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Salty taste is one of the five basic tastes and is often elicited by NaCl. Because excess sodium intake is associated with many health problems, it could be useful to have salt taste enhancers that are not sodium based. In this study, the regulation of NaCl-induced responses was investigated in cultured human fungiform taste papillae (HBO) cells with five arginyl dipeptides: Ala-Arg (AR), Arg-Ala (RA), Arg-Pro (RP), Arg-Glu (RE), and Glu-Arg (ER); and two non-arginyl dipeptides: Asp-Asp (DD) and Glu-Asp (ED). AR, RA, and RP significantly increased the number of cell responses to NaCl, whereas no effect was observed with RE, ER, DD, or ED. We also found no effects with alanine, arginine, or a mixture of both amino acids. Pharmacological studies showed that AR significantly increased responses of amiloride-sensitive but not amiloride-insensitive cells. In studies using small interfering RNAs (siRNAs), responses to AR were significantly decreased in cells transfected with siRNAs against epithelial sodium channel ENaCα or ENaCδ compared to untransfected cells. AR dramatically increased NaCl-elicited responses in cells transfected with NHE1 siRNA but not in those transfected with ENaCα or ENaCδ siRNAs. Altogether, AR increased responses of amiloride-sensitive cells required ENaCα and ENaCδ.

Analysis of single-cell RNA-Seq data can provide insights into the specific functions of individual cell types that compose complex tissues. Here, we examined gene expression in two distinct subpopulations of mouse taste cells: Tas1r3-expressing type II cells and physiologically identified type III cells. Our RNA-Seq libraries met high quality control standards and accurately captured differential expression of marker genes for type II (e.g. the Tas1r genes, Plcb2, Trpm5) and type III (e.g. Pkd2l1, Ncam, Snap25) taste cells. Bioinformatics analysis showed that genes regulating responses to stimuli were up-regulated in type II cells, while pathways related to neuronal function were up-regulated in type III cells. We also identified highly expressed genes and pathways associated with chemotaxis and axon guidance, providing new insights into the mechanisms underlying integration of new taste cells into the taste bud. We validated our results by immunohistochemically confirming expression of selected genes encoding synaptic (Cplx2 and Pclo) and semaphorin signalling pathway (Crmp2, PlexinB1, Fes and Sema4a) components. The approach described here could provide a comprehensive map of gene expression for all taste cell subpopulations and will be particularly relevant for cell types in taste buds and other tissues that can be identified only by physiological methods.

The past decade has witnessed a consolidation and refinement of the extraordinary progress made in taste research. This Review describes recent advances in our understanding of taste receptors, taste buds, and the connections between taste buds and sensory afferent fibres. The article discusses new findings regarding the cellular mechanisms for detecting tastes, new data on the transmitters involved in taste processing and new studies that address longstanding arguments about taste coding.

The perception of sour taste in mammals is important for its basic modality properties and avoiding toxic substances. We explore a biomimetic bioelectronic tongue, which integrate MEA (microelectrode array) and taste receptor cell for acid detection as a switch. However, the acid-sensing mechanism and coding of the taste receptor cells in the periphery is not well understood, with long-standing debate. Therefore, we firstly construct a Hodgkin-Huxley type mathematical model of whole-cell acid-sensing taste receptor cells based on the electrophysiologic patch clamp recordings with different acid sensitive receptor expressing and different acidic stimulations. ASICs and PKDL channels are two most promising candidates for acidic sensation. ASICs channels contribute to the On response, and PKDL channels coding the Offset stimulations respectively, which function as a pair for switch. Therefore, with the advantage of effective and noninvasive detection for MEA, a sour taste biosensor based on MEA and taste receptor cells was designed and established to detect sour response from the elementary acid sensitive taste receptor cells during and after stimulus. From simulation and extracelluar potential recordings, we found the biomimetic bioelectronic tongue was acid-sensitive, as acid stimulation pH decrease, the firing frequency significantly increase. Furthermore, this reliable and effective MEA based bioelectronic tongue functioned as a switch for stimulation On and Off. This study provided a powerful platform to recognize sour stimulation and help elucidate the sour taste sensation and coding mechanism., (Copyright © 2016 Elsevier B.V. All rights reserved.)

At present, developing an efficient assay method for truly reflecting the real feelings of gustatory tissues is of great importance. In this study, a novel biosensor was fabricated to investigate the kinetic characteristics of the receptors in taste bud tissues sensing bitter substances for the first time. Porcine taste bud tissues were used as the sensing elements, and the sandwich-type sensing membrane was fixed onto a glassy carbon electrode for assembling the biosensor. With the developed sensor, the response currents induced by sucrose octaacetate, denatonium benzoate, and quercetin stimulating corresponding receptors were determined. The results demonstrated that the interaction between the analyst with their receptors were fitting to hyperbola (R 2 =0.9776, 0.9980 and 0.9601), and the activation constants were 8.748×10 -15 mol/L, 1.429×10 -12 mol/L, 6.613×10 -14 mol/L, respectively. The average number of receptors per cell was calculated as 1.75, 28.58, and 13.23, while the signal amplification factors were 1.08×10 4 , 2.89×10 3 and 9.76×10 4 . These suggest that the sensor can be used to quantitatively describe the interaction characteristics of cells or tissue receptors with their ligands, the role of cellular signaling cascade, the number of receptors, and the signal transmission pathways., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The connections between taste receptor cells (TRCs) and innervating gustatory neurons are formed in a mutually dependent manner during development. To investigate whether a change in the ratio of cell types that compose taste buds influences the number of innervating gustatory neurons, we analyzed the proportion of gustatory neurons that transmit sour taste signals in adult Skn-1a -/- mice in which the number of sour TRCs is greatly increased. We generated polycystic kidney disease 1 like 3-wheat germ agglutinin (pkd1l3-WGA)/Skn-1a +/+ and pkd1l3-WGA/Skn-1a -/- mice by crossing Skn-1a -/- mice and pkd1l3-WGA transgenic mice, in which neural pathways of sour taste signals can be visualized. The number of WGA-positive cells in the circumvallate papillae is 3-fold higher in taste buds of pkd1l3-WGA/Skn-1a -/- mice relative to pkd1l3-WGA/Skn-1a +/+ mice. Intriguingly, the ratio of WGA-positive neurons to P2X 2 -expressing gustatory neurons in nodose/petrosal ganglia was similar between pkd1l3-WGA/Skn-1a +/+ and pkd1l3-WGA/Skn-1a -/- mice. In conclusion, an alteration in the ratio of cell types that compose taste buds does not influence the number of gustatory neurons that transmit sour taste signals., (Copyright © 2017. Published by Elsevier B.V.)

Salt reception by taste cells is still the less understood transduction process occurring in taste buds, the peripheral sensory organs for the detection of food chemicals. Although there is evidence suggesting that the epithelial sodium channel (ENaC) works as sodium receptor, yet it is not clear how salt-detecting cells signal the relevant information to nerve endings. Taste cells responding to sweet, bitter, and umami substances release ATP as neurotransmitter through a nonvesicular mechanism. Three different channel proteins have been proposed as conduit for ATP secretion: pannexin channels, connexin hemichannels, and calcium homeostasis modulator 1 (CALHM1) channels. In heterologous expression systems, these channels mediate outwardly rectifying membrane currents with distinct biophysical and pharmacological properties. I therefore tested whether also salt-detecting taste cells were endowed with these currents. To this aim, I applied the patch-clamp techniques to single cells in isolated taste buds from rat fungiform papillae. Salt-detecting cells were functionally identified by exploiting the effect of amiloride, which induces a current response by shutting down ENaCs. I looked for the presence of outwardly rectifying currents by using appropriate voltage-clamp protocols and specific pharmacological tools. I found that indeed salt-detecting cells possessed these currents with properties consistent with the presence, at least in part, of CALHM1 channels. Unexpectedly, CALHM1-like currents in taste cells were potentiated by known blockers of pannexin, suggesting a possible inhibitory action of this protein on CALMH1. These findings indicate that communication between salt-detecting cells and nerve endings might involve ATP release by CALMH1 channels., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: It has been reported that stress can cause anhedonia, a core symptom of depression, and also affect taste responses of the stressed subjects. Anhedonia refers to a reduction of the ability to experience pleasure, which can be detected by decreased response to palatable food in rats. The present study was conducted to examine if stress-induced anhedonia is accompanied by changes in gene expression for taste., Design: For anhedonia test, rats had free choices of cookies, a palatable food, and chow for 1h following 1h of daily restraint sessions. To examine the development of behavioral depression by restraint stress, ambulatory activity and forced swim tests were performed. Taste cells were harvested from the circumvallate papillae of rats on the 1st, 3rd and 7th day of stress exposure and subjected to the analysis of gene expression for taste., Results: One hour of daily stress exposure did not affect chow intake during the entire experimental period. However, from day 2 cookie intake was suppressed, suggesting the development of anhedonia. Ambulatory activity was significantly decreased, and immobility during forced swim test was increased, after the 7th day of stress exposure, but not before. 5-HT1A mRNA expression, but not T1R2, T1R3, T2R6, α-gustducin or PLCβ2 mRNA expression, appeared to be decreased after the 3rd day of stress exposure., Conclusion: Reduced expression of 5-HT1A in the taste cells, possibly leading to a reduced processing of taste information for palatable food, may additively contribute to the development of anhedonia as a pre-symptomatic feature of depression in stressed subjects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The taste of sugar elicits cephalic-phase insulin release (CPIR), which limits the rise in blood glucose associated with meals. Little is known, however, about the gustatory mechanisms that trigger CPIR. We asked whether oral stimulation with any of the following taste stimuli elicited CPIR in mice: glucose, sucrose, maltose, fructose, Polycose, saccharin, sucralose, AceK, SC45647, or a nonmetabolizable sugar analog. The only taste stimuli that elicited CPIR were glucose and the glucose-containing saccharides (sucrose, maltose, Polycose). When we mixed an α-glucosidase inhibitor (acarbose) with the latter three saccharides, the mice no longer exhibited CPIR. This revealed that the carbohydrates were hydrolyzed in the mouth, and that the liberated glucose triggered CPIR. We also found that increasing the intensity or duration of oral glucose stimulation caused a corresponding increase in CPIR magnitude. To identify the components of the glucose-specific taste-signaling pathway, we examined the necessity of Calhm1, P2X2+P2X3, SGLT1, and Sur1. Among these proteins, only Sur1 was necessary for CPIR. Sur1 was not necessary, however, for taste-mediated attraction to sugars. Given that Sur1 is a subunit of the ATP-sensitive K + channel (K ATP ) channel and that this channel functions as a part of a glucose-sensing pathway in pancreatic β-cells, we asked whether the K ATP channel serves an analogous role in taste cells. We discovered that oral stimulation with drugs known to increase (glyburide) or decrease (diazoxide) K ATP signaling produced corresponding changes in glucose-stimulated CPIR. We propose that the K ATP channel is part of a novel signaling pathway in taste cells that mediates glucose-induced CPIR., (Copyright © 2017 the American Physiological Society.)

The gustatory system of the sea catfish Plotosus japonicus, like that of other catfishes, is highly developed. To clarify the details of the morphology of the peripheral gustatory system of Plotosus, we used whole-mount immunohistochemistry to investigate the distribution and innervation of the taste buds within multiple organs including the barbels, oropharyngeal cavity, fins (pectoral, dorsal, and caudal), and trunk. Labeled taste buds could be observed in all the organs examined. The density of the taste buds was higher along the leading edges of the barbels and fins; this likely increases the chance of detecting food. In all the fins, the taste buds were distributed in linear arrays parallel to the fin rays. Labeling of nerve fibers by anti-acetylated tubulin antibody showed that the taste buds within each sensory field are innervated in different ways. In the barbels, large nerve bundles run along the length of the organ, with fascicles branching off to innervate polygonally organized groups of taste buds. In the fins, nerve bundles run along the axis of fin rays to innervate taste buds lying in a line. In each case, small fascicles of fibers branch from large bundles and terminate within the basal portions of the taste buds. Serotonin immunohistochemistry demonstrated that most of the taste buds in all the organs examined contained disk-shaped serotonin-immunopositive cells in their basal region. This indicates a similar organization of the taste buds, in terms of the existence of serotonin-immunopositive basal cells, across the different sensory fields in this species., (© 2017 S. Karger AG, Basel.)

The sense of taste is achieved by cooperation of many signaling molecules expressed in taste cells, which code and transmit information on quality and intensity of taste to the nervous system. Viral vector-mediated gene transfer techniques have been proven to be useful to study and control function of a gene product in vivo However, there is no transduction method for taste cells in live animals. Here, we have established a method for inducing foreign gene expression in mouse taste cells in vivo by recombinant adeno-associated virus (AAV) vector. First, using enhanced green fluorescent protein (EGFP) as a reporter, we screened 6 AAV serotypes along with a recombinant lentivirus vector for their ability to transduce taste cells. One week after viral injection into the submucosa of the tongue, EGFP expression in fungiform taste cells was observed only in animals injected with AAV-DJ, a synthetic serotype. Next, time course of AAV-DJ-mediated EGFP expression in fungiform taste cells was evaluated. Intragemmal EGFP signals appeared after a delay, rapidly increased until 7 days postinjection, and gradually decreased over the next few weeks probably because of the cell turnover. Finally, the taste cell types susceptible to AAV-DJ transduction were characterized. EGFP expression was observed in PLCβ2-immunoreactive type II and aromatic l-amino acid decarboxylase (AADC)-immunoreactive type III taste cells as well as in cells immunonegative for both PLCβ2 and AADC, demonstrating that AAV-DJ does not discriminate functional taste cell types. In conclusion, the method established in this study will be a promising tool to study the mechanism of taste., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240-360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.

During the gestation and the lactating periods the gustatory papillae contain taste buds that respond to different flavors and aversive stimuli. The current study analyzed the effects of pre-and neonatal undernutrition on the circumvallate papillae of rats at 12, 20, and 30days of age. Early undernourishment occurred from gestational days G6 to G19 when dams received low percentages of food followed by a balanced diet from G20-21. After birth pups were underfed by rotating two lactating dams every 12h; in one of them, the nipples were tied. The pups were weaned at 25days of age, and then given an ad libitum diet. Under anesthesia the tongues were removed and stained with the hematoxylin-eosin (H-E) procedure. The results indicated that young underfed rats had significantly body weight reductions. The tongue measurements in underfed rats showed reduced total area and length of the anterior portion, but there were negligible effects on the posterior portion. The circumvallate papillae in underfed rats was significantly reduced in major length, major diameter, and total and upper areas, but unaffected in the lateral wall trench region. The taste bud areas and minor diameter were unaffected by undernutrition, but there were significant reductions in the total number of visible taste buds and the major diameter, delayed opening of taste bud pores, and an increased number of closed pores were also observed. These alterations by undernutrition reflect the vulnerability of structures in the gustatory oral cavity and suggest a possible interference with the receptorś activation, and transduction and perhaps with the taste encoding of signals to generate the gustatory sensory and hedonic responses., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Taste buds are sensory organs in jawed vertebrates, composed of distinct cell types that detect and transduce specific taste qualities. Taste bud cells differentiate from oropharyngeal epithelial progenitors, which are localized mainly in proximity to the forming organs. Despite recent progress in elucidating the molecular interactions required for taste bud cell development and function, the cell behavior underlying the organ assembly is poorly defined. Here, we used time-lapse imaging to observe the formation of taste buds in live zebrafish larvae. We found that tg(fgf8a.dr17)-expressing cells form taste buds and get rearranged within the forming organs. In addition, differentiating cells move from the epithelium to the forming organs and can be displaced between developing organs. During organ formation, tg(fgf8a.dr17) and type II taste bud cells are displaced in random, directed or confined mode relative to the taste bud they join or by which they are maintained. Finally, ascl1a activity in the 5-HT/type III cell is required to direct and maintain tg(fgf8a.dr17)-expressing cells into the taste bud. We propose that diversity in displacement modes of differentiating cells acts as a key mechanism for the highly dynamic process of taste bud assembly., (© 2016. Published by The Company of Biologists Ltd.)

Objective: To elucidate the regeneration process of fungiform taste buds after severing the chorda tympani nerve (CTN) by confocal laser scanning microscopy in vivo., Methods: In 7 consecutive patients whose CTN was severed during tympanoplasty, an average of 10 fungiform papillae in the midlateral region of the tongue were periodically observed, and the number of taste buds was counted until 12 to 24 months after surgery. Gustatory function was assessed by EGM., Results: EGM thresholds showed no response within 1 month after surgery in any patient. All taste buds had disappeared until 13 to 71 days after surgery. Regenerated taste buds were first detected 5 to 8 months after surgery in 5 of the 7 patients. EGM thresholds recovered to their preoperative values in 2 patients. In these 2 patients, the number of regenerated taste buds gradually increased in combination with a recovered taste function. However, a time lag existed between taste bud regeneration and taste function recovery. EGM thresholds did not recover in the other 3 patients with regenerated taste buds, suggesting that these taste buds were immature without gustatory function., Conclusion: The long-term regeneration process of fungiform taste buds could be clarified using confocal laser scanning microscopy., (© The Author(s) 2015.)

Molecular labeling in whole-mount tissues provides an efficient way to obtain general information about the formation, maintenance, degeneration, and regeneration of many organs and tissues. However, labeling of lingual taste buds in whole tongue tissues in adult mice has been problematic because of the strong permeability barrier of the tongue epithelium. In this study, we present a simple method for labeling taste buds in the intact tongue epithelial sheet of an adult mouse. Following intralingual protease injection and incubation, immediate fixation of the tongue on mandible in 4% paraformaldehyde enabled the in situ shape of the tongue epithelium to be well maintained after peeling. The peeled epithelium was accessible to taste bud labeling with a pan-taste cell marker, keratin 8, and a type II taste cell marker, α-gustducin, in all three types of taste papillae, that is, fungiform, foliate, and circumvallate. Overnight incubation of tongue epithelial sheets with primary and secondary antibodies was sufficient for intense labeling of taste buds with both fluorescent and DAB visualizations. Labeled individual taste buds were easy to identify and quantify. This protocol provides an efficient way for phenotypic analyses of taste buds, especially regarding distribution pattern and number.

Taste signals are received by taste buds. To better understand the taste reception system, expression patterns of taste-related molecules are determined by in situ hybridization (ISH) analyses at the histological level. Nevertheless, even though ISH is essential for determining mRNA expression, few taste bud markers can be applied together with ISH. Ulex europaeus agglutinin-1 (UEA-1) appears to be a reliable murine taste bud marker based on immunohistochemistry (IHC) analyses. However, there is no evidence as to whether UEA-1 can be used for ISH. Thus, the present study evaluated UEA-1 using various histochemical methods, especially ISH. When lectin staining was performed after ISH procedures, UEA-1 clearly labeled taste cellular membranes and distinctly indicated boundaries between taste buds and the surrounding epithelial cells. Additionally, UEA-1 was determined as a taste bud marker not only when used in single-colored ISH but also when employed with double-labeled ISH or during simultaneous detection using IHC and ISH methods. These results suggest that UEA-1 is a useful marker when conducting analyses based on ISH methods. To clarify UEA-1 staining details, multi-fluorescent IHC (together with UEA-1 staining) was examined, resulting in more than 99% of cells being labeled by UEA-1 and overlapping with KCNQ1-expressing cells., (© 2016 The Histochemical Society.)

G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Responses in the amiloride-insensitive (AI) pathway, one of the two pathways mediating salty taste in mammals, are modulated by the size of the anion of a salt. This "anion effect" has been hypothesized to result from inhibitory transepithelial potentials (TPs) generated across the lingual epithelium as cations permeate through tight junctions and leave their larger and less permeable anions behind (Ye et al., 1991). We tested directly the necessity of TPs for the anion effect by measuring responses to NaCl and Na-gluconate (small and large anion sodium salts, respectively) in isolated taste cells from mouse circumvallate papillae. Using calcium imaging, we identified AI salt-responsive type III taste cells and demonstrated that they compose a subpopulation of acid-responsive taste cells. Even in the absence of TPs, many (66%) AI salt-responsive type III taste cells still exhibited the anion effect, demonstrating that some component of the transduction machinery for salty taste in type III cells is sensitive to anion size. We hypothesized that osmotic responses could explain why a minority of type III cells (34%) had AI salt responses but lacked anion sensitivity. All AI type III cells had osmotic responses to cellobiose, which were significantly modulated by extracellular sodium concentration, suggesting the presence of a sodium-conducting osmotically sensitive ion channel. However, these responses were significantly larger in AI type III cells that did not exhibit the anion effect. These findings indicate that multiple mechanisms could underlie AI salt responses in type III taste cells, one of which may contribute to the anion effect., Significance Statement: Understanding the mechanisms underlying salty taste will help inform strategies to combat the health problems associated with NaCl overconsumption by humans. Of the two pathways underlying salty taste in mammals, the amiloride-insensitive (AI) pathway is the least understood. Using calcium imaging of isolated mouse taste cells, we identify two separate populations of AI salt-responsive type III taste cells distinguished by their sensitivity to anion size and show that these cells compose subpopulations of acid-responsive taste cells. We also find evidence that a sodium-conducting osmotically sensitive mechanism contributes to salt responses in type III taste cells. Our data not only provide new insights into the transduction mechanisms of AI salt taste but also have important implications for general theories of taste encoding., (Copyright © 2016 the authors 0270-6474/16/361942-12$15.00/0.)

Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn(2+)-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K(+) current. We identify KIR2.1 as the acid-sensitive K(+) channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of Kir2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K(+) current in amplifying the sensory response, entry of protons through the Zn(2+)-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K(+) channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of oral diseases. Mammalian taste buds are the structural unit for detecting taste stimuli in the oral cavity. The effects of arecoline on taste bud morphology are poorly understood. Arecoline was injected intraperitoneally (IP) into C57BL/6 mice twice daily for 1-4 weeks. After arecoline treatment, the vallate papillae were processed for electron microscopy and immunohistochemistry analysis of taste receptor proteins (T1R2, T1R3, T1R1, and T2R) and taste associated proteins (α-gustducin, PLCβ2, and SNAP25). Body weight, food intake and water consumption were recorded. A 2-bottle preference test was also performed. The results demonstrated that 1) arecoline treatment didn't change the number and size of the taste buds or taste bud cells, 2) electron microscopy revealed the change of organelles and the accumulation of autophagosomes in type II cells, 3) immunohistochemistry demonstrated a decrease of taste receptor T1R2- and T1R3-expressing cells, 4) the body weight and food intake were markedly reduced, and 5) the sweet preference behavior was reduced. We concluded that the long-term injection of arecoline alters the morphology of type II taste bud cells, retards the growth of mice, and affects discrimination competencies for sweet tastants., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The sense of taste is mediated by multicellular taste buds located within taste papillae on the tongue. In mice, individual taste buds reside in fungiform papillae, which develop at mid-gestation as epithelial placodes in the anterior tongue. Taste placodes comprise taste bud precursor cells, which express the secreted factor sonic hedgehog (Shh) and give rise to taste bud cells that differentiate around birth. We showed previously that epithelial activation of β-catenin is the primary inductive signal for taste placode formation, followed by taste papilla morphogenesis and taste bud differentiation, but the degree to which these later elements were direct or indirect consequences of β-catenin signaling was not explored. Here, we define discrete spatiotemporal functions of β-catenin in fungiform taste bud development. Specifically, we show that early epithelial activation of β-catenin, before taste placodes form, diverts lingual epithelial cells from a taste bud fate. By contrast, β-catenin activation a day later within Shh(+) placodes, expands taste bud precursors directly, but enlarges papillae indirectly. Further, placodal activation of β-catenin drives precocious differentiation of Type I glial-like taste cells, but not other taste cell types. Later activation of β-catenin within Shh(+) precursors during papilla morphogenesis also expands taste bud precursors and accelerates Type I cell differentiation, but papilla size is no longer enhanced. Finally, although Shh regulates taste placode patterning, we find that it is dispensable for the accelerated Type I cell differentiation induced by β-catenin., (© 2015. Published by The Company of Biologists Ltd.)

Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration.

Inflammatory cytokines are important regulators of metabolism and food intake. Over production of inflammatory cytokines during bacterial and viral infections leads to anorexia and reduced food intake. However, it remains unclear whether any inflammatory cytokines are involved in the regulation of taste reception, the sensory mechanism governing food intake. Previously, we showed that tumor necrosis factor (TNF), a potent proinflammatory cytokine, is preferentially expressed in a subset of taste bud cells. The level of TNF in taste cells can be further induced by inflammatory stimuli. To investigate whether TNF plays a role in regulating taste responses, in this study, we performed taste behavioral tests and gustatory nerve recordings in TNF knockout mice. Behavioral tests showed that TNF-deficient mice are significantly less sensitive to the bitter compound quinine than wild-type mice, while their responses to sweet, umami, salty, and sour compounds are comparable to those of wild-type controls. Furthermore, nerve recording experiments showed that the chorda tympani nerve in TNF knockout mice is much less responsive to bitter compounds than that in wild-type mice. Chorda tympani nerve responses to sweet, umami, salty, and sour compounds are similar between TNF knockout and wild-type mice, consistent with the results from behavioral tests. We further showed that taste bud cells express the two known TNF receptors TNFR1 and TNFR2 and, therefore, are potential targets of TNF. Together, our results suggest that TNF signaling preferentially modulates bitter taste responses. This mechanism may contribute to taste dysfunction, particularly taste distortion, associated with infections and some chronic inflammatory diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Studies over the last 8 years have identified 3 potential channels that appear to release ATP from Type II cells in response to taste stimuli. These studies have taken different methodological approaches but have all provided data supporting their candidate channel as the ATP release channel. These potential channels include Pannexin 1, Connexins (30 and/or 43), and most recently, the Calhm1 channel. Two papers in this issue of Chemical Senses provide compelling new evidence that Pannexin 1 is not the ATP release channel. Tordoff et al. did a thorough behavioral analysis of the Pannexin1 knock out mouse and found that these animals have the same behavioral responses as wild type mice for 7 different taste stimuli that were tested. Vandenbeuch et al. presented an equally thorough analysis of the gustatory nerve responses in the Pannexin1 knock out mouse and found no differences compared with controls. Thus when the role of Pannexin 1 is analyzed at the systems level, it is not required for normal taste perception. Further studies are needed to determine the role of this hemichannel in taste cells., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The pharyngeal arches (PAs) generate cranial organs including the tongue. The taste placodes, formed in particular locations on the embryonic tongue surface, differentiate into taste buds harbored in distinct gustatory papillae. The developing tongue also has a complex supply of cranial nerves through each PA. However, the relationship between the PAs and taste bud development is not fully understood., Results: Ripply3 homozygous mutant mice, which have impaired third/fourth PAs, display a hypoplastic circumvallate papilla and lack taste buds, although the taste placode is normally formed. Formation of the glossopharyngeal ganglia is defective and innervation toward the posterior tongue is completely missing in Ripply3 mutant embryos at E12.5. Moreover, the distribution of neuroblasts derived from the epibranchial placode is severely, but not completely, atenuated, and the neural crest cells are diminished in the third PA region of Ripply3 mutant embryos at E9.5-E10.5. In Tbx1 homozygous mutant embryos, which exhibit another type of deficiency in PA development, the hypoplastic circumvallate papilla is observed along with abnormal formation of the glossopharyngeal ganglia and severely impaired innervation., Conclusions: PA deficiencies affect multiple aspects of taste bud development, including formation of the cranial ganglia and innervation to the posterior tongue., (© 2015 Wiley Periodicals, Inc.)

Sour taste is detected by taste receptor cells that respond to acids through yet poorly understood mechanisms. The cells that detect sour express the protein PKD2L1, which is not the sour receptor but nonetheless serves as a useful marker for sour cells. By use of mice in which the PKD2L1 promoter drives expression of yellow fluorescent protein, we previously reported that sour taste cells from circumvallate papillae in the posterior tongue express a proton current. To establish a correlation between this current and sour transduction, we examined its distribution by patch-clamp recording. We find that the current is present in PKD2L1-expressing taste cells from mouse circumvallate, foliate, and fungiform papillae but not in a variety of other cells, including spinal cord neurons that express PKD2L1. We describe biophysical properties of the current, including pH-dependent Zn(2+) inhibition, lack of voltage-dependent gating, and activation at modest pH values (6.5) that elicit action potentials in isolated cells. Consistent with a channel that is constitutively open, the cytosol of sour taste cells is acidified. These data define a functional signature for the taste cell proton current and indicate that its expression is mostly restricted to the subset of taste cells that detect sour., (© FASEB.)

Taste perception begins in the oral cavity by interactions of taste stimuli with specific receptors. Specific subsets of taste receptor cells (TRCs) are activated upon tastant stimulation and transmit taste signals to afferent nerve fibers and ultimately to the brain. How specific TRCs impinge on the innervating nerves and how the activation of a subset of TRCs leads to the discrimination of tastants of different qualities and intensities is incompletely understood. To investigate the organization of taste circuits, we used gene targeting to express the transsynaptic tracer barley lectin (BL) in the gustatory system of mice. Because TRCs are not synaptically connected with the afferent nerve fibers, we first analyzed tracer production and transfer within the taste buds (TBs). Surprisingly, we found that BL is laterally transferred across all cell types in TBs of mice expressing the tracer under control of the endogenous Tas1r1 and Tas2r131 promotor, respectively. Furthermore, although we detected the BL tracer in both ganglia and brain, we also found local low-level Tas1r1 and Tas2r131 gene, and thus tracer expression in these tissues. Finally, we identified the Tas1r1 and Tas2r131-expressing cells in the peripheral and CNS using a binary genetic approach. Together, our data demonstrate that genetic transsynaptic tracing from bitter and umami receptor cells does not selectively label taste-specific neuronal circuits and reveal local taste receptor gene expression in the gustatory ganglia and the brain., Significance Statement: Previous papers described the organization of taste pathways in mice expressing a transsynaptic tracer from transgenes in bitter or sweet/umami-sensing taste receptor cells. However, reported results differ dramatically regarding the numbers of synapses crossed and the reduction of signal intensity after each transfer step. Nevertheless, all groups claimed this approach appropriate for quality-specific visualization of taste pathways. In the present study, we demonstrate that genetic transsynaptic tracing originating from umami and bitter taste receptor cells does not selectively label taste quality-specific neuronal circuits due to lateral transfer of the tracer in the taste bud and taste receptor expression in sensory ganglia and brain. Moreover, we visualized for the first time taste receptor-expressing cells in the PNS and CNS., (Copyright © 2015 the authors 0270-6474/15/359717-13$15.00/0.)

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller & Reedy's 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller & Reedy's original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller & Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.

Five fundamental taste qualities (sweet, bitter, salty, sour, umami) are sensed by dedicated taste cells (TCs) that relay quality information to gustatory nerve fibers. In peripheral taste signaling pathways, ATP has been identified as a functional neurotransmitter, but it remains to be determined how specificity of different taste qualities is maintained across synapses. Recent studies demonstrated that some gut peptides are released from taste buds by prolonged application of particular taste stimuli, suggesting their potential involvement in taste information coding. In this study, we focused on the function of glucagon-like peptide-1 (GLP-1) in initial responses to taste stimulation. GLP-1 receptor (GLP-1R) null mice had reduced neural and behavioral responses specifically to sweet compounds compared to wild-type (WT) mice. Some sweet responsive TCs expressed GLP-1 and its receptors were expressed in gustatory neurons. GLP-1 was released immediately from taste bud cells in response to sweet compounds but not to other taste stimuli. Intravenous administration of GLP-1 elicited transient responses in a subset of sweet-sensitive gustatory nerve fibers but did not affect other types of fibers, and this response was suppressed by pre-administration of the GLP-1R antagonist Exendin-4(3-39). Thus GLP-1 may be involved in normal sweet taste signal transmission in mice., (© FASEB.)

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.

Implication of the long-chain fatty acid (LCFA) receptor GPR120, also termed free fatty acid receptor 4, in the taste-guided preference for lipids is a matter of debate. To further unravel the role of GPR120 in the "taste of fat", the present study was conducted on GPR120-null mice and their wild-type littermates. Using a combination of morphological [i.e., immunohistochemical staining of circumvallate papillae (CVP)], behavioral (i.e., two-bottle preference tests, licking tests and conditioned taste aversion) and functional studies [i.e., calcium imaging in freshly isolated taste bud cells (TBCs)], we show that absence of GPR120 in the oral cavity was not associated with changes in i) gross anatomy of CVP, ii) LCFA-mediated increases in intracellular calcium levels ([Ca(2+)]i), iii) preference for oily and LCFA solutions and iv) conditioned avoidance of LCFA solutions. In contrast, the rise in [Ca(2+)]i triggered by grifolic acid, a specific GPR120 agonist, was dramatically curtailed when the GPR120 gene was lacking. Taken together, these data demonstrate that activation of lingual GPR120 and preference for fat are not connected, suggesting that GPR120 expressed in TBCs is not absolutely required for oral fat detection in mice., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Many studies have demonstrated that chronic exposure to nicotine, one of the main components of tobacco smoke, has profound effects on the functionality of the mammalian taste system. However, the mechanisms underlying nicotine action are poorly understood. In particular no information is available on the chronic effect of nicotine on the functioning of taste cells, the peripheral detectors which transduce food chemicals into electrical signals to the brain. To address this issue, I studied the membrane properties of rat fungiform taste cells and evaluated the effect of long-term exposure to nicotine on the amiloride-sensitive sodium currents (ASSCs). These currents are mediated by the epithelial sodium channels (ENaC) thought to be important, at least in part, in the transduction of salty stimuli. Patch-clamp recording data indicated that ASSCs in taste cells from rats chronically treated with nicotine had a reduced amplitude compared to controls. The pharmacological and biophysical analysis of ASSCs revealed that amplitude reduction was not dependent on changes in amiloride sensitivity or channel ionic permeability, but likely derived from a decrease in the activity of ENaCs. Since these channels are considered to be sodium receptors in taste cells, my results suggest that chronic exposure to nicotine hampers the capability of these cells to respond to sodium ions. This might represent a possible cellular mechanism underlying the reduced taste sensitivity to salt typically found in smokers., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

The mammalian taste system is responsible for sensing and responding to the five basic taste qualities: sweet, sour, bitter, salty and umami. Previously, we showed that each taste is detected by dedicated taste receptor cells (TRCs) on the tongue and palate epithelium. To understand how TRCs transmit information to higher neural centres, we examined the tuning properties of large ensembles of neurons in the first neural station of the gustatory system. Here, we generated and characterized a collection of transgenic mice expressing a genetically encoded calcium indicator in central and peripheral neurons, and used a gradient refractive index microendoscope combined with high-resolution two-photon microscopy to image taste responses from ganglion neurons buried deep at the base of the brain. Our results reveal fine selectivity in the taste preference of ganglion neurons; demonstrate a strong match between TRCs in the tongue and the principal neural afferents relaying taste information to the brain; and expose the highly specific transfer of taste information between taste cells and the central nervous system.

Taste is one of the fundamental senses, and it is essential for our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Taste buds, which are clusters of neuroepithelial receptor cells, are housed in highly organized structures called taste papillae in the oral cavity. Whereas the overall structure of the taste periphery is conserved in almost all vertebrates examined to date, the anatomical, histological, and cell biological, as well as potentially the molecular details of taste buds in the oral cavity are diverse across species and even among individuals. In mammals, several types of gustatory papillae reside on the tongue in highly ordered arrangements, and the patterning and distribution of the mature papillae depend on coordinated molecular events in embryogenesis. In this review, we highlight new findings in the field of taste development, including how taste buds are patterned and how taste cell fate is regulated. We discuss whether a specialized taste bud stem cell population exists and how extrinsic signals can define which cell lineages are generated. We also address the question of whether molecular regulation of taste cell renewal is analogous to that of taste bud development. Finally, we conclude with suggestions for future directions, including the potential influence of the maternal diet and maternal health on the sense of taste in utero., (© 2015 Elsevier Inc. All rights reserved.)

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste., (Copyright © 2015 the American Physiological Society.)

Dietary lipids are usually responsible of several metabolic disorders. Recent compelling evidences suggest that there is a sixth taste modality, destined for the detection of oro-gustatory fats. The lipid-binding glycoprotein CD36, expressed by circumvallate papillae (CVP) of the mouse tongue, has been shown to be implicated in oro-gustatory perception of dietary lipids. We demonstrate that linoleic acid (LA) by activating sPLA2, cPLA2 and iPLA2 via CD36, produced arachidonic acid (AA) and lyso-phosphatidylcholine (Lyso-PC) which triggered Ca(2+) influx in CD36-positive taste bud cells (TBC), purified from mouse CVP. LA induced the production of Ca(2+) influx factor (CIF). CIF, AA and Lyso-PC exerted different actions on the opening of store-operated Ca2+ (SOC) channels, constituted of Orai proteins and regulated by STIM1, a sensor of Ca(2+) depletion in the endoplasmic reticulum. We observed that CIF and Lyso-PC opened Orai1 channels whereas AA-opened Ca(2+) channels were composed of Orai1/Orai3. STIM1 was found to regulate LA-induced CIF production and opening of both kinds of Ca(2+) channels. Furthermore, Stim1(-/-) mice lost the spontaneous preference for fat, observed in wild-type animals. Our results suggest that fatty acid-induced Ca(2+) signaling, regulated by STIM1 via CD36, might be implicated in oro-gustatory perception of dietary lipids and the spontaneous preference for fat. Other cell types are involved in, and external factors can influence this preference., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5(+)) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5(+) or Lgr6(+) cells from taste tissue can generate continuously expanding 3D structures ("organoids"). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2'-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5(+) cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6(+) cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5(+) or Lgr6(+) cells, validating the use of this model for the study of taste cell generation.

The location and distribution of mucosal sensory structures of the crocodilian oral cavity are poorly understood. Although there are several descriptions of these structures in adults, nothing is known about their development. The purpose of this study was to document location, morphology, and relative abundance of these mucosal sensory structures in both hatchling and subadult alligators. Numerous mucosal sensory structures and pale staining dome-shaped papillae were observed only in the upper palate and tongue. In hatchlings, these papillae, which house either mechanoreceptive or chemosensory (taste buds) structures, were larger and more prevalent on the tongue than the upper palate. In the subadult, however, these papillae housed primarily mechanoreceptive structures and possibly degenerate taste buds. Although the presence of the mechanoreceptive structures in the palates of the suabadult alligator are to be expected, the loss of most taste buds is hitherto undocumented. Thus, there is morphological support for an ontogenetic shift in the role of the sensory palate, from a prey detection gustatory sensory system in hatchlings to a prey-manipulative mechanoreceptive system in subadults., (© 2014 Wiley Periodicals, Inc.)

We isolated chicken taste buds and used a real-time Ca2+ imaging technique to investigate the functions of the taste cells. With RT-PCR, we found that isolated chicken taste bud-like cell subsets express chicken gustducin messenger RNA. Immunocytochemical techniques revealed that the cell subsets were also immunopositive for chicken gustducin. These results provided strong evidence that the isolated cell subsets contain chicken taste buds. The isolated cell subsets were spindle-shaped and approximately 61-75 μm wide and 88-98 μm long, and these characteristics are similar to those of sectional chicken taste buds. Using Ca2+ imaging, we observed the buds' response to 2 mmol/L quinine hydrochloride (a bitter substance) and their response to a mixture of 25 mmol/L L-glutamic acid monopotassium salt monohydrate and 1 mmol/L inosine 5'-monophosphate disodium salt, umami substances. The present study is the first morphological demonstration of isolated chicken taste buds, and our results indicate that the isolated taste buds were intact and functional approaches for examining the taste senses of the chicken using Ca2+ imaging can be informative., (© 2014 Japanese Society of Animal Science.)

Animal food intake is primarily controlled by appetite, which is affected by food quality, environment, and the management and status of animal health. Sensing of taste is mediated by taste receptor cells and is central to appetite. Taste receptor cells possess distinctive physiological characteristics that permit the recognition of various stimuli in foods. Thus, cultures of porcine circumvallate papillae cells provide a model for identification of the molecular and functional characteristics of taste receptor cells. In this study, we described the isolation and culture of porcine circumvallate papillae, using tissue explants and enzymatic digestion, and showed continuous viability and expression of pivotal taste marker proteins for more than 9 passages. In addition, cultured cells showed dramatic rises in intracellular calcium upon stimulation with several taste stimuli (sweet, umami, bitter, and fat). These cultures of porcine taste receptor cells provide a useful model for assessing taste preferences of pigs and may elucidate interactions between various taste stimuli., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Background: Taste buds contain ∼60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I, glial cells; II, bitter/sweet/umami receptor cells; and III, sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turn over rapidly suggesting that Shh+ cells are post-mitotic precursors of some or all taste cell types., Results: To fate map Shh-expressing cells, mice carrying ShhCreER(T2) and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III)., Conclusions: Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds., (Copyright © 2014 Wiley Periodicals, Inc.)

Systemic blockade of serotonin (5HT) reuptake with paroxetine has been shown to increase sensitivity to sucrose and quinine in humans. Here, using a 2-response operant taste detection task, we measured the effect of paroxetine and the 5HT1A receptor antagonist WAY100635 on the ability of rats to discriminate sucrose, NaCl, and citric acid from water. After establishing individual psychometric functions, 5 concentrations of each taste stimulus were chosen to represent the dynamic portion of the concentration-response curve, and the performance of the rats to these stimuli was assessed after vehicle, paroxetine (7mg/kg intraperitoneally), and WAY100635 (0.3mg/kg subcutaneously; 1mg/kg intravenously) administration. Although, at times, overall performance across concentrations dropped, at most, 5% from vehicle to drug conditions, no differences relative to vehicle were seen on the parameters of the psychometric function (asymptote, slope, or EC50) after drug administration. In contrast to findings in humans, our results suggest that modulation of 5HT activity has little impact on sucrose detectability at perithreshold concentrations in rats, at least at the doses used in this task. In the rat model, the purported paracrine/neurocrine action of serotonin in the taste bud may work in a manner that does not impact overt taste detection behavior., (© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Carbonic anhydrase VI (CA VI) is a secretory isozyme of the α-CA gene family. It is highly expressed in the salivary and mammary glands and secreted into saliva and milk. Although CA VI was first described as a gustatory protein, its exact functional roles have remained enigmatic. Interestingly, polymorphism of the CA6 gene was recently linked to bitter taste perception in humans. In this study, we compared the preference of Car6⁻/⁻ and wild-type mice for different taste modalities in an IntelliCage monitoring environment. Morphologies of taste buds, tongue papillae, and von Ebner's glands were evaluated by light microscopy. Cell proliferation and rate of apoptosis in tongue specimens were examined by Ki67 immunostaining and fluorescent DNA fragmentation staining, respectively., Results: The behavioral follow up of the mice in an IntelliCage system revealed that Car6⁻/⁻ mice preferred 3 μM quinine (bitter) solution, whereas wild type mice preferred water. When the quinine concentration increased, both groups preferentially selected water. Histological analysis, Ki67 immunostaining and detection of apoptosis did not reveal any significant changes between tongue specimens of the knockout and wild type mice., Conclusions: Our knockout mouse model confirms that CA VI is involved in bitter taste perception. CA VI may be one of the factors which contribute to avoidance of bitter, potentially harmful, substances.

Taste buds (sensory structures embedded in oral epithelium) show a remarkable diversity of transmitters synthesized and secreted locally. The known transmitters accumulate in a cell type selective manner, with 5-HT and noradrenaline being limited to presynaptic cells, GABA being synthesized in both presynaptic and glial-like cells, and acetylcholine and ATP used for signalling by receptor cells. Each of these transmitters participates in local negative or positive feedback circuits that target particular cell types. Overall, the role of ATP is the best elucidated. ATP serves as a principal afferent transmitter, and also is the key trigger for autocrine positive feedback and paracrine circuits that result in potentiation (via adenosine) or inhibition (via GABA or 5-HT). While many of the cellular receptors and mechanisms for these circuits are known, their impact on sensory detection and perception remains to be elaborated in most instances. This brief review examines what is known, and some of the open questions and controversies surrounding the transmitters and circuits of the taste periphery., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

We have previously reported that hair follicles contain multipotent stem cells, which express nestin and participate in follicle growth at anagen as well as in the extension of the follicle sensory nerve. The nestin-driven green fluorescent protein (ND-GFP) transgenic mouse labels all nestin-expressing cells with GFP. The hair follicle nestin-GFP cells can differentiate into neurons, Schwann cells, and other cell types. In this study, we describe nestin-expressing multipotent stem cells in the fungiform papilla in the tongue. The nestin-expressing multipotent stem cells in the fungiform papilla are located around a peripheral sensory nerve immediately below the taste bud and co-express the neural crest cell marker p75(NTR) . The fungiform papilla cells formed spheres in suspension culture in DMEM-F12 medium supplemented with basic fibroblast growth factor (bFGF). The spheres consisted of nestin-expressing cells that co-expressed the neural crest marker p75(NTR) and which developed expression of the stem cell marker CD34. P75(NTR), CD34 and nestin co-expression suggested that nestin-expressing cells comprising the fungiform papilla spheres were in a relatively undifferentiated state. The nestin-expressing cells of these spheres acquired the following markers: β III tubulin typical of nerve cells; GFAP typical of glial cells; K15 typical of keratinocytes; and smooth-muscle antigen (SMA), after transfer to RPMI 1640 medium with 10% fetal bovine serum (FBS), suggesting they differentiated into multiple cell types. The results of the current study indicate nestin-expressing fungiform papilla cells and the nestin-expressing hair follicle stem cells have common features of cell morphology and ability to differentiate into multiple cell types, suggesting their remarkable similarity., (© 2013 Wiley Periodicals, Inc.)

We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats.

Investigating molecular mechanisms underlying human taste sensation requires functionally dedicated and at the same time proliferating human taste cells. Here, we isolated viable human fungiform taste papillae cells from biopsy samples, adenovirally transduced proliferation promoting genes, and obtained stably proliferating cell lines. Analysis of gene expression of 1 human taste cell line termed HTC-8 revealed that these cells express 13 TAS2R bitter taste receptor genes, CD36, OXTR encoding oxytocin receptor, as well as genes implicated with signal transduction and cell fate control. Bitter tastants triggered functionally distinct signaling pathways in HTC-8 cells. Salicin elicited phospholipase C-dependent calcium signaling and no cell depolarization. In contrast, stimulation with saccharin, aristolochic acid, or phenylthiocarbamide triggered cell depolarization and phospholipase C-independent calcium influx. Simultaneous stimulation with salicin and saccharin revealed that saccharin can enhance the phospholipase C-dependent response to salicin indicating crosstalk of signaling pathways. Our results show that HTC-8 cells are programmed to bitter taste reception but are also responsive to fatty acids, oxytocin, and somatosensory stimuli, whereas HTC-8 cells are insensitive to compounds representing other basic taste qualities.

We previously showed that the hypertonicity of taste stimulating solutions modified tonic responses, the quasi-steady state component following the transient (phasic) component of each integrated taste nerve response. Here we show that the hypertonicity opens tight junctions surrounding taste receptor cells in a time-dependent manner and modifies whole taste nerve responses in bullfrogs. We increased the tonicity of stimulating solutions with non-taste substances such as urea or ethylene glycol. The hypertonicity enhanced phasic responses to NaCl>0.2M, and suppressed those to NaCl<0.1M, 1mM CaCl2, and 1mM bitter substances (quinine, denatonium and strychnine). The hypertonicity also enhanced the phasic responses to a variety of 0.5M salts such as LiCl and KCl. The enhancing effect was increased by increasing the difference between the ionic mobilities of the cations and anions in the salt. A preincubation time >20s in the presence of 1M non-taste substances was needed to elicit both the enhancing and suppressing effects. Lucifer Yellow CH, a paracellular marker dye, diffused into bullfrog taste receptor organs in 30s in the presence of hypertonicity. These results agreed with our proposed mechanism of hypertonic effects that considered the diffusion potential across open tight junctions., (Copyright © 2014 Elsevier B.V. All rights reserved.)