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Biological processes linked to intramuscular inflammation during myogenous temporomandibular disorder (TMDM) are largely unknown. We mimicked this inflammation by intra-masseteric muscle (MM) injections of complete Freund’s adjuvant (CFA) or collagenase type 2 (Col), which emulates tissue damage. CFA triggered mechanical hypersensitivity at 1d post-injection was mainly linked to processes controlling chemotactic activity of monocytes and neutrophils. At 5d post-CFA, when hypersensitivity was resolved, there was minimal inflammation whereas tissue repair processes were pronounced. Low dose Col (0.2U) also produced acute orofacial hypersensitivity that was linked to tissue repair, but not inflammatory processes. High dose Col (10U) triggered prolonged orofacial hypersensitivity with inflammatory processes dominating at 1d post-injection. At pre-resolution time point (6d), tissue repair processes were underway and a significant increase in pro-inflammatory gene expressions compared to 1d post-injection were detected. RNA-seq and flow cytometry showed that immune processes in MM were linked to accumulation of macrophages, natural killer and natural killer T cells, dendritic cells and T-cells. Altogether, CFA and Col treatments induced different immune processes in MM. Importantly, orofacial hypersensitivity resolution was preceded with repairs of muscle cell and extracellular matrix, an elevation in immune system gene expression and accumulation of distinct immune cells in MM.

Pain development and resolution patterns in many diseases are sex-dependent. This study aimed to develop pain models with sex-dependent resolution trajectories, and identify factors linked to resolution of pain in females and males. Using different intra-plantar (i.pl.) treatment protocols with prolactin (PRL), we established models with distinct, sex-dependent patterns for development and resolution of pain. An acute PRL-evoked pain trajectory, in which hypersensitivity is fully resolved within 1 day, showed substantial transcriptional changes after pain-resolution in female and male hindpaws and in the dorsal root ganglia (DRG). This finding supports the notion that pain resolution is an active process. Prolonged treatment with PRL high dose (1μg) evoked mechanical hypersensitivity that resolved within 5–7 days in mice of both sexes and exhibited a pro-inflammatory transcriptional response in the hindpaw, but not DRG, at the time point preceding resolution. Flow cytometry analysis linked pro-inflammatory responses in female hindpaws to macrophages/monocytes, especially CD11b(+)/CD64(+)/MHCII(+) cell accumulation. Prolonged low dose PRL (0.1μg) treatment caused non-resolving mechanical hypersensitivity only in females. This effect was independent of sensory neuronal PRLR and was associated with a lack of immune response in the hindpaw, although many genes underlying tissue damage were affected. We conclude that different i.pl. PRL treatment protocols generates distinct, sex-specific pain hypersensitivity resolution patterns. PRL-induced pain resolution is preceded by a pro-inflammatory macrophage/monocyte-associated response in the hindpaws of mice of both sexes. On the other hand, the absence of a peripheral inflammatory response creates a permissive condition for PRL-induced pain persistency in females.

Objective Migraine is three times more common in women. CGRP plays a critical role in migraine pathology and causes female-specific behavioral responses upon meningeal application. These effects are likely mediated through interactions of CGRP with signaling systems specific to females. Prolactin (PRL) levels have been correlated with migraine attacks. Here, we explore a potential interaction between CGRP and PRL in the meninges. Methods Prolactin, CGRP, and receptor antagonists CGRP8-37 or Δ1-9-G129R-hPRL were administered onto the dura of rodents followed by behavioral testing. Immunohistochemistry was used to examine PRL, CGRP and Prolactin receptor (Prlr) expression within the dura. Electrophysiology on cultured and back-labeled trigeminal ganglia (TG) neurons was used to assess PRL-induced excitability. Finally, the effects of PRL on evoked CGRP release from ex vivo dura were measured. Results We found that dural PRL produced sustained and long-lasting migraine-like behavior in cycling and ovariectomized female, but not male rodents. Prlr was expressed on dural afferent nerves in females with little-to-no presence in males. Consistent with this, PRL increased excitability only in female TG neurons innervating the dura and selectively sensitized CGRP release from female ex vivo dura. We demonstrate crosstalk between PRL and CGRP systems as CGRP8-37 decreases migraine-like responses to dural PRL. Reciprocally, Δ1-9-G129R-hPRL attenuates dural CGRP-induced migraine behaviors. Similarly, Prlr deletion from sensory neurons significantly reduced migraine-like responses to dural CGRP. Interpretation This CGRP-PRL interaction in the meninges is a mechanism by which these peptides could produce female-selective responses and increase the prevalence of migraine in women. This article is protected by copyright. All rights reserved.

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Understanding masseter muscle (MM) innervation is critical for the study of cell-specific mechanisms of pain induced by temporomandibular disorder or after facial surgery. Here, we identified trigeminal (TG) sensory neuronal subtypes (MM TG neurons) innervating masseter muscle fibers, masseteric fascia, tendons, and adjusted tissues. A combination of patch clamp electrophysiology and immunohistochemistry on TG neurons backtraced from reporter mouse MM found 9 distinct subtypes of MM TG neurons. Of these neurons, 24% belonged to non-peptidergic IB-4+/TRPA1- or IB-4+/TRPA1+ groups, while two TRPV1+ small-sized neuronal groups were classified as peptidergic/CGRP+ One small-sized CGRP+ neuronal group had a unique electrophysiological profile and were recorded from Nav1.8- or trkC+ neurons. The remaining CGRP+ neurons were medium-sized, could be divided into Nav1.8-/trkC- and Nav1.8low/trkC+ clusters, and showed large 5HT-induced current. The final two MM TG neuronal groups were trkC+ and had no Nav1.8 and CGRP. Among MM TG neurons, TRPV1+/CGRP- (somatostatin+), tyrosine hydroxylase+ (C-LTMR), TRPM8+, MrgprA3+ or trkB+ (Aδ-LTMR) subtypes have not been detected. Masseteric muscle fibers, tendons and masseteric fascia in mice and the common marmoset, a new world monkey, were exclusively innervated by either CGRP+/NFH+ or CGRP-/NFH+ medium-to-large neurons, which we found using a Nav1.8-YFP reporter, and labeling with CGRP, TRPV1, neurofilament heavy chain (NFH) and pgp9.5 antibodies. These nerves were mainly distributed in tendon and at junctions of deep-middle-superficial parts of MM. Overall, the data presented here demonstrates that MM is innervated by a distinct subset of TG neurons, which have unique characteristics and innervation patterns.Significance StatementIdentification of sensory neuron subtypes innervating masseter muscle (MM) will enable the study of cell-specific mechanisms of masticatory myofascial pain, including temporomandibular disorder and after restorative surgeries involving MM. Combining back tracing from MM, patch-clamp electrophysiology, and immunohistochemistry with sensory neuronal markers on mouse and nonhuman primate tissues, we identified trigeminal neuronal groups innervating MM (MM TG neurons). MM and adjacent tissues are innervated by 9 distinct types of TG neurons, some of which are substantially different from L3-L5 DRG neurons. However, MM fibers, tendon, muscle-tendon junction, and fascia in mice and common marmosets are exclusively innervated by medium-to-large neurons. These neurofilament heavy chain-positive sensory nerve fibers are mainly distributed in tendon and at junctions between deep, middle, and superficial parts of MM.

Sensory neurones exhibit sex-dependent responsiveness to prolactin (PRL). This could contribute to sexual dimorphism in pathological pain conditions. The present study aimed to determine the mechanisms underlying sex-dependent PRL sensitivity in sensory neurones. A quantitative reverse transcriptase-polymerase chain reaction shows that prolactin receptor (Prlr) long and short isoform mRNAs are expressed at comparable levels in female and male mouse dorsal root ganglia (DRG). In Prlrcre/+ ;Rosa26LSL-tDTomato/+ reporter mice, percentages of Prlr+ sensory neurones in female and male DRG are also similar. Characterisation of Prlr+ DRG neurones using immunohistochemistry and electrophysiology revealed that Prlr+ DRG neurones are mainly peptidergic nociceptors in females and males. However, sensory neurone type-dependent expression of Prlr is sex dimorphic. Thus, Prlr+ populations fell into three small- and two medium-large-sized sensory neuronal groups. Prlr+ DRG neurones are predominantly medium-large sized in males and are proportionally more comprised of small-sized sensory neurones in females. Specifically, Prlr+ /IB4+ /CGRP+ neurones are four- to five-fold higher in numbers in female DRG. By contrast, Prlr+ /IB4- /CGRP+ /5HT3a+ /NPYR2- are predominant in male DRG. Prlr+ /IB4- /CGRP- , Prlr+ /IB4- /CGRP+ and Prlr+ /IB4- /CGRP+ /NPYR2+ neurones are evenly encountered in female and male DRG. These differences were confirmed using an independently generated single-cell sequencing dataset. Overall, we propose a novel mechanism by which sensory neurone type-dependent expression of Prlr could explain the unique sex dimorphism in responsiveness of nociceptors to PRL.

Peptidergic sensory neurons play a critical role in nociceptive pathways. To precisely define the function and plasticity of sensory neurons in detail, new tools such as transgenic mouse models are needed. We employed electrophysiology and immunohistochemistry to characterize in detail dorsal root ganglion (DRG) neurons expressing an inducible CGRPcre-ER (CGRP-cre+); and compared them to DRG neurons expressing Nav1.8cre (Nav1.8-cre+), TRPV1cre (TRPV1-cre+) and TRPV1-GFP (V1-GFP+). Tamoxifen effectively induced CGRPcre-ER production in DRG. ≈87% of CGRPcre-ER-expressing neurons were co-labeled CGRP antibody. Three small and two medium-large-sized (5HT3a+/NPY2R- and NPY2R+) neuronal groups with unique electrophysiological profiles were CGRP-cre+. Nav1.8-cre+ neurons were detected in all CGRP-cre+ groups, as well as in 5 additional neuronal groups: MrgprD+/TRPA1-, MrgprD+/TRPA1+, TRPV1+/CGRP-, vGLUT3+ and ≈30% of trkC+ neurons. Differences between TRPV1cre and Nav1.8cre reporters were that unlike TRPV1-cre+, Nav1.8-cre+ expression was detected in non-nociceptive vGLUT3+ and trkC+ populations. Many TRPV1-cre+ neurons did not respond to capsaicin. In contrast, V1-GFP+ neurons were in 4 groups, each of which was capsaicin-sensitive. Finally, none of the analyzed reporter lines showed cre-recombination in trkB+, calbindin+, 70% of trkC+ or parvalbumin+ neurons, which together encompassed ≈20% of Nav1.8-cre- DRG neurons. The data presented here increases our knowledge of peptidergic sensory neuron characteristics, while showing the efficiency and specificity manipulation of peptidergic neurons by the CGRPcre-ER reporter. We also demonstrate that manipulation of all C- and A-nociceptors is better achieved with TRPV1-cre reporter. Finally, the described approach for detailed characterization of sensory neuronal groups can be applied to a variety of reporter mice.