at San Antonio (UTHSCSA), San Antonio, TX, 78229, USA
Exing Wang
Center for Pain Therapeutics and Addiction Research, The School of Dentistry, The University of Texas Health Science Center at San Antonio (UTHSCSA), 7703 Floyd Curl Drive, San Antonio, TX, 78229-3900, USA
Yufang Jin, Zhao Lai, Lindsey Macpherson, Alexei Tumanov, Yi Zou & Armen N. Akopian
Departments of Cellular and Integrative Physiology, The School of Medicine, UTHSCSA, San Antonio, TX, 78229, USA
Armen N. Akopian
The University of Florida, Gainesville, FL, 32611, USA
Kyle Allen, Basak Ayaz, Bruna Balbino de Paula, Robert Caudle, Yenisel Cruz-Almeida, M.Franklin Dolwick, Danielle Freburg-Hoffmeister, Janak Gaire, Priya Kulkarni, Abbas Muhammad, Kevin Otto, Folly Patterson, Shane Priester, Hernan Prieto, Margarete Ribeiro Dasilva, Kaitlin Southern, Michael Strinden & Airam Vivanco-Estela
The University of Pittsburgh, Pittsburgh, PA, 15260, USA
Alejandro Almarza, Mairobys Soccorro Gonzalez & Joshua Stover
Baylor College of Medicine, Houston, TX, 77030, USA
Benjamin Arenkiel, Yangjin Bae, Dellina Lane Carpio, Racel Cela, Ibdanelo Cortez, Zelong Dou, Nele Haelterman, Zixue Jin, Brendan Lee, Camilla Majano, Russell Ray, Oscar Ruiz, Luis Tovias Sanchez, Kim Worley & Jiansen Yan
The University of California San Diego, La Jolla, CA, 92093, USA
Anita Bandrowski, Maryann Martone & Marlena Pela
Wake Forest Medical School, Winston-Salem, NC, 27101, USA
Mario Danilo Boada
The University of Pennsylvania, Philadelphia, PA, 19104, USA
Jacqueline Boccanfuso & Joost Wagenaar
Michigan Neuroscience Institute, The University of Michigan, Ann Arbor, MI, 48109-2800, USA
Jyl Boline & Dawen Cai
The University of California – Irvine, Irvine, CA, 92697, USA
Rui Chen
Duke University, Durham, NC, 27708, USA
Yong Chen, Chris Donnelly, Benjamin Goolsby, Sudhish Jayachandran, Alexus Roberts & Shad Smith
The University of Michigan, Ann Arbor, MI, 48109, USA
Brian Constantinescu, Joshua Emrick, Akash Gandhi, Stacey Greene, Tristan Maerz, Michael Newton, Sienna Perry & Elizabeth Ronan
Karolinska Institute, Stockholm, Sweden
Malin Ernberg
Rush University, Chicago, IL, 60612, USA
Spencer Fullam, Shingo Ishihara, Frank Ko, Jun Li, Anne-Marie Malfait, Rachel Miller, Alia Obeidat & Lai Wang
National Institutes of Health (NIH), Bethesda, MD, 20892, USA
Michael Iadarola
The University of Virginia, Charlottesville, VA, 22903, USA
Azeez Ishola & Joshua Wythe
Emory University, Atlanta, GA, 30322, USA
Yona Levites
Scripps Research, La Jolla, CA, 92037, USA
Martin Lotz, Merissa Olmer & Hannah Swahn
Hospital of Special Surgery, New York, NY, 10021, USA
Bella Mehta & Miguel Otero
Northwestern University, Evanston, IL, 60208, USA
Richard Miller & Dongjun Ren
The Rockefeller University, New York, NY, 10065, USA
Dana Orange
The University of Texas at Dallas, Richardson, TX, 75080, USA
Theodore Price
Rock Maple Science LLC, Hinesburg, VT, 05461, USA
Sue Tappan

Authors

Jessie J. Alfaro
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Exing Wang
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Armen N. Akopian
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Consortia

R. E.-JOIN Consortium Investigators

Kyle Allen
, Kyle Allen
, Alejandro Almarza
, Benjamin Arenkiel
, Basak Ayaz
, Yangjin Bae
, Bruna Balbino de Paula
, Anita Bandrowski
, Mario Danilo Boada
, Jacqueline Boccanfuso
, Jyl Boline
, Dellina Lane Carpio
, Dawen Cai
, Robert Caudle
, Racel Cela
, Rui Chen
, Yong Chen
, Brian Constantinescu
, Ibdanelo Cortez
, Yenisel Cruz-Almeida
, M.Franklin Dolwick
, Chris Donnelly
, Zelong Dou
, Joshua Emrick
, Malin Ernberg
, Danielle Freburg-Hoffmeister
, Spencer Fullam
, Janak Gaire
, Akash Gandhi
, Benjamin Goolsby
, Stacey Greene
, Nele Haelterman
, Michael Iadarola
, Shingo Ishihara
, Azeez Ishola
, Sudhish Jayachandran
, Yufang Jin
, Zixue Jin
, Frank Ko
, Priya Kulkarni
, Zhao Lai
, Brendan Lee
, Yona Levites
, Jun Li
, Martin Lotz
, Lindsey Macpherson
, Tristan Maerz
, Camilla Majano
, Anne-Marie Malfait
, Maryann Martone
, Bella Mehta
, Rachel Miller
, Richard Miller
, Abbas Muhammad
, Michael Newton
, Alia Obeidat
, Merissa Olmer
, Dana Orange
, Miguel Otero
, Kevin Otto
, Folly Patterson
, Marlena Pela
, Sienna Perry
, Theodore Price
, Shane Priester
, Hernan Prieto
, Russell Ray
, Dongjun Ren
, Margarete Ribeiro Dasilva
, Alexus Roberts
, Elizabeth Ronan
, Oscar Ruiz
, Shad Smith
, Mairobys Soccorro Gonzalez
, Kaitlin Southern
, Joshua Stover
, Michael Strinden
, Hannah Swahn
, Sue Tappan
, Luis Tovias Sanchez
, Alexei Tumanov
, Airam Vivanco-Estela
, Joost Wagenaar
, Exing Wang
, Lai Wang
, Kim Worley
, Joshua Wythe
, Jiansen Yan
& Yi Zou

Contributions

J.J.A. and E.W.: methodology, investigation, visualization. J.J.A., E.W., and A.N.A.: analysis, conceptualization. J.J.A. and A.N.A.: research design. J.J.A., E.W., RE-JOIN and A.N.A.: discussion of results. J.J.A. and A.N.A: drafted the manuscript. J.J.A., E.W., RE-JOIN and A.N.A: prepared final version of the manuscript. A.N.A.: resources, supervision, funding acquisition. All authors reviewed the final version of the manuscript.

Corresponding author

Correspondence to Armen N. Akopian.

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Competing interests

The authors declare no competing interests.

Ethical approval

The reporting in the manuscript follows the recommendations in the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). All experimental protocols were approved by the UTHSCSA IACUC committee. Protocol numbers is 20220069AR.

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Alfaro, J.J., Wang, E., R. E.-JOIN Consortium Investigators. et al. Identification of sensory fiber types in mouse temporomandibular joint tissues. Sci Rep 15, 32210 (2025). https://doi.org/10.1038/s41598-025-17508-x

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Received: 20 May 2025
Accepted: 25 August 2025
Published: 01 September 2025
DOI: https://doi.org/10.1038/s41598-025-17508-x

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Abstract

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Introduction

Results

TMJ tissues examined in this study

Myelinated and unmyelinated sensory afferents in TMJ tissues

Peptidergic and Htr3a-positive sensory afferents in TMJ tissues

Presence of non-peptidergic—MrgprD, MrgprA3, MrgprC11, somatostatin and parvalbumin—sensory afferents in TMJ tissues

Innervation of vasculature in TMJ tissues

Discussion

Materials and methods

Ethical approval

Mouse lines

Tissue collection and processing

Immunohistochemistry (IHC)

Counting and imaging of fibers

Statistical analyses

Data availability

References

Acknowledgements

Funding

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Authors and Affiliations

Consortia

R. E.-JOIN Consortium Investigators

Contributions

Corresponding author

Ethics declarations

Competing interests

Ethical approval

Additional information

Publisher’s note

Supplementary Information

Supplementary Information.

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