Expression of Neurotrophic Factors and Their Receptors in Rat Posterior Taste Bud Cells

Taste is an important sense in survival and growth of animals. The growth and maintenance of taste buds, the receptor organs of taste sense, are under the regulation of various neurotrophic factors. But the distribution aspect of neurotrophic factors and their receptors in distinct taste cell types are not clearly known. The present research was designed to characterize mRNA expression pattern of neurotrophic factors and their receptors in distinct type of taste cells. In male 45-60 day-old Sprague-Dawley rats, epithelial tissues with and without circumvallate and folliate papillaes were dissected and homogenized, and mRNA expressions for neurotrophic factors and their receptors were determined by RT-PCR. The mRNA expressions of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), receptor tyrosine kinase B (TrkB), exclusion of nerve growth factor (NGF), neurotrophin-4/5 (NT4/5), receptor tyrosine kinase A (TrkA), receptor tyrosine kinase C (TrkC), and p75NGFR were observed in some population of taste cell. In support of this result and to characterize which types of taste cells express NT3, BDNF, or TrkB, we examined mRNA expressions of NT3, BDNF, or TrkB in the PLCbeta2 (a marker of Type II cell)- and/or SNAP25 (a marker of Type III cell)-positive taste cells by a single taste cell RT-PCR and found that the ratio of positively stained cell numbers were 17.4, 6.5, 84.1, 70.3, and 1.4% for PLCbeta2, SNAP25, NT3, BDNF, and TrkB, respectively. In addition, all of PLCbeta2- and SNAP25-positive taste cells expressed NT3 mRNA, except for one taste bud cell. The ratios of NT3 mRNA expressions were 100% and 91.7% in the SNAP25- and PLCbeta2-positive taste cells, respectively. However, two TrkB-positive taste cells co-expressed neither PLCbeta2 nor SNAP 25. The results suggest that the most of type II or type III cells express BDNF and NT3 mRNA, but the expression is shown to be less in type I taste cells.

Células neuroendocrinas en yemas gustativas primordiales / Neuroendocrine cells in primordial taste buds

El objetivo del trabajo fue identificar la presencia de células neuroendócrinas en yemas gustativas primordiales (fetales) humanas. Fueron utilizadas 15 lenguas fetales humanas obtenidas de abortos espontáneos (tiempo de gestación 23 semanas) de pacientes ingresados en el Hospital General, Chihuahua, México. Una muestra representativa del ápex lingual fue embebida en parafina y cortada a 3 micras para ser procesadas con la técnica inmunohitoquímica utilizando los siguientes anticuerpos: Anti-sinaptofisina; anti-proteína neurofilamento; anti-cromogranina; anti-citoqueratina 20; y anti proteínas-S100. El protocolo de investigación fue aprobado por los comités de bioética de las instituciones participantes. Se obtuvo la autorización de los padres para la utilización del material biológico. Las células gustativas fetales fueron fuertemente positivas a cromogranina, y negativas a sinaptofisina. Las papilas gustativas fetales humanas mostraron inmunorreactividad positiva contra citoqueratina 20, mientras que fibras nerviosas intragemulares mostraron inmunorreactividad contra anticuerpos anti-proteína neurofilamento. Las fibras nerviosas subyacentes al epitelio gustativo fueron positivas a proteína S100. Se muestra evidencia inmunohistoquímica de la presencia de células neuroendócrinas gustativa en yemas gustativas linguales primordiales humanas. Esto sugiere una probable participación neuroendocrina o paracrina en el desarrollo de las yemas gustativas humanas.

The objective was to identify the presence of neuroendocrine cells in primary taste buds (fetal) cells. We used 15 human embryionic fetal tongues derived from human spontaneous abortions (23 weeks gestation time) of patients admitted to the General Hospital, Chihuahua Mexico. A representative sample of lingual apex was embedded in paraffin and cut to 3 microns processed by immune histochemical technique using the following antibodies: anti-synaptophysin, neuro filament anti-protein, anti chromogranin; anti-citokeratin 20 and S100 anti proteins. Research was approved by bioethics committees of the participating institutions. Permission was obtained from the parents to use the biological material. The taste bud fetal cells were significantly positive for chromogranin and synaptophysin negative. The human fetal taste buds showed positive immunoreactivity against cyto keratin 20, while nerve fibers underlying the gustatory epithelium were positive for S100 protein. Immunohistochemical evidence shows the presence of neuro endocrine cells in human primordial taste bud papilla. This suggests a probable neuro endocrine or paracrine participation in the development of human taste buds.

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCbeta2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Immunohistochemical Study on the Calretinin and Neuronal Nitric Oxide Synthase in the Rat Tongue / 대한해부학회지

To examine the distribution of calretinin and neuronal nitric oxide synthase (nNOS) in tongue, immunohistochemistry was conducted to cryosections of vallate, foliate and fungiform papillae of rats.Immunohistochemistry revealed that nerve fibers around taste buds and ganglia cells contained calretinin and nNOS each. In vallate and foliate papillae, calretinin nerve fibers formed subgemmal nerve plexus under the basal lamina, and branched intragemmal nerve fibers. Under the fungiform papillae, thick calretinin nerve bundles were found. Tthese nerve fibers formed subgemmal nerve plexus under the basal lamina, and some varicose fibers were located in taste bud. Ganglia cells, 20~40 mm in diameter, formed groups under the vallate papilla and showed strong immunoreactivity for nNOS. Their nerve fibers branched into blood vessels. Ganglia cells around serous gland also contained nNOS and their nerve fibers branched into acini, ducts and blood vessels. Based on these findings, it is postulated that calretinin nerve fibers function as a somatosensory or taste sensory nerves in taste papillae. The intralingual nNOS ganglia cells may function as vasodilator and control the secretion of serous salivary glands.

Immuno-electronmicroscopic study on the serotoninergic taste cells and calcitonin gene-related peptide nerve fibers in mouse taste buds / 대한해부학회지

To investigate the distribution, ultrastructure and synapsis of serotoninergic cells and CGRP nerve fibers in mammalian taste buds, immunohistochemistry and electronmicroscopy were applied to mice vallate papillae. In normal mice, 1~2 serotonin immunoreactive cells were present in each taste bud section. After preloading 5-HTP, 3~6 cells showed strong immunoreactivity for serotonin. These cells were elongated, and their cytoplasm extended from the taste pore to the base of the taste bud. CGRP nerve fibers formed thick subgemmal nerve plexus under the basal lamina, and branched varicose perigemmal and intragemmal nerve fibers. Under the electron-microscope, three types of taste cells; dark cell, light cell and basal cells, were identified by their shape, location and electrical densities. Immuno-electronmicroscopy revealed that serotoninergic cells were dark cells. CGRP nerve fibers were located in and around taste buds, but the synaptic contacts with taste cells was not found. These findings indicate that mice taste cells are consisted of dark cell, light cell and basal cells, and dark cells contain serotonin. And, CGRP nerve fibers in taste buds may function as general sensory fibers.

Immunohistochemical study on the dopaminergic and norepinephrinergic taste cells in rat taste buds / 대한해부학회지

Immunohistochemistry was applied to rat circumvallate papilla to localize the dopamine and norepinephrine in taste bud and to investigate the effect of preloading L-DOPA, dopamine and norepinephrine into taste cells. Dopamine and norepinephrine immunohistochemistry demonstrated that two to four taste cells except basal cells were weakly immunopositive for these neurotransmitters. Immunoreactive cells were elongated and their cell processes extended from the taste pore to the base of the taste bud. After pretreating animals with L-DOPA, four to six taste bud cells showed strong immunoreactivity for dopamine, but weak immunoreactivity for norepinephrine. Administration of dopamine or norepinephrine did not alter the number or intensity of immunoreactive cells in taste bud. These findings indicated that mammalian taste cells normally contain dopamine and norepinephrine, and that taste cells can take up L-DOPA and convert it to dopamine. Based on these findings, we postulate that norepinephrine functions as neurotransmitters or neuromodulators in taste sensory transmission.

Immunohistochemical Study on Cfokeratin Expression in Lingual Epithelium of Human Fetus / 대한해부학회지

The human oral mucosa has noncornified lining epithelium, cornified masticatory epithelium, and complex epithelium. The epithelium of human tongue shows diverse morphological variations from one site to another, and conflicting reports exist in the literature concerning the type of lingual epithelium. Cytokeratin[CK] have been shown to characterize different type of epithelia. In the present study to clarify intermediate filament patterns of tongue mucosa and lingual gland in human fetus from second trimester of pregnancy and adult, cytokeratin expression was investigated immunohistochemically using antibodies for cytokeratins in the dorsal surface of tongue, taste bud, and lingual gland, and comparison with the expression between fetus and adult was made. The epithelium of the fetal lingual papillae consisted of 4 to 6 layered stratified cells, and that of the inferior surface of tongue consisted of 6-8 layered stratified cells in PAS-hematoxylin stain. The lingual gland was well differentiated and the alveolus was strongly positive to PAS reaction. The dorsal surface of the adult tongue was composed of lingual papillae. The papillae was covered by keratinized stratified squamous epithelium and interpapillary area by nokeratinized epithelium in hematoxylin-eosin stain. The taste bud was present in the fungiform papilla. The lingual gland appeared among the muscle layers near the inferior surtace and was composed of mucous and serous cells. By immunohistochemical stain, the epithelium of the lingual papillae exhibited various staining-intensities for cytokeratin antibodies, and showed same staining patterns bosh epithelium in tip of papilla and interpapillary area in fetus. However, the dorsal epithelium of the adult tongue showed different staining patterns between tip of the papilla and interpapillary area. In fetal lingual papilla CK7 and CK8 were expressed in superficial cells, AE8 in intermediate and superficial cells, CKl4 in basal cells. MNFI116 and AE3 showed a strong reaction in basal and suprabasal cells. The epithelium of the inferior lingual surface reacted positively with AE8 and CK14, MNF116, and AE3. CK7 and CK8, however, were not reacted. In adult lingual papilla CK10 was expressed in superficial cells of the tip of the papilla, AE8 in suprabasal cells of interpapillary epithelium, CKl4 in basal cells of papillary and interpapillary epithelium, CKl9 in superficial cells of interpapillary epithelium, MNFI116 and AE3 in suprabasal cells of papillary and interpapillary epithelium. However, CK7, CK8, CK18, and 5D3 were not expressed in the epithelium of the dorsal tongue. The cells of taste bud in fetus showed positive reactions for CK7, CK8, MNF116, and AE3, but negative reactions with CK10 and AE8. The cells of taste bud in adult were stained with CK7, CK8, CK18, and MNF116, but not stained with CK10 and AE8. In lingual gland of fetus, CK7, CK8, CKl8, 5D3, MNF116, and AE3 were expressed in alveolar cells. Only CK10 gave a negative staining in ductal cells. The mucous cells of the adult lingual gland were reacted with CK7, CK10, CK18, CK19, and MNF116, and the serous cell with CK7, CK19, and MNF116. The ductal cells of the adult lingual gland were stained with CK7, AE8, CK18, CKl9, 5D3, MNF116, and AE3. CK14 was expressed in the cells of intralubular ductule, not in the ductal cells. By electron microscopy, the epithelia of both dorsal and inferior lingual surfaces in fetus consisted of nonkeratinized stratified squamous epithelium. A cell with clear cytoplasm and some dense granules was noted among the basal cells. These results indicate that the epithelium of fetal lingual papillae is non-keratinized type and the epithelium of the papillary tip is keratinized type and interpapillary epithelium is nonkeratinized type in adult, and suggest that the superficial cell containing cytokeratins 7 and 8 in dorsal lingual epithelium of fetus has a similar role to the periderm of fatal skin.

Development and Growth of Tongue in Korean Fetuses

We examined sixty-three human embryos ranged from three weeks to eight weeks of fertilization age and 117 human fetuses from eleven weeks to fourty weeks of gestational age. Anatomical structure of developing tongue could be classified into eight developmental stages. The first is the sgage of mesial swelling of tongue primordium in the fertilization age of 28~40 days (Streeter stage 13~16), the second is the stage of lateral swelling of tongue primordium in the fertilization age of 41~46 days (Streeter stage 17~18), the third is the sgage of vertical positioning of tongue in the fertilization age of 47~53 days (Streeter stage 19~21), the fourth is the transitional stage of tongue from vertical position to horizontal position in the fertilization age of 54~56 days (Streeter stage 22~23), the fifth is the stage horizontal positioning of tongue in the gestational age of 11 weeks, the sixth is the stage of protrusion of tongue in the gestational age of 12 weeks, the seventh is the stage of maturation of tongue muscle in the gestational age of 7-10 months. The development of tongue papilla characteristically progresses into three stages. The first stage is the epithelial ingrowth for the crypt formation, the second stage is the anatomical formation of vallate, fungiform and filiform papillae, and the third stage is the differentiation of taste buds in the vallate and fungiform papillae or the formation of thick spike-like keratinization at the tip of filiform papilla. We observed that the tongue primordium mainly derived from occipital myotome developed more repidly than other oro-facial structures, so it transitionally occuied the spaces of the pharynx and the posterior nasal cavity, and directly affected the formation of palate and the growth of maxilla and mandible. Whereas the tongue papilla development showed continuous developmental sequences during the fetal period.