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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.

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A sensitive period for the neural induction of taste buds.

Taste buds mature postnatally in the vallate papilla of the rat and reach a mean number of 610 by day 90. Although taste buds are neurotrophically dependent, the presence of widespread bilateral innervation permits more than 80% of the 610 vallate taste buds to survive after one IXth nerve is removed in adults. However, after a IXth nerve is removed at 0-3 d postpartum, about two-thirds of the vallate taste buds fail to develop. In the present investigation, the timing of the neural induction of taste buds was examined by unilaterally removing the IXth nerve at 12 different postnatal ages, from 0 to 75 d. Unilateral denervation revealed the existence of a sensitive period that is maximal from 0 to 10 d, when unilateral or bilateral interruption of the IXth nerve profoundly impairs the formation of taste buds. The number of taste buds that form is nonlinearly dependent upon the number of axons; at low levels of innervation, a doubling of the number of myelinated axons quintuples the number of taste buds. Thus, taste axons interact synergistically. In studying regeneration, we found that axons of both neonatal and adult IXth nerves elongate approximately 1.8 mm/d. Taste buds were re-formed more rapidly and a higher proportion were bilaterally innervated when regenerating axons and the sites of former taste buds were numerous. The proportion of bilaterally innervated taste buds could be approximated from the likelihood of random overlap of axons from the right and left IXth nerves. The greater ease with which taste buds are re-formed than developed suggests that taste bud regeneration does not recapitulate taste bud development.

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Analysis of cell lineage relationships in taste buds.

Taste buds are a heterogeneous population of cells exhibiting diverse morphological and biochemical characteristics. Because taste buds arise from multiple progenitors, the different types of taste cells may represent distinct lineages. The present study was undertaken to determine the following: (1) how many progenitors contribute to a taste bud, and (2) whether the specific subpopulation of serotonin-immunoreactive (IR) taste cells are related by lineage to a restricted set of progenitor cells. These questions were addressed using cell lineage analysis of taste buds from H253 X-inactivation mosaic mice. After random X-inactivation of the lacZ transgene, the tongue of hemizygous female mice displays discrete patches of epithelial cells, which are either beta-galactosidase (beta-gal) positive or beta-gal negative. By analyzing the proportion of the two differently stained cell populations in taste buds located at the boundary between positive and negative epithelial patches, we can determine the minimum number of progenitors that may contribute to the formation of a taste bud. The presence of taste buds containing only 6-12% labeled cells indicates that at least eight progenitors contribute to an average taste bud of 55 cells, assuming progenitors contribute equally to the cell population. Cell lineage analysis of serotonin-IR taste cells in such mixed taste buds suggests that this subpopulation likely arises from only one to two progenitors and often is related by lineage. Thus, at least some of the cell types in a taste bud represent distinct lineages of cells and are not merely phenotypic stages as a cell progresses from a young to a mature state.

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Immunolocalization of different forms of neural cell adhesion molecule (NCAM) in rat taste buds.

Taste buds consist of approximately 100 taste cells, including three morphological types of short receptor cells which synapse on the peripheral gustatory nerves. Some of the receptor cells produce neural cell adhesion molecule (NCAM), which may play a role in formation of specific connections in this system. Antibodies directed against different forms of NCAM were utilized in an attempt to define not only the distribution, but also the type of NCAM within taste buds. Within each taste bud approximately 10% of the taste cells exhibit abundant immunoreactivity for 180 kD (ld) or 140 kD (sd) forms of NCAM (i.e., those with an intracellular domain) along virtually the entire surface of the cell. Ultrastructural analysis reveals that these abundantly immunoreactive taste cells are of the intermediate morphological type, although not all of the intermediate taste cells within any bud are immunoreactive. In addition, the ultrastructural studies show that punctate (ld/sd) NCAM-immunoreactivity occurs on the membranes of taste cells and nerve fibers throughout each taste bud. The embryonic form of NCAM (E-NCAM), rich in polysialic acid residues, is present only in association with nerve fibers and other unidentified elongate, thin profiles of a few taste buds. The nerve plexus beneath the gustatory epithelium is also rich in NCAM-immunoreactivity. These fibers occasionally reveal immunoreactivity indicative of only the 120 kD (ssd) form of NCAM, typical of glial cells.

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Distribution of cytokeratin filaments and vimentin in developing human taste buds.

Taste buds in humans originate from approximately the 8th postovulatory week under the influence of ingrowing nerve fibers. Since they develop from local epithelium, it is of interest whether or not prospective taste cells maintain or develop characteristics of epithelial cells that are different from those of the adjacent epithelium during differentiation. The aim of this study was to monitor changes of the distribution of the cytokeratin filaments (CKs) 8, 18, 19 and 20 ("gastrointestinal" type), CK 7 ("ductal" type), and CK 13 (maturation "mucosa type"), as well as vimentin in developing human taste buds and adjacent squamous epithelium. With the exception of CK13, which remains negative in taste bud anlagen and adult taste buds, all cytokeratins tested were present in taste cells. With the progress of development, the distribution of CKs becomes more and more restricted to taste cells and salivatory ducts as well as Ebner gland cells. Only CK20 is exclusively specific to taste bud anlagen and sometimes to individual bipolar cells occurring in early stages (week 8-9). Vimentin was located mainly in mesodermal derivatives but also in perigemmal epithelial cells during all stages of development. The occurrence of vimentin in "borderline" epithelia that interface with underlying connective tissue, i.e., in a region of discontinuity, may be associated with particular events in development, cell migration or even dedifferentiation.

Adult↗

The neural differentiation gene Mash-1 has a distinct pattern of expression from the taste reception-related genes gustducin and T1R2 in the taste buds.

Taste bud cells have a limited lifespan and are continuously replaced just like other epithelial cells. Although there is some evidence that taste buds may arise from the local epithelium, taste receptor cells have neuronal properties. This implies that there must be a critical stage at which the epithelial precursor cells for taste receptor cells start to exhibit neural properties during the differentiation of the taste receptor cells. The expression of the neural-specific transcription factors Mash-1 and Prox-1 in the nervous system is transient and precedes neuronal differentiation. Therefore, we examined the expression of Mash-1 and Prox-1 in the epithelium of circumvallate papillae of the tongue in order to clarify the localization of the precursor cells with neural properties and observed that both expressions are restricted to the taste buds. Two-colour in situ hybridization showed that the signals for Mash-1 did not overlap those for taste receptor cell-specific genes such as gustducin and T1R2. In the process of development and regeneration of the taste buds, the expression of Mash-1 preceded that of gustducin and T1R2. These observations suggest that Mash-1 could be a candidate for a marker of immature taste receptor cells, including the cells that express gustducin and/or T1R2 at a later stage.

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Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses.

The ultrastructural features of murine vallate taste bud cells and their associated synapses have been examined in thin and thick sections with conventional transmission electron microscopy and high-voltage electron microscopy. Computer-assisted reconstructions from serial sections were utilized to aid in visualization of taste bud cell-nerve fiber synapses. We have classified taste bud cells on the basis of previously established criteria-namely, size of the nucleus, shape and density of chromatin, density of cytoplasm, and presence or absence of dense-cored or clear vesicles, other cytoplasmic organelles, and synaptic foci. Both dark cells and light cells are present, as well as cells with intermediate morphological characteristics. Synapses were observed from taste bud cells onto nerve fiber processes. In virtually all instances, synapses are associated with the nuclear region of the taste cell. These synapses are characterized by the presence of 40-70 nm clear vesicles embedded in a thickened presynaptic membrane separated from the postsynaptic membrane by a 16-30 nm cleft. Synapses are not unique to any particular cell type. Dark, intermediate, and light cells all synapse onto nerve fibers. Two general types of synapses exist: spot (or macular) and fingerlike. In the latter, the postsynaptic region of the neuronal process protrudes into an invagination of the taste cell membrane. Differences in synaptic morphology are not correlated with taste cell type. In some cases a single taste cell was observed to possess both macular and fingerlike synapses adjacent to one another, forming a synaptic complex onto a single neuronal process. On the basis of the presence of synaptic contacts, we conclude that both "dark" and "light" cells are gustatory receptors.

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Desquamation on taste buds.

Taste bud surface cells suffer from a desquamation process that modifies the pores' shape and size and the amount of amorphous substance in the pits region. We have studied this phenomenon-which probably plays an important functional role-by light, electron and scanning microscopy.

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Variations in human taste bud density and taste intensity perception.

Some variations in human taste sensitivity may be due to different numbers of taste buds among subjects. Taste pores were counted on the tongue tips of 16 people with videomicroscopy, and the subjects were divided into two groups (N = 8) by the rank order of their taste bud densities. The "higher" density group averaged 374 +/- 134 taste pores/cm2, while the "lower" density group averaged 135 +/- 43 tp/cm2. The higher density group had an average fungiform papilla density which was 1.8 times greater than the lower density group and an average of 1.5 times more taste pores/papilla. The subjects also rated the intensity for 4 suprathreshold concentrations of 5 taste stimuli placed on the same region of the tongue where taste pores were counted. The group with higher taste bud densities gave significantly higher average intensity ratings for sucrose (196%), NaCl (135%) and PROP (142%), but not for citric acid (118%) and quinine HCl (110%) than the lower density group. Thus, the subjects with higher fungiform taste bud densities also reported some tastes as more intense than subjects with fewer fungiform taste buds.

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Fingerprinting taste buds: intermediate filaments and their implication for taste bud formation.

Intermediate filaments in taste organs of terrestrial (human and chick) as well as aquatic (Xenopus laevis) species were detected using immunohistochemistry and electron microscopy. During development, the potential importance of the interface between the taste bud primordium and non-gustatory adjacent tissues is evidenced by the distinct immunoreactivity of a subpopulation of taste bud cells for cytokeratins and vimentin. In human foetuses, the selective molecular marker for taste bud primordia, cytokeratin 20, is not detectable prior to the ingrowth of nerve fibres into the epithelium, which supports the hypothesis that nerve fibres are necessary for initiating taste bud development. Another intermediate filament protein, vimentin, occurs in derivatives of mesoderm, but usually not in epithelium. In humans, vimentin immunoreactivity is expressed mainly in border (marginal) epithelial cells of taste bud primordia, while in chick, vimentin expression occurs in most taste bud cells, whereas non-gustatory epithelium is vimentin immunonegative. Our chick data suggest a relationship between the degree of vimentin expression and taste bud cell proliferation especially during the perihatching period. It is suggested that surrounding epithelial cells (human) and mesenchymal cells (chick) may be contributing sources of developing taste buds. The dense perinuclear network of intermediate filaments especially in dark (i.e. non-sensory) taste disc cells of Xenopus indicates that vimentin filaments also might be associated with cells of non-gustatory function. These results indicate that the mechanisms of taste bud differentiation from source tissues may differ among vertebrates of different taxa.

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Location of taste buds in intact taste papillae by a selective staining method.

Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.

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Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development.

Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7-9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7-8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events.

Adult↗

Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes.

Taste buds are constituted of several kinds of cells which have distinct characteristics and play different roles. In this study, we have established an in vitro culture system by optimizing the method for isolating the cells and by selecting culture media and reagents effective for cell viability and adhesion. As a result, the taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium. The cells retained molecular markers for both the cytoskeleton and intracellular signaling such as cytokeratin 8 and phospholipase Cbeta2. In addition, three intracellular signaling molecules, gustducin, phospholipase Cbeta2, and inositol 1,4,5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo, although the ratio of signaling molecule-positive cells vs. total cells was somewhat lower in the culture than in vivo. Next, we tried several methods to introduce foreign genes into the cells, and obtained a greater than 90% efficiency of introduction using an adenovirus vector. Finally, we show that an exogenously expressed myc-tagged alpha1A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular [Ca(2+)] increase in about half of the infected cells. These results suggest that taste bud cells after 3 days of culture retain characteristic molecular markers, and may prove useful for describing the molecular and physiological features of taste bud cells, and that these cells can be further manipulated by adenovirus-mediated gene introduction.

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Glycoconjugate in rat taste buds.

The taste buds of the fungiform papillae, circumvallate papilla, foliate papillae, soft palate and epiglottis of the rat oral cavity were examined by lectin histochemistry to elucidate the relationships between expression of glycoconjugates and innervation. Seven out of 21 lectins showed moderate to intense staining in at least more than one taste bud. They were succinylated wheat germ agglutinin (s-WGA). Dolichos biflorus agglutinin (DBA), Bandeiraea simplicifolia lectin-I (BSL-I), Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I) and Phaseolus vulgaris agglutinin-L (PHA-L). UEA-I and BSL-I showed moderate to intense staining in all of the taste buds examined. They strongly stained the taste buds of the epiglottis, which are innervated by the cranial nerve X. UEA-I intensely stained the taste buds of the fungiform papillae and soft palate, both of which are innervated by the cranial nerve VII. The taste buds of circumvallate papilla and foliate papillae were innervated by the cranial nerve IX and strongly stained by BSL-I. Thus, UEA-I and BSL-I binding glycoconjugates, probably alpha-linked fucose and alpha-D-galactose, respectively, might be specific for taste buds. Although the expression of these glycoconjugates would be related to the innervation of the cranial nerve X, the differential expression of alpha-linked fucose and alpha-D-galactose might be related to the innervation of the cranial nerve VII and IX, respectively.

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Membrane properties of two types of basal cells in Necturus taste buds.

Necturus taste buds contain two types of basal cells: presumptive stem cells and Merkel-like basal cells. Both types of basal cells are small round cells located at the base of the taste bud, indistinguishable from each other with light microscopy. However, with electron microscopy, autoradiography, or immunocytochemistry, these two types of basal cells can be easily distinguished. We isolated basal cells from taste buds, characterized their voltage-dependent currents using gigaseal whole-cell recordings, and processed the cells for electron microscopy or immunocytochemistry. We were able to distinguish two cell types electrophysiologically and to correlate cell type with membrane properties. Isolated Merkel-like basal cells had several voltage-activated currents: transient, TTX-sensitive, inward Na+ current; sustained, saturating outward K+ current; and slowly inactivating inward Ca2+ current. These currents are similar to those observed in taste receptor cells. In contrast, presumptive stem cells from Necturus taste buds only had outward K+ currents.

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The rate and locus of development of rat vallate taste buds.

Several hundred taste buds develop and mature in the trench walls of the rat's vallate papilla during the first 3 months after birth. The objective of this investigation of taste bud development was to determine: (i) whether the vallate papilla has local growth zones where new taste buds form, (ii) whether new taste buds arise by the division of mature taste buds, (iii) how many days are required for a new taste bud to mature, and (iv) whether a taste pore forms as the taste bud reaches a critical volume. Camera lucida drawings were made of more than 4000 iron hematoxylin-stained, serially sectioned, vallate taste buds. The relative abundance of immature taste buds declined exponentially with age, from 18% of the mature taste buds at day 15 to 2% at day 90. At days 21, 33 and 45 most of the immature taste buds (those lacking a taste pore) were located in growth zones at the anterior and posterior extremes of the vallate trench. A mean of 10.5 days was required for the maturation of each cohort of immature taste buds present at days 15, 21, 33 and 45. Vallate taste buds were added de novo; fission of mature taste buds was rare. Taste buds varied widely in the taste bud volume at which a pore formed and in the final volume of the taste bud.

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