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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.)

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.)

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.)

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.)

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.)

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

Taste buds contain two types of taste receptor cells, inositol 1,4,5-triphosphate receptor type 3-immunoreactive cells (type II cells) and synaptosomal-associating protein-25-immunoreactive cells (type III cells). We investigated their postnatal development in mouse fungiform taste buds immunohistochemically and electrophysiologically. The cell density, i.e. the number of cells per taste bud divided by the maximal area of the horizontal cross-section of the taste bud, of type II cells increased by postnatal day (PD)49, where as that of type III cells was unchanged throughout the postnatal observation period and was equal to that of the adult cells at PD1. The immunoreactivity of taste bud cell subtypes was the same as that of their respective subtypes in adult mice throughout the postnatal observation period. Almost all type II cells were immunoreactive to gustducin at PD1, and then the ratio of gustducin-immunoreactive type II cells to all type II cells decreased to a saturation level, ∼60% of all type II cells, by PD15. Type II and III cells generated voltage-gated currents similar to their respective adult cells even at PD3. These results show that infant taste receptor cells are as excitable as those of adults and propagate in a subtype-dependent manner. The relationship between the ratio of each taste receptor cell subtype to all cells and taste nerve responses are discussed., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

We show here the expression, permeability and pharmacology of a voltage-gated channel in certain taste bud cells (TBCs) which is known to be permeable to Lucifer Yellow CH (LY) and known to release ATP as a neurotransmitter in response to taste substances. LY dissolved in a 200 mM K(+)-containing solution label TBCs immunoreactive to PLCβ2, a phospholipase subtype, but not the TBC subtype immunoreactive to SNAP-25, a SNARE protein. In addition to these subtypes, LY also labelled a few of the non-immunoreactive TBCs. Monovalent and divalent anion probes with molar mass less than 1200 also label PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs, whereas a cation probe, rhodamine B, labels the cell membrane of TBCs nonselectively and K(+) independently. The number of LY-labelled TBCs is decreased by 5 μM DIDS (4,4'-diisothiocyanostilbene-2-2'disulfonate), 1mM octanol and 10(-5)M H(+), but not by 10 μM carbenoxolone, 2mM probenecid, 10mM TEA, or 30 μM flufenamic acid. PLCβ2-immunoreactive TBCs and a few non-immunoreactive TBCs generate a TEA-insensitive outwardly rectifying current. DIDS decreases this current in magnitude with IC(50) of ~0.4 μM in a voltage-independent manner. Also 10(-5)M H(+) and 1mM octanol decreases the current magnitude, but 10 μM carbenoxolone and 2mM probenecid do not. These results show that the LY-permeable channel preferably permeates anions and occurs not only on PLCβ2-immunoreactive TBCs but also on certain non-immunoreactive TBCs. Also the results show that the pharmacology of the LY-permeable channel is different from hemichannels reported. The discussion focuses on the pharmacology and the role of the LY-permeable channel., (Copyright © 2010 Elsevier B.V. All rights reserved.)

In mice, BDNF provided by the developing taste epithelium is required for gustatory neuron survival following target innervation. However, we find that expression of BDNF, as detected by BDNF-driven β-galactosidase, begins in the cranial ganglia before its expression in the central (hindbrain) or peripheral (taste papillae) targets of these sensory neurons, and before gustatory ganglion cells innervate either target. To test early BDNF function, we examined the ganglia of bdnf null mice before target innervation, and found that while initial neuron survival is unaltered, early neuron development is disrupted. In addition, fate mapping analysis in mice demonstrates that murine cranial ganglia arise from two embryonic populations, i.e., epibranchial placodes and neural crest, as has been described for these ganglia in non-mammalian vertebrates. Only placodal neurons produce BDNF, however, which indicates that prior to innervation, early ganglionic BDNF produced by placode-derived cells promotes gustatory neuron development., (Copyright © 2011 Wiley-Liss, Inc.)

Mammalian taste bud cells (TBCs) consist of several cell types equipped with different taste receptor molecules, and hence the ratio of cell types in a taste bud constitutes the taste responses of the taste bud. Here we show that the population of immunohistochemically identified cell types per taste bud is proportional to the number of total TBCs in the taste bud or the area of the taste bud in fungiform papillae, and that the proportions differ among cell types. This result is applicable to soft palate taste buds. However, the density of almost all cell types, the population of cell types divided by the area of the respective taste buds, is significantly higher in soft palates. These results suggest that the turnover of TBCs is regulated to keep the ratio of each cell type constant, and that taste responsiveness is different between fungiform and soft palate taste buds., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Electrical stimulation of parasympathetic nerve (PSN) efferent fibers in the glossopharyngeal nerve induced a slow depolarizing synaptic potential (DSP) in frog taste cells under hypoxia. The objective of this study is to examine the interaction between a gustatory depolarizing receptor potential (GDRP) and a slow DSP. The amplitude of slow DSP added to a tastant-induced GDRP of 10 mV was suppressed to 60% of control slow DSPs for NaCl and acetic acid stimulations, but to 20-30% for quinine-HCl (Q-HCl) and sucrose stimulations. On the other hand, when a GDRP was induced during a prolonged slow DSP, the amplitude of GDRPs induced by 1 M NaCl and 1 M sucrose was suppressed to 50% of controls, but that by 1 mM acetic acid and 10 mM Q-HCl unchanged. It is concluded that the interaction between GDRPs and efferent-induced slow DSPs in frog taste cells under hypoxia derives from the crosstalk between a gustatory receptor current across the receptive membrane and a slow depolarizing synaptic current across the proximal subsynaptic membrane of taste cells.

Type III cells in taste buds form chemical synapses with intragemmal afferent nerve fibers and are characterized by the presence of membrane-bound vesicles in the cytoplasm. Although the vesicles differ in shape and size among species, they are primarily categorized into small clear (40 nm in diameter) and large dense-cored (90-200 nm) types. As such vesicles tend to be closely juxtaposed to the synaptic membrane of the cells, it is reasonable to consider that the vesicles include transmitter(s) towards the gustatory nerve. In the guinea-pig taste bud, stimulation with various taste substances (sucrose, sodium chloride, quinine hydrochloride, or monosodium L-glutamate) causes ultrastructural alterations of the type III cells. At the synapse, the presynaptic plasma membrane often displays invaginations of 90 nm in a mean diameter towards the cytoplasm, which indicates the dense-cored vesicles opening into the synaptic cleft by means of exocytosis. The vesicles are also exocytosed at the non-synaptic region into the intercellular space. These findings strongly suggest that the transmitters presumably contained in the vesicles are released to conduct the excitement of the type III cells to the nerves and also to exert their paracrine effects upon the surroundings, such as the Ebner's salivary gland, acting as local hormones.

The present study evaluates the central circuits that are synaptically engaged by very small subsets of the total population of geniculate ganglion cells to test the hypothesis that taste ganglion cells are heterogeneous in terms of their central connections. We used transsynaptic anterograde pseudorabies virus labeling of fungiform taste papillae to infect single or small numbers of geniculate ganglion cells, together with the central neurons with which they connect, to define differential patterns of synaptically linked neurons in the taste pathway. Labeled brain cells were localized within known gustatory regions, including the rostral central subdivision (RC) of the nucleus of the solitary tract (NST), the principal site where geniculate axons synapse, and the site containing most of the cells that project to the parabrachial nucleus (PBN) of the pons. Cells were also located in the rostral lateral NST subdivision (RL), a site of trigeminal and sparse geniculate input, and the ventral NST (V) and medullary reticular formation (RF), a caudal brainstem pathway leading to reflexive oromotor functions. Comparisons among cases, each with a random, very small subset of labeled geniculate neurons, revealed "types" of central neural circuits consistent with a differential engagement of either the ascending or the local, intramedullary pathway by different classes of ganglion cells. We conclude that taste ganglion cells are heterogeneous in terms of their central connectivity, some engaging, predominantly, the ascending "lemniscal," taste pathway, a circuit associated with higher order discriminative and homeostatic functions, others engaging the "local," intramedullary "reflex" circuit that mediates ingestion and rejection oromotor behaviors.

In many sensory systems, stimulus sensitivity is dynamically modulated through mechanisms of peripheral adaptation, efferent input, or hormonal action. In this way, responses to sensory stimuli can be optimized in the context of both the environment and the physiological state of the animal. Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the mechanisms for modulating taste sensitivity are poorly understood. In this study, we report that glucagon-like peptide-1 (GLP-1) signaling in taste buds modulates taste sensitivity in behaving mice. We find that GLP-1 is produced in two distinct subsets of mammalian taste cells, while the GLP-1 receptor is expressed on adjacent intragemmal afferent nerve fibers. GLP-1 receptor knockout mice show dramatically reduced taste responses to sweeteners in behavioral assays, indicating that GLP-1 signaling normally acts to maintain or enhance sweet taste sensitivity. A modest increase in citric acid taste sensitivity in these knockout mice suggests GLP-1 signaling may modulate sour taste, as well. Together, these findings suggest a novel paracrine mechanism for the regulation of taste function.

The act of eating requires a decision by an animal to place food in its mouth. The reasons to eat are varied and include hunger as well as the food's expected reward value. Previous studies of tastant processing in the rat primary gustatory cortex (GC) have used either anesthetized or awake behaving preparations that yield somewhat different results. Here we have developed a new preparation in which we explore the influences of intra-oral and non-contingent tastant delivery on rats' behavior and on their GC neural responses. We recorded single-unit activity in the rat GC during two sequences of tastant deliveries, PRE and POST, which were separated by a waiting period. Six tastants ranging in hedonic value from sucrose to quinine were delivered in the first two protocols called 4TW and L-S. In the third one, the App L-S protocol, only hedonically positive tastants were used. In the 4TW protocol, tastants were delivered in blocks whereas in the two L-S protocols tastants were randomly interleaved. In the 4TW and L-S protocols the probability of ingesting tastants in the PRE sequence decreased exponentially with the trial number. Moreover, in both protocols this decrease was greater in the POST than in the PRE sequence likely because the subjects learned that unpleasant tastants were to be delivered. In the App L-S protocol the decrease in ingestion was markedly slower than in the other protocols, thus supporting the hypothesis that the decrease in appetitive behavior arises from the non-contingent intra-oral delivery of hedonically negative tastants like quinine. Although neuronal responses in the three protocols displayed similar variability levels, significant differences existed between the protocols in the way the variability was partitioned between chemosensory and non-chemosensory neurons. While in the 4TW and L-S protocols the former population displayed more changes than the latter, in the App L-S protocol variability was homogeneously distributed between the two populations. We posit that these tuning changes arise, at least in part, from compounds released upon ingestion, and also from differences in areas of the oral cavity that are bathed as the animals ingest or reject the tastants.

Two voltage-dependent potassium channels, KCNQ1 and KCNH2, are expressed in the taste buds and were identified as strong candidates involved in the repolarization of taste receptor cells expressing phospholipase C-beta2 and TRPM5 (beta2/M5-TRCs). In cell type-specific expression, KCNQ1 was expressed in most taste bud cells, including beta2/M5-TRCs, whereas KCNH2 was expressed in a subset of beta2/M5-TRCs with no correlation with their taste modality, such as sweet or bitter taste reception. Expression of KCNH2 was restricted to young beta2/M5-TRCs. These results suggest that taste bud cells other than beta2/M5-TRCs are depolarized by some stimuli and also that beta2/M5-TRCs have cell age-dependent molecular mechanisms of repolarization.

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds--the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.

Little is known about subgemmal neurogenous plaques in the foliate papillae of tongue, which prompted the present investigation. The plaques were immunohistochemically studied in biopsies from 16 adults with the use of neural, stromal, basement membrane, and cell-cycle markers. They displayed a zonal pattern of organization. The neurofibroma-like superficial zone expanded lamina propria and was contiguous to the epithelium covering the papillae. It consisted of tangled composites of S-100 protein-positive spindled cells and fibrils stained for protein gene product 9.5 and neurofilament protein. The composites also expressed the CD56 antigen, could be traced into overlying taste buds, were associated with abundant laminin, and were intermingled with scattered dendritic cells expressing factor XIIIalpha and with mast cells decorated on staining for CD117. Similar composites, but arranged in fascicles and invested by epithelial membrane antigen-positive and collagen IV-positive sheaths, characterized the deep zone of the plaques. Intrafascicular CD57-positive myelin-like annuli and CD34-positive spindled cells were also features of this zone. The sheaths and, most often, the CD57-positive annuli and CD34-positive cells were progressively spread apart toward the intermediate zone of the plaques and were lost superficially. Ki67 and Bcl-1 were not expressed in the plaques. The results suggest that composites of Schwann cells and unmyelinated axons make up the superficial bulk of the plaques, whereas perineurial cells, endoneurial fibroblasts, and myelinated axons are present more deeply. It is possible that the composites achieve neuroeffector relationships and are not neoplastic. Trophic influences from gustatory nerve fibers could play a role in the development of the plaques.

The role of basolateral Na(+)-H(+) exchanger isoform-1 (NHE-1) was investigated in neural adaptation of rat taste responses to acidic stimuli, by direct measurement of intracellular pH (pH(i)) in polarized taste receptor cells (TRCs) and by chorda tympani (CT) taste nerve recordings. In TRCs perfused with CO(2)/HCO(3)(-)-free solution (pH 7.4), removal of basolateral Na(+) decreased pH(i) reversibly and zoniporide, a specific NHE-1 blocker, inhibited the Na(+)-induced changes in pH(i). The spontaneous rate of TRC pH(i) recovery from NH(4)Cl pulses was inhibited by basolateral zoniporide with a K(i) of 0.33microm. Exposure to basolateral ionomycin, reversibly increased TRC Ca(2+), resting pH(i), and the spontaneous rate of pH(i) recovery from an NH(4)Cl pulse. These effects of Ca(2+) on pH(i) were blocked by zoniporide. In in vivo experiments, topical lingual application of zoniporide increased the magnitude of the CT responses to acetic acid and CO(2), but not to HCl. Topical lingual application of ionomycin did not affect the phasic part of the CT responses to acidic stimuli, but decreased the tonic part by 50% of control over a period of about 1 min. This increased adaptation in the CT response was inhibited by zoniporide. Topical lingual application of 8-CPT-cAMP increased the CT responses to HCl, but not to CO(2), and acetic acid. In the presence of cAMP, ionomycin increased sensory adaptation to HCl, CO(2), and acetic acid. Thus, cAMP and Ca(2+) independently modulate CT responses to acidic stimuli. While cAMP enhances TRC apical H(+) entry and CT responses to strong acid, an increase in Ca(2+) activates NHE-1, and increases neural adaptation to all acidic stimuli.

The superior laryngeal nerve (SLN) contains CO2-sensitive fibers. In the laryngeal epithelium, two candidates for CO2 reception have been identified, namely the intraepithelial free nerve endings and the taste buds. To elucidate the contribution of free nerve endings to CO2 reception, electrophysiological activities were recorded during various stages of regeneration of nerve endings following SLN-crush in rats. The left SLN was crushed surgically and maintained from 4 to 40 days for regeneration of nerve endings. Laryngeal sections were processed for immunohistochemical staining of protein gene product 9.5 to observe regeneration of free nerve endings and taste buds in the epithelium. By day 4 after SLN-crush, both the free nerve endings and taste buds had disappeared. Regeneration of the free nerve endings was recognized from day 8, while that of the taste buds started at day 16. On day 40, the number of taste buds on SLN-crush side was similar to that on the untreated side. Electrophysiological recording of SLN throughout the regeneration period (excluding day 4), showed response to intralaryngeal 9% CO2 (stimulation or inhibition) whether or not taste buds were present. Our results showed intralaryngeal CO2 reception without taste bud involvement, indicating that the free nerve endings in the laryngeal epithelium are receptive to intralaryngeal CO2.

Taste cells located in taste buds respond to gustatory stimuli using a complex arrangement of ion channels, receptor molecules and signaling cascades. Previous electrophysiological experiments have shown that sour taste (essentially a taste of protons) is mediated, at least in part, by apically located amiloride-sensitive channels in the rat. Here, the molecular cloning of sour-taste receptor genes is described. Using a combination of homology screening and functional expression approaches, we screened a rat circumvallate papilla cDNA library and identified acid-sensing ion channel-2a (ASIC2a) and ASIC2b, amiloride-sensitive proton-activated cation channels. In situ hybridization and reverse transcription-polymerase chain reaction experiments showed that ASIC2a and ASIC2b transcripts were localized in taste cells. Immunohistochemical and immunoprecipitation studies revealed that both channels were expressed in a subset of taste cells and that some of the cells expressed ASIC2a/ASIC2b heteromeric assemblies. Immunoelectron microscopic analyses demonstrated that some of the ASIC2a-immunopositive cells had the characteristics of type III cells, which make synaptic contacts with gustatory afferent neurons. According to our electrophysiological studies, stimulation by acetic acid generated larger inward currents in ASIC2a- or ASIC2a/ASIC2b-expressing oocytes than those induced by hydrochloric acid at the same proton concentration and these findings are in good agreement with the well-known taste phenomenon that acetic acid is more sour than hydrochloric acid at equal pH. Taken together, the present results strongly suggest that mucosal protons dissociated from sour-taste substances induce taste cell depolarization through the activation of the ASIC2a and ASIC2a/ASIC2b channels, which leads to transmitter release onto gustatory neurons.

In an attempt to determine the chemosensory cues, if any, provided by fats in the oral cavity, we have performed patch-clamp recordings on isolated rat taste receptor cells during application of free fatty acids. Cis-polyunsaturated fatty acids, when applied extracellularly, inhibit delayed-rectifying K+ channels. In a subset of cells, these fatty acids also enhance inwardly rectifying K+ currents. Saturated, monounsaturated, and trans-polyunsaturated fatty acids have no significant effect on K+ currents. These effects do not involve activation of G protein-mediated pathways, including protein kinase C and protein kinase A, lipoxygenase pathways, cyclooxygenase pathways, or cytochrome P-450 pathways, consistent with direct effects on these ion channels or closely associated proteins. The net effect of fatty acids is to prolong stimulus-induced depolarizations of taste receptor cells, and we propose the effects on K+ channels represent the mechanism by which fats are detected by receptor cells in the oral cavity.

1. Taste cells in intact taste buds in slices of Necturus lingual epithelium were impaled with microelectrodes for intracellular recording. Two types of cells were investigated: taste receptor cells and basal cells. 2. Impaling cells in the apical end of taste buds resulted in intracellular records from taste receptor cells. Applying short pulses (100- to 200-ms duration) of 140 mM KCl solution to the apical pore elicited receptor potentials in the taste receptor cells. 3. Impaling cells in the base of the taste bud resulted in intracellular records from taste receptor cells and basal cells. KCl applied to the taste pore elicited responses in the basal region that varied greatly in both magnitude and time of onset. The latency of these responses (time of onset compared with the onset of the receptor potential) ranged from 0 to hundreds of milliseconds. 4. Impaled cells were identified by injecting Lucifer yellow after recording KCl responses for 21 cells. KCl responses recorded from identified basal cells all had latencies of greater than 75 ms. KCl responses from identified receptor cells all had latencies of less than 75 ms. 5. One explanation for the long latency of KCl responses recorded in basal cells is that the responses represent postsynaptic potentials. In agreement with this interpretation, long-latency responses, but not short-latency responses, were reversibly reduced by the Ca antagonist Cd (1 mM, 10- to 20-min bath exposure). 6. Long-latency responses also differed from short-latency responses in their voltage dependence. Short-latency responses had the same voltage dependence as apically recorded receptor potentials, increasing with hyperpolarization from resting potential with an extrapolated reversal potential near 0 mV. Long-latency responses were much less dependent on voltage in this range. 7. We measured the spread of exogenously applied KCl with potassium-sensitive electrodes. Long-latency responses were not generated by diffusion of applied KCl to the basal region of the taste bud. A small transient increase in extracellular potassium occurred at the base of the taste bud after chemostimulation at the apical pore. This increase was due to depolarization-evoked release of potassium from taste cells and did not cause the long-latency responses in basal cells. 8. We conclude that short-latency (less than 75 ms) responses recorded from cells situated in the bases of taste buds are electrotonically conducted receptor potentials generated at the apical region. Long-latency (greater than 75 ms) responses are consistent with recording postsynaptic responses in basal cells.

In the rat, calbindin-like immunoreactivity was observed at both the light and electron microscopic levels within the chemoreceptor neurons of the vomeronasal organ (VNO) and both intragemmal cells and associated nerve fibers of the circumvallate taste buds. All VNO neurons were immunoreactive. Only a subset of intragemmal taste cells was immunoreactive; associated immunoreactive nerve fibers were apposed to both labeled and unlabeled cells but no synaptic contacts were observed.

Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-microns opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10-100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50-900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250 mM, the maximal collective spike rate of a bud was in the range of 6-10 sec-1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5-2 sec-1. Buds of pure phasic response were also observed. Amiloride (30 microM) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 microM). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud.(ABSTRACT TRUNCATED AT 250 WORDS)

The recent application of precise biochemical and electrophysiological techniques to studies of taste cells has brought new insights into the cellular mechanisms of taste transduction. They have revealed that taste cells use a variety of mechanisms for transduction, including apically located ion channels, ligand-gated channels, and receptors coupled to second messenger systems.

To assess the role of phosphatidylinositol turnover in taste transduction we have visualized, in rat tongue, ATP-dependent endoplasmic reticular accumulation of 45Ca2+, inositol 1,4,5-trisphosphate receptor binding sites, and phosphatidylinositol turnover monitored by autoradiography of [3H]cytidine diphosphate diacylglycerol formed from [3H]cytidine. Accumulated 45Ca2+, inositol 1,4,5-trisphosphate receptors, and phosphatidylinositol turnover are selectively localized to apical areas of the taste buds of circumvallate papillae, which are associated with bitter taste. Further evidence for a role of phosphatidylinositol turnover in bitter taste is our observation of a rapid, selective increase in mass levels of inositol 1,4,5-trisphosphate elicited by low concentrations of denatonium, a potently bitter tastant.

New technologies in neurophysiology and ultrastructural research are bringing about rapid advances in our understanding of taste, particularly at the cellular level. The model of chemosensory processing in the taste bud presented here can now be explored in great detail. The synaptic organization of the taste bud indicates a potential for intriguing peripheral integrative mechanisms, including cross-talk between taste cells, summation of chemoreceptor responses by interneurons (basal cells) in the taste bud, and centrifugal control of taste buds via efferent input from the CNS. Figure 2 summarizes these findings. The existence of voltage-gated ionic channels on taste cells and their unequal distribution in apical and basolateral membrane suggests mechanisms for chemosensory transduction: A primary event in the transduction process for many taste stimuli is likely to be the closure of apical potassium channels, thus leading to a depolarizing receptor potential. The closure of these apical potassium channels is probably mediated via cyclic nucleotides or intracellular Ca2+.

Isolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential held at -80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringer's solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110 mM Na in the external and 10 mM Na in the internal solution, the inhibition constant of amiloride was (at -80 mV) near 0.3 microM. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability by small concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, which in situ express these channels in their apical membrane, may be competent to detect the taste quality "salty." The same cells also express TTX-blockable voltage-gated Na channels.