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New Findings: What is the central question of this study? Loss of taste or inability to distinguish between different tastes progresses with age. The purpose was to evaluate the age-dependent changes in taste by studying the electrophysiological properties of taste receptor cells. What is the main finding and its importance? Ageing decreased the voltage-gated Na + and K + current densities of type III cells (sour and/or salt receptor cells) but did not affect the current densities in type II cells. At the peripheral levels, the excitability of type III cells was reduced due to ageing, which may affect the signal transduction to taste nerves., Abstract: The loss of taste due to normal ageing in mammals is assumed to be caused by the ageing of taste receptor cells. We examined the electrophysiological properties of taste receptor cells in the fungiform taste buds of ∼20-month-old mice in situ and subsequently identified their cell types with immunological markers: the inositol 1,4,5-trisphosphate (IP 3 ) receptor (IP 3 R3) for type II cells and a SNARE protein, synaptosomal-associated protein 25 (SNAP-25), for type III cells. Other cells are referred to as non-immunoreactive cells (non-IRCs). Cell types of some cells that could not be identified using cell-type markers were identified based on the electrophysiological feature of the respective cell types. All cell types generated action potentials and a variety of voltage-gated currents. The type II cells mainly expressed tetraethylammonium (TEA)-insensitive and slowly activating outwardly rectifying currents and generated tail currents in repolarization. In contrast, the type III cells expressed TEA-sensitive and faster activating K + currents and did not generate tail currents. These cell type-specific characteristics of voltage-gated currents in ∼20-month-old mice were similar to their respective cell types in ∼2-month-old mice. Also, we showed an age-dependent decrease in Na + and K + current densities in type III cells and an age-dependent increase in outwardly rectifying current density in non-IRCs. Ageing did not affect the voltage-gated current densities in type II cells. The decreased Na + and K + current densities, i.e. the decreased excitability of type III cells, due to ageing may affect the signal transduction to taste nerves., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

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.)

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.)

The tonicity of taste stimulating solutions has been usually ignored, though taste substances themselves yielded the tonicity. We investigated the effect of hypertonicity on bullfrog taste nerve responses to inorganic salts by adding nonelectrolytes such as urea and sucrose that elicited no taste nerve responses. Here, we show that hypertonicity alters bullfrog taste nerve-response magnitude and firing pattern. The addition of urea or sucrose enhances the taste nerve-response magnitude to NaCl and shifts the concentration-response curve to the left. The effect of hypertonicity on responses to CaCl(2) is bimodal; hypertonicity suppresses CaCl(2) responses at concentrations less than ~30 mM and enhances them at concentrations greater than ~30 mM. The hypertonicity also enhances response magnitude to other monovalent salts. The extent of the enhancing effects depends on the difference between the mobility of the cation and anion in the salt. We quantitatively suggest that both the enhancing and suppressing effects result from the magnitude and direction of local circuit currents generated by diffusion potentials across tight junctions surrounding taste receptor cells.

Taste buds endure extreme changes in temperature, pH, osmolarity, so on. Even though taste bud cells are replaced in a short span, they contribute to consistent taste reception. Each taste bud consists of about 50 cells whose networks are assumed to process taste information, at least preliminarily. In this article, we describe a neural network model inspired by the taste bud cells of mice. It consists of two layers. In the first layer, the chemical stimulus is transduced into an irregular spike train. The synchronization of the output impulses is induced by the irregular spike train at the second layer. These results show that the intensity of the chemical stimulus is encoded as the degree of the synchronization of output impulses. The present algorithms for signal processing result in a robust chemical-sensing system.

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.)

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

Taste bud cells (TBCs) on soft palates differ from those on tongues in innervation and chemosensitivity. We investigated voltage-gated channels involved in the taste responses of TBCs on mouse soft palates under in-situ tight-seal voltage/current-clamp conditions. Under the cell-attached mode, TBCs spontaneously fired action currents, which were blocked by application of 1 microM TTX to TBC basolateral membranes. Firing frequencies increased in response to taste substances applied to TBC receptor membranes. Under the whole-cell clamp mode, as expected, TBCs produced various voltage-gated currents such as TTX-sensitive Na+ currents (INa), outward currents (Iout) including TEA-sensitive and insensitive currents, inward rectifier K+ currents (Iir), and Ca2+ currents including T-type, P/Q-type, and L-type Ca2+ currents. We classified TBCs into three types based on the magnitude of their voltage-gated Na+ currents and membrane capacitance. HEX type (60% of TBCs examined) was significantly larger in Na+ current magnitude and smaller in membrane capacitance than LEX type (23%). NEX type (17%) had no Na+ currents. HEX type was equally distributed within single taste buds, while LEX type was centrally distributed, and NEX type was peripherally distributed. There were correlations between these electrophysiological cell types and morphological cell types determined by three-dimensional reconstruction. The present results show that soft palate taste buds contain TBCs with different electrophysiological properties, and suggest that their co-operation is required in taste transduction.

Single taste buds in mouse fungiform papillae consist of approximately 50 elongated cells (TBCs), where fewer than three TBCs have synaptic contacts with taste nerves. We investigated whether the non-innervated TBCs were chemosensitive using a voltage-sensitive dye, tetramethylrhodamine methyl ester (TMRM), under in situ optical recording conditions. Prior to the optical recordings, we investigated the magnitude and polarity of receptor potentials under in situ whole-cell clamp conditions. In response to 10 mM HCl, several TBCs were depolarized by approximately 25 mV and elicited action potentials, while other TBCs were hyperpolarized by approximately 12 mV. The TBCs eliciting hyperpolarizing receptor potentials also generated action potentials on electrical stimulation. A mixture of 100 mM NaCl, 10 mM HCl and 500 mM sucrose depolarized six TBCs and hyperpolarized another three TBCs out of 13 identified TBCs in a taste bud viewed by optical section. In an optical section of another taste bud, 1 M NaCl depolarized five TBCs and hyperpolarized another two TBCs out of 11 identified TBCs. The number of chemosensitive TBCs was much larger than the number of innervated TBCs in a taste bud, indicating the existence of chemosensitivity in non-innervated TBCs. There was a tendency for TBCs eliciting the same polarity of receptor potential to occur together in taste buds. We discuss the role of non-innervated TBCs in taste information processing.

The suppression mechanism of NH3 and CO2 on bullfrog taste nerve responses to 0.1 mM quinine was investigated by applying them directly on the tongue surface (surface application) or at the tongue interstices through the lingual artery (interstitial application). The surface application of NH3 and CO2 reversibly suppressed the taste nerve responses with IC50 values of 0.37 mM and 2.2 mM, respectively, whereas their hydrates were ineffective. The interstitial application of NH3 reversibly suppressed the taste nerve responses. The surface application of CO2 recovered the suppressed responses to quinine. The 4 s application of CO2 prior to that of 0.1 mM quinine had the maximum effect. These results show that NH3-induced alkalosis and CO2-induced acidosis of taste cells, taste nerve endings, or tongue interstices surrounding them suppressed the taste nerve responses, and that the neutralization of their intracellular pH recovered these responses. The time-dependent recovery suggests that the neutralization at a layer inside taste cells, taste nerve endings, or the interstices surrounding them is critical in taste transduction., (Copyright 1998 Elsevier Science B.V.)

We investigated the taste responses of single taste-bud cells (TBCs) in mice by applying stimuli only on receptor membranes acclimated to deionized water under tight-seal cell-attached voltage-clamp conditions, while their basolateral membranes were irrigated with a physiological saline solution. For this irrigation, we developed a new method: a peeled-tongue epithelium with TBCs mounted on a recording chamber where the peeled epithelium separated the irrigating solutions for each membrane as it separated in situ. Although no quinine-elicited action potentials had been reported, TBCs elicited a long-lasting train of biphasic currents derived from the action potentials in response to 10 mM quinine, in addition to responses to 10 mM HCl, or 200 mM NaCl dissolved in deionized water. These results indicate that quinine as well as HCl and NaCl depolarizes TBCs and generate action potentials. Under whole-cell recording conditions, TBCs generated action potentials, and voltage-gated currents such as LVA and HVA Ca currents, TTX-sensitive Na currents, and TEA/4-AP-sensitive K currents on depolarization. These voltage-gated channels were shown to exist predominantly on the basolateral membranes. We discussed the receptor mechanisms and the role of taste substance-elicited action potentials.

We investigated the effects of intracellular pH values (pHi) on taste nerve responses of clawed toads and eels. (1) CO2, NH3 or trimethylamine reversibly suppressed the taste nerve responses of clawed toads to various amino acids, CaCl2 and caffeine. IC50 values of the suppression of the responses to 0.1 mM L-proline were as follows: approximately 10 mM for CO2, approximately 0.3 mM for NH3, approximately 0.2 mM for trimethylamine. (2) Cross-adaptation experiments showed that L-proline, caffeine and CaCl2 stimulated different receptor sites from each other, indicating the suppressive effect was non-specific. (3) Although CO2, NH3 or trimethylamine yielded the charged molecules, HCO3-, CO3(2-), NH4+ or trimethylammonium on hydration, none of these charged species suppressed taste responses. (4) NH3 increased the threshold concentration of L-proline by e-fold per 0.37 mM NH3 and decreased the maximum response to L-proline with increasing NH3 concentration. (5) The taste nerve responses of eels to 0.1 mM L-arginine, a potent stimulus on eel taste receptors, were similarly suppressed by NH3 with an IC50 value of approximately 0.3 mM. (6) These results indicated that these uncharged species changed pHi, which suppressed the taste responses. CO2-induced acidosis and NH3- or trimethylamine-induced alkalosis are likely to inhibit activities of ion channels or enzymes involved in taste transduction mechanisms.

Taste responses of bullfrogs to various pungent compounds and taste substances were electrophysiologically recorded from glossopharyngeal nerves. The threshold concentrations were approximately 10(-7) M for piperine, approximately 10(-6) M for capsaicin and approximately 10(-4) M for allyl isothiocyanate. At any concentration examined, piperine was more potent than capsaicin. Both piperine and capsaicin elicited desensitizing responses, but the taste receptors recovered from the desensitization within 10 min after washing with deionized water. Cross-adaptation experiments revealed that capsaicin only partially desensitizes receptors for piperine, L-leucine, HCl or quinine. Perfusion of the lingual artery with a solution containing no added Ca decreased the responses to capsaicin. Such a solution has been shown to suppress the taste nerve responses by blocking synaptic transmissions between taste cells and taste nerves [8]. These results suggest that the gustatory effects of capsaicin are different from its pharmacological effects on sensory neurons. It is likely that capsaicin and other pungent compounds, when they act as seasonings, stimulate taste cells rather than the free nerve endings of the sensory neurons.

The carp gustatory responses to various salts and those to distilled water after adaptation of the receptors to the salts were recorded from the palatine nerve under varying conditions. (a) As far as Na4Fe(CN)6 solution prepared freshly was used, any peak in the dose-response curve was not observed, while the solution stored overnight induced a large peak response at the low concentration region as Konishi reported. The magnitude of the water response after adaptation to the stored solution was practically equal to that after adaptation to the fresh solution, suggesting that the receptor site for the water response is different from that for the dilute salts. (b) The responses to salts depended largely on the species of both cations and anions of the salts. The responses were deceased with an increase in the lyotropic number of the anions and increased with an increase in the number above 11. The response to distilled water was practically independent of the species of both monovalent cations and anions of the salts used for the adaptation. The pH dependence of the response to 10 mM NaCl was largely different from that to distilled water after adaptation to 10 mM NaCl. (c) The water response was suppressed by the presence of electrolytes in stimulating solution; the data obtained with different species of salts were described by a single curve as a function of the ionic strength. (d) The mechanism to explain how distilled water leads to depolarization of the taste cell was discussed in terms of the electric double-layer potential.

The effects of changed ionic environments on the taste responses to amino acids were examined by recording the activity of the palatine nerve. The responses to glycine, L-proline and L-arginine dissolved in deionized water were greatly reduced after ions were removed from the surface of the palatine epithelium by treatment with 5 mM EDTA. The addition of various species of salts to amino acid solutions caused reversible recovery of the responses. The response to 0.1 mM glycine increased with an increase in salt concentration in amino acid solutions and reached saturation level. The effects of salts of divalent cations (CaCl2 and MgCl2) appeared at much lower concentration (10(-7) M) than those of salts of monovalent cations (NaCl and KCl) (10(-4) M), suggesting that cations support the taste response. All the organic cations examined, including those of large molecular weights (choline, Tris, bis-Tris, bis-Tris propane, tetraethylammonium, triethanolamine and D-glucosamine), also supported the taste responses. The results obtained led to a conclusion that not a specific cation but various species of cations can support the eel taste responses. It is suggested that the cations do not act as current carrying ions to depolarize the taste cells but that the binding of the cations to the receptor membrane plays an essential role in taste reception.

Salts carrying impermeable organic cations elicited large responses in the frog and rat taste nerves. Amiloride suppressed the responses of the frog and rat taste nerves to NaCl, KCl and the organic salts to a similar extent. The estimated equilibrium potentials for Na+ and K+ at the thresholds determined were more negative than the resting potentials of the taste cells. The results obtained suggest that the taste responses to salts including NaCl are not induced by entry of cations via apical membranes of taste cells.

Synergism between 5'-nucleotides and L-amino acids in the rat taste nerve responses was investigated. The synergism was examined between low concentration of the nucleotides which induce only a negligibly small response and various concentrations of amino acids. A marked synergism was found between 5'-nucleotides and all the amino acids examined. Purine 5'-nucleotides such as GMP, deoxy GMP, GDP, GTP, AMP or IMP exhibited the synergistic effect on the responses to amino acids, while pyrimidine 5'-nucleotides such as CMP or UMP exhibited practically no synergistic effect. The presence of GMP led to a shift of the concentration-response curves for amino acids to a lower concentration region without affecting the response of the saturation level. These curves were analyzed under the assumption that there exist two types of receptor sites with different dissociation constants for each amino acid. The results suggest that GMP leads to a decrease in dissociation constants, especially for high affinity sites without affecting maximal responses. Monosodium glutamate (Glu) itself, which is known as a typical 'flavor potentiator', exhibited no enhancing effect on the responses to 4 primary taste stimuli. The mechanism of how Glu and the 5'-nucleotides 'potentiate' flavor of food is discussed.

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.

The gustatory nerve responses of the aquatic toad Xenopus laevis to salts, acids, amino acids, and bitter substances were recorded. 1) The gustatory receptors were sensitive to amino acids and bitter substances. The thresholds were 10(-7) M for Arg, 3 X 10(-9) M for strychnine, and 3 X 10(-8) M for quinine, 200-20,000 times lower than the thresholds for the respective stimuli in the bullfrog. 2) The basic and the neutral amino acids were effective whereas the acidic ones were not. Relations between the responses and log stimulus concentrations for the basic amino acids were linear in a wide concentration range whereas those for the neutral ones were of S shape. Cross-adaptation did not occur among pairs of a basic amino acid and a neutral one. 3) Responses to the basic amino acids and the basic bitter substances were suppressed by the addition of salts to the stimulating solutions, while those to the neutral and the acidic substances were not suppressed.

Properties of the receptor sites for L-amino acids in taste cells of the bullfrog (Rana catesbeiana) were examined by measuring the neural activities of the glossopharyngeal nerve under various conditions. (a) The frogs responded to 12 amino acids, but the responses to the amino acids varied with individual frogs under natural conditions. The frog tongues, however, exhibited similar responses after an alkaline treatment that removes Ca2+ from the tissue. The variation in the responses under natural conditions was apparently due to the variation in the amount of Ca2+ bound to the receptor membrane. (b) The responses to hydrophilic L-amino acids (glycine, L-alanine, L-serine, L-threonine, L-cysteine, and L-proline) were of a tonic type, but those to hydrophobic L-amino acids (L-valine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine, and L-tyrptophan) were usually composed of both phasic and tonic components. (c) The properties of the tonic component were quite different from those of the phasic component: the tonic component was largely enhanced by the alkaline treatment and suppressed by the acidic treatment that increases binding of Ca2+ to the tissue. Also, the tonic component was suppressed by the presence of low concentrations of salts, or the action of pronase E, whereas the phasic component was unchanged under these conditions. These properties of the phasic component were quite similar to those of the response to hydrophobic substances such as quinine. These results suggest that the hydrophilic L-amino acids stimulate receptor protein(s) and that the hydrophobic L-amino acids stimulate both the receptor protein and a receptor site similar to that for quinine. (d) On the basis of the suppression of the responses to amino acids by salts, the mechanism of generation of the receptor potential is discussed.