Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

The role of O-GlcNAcylation mediated by OGT during tooth development.

To understand the mechanisms underlying tooth morphogenesis, we examined the developmental roles of important posttranslational modification, O-GlcNAcylation, which regulates protein stability and activity by the addition and removal of a single sugar (O-GlcNAc) to the serine or threonine residue of the intracellular proteins. Tissue and developmental stage-specific immunostaining results against O-GlcNAc and O-GlcNAc transferase (OGT) in developing tooth germs would suggest that O-GlcNAcylation is involved in tooth morphogenesis, particularly in the cap and secretory stage. To evaluate the developmental function of OGT-mediated O-GlcNAcylation, we employed an in vitro tooth germ culture method at E14.5, cap stage before secretory stage, for 1 and 2 days, with or without OSMI-1, a small molecule OGT inhibitor. To examine the mineralization levels and morphological changes, we performed renal capsule transplantation for one and three weeks after 2 days of in vitro culture at E14.5 with OSMI-1 treatment. After OGT inhibition, morphological and molecular alterations were examined using histology, immunohistochemistry, real-time quantitative polymerase chain reaction, in situ hybridization, scanning electron microscopy, and ground sectioning. Overall, inhibition of OGT resulted in altered cellular physiology, including proliferation, apoptosis, and epithelial rearrangements, with significant changes in the expression patterns of ß-catenin, fibroblast growth factor 4 (fgf4), and sonic hedgehog (Shh). Moreover, renal capsule transplantation and immunolocalizations of Amelogenin and Nestin results revealed that OGT-inhibited tooth germs at cap stage exhibited with structural changes in cuspal morphogenesis, amelogenesis, and dentinogenesis of the mineralized tooth. Overall, we suggest that OGT-mediated O-GlcNAcylation regulates cell signaling and physiology in primary enamel knot during tooth development, thus playing an important role in mouse molar morphogenesis.

Developmental function of Piezo1 in mouse submandibular gland morphogenesis.

Mechanically activated factors are important in organogenesis, especially in the formation of secretory organs, such as salivary glands. Piezo-type mechanosensitive ion channel component 1 (Piezo1), although previously studied as a physical modulator of the mechanotransduction, was firstly evaluated on its developmental function in this study. The detailed localization and expression pattern of Piezo1 during mouse submandibular gland (SMG) development were analyzed using immunohistochemistry and RT-qPCR, respectively. The specific expression pattern of Piezo1 was examined in acinar-forming epithelial cells at embryonic day 14 (E14) and E16, which are important developmental stages for acinar cell differentiation. To understand the precise function of Piezo1 in SMG development, siRNA against Piezo1 (siPiezo1) was employed as a loss-of-function approach, during in vitro organ cultivation of SMG at E14 for the designated period. Alterations in the histomorphology and expression patterns of related signaling molecules, including Bmp2, Fgf4, Fgf10, Gli1, Gli3, Ptch1, Shh, and Tgfß-3, were examined in acinar-forming cells after 1 and 2 days of cultivation. Particularly, altered localization patterns of differentiation-related signaling molecules including Aquaporin5, E-cadherin, Vimentin, and cytokeratins would suggest that Piezo1 modulates the early differentiation of acinar cells in SMGs by modulating the Shh signaling pathway.

Can the subject reliably reproduce maximum voluntary contraction of temporalis and masseter muscles in surface EMG?

OBJECTIVE: To evaluate the repeatability of the surface EMG variables of myoelectric signals from the masseter and temporalis muscles according to three methods to induce maximum voluntary contraction (MVC) in healthy adults. METHODS: Thirty healthy young subjects performed the following three MVC tasks three times each in three sessions on the same day without replacing surface electrodes: clenching the teeth (MVCIC) and biting down on two cotton rolls bilaterally with the posterior teeth (MVCBP) or first molars (MVCB6). RESULTS: The intra-class correlation coefficient (ICC) of the amplitudes during the three MVC tasks ranged from 65 to 79%. The ICCs of the spectral variables ranged from 78 to 86%. The ICCs of the asymmetry index of the masseter ranged from 77 to 86%, and those of the activity index ranged from 68 to 90%. CONCLUSION: The surface EMG measurements according to the three MVC methods exhibited good to excellent reproducibility.

Gene profiling in dorso-ventral patterning of mouse tongue development.

BACKGROUND: The tongue is a muscular fleshy organ in the oral cavity that is anatomically divided into the dorsal, ventral, anterior, and posterior part. The intricate tissue organisation and diverse origins of the tongue make it a complex organ of the oral cavity. OBJECTIVES: To reveal the signalling molecules involved in the formation of the dorsal and ventral parts of the tongue through microarray analysis. METHODS: Dorsal and ventral tongue tissues were isolated from embryonic day 14 mice by micro-dissection. RNA was extracted from the dorsal and ventral tongue tissues separately for microarray analysis. Microarray data were confirmed by quantitative reverse transcription polymerase chain reaction and whole-mount in situ hybridisation. RESULTS: Microarray analysis revealed expression of 33,793 genes. Of these, 931 genes were found to be equally expressed in both the dorsal and ventral parts of the tongue. On limiting the fold-change cut-off to over 1.5-fold, 725 genes were expressed over 1.5-fold in the ventral part and 1,672 in the dorsal part of the tongue. The qPCR and whole-mount in situ hybridisation revealed the expressions of angiopoietin 2 (Angpt2), fibroblast growth factor 18 (Fgf18), mesenchyme homeobox gene1 (Meox1), and SPARC-related modular calcium binding 2 (Smoc2) in the ventral part of the tongue. CONCLUSIONS: Numerous signalling molecules can be selected from our microarray results to examine their roles in tongue development and disease model systems. In the near future, the selection of candidate genes and their functional evaluations will be performed through loss- and gain-of-function mutation studies.

The effect of mucosal dryness on the electrogustometric threshold.

Electrogustometry (EGM) is one of the most useful diagnostic tools widely used to evaluate the taste function by measuring the perception threshold to electrogustatory stimuli on the tongue. However, the effects of oral environments on electrogustometric threshold (EGMT) remain to be established despite its simple applicability. Thus, this study aims to determine the effect of mucosal dryness on EGMT in 68 healthy subjects. The experiment was conducted in two different conditions. First, the baseline EGMT was measured when the dryness of the tongue surface was normal. Second, the EGMT was remeasured after the tongue was intentionally desiccated. The current study showed that the mean of the EGMT was significantly increased when the tongue was desiccated, possibly indicating the reduced sensitivity to electrogustatory stimuli. Such an alteration may be related to the disturbed EGM electrical circuit through the dried mucosa with enhanced impedance. Thus, these findings suggested that mucosal dryness should be considered for better evaluation of gustatory function using EGM.

Facilitation of Reparative Dentin Using a Drug Repositioning Approach With 4-Phenylbutric Acid.

For hard tissue formation, cellular mechanisms, involved in protein folding, processing, and secretion play important roles in the endoplasmic reticulum (ER). In pathological and regeneration conditions, ER stress hinders proper formation and secretion of proteins, and tissue regeneration by unfolded protein synthesis. 4-Phenylbutyric acid (4PBA) is a chemical chaperone that alleviates ER stress through modulation in proteins folding and protein trafficking. However, previous studies about 4PBA only focused on the metabolic diseases rather than on hard tissue formation and regeneration. Herein, we evaluated the function of 4PBA in dentin regeneration using an exposed pulp animal model system via a local delivery method as a drug repositioning strategy. Our results showed altered morphological changes and cellular physiology with histology and immunohistochemistry. The 4PBA treatment modulated the inflammation reaction and resolved ER stress in the early stage of pulp exposure. In addition, 4PBA treatment activated blood vessel formation and TGF-ß1 expression in the dentin-pulp complex. Micro-computed tomography and histological examinations confirmed the facilitated formation of the dentin bridge in the 4PBA-treated specimens. These results suggest that proper modulation of ER stress would be an important factor for secretion and patterned formation in dentin regeneration.

The effects of 4-Phenylbutyric acid on ER stress during mouse tooth development.

Introduction: During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4-Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice. Methods: We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The in vitro cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis. Results and Discussion: Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration.

Management of Chronic Idiopathic Pain in Patients With Dental Implant Without a Clear Pathological Lesion: A Retrospective Study.

Non-nociceptive, persistent idiopathic facial pain (PIFP) is a poorly localized, continuous dull pain that occurs even in the absence of apparent pathological lesions or clinical neurologic deficiency. This study aimed to investigate the disease characteristics of PIFP that developed after dental implant treatment. The clinical characteristics of pain as well as treatment method and outcomes were retrospectively analyzed in 20 patients diagnosed with PIFP. The patients developed pain either after implant fixation or prosthetic treatment. In most patients, the pain persisted not only around the implant region but also at a distant site from the related implant (13/20, 65%). Many patients desired removal of the implants to manage the pain although the pain was not considered to be related to the implant treatment. In 12 patients, the related implants were removed, but 67% (n = 8/12) of the patients still experienced chronic pain after implant removal. Medication helped decrease the pain in most patients (n = 17). Pregabalin and clonazepam showed relatively higher efficiency than other medications for controlling the pain. The results showed that although the onset of PIFP was related to dental implant treatment, implant removal could not be considered a reliable option for the management of PIFP. Although medication controls the pain at least partially, complete pain control with medication should not be expected. These results demonstrate that an accurate diagnosis of PIFP is important for the selection of appropriate treatment.

Facilitating Reparative Dentin Formation Using Apigenin Local Delivery in the Exposed Pulp Cavity.

Apigenin, a natural product belonging to the flavone class, affects various cell physiologies, such as cell signaling, inflammation, proliferation, migration, and protease production. In this study, apigenin was applied to mouse molar pulp after mechanically pulpal exposure to examine the detailed function of apigenin in regulating pulpal inflammation and tertiary dentin formation. In vitro cell cultivation using human dental pulp stem cells (hDPSCs) and in vivo mice model experiments were employed to examine the effect of apigenin in the pulp and dentin regeneration. In vitro cultivation of hDPSCs with apigenin treatment upregulated bone morphogenetic protein (BMP)- and osteogenesis-related signaling molecules such as BMP2, BMP4, BMP7, bone sialoprotein (BSP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN) after 14 days. After apigenin local delivery in the mice pulpal cavity, histology and cellular physiology, such as the modulation of inflammation and differentiation, were examined using histology and immunostainings. Apigenin-treated specimens showed period-altered immunolocalization patterns of tumor necrosis factor (TNF)-α, myeloperoxidase (MPO), NESTIN, and transforming growth factor (TGF)-ß1 at 3 and 5 days. Moreover, the apigenin-treated group showed a facilitated dentin-bridge formation with few irregular tubules after 42 days from pulpal cavity preparation. Micro-CT images confirmed obvious dentin-bridge structures in the apigenin-treated specimens compared with the control. Apigenin facilitated the reparative dentin formation through the modulation of inflammation and the activation of signaling regulations. Therefore, apigenin would be a potential therapeutic agent for regenerating dentin in exposed pulp caused by dental caries and traumatic injury.

Resveratrol enhances bone formation by modulating inflammation in the mouse periodontitis model.

OBJECTIVE: To evaluate the effect of resveratrol on periodontal bone regeneration after local delivery and to determine its effect on inflammatory mediators. BACKGROUND: Resveratrol is considered an anti-inflammatory polyphenolic stilbene involved in the modulation of inflammation. MATERIALS AND METHODS: Periodontitis was induced in mouse molars using a 5-day ligature model followed by the left second molar extraction and 50 µM resveratrol treatment for 1 and 2 weeks. We then examined specimens treated for 1 week histologically and with immunostaining. Microfocus-computed tomography (micro-CT) was used to examine the bone volume formation. RESULTS: After 1 week of treatment, proinflammatory cytokine levels (TNF-alpha and IL6), cells exhibiting neutrophil and macrophage marker (MPO), cell proliferation marker (Ki67), and preosteoblastic marker (RUNX2) reactivity decreased in the resveratrol-treated specimens compared to the control group. In contrast, we observed a higher number of CD31-, F4/80-, and osteocalcin- (OCN-) positive cells in the resveratrol-treated specimens. After 2 weeks, micro-CT confirmed an increased bone mass in the region of the extraction socket in the resveratrol-treated group. CONCLUSION: After 1 week, the resveratrol-treated specimens revealed evidence of inflammation modulation compared to the control group. These data suggest that resveratrol not only affects inflammation control but also is useful for treating periodontitis-related tissue defects and bone regeneration.

Implications of the specific localization of YAP signaling on the epithelial patterning of circumvallate papilla.

Circumvallate papilla (CVP) is a distinctively structured with dome-shaped apex, and the surrounding trench which contains over two hundred taste buds on the lateral walls. Although CVP was extensively studied to determine the regulatory mechanisms during organogenesis, it still remains to be elucidated the principle mechanisms of signaling regulations on morphogenesis including taste buds formation. The key role of Yes-associated protein (YAP) in the regulation of organ size and cell proliferation in vertebrates is well understood, but little is known about the role of this signaling pathway in CVP development. We aimed to determine the putative roles of YAP signaling in the epithelial patterning during CVP morphogenesis. To evaluate the precise localization patterns of YAP and other related signaling molecules, including ß-catenin, Ki67, cytokeratins, and PGP9.5, in CVP tissue, histology and immunohistochemistry were employed at E16 and adult mice. Our results suggested that there are specific localization patterns of YAP and Wnt signaling molecules in developing and adult CVP. These concrete localization patterns would provide putative involvements of YAP and Wnt signaling for proper epithelial cell differentiation including the formation and maintenance of taste buds.

Facilitation of Bone Healing Processes Based on the Developmental Function of Meox2 in Tooth Loss Lesion.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson's trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1ß, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Ginsenoside F2 attenuates chronic-binge ethanol-induced liver injury by increasing regulatory T cells and decreasing Th17 cells.

BACKGROUND: Recently, beneficial roles of ginsenoside F2 (GF2), a minor constituent of Panax ginseng, have been demonstrated in diverse inflammatory diseases. However, its roles in alcoholic liver inflammation and injury have not been clearly understood. Here, we investigated the underlying mechanism by which GF2 ameliorated alcoholic liver injury. METHODS: To induce alcoholic liver injury, C57BL/6J wild type (WT) or interleukin (IL)-10 knockout (KO) mice were orally administered with ethanol (3 g/kg) or ethanol-containing GF2 (50 mg/kg) for 2 wk. Liver injury and infiltration of macrophages and neutrophils were evaluated by serum biochemistry and immunohistochemistry, respectively. The changes of hepatic immune cells were assessed by flow cytometry and polymerase chain reaction analysis. In vitro differentiation of naïve T cells was performed. RESULTS: GF2 treatment significantly attenuated alcoholic liver injury, in which infiltrations of inflammatory macrophages and neutrophils were decreased. Moreover, the frequencies of Foxp3+ regulatory T cells (Tregs) increased but IL-17-producing T (Th17) cells decreased in GF2-treated mice compared to controls. Furthermore, the mRNA expression of IL-10 and Foxp3 was significantly increased, whereas IL-17 mRNA expression was suppressed in GF2-treated mice. However, these beneficial roles of GF2 were not observed in GF2-treated IL-10 KO mice, suggesting a critical role of IL-10. Similarly, GF2 treatment suppressed differentiation of naïve T cells into Th17 cells by inhibiting RORγt expression and stimulating Foxp3 expression. CONCLUSION: The present study suggests that GF2 treatment attenuates alcoholic liver injury by increasing IL-10 expression and Tregs and decreasing IL-17 expression and Th17 cells.

Experimental Animal Model Systems for Understanding Salivary Secretory Disorders.

Salivary secretory disorders are life-disrupting pathologic conditions with a high prevalence, especially in the geriatric population. Both patients and clinicians frequently feel helpless and get frustrated by the currently available therapeutic strategies, which consist mainly of palliative managements. Accordingly, to unravel the underlying mechanisms and to develop effective and curative strategies, several animal models have been developed and introduced. Experimental findings from these models have contributed to answer biological and biomedical questions. This review aims to provide various methodological considerations used for the examination of pathological fundamentals in salivary disorders using animal models and to summarize the obtained findings. The information provided in this review could provide plausible solutions for overcoming salivary disorders and also suggest purpose-specific experimental animal systems.

Stage-specific expression patterns of ER stress-related molecules in mice molars: Implications for tooth development.

The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.

The correlation of gagging threshold with intra-oral tactile and psychometric profiles in healthy subjects: A pilot study.

Excessive gag reflex could be problematic for adequate dental care. Although various factors may increase the susceptibility to gagging, its contributing factors have not been fully determined. This study aimed to determine whether gag reflex was associated with tactile sensitivity and psychological characteristics. Fifteen volunteers of healthy males and females each were recruited for this study. After completing a questionnaire describing the self-perceived gag reflex activity, a disposable saliva ejector was inserted along the palate into the mouth until gagging was evoked. The ratio of the insertion depth to the palatal length was used as an index for the gagging threshold. The two-point discrimination (TPD) and Semmes-Weinstein monofilament (SWM) tests were performed to assess the tactile sensitivity of the palatal regions (hard palate, anterior and posterior soft palate). The Symptom Checklist-90-Revised was used to investigate the relationship between the gagging threshold and the psychological status. Our findings showed that the gagging threshold had a significant positive correlation with the TPD and SWM thresholds on the hard palate. The psychological profiles of psychoticism and hostility score were also significantly correlated with the gagging threshold. However, there were no significant differences in the tactile and gagging thresholds, as well as the psychological profiles, between males and females. Our results suggested that the tactile sensitivity of the anterior palate is a determining factor for the gagging threshold and implied that the initial response of the oral entry site to stimulation may lead to the development of gag reflex.

Vesicular Glutamate Transporter 1 (VGLUT1)- and VGLUT2-containing Terminals on the Rat Jaw-closing γ-Motoneurons.

Currently, compared to jaw-closing (JC) α-motoneurons, the information on the distribution and morphology of glutamatergic synapses on the jaw-closing (JC) γ-motoneurons, which may help elucidate the mechanism of isometric contraction of the JC muscle, is very limited. This study investigated the distribution and ultrastructural features of vesicular glutamate transporter 1 (VGLUT1)- and VGLUT2-immunopositive (+) axon terminals (boutons) on JC γ-motoneurons by retrograde tracing with horseradish peroxidase, electron microscopic immunocytochemistry, and quantitative analysis. About 35% of the boutons on identified JC γ-motoneurons were VGLUT+, and of those, 99% were VGLUT2+. The fraction of VGLUT1+ boutons of all boutons and the percentage of membrane of JC γ-motoneurons covered by these boutons were significantly lower than those for the JC α-motoneurons, revealed in our previous work. The bouton volume, mitochondrial volume, and active zone area of the VGLUT2+ boutons on the JC γ-motoneurons were uniformly small. These findings suggest that the JC γ-motoneurons, in contrast to the JC α-motoneurons, receive generally weak glutamatergic synaptic input almost exclusively from VGLUT2+ premotoneurons that form direct synapse with motoneurons.