Ascl1-expressing cell differentiation in initially developed taste buds and taste organoids.

Mammalian taste bud cells are composed of several distinct cell types and differentiated from surrounding tongue epithelial cells. However, the detailed mechanisms underlying their differentiation have yet to be elucidated. In the present study, we examined an Ascl1-expressing cell lineage using circumvallate papillae (CVP) of newborn mice and taste organoids (three-dimensional self-organized tissue cultures), which allow studying the differentiation of taste bud cells in fine detail ex vivo. Using lineage-tracing analysis, we observed that Ascl1 lineage cells expressed type II and III taste cell markers both CVP of newborn mice and taste organoids. However, the coexpression rate in type II cells was lower than that in type III cells. Furthermore, we found that the generation of the cells which express type II and III cell markers was suppressed in taste organoids lacking Ascl1-expressing cells. These findings suggest that Ascl1-expressing precursor cells can differentiate into both type III and a subset of type II taste cells.

Msx1 is essential for proper rostral tip formation of the mouse mandible.

The right and left mandibular processes derived from the first branchial arch grow toward the midline and fuse to create the rostral tip region of the mandible during mandibular development. Severe and mild cases of failure in this process results in rare median cleft of the lower lip and cleft chin, respectively. The detailed molecular mechanisms of mandibular tip formation are unknown. We hypothesize that the Msx1 gene is involved in mandibular tip development, because Msx1 has a central role in other craniofacial morphogenesis processes, such as teeth and the secondary palate development. Normal Msx1 expression was observed in the rostral end of the developing mandible; however, a reduced expression of Msx1 was observed in the soft tissue of the mandibular tip than in the lower incisor bud region. The rostral tip of the right and left mandibular processes was unfused in both control and Msx1-null (Msx1-/-) mice at embryonic day (E) 12.5; however, a complete fusion of these processes was observed at E13.5 in the control. The fused processes exhibited a conical shape in the control, whereas the same region remained bifurcated in Msx1-/-. This phenotype occurred with 100% penetrance and was not restored at subsequent stages of development. Furthermore, Meckel's cartilage in addition to the outline surface soft tissues was also unfused and bifurcated in Msx1-/- from E14.5 onward. The expression of phosho-Smad1/5, which is a mediator of bone morphogenetic protein (Bmp) signaling, was downregulated in the mandibular tip of Msx1-/- at E12.5 and E13.5, probably due to the downregulated Bmp4 expression in the neighboring lower incisor bud. Cell proliferation was significantly reduced in the midline region of the mandibular tip in Msx1-/- at the same developmental stages in which downregulation of pSmad was observed. Our results indicate that Msx1 is indispensable for proper mandibular tip development.

Fibrillin-1 regulates periostin expression during maintenance of periodontal homeostasis.

Background/purpose: Human periodontal ligament consists of elastic system fibers, mainly fibrillin-1 (FBN1). Periostin (POSTN) maintains periodontal homeostasis. A previous study showed that the expression of Postn in periodontal ligament cells was decreased in mice underexpressing Fbn1. However, the relationship between FBN1 and POSTN is not fully understood in the context of mechanical stress. FBN1 contributes to transforming growth factor ß1 (TGF-ß1) activation; TGF-ß1 upregulates the expression of POSTN in human periodontal ligament cells. This study examined whether FBN1 contributed to the maintenance of periodontal homeostasis in cultured human periodontal ligament cells. Materials and methods: Human periodontal ligament fibroblasts (HPDLFs) were exposed to mechanical force via centrifugation. The expression of POSTN was examined by quantitative reverse transcription polymerase chain reaction. The phosphorylation of Smad2 in the TGF-ß/Smad signaling pathway was monitored by western blotting. Results: The expression levels of FBN1 and POSTN were not significantly decreased by centrifugation. However, the expression of POSTN after centrifugation significantly decreased upon knockdown of FBN1. The phosphorylation of Smad2 after centrifugation was decreased, regardless of FBN1 knockdown. Supplementation with 0.1 ng/ml recombinant human TGF-ß1 rescued POSTN expression after centrifugation in HPDLFs upon knockdown of FBN1. Conclusion: FBN1 regulates the expression of POSTN to maintain periodontal homeostasis via TGF-ß/Smad signaling during centrifugation.

Cisplatin induces TRPA1-mediated mechanical allodynia in the oral mucosa.

OBJECTIVE: Cisplatin, a platinum-based anticancer drug, produces reactive oxygen species (ROS) in many cell types and induces mechanical allodynia in the hands and/or feet (chemotherapy-induced painful neuropathy: CIPN). In this study, we examined the possibility of inducing neuropathy in the oral region using oral keratinocytes and rats. METHODS: Human oral keratinocytes (HOKs) were used to evaluate ROS generation after cisplatin application by a ROS-reactive fluorescent assay. In rats, after cisplatin administrations (two times), the trigeminal ganglion (TG) was investigated by electron microscopy and quantitative RT-PCR. Using our proprietary assay system, oral pain-related behaviors were observed in cisplatin-treated rats. RESULTS: In rats, cisplatin administration reduced food intake and body weight. In electron microscopic analysis, glycogen granules in the TG were depleted following administration, although organelles were intact. In HOK cells, cisplatin significantly increased ROS generation with cell death, similar to glycolysis inhibitors. Cisplatin administration did not show any effects on Trpa1 mRNA levels in the TG. However, the same procedure induced hypersensitivity to mechanical stimulation and the TRPA1 agonist allyl isothiocyanate in the oral mucosa. Mechanical hypersensitivity was inhibited by the antioxidative drug α-lipoic acid and the TRPA1 antagonist HC-030031, similar to that of the hind paw. CONCLUSION: The present findings suggest that cisplatin induces TRPA1-mediated CIPN due to ROS generation in the oral region. This study will provide a better understanding of persistent oral pain in cancer patients.

Myogenic differentiation 1 and transcription factor 12 activate the gene expression of mouse taste receptor type 1 member 1.

OBJECTIVES: Myogenic differentiation 1 (Myod1) is involved in the expression of taste receptor type 1 member 1 (Tas1r1) during myogenic differentiation. Further, the target genes of Myod1 participate in transcriptional control, muscle development, and synaptic function. We examined, for the first time, the function of Myod1 in the transcriptional regulation of Tas1r1. METHODS: ENCODE chromatin immunoprecipitation and sequencing (ChIP-seq) data of myogenically differentiated C2C12 cells were analyzed to identify the Myod1 and transcription factor 12 (Tcf12) binding sites in the Tas1r1 promoter region. Luciferase reporter assays, DNA affinity precipitation assays, and co-immunoprecipitation assays were also performed to identify the functions of Myod1, Tcf12, and Krüppel-like factor 5 (Klf5). RESULTS: Based on ENCODE ChIP-seq, Myod1 bound to the Tas1r1 promoter region containing E-boxes 1-3. Luciferase reporter assays revealed that site-directed E-box1 mutations significantly reduced promoter activation induced by Myod1 overexpression. According to the DNA affinity precipitation assay and co-immunoprecipitation assay, Myod1 formed a heterodimer with Tcf12 and bound to E-box1. Further, Klf5 bound to the GT box near E-box1, activating Tas1r1 expression. CONCLUSIONS: During myogenic differentiation, the Myod1/Tcf12 heterodimer, in collaboration with Klf5, binds to E-box1 and activates Tas1r1 expression.

Msx1 Heterozygosity in Mice Enhances Susceptibility to Phenytoin-Induced Hypoxic Stress Causing Cleft Palate.

OBJECTIVE: Cleft palate is among the most frequent congenital defects in humans. While gene-environment multifactorial threshold models have been proposed to explain this cleft palate formation, only a few experimental models have verified this theory. This study aimed to clarify whether gene-environment interaction can cause cleft palate through a combination of specific genetic and environmental factors. METHODS: Msx1 heterozygosity in mice (Msx1+/-) was selected as a genetic factor since human MSX1 gene mutations may cause nonsyndromic cleft palate. As an environmental factor, hypoxic stress was induced in pregnant mice by administration of the antiepileptic drug phenytoin, a known arrhythmia inducer, during palatal development from embryonic day (E) 11 to E14. Embryos were dissected at E13 for histological analysis or at E17 for recording of the palatal state. RESULTS: Phenytoin administration downregulated cell proliferation in palatal processes in both wild-type and Msx1+/- embryos. Bone morphogenetic protein 4 (Bmp4) expression was slightly downregulated in the anterior palatal process of Msx1+/- embryos. Although Msx1+/- embryos do not show cleft palate under normal conditions, phenytoin administration induced a significantly higher incidence of cleft palate in Msx1+/- embryos compared to wild-type littermates. CONCLUSION: Our data suggest that cleft palate may occur because of the additive effects of Bmp4 downregulation as a result of Msx1 heterozygosity and decreased cell proliferation upon hypoxic stress. Human carriers of MSX1 mutations may have to take more precautions during pregnancy to avoid exposure to environmental risks.

Mash1-expressing cells may be relevant to type III cells and a subset of PLCß2-positive cell differentiation in adult mouse taste buds.

Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1CreERT2::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L-amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C ß2 (PLCß2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats.

OBJECTIVE: Pain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DESIGN: Expression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. RESULTS: After HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 µM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). CONCLUSIONS: These results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.

Krüppel-like factor 5 (Klf5) regulates expression of mouse T1R1 amino acid receptor gene (Tas1r1) in C2C12 myoblast cells.

T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds.

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers-aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)-were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)-markers of type II taste bud cells-were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.

Hey1 and Hey2 are differently expressed during mouse tooth development.

The Hey family (also known as Chf, Herp, Hesr, and Hrt) is a set of Hairy/Enhancer of Split-related basic helix-loop-helix type transcription factors. Hey1, Hey2, and HeyL have been identified in mammals. Although Hey proteins are known to regulate cardiovascular development, muscle homeostasis, osteogenesis, neurogenesis, and oncogenesis, their roles in tooth development have been largely obscure. Therefore, this study aimed to clarify detailed spatiotemporal expression patterns of Hey1 and Hey2 in developing molars and incisors of mice by section in situ hybridization. Hey1 and Hey2 were not significantly expressed in tooth germs at epithelial thickening, bud, and cap stages during molar development. In the dental epithelium in molars at the bell stage and incisors, Hey2 transcripts were restricted to the undifferentiated inner enamel epithelium and down-regulated in preameloblasts and ameloblasts. On the other hand, Hey1 was mainly expressed in preameloblasts and down-regulated in differentiated ameloblasts. Both genes were not significantly expressed in other dental epithelial tissues, including the outer enamel epithelium, stellate reticulum, and stratum intermedium cells. In the dental mesenchyme, Hey1 was intensely transcribed in the subodontoblastic layer of the dental pulp in both molars and incisors, whereas Hey2 was barely detectable in mesenchymal components. Our data implied that Hey2 function is restricted to transient amplifying cells of the ameloblast cell lineage and that Hey1 plays a role in the composition of the subodontoblastic layer, in addition to ameloblast differentiation. These findings provide novel clues for the better understanding of tooth development.

Lysophosphatidylcholine acyltransferase 4 is involved in chondrogenic differentiation of ATDC5 cells.

Glycerophospholipids have important structural and functional roles in cells and are the main components of cellular membranes. Glycerophospholipids are formed via the de novo pathway (Kennedy pathway) and are subsequently matured in the remodeling pathway (Lands' cycle). Lands' cycle consists of two steps: deacylation of phospholipids by phospholipases A2 and reacylation of lysophospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play key roles in the maturation and maintenance of the fatty acid composition of biomembranes, and cell differentiation. We examined whether LPLATs are involved in chondrogenic differentiation of ATDC5 cells, which can differentiate into chondrocytes. Lysophosphatidylcholine acyltransferase 4 (LPCAT4) mRNA expression and LPCAT enzymatic activity towards 18:1-, 18:2-, 20:4-, and 22:6-CoA increased in the late stage of chondrogenic differentiation, when mineralization occurred. LPCAT4 knockdown decreased mRNA and protein levels of chondrogenic markers as well as Alcian blue staining intensity and alkaline phosphatase activity in ATDC5 cells. These results suggest that LPCAT4 plays important roles during the transition of chondrocytes into hypertrophic chondrocytes and/or a mineralized phenotype.

ADAM17 regulates TNF-α expression upon lipopolysaccharide stimulation in oral keratinocytes.

A disintegrin and metalloprotease 17 (ADAM17) is a tumor necrosis factor (TNF)-converting enzyme and was first identified as the enzyme that cleaves the prodomain of TNF-α, a proinflammatory cytokine that plays a central role in immune regulation and a variety of inflammatory responses in destructive periodontal disease. The aim of the present study was to verify the presence of ADAM17 in the gingival epithelium and elucidate its involvement in the release of TNF-αin oral keratinocytes. Immunohistochemical analyses of ADAM17 were performed in gingival tissues obtained from patients and in human oral keratinocytes (HOKs). Additionally, levels of TNF-α and ADAM17 in HOKs exposed to lipopolysaccharide (LPS) were assessed using enzyme-linked immunosorbent assays. Moreover, the effects of ADAM17 inhibitor, matrix metalloproteinase (MMP) inhibitor, and ADAM17 siRNA on TNF-α concentration were assessed. Strong immunoreactivity for ADAM17 was observed in the epithelium of the inflamed gingival tissues and in HOKs. Furthermore, treatment with either ADAM17 inhibitor or ADAM17 siRNA inhibited the generation of TNF-α induced by LPS in HOKs. The present study demonstrates that ADAM17 is strongly expressed in the epithelium of gingival tissues and suggests that ADAM17 may be a key enzyme that regulates the generation of TNF-α in oral keratinocytes.

Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts.

Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp.

Asporin in compressed periodontal ligament cells inhibits bone formation.

OBJECTIVE: During orthodontic tooth movement, bone resorption and inhibition of bone formation occur on the compressed side, thereby preventing ankylosis. Periodontal ligament (PDL) cells control bone metabolism and inhibition of bone formation on the compressed side by secreting bone-formation inhibitory factors such as asporin (ASPN) or sclerostin (encoded by SOST). The aim of this study was to identify the inhibitory factors of bone formation in PDL cells. DESIGN: In vitro, the changes in expression of ASPN and SOST and subsequent protein release in human PDL (hPDL) cells were assessed by semi-quantitative polymerase chain reaction (PCR), real-time PCR, and immunofluorescence in hPDL cells subjected to centrifugal force using a centrifuge (45, 90, 135, and 160 × g). In vivo, we applied a compressive force using the Waldo method in rats, and examined the distribution of ASPN or sclerostin by immunohistochemistry. RESULTS: In vitro, hPDL cells subjected to 90 × g for 24h demonstrated upregulated ASPN and downregulated SOST expressions, which were confirmed by immunofluorescent staining. In addition, the formation of mineralized tissue by human osteoblasts was significantly inhibited by the addition of medium from hPDL cells cultured during compressive force as well as the addition of equivalent amounts of ASPN peptide. In vivo, asporin-positive immunoreactive PDL cells and osteoclasts were found on the compressed side, whereas few sclerostin-positive PDL cells were observed. CONCLUSIONS: PDL cells subjected to an optimal compressive force induce the expression and release of ASPN, which inhibits bone formation during orthodontic tooth movement on the compressed side.

A spatial association between odontomas and the gubernaculum tracts.

OBJECTIVES: The purpose of this study was to identify the spatial relationship and/or association between odontomas and the gubernaculum tract or the dental sac and the characteristic findings for radiographic diagnosis of odontomas. STUDY DESIGN: The visualizations of the odontomas and the gubernaculum tract were retrospectively analyzed using cone beam computed tomography or multidetector computed tomography. RESULTS: Most of odontomas were within the gubernaculum tract or dental sac of unerupted permanent teeth on computed tomography. In some odontomas, the gubernaculum tract existed as a well-defined low density tract extending from the top of odontomas on computed tomography. CONCLUSIONS: A close spatial relationship and/or association between odontomas and the gubernaculum tract or dental sac on computed tomography may be used as one of the criteria for radiographic diagnosis of odontomas. Development of odontomas may be associated with the gubernaculum tract or dental sac of unerupted permanent teeth.

Muscle regulatory factors regulate T1R3 taste receptor expression.

T1R3 is a T1R class of G protein-coupled receptors, composing subunit of the umami taste receptor when complexed with T1R1. T1R3 was originally discovered in gustatory tissue but is now known to be expressed in a wide variety of tissues and cell types such the intestine, pancreatic ß-cells, skeletal muscle, and heart. In addition to taste recognition, the T1R1/T1R3 complex functions as an amino acid sensor and has been proposed to be a control mechanism for the secretion of hormones, such as cholecystokinin, insulin, and duodenal HCO3(-) and activates the mammalian rapamycin complex 1 (MTORC1) to inhibit autophagy. T1R3 knockout mice have increased rate of autophagy in the heart, skeletal muscle and liver. Thus, T1R3 has multiple physiological functions and is widely expressed in vivo. However, the exact mechanisms regulating T1R3 expression are largely unknown. Here, we used comparative genomics and functional analyses to characterize the genomic region upstream of the annotated transcriptional start of human T1R3. This revealed that the T1R3 promoter in human and mouse resides in an evolutionary conserved region (ECR). We also identified a repressive element located upstream of the human T1R3 promoter that has relatively high degree of conservation with rhesus macaque. Additionally, the muscle regulatory factors MyoD and Myogenin regulate T1R3 expression and T1R3 expression increases with skeletal muscle differentiation of murine myoblast C2C12 cells. Taken together, our study raises the possibility that MyoD and Myogenin might control skeletal muscle metabolism and homeostasis through the regulation of T1R3 promoter activity.

Detection and imaging characteristics of the gubernacular tract in children on cone beam and multidetector computed tomography.

PURPOSE: To elucidate the appearance and imaging characteristics of the gubernacular tract (GT) during the growth stage of children. Furthermore, this study evaluated the significance of the appearance of the GT. STUDY DESIGN: The visualizations of the GT were retrospectively analyzed by using panoramic radiographs and computed tomography (CT) in children. RESULTS: In patients with normal eruption who had unerupted permanent teeth, except maxillary central supernumerary teeth, the GT was clearly visualized as a well-defined low-density tract on CT but not on panoramic radiographs. In patients with obstructive eruption, including impaction, the GT was deformed and not visible on CT. CONCLUSIONS: This paper describes the frequency of detection and appearance of the GT in unerupted teeth. Preliminary data suggest that any alteration to the GT may be used to predict abnormal eruption of permanent teeth.

Novel methods of applying direct chemical and mechanical stimulation to the oral mucosa for traditional behavioral pain assays in conscious rats.

BACKGROUND: Stomatitis induces severe and painful hypersensitivity to pungency and physical contact during meals. Many studies have used anesthetized animals to examine evoked nociception in the oral mucosa, but no reports have used traditional behavioral assays to evaluate nociception in conscious animals. NEW METHODS: We developed two new methods of applying chemical or mechanical stimulation directly to the oral mucosa of the mandibular vestibule of conscious rats. Nociceptive evaluations were performed by measuring facial grooming time and the head withdrawal threshold to von Frey stimulations. (1) For the intraoral dropping method, rat mucosa was transiently exposed by hand, and a drop of a pungent solution was applied. (2) For the stable intraoral opening method, rat mucosa was long-term exposed following piercing surgery of the mental skin after habitual training for 2-3 weeks. RESULTS: In the intraoral dropping method, the application of 100 µM capsaicin or 100 mM allyl isothiocyanate prolonged mouth-rubbing time. Capsaicin-induced mouth-rubbing time was further enhanced following the development of an acetic acid-induced ulcer. The stable intraoral opening method enabled stable measurements of the mechanical withdrawal threshold in the oral mucosa of conscious rats. Ulcer development decreased the mechanical threshold, whereas topical lidocaine treatment increased the threshold. COMPARISON WITH EXISTING METHODS: These new methods enable the evaluations of motivational nocifensive behaviors in response to intraoral stimulations without any anesthetic effects. CONCLUSIONS: The intraoral dropping and stable intraoral opening methods can be used in combination with traditional behavioral assays to evaluate nociception in the oral mucosa of conscious rats.

Expression of GAD67 and Dlx5 in the taste buds of mice genetically lacking Mash1.

It has been reported that a subset of type III taste cells express glutamate decarboxylase (GAD)67, which is a molecule that synthesizes gamma-aminobutyric acid (GABA), and that Mash1 could be a potential regulator of the development of GABAnergic neurons via Dlx transcription factors in the central nervous system. In this study, we investigated the expression of GAD67 and Dlx in the embryonic taste buds of the soft palate and circumvallate papilla using Mash1 knockout (KO)/GAD67-GFP knock-in mice. In the wild-type animal, a subset of type III taste cells contained GAD67 in the taste buds of the soft palate and the developing circumvallate papilla, whereas GAD67-expressing taste bud cells were missing from Mash1 KO mice. A subset of type III cells expressed mRNA for Dlx5 in the wild-type animals, whereas Dlx5-expressing cells were not evident in the apical part of the circumvallate papilla and taste buds in the soft palate of Mash1 KO mice. Our results suggest that Mash1 is required for the expression of GAD67 and Dlx5 in taste bud cells.