THE EFFECT OF TEMPERATURE ON THE TURNOVER OF TASTE BUD CELLS IN CATFISH

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14°C, 18°C, 22°C and 30°C dechlorinated tap water were injected with [³H]thymidine (3.0 μCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14°C, 18°C, 22°C and 30°C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

RENEWAL OF TASTE BUD CELLS IN RAT CIRCUMVALLATE PAPILLAE

The life span of taste bud cells in rat circumvallate papillae was measured by autoradiography after labeling them with a pulse of [³H]thymidine. Specimens of circumvallate papillae were taken daily 1·5-18·5 days after the isotope was administered; thereafter, specimens were taken on alternate days until 25·5 days. For each time interval, the number of labeled cell nuclei was counted in 200-450 taste buds and plotted as the ratio of labeled cells/taste bud v. time after injection of [³H]TdR. In all, 6958 taste buds were counted. The total number of labeled cells (dark plus light) per taste bud reached peaks at 6·5, 13·5 and 20·5 days. The curve for the number of labeled dark cells/bud had essentially the same shape as that for total cells. The number of labeled light cells/bud reached a modest peak at 6·5 days and slowly declined to a plateau for the remainder of the experiment. The data show that an average of 2 days elapsed after injection before labeled dark cells entered the bud and they spent an average of 7 days in the non-proliferating taste bud compartment; thus, the life span of the dark cell was 9 days. The life span of the light cell was difficult to estimate quantitatively, but this cell type was labeled at a much slower rate than dark cells and is assumed to have a significantly longer tenure in the taste bud.     

Maintenance of rat taste buds in primary culture

The differentiated taste bud is a complex end organ consisting of multiple cell types with various morphological, immunocytochemical and electrophysiological characteristics. Individual taste cells have a limited lifespan and are regularly replaced by a proliferative basal cell population. The specific factors contributing to the maintenance of a differentiated taste bud are largely unknown. Supporting isolated taste buds in culture would allow controlled investigation of factors relevant to taste bud survival. Here we describe the culture and maintenance of isolated rat taste buds at room temperature and at 37 degrees C. Differentiated taste buds can be sustained for up to 14 days at room temperature and for 3-4 days at 37 degrees C. Over these periods individual cells within the cultured buds maintain an elongated morphology. Further, the taste cells remain electrically excitable and retain various proteins indicative of a differentiated phenotype. Despite the apparent health of differentiated taste cells, cell division occurs for only a short period following plating, suggesting that proliferating cells in the taste bud are quickly affected by isolation and culture.     

The timing of alpha-gustducin expression during cell renewal in rat vallate taste buds

The G protein subunit alpha-gustducin is expressed in a subset of light (Type II) but not in dark (Type I) cells in rat vallate taste buds. The thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) is incorporated into DNA during the S-phase of the cell cycle and can be used to determine the time of origin of a cell. In this study, 31 rats were injected with BrdU (50 mg/kg i.p.) and perfused at various times, from 2.5 to 10.5 days, following BrdU administration. Vallate papillae were embedded in polyester wax, cut into 4 microm transverse sections, and characterized with antibodies to BrdU and alpha-gustducin. Sections were processed for indirect immunofluorescence or with an immunoperoxidase procedure. From immunoperoxidase material on 21 rats, counts of alpha-gustducin- and BrdU-labeled cells were obtained from 300-800 taste bud profiles at each survival time; a total of 4122 taste bud profiles were examined. Cells with nuclei immunoreactive for BrdU occurred within the taste buds at 2.5 days and double-labeled cells were clearly evident at 3.5 days; a small number of double-labeled cells were seen as early as 2.5 days. Double-labeled cells reached a peak at 6.5 days and did not decline significantly by 10.5 days. Cells labeled for BrdU but not alpha-gustducin peaked at 5.5 days and showed a significant decline by 8.5 days. These latter cells included light cells not expressing alpha- gustducin and dark cells, which have previously been shown to have a shorter life span than light cells. These data suggest that expression of alpha-gustducin appears very early in a cell's life span and that these cells are longer lived than many of the cells that do not express this G protein.      

Morphometry and cellular dynamics of denervated fungiform taste buds in the hamster

For most species and gustatory papillae denervation resultsin a virtual disappearance of taste buds. This is not the casefor hamster fungiform papillae, which contain taste buds thatsurvive denervation. To characterize these taste buds, in thisstudy, counts and measurements were made of all buds on theanterior 3 mm of the hamster tongue at 36 or 91 days after resectingthe chorda/lingual nerve. Taste bud numbers were, at both timeperiods, unaffected by denervation. However, bud dimensionswere affected with denervated buds 25–30% smaller thancontrol ones. Counts of taste bud cells indicated that decreasesin bud size may result from shrinkage, but not a loss of cells.Tritiated thymidine autoradiography was used to evaluate whetherdenervation influences the mitotic activity or the migratorypattern of bud cells. For every animal, the average number oflabelled cells per bud was slightly lower on the denervatedthan the control side of the tongue. However, when labelledcell positions were evaluated at 0.25, 3 and 6 days after thymidine,the distances from the sides of the bud increased at increasingtimes after injection for both the innervated and the denervatedbuds. Stem cells were located laterally or basally in the bud.Labelled cells that migrated into the centers of the buds werefew and seen only at 6 days post-injection time in both controland experimental buds. The moderate effects of denervation ontaste bud sizes and mitotic activities may indicate a generalizedatrophy. Remarkably intact were taste bud numbers and the migratorypatterns of cells, features of anterior tongue taste buds inthe hamster that are relatively invulnerable to resection ofthe chorda /lingual nerve.     

Apoptosis in mouse taste buds after denervation

Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing. Received: 20 November 1995 / Accepted: 15 May 1996     

RENEWAL OF CELLS WITHIN TASTE BUDS

Colchicine blocks mitotic division of the epithelial cells surrounding the taste bud of the rat tongue. Response to chemical stimulation decreases 50 per cent 3 hours after colchicine injection as measured by recording the electrical activity from the taste nerve bundle. Radioautography, using tritiated thymidine, shows that those epithelial cells surrounding the taste bud divide and that some of the daughter cells enter the taste bud and slowly move toward the center. The life span of the average cell is about 250 ± 50 hours, although some cells have a much shorter and others a much longer life span. These studies suggest that the cells within the taste bud, as well as the nerves, undergo considerable change with time. Corresponding changes in function are considered.     

Changes in the cell population of taste buds during degeneration and regeneration of their sensory innervation

Summary Taste buds of rabbit foliate papillae were observed in control, after denervation and during reinnervation by the glossopharyngeal nerve. In control, taste bud cells could be divided into three groups according to their shapes and staining characteristics. Most of the cells were identified as either dark (corresponding to gustatory) or light (corresponding to supporting) cells. However, some cells were encountered which could not readily be placed in either group; they have been termed intermediate cells. Nine to twelve hours after axotomy, wandering cells were observed in many of the taste buds. Thereafter taste buds gradually decreased in size and disappeared, for the most part, by the 14th postoperative day. It was found that dark cells disappeared first, then at a later stage the light cells also disappeared. During reinnervation, dark cells were first to appear about 40 days after the operation and light cells were not seen till about 9 days later.From the observations, it is concluded that the dark cells of the taste bud differentiate from epithelial cells under the influence of nerves and mature into light cells through intermediate cells.     

Distribution of vimentin in the developing chick taste bud during the perihatching period.

The tissue environment within which taste bud cells develop has not been wholly elaborated. Previous studies of taste bud development in vertebrates, including the avian chick, have suggested that taste bud cells could arise from one, or several tissue sources (e.g. crest-mesenchyme, local ectoderm or endoderm). Thus, molecular markers which are present in gemmal as well as interfacing (peribud epithelium; mesenchyme-epithelium) regions, and their degree of expression during stages of taste bud development, are of special interest. The intermediate filament protein, vimentin, occurs in mesenchymal and mesodermally-derived (e.g. endothelial, fibroblast) cells as well as highly proliferating epithelium (e.g. tumors). The present study in chick gustatory tissue utilized antibodies against vimentin and the avidin-biotin-peroxidase technique to evaluate vimentin immunoreactivity (IR) within a timeframe which includes: 1) early stages of the taste bud primordium [embryonic days (E)17-E18)]; 2) the beginning of an accelerated bud cell proliferation at the time of initial, taste bud pore opening [around E19]; 3) attaining the adult complement of taste buds [around posthatch (H) day 1], and 4) completed organogenesis (H 17). During this time span, vimentin-IR was characterized in a region including and sometimes bridging taste bud and subepithelial connective tissue, whereas non-gustatory surrounding epithelium and salivary glands were vimentin-immuno-negative. Intragemmally, the proportion of vimentin-IR cells as related to total taste bud cells peaked at E19. These results indicate that vimentin expression, in part, is related to the onset of taste bud cell proliferation and suggest that mesenchyme could be one source of taste bud cells. Secondly, fibronectin, an extracellular matrix component of the epithelial basement membrane interface with mesenchyme, was expressed at or near the apical surfaces of taste bud cells projecting into the bud lumen, and in the basal gemmal region suggesting the possible role of fibronectin as a chemotactic anchor for differentiating and migrating taste bud receptor cells. Lastly, neuron-specific enolase-IR indicates that axonal varicosities are already present intragemmally at E17-E18, that is, during the incipient period of identifiable taste bud primordia.     

Distribution of vasoactive intestinal peptide-like immunoreactivity in the taste organs of teleost fish and frog

Summary Using immunohistochemistry, vasoactive intestinal peptide (VIP) was visualized in taste bud cells of the carp, Cyprinus carpio, and the European catfish, Silurus glanis, by means of light and electron microscopy. Intracellular membrane systems, presumably smooth endoplasmic reticulum, of light (sensory) cells, but not of dark (supporting) cells and basal cells, were densely labelled with antibody. In the frog (four species: Rana temporaria, R. ridibunda, R. arvalis, R. pipiens), taste bud cells did not label. However, the dense basal nerve fibre plexus, some subepithelial ganglionic cells, but no ascending intragemmal fibres, were immunoreactive. In fish, the results support evidence that VIP is involved in the modulation of taste transduction at the level of receptor cells. In the frog, an indirect, possibly vasodilatatory effect on taste perception may be considered.     

Functional Cell Types in Taste Buds Have Distinct Longevities

Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8–12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2′-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.     

Surface morphology of taste buds in catfish barbels

Summary External taste buds abound on barbels of the adult catfish Corydoras arcuatus. When examined by scanning electron microscopy, they are visualized as a series of punctate, conical elevations projecting from the general surface epithelium. All taste buds were found to be of one type. Both their external and internal surface features could be clearly elucidated on intact barbels and in barbels fractured transversely at various positions along their length. An extensive nerve terminal network penetrates the base of each taste bud. Two populations of elongated cells bearing prominent microvilli project through the central pore at the tip of each bud. One set of microvilli is thicker, longer and more club-shaped than its counterpart. While both are randomly distributed within each central pore, the small, short microvilli appear to outnumber the larger ones. A third population of cells, devoid of any apical microvilli, was also seen in some of the taste buds examined internally. These cells do not project to the external surface and are interpreted as basal cells described in previous light and transmission electron microscope studies of taste buds in other vertebrate species. The functional significance of some of these morphological findings is discussed.Supported by grants from the Medical Research Council of Canada and the Muscular Dystrophy Association of CanadaThe excellent technical assistance of Mr. F.T. McConnell is gratefully acknowledged     

Fine structure and distribution of epidermal projections associated with taste buds on the oral papillae in some loricariid catfishes (Siluroidei: Loricariidae)

Two morphologically distinct structures occur on the surfaces of the oral papillae in several loricariid catfish species; namely, (1) typical vertebrate taste buds composed of receptor and sustentacular cells and (2) brushlike projections, termed epidermal brushes, that represent specialized epidermal cells containing keratin. Both of these structures were studied with the combined use of light microscopy and scanning and transmission electron microscopy. The general body surface, fins, and rostral cutaneous processes of some loricariid catfishes are covered with taste or terminal buds but lack the epidermal brushes. It is suggested that the epidermal brushes found on the oral papillae serve as protective devices for the taste buds and as abrasive surfaces for substrate scraping during feeding. The taste buds on the oral papillae may detect any gustatory stimuli from the resulting substrate disturbance. Comparative studies reveal many differences in the number and spatial arrangement of these two structures on the oral papillae among the several species of the Loricariidae examined. These differences may represent functional adaptations to the various modes of life in the Loricariidae.     

哺乳动物味觉的细胞生物学

味觉对于生命具有重要作用,在一定程度上决定了动物对食物的选择。哺乳动物味觉识别主要依赖于舌味蕾中的味细胞,味蕾由50～100个极化的神经上皮细胞聚集而成。通过对味蕾细胞的分析显示,味蕾是一种精巧的单元结构。这篇文章综述了味蕾细胞的形态、结构功能、细胞生物学活性以及味觉信息的传导。     

The fine structural effect of sialectomy on the taste bud cells in the rat.

Taste buds in the rat and other mammals share a secretory activity with their transduction function as taste receptor. The present work shows the effect of bilateral removal of the main salivary glands on taste bud cells' components related to secretion in the vallate papilla of the rat. In the sialectomized rats remarkable changes were evidence in the dark and intermediate types of taste bud cells, which are known to be the secretory components. Such changes involve hypertrophy of either the protein synthetizing machinery, the smooth endoplasmic reticulum or the Golgi complex. Lucent and coated vesicles associated to Golgi cisternae increased in number but the amount of dense-core vesicles (secretory vesicles) at the apical cytoplasm of cells decreased. Images of exocytosis of secretory products were observed. The hypertrophy of Golgi complex components was clearly detected with the OsO4 impregnation method for light and electron microscopy. Alteration in the acid phosphatase activity of taste bud cells was not observed in the sialectomized rats. These findings suggest that sialectomy stimulates the entire secretory cycle of dark and intermediate taste bud cells. The light taste bud cells, which are not engaged in secretion, are hardly affected by the treatment. Although taste buds in mammals are neuro-dependent structures, present evidence indicates that they are also sensitive to non-neural influences.     

Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chordalingual nerve

In normal rats there is one taste bud on the apical surfaceof each fungiform papilla. These taste buds are innervated bythe chorda tympani proper nerve (CT). According to general consensus,after cutting the nerve the taste buds should disappear. Inthis study, performed on 24 rats divided in six groups, theCT nerve on the left side (singly denervated) and the combinedchorda-lingual (CT-L) nerve on the other side (doubly denervatedwere permanently interrupted. The animals were sacrificed after5, 10, 20, 35,60 and 100 days and their tongues serially sectionedfor light microscope examiation. Some papillae were examinedunder an electron microscope. The papillae were categorizedinto three groups: papillae with a normal looking taste bud,with an abnormal looking taste bud and without a taste bud.The results showed a substantial number of papillae with a normallooking taste bud present at all time intervals in all animals.More specifically, on the singly denervated side the proportionof normal looking taste buds stayed below 10% until day 60,when it increased to 15% and to 23% on day 100. The proportionof abnormal looking taste buds decreased from above 92% by day5 to 49% on day 100. The percentage of fungiform papillae withoutsigns of a taste bud was relatively low on the singly denervatedside at times (1, 5, 16, 29, 34 and 28%). On the doubly denervatedside fewer than than 4% normal looking taste buds were foundthroughout the time period. The proportion of abnormal lookingtaste buds decreased from {small tilde} 96% by day 5 to 35%on day 100. A significantly higher proportion of papillae withno taste bud was observed on this side from day 10 onwards.(1, 29, 32, 52, 60 and 63%). The reasons for the differencein tast bud number between the doubly and singly denervatedsides are unknown, but it is possible that collaterals fromother (non-gustatory) nerves have an ability, although limited,to induce and maintain fungiform taste buds. In other words,the capacity to induce taste bud formation is not limited exclusivelyto gustatory nerves.     

Glycoconjugates and keratin 18 define subsets of taste cells

Summary Sections of neonatal, normal adult and denervated adult rat tongue were examined with lectin histochemistry. Attention was focused upon intragemmal cells (cells within the taste bud) and the surrounding perigemmal cells. Informative staining patterns were observed with four of 12 lectins:Ulex europaeus (UEA-I),Bauhinia purpurea (BPA),Helix pomatia (HPA) andLotus tetragonolobus (LTA) agglutinins. In normal adult tongues, BPA bound to those lingual epithelial cells lacking contact with the basal lamina. After they formed, vallate taste buds were laterally surrounded by distinctive BPA-positive cells. HPA reacted selectively with 28% and LTA with 23% of the intragemmal cells in vallate/foliate taste buds. In double-stained taste buds there was, a statistically significant overlap of LTA-positive cells and keratin 18-positive cells. The overlap between HPA binding and keratin 18 was more marked: double-stained cells comprized 67% of all stained cells. During taste bud development in neonates keratin 18 synthesis preceded HPA binding. In contrast, during the replacement of adult taste cells, keratin 18 synthesis and HPA binding were generally concurrent. Keratin 18 and HPA probably identify the same subset of older taste receptor cells. HPA may bind to glycoconjugates on the surface of keratin 18-positive cells. In denervated adult tongue the loss of all UEA-I-positive or BPA-positive perigemmal cells suggests that perigemmal as well as intragemmal cells are nerve-dependent.     

Insulin-Like Growth Factors Are Expressed in the Taste System,but Do Not Maintain Adult Taste Buds

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1r^lox/lox). While K14-Cre::Igf1r^lox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1r^lox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C β2- (PLCβ2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1r^lox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1r^lox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.     

Espin cytoskeletal proteins in the sensory cells of rodent taste buds

Espins are multifunctional actin-bundling proteins that are highly enriched in the microvilli of certain chemosensory and mechanosensory cells, where they are believed to regulate the integrity and/or dimensions of the parallel-actin-bundle cytoskeletal scaffold. We have determined that, in rats and mice, affinity purified espin antibody intensely labels the lingual and palatal taste buds of the oral cavity and taste buds in the pharyngo-laryngeal region. Intense immunolabeling was observed in the apical, microvillar region of taste buds, while the level of cytoplasmic labeling in taste bud cells was considerably lower. Taste buds contain tightly packed collections of sensory cells (light, or type II plus type III) and supporting cells (dark, or type I), which can be distinguished by microscopic features and cell type-specific markers. On the basis of results obtained using an antigen-retrieval method in conjunction with double immunofluorescence for espin and sensory taste cell-specific markers, we propose that espins are expressed predominantly in the sensory cells of taste buds. In confocal images of rat circumvallate taste buds, we counted 21.5 ± 0.3 espin-positive cells/taste bud, in agreement with a previous report showing 20.7 ± 1.3 light cells/taste bud when counted at the ultrastructural level. The espin antibody labeled spindle-shaped cells with round nuclei and showed 100% colocalization with cell-specific markers recognizing all type II [inositol 1,4,5-trisphosphate receptor type III (IP₃R₃)_, α-gustducin, protein-specific gene product 9.5 (PGP9.5)] and a subpopulation of type III (IP₃R₃, PGP9.5) taste cells. On average, 72%, 50%, and 32% of the espin-positive taste cells were labeled with antibodies to IP₃R₃, α-gustducin, and PGP9.5, respectively. Upon sectional analysis, the taste buds of rat circumvallate papillae commonly revealed a multi-tiered, espin-positive apical cytoskeletal apparatus. One espin-positive zone, a collection of ～3 μm-long microvilli occupying the taste pore, was separated by an espin-depleted zone from a second espin-positive zone situated lower within the taste pit. This latter zone included espin-positive rod-like structures that occasionally extended basally to a depth of 10–12 μm into the cytoplasm of taste cells. We propose that the espin-positive zone in the taste pit coincides with actin bundles in association with the microvilli of type II taste cells, whereas the espin-positive microvilli in the taste pore are the single microvilli of type III taste cells.