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PubMed1976

Biochemical studies of taste sensation. III. Preparation of a suspension of bovine taste bud cells and their labeling with a fluorescent probe.

A method to prepare suspensions of taste bud cells is described. Bovine circumvallate papillae, which contain most of the taste buds in this animal, are incubated in collagenase-containing medium and the epidermal sidewall tissue is then dissected from the inner gelatinous dermis. The sidewall tissue, which contains the taste buds, is gently homogenized by manual operation of an all-glass homogenizer with a loose-fitting pestle. The suspended material is separated on a discontinous Ficoll gradient (2%, 8%, 10%, 12% w/w). The material banding at the 8-2% interface is greatly enriched in spindle-shaped cells that are morphologically similar to taste bud cells as they appear in situ. These cells are not seen when the procedure is done with tissues devoid of taste buds, namely the upper surface of the circumvallate papilla or epithelium from the intermolar eminence. Fluorescence analysis indicates that the hydrophobic probe, 8-anilino-1-naphthalenesulfonate (ANS), binds to relatively nonpolar sites in the suspension. It is postulated that the probe is adsorbing onto the surface membrane of the cell. These preparations may be useful in studying specificity and transduction in taste sensation.

Anilino Naphthalenesulfonates↗

PubMed1997

Chicken transcription factor AP-2: cloning, expression and its role in outgrowth of facial prominences and limb buds.

Embryonic facial development in chick embryos involves a sequential activation of genes that control differential growth and patterning of the beak. In the present study we isolate one such gene, the transcription factor, AP-2, that is known to be expressed in the face of mouse embryos. The protein sequence of chick AP-2alpha is 94% homologous to human and mouse AP-2. Wholemount in situ hybridization with a probe for chick AP-2 identifies expression from primitive streak stages up to stage 28. The most striking expression patterns in the head are during neural crest cell migration when AP-2 transcripts follow closely the tracts previously mapped for neural crest cells. Later, expression in the facial mesenchyme is strongest in the frontonasal mass and lateral nasal prominences and is downregulated in the maxillary and mandibular prominences. Once limb buds are visible, high expression is seen in the distal mesenchyme but not in the apical ectodermal ridge. The expression patterns of AP-2 in stage 20 embryos suggested that the gene may be important in "budding out" of facial prominences and limb buds. We implanted beads soaked in retinoic acid in the right nasal pit of stage 20 embryos resulting in a specific inhibition of outgrowth of the frontonasal mass and lateral nasal prominences. AP-2 expression was completely down-regulated in the lateral nasal within 8 hr of bead application. In addition, the normal up-regulation of AP-2 in the frontonasal mass did not occur following retinoic-acid treatment. There was an increase in programmed cell death around the right nasal pit that accompanied the down-regulation of AP-2. Prominences whose morphogenesis were not affected by retinoic acid did not have altered expression patterns. We removed the apical ectodermal ridge in stage 20 limb buds and found that AP-2 expression was partially downregulated 4 hr following ridge removal and completely downregulated 8 hr following stripping. Application of an FGF-4 soaked bead to the apex of the limb bud maintained AP-2 expression. Thus AP-2 is involved in outgrowth and could be regulated by factors such as FGFs that are present in the ectoderm of both the face and limb.

Amino Acid Sequence↗

PubMed2004

Expression of P2Y1 receptors in rat taste buds.

Extracellular nucleotides such as ATP are the signaling molecules which bind to membrane receptors (P2X ligand-gated ion channels and G-protein-coupled P2Y families). In the gustatory system, it is known that P2X receptors are expressed exclusively in nerve fibers innervating the taste buds. Also, P2Y receptors are suggested to play some important roles in taste signal transductions on the basis of the physiological studies. In the present study, we examined the expression patterns of P2Y1 receptor subtype by using reverse transcript polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. RT-PCR analyses showed that P2Y1 receptor mRNAs appeared in circumvallate papillae. P2Y1 receptor mRNA was detected in a subset of taste bud cells by in situ hybridization. By immunohistochemical analyses, P2Y1 receptor was detected in a subset of taste bud cells of fungiform, foliate, and circumvallate papillae. We showed that ATP induced a biphasic intracellular Ca2+ increase in taste cells by a Ca2+ imaging method. Furthermore, we showed by double-immunolabeling methods that P2Y1-expressing cells coexpressed both IP3R3 and SNAP-25. These results suggest that ATP may activate P2Y receptors resulting in Ca2+ release from internal stores via IP3R3. Since many SNAP-25-immunoreactive taste bud cells coexpressed P2Y1 immunoreactivity, it is suggested that P2Y1-expressing cells may possess synapses with afferent nerve fibers. The results of the present study suggest that P2Y1 receptor may play some roles in ATP-mediated signal transductions between taste bud cells and afferent taste fibers.

Adenosine Triphosphate↗

PubMed1998

Innervation of developing human taste buds. An immunohistochemical study.

Morphological changes in developing human gustatory papillae during the 6th to the 23rd postovulatory week have been studied. The general innervation pattern of taste papillae and taste bud primordia was revealed immunohistochemically using antibodies against protein gene product 9.5 (PGP9.5), neurofilament H (NFH), neurofilament L (NFL), neurone-specific enolase (NSE), and tubulin. The autonomic and somatosensory nerve supply has been investigated using antibodies against substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), neuropeptide Y (NPY), the neuronal form of nitric oxide synthase (n-NOS), and, enzyme histochemically, NADPH-diaphorase. Nerve fibers approach the basal membrane of the lingual epithelium around the 7th postovulatory week and invade the epithelium of papilla-like structures at the 8th week, but some also penetrate the basal membrane of the non-papillary epithelium. They are in close contact with slender epithelial cells that are considered to be the taste bud's progenitor cells. Early human taste buds situated at the anterior part of the tongue do not necessarily require a dermal (later fungiform) papilla. The NADPH-diaphorase reaction revealed positive results in dermal nerve fibers, but the immunohistochemical reaction against n-NOS was negative. Immunohistochemical detection of neuropeptides and vasoactive substances rendered negative results for developmental stages of 7-18 postovulatory weeks. By the 18th week, only SP was detected in dermal papillae, but not in the vicinity of taste buds' primordia. Thus, autonomic and somatosensory nerves seem not to play a key role in formation and maintenance of early human taste buds.

Biomarkers↗

PubMed1999

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↗

PubMed2005

Are there efferent synapses in fish taste buds?

In fish, nerve fibers of taste buds are organized within the bud's nerve fiber plexus. It is located between the sensory epithelium consisting of light and dark elongated cells and the basal cells. It comprises the basal parts and processes of light and dark cells that intermingle with nerve fibers, which are the dendritic endings of the taste sensory neurons belonging to the cranial nerves VII, IX or X. Most of the synapses at the plexus are afferent; they have synaptic vesicles on the light (or dark) cells side, which is presynaptic. In contrast, the presumed efferent synapses may be rich in synaptic vesicles on the nerve fibers (presynaptic) side, whereas the cells (postsynaptic) side may contain a subsynaptic cistern; a flat compartment of the smooth endoplasmic reticulum. This structure is regarded as a prerequisite of a typical efferent synapse, as occurring in cochlear and vestibular hair cells. In fish taste buds, efferent synapses are rare and were found only in a few species that belong to different taxa. The significance of efferent synapses in fish taste buds is not well understood, because efferent connections between the gustatory nuclei of the medulla with taste buds are not yet proved.

Animals↗

PubMed1991

The distribution of fungiform papillae and taste buds on the human tongue.

Investigations on monkeys have shown that the application of the acidic dye Ponceau S red or the basic dye Alcian blue to the tongue surface facilitates identification of fungiform papillae and taste buds. Both of these dyes were now used in varying degrees of acidity on fixed and unfixed human cadaveric tongues in an attempt to determine the regional distribution of papillae and buds. Satisfactory staining was obtained with acidic Ponceau S red in 10% formalin and 10% trichloracetic acid (pH 2.5). For six tongues, the number of fungiform papillae ranged from 171 to 253 (mean 195) and these were located predominantly at the tip. Of the fungiform papillae, 67% had no staining of taste bud pores. The average number of visible taste pores on the other fungiform papillae was 3 (range 1-21). The correlation between the number of stained taste pores and underlying taste buds was confirmed using serial histological sections of 90 fungiform papillae. This work has shown that a mean of 193 taste buds are carried on fungiform papillae of the human tongue and that 87% of these are located in the anterior 2 cm.

Adolescent↗

PubMed1985

Distribution of calmodulin in taste buds.

Calmodulin is higher in particulate fractions from bovine taste buds containing taste bud membranes which specifically bind sweet tastants compared to corresponding fractions from control non-taste bud bearing lingual epithelial tissue. As biochemical purity (i.e., membrane enzyme marker activity) of these membrane enriched fractions increased (P4B greater than P3B greater than P2B) calmodulin correspondingly increased (P4B greater than P3B greater than P2B); these increases also correlated with increased membrane purity as demonstrated by electron microscopy. All PB subfractions from taste buds contained a greater membrane concentration than those from PD subfractions and calmodulin was significantly increased in each corresponding subfraction. The presence of calmodulin in taste bud membranes, its correlation with membrane purification and reports that numerous drugs which induce taste loss are potent inhibitors of calmodulin suggest a role for calmodulin in taste function.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1990

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.

Arousal↗

PubMed1996

Whole-cell recording from non-dissociated taste cells in mouse taste bud.

A method for the whole-cell recording from non-dissociated taste cells within mouse taste bud is described. The lingual epithelial sheet containing the taste buds was peeled free from the tongue by injecting a proteolytic enzyme, elastase, under the lingual epithelium and by incubating it in normal Tyrode solution at 30 degrees C. The preparation consisting of a taste bud and a small piece of the lingual epithelium was obtained by further the incubation in divalent cation-free Tyrode solution. After holding the small piece of the epithelium by a holding pipette loaded with continuous negative pressure for keeping the orientation of the taste bud, whole-cell configuration was established in a non-dissociated taste cell within the taste bud with a patch pipette containing Lucifer Yellow. Taste stimuli or blockers were applied from the third pipette placed near the taste pore under the continuous flow of bathing solution. Under this condition, we could simultaneously accomplish patch-clamping, visualization of taste cell morphology, localized taste stimulation and maintenance of microenvironment around the taste organ. Rapid responses to a relatively high concentration of salt stimuli were also obtained.

Amiloride↗

PubMed2004

Neurotrophin receptors in taste buds of adult zebrafish (Danio rerio).

TrkB plays crucial roles in the development and maintenance of taste buds in mammals. In this study we investigated the presence and cell localization of Trks (TrkA, TrkB and TrkC) in taste buds of the zebrafish (Danio rerio) using Western blot and immunohistochemistry. Proteins of 140 and 145 kDa, identified as full-length TrkA and TrkB, were detected. Conversely, the anti-TrkC antibody recognized a protein lower than expected (100 kDa). In agreement with these results the sensory cells of taste buds, displayed TrkA- and TrkB-like, but not TrkC-like, immunoreactivity. TrkA and TrkB co-existed in the same taste buds, but remains to be clarified whether or not they are co-expressed in the same cells. Present results demonstrate that as for mammals neurotrophins might play a role in sensory cells of the teleostean taste buds.

Animals↗

PubMed2006

Taste bud contains both short-lived and long-lived cell populations.

Taste bud cells undergo continual turnover even in adulthood, and their average lifespan has been estimated as approximately 10 days. However, it is not clear whether this figure can be applied to all the different cell types contained in a taste bud. Here, we describe the age and life cycle of taste bud cells in rat circumvallate papillae, and indicate that the lifespan is heterogeneous, ranging from 2 days to over 3 weeks. Taste bud cells were incorporated from the basal proliferative layer in 1-2 days after birth. After incorporation, approximately half of the cells were eliminated within 2-3 days, and the remaining half were maintained with gradual decrease, suggesting that there are at least two types of cells; short-lived cells and long-lived cells. Moreover, above 10% of the incorporated cells were maintained at 3 weeks. In order to gain information about the relationship between the cell functions and the cell age, we carried out double-labeling experiments using 5-bromo-2'-deoxyuridine and each of two markers for in situ hybridization: mammalian achaete-scute homolog 1 (Mash1) and phospholipase C beta 2 (PLCbeta2) as markers of early differentiation and functional taste signaling, respectively. Mash1 expression began immediately after the incorporation and reached a maximum at 5-6 days after birth. Fewer but distinct Mash1-positive cells were still observed after 3 weeks. PLCbeta2 expression was observed from day 5, reached a maximum at day 12, and continued over 3 weeks. Taken together, a taste bud contains both short-lived and long-lived cells: the short-lived cells are eliminated in a time course similar to the surrounding epithelial cells, and the long-lived cells including taste receptor cells have a lifespan longer than the previous estimation.

Analysis of Variance↗

PubMed2003

Ephrin-A2 regulates position-specific cell affinity and is involved in cartilage morphogenesis in the chick limb bud.

In the developing limb bud, mesenchymal cells show position-specific affinity, suggesting that the positional identity of the cells is represented as their surface properties. Since the affinity is regulated by glycosylphosphatidylinositol (GPI)-anchored cell surface proteins, and by EphA4 receptor tyrosine kinase, we hypothesized that the GPI-anchored ligand, the ephrin-A family, also contributes to the affinity. Here, we describe the role of ephrin-A2 in the chick limb bud. Ephrin-A2 protein is uniformly distributed in the limb bud during early limb development. As the limb bud grows, expression of ephrin-A2 is strong in its proximal-to-intermediate regions, but weak distally. The position-dependent expression is maintained in vitro, and is regulated by FGF protein, which is produced in the apical ectodermal ridge. To investigate the role of ephrin-A2 in affinity and in cartilage morphogenesis of limb mesenchyme, we ectopically expressed ephrin-A2 in the limb bud using the retrovirus vector, RCAS. Overexpressed ephrin-A2 modulated the affinity of the mesenchymal cells that differentiate into autopod elements. It also caused malformation of the autopod skeleton and interfered with cartilage nodule formation in vitro without inhibiting chondrogenesis. These results suggest that ephrin-A2 regulates the position-specific affinity of limb mesenchyme and is involved in cartilage pattern formation in the limb.

Animals↗

PubMed1998

Intracellular free calcium concentration in human taste bud cells increases in response to taste stimuli.

We examined changes of intracellular free calcium concentration [Ca2+]i elicited by taste stimuli of sucrose, denatonium and NaCl in the taste buds of seven human fungiform papillae. In one taste bud we observed an increase in [Ca2+]i induced by only NaCl. In another bud an increase of [Ca2+]i in response to both NaCl and sucrose was found. The Ca2+ responses to NaCl and sucrose occurred in differential areas within the one taste bud. In the other five fungiform papillae [Ca2+]i was not changed by the taste stimuli. These results suggest that an increase of [Ca2+]i participates in taste transduction mechanisms for sucrose and NaCl, and that taste cells in one taste bud may respond to differential stimuli.

Adult↗

PubMed1975

On the ultrastructure and permeability of taste buds of the marine teleost Ciliata mustela.

The abundant taste buds of the barbels and free fin rays of the five bearded rockling, Ciliata mustela contain an average of 100-150 cells, falling into two types. Tubule-containing cells ('t-cells'), tentatively identified as receptor cells, and each surrounded by fibril-containing cells ('f-cells') in the central part of the bud. t-Cells also occur in two concentric shells separated by indifferent epithelial cells at the periphery of the bud. f-Cells are characterized by their concentrations of fine fibrils, and by granules or vesicles of 180-190 mmu diameter. The 100 or so receptor cells in a taste bud are innervated by some 250 axons. Lanthanum penetrates more deeply into the extracellular space of taste buds than into the extracellular space of the general epithelium, perhaps indicating that a greater area than the mere protruding tip of receptor cells may be accessible to chemical stimulation. Degenerating cells may provide an important route of entry for such external agents.

Animals↗

PubMed1998

Amphibians provide new insights into taste-bud development.

Until recently, the predominant model of taste-bud development was one of neural induction: ingrowing sensory fibers were thought to induce taste-bud differentiation late in embryonic development. Recent experimental studies, however, show that the development of taste buds is independent of their innervation. In amphibian embryos, the ability to generate taste buds is an intrinsic feature of the oropharyngeal epithelium long before the region becomes innervated. These studies indicate that patterning of the oropharyngeal epithelium occurs during gastrulation, and suggest that taste buds or their progenitors play the dominant role in the development of their own innervation.

Amphibians↗

PubMed2001

Shh and Ptc are associated with taste bud maintenance in the adult mouse.

In mammals, taste receptor cells are organized into taste buds on tongue. Taste buds are trophically maintained by taste neurons and under continuous renewal, even in adults. We found that the receptor for Sonic hedgehog (Shh), Patched1 (Ptc), was expressed around taste buds where cells were proliferating, and that Shh was expressed within basal cells of taste buds. Denervation caused the loss of Shh and Ptc expression before the degeneration of taste buds.

Animals↗

PubMed2003

Expression of rigf, a member of avian VEGF family, correlates with vascular patterning in the developing chick limb bud.

In a differential display screening for genes regulated by retinoic acid in the developing chick limb bud, we have isolated a novel gene, termed rigf, retinoic-acid induced growth factor, that encodes a protein belonging to the vascular endothelial growth factor (VEGF) family. Rigf transcripts were found in the posterior region of the limb bud in a region-specific manner as well as in other embryonic tissues and regions, including the notochord, head and trunk mesenchyme, retinal pigment epithelium, and branchial arches. Several manipulations revealed that retinoic acid and sonic hedgehog signaling pathways regulate rigf expression in the limb bud. VEGF family members, which promote the migration, differentiation and proliferation of endothelial cells in both blood and lymphatic vessels, are important factors for the formation of blood and lymphatic vasculatures during development. We demonstrated that the anterior border of the rigf expression domain in the limb bud corresponds with the position of the primary central artery (the subclavian artery in the forelimb), which is a main artery for supplying blood to the limb. These observations taken together with results from some experimental manipulations suggest that the limb tissue attracts blood vessels into the limb bud and that rigf is involved in the pattern formation of blood vessels in the limb.

Amino Acid Sequence↗