Search

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K + currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K + currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K + currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K + currents which were completely inhibited by 10 mM TEA, whereas IP 3 R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K + currents were 17.9 ± 4.5 (n = 17) and - 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K + channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K + channel phosphorylation likely affects the signal transduction of taste., (© 2024. The Author(s).)

Taste substances are detected by taste receptor cells in the taste buds in the oral epithelium. Individual taste receptor cells contribute to evoking one of the five taste qualities: sweet, umami, bitter, sour, and salty (sodium). They are continuously replaced every few weeks by new ones generated from local epithelial stem cells. A POU transcription factor, Pou2f3 (also known as Skn-1a), regulates the generation and differentiation of sweet, umami, and bitter cells. However, the molecular mechanisms underlying terminal differentiation into these Pou2f3-dependent taste receptor cells remain unknown. To identify the candidate molecules that regulate the differentiation of these taste receptor cells, we searched for taste receptor type-specific transcription factors using RNA-sequence data of sweet and bitter cells. No transcription factor gene showing higher expression in sweet cells than in bitter cells was found. Eyes absent 1 (Eya1) was identified as the only transcription factor gene showing higher expression in bitter cells than in sweet cells. In situ hybridization revealed that Eya1 was predominantly expressed in bitter cells and also in the putative immature/differentiating taste bud cells in circumvallate and fungiform papillae and soft palate. Eya1 is a candidate molecule that regulates the generation and differentiation of bitter cells.

Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1 CreERT2 ::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L -amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C β2 (PLCβ2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.

Sox2 is proposed to regulate the differentiation of bipotential progenitor cells into taste bud cells. However, detailed expression of Sox2 remains unclear. In this report, Sox2 expression during taste bud development in the fungiform (FF), circumvallate (CV) and soft palate (SP) areas is examined together with Prox1. First, we immunohistochemically checked Prox1 expression in adults and found that almost all taste bud cells are Prox1-positive. During FF development, intense Sox2 expression was restricted to taste bud primordia expressing Prox1 at E12.5. However, at E14.5, Sox2 was intensely expressed outside the developing taste buds resolving to perigemmal Sox2 expression in adults. In the SP, at E14.5, taste bud primordia emerged as Prox1-expressing cell clusters. However, intense Sox2 expression was not restricted to taste bud primordia but was detected widely in the epithelium. During development, Sox2 expression outside developing taste buds was generally down-regulated but was retained in the perigemmal region similarly to that in the FF. In the CV, the initial stage of taste bud development remained unclear because of the lack of taste bud primordia comparable to that in the FF and SP. Here, we show that Prox1-expressing cells appear in the apical epithelium at E12.5, in the inner trench wall at E17.5 and in the outer trench wall at E18.5. Sox2 was again not restricted to developing taste bud cells expressing Prox1 during CV development. The expression patterns support that Sox2 does not serve as a cell fate selector between taste bud cells and surrounding keratinocytes but rather may contribute to them both.

Synaptogyrins are conserved components of the exocytic apparatus and function as regulators of Ca(2+)-dependent exocytosis. The synaptogyrin family comprises three isoforms: two neuronal (synaptogyrin-1 and -3) and one ubiquitous (synaptogyrin-2) form. Although the expression patterns of the exocytic proteins synaptotagmin-1, SNAP-25, synaptobrevin-2 and synaptophysin have been elucidated in taste buds, the function and expression pattern of synaptogyrin-1 in rat gustatory tissues have not been determined. Therefore, we examined the expression patterns of synaptogyrin-1 and several cell-specific markers of type II and III cells in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays and immunoblot analysis revealed the expression of synaptogyrin-1 mRNA and its protein in circumvallate papillae. In fungiform, foliate and circumvallate papillae, the antibody against synaptogyrin-1 immunolabeled a subset of taste bud cells and intra- and subgemmal nerve processes. Double-labeling experiments revealed the expression of synaptogyrin-1 in most taste cells immunoreactive for aromatic L-amino acid decarboxylase and the neural cell adhesion molecule. A subset of synaptogyrin-1-immunoreactive taste cells also expressed phospholipase Cβ2, gustducin, or sweet taste receptor (T1R2). In addition, most synaptogyrin-1-immunoreactive taste cells expressed synaptobrevin-2. These results suggest that synaptogyrin-1 plays a regulatory role in transmission at the synapses of type III cells and is involved in exocytic function with synaptobrevin-2 in a subset of type II cells in rat taste buds.

The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

Acetylcholine (ACh) is well established as a neurotransmitter and/or neuromodulator in various organs. Previously, it has been shown by Ogura (J Neurophysiol 87:2643-2649, 2002) that in both physiological and immunohistochemical studies the muscarinic acetylcholine (ACh) receptor is present in taste receptor cells. However, it has not been determined if ACh is released locally from taste receptor cells and/or surrounding nerve fibers. In this study we investigated the sites of ACh release in mouse taste tissue using the antisera against vesicular ACh transporter (VAChT), a key element of ACh-containing vesicles. Our data show that VAChT-immunoreactivity is present in many taste receptor cells, including cells expressing the transient receptor potential channel M5 (TRPM5). In taste cells, VAChT-immunoreactivity was colocalized with the immunoreactivity to choline-acetyltransferase (ChAT), which synthesizes ACh. Additionally, enhanced green fluorescent protein (eGFP) was detected in the taste cells of BAC-transgenic mice, in which eGFP was placed under the control of endogenous ChAT transcriptional regulatory elements (ChAT(BAC)-eGFP mice). Furthermore, many ChAT-immunolabeled taste cells also reacted to an antibody against the vesicle-associated membrane protein synaptobrevin-2. These data suggest that ACh-containing vesicles are present in taste receptor cells and ACh release from taste cells may play a role in autocrine and/or paracrine cell-to-cell communication. In addition, certain nerve fibers surrounding or within taste buds were immunoreactive for the VAChT antibody. Some of these fibers were also immunolabeled with antibody against calcitonin gene-related peptide (CGRP), a marker for trigeminal peptidergic fibers. Thus, functions of taste receptor cells could be modulated by trigeminal fibers via ACh release as well.

Glutamate is one candidate for the neurotransmitters and/or neuromodulators involved in taste signaling in taste buds. Group II metabotropic glutamate receptors (mGluRs: mGluR2 and mGluR3) are known to function as presynaptic receptors that regulate the release of glutamate and/or other neurotransmitters in the central nervous system. Group II mGluRs are negatively linked to adenylyl cyclase through Galphai subunits and thereby reduce the turnover of cAMP. In rat taste tissues, a subset of adenylyl-cyclase-8-expressing taste cells coexpress the Galphai subunits gustducin and Galphai2. However, the expression patterns of group II mGluRs in rat taste tissues have not yet been elucidated. We have therefore examined the expression patterns of mGluR2, mGluR3, and gustducin in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays have revealed that mGluR2 and mGluR3 mRNAs are expressed in the circumvallate papillae. In situ hybridization analyses have detected positive signals for mGluR2 and mGluR3 mRNAs only in the circumvallate taste buds. Among the fungiform, foliate, and circumvallate papillae, an antibody against mGluR2/3 labels a subset of taste bud cells and nerve fibers immediately beneath the taste lingual epithelium. Double-labeling experiments have demonstrated that mGluR2/3-positive cells coexpress gustducin. These results indicate that mGluR2 and mGluR3 are coupled to Galphai subunits and play roles in glutamate-mediated signaling in taste transductions.

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner's gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.

Recent functional evidence indicates that mouse taste cells express P2Y receptors coupled to IP(3) production and Ca(2+) mobilization. Our studies of the expression profile of particular P2Y isoforms in the taste tissue of the mouse have revealed that ATP and UTP equipotently mobilize intracellular Ca(2+) at saturating concentrations, suggesting that common receptors for both nucleotides, i.e., P2Y(2) and P2Y(4) subtypes, might be involved. Reverse transcription/polymerase chain reaction and immunohistochemistry have confirmed the presence of P2Y(2) and P2Y(4) receptors in a population of taste bud cells from the circumvallate and foliate papillae. Transcripts for the P2Y(1) and P2Y(6) isoforms have also been detected in taste tissue preparations, this observation being consistent with the ADP and UDP responsiveness of taste cells. Together, our data suggest that P2Y(2) and P2Y(4) receptors play a predominant role in mediating taste cell responses to ATP and UTP.

The morphology of viable taste disks of the frog was explored with multi-photon microscopy. In order to identify single sensory or supporting cells within the tissue, we searched for fluorescent dyes that stained subsets of the cell population or possibly cell types. Some cell types indeed stained preferentially with certain fluorescent dyes. A subset of glia-like cells (type Ic) stained with BCECF, a H+-sensitive dye, and indo-1, a Ca2+-sensitive dye, both presented in the membrane-permeant ester form. BCECF-ester also stained the dendrites of type III receptor cells, but indo-1 ester did not. Receptor cells of type II stained with MQAE, a positively charged Cl- -sensitive dye. A subset of type II cells accumulated amiloride, a positively charged fluorescent diuretic. Certain supporting cells, i.e., wing cells (type Ib) and glia-like cells (type Ic), were labeled by negatively charged dyes, e.g., calcium green-1 dextran. Mucus cells (type Ia) were stained with only two of the 19 dyes examined, and Merkel-like basal cells (type IV) were stained only with a membrane-labeling voltage-sensitive dye, presumably by endocytosis. No dye was found which would stain all types of cells or all receptor cells. This finding reveals a potential problem for future functional imaging aiming at population responses, as the responses of unstained cells then would remain unobserved. Specificity of dyes with respect to cell types was sufficient to identify supporting cells and receptor cells. Cell shape could then be reconstructed, using optical slicing and rendering techniques. Thus populations of dye-loaded elongated cells, especially types Ic, II and III, could for the first time be visualized in three dimensions.

NeuroD, a basic helix-loop-helix transcription factor, has been shown to play a role in the differentiation of neurons, olfactory cells, and neuroendocrine tissues. Since the taste buds have characteristics of typical paraneurons, we examined the expression of NeuroD in the taste buds of mice. By RT-PCR analysis, NeuroD mRNA was found to be expressed in the epithelium of circumvallate papillae-containing taste buds, but not in the lingual epithelium lacking them. NeuroD immunoreactivity was detected in a subset of taste bud cells in the circumvallate, foliate, and fungiform papillae and in the soft palate. NeuroD-expressing cells had a spindle-like shape, first appeared at postnatal day 3, and increased in number during postnatal development. After bisection of the glossopharyngeal nerves, NeuroD-expressing cells decreased in number at day 4 and disappeared from the trench wall of the circumvallate papillae by day 14. A few NeuroD-expressing taste buds reappeared at postoperative day 28. Denervation and regeneration experiments showed that expression of NeuroD in the taste buds was dependent upon gustatory innervation. Double immunolabeling with gustducin or with neural cell adhesion molecule (NCAM) showed that NeuroD-expressing cells did not express NCAM, but did express gustducin. These results suggest that NeuroD is expressed in a mature cell type, type-II cells, but not in type-III cells.

Keratin filaments of epithelial- and taste-bud cells in the circumvallate papillae of adult and developing mice were studied by immunocytochemistry using monoclonal antikeratin antibodies (PKK2 and PKK3) and by conventional electron microscopy. Elongated cells (type-I, -II, and -III cells) of the taste buds were stained by PKK3 antibody, which reacts with 45-kdalton keratin, whereas basal cells of the taste buds and surrounding epithelial cells showed negative staining with PKK3. Such PKK3-reactive cells occurred at 0 day after birth, when taste-buds first appeared in the dorsal surface epithelium of the papillae. Thus 45-kdalton keratin seems to be an excellent immunocytochemical marker for identifying taste-bud cells. Epithelial cells in all layers of the trench wall and basal layer cells of the dorsal surface contained densely aggregated bundles of keratin filaments that reacted with PKK2 antibody, but not with PKK3. In contrast, taste-bud cells and spinous and granular layer cells of the dorsal surface possessed loose aggregated bundles of filaments that reacted with PKK3, but not with PKK2. These results suggest that the aggregation and distribution pattern of keratin filaments may reflect differences in the keratin subtypes that comprise these filaments.

It is believed that differentiation and maintenance of taste buds in vertebrates is dependent on the trophic function of their sensory nerve supply. In the present work, colchicine was injected into the circumvallate papilla of the rat. This produced a reversible blockade of neuroplasmic transport and disappearance of taste buds. Colchicine inhibited the further differentiation of bud cells, but apparently did not change the life cycle of the cells present already at the time of injection. It is speculated that the neurotrophic factors in this particular cell system are effective to induce cell differentiation only.

Fluorescence histochemistry reveals that in the frog's taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 mmug. Following reserpine treatment the yellow fluroescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin). The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700-1000 angstrom in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions of nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustary cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.

The taste buds on the barbels in three species of teleosts (Cyprinus carpio, Misgurnus anguillicaudatus, Parasilurus asotus) were studied by means of fluorescence- and electron microscopy. Intensely yellow-fluorescent cells, which are disk-shaped and located exclusively in a basal position, are observed in the barbel-buds of all fishes examined. The basal cells contain a large number of small clear vesicles approximately 40-60 nm in diameter, which show a tendency to aggregate in the cytoplasm facing the junction of the nerve terminals; chemically transmitting synapses are seen in the latter region. It is suggested from the present observations that the basal cells in the barbel-bud may originate from Schwann cells and have a dual function both as mechanoreceptors and paracrine elements. Since the administration of 5,6-DHT results in an appearance of small dense vesicles among the small clear vesicles, the possibility exists that the basal cell may be capable of taking up monoamines and storing them in the small clear vesicles.

A method to isolate taste buds from the foliate papillae of the rabbit tongue is described. The method comprises (a) separation of the epidermis from the dermal layer after treatment with dilute acetic acid, and (b) mechanical removal of the taste buds from the epithelium with the use of a surgical needle. The procedure yields taste buds that are morphologically well preserved, and in quantities sufficient to enable a detailed biochemical characterization. Preliminary tests have shown the taste buds to have biochemical properties clearly distinct from those of the adjacent epithelium. The method may provide a basis for studying the molecular mechanism of taste perception in greater detail.

The taste bud of the human fungiform papilla was examined by electron microscopy. Typical type I, type II, and type III cells were found along with contact sites with nerve endings. Vesicles in nerve fibers contacting type I and type II cells suggest that these cells may receive efferent impulses, whereas vesicles and granules in type III cells adjacent to (afferent) nerve fibers support the view that type III cells are sensory receptors. All of these features are virtually indistinguishable from those previously reported in fungiform taste buds of other mammals.