Degenerative and regenerative peripheral processes are associated with persistent painful chemotherapy-induced neuropathies in males and females.

This study aimed to investigate the time course of gene expression changes during the progression of persistent painful neuropathy caused by paclitaxel (PTX) in male and female mouse hind paws and dorsal root ganglia (DRG). Bulk RNA-seq was used to investigate the gene expression changes in the paw and DRG collected at 1, 16, and 31 days post-PTX. At these time points, differentially expressed DEGs were predominantly related to reduction or increase in epithelial, skin, bone, and muscle development and to angiogenesis, myelination, axonogenesis, and neurogenesis. These processes were accompanied by regulation of DEGs related to cytoskeleton, extracellular matrix organization and cellular energy production. This gene plasticity during persistent painful neuropathy progression likely represents biological processes linked to tissue regeneration and degeneration. Unlike regeneration/degeneration, gene plasticity related to immune processes was minimal at 1-31 days post-PTX. It was also noted that despite similarities in biological processes and pain chronicity in males and females, specific DEGs showed dramatic sex-dependency. The main conclusions of this study are that gene expression plasticity in paws and DRG during PTX neuropathy progression relates to tissue regeneration and degeneration, minimally affects the immune system processes, and is heavily sex-dependent at the individual gene level.

Associations of tissue damage induced inflammatory plasticity in masseter muscle with the resolution of chronic myalgia.

Gene plasticity during myogenous temporomandibular disorder (TMDM) development is largely unknown. TMDM could be modeled by intramuscular inflammation or tissue damage. To model inflammation induced TMDM we injected complete Freund's adjuvant (CFA) into masseter muscle (MM). To model tissue damage induced TMDM we injected extracellular matrix degrading collagenase type 2 (Col). CFA and Col produced distinct myalgia development trajectories. We performed bulk RNA-seq of MM to generate gene plasticity time course. CFA initiated TMDM (1d post-injection) was mainly linked to chemo-tacticity of monocytes and neutrophils. At CFA-induced hypersensitivity post-resolution (5d post-injection), tissue repair processes were pronounced, while inflammation was absent. Col (0.2U) produced acute hypersensitivity linked to tissue repair without inflammatory processes. Col (10U) generated prolonged hypersensitivity with inflammatory processes dominating initiation phase (1d). Pre-resolution phase (6d) was accompanied with acceleration of expressions for tissue repair and pro-inflammatory genes. Flow cytometry showed that immune processes in MM was associated with accumulations of macrophages, natural killer, dendritic and T-cells, further confirming our RNA-seq findings. Altogether, CFA and Col treatments induced different immune processes in MM. Importantly, TMDM resolution was preceded with muscle cell and extracellular matrix repairs, an elevation in immune system gene expressions and distinct immune cell accumulations in MM.

Sensory innervation of masseter, temporal and lateral pterygoid muscles in common marmosets.

Myogenous temporomandibular disorders is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate-common marmosets. Sensory nerves were localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were primarily innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and lowest in MM. All C-fibers (pgp9.5+/NFH-) observed in masticatory muscles were peptidergic (CGRP+) and lacked mrgprD and CHRNA3, a silent nociceptive marker. TrpV1 was register in 17% of LPM nerves. All fibers in masticatory muscles were labeled with GFAP+, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP+/NFH+) in TM and LPM compared to MM. MM, TM and LPM NFH+ fibers contained different percentages of trkC+ and parvalbumin+, but not trkB+ fibers. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have similarities and differences in innervation patterns.

Lingual innervation in male and female marmosets.

Several gaps in knowledge exists in our understanding of orofacial pain. Some of these include type of peripheral sensory innervation in specific tissues, differences in innervation between sexes and validation of rodent studies in higher order species. The current study addresses these gaps by validating mouse studies for sensory innervation of tongue tissue in non-human primates as well as assesses sex-specific differences. Tongue and trigeminal ganglia were collected from naïve male and female marmosets and tested for nerve fibers using specific markers by immunohistochemistry and number of fibers quantified. We also tested whether specific subgroups of nerve fibers belonged to myelinating or non-myelinating axons. We observed that similar to findings in mice, marmoset tongue was innervated with nerve filaments expressing nociceptor markers like CGRP and TRPV1 as well as non-nociceptor markers like TrkB, parvalbumin (PV) and tyrosine hydroxylase (TH). Furthermore, we found that while portion of TrkB and PV may be sensory fibers, TH-positive fibers were primarily sympathetic nerve fibers. Moreover, number of CGRP, TrkB and TH-positive nerve fibers were similar in both sexes. However, we observed a higher proportion of myelinated TRPV1 positive fibers in females than in males as well as increased number of PV + fibers in females. Taken together, the study for the first time characterizes sensory innervation in non-human primates as well as evaluates sex-differences in innervation of tongue tissue, thereby laying the foundation for future orofacial pain research with new world smaller NHPs like the common marmoset.

Transcriptional profiles of non-neuronal and immune cells in mouse trigeminal ganglia.

Non-neuronal cells constitute 90%-95% of sensory ganglia. These cells, especially glial and immune cells, play critical roles in the modulation of sensory neurons. This study aimed to identify, profile, and summarize the types of trigeminal ganglion (TG) non-neuronal cells in naïve male mice using published and our own data generated by single-cell RNA sequencing, flow cytometry, and immunohistochemistry. TG has five types of non-neuronal cells, namely, glial, fibroblasts, smooth muscle, endothelial, and immune cells. There is an agreement among publications for glial, fibroblasts, smooth muscle, and endothelial cells. Based on gene profiles, glial cells were classified as myelinated and non-myelinated Schwann cells and satellite glial cells. Mpz has dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2+ fibroblasts located throughout TG were distinguished. TG smooth muscle and endothelial cells in the blood vessels were detected using well-defined markers. Our study reported three types of macrophages (Mph) and four types of neutrophils (Neu) in TG. Mph were located in the neuronal bodies and nerve fibers and were sub-grouped by unique transcriptomic profiles with Ccr2, Cx3cr1, and Iba1 as markers. A comparison of databases showed that type 1 Mph is similar to choroid plexus-low (CPlo) border-associated Mph (BAMs). Type 2 Mph has the highest prediction score with CPhi BAMs, while type 3 Mph is distinct. S100a8+ Neu were located in the dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r, Ly6G, Ngp, Elane, and Mpo. Integrative analysis of published datasets indicated that Neu-1, Neu-2, and Neu-3 are similar to the brain Neu-1 group, while Neu-4 has a resemblance to the monocyte-derived cells. Overall, the generated and summarized datasets on non-neuronal TG cells showed a unique composition of myeloid cell types in TG and could provide essential and fundamental information for studies on cell plasticity, interactomic networks between neurons and non-neuronal cells, and function during a variety of pain conditions in the head and neck regions.

Understanding painful versus non-painful dental pain in female and male patients: A transcriptomic analysis of human biopsies.

Dental pain from apical periodontitis is an infection induced-orofacial pain condition that presents with diversity in pain phenotypes among patients. While 60% of patients with a full-blown disease present with the hallmark symptom of mechanical allodynia, nearly 40% of patients experience no pain. Furthermore, a sexual dichotomy exists, with females exhibiting lower mechanical thresholds under basal and diseased states. Finally, the prevalence of post-treatment pain refractory to commonly used analgesics ranges from 7-19% (∼2 million patients), which warrants a thorough investigation of the cellular changes occurring in different patient cohorts. We, therefore, conducted a transcriptomic assessment of periapical biopsies (peripheral diseased tissue) from patients with persistent apical periodontitis. Surgical biopsies from symptomatic male (SM), asymptomatic male (AM), symptomatic female (SF), and asymptomatic female (AF) patients were collected and processed for bulk RNA sequencing. Using strict selection criteria, our study found several unique differentially regulated genes (DEGs) between symptomatic and asymptomatic patients, as well as novel candidate genes between sexes within the same pain group. Specifically, we found the role of cells of the innate and adaptive immune system in mediating nociception in symptomatic patients and the role of genes involved in tissue homeostasis in potentially inhibiting nociception in asymptomatic patients. Furthermore, sex-related differences appear to be tightly regulated by macrophage activity, its secretome, and/or migration. Collectively, we present, for the first time, a comprehensive assessment of peripherally diseased human tissue after a microbial insult and shed important insights into the regulation of the trigeminal system in female and male patients.

Transcriptional Profiles of Non-neuronal and Immune Cells in Mouse Trigeminal Ganglia.

Non-neuronal cells constitute 90-95% of sensory ganglia. These cells play critical roles in modulation of nociceptive signal transmissions by sensory neurons. Accordingly, the aim of this review-study was to identify, profile and summarize TG non-neuronal cell types in naïve male mice using published and our own data generated by single-cell RNA sequencing (scRNA-seq), flow cytometry (FC) and immunohistochemistry (IHC). TG contains 5 types of non-neuronal cells: glial, fibroblasts, smooth muscle, endothelial and immune cells. There is agreement among publications for glial, fibroblasts, smooth muscle and endothelial cells. Based on gene profiles, glial cells were classified as Schwann cells and satellite glial cells (SGC). Mpz had dominant expression in Schwann cells, and Fabp7 is specific for SCG. Two types of Col1a2 + fibroblasts located throughout TG were distinguished using gene profiles. TG smooth muscle and endothelial cells representing blood vessels were detected with well recognized markers. Our study split reported single TG immune cell group into 3 types of macrophages and 4 types of neutrophils. Macrophages were located among neuronal bodies and nerve fibers, and were sub-grouped by unique transcriptomic profiles and using Ccr2 , Cx3cr1 and Iba1 as markers. S100a8 + neutrophils were located in dura surrounding TG and were sub-grouped by clustering and expressions of Csf3r , Ly6G, Ngp, Elane and Mpo . Overall, generated and summarized here dataset on non-neuronal TG cells could provide essential and fundamental information for studies on cell plasticity, interactomic network between neurons and non-neuronal cells and function during variety of pain conditions in the head and neck region.

Association of inflammation and tissue damage induced biological processes in masseter muscle with the resolution of chronic myalgia.

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.

Sensory innervation of masseter, temporal and lateral pterygoid muscles in common marmosets.

Myogenous temporomandibular disorders (TMDM) is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), medial pterygoid (MPM) and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate - common marmosets. Sensory nerves are localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were predominantly innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and minimal (6-8%) in MM. All C-fibers (pgp9.5+/NFH-) observed in masticatory muscles were peptidergic (CGRP+) and lacked mrgprD, trpV1 and CHRNA3, a silent nociceptive marker. All fibers in masticatory muscles were labeled with GFAP+, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP+/NFH+) in TM and LPM compared to MM. Almost all A-fibers in MM expressed trkC, with some of them having trkB and parvalbumin. In contrast, a lesser number of TM and LPM nerves expressed trkC, and lacked trkB. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have distinct and different innervation patterns.

Characterization of Fragile X Mental Retardation Protein expression in human nociceptors and their axonal projections to the spinal dorsal horn.

Fragile X Mental Retardation Protein (FMRP) regulates activity-dependent RNA localization and local translation to modulate synaptic plasticity throughout the central nervous system. Mutations in the FMR1 gene that hinder or ablate FMRP function cause Fragile X Syndrome (FXS), a disorder associated with sensory processing dysfunction. FXS premutations are associated with increased FMRP expression and neurological impairments including sex dimorphic presentations of chronic pain. In mice, FMRP ablation causes dysregulated dorsal root ganglion (DRG) neuron excitability and synaptic vesicle exocytosis, spinal circuit activity, and decreased translation-dependent nociceptive sensitization. Activity-dependent, local translation is a key mechanism for enhancing primary nociceptor excitability that promotes pain in animals and humans. These works indicate that FMRP likely regulates nociception and pain at the level of the primary nociceptor or spinal cord. Therefore, we sought to better understand FMRP expression in the human DRG and spinal cord using immunostaining in organ donor tissues. We find that FMRP is highly expressed in DRG and spinal neuron subsets with substantia gelatinosa exhibiting the most abundant immunoreactivity in spinal synaptic fields. Here, it is expressed in nociceptor axons. FMRP puncta colocalized with Nav1.7 and TRPV1 receptor signals suggesting a pool of axoplasmic FMRP localizes to plasma membrane-associated loci in these branches. Interestingly, FMRP puncta exhibited notable colocalization with calcitonin gene-related peptide (CGRP) immunoreactivity selectively in female spinal cord. Our results support a regulatory role for FMRP in human nociceptor axons of the dorsal horn and implicate it in the sex dimorphic actions of CGRP signaling in nociceptive sensitization and chronic pain.

Trigeminal neurons control immune-bone cell interaction and metabolism in apical periodontitis.

Apical periodontitis (AP) is an inflammatory disease occurring following tooth infection with distinct osteolytic activity. Despite increasing evidence that sensory neurons participate in regulation of non-neuronal cells, their role in the development of AP is largely unknown. We hypothesized that trigeminal ganglia (TG) Nav1.8+ nociceptors regulate bone metabolism changes in response to AP. A selective ablation of nociceptive neurons in Nav1.8Cre/Diphtheria toxin A (DTA)Lox mouse line was used to evaluate the development and progression of AP using murine model of infection-induced AP. Ablation of Nav1.8+ nociceptors had earlier progression of AP with larger osteolytic lesions. Immunohistochemical and RNAscope analyses demonstrated greater number of macrophages, T-cells, osteoclast and osteoblast precursors and an increased RANKL:OPG ratio at earlier time points among Nav1.8Cre/ DTALox mice. There was an increased expression of IL-1α and IL-6 within lesions of nociceptor-ablated mice. Further, co-culture experiments demonstrated that TG neurons promoted osteoblast mineralization and inhibited osteoclastic function. The findings suggest that TG Nav1.8+ neurons contribute to modulation of the AP development by delaying the influx of immune cells, promoting osteoblastic differentiation, and decreasing osteoclastic activities. This newly uncovered mechanism could become a therapeutic strategy for the treatment of AP and minimize the persistence of osteolytic lesions in refractory cases.

A Female-Specific Role for Calcitonin Gene-Related Peptide (CGRP) in Rodent Pain Models.

We aimed to investigate a sexually dimorphic role of calcitonin gene-related peptide (CGRP) in rodent models of pain. Based on findings in migraine where CGRP has a preferential pain-promoting effect in female rodents, we hypothesized that CGRP antagonists and antibodies would attenuate pain sensitization more efficaciously in female than male mice and rats. In hyperalgesic priming induced by activation of interleukin 6 signaling, CGRP receptor antagonists olcegepant and CGRP8-37 both given intrathecally, blocked, and reversed hyperalgesic priming only in females. A monoclonal antibody against CGRP, given systemically, blocked priming specifically in female rodents but failed to reverse it. In the spared nerve injury model, there was a transient effect of both CGRP antagonists, given intrathecally, on mechanical hypersensitivity in female mice only. Consistent with these findings, intrathecally applied CGRP caused a long-lasting, dose-dependent mechanical hypersensitivity in female mice but more transient effects in males. This CGRP-induced mechanical hypersensitivity was reversed by olcegepant and the KCC2 enhancer CLP257, suggesting a role for anionic plasticity in the dorsal horn in the pain-promoting effects of CGRP in females. In spinal dorsal horn slices, CGRP shifted GABAA reversal potentials to significantly more positive values, but, again, only in female mice. Therefore, CGRP may regulate KCC2 expression and/or activity downstream of CGRP receptors specifically in females. However, KCC2 hypofunction promotes mechanical pain hypersensitivity in both sexes because CLP257 alleviated hyperalgesic priming in male and female mice. We conclude that CGRP promotes pain plasticity in female rodents but has a limited impact in males.SIGNIFICANCE STATEMENT The majority of patients impacted by chronic pain are women. Mechanistic studies in rodents are creating a clear picture that molecular events promoting chronic pain are different in male and female animals. We sought to build on evidence showing that CGRP is a more potent and efficacious promoter of headache in female than in male rodents. To test this, we used hyperalgesic priming and the spared nerve injury neuropathic pain models in mice. Our findings show a clear sex dimorphism wherein CGRP promotes pain in female but not male mice, likely via a centrally mediated mechanism of action. Our work suggests that CGRP receptor antagonists could be tested for efficacy in women for a broader variety of pain conditions.

Sex-dependent pain trajectories induced by prolactin require an inflammatory response for pain resolution.

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 (1 µg) 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 (0.1 µg) 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.

Sex Differences in Nociceptor Translatomes Contribute to Divergent Prostaglandin Signaling in Male and Female Mice.

BACKGROUND: There are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences. METHODS: We used translating ribosome affinity purification sequencing and behavioral pharmacology to test the hypothesis that in Nav1.8-positive neurons, most of which are nociceptors, translatomes would differ by sex. RESULTS: We found 80 genes with sex differential expression in the whole DRG transcriptome and 66 genes whose messenger RNAs were sex differentially actively translated (translatome). We also identified different motifs in the 3' untranslated region of messenger RNAs that were sex differentially translated. In further validation studies, we focused on Ptgds, which was increased in the translatome of female mice. The messenger RNA encodes the prostaglandin PGD2 synthesizing enzyme. We observed increased PTGDS protein and PGD2 in female mouse DRG. The PTGDS inhibitor AT-56 caused intense pain behaviors in male mice but was only effective at high doses in female mice. Conversely, female mice responded more robustly to another major prostaglandin, PGE2, than did male mice. PTGDS protein expression was also higher in female cortical neurons, suggesting that DRG findings may be generalizable to other nervous system structures. CONCLUSIONS: Our results demonstrate sex differences in nociceptor-enriched translatomes and reveal unexpected sex differences in one of the oldest known nociceptive signaling molecule families, the prostaglandins.

Prolactin signaling modulates stress-induced behavioral responses in a preclinical mouse model of migraine.

OBJECTIVE: The aim of this study was to determine if prolactin signaling modulates stress-induced behavioral responses in a preclinical migraine model. BACKGROUND: Migraine is one of the most complex and prevalent disorders. The involvement of sex-selective hormones in migraine pathology is highly likely as migraine is more common in women and its frequency correlates with reproductive stages. Prolactin has been shown to be a worsening factor for migraine. Normally prolactin levels are low; however levels can surge during stress. Dopamine receptor agonists, which suppress pituitary prolactin release, are an effective migraine treatment in a subset of patients. Previously, we showed that administration of prolactin onto the dura mater induces female-specific behavioral responses, suggesting that prolactin may play a sex-specific role in migraine. METHODS: The effects of prolactin signaling were assessed using a preclinical migraine model we published recently in which behavioral sensitization is induced by repeated stress. Plasma prolactin levels were assessed in naïve and stressed CD-1 mice (n = 3-5/group) and transgenic mice with conditional deletion of the Prlr in Nav1.8-positive sensory neurons (Prlr conditional knock-out [CKO]; n = 3/group). To assess the contribution of prolactin release during stress, naïve or stressed male and female CD-1 mice were treated with the prolactin release inhibitor bromocriptine (2 mg/kg; n = 7-12/group) or vehicle for 5 days (8-12/group) and tested for facial hypersensitivity following stress. Additionally, the contribution of ovarian hormones in regulating the prolactin-induced responses was assessed in ovariectomized female CD-1 mice (n = 6-10/group). Furthermore, the contribution of Prlr activation on Nav1.8-positive sensory neurons was assessed. Naïve or stressed male and female Prlr CKO mice and their control littermates were tested for facial hypersensitivity (n = 8-9/group). Immunohistochemistry was used to confirm loss of Prlr in Nav1.8-positive neurons in Prlr CKO mice. The total sample size is n = 245; the full analysis sample size is n = 221. RESULTS: Stress significantly increased prolactin levels in vehicle-treated female mice (39.70 ± 2.77; p < 0.0001). Bromocriptine significantly reduced serum prolactin levels in stressed female mice compared to vehicle-treated mice (-44.85 ± 3.1; p < 0.0001). Additionally, no difference was detected between female stressed mice that received bromocriptine compared to naïve mice treated with bromocriptine (-0.70 ± 2.9; p = 0.995). Stress also significantly increased serum prolactin levels in male mice, although to a much smaller extent than in females (0.61 ± 0.08; p < 0.001). Bromocriptine significantly reduced serum prolactin levels in stressed males compared to those treated with vehicle (-0.49 ± 0.08; p = 0.002). Furthermore, bromocriptine attenuated stress-induced behavioral responses in female mice compared to those treated with vehicle (maximum effect observed on day 4 post stress [0.21 ± 0.08; p = 0.03]). Bromocriptine did not attenuate stress-induced behavior in males at any timepoint compared to those treated with vehicle. Moreover, loss of ovarian hormones did not affect the ability of bromocriptine to attenuate stress responses compared to vehicle-treated ovariectomy mice that were stressed (maximum effect observed on day 4 post stress [0.29 ± 0.078; p = 0.013]). Similar to CD-1 mice, stress increased serum prolactin levels in both Prlr CKO female mice (27.74 ± 9.96; p = 0.047) and control littermates (28.68 ± 9.9; p = 0.041) compared to their naïve counterparts. There was no significant increase in serum prolactin levels detected in male Prlr CKO mice or control littermates. Finally, conditional deletion of Prlr from Nav1.8-positive sensory neurons led to a female-specific attenuation of stress-induced behavioral responses (maximum effect observed on day 7 post stress [0.32 ± 0.08; p = 0.007]) compared to control littermates. CONCLUSION: These data demonstrate that prolactin plays a female-specific role in stress-induced behavioral responses in this preclinical migraine model through activation of Prlr on sensory neurons. They also support a role for prolactin in migraine mechanisms in females and suggest that modulation of prolactin signaling may be an effective therapeutic strategy in some cases.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system.

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.

Identification of Trigeminal Sensory Neuronal Types Innervating Masseter Muscle.

Understanding masseter muscle (MM) innervation is critical for the study of cell-specific mechanisms of pain induced by temporomandibular disorder (TMDs) or after facial surgery. Here, we identified trigeminal (TG) sensory neuronal subtypes (MM TG neurons) innervating MM fibers, masseteric fascia, tendons, and adjusted tissues. A combination of patch clamp electrophysiology and immunohistochemistry (IHC) on TG neurons back-traced from reporter mouse MM found nine 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 (TH)+ (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.

Redefining the Role of Lymphotoxin Beta Receptor in the Maintenance of Lymphoid Organs and Immune Cell Homeostasis in Adulthood.

Lymphotoxin beta receptor (LTßR) is a promising therapeutic target in autoimmune and infectious diseases as well as cancer. Mice with genetic inactivation of LTßR display multiple defects in development and organization of lymphoid organs, mucosal immune responses, IgA production and an autoimmune phenotype. As these defects are imprinted in embryogenesis and neonate stages, the impact of LTßR signaling in adulthood remains unclear. Here, to overcome developmental defects, we generated mice with inducible ubiquitous genetic inactivation of LTßR in adult mice (iLTßRΔ/Δ mice) and redefined the role of LTßR signaling in organization of lymphoid organs, immune response to mucosal bacterial pathogen, IgA production and autoimmunity. In spleen, postnatal LTßR signaling is required for development of B cell follicles, follicular dendritic cells (FDCs), recruitment of neutrophils and maintenance of the marginal zone. Lymph nodes of iLTßRΔ/Δ mice were reduced in size, lacked FDCs, and had disorganized subcapsular sinus macrophages. Peyer`s patches were smaller in size and numbers, and displayed reduced FDCs. The number of isolated lymphoid follicles in small intestine and colon were also reduced. In contrast to LTßR-/- mice, iLTßRΔ/Δ mice displayed normal thymus structure and did not develop signs of systemic inflammation and autoimmunity. Further, our results suggest that LTßR signaling in adulthood is required for homeostasis of neutrophils, NK, and iNKT cells, but is dispensable for the maintenance of polyclonal IgA production. However, iLTßRΔ/Δ mice exhibited an increased sensitivity to C. rodentium infection and failed to develop pathogen-specific IgA responses. Collectively, our study uncovers new insights of LTßR signaling in adulthood for the maintenance of lymphoid organs, neutrophils, NK and iNKT cells, and IgA production in response to mucosal bacterial pathogen.

Meningeal CGRP-Prolactin Interaction Evokes Female-Specific Migraine Behavior.

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. ANN NEUROL 2021;89:1129-1144.

Transcriptomic sex differences in sensory neuronal populations of mice.

Many chronic pain conditions show sex differences in their epidemiology. This could be attributed to sex-dependent differential expression of genes (DEGs) involved in nociceptive pathways, including sensory neurons. This study aimed to identify sex-dependent DEGs in estrous female versus male sensory neurons, which were prepared by using different approaches and ganglion types. RNA-seq on non-purified sensory neuronal preparations, such as whole dorsal root ganglion (DRG) and hindpaw tissues, revealed only a few sex-dependent DEGs. Sensory neuron purification increased numbers of sex-dependent DEGs. These DEG sets were substantially influenced by preparation approaches and ganglion types [DRG vs trigeminal ganglia (TG)]. Percoll-gradient enriched DRG and TG neuronal fractions produced distinct sex-dependent DEG groups. We next isolated a subset of sensory neurons by sorting DRG neurons back-labeled from paw and thigh muscle. These neurons have a unique sex-dependent DEG set, yet there is similarity in biological processes linked to these different groups of sex-dependent DEGs. Female-predominant DEGs in sensory neurons relate to inflammatory, synaptic transmission and extracellular matrix reorganization processes that could exacerbate neuro-inflammation severity, especially in TG. Male-selective DEGs were linked to oxidative phosphorylation and protein/molecule metabolism and production. Our findings catalog preparation-dependent sex differences in neuronal gene expressions in sensory ganglia.