Mouse Trigeminal Ganglia Research Articles

Elucidation of neuronal activity in mouse models of temporomandibular joint injury and inflammation by in vivo GCaMP Ca2+ imaging of intact trigeminal ganglion neurons.

Patients with temporomandibular disorders (TMDs) typically experience facial pain and discomfort or tenderness in the temporomandibular joint (TMJ), causing disability in daily life. Unfortunately, existing treatments for TMD are not always effective, creating a need for more advanced, mechanism-based therapies. In this study, we used in vivo GCaMP3 Ca2+ imaging of intact trigeminal ganglia (TG) to characterize functional activity of the TG neurons in vivo, specifically in mouse models of TMJ injury and inflammation. This system allows us to observe neuronal activity in intact anatomical, physiological, and clinical conditions and to assess neuronal function and response to various stimuli. We observed a significant increase in spontaneously and transiently activated neurons responding to mechanical, thermal, and chemical stimuli in the TG of mice with TMJ injection of complete Freund adjuvant or with forced mouth opening (FMO). An inhibitor of the calcitonin gene-related peptide receptor significantly attenuated FMO-induced facial hypersensitivity. In addition, we confirmed the attenuating effect of calcitonin gene-related peptide antagonist on FMO-induced sensitization by in vivo GCaMP3 Ca2+ imaging of intact TG. Our results contribute to unraveling the role and activity of TG neurons in the TMJ pain, bringing us closer to understanding the pathophysiological processes underlying TMJ pain after TMJ injury. Our study also illustrates the utility of in vivo GCaMP3 Ca2+ imaging of intact TG for studies aimed at developing more targeted and effective treatments for TMJ pain.

The Influence of Sex Steroid Hormone Fluctuations on Capsaicin-Induced Pain and TRPV1 Expression.

Sexual dimorphism among mammals includes variations in the pain threshold. These differences are influenced by hormonal fluctuations in females during the estrous and menstrual cycles of rodents and humans, respectively. These physiological conditions display various phases, including proestrus and diestrus in rodents and follicular and luteal phases in humans, distinctly characterized by varying estrogen levels. In this study, we evaluated the capsaicin responses in male and female mice at different estrous cycle phases, using two murine acute pain models. Our findings indicate that the capsaicin-induced pain threshold was lower in the proestrus phase than in the other three phases in both pain assays. We also found that male mice exhibited a higher pain threshold than females in the proestrus phase, although it was similar to females in the other cycle phases. We also assessed the mRNA and protein levels of TRPV1 in the dorsal root and trigeminal ganglia of mice. Our results showed higher TRPV1 protein levels during proestrus compared to diestrus and male mice. Unexpectedly, we observed that the diestrus phase was associated with higher TRPV1 mRNA levels than those in both proestrus and male mice. These results underscore the hormonal influence on TRPV1 expression regulation and highlight the role of sex steroids in capsaicin-induced pain.

Contribution of inwardly rectifying potassium channel 4.1 in orofacial neuropathic pain: Regulation of pannexin 3 via the reactive oxygen species-activated P38 MAPK signal pathway.

The involvement of inwardly rectifying potassium channel 4.1 (Kir4.1) in neuropathic pain has been established. However, there is limited understanding of the downstream mechanism through which Kir4.1 contributes to orofacial neuropathic pain. The objective of this study was to examine the regulation of Kir4.1 on the expression of pannexin 3 (Panx3) in the trigeminal ganglion (TG) and the underlying mechanism in the context of orofacial neuropathic pain caused by chronic constriction injury of the infraorbital nerve (CCI-ION). The study observed a significant increase in Panx3 expression in the TG of mice with CCI-ION. Inhibition of Panx3 in the TG of CCI-ION mice resulted in alleviation of orofacial mechanical allodynia. Furthermore, conditional knockdown (CKD) of Kir4.1 in the TG of both male and female mice led to mechanical allodynia and upregulation of Panx3 expression. Conversely, overexpression of Kir4.1 decreased Panx3 levels in the TG and relieved mechanical allodynia in CCI-ION mice. In addition, silencing Kir4.1 in satellite glial cells (SGCs) decreased Panx3 expression and increased the phosphorylation of P38 MAPK. Moreover, silencing Kir4.1 in SGCs increased the levels of reactive oxygen species (ROS). The elevated phosphorylation of P38 MAPK resulting from Kir4.1 silencing was inhibited by using a superoxide scavenger known as the tempol. Silencing Panx3 in the TG in vivo attenuated the mechanical allodynia caused by Kir4.1 CKD. In conclusion, these findings suggest that the reduction of Kir4.1 promotes the expression of Panx3 by activating the ROS-P38 MAPK signalling pathway, thus contributing to the development of orofacial neuropathic pain.

MicroRNA-6954-3p Downregulation Contributes to Orofacial Neuropathic Pain in Mice Via Targeting Voltage-Gated Sodium Channel β2 Subunit Protein

The voltage-gated sodium channel β2 subunit protein (SCN2B) plays a crucial role in neuropathic pain. However, the role and mechanisms of SCN2B in orofacial neuropathic pain are still unclear. This study aimed to investigate the upstream regulatory mechanisms of SCN2B in the trigeminal ganglion (TG) underlying orofacial neuropathic pain. Chronic constriction injury of the infraorbital nerve (CCI-ION) of mice was performed to establish the model of orofacial neuropathic pain. Von Frey filament test was performed to detect the head withdrawal threshold (HWT) of mice. Quantitative reverse transcription-polymerase chain, western blotting (WB), fluorescence in situ hybridization, and immunofluorescence (IF) staining were used to detect the expression and distribution of SCN2B and miR-6954-3p in the TG of mice. A luciferase activity assay was carried out to prove the binding between SCN2B messenger ribonucleic acid (mRNA) and miR-6954-3p. After the CCI-ION surgery, the levels of Scn2b mRNA and protein significantly increased and miR-6954-3p decreased in the TG of mice with decreasing HWT. IF staining revealed that SCN2B was expressed specifically in the TG neurons. Silencing SCN2B in the TG of CCI-ION mice significantly increased the HWT. Importantly, the 3′-untranslated region of Scn2b mRNA was proved to bind with miR-6954-3p. Fluorescence in situ hybridization and IF staining demonstrated that miR-6954-3p was expressed in TG neurons and co-expressed with SCN2B. Furthermore, intraganglionic injection of miR-6954-3p agomir into the TG of CCI-ION mice resulted in the downregulation of SCN2B and increased the HWT. These findings suggest that the downregulation of miR-6954-3p in the TG promotes orofacial neuropathic pain by promoting SCN2B expression following trigeminal nerve injury. PerspectiveThis study points to the important role of SCN2B in orofacial neuropathic pain. Furthermore, miR-6954-3p is proven to regulate the expression of SCN2B by binding to the 3′-untranslated region of Scn2b mRNA. These findings indicate that SCN2B and miR-6954-3p are potential therapeutic targets for the treatment of orofacial neuropathic pain.

Abstract 6652: Advanced glioblastoma immunotherapy: Attenuated herpes oncolytic virus armed with anti-PD-1 antibody and IL-12

Abstract Background: Glioblastoma (GBM), the most common and deadly primary brain tumor in adults, has limited treatment options with poor outcomes. The urgent need for innovative treatments has spurred research into immunotherapy and oncolytic virus therapy as promising alternatives. Herein, we have developed a new generation oncolytic herpes simplex virus (oHSV) armed with IL-12 and anti-PD-1 antibody to provide a synergistic anti-GBM efficacy for immune-oncolytic therapy. Methods: MVR-C5252, a novel oHSV, was created by genetic engineering, which included the removal of a 15-kb internal repeat (IR) region and both copies of the γ34.5 genes, along with the insertion of two exogenous genes encoding the anti-PD-1 antibody and IL-12. MVR-C5252 was characterized and assessed for its anti-GBM activities through a series of experiments as follows: 1) Safety assessments, including evaluations of neurotoxicity and latency-reactivation characteristics, were conducted in Balb/C mice. 2) Anti-tumor activities were evaluated in cell cultures, subcutaneous and orthotopic xenograft mouse models, and immunocompetent mouse models. 3) Mechanistic studies were performed to uncover the mode of action of MVR-C5252, with a specific focus on its ability to induce cell death in GBM cells and activate the immune response in vivo. Results: 1) In vitro studies showed that MVR-C5252 is highly replication attenuated but with higher cell-killing activity in GBM cells compared to the first generation oHSV, R3616, which has γ34.5 deletions only. 2) Mechanistic studies showed that MVR-C5252 infection increased cell death by reducing CNTNRα expression, which typically prevents caspase 3 activation and has an anti-apoptotic effect in glioma cells. 3) Intracranial injection of MVR-C5252 revealed a remarkable safety profile, with over an 800-fold reduction in virulence compared to wild-type HSV-1. Furthermore, the mouse trigeminal ganglion (TG) infection model suggested that MVR-C5252 was unable to establish latency or reactivate in the TG. 4) In vivo subcutaneous and orthotopic xenograft mouse models showed that compared to the R3616 group, enhanced anti-tumor activity and extended survival were observed in the MVR-C5252 group. Additionally, in the syngeneic mice model, MVR-C5252 exhibited superior anti-tumor activity and immune activation over the backbone virus. Conclusions: With the unique combination of safety, potent anti-tumor activity, and immune-stimulatory properties, MVR-C5252 makes it a big potential candidate to address the pressing need for effective treatment strategies of GBM. Citation Format: Lei Wang, Xusha Zhou, Xiaoqing Chen, Yuanyuan Liu, Yanxin Zheng, Runbin Yan, Yonghong Liu, Jing Zhao, Grace Guoying Zhou. Advanced glioblastoma immunotherapy: Attenuated herpes oncolytic virus armed with anti-PD-1 antibody and IL-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6652.

Neuronal miR-9 promotes HSV-1 epigenetic silencing and latency by repressing Oct-1 and Onecut family genes

Herpes simplex virus 1 (HSV-1) latent infection entails repression of viral lytic genes in neurons. By functional screening using luciferase-expressing HSV-1, we identify ten neuron-specific microRNAs potentially repressing HSV-1 neuronal replication. Transfection of miR-9, the most active candidate from the screen, decreases HSV-1 replication and gene expression in Neuro-2a cells. Ectopic expression of miR-9 from lentivirus or recombinant HSV-1 suppresses HSV-1 replication in male primary mouse neurons in culture and mouse trigeminal ganglia in vivo, and reactivation from latency in the primary neurons. Target prediction and validation identify transcription factors Oct-1, a known co-activator of HSV transcription, and all three Onecut family members as miR-9 targets. Knockdown of ONECUT2 decreases HSV-1 yields in Neuro-2a cells. Overexpression of each ONECUT protein increases HSV-1 replication in Neuro-2a cells, human induced pluripotent stem cell-derived neurons, and primary mouse neurons, and accelerates reactivation from latency in the mouse neurons. Mutagenesis, ChIP-seq, RNA-seq, ChIP-qPCR and ATAC-seq results suggest that ONECUT2 can nonspecifically bind to viral genes via its CUT domain, globally stimulate viral gene transcription, reduce viral heterochromatin and enhance the accessibility of viral chromatin. Thus, neuronal miR-9 promotes viral epigenetic silencing and latency by targeting multiple host transcription factors important for lytic gene activation.

Inhibition of miR-144-3p/FOXO1 Attenuates Diabetic Keratopathy Via Modulating Autophagy and Apoptosis.

Diabetic keratopathy (DK) is a vision-threatening disease that occurs in people with diabetes. Mounting evidence indicates that microRNAs (miRNAs) are indispensable in nerve regeneration within DK. Herein, the role of miRNAs associated with DK, especially focusing on autophagy and apoptosis regulation, was investigated. To identify differentially expressed miRNAs, we performed miRNA sequencing on trigeminal ganglion (TG) tissues derived from streptozotocin-induced type 1 diabetic mellitus (T1DM) and normal mice. MiR-144-3p was chosen for the subsequent experiments. To explore the regulatory role of miR-144-3p in DK, miRNA antagomir was utilized to inhibit miR-144-3p expression. Bioinformatic tools were used to predict the target genes of miR-144-3p, and a dual-luciferase reporter assay was then applied for validation. Autophagy and apoptosis activities were measured utilizing TUNEL staining, immunofluorescence staining, and Western blotting. Overall, 56 differentially expressed miRNAs were detected in diabetic versus control mice. In the diabetic mouse TG tissue, miR-144-3p expression was aberrantly enhanced, whereas decreasing its expression contributed to improved diabetic corneal re-epithelialization and nerve regeneration. Fork-head Box O1 (FOXO1) was validated as a target gene of miR-144-3p. Overexpression of FOXO1 could prevent both inadequate autophagy and excessive apoptosis in DK. Consistently, a specific miR-144-3p inhibition enhanced autophagy and prevented apoptosis in DK. In this study, our research confirmed the target binding relationship between miR-144-3p and FOXO1. Inhibiting miR-144-3p might modulate autophagy and apoptosis, which could generate positive outcomes for corneal nerves via targeting FOXO1 in DK.

TET1-mediated epigenetic regulation of tumor necrosis factor-α in trigeminal ganglia contributes to chronic temporomandibular joint pain

Chronic temporomandibular joint (TMJ) pain profoundly affects patients' quality of life. Trigeminal tumor necrosis factor-α (TNFα) plays a pivotal role in mediating TMJ pain in mice, yet the underlying epigenetic mechanisms remain enigmatic. To unravel these epigenetic intricacies, we employed a multifaceted approach. Hydroxymethylated DNA immunoprecipitation (hMeDIP) and chromatin immunoprecipitation (ChIP) followed by qPCR were employed to investigate the demethylation of TNFα gene (Tnfa) and its regulation by ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in a chronic TMJ pain mouse model. The global levels of 5-hydroxymethylcytosine (5hmc) and percentage of 5hmc at the Tnfa promoter region were measured in the trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) following complete Freund's adjuvant (CFA) or saline treatment. TET1 knockdown and pain behavioral testing were conducted to ascertain the role of TET1-mediated epigenetic regulation of TNFα in the pathogenesis of chronic TMJ pain. Our finding revealed an increase in 5hmc at the Tnfa promoter region in both TG and Sp5C of CFA-treated mice. TET1 was upregulated in the mouse TG, and the ChIP result showed TET1 direct binding to the Tnfa promoter, with higher efficiency in the CFA-treated group. Immunofluorescence revealed the predominant expression of TET1 in trigeminal neurons. TET1 knockdown in the TG significantly reversed CFA-induced TNFα upregulation and alleviated chronic TMJ pain. In conclusion, our study implicates TET1 as a vital epigenetic regulator contributing to chronic inflammatory TMJ pain via trigeminal TNFα signaling. Targeting TET1 holds promise for epigenetic interventions in TMJ pain management.

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.

Cross-species gene expression patterns of purinergic signaling in the human and mouse trigeminal ganglion

Purinergic signaling system plays a pivotal role in the trigeminal ganglion (TG) which is a primary sensory tissue in vertebrate nervous systems involving orofacial nociception and peripheral sensitization. Despite previous efforts to reveal the expression patterns of purinergic components in the mouse TG, it is still unknown the interspecies differences between human and mouse. In this study, we provide a comprehensive transcriptome profile of the purinergic signaling system across diverse cell types and neuronal subpopulations within the human TG, systematically comparing it with mouse TG. In addition, the evolutionary conservation and species-specific expression patterns of the purinergic components are also discussed. We propose that the data can improve our understanding of purinergic signaling in the peripheral nervous system and facilitate the identification of novel therapeutic targets.

Phosphorylation of the AMPARs regulated by protein kinase C (PKC) and protein interacting with C-kinase 1 (PICK1) contribute to orofacial neuropathic pain

The α-amino-3-hydroxy-5-methylisoxazole-4-isoxazolepropionic acid receptor (AMPAR) has been recognized to play a vital role in the development of neuropathic pain. Recent studies have indicated that protein kinase C (PKC) and protein interacting with C-kinase 1 (PICK1) are involved in the phosphorylation of AMPARs. However, whether PKC and PICK1 were involved in the AMPAR phosphorylation in the trigeminal ganglion (TG) to participate in orofacial neuropathic pain remains enigmatic. A behavioral test was utilized to evaluate the head withdrawal threshold (HWT) after chronic constriction injury of the infraorbital nerve (CCI-ION). The distribution and expression of GluA1, GluA2, PKC, and PICK1 were examined in the trigeminal ganglion (TG) by immunofluorescence, real-time reverse transcription-quantitative polymerase chain reaction, immunoblotting, and co-immunoprecipitation. Intra-ganglionic injections of drugs were performed to investigate the regulation mechanism. The present study demonstrated that CCI-ION-induced mechanical allodynia was maintained over at least 21 days. GluA1 and GluA2 were mainly expressed in the neurons. Trigeminal nerve injury potentiated the phosphorylation of GluA1, GluA2, and PKC in the TG, which was prevented by inhibiting PKC with chelerythrine chloride. Additionally, PICK1 colocalized and interacted with GluA2 in the TG. Following blocking PICK1 with FSC-231, the phosphorylation of GluA2 decreased. Finally, inhibition of PKC and PICK1 both alleviated mechanical allodynia in the whisker pad of CCI-ION mice. In conclusion, activation of PKC and PICK1 contribute to orofacial allodynia by regulating AMPAR phosphorylation in the TG of male mice, which provides potential therapeutic targets for alleviating orofacial neuropathic pain.

The G protein-coupled estrogen receptor of the trigeminal ganglion regulates acute and chronic itch in mice.

Itch is an unpleasant sensation that severely impacts the patient's quality of life. Recent studies revealed that the G protein-coupled estrogen receptor (GPER) may play a crucial role in the regulation of pain and itch perception. However, the contribution of the GPER in primary sensory neurons to the regulation of itch perception remains elusive. This study aimed to investigate whether and how the GPER participates in the regulation of itch perception in the trigeminal ganglion (TG). Immunofluorescence staining results showed that GPER-positive (GPER+ ) neurons of the TG were activated in both acute and chronic itch. Behavioral data indicated that the chemogenetic activation of GPER+ neurons of the TG of Gper-Cre mice abrogated scratching behaviors evoked by acute and chronic itch. Conversely, the chemogenetic inhibition of GPER+ neurons resulted in increased itch responses. Furthermore, the GPER expression and function were both upregulated in the TG of the dry skin-induced chronic itch mouse model. Pharmacological inhibition of GPER (or Gper deficiency) markedly increased acute and chronic itch-related scratching behaviors in mouse. Calcium imaging assays further revealed that Gper deficiency in TG neurons led to a marked increase in the calcium responses evoked by agonists of the transient receptor potential ankyrin A1 (TRPA1) and transient receptor potential vanilloid V1 (TRPV1). Our findings demonstrated that the GPER of TG neurons is involved in the regulation of acute and chronic itch perception, by modulating the function of TRPA1 and TRPV1. This study provides new insights into peripheral itch sensory signal processing mechanisms and offers new targets for future clinical antipruritic therapy.

Culture of Primary Neurons from Dissociated and Cryopreserved Mouse Trigeminal Ganglion.

Corneal nerves originate from the ophthalmic branch of the trigeminal nerve, which enters the cornea at the limbus radially from all directions toward the central cornea. The cell bodies of the sensory neurons of trigeminal nerve are located in the trigeminal ganglion (TG) while the axons are extended into the three divisions including ophthalmic branch that supplies corneal nerves. Study of primary neuronal cultures established from the TG fibers can therefore provide a knowledge basis for corneal nerve biology and potentially be developed as an in vitro platform for drug testing. However, setting up primary neuron cultures from animal TG has been dubious with inconsistency among laboratories due to a lack of efficient isolation protocol, resulting in low yield and heterogenous cultures. In this study, we employed a combined enzymatic digestion with collagenase and TrypLE to dissociate mouse TG while preserving nerve cell viability. A subsequent discontinuous Percoll density gradient followed by mitotic inhibitor treatment effectively diminished the contamination of non-neuronal cells. Using this protocol, we reproducibly generated high yield and homogenous primary TG neuron cultures. Similar efficiency of nerve cell isolation and culture was further obtained for TG tissue cryopreserved for short (1 week) and long duration (3 months), compared to freshly isolated tissues. In conclusion, this optimized protocol shows a promising potential to standardize TG nerve culture and generate a high-quality corneal nerve model for drug testing and neurotoxicity studies.

Single-nucleus transcriptome analysis reveals transcriptional profiles of circadian clock and pain related genes in human and mouse trigeminal ganglion.

Clinical studies have revealed the existence of circadian rhythms in pain intensity and treatment response for chronic pain, including orofacial pain. The circadian clock genes in the peripheral ganglia are involved in pain information transmission by modulating the synthesis of pain mediators. However, the expression and distribution of clock genes and pain-related genes in different cell types within the trigeminal ganglion, the primary station of orofacial sensory transmission, are not yet fully understood. In this study, data from the normal trigeminal ganglion in the Gene Expression Omnibus (GEO) database were used to identify cell types and neuron subtypes within the human and mouse trigeminal ganglion by single nucleus RNA sequencing analysis. In the subsequent analyses, the distribution of the core clock genes, pain-related genes, and melatonin and opioid-related genes was assessed in various cell clusters and neuron subtypes within the human and mouse trigeminal ganglion. Furthermore, the statistical analysis was used to compare the differences in the expression of pain-related genes in the neuron subtypes of trigeminal ganglion. The present study provides comprehensive transcriptional profiles of core clock genes, pain-related genes, melatonin-related genes, and opioid-related genes in different cell types and neuron subtypes within the mouse and human trigeminal ganglion. A comparative analysis of the distribution and expression of the aforementioned genes was conducted between human and mouse trigeminal ganglion to investigate species differences. Overall, the results of this study serve as a primary and valuable resource for exploring the molecular mechanisms underlying oral facial pain and pain rhythms.

Circadian rhythm of PERIOD2::LUCIFERASE expression in the trigeminal ganglion of mice.

The trigeminal nerve conveys delicate sensations such as warmth, pain, and tactile pressure in the oral and facial regions, and most trigeminal afferent cell bodies are located in the trigeminal ganglion. Our previous study has shown that sensations in trigeminal nerve innervated areas, specifically in the maxillofacial region, exhibit diurnal variation and that sensitivity changes time-dependently. In this study, we aimed to clarify the rhythm of expression of clock gene in the trigeminal ganglion of mice to elucidate the mechanism of circadian regulation in the same area. Immunohistochemistry examined the expression of the PER2 protein in the suprachiasmatic nucleus and trigeminal ganglion of wild-type mice. To measure gene expression as bioluminescence, PERIOD2::LUCIFERASE knock-in (PER2::LUC) mice were used. Unilateral trigeminal ganglion and brain sections including the suprachiasmatic nucleus were incubated ex vivo. Bioluminescence levels were then measured using a highly sensitive photodetector. The same experiments were then conducted with Cry1 gene-deficient (Cry1-/- ) or Cry2 gene-deficient (Cry2-/- ) mice. In the trigeminal ganglion, immunohistochemistry localized PER2 protein expression within the neuronal cell body. Mouse trigeminal ganglion ex vivo tissues showed distinct circadian oscillations in PER2::LUC levels in all genotypes, wild-type, Cry1-/- , and Cry2-/- . The period was shorter in the trigeminal ganglion than in the suprachiasmatic nucleus; it was shorter in Cry1-/- and longer in Cry2-/- mice than in the wild-type mice. The expression of Per2 in neurons of the trigeminal ganglion in ex vivo culture and the oscillation in a distinct circadian rhythm suggests that the trigeminal ganglion is responsible for the relay of sensory inputs and temporal gating through autonomous circadian oscillations.

The cellular basis of mechanosensation in mammalian tongue.

Mechanosensory neurons that innervate the tongue provide essential information to guide feeding, speech, and social grooming. We use invivo calcium imaging of mouse trigeminal ganglion neurons to identify functional groups of mechanosensory neurons innervating the anterior tongue. These sensory neurons respond to thermal and mechanical stimulation. Analysis of neuronal activity patterns reveal that most mechanosensory trigeminal neurons are tuned to detect moving stimuli across the tongue. Using an unbiased, multilayer hierarchical clustering approach to classify pressure-evoked activity based on temporal response dynamics, we identify five functional classes of mechanosensory neurons with distinct force-response relations and adaptation profiles. These populations are tuned to detect different features of touch. Molecular markers of functionally distinct clusters are identified by analyzing cluster representation in genetically marked neuronal subsets. Collectively, these studies provide a platform for defining the contributions of functionally distinct mechanosensory neurons to oral behaviors crucial for survival in mammals.

OnabotulinumtoxinA effects on trigeminal nociceptors.

OnabotulinumtoxinA (onabotA) is approved globally for prevention of chronic migraine; however, the classical mechanism of action of onabotA in motor and autonomic neurons cannot fully explain the effectiveness of onabotulinumtoxinA in this sensory neurological disease. We sought to explore the direct effects of onabotulinumtoxinA on mouse trigeminal ganglion sensory neurons using an inflammatory soup-based model of sensitization. Primary cultured trigeminal ganglion neurons were pre-treated with inflammatory soup, then treated with onabotulinumtoxinA (2.75 pM). Treated neurons were used to examine transient receptor potential vanilloid subtype 1 and transient receptor potential ankyrin 1 cell-surface expression, calcium influx, and neuropeptide release. We found that onabotulinumtoxinA cleaved synaptosomal-associated protein-25 kDa in cultured trigeminal ganglion neurons; synaptosomal-associated protein-25 kDa cleavage was enhanced by inflammatory soup pre-treatment, suggesting greater uptake of toxin under sensitized conditions. OnabotulinumtoxinA also prevented inflammatory soup-mediated increases in TRPV1 and TRPA1 cell-surface expression, without significantly altering TRPV1 or TRPA1 protein expression in unsensitized conditions. We observed similar inhibitory effects of onabotulinumtoxinA on TRP-mediated calcium influx and TRPV1- and TRPA1-mediated release of calcitonin gene-related peptide and prostaglandin 2 under sensitized, but not unsensitized control, conditions. Our data deepen the understanding of the sensory mechanism of action of onabotulinumtoxinA and support the notion that, once endocytosed, the cytosolic light chain of onabotulinumtoxinA cleaves synaptosomal-associated protein-25 kDa to prevent soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated processes more generally in motor, autonomic, and sensory neurons.

Molecular taxonomy of nociceptors and pruriceptors.

Molecular taxonomy of nociceptors and pruriceptors.

Remodeling of Blood Vessels by Binge Alcohol Intake in the Mouse Trigeminal Ganglia

Remodeling of Blood Vessels by Binge Alcohol Intake in the Mouse Trigeminal Ganglia

Enhancement of allyl isothiocyanate-evoked responses of mouse trigeminal ganglion cells by the kokumi substance γ-glutamyl-valyl-glycine (γ-EVG) through activation of the calcium-sensing receptor (CaSR)

Some γ-glutamyl peptides including glutathione (γ-Glu-Cys-Gly) and γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly= γ-EVG) are reported to increase the intensity of basic tastes, such as salty, sweet, and umami, although they have no taste themselves at tested concentrations. The mechanism of action of γ-glutamyl peptides is not clearly understood, but the calcium sensing receptor (CaSR) may be involved. Glutathione and γ-EVG enhance the pungency of some spices, and the present study investigated the effects of γ-EVG on the responses of trigeminal ganglion (TG) cells to thermosensitiveTRP channel agonists. Single-cell RT-PCR revealed that most CaSR-expressing cells co-expressed TRPV1 (sensitive to capsaicin) and TRPA1 (sensitive to allyl isothiocyanate= AITC). Intracellular Ca2+ imaging showed that pretreatment with γ-EVG excited 7% of trigeminal ganglion (TG) cells and increased the amplitude of their responses to AITC, but not to capsaicin or menthol. The enhancing effect of γ-EVG was prevented by a CaSR inhibitor. The results indicate that γ-EVG increases AITC pungency by activating a subset of trigeminal ganglion cells that co-express CaSR and TRPA1.