Kiyonori Yoshii

In situ tight-seal recordings of taste substance-elicited action currents and voltage-gated Ba currents from single taste bud cells in the peeled epithelium of mouse tongue.

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.

Functional expression of ionotropic purinergic receptors on mouse taste bud cells

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 μm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca2+ imaging showed that similarly applied 1 μm ATP, 30 μm BzATP (a P2X7 agonist), or 1 μm 2MeSATP (a P2Y1 and P2Y11 agonist) increased intracellular Ca2+ concentration, but 100 μm UTP (a P2Y2 and P2Y4 agonist) and α,β‐meATP (a P2X agonist except for P2X2, P2X4 and P2X7) did not. RT‐PCR suggested the expression of P2X2, P2X4, P2X7, P2Y1, P2Y13 and P2Y14 among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X2. 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 μm PPADS (a non‐selective P2 blocker) and 1 μm KN‐62 (a P2X7 blocker). These results showed for the first time the functional expression of P2X2 and P2X7 on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

Optical recordings of taste responses from fungiform papillae of mouse in situ.

1 Single taste buds in mouse fungiform papillae consist of ≈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. 2 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 ≈25 mV and elicited action potentials, while other TBCs were hyperpolarized by ≈12 mV. The TBCs eliciting hyperpolarizing receptor potentials also generated action potentials on electrical stimulation. 3 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. 4 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.

A method for in-situ tight-seal recordings from single taste bud cells of mice

In order to investigate taste transduction mechanisms, we developed a method to irrigate the receptor and basolateral membranes of mouse taste bud cells with different solutions under tight-seal recording conditions. A peeled tongue epithelium with taste bud cells was mounted on a recording platform designed to separate irrigating solutions for each membrane. The mucosal surface (receptor membrane side) of the peeled epithelium facing an inner chamber was always irrigated with deionized water or stimulating solutions, and the serosal surface (basolateral membrane side) was irrigated with a physiological saline solution. A recording electrode was placed on the basolateral membrane of a taste bud cell under an upright-microscope with a x 40-water-immersed objective. Investigated taste bud cells generated action potentials, and 1 M glucose, 200 mM NaCl, and 10 mM quinine elicited inward current. Irrigation with deionized water for more than 1 h had no effect. The resistance of the peeled tongue epithelium was 1570 +/- 343 omega cm2. These results show that the peeled tongue epithelium protects basolateral membranes from deionized water or stimulating solutions as the tongue epithelium does in situ and that this method is suitable to investigate the role of each membrane in taste transduction.

Quantitative analysis of taste bud cell numbers in fungiform and soft palate taste buds of mice

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.

Lucifer Yellow slows voltage-gated Na+ current inactivation in a light-dependent manner in mice.

Lucifer Yellow CH (LY), a membrane‐impermeant fluorescent dye, has been used in electrophysiological studies to visualize cell morphology, with little concern about its pharmacological effects. We investigated its effects on TTX‐sensitive voltage‐gated Na+ channels in mouse taste bud cells and hippocampal neurons under voltage‐clamp conditions. LY applied inside cells irreversibly slowed the inactivation of Na+ currents upon exposure to light of usual intensities. The inactivation time constant of Na+ currents elicited by a depolarization to −15 mV was increased by fourfold after a 5 min exposure to halogen light of 3200 lx at source (3200 lx light), and sevenfold after a 1‐min exposure to 12 000 lx light. A fraction of the Na+ current became non‐inactivating following the exposure. The non‐inactivating current was ≈ 20 % of the peak total Na+ current after a 5 min exposure to 3200 lx light, and ≈ 30 % after a 1 min exposure to 12 000 lx light. Light‐exposed LY shifted slightly the current‐voltage relationship of the peak Na+ current and of the steady‐state inactivation curve, in the depolarizing direction. A similar light‐dependent decrease in kinetics occurred in whole‐cell Na+ currents of cultured mouse hippocampal neurones. Single‐channel recordings showed that exposure to 6500 lx light for 3 min increased the mean open time of Na+ channels from 1.4 ms to 2.4 ms without changing the elementary conductance. The pre‐incubation of taste bud cells with 1 mM dithiothreitol, a scavenger of radical species, blocked these LY effects. These results suggest that light‐exposed LY yields radical species that modify Na+ channels.

Voltage-gated channels involved in taste responses and characterizing taste bud cells in mouse soft palates

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.

Phenytoin, an antiepileptic drug, competitively blocked non-NMDA receptors produced by Xenopus oocytes

The blocking effect of phenytoin (PHT) and other antiepileptic agents on the glutamate receptors was investigated under voltage-clamp conditions using Xenopus oocytes that translated ddY-stock mouse brain mRNA. PHT shifted the concentration-response curve for kainate to right without significant block on the maximum response obtained, indicating that PHT competitively blocks the non-NMDA receptors. An apparent dissociation constant of PHT was 1.9 x 10(-4) M. The block was voltage-independent. Other antiepileptic agents examined hardly blocked the kainate responses except phenobarbital that blocked the responses. The possibility that the competitive block constitutes a part of the antiepileptic action of PHT was discussed.

Open channel block of NMDA receptors by conformationally restricted analogs of milnacipran and their protective effect against NMDA-induced neurotoxicity

We investigated the blocking effect of the conformationally restricted analogs of milnacipran on NMDA receptors by recording the whole‐cell currents of Xenopus oocytes injected with rat brain mRNA and the single channel currents of cultured hippocampal neurons under voltage‐clamp conditions. Their protective effect against excitotoxicity was also investigated on cultured cortex neurons. All conformationally restricted analogs examined blocked activated NMDA receptors, though their structures were quite different from known NMDA receptor blockers. The analogs with a (1S, 2R, 1′S)‐configuration such as PPDC ((1S, 2R)‐1‐phenyl‐2[(S)‐1‐aminopropyl]‐N,N‐diethylcyclopropanecarboxamide) had lower IC50 values than those with other configurations. The empirical Hill coefficients for each compound were close to unity, indicating a 1:1 stoichiometry for the block. PPDC decreased the maximum responses to both N‐methyl d‐aspartate (NMDA) and glycine without altering their dissociation constants. The blocking effect was enhanced on hyperpolarization. PPDC had no effects on other glutamate receptor subtypes (AMPA, kainate, and metabotropic glutamate receptors) or other neurotransmitter receptors (GABAA, 5HT2C, and AChM1 receptors) produced by the oocytes. PPDC decreased the mean open time of NMDA receptors without decreasing their elementary conductance. The microscopic blocking rate constant was 2.8 × 107 M−1s−1. The macroscopic unblocking rate constant of PPDC was much faster than that of MK‐801. Only the analogs with the (1S, 2R, 1′S)‐configuration protected the cultures against NMDA‐induced neurotoxicity, though they failed to protect against kainate‐induced neurotoxicity. These results show that conformationally restricted analogs, at least PPDC, selectively blocked open channels of NMDA receptors. Synapse 31:87–96, 1999.

Subtype‐dependent postnatal development of taste receptor cells in mouse fungiform taste buds

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.