Yoshitaka Ohtubo

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.

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.

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.

Cell-type-dependent action potentials and voltage-gated currents in mouse fungiform taste buds.

Taste receptor cells fire action potentials in response to taste substances to trigger non‐exocytotic neurotransmitter release in type II cells and exocytotic release in type III cells. We investigated possible differences between these action potentials fired by mouse taste receptor cells using in situ whole‐cell recordings, and subsequently we identified their cell types immunologically with cell‐type markers, an IP3 receptor (IP3R3) for type II cells and a SNARE protein (SNAP‐25) for type III cells. Cells not immunoreactive to these antibodies were examined as non‐IRCs. Here, we show that type II cells and type III cells fire action potentials using different ionic mechanisms, and that non‐IRCs also fire action potentials with either of the ionic mechanisms. The width of action potentials was significantly narrower and their afterhyperpolarization was deeper in type III cells than in type II cells. Na+ current density was similar in type II cells and type III cells, but it was significantly smaller in non‐IRCs than in the others. Although outwardly rectifying current density was similar between type II cells and type III cells, tetraethylammonium (TEA) preferentially suppressed the density in type III cells and the majority of non‐IRCs. Our mathematical model revealed that the shape of action potentials depended on the ratio of TEA‐sensitive current density and TEA‐insensitive current one. The action potentials of type II cells and type III cells under physiological conditions are discussed.

Time-dependent expression of hypertonic effects on bullfrog taste nerve responses to salts and bitter substances.

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.

Network model of chemical-sensing system inspired by mouse taste buds

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.

Stochastic Synchronization and Array-Enhanced Coherence Resonance in a Bio-inspired Chemical Sensor Array

The purpose of our project is to establish how to design brain-inspired chemical sensor arrays from physiological, theoretical, and engineering points of view. In a previous work, a computational model for chemical sensor arrays has been proposed inspired from physiological properties of mouse taste bud cells. The computational model consists of three functional parts: the chemical sensor, the random pulse generator, and the stochastic synchronizer. The model converts the concentration of chemical substances into the degree of stochastic synchronization. We have implemented the chemical sensor array as a silicon devices. We discuss about functional significance of our device in view of noise-induced nonlinear phenomena: stochastic synchronization and array-enhanced coherence resonance.