Quantitative study of fungiform papillae and taste buds on the cat's tongue

The number of fungiform papillae has been counted on the tongues of six adult cats and of kittens both at birth and aged 2 and 4 months. Papillae were sampled from different regions of the tongue, and their size and the number of taste buds they contained were determined using histological sections taken parallel to the tongue surface. There were approximately 250 fungiform papillae on the tongues of the adult cats, the papillae were most numerous at the tip of the tongue, and there was no significant difference between the number of papillae on each side. The size of the papillae increased from a mean maximum diameter of 0.28 mm at the tip of the tongue to 0.48 mm at the back; the mean number of taste buds increased correspondingly from 6.9 to 16.6. The kitten tongues had a number and distribution of fungiform papillae similar to that found in the adults. In the neonate, papillae were smaller and contained fewer taste buds; these parameters increased with the corresponding increase in tongue size in the 2- and 4-month-old kittens.     

Expression of alpha-gustducin in the circumvallate papillae of taste buds of diabetic rats

Taste impairment is a complication of Diabetes mellitus and some studies have shown this taste disorder in diabetes. Diabetes can decrease the ability of individuals to detect and recognize sweet, salty and bitter tastes. alpha-Gustducin is a transducin-like G-protein selectively expressed in 20 - 30% of taste receptor cells, which has been shown to be involved in bitter, sweet and umami taste responses. The present study was performed to explore the protein and mRNA expression of alpha-gustducin in the taste buds of diabetic and control rat circumvallate papillae. Our results showed that the positive expression of alpha-gustducin in diabetic rat taste bud cells is higher than that in normal controls as shown by both immunohistochemistry and RT-PCR. There may be some variant of bitter, sweet or umami taste transduction during diabetes and that taste transduction variant may be one cause of diabetic taste impairment.     

Differentiation of alpha-gustducin in taste buds of the mouse soft palate and fungiform papillae

We used alpha-gustducin, a type II taste-cell-specific G protein, to investigate the onset of taste transduction and its relation to the development of the soft palate (SP) and fungiform (FF) papillae taste buds in the mouse. Paraffin wax embedded sections were prepared from the SP and anterior region of the tongue of the mouse from birth until postnatal day (PD) 63. No alpha-gustducin-immunoreactive cells were observed on the day of birth. One day later, alpha-gustducin was immunolocalised in taste buds with pores with a relatively higher frequency recorded in the SP as compared with the FF papillae. The immunoreactive cells were spindle shaped with elongated processes extending from the base to the pore of the taste buds. On PD 7, the number of taste buds containing alpha-gustducin-immunoreactive cells in the SP was three times greater than that of FF papillae. Our results indicate that taste transduction is essentially acquired from the time of birth. Moreover, the onset of taste transduction by the SP taste buds developed earlier than that achieved by taste buds in the FF papillae.     

Apical CD36 immunolocalization in human and porcine taste buds from circumvallate and foliate papillae

CD36 is the receptor for long chain fatty acids (LCFA), and is expressed in lingual taste cells from rodents. In these animals, CD36 has been proposed to play an important role in oral detection of LCFA, and subsequently, determines their dietary fat preference. Humans also seem to detect LCFA in the oral cavity, however, information on the molecular mechanism of this human orosensory LCFA recognition is currently lacking. The aim of our study was to investigate whether CD36 is also expressed in lingual human and porcine taste buds cells. Using fluorescence immunohistochemistry, apical CD36 expression was revealed in human and porcine taste bud cells from circumvallate and foliate papillae. These data suggest CD36 as the putative orosensory receptor for dietary LCFA in human, and, therefore, may be involved in our preference for fatty foods.     

Expression of BDNF and TrkB in mouse taste buds after denervation and in circumvallate papillae during development

BDNF (brain-derived neurotrophic factor) is a member of the neurotrophin family which affects the proliferation and survival of neurons. Using an immunocytochemical method, we examined the expression of BDNF and its receptor, TrkB, in the taste bud cells of the circumvallate papillae of normal mice and of mice after transection of the glossopharyngeal nerves. We additionally observed the expression of BDNF and TrkB in the developing circumvallate papillae of late prenatal and early postnatal mice. In normal untreated mice, BDNF was expressed in most of the taste bud cells; TrkB was detected in the plasma membrane of taste bud cells and in the nerve fibers. Double-labeling studies showed that BDNF and NCAM (neural cell adhesion molecule) or TrkB and NCAM colocalized in some of the taste bud cells, but that most taste bud cells were immunopositive for only BDNF or TrkB. NCAM-immunoreactive cells are known to be type-III cells, which have afferent synaptic contacts with the nerve terminals. Five days after denervation, the number of taste buds and nerve fibers markedly decreased; however, the remaining taste bud cells still expressed BDNF and TrkB. By 10 days after denervation, most of the taste buds had disappeared, and there were a few TrkB-immunoreactive nerve fibers in the connective tissue core. By 4 weeks after denervation, numerous TrkB-immunoreactive nerve fibers had invaded the papillae, and a few taste buds expressing BDNF and TrkB had regenerated. At E (embryonic day) 15 during development, the circumvallate papillae appeared, and then TrkB-immunoreactive nerve fibers entered the connective tissue core, and some of these fibers further invaded among the dorsal epithelial cells of the papillae. TrkB-immunoreactive oval-shaped cells were occasionally found in the dorsal epithelium. Such TrkB-immunoreactive nerve fibers and cells were also observed at E16-18. However, BDNF was not expressed in the papillae through the late prenatal days of E15 to E18. At P (postnatal day) 0, a cluster of BDNF-and TrkB-immunoreactive cells appeared in the dorsal epithelium of the papillae, and was presumed to be primitive taste buds. We conclude that TrkB-immunoreactive nerve fibers are necessary for papillary and taste bud formation during development and for the regeneration of taste buds after denervation. BDNF in the taste bud cells may act as a neurotrophic factor for innervating sensory neurons--through TrkB receptors of the axons of those neurons, and also may exert autocrine and paracrine trophic actions on neighboring taste bud cells by binding to their TrkB receptors.     

Exocytotic release of neurotransmitter substances from nerve endings in the taste buds of rat circumvallate papillae

Exocytotic release of neurotransmitters from nerve endings was demonstrated ultrastructurally in the taste buds of rat circumvallate papillae by stimulation of high K+ and Ca2+ Ringer perfusion and application of tannic acid-Ringer incubation (TARI) method. Omega-shaped images of large cored vesicles and small clear vesicles, indicating exocytotic release of their contents, were found only in the non-synaptic sites. Occasionally exocytosis occurred at sites facing other nerve fibers. Many coated pits were also seen, which presumably represent membrane retrieval at a later stage of exocytosis. It is likely that the taste buds receive more than one type of innervation.     

Distribution of taste and general sensory nerve endings in fungiform papillae of the hamster

Sensory endings of chorda tympani and lingual (trigeminal) nerve fibers were identified by selective denervation and localized within specific regions of fungiform pipillae in the hamster. The chorda tympani was resected from the middle ear and the peripheral fibers were allowed to degenerate for 1, 3, or 8 days prior to perfusion-fixation and electron-microscopic examination of the anterior tongue. Taste buds were virtually devoid of intact nerves by 3 days following chorda tympani denervation. Remnants of the fibers were restricted to taste buds. Lingual fibers, on the other hand, persist in normal numbers after chorda tympani resection and populate perigemmal areas of connective tissue and extragemmal areas located apically in the squamous, nontaste epithelium surrounding the taste bud. This study provides evidence of a segregation of chorda tympani fibers in the taste bud and lingual nerve fibers in the apical fungiform papilla. The lingual nerve-epithelial arrangement and superficial location, near the least cornified area of the tongue, may be well suited for relatively sensitive somatosensation, possibly mechanoreception. Thus, the apical fungiform papilla appears to be a site where both taste and tactile oral stimuli interact with receptors.     

Long-term effects of nicotine on rat fungiform taste buds

Nicotine, an alkaloid found in tobacco smoke, has been recognized as capable of inducing changes in taste functionality in conditions of chronic exposure. The mechanisms underlying these sensory alterations, however, are currently unknown. We addressed this issue by studying the long-term effects of nicotine on the anatomical features of taste buds, the peripheral end-organs of taste, in rat fungiform papillae. Nicotine was administered to rats via drinking water over a period of 3 weeks, which represents a standard method to achieve chronic drug exposure in laboratory animals. We found that prolonged administration of nicotine induced a significant reduction in the size of fungiform taste buds, without affecting their total number on the rat tongue. Morphometric measurements as well as evaluations of taste cell membrane capacitance suggested that the reduced size of taste organs was determined by a decrease in the number of cells per taste bud. In addition, chronic treatment with nicotine caused an increase in the relative density of cells expressing gustducin, a specific G protein alpha-subunit found in some taste cells and involved in bitter/sweet transduction. Interestingly, changes in the expression pattern of gustducin turned out to be more pronounced in periadolescent/adolescent than in adult rats. As a whole, our data indicate that long-term nicotine administration induces significant changes in the anatomical properties of taste buds in rat fungiform papillae. These changes could have a profound impact on the sensory information relayed to the brain; therefore, they may be responsible, at least in part, for the alterations in taste functionality observed during chronic nicotine exposure, a condition found in regular smokers.     

Patterns of immunoreactivity specific for gustducin and for NCAM differ in developing rat circumvallate papillae and their taste buds

α-Gustducin and neural cell adhesion molecule (NCAM) are molecules previously found to be expressed in different cell types of mammalian taste buds. We examined the expression of α-gustducin and NCAM during the morphogenesis of circumvallate papillae and the formation of their taste buds by immunofluorescence staining and laser-scanning microscopy of semi-ultrathin sections of fetal and juvenile rat tongues. Images obtained by confocal laser scanning microscopy in transmission mode were also examined to provide outlines of histology and cell morphology. Morphogenesis of circumvallate papillae had already started on embryonic day 13 (E13) and was evident as the formation of placode. By contrast, taste buds in the circumvallate papillae started to appear between postnatal day 0 (P0) and P7. Although no cells with immunoreactivity specific for α-gustducin were detected in fetuses from E13 to E19, cells with NCAM-specific immunoreactivity were clearly apparent in the entire epithelium of the circumvallate papillary placode, the rudiment of each circumvallate papilla and the developing circumvallate papilla itself from E13 to E19. However, postnatally, both α-gustducin and NCAM became concentrated within taste cells as the formation of taste buds advanced. After P14, neither NCAM nor α-gustducin was detectable in the epithelium around the taste buds. In conclusion, α-gustducin appeared in the cytoplasm of taste cells during their formation after birth, while NCAM appeared in the epithelium of the circumvallate papilla-forming area. However, these two markers of taste cells were similarly distributed within mature taste cells.     

Bovine circumvallate taste buds: taste cell structure and immunoreactivity to alpha-gustducin

The taste buds of bovine circumvallate papillae were investigated under light and electron microscopy both by histological and immunohistochemical methods. Taste buds existed in the inner epithelium of the trench of the papillae. Under electron microscopy, two types of taste cells, type I and type II, could be classified according to the existence of dense-cored vesicles and cytoplasmic density. Type I had electron-lucent cytoplasm and possessed many electron-dense cored vesicles in the apical cytoplasm. It was considered that the electron-dense materials of the vesicles were released and constituted the pore substance. This type of cell possessed long and thick apical processes in the taste pore. Type II had denser electron cytoplasm compared with that of type I and possessed many electron-lucent vesicles in the apical cytoplasm. This type of cell possessed microvilli in the taste pore. To know the immunoreactivity to alpha-gustducin in bovine circumvallate taste buds, we used the immunoblotting method and the immunohistochemical method. The alpha-gustducin reaction band at 40 kDa was displayed in the specimen of Western blots. The immunohistochemical property of the antiserum to alpha-gustducin was investigated by using the avidin-biotin complex (ABC) method and the 1.4-nm gold and silver enhancement methods. A subset of taste cells showed the immunoreactivity under light microscopy. The electron microscopic specimens with the 1.4-nm gold and silver enhancement method revealed that only type II cells exhibited the alpha-gustducin immunoreactivity.     

Unilateral nasal obstruction alters sweet taste preference and sweet taste receptors in rat circumvallate papillae

Nasal obstruction causes mouth breathing, and affects the growth and development of craniofacial structures, muscle function in the stomatognathic system, and the taste perceptive system. However, the detailed mechanism underlying the effects of nasal obstruction on taste perception has not been fully elucidated. In this study, we investigated this mechanism using the two-bottle taste preference test, immunohistological analysis, and quantification of the mRNA expression of taste-related molecules in the circumvallate papillae. Neonatal male Wistar rats were divided randomly into control and experimental groups. Rats in the experimental group underwent unilateral nasal obstruction by cauterization of the external nostril at the age of 8 days. Arterial oxygen saturation (SpO₂) was recorded in awake rats using collar clip sensors. Taste preference for five basic taste solutions was evaluated. Immunohistochemical analysis and quantitative real-time polymerase chain reaction (RT-PCR) were conducted to evaluate the expressions of taste-related molecules in the taste cells of the circumvallate papillae. Body weights were similar between the two groups throughout the experimental period. The SpO₂ in the 7- to 12-week-old rats in the experimental group was significantly lower than that in the age-matched rats in the control group. In the two-bottle taste preference test, the sensitivities to sweet taste decreased in the experimental group. The mRNA expression of T1R2, T1R3, α-gustducin, and PLCβ2 was significantly lower in the experimental group than in the control group as determined by quantitative RT-PCR, and the immunohistochemical staining for α-gustducin and PLCβ2 was less prominent. These findings suggest that nasal obstruction may affect sweet taste perception via the reduced expression of taste-related molecules in the taste cells in rat circumvallate papillae.     

Immunohistochemical identification of cells expressing steroidogenic enzymes cytochrome P450scc and P450 aromatase in taste buds of rat circumvallate papillae

The present study demonstrated for the first time the localizations and patterns of expression of key enzymes for steroidogenesis, cytochrome P450 side-chain-cleavage (P450scc), and P450 aromatase in the taste buds of rat circumvallate papillae, using immunoblot analyses and immunohistochemistry. Immunoblot analyses showed that proteins with a molecular weight close to that of rat adrenal cytochrome P450scc and a molecular weight close to that of rat ovary cytochrome P450 aromatase were present in the rat circumvallate papillae. In immunohistochemistry, antibodies against cytochrome P450scc and P450 aromatase yielded the labelings of a subset of taste bud cells. In the double immunolabeling of P450scc and alpha-gustducin or phospholipase C beta2(PLCbeta2), which were considered as markers of a majority of type II cells, P450scc was co-expressed in a subset of alpha-gustducin or PLCbeta2, but did not co-express neural adhesion molecule (NCAM), a marker of major type III cells. Further double immunolabeled studies showed that P450 aromatase was co-expressed in a subset of alpha-gustducin or PLCbeta2, but did not co-express PGP9.5, a marker of a majority of type III cells. The selective localization of cytochrome P450scc and P450 aromatase strongly suggests that estrogen biosynthesis from cholesterol might occur in a subset of type II cells of the rat taste buds. Although the full significance of estrogen in the taste bud function is not yet understand, estrogen appears to be an important regulator of taste transduction, as is the case with ATP (Finger et al., 2005), which further supports the centrality of taste cells in the life of taste buds.     

Effects of streptozotocin-induced diabetes on taste buds in rat vallate papillae

Some studies have documented taste changes in patients with diabetes mellitus (DM). In order to understand the relationships between taste disorders caused by DM and the innervation and morphologic changes in the taste buds, we studied the vallate papillae and their taste buds in rats with DM. DM was induced in these rats with streptozotocin (STZ), which causes the death of beta cells of the pancreas. The rats were sacrificed and the vallate papillae were dissected for morphometric and quantitative immunohistochemical analyses. The innervations of the vallate papillae and taste buds in diabetic and control rats were detected using immunohistochemistry employing antibodies directed against protein gene product 9.5 (PGP 9.5) and calcitonin gene-related peptide (CGRP). The results showed that PGP 9.5- and CGRP-immunoreactive nerve fibers in the trench wall of diabetic vallate papillae, as well as taste cells in the taste buds, gradually decreased both intragemmally and intergemmally. The morphometry revealed no significant difference in papilla size between the control and diabetic groups, but there were fewer taste buds per papilla (per animal). The quantification of innervation in taste buds of the diabetic rats supported the visual assessment of immunohistochemical labeling, that the innervation of taste cells was significantly reduced in diabetic animals. These findings suggest that taste impairment in diabetic subjects may be caused by neuropathy defects and/or morphological changes in the taste buds.