Characteristics of A-type voltage-gated K+ currents expressed on sour-sensing type III taste receptor cells in mice.

Sour taste is detected by type III taste receptor cells that generate membrane depolarization with action potentials in response to HCl applied to the apical membranes. The shape of action potentials in type III cells exhibits larger afterhyperpolarization due to activation of transient A-type voltage-gated K+ currents. Although action potentials play an important role in neurotransmitter release, the electrophysiological features of A-type K+ currents in taste buds remain unclear. Here, we examined the electrophysiological properties of A-type K+ currents in mouse fungiform taste bud cells using in-situ whole-cell patch clamping. Type III cells were identified with SNAP-25 immunoreactivity and/or electrophysiological features of voltage-gated currents. Type III cells expressed A-type K+ currents which were completely inhibited by 10 mM TEA, whereas IP3R3-immunoreactive type II cells did not. The half-maximal activation and steady-state inactivation of A-type K+ currents were 17.9 ± 4.5 (n = 17) and - 11.0 ± 5.7 (n = 17) mV, respectively, which are similar to the features of Kv3.3 and Kv3.4 channels (transient and high voltage-activated K+ channels). The recovery from inactivation was well fitted with a double exponential equation; the fast and slow time constants were 6.4 ± 0.6 ms and 0.76 ± 0.26 s (n = 6), respectively. RT-PCR experiments suggest that Kv3.3 and Kv3.4 mRNAs were detected at the taste bud level, but not at single-cell levels. As the phosphorylation of Kv3.3 and Kv3.4 channels generally leads to the modulation of cell excitability, neuromodulator-mediated A-type K+ channel phosphorylation likely affects the signal transduction of taste.

An electrophysiological method for evaluation of topical antipruritic drugs on itch-related neuronal activities in the spinal cord in hairless mice.

To evaluate the effects of antipruritic drugs, it is important to determine whether the neural responses induced by physiological itch stimuli are suppressed. Although there are several behavioral assessments for topical antipruritic drugs applied to the skin, there are few established methods at neuronal levels using in vivo electrophysiological recordings for predicting local efficacy of antipruritic drugs for cutaneous application. To establish an assessment of topical antipruritic drugs applied to skin using in vivo extracellular recording from neurons in the superficial dorsal horn, we examined the relationships between itch-related biting behavior and spinal neuronal responses elicited by intradermal injection of pruritogen serotonin (5-HT) in hairless mice. The efficacy of topical occlusive application of local anesthetics was also evaluated by an in vivo electrophysiological method. 5-HT significantly increased the firing frequency in spinal neurons. The spinal firing frequency time course was similar to that of the biting behavior after the 5-HT injections. The 5-HT-induced spinal responses were significantly decreased by topical occlusive application of lidocaine or a Nav 1.7 channel blocker to the calf. The intradermal 5-HT injection-induced spinal neuronal responses appeared to be suppressed by topical occlusive application of lidocaine or a Nav1.7 channel blocker. The electrophysiological method for evaluating topical antipruritic drugs may be beneficial in assessing local effects on the skin.

Taste Receptor Cells Generate Oscillating Receptor Potentials by Activating G Protein-Coupled Taste Receptors.

The receptor potentials of taste receptor cells remain unclear. Here, we demonstrate that taste receptor cells generate oscillating depolarization (n = 7) with action potentials in response to sweet, bitter, umami, and salty taste substances. At a lower concentration of taste substances, taste receptor cells exhibited oscillations in membrane potentials with a low frequency and small magnitude of depolarization. Although the respective waves contained no or 1-2 action potentials, the taste receptor cells generated action potentials continuously in the presence of taste stimuli. Both the frequency and magnitude of oscillations increased when the concentration was increased, to 0.67-1.43 Hz (n = 3) and Δ39-53 mV (n = 3) in magnitude from -64.7 ± 4.2 to -18.7 ± 5.9 mV, which may activate the ATP-permeable ion channels. In contrast, a sour tastant (10-mM HCl) induced membrane depolarization (Δ19.4 ± 9.5 mV, n = 4) with action potentials in type III taste receptor cells. Interestingly, NaCl (1 M) taste stimuli induced oscillation (n = 2) or depolarization (Δ10.5 ± 5.7 mV at the tonic component, n = 9). Our results indicate that the frequency and magnitude of oscillations increased with increasing taste substance concentrations. These parameters may contribute to the expression of taste "thickness."

Diurnal rhythm regulates the frequency of carbachol-induced beta oscillation via inhibitory neural system in rat hippocampus.

Animals have a diurnal rhythm with a cycle of approximately 1 day modulated by light information. This rhythm modulates memory performance. Relatedly, the hippocampal neural circuit has a dynamic property that can induce theta, beta, and gamma brain waves. However, the associated between the diurnal rhythm and these waves has not yet been elucidated. Carbachol, a cholinergic agent, can induce oscillations (e.g., beta waves) in rat hippocampal slices. In this study, we investigate the diurnal changes in the dynamic properties of hippocampal neuronal circuits using carbachol-induced beta oscillations (CIBOs). The hippocampal slices were made from rats adapted to 12-h-each light and dark conditions. The frequency of CIBO was significantly decreased more at midnight than at midday. There was no significant difference both under 12-h-each dark/dark condition and in the shuffled data of diurnal condition. The frequency at midday was significantly decreased by the application of SR95531 (gabazine) and bicuculline gamma-aminobutyric acid (GABA)-A receptor antagonists. In paired-pulse inhibition (PPI) experiments, the PPI ratio at midnight was larger than that at midday. The PPI ratio reflects the degree of recurrent inhibition. The expression level of Glutamate decarboxylase 65, an enzyme that synthetizes GABA, was significantly higher at midnight than at midday. These results suggest that the CIBO frequency can be modulated by diurnal changes of hippocampal inhibitory neurons, and the modulation may lead to a diurnal change in memory performance. Supplementary Information: The online version contains supplementary material available at 10.1007/s11571-021-09736-4.

Slow recovery from the inactivation of voltage-gated sodium channel Nav1.3 in mouse taste receptor cells.

Action potentials play an important role in neurotransmitter release in response to taste. Here, I have investigated voltage-gated Na+ channels, a primary component of action potentials, in respective cell types of mouse fungiform taste bud cells (TBCs) with in situ whole-cell clamping and single-cell RT-PCR techniques. The cell types of TBCs electrophysiologically examined were determined immunohistochemically using the type III inositol 1,4,5-triphoshate receptor as a type II cell marker and synaptosomal-associated protein 25 as a type III cell marker. I show that type II cells, type III cells, and TBCs not immunoreactive to these markers (likely type I cells) generate voltage-gated Na+ currents. The recovery following inactivation of these currents was well fitted with double exponential curves. The time constants in type III cells (~20 ms and ~ 1 s) were significantly slower than respective time constants in other cell types. RT-PCR analysis indicated the expression of Nav1.3, Nav1.5, Nav1.6, and ß1 subunit mRNAs in TBCs. Pharmacological inhibition and single-cell RT-PCR studies demonstrated that type II and type III cells principally express tetrodotoxin (TTX)-sensitive Nav1.3 channels and that ~ 30% of type I cells express TTX-resistant Nav1.5 channels. The auxiliary ß1 subunit that modulates gating kinetics was rarely detected in TBCs. As the ß1 subunit co-expressed with an α subunit is known to accelerate the recovery from inactivation, it is likely that voltage-gated Na+ channels in TBCs may function without ß subunits. Slow recovery from inactivation, especially in type III cells, may limit high-frequency firing in response to taste substances.

Age-related electrophysiological changes in mouse taste receptor cells.

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 (IP3 ) receptor (IP3 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.

Quantitative Analysis of Taste Bud Cell Numbers in the Circumvallate and Foliate Taste Buds of Mice.

A mouse single taste bud contains 10-100 taste bud cells (TBCs) in which the elongated TBCs are classified into 3 cell types (types I-III) equipped with different taste receptors. Accordingly, differences in the cell numbers and ratios of respective cell types per taste bud may affect taste-nerve responsiveness. Here, we examined the numbers of each immunoreactive cell for the type II (sweet, bitter, or umami receptor cells) and type III (sour and/or salt receptor cells) markers per taste bud in the circumvallate and foliate papillae and compared these numerical features of TBCs per taste bud to those in fungiform papilla and soft palate, which we previously reported. In circumvallate and foliate taste buds, the numbers of TBCs and immunoreactive cells per taste bud increased as a linear function of the maximal cross-sectional taste bud area. Type II cells made up approximately 25% of TBCs irrespective of the regions from which the TBCs arose. In contrast, type III cells in circumvallate and foliate taste buds made up approximately 11% of TBCs, which represented almost 2 times higher than what was observed in the fungiform and soft palate taste buds. The densities (number of immunoreactive cells per taste bud divided by the maximal cross-sectional area of the taste bud) of types II and III cells per taste bud are significantly higher in the circumvallate papillae than in the other regions. The effects of these region-dependent differences on the taste response of the taste bud are discussed.

A subset of taste receptor cells express biocytin-permeable channels activated by reducing extracellular Ca2+ concentration.

Taste receptor cells (type II cells) transmit taste information to taste nerve fibres via ATP-permeable channels, including calcium homeostasis modulator (CALHM), connexin and/or pannexin1 channels, via the paracrine release of adenosine triphosphate (ATP) as a predominant transmitter. In the present study, we demonstrate that extracellular Ca2+ -dependent biocytin-permeable channels are present in a subset of type II cells in mouse fungiform taste buds using biocytin uptake, immunohistochemistry and in situ whole-cell recordings. Type II cells were labelled with biocytin in an extracellular Ca2+ concentration ([Ca2+ ]out )-sensitive manner. We found that the ratio of biocytin-labelled type II cells to type II cells per taste bud was approximately 20% in 2 mM Ca2+ saline, and this ratio increased to approximately 50% in nominally Ca2+ -free saline. The addition of 300 µM GdCl3 , which inhibits various channels including CALHM1 channels, significantly inhibited biocytin labelling in nominally Ca2+ -free saline, whereas the addition of 20 µM ruthenium red did not. Moreover, Cs+ -insensitive currents increased in nominally Ca2+ -free saline in approximately 40% of type II cells. These increased currents appeared at a potential of above -35 mV, reversed at approximately +10 mV and increased with depolarization. These results suggest that biocytin labels type II cells via ion channels activated by [Ca2+ ]out reduction, probably "CALHM-like" channels, on the basolateral membrane and that taste receptor cells can be categorized into two groups based on differences in the expression levels of [Ca2+ ]out -dependent biocytin-permeable channels. These data indicate electrophysiological and pharmacologically relevant properties of biocytin-permeable channels and suggest their contributions to taste signal transduction.

Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca2+ imaging, we supravitally labeled type II cells (phospholipase C ß2 [PLCß2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca2+ in the bathing solution. The addition of 1 µM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 µM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca2+ from intracellular stores. We also discuss the underlying mechanism of the Ca2+ response and the role of M3 in type III cells.

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.

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 (IP3 R3) 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.

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.

Hypertonicity augments bullfrog taste nerve responses to inorganic salts.

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.

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.

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.

Dye-permeable, voltage-gated channel on mouse fungiform taste bud cells.

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 PLCß2, 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 PLCß2-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. PLCß2-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 PLCß2-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.

Functional expression of M3, a muscarinic acetylcholine receptor subtype, in taste bud cells of mouse fungiform papillae.

Taste bud cells (TBCs) express various neurotransmitter receptors assumed to facilitate or modify taste information processing within taste buds. We investigated the functional expression of muscarinic acetylcholine receptor (mAChR) subtypes, M1-M5, in mouse fungiform TBCs. ACh applied to the basolateral membrane of TBCs elevates the intracellular Ca(2+) level in a concentration-dependent manner with the 50% effective concentration (EC(50)) of 0.6 microM. The Ca(2+) responses occur in the absence of extracellular Ca(2+) and are inhibited by atropine, a selective antagonist against mAChRs. The order of 50% inhibitory concentration (IC(50)) examined with a series of antagonists selective to mAChR subtypes shows the expression of M3 on TBCs. Perforated whole-cell voltage clamp studies show that 1 microM ACh blocks an outwardly rectifying current and that 100 nM atropine reverses the block. Reverse transcriptase-mediated polymerase chain reaction studies suggest the expression of M3 but not the other mAChR subtypes. Immunohistochemical studies show that phospholipase Cbeta-immunoreactive TBCs and synaptosome-associated protein of 25 kDa-immunoreactive nerve endings are immunoreactive to a transporter that packs ACh molecules into synaptic vesicles (vesicular acetylcholine transporter). These results show that M3 occurs on a few fungiform TBCs and suggest that a few nerve endings, and probably a few TBCs, release ACh by exocytosis. The role of ACh in taste responses is discussed.

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 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.

Effects of endomorphin on substantia gelatinosa neurons in rat spinal cord slices.

1. Whole-cell patch recordings were made from substantia gelatinosa (SG) neurons in transverse lumbar spinal cord slices of 15- to 30-day-old rats. 2. Endomorphin 1 (EM-1) or EM-2 (

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 electro-physiological 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-damp 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 Ix at source (3200 Ix light), and sevenfold after a 1-min exposure to 12,000 Ix light. A fraction of the Na+ current became non-inactivating following the exposure. The non-inactivating current was approximately 20 % of the peak total Na+ current after a 5 min exposure to 3200 Ix light, and approximately 30 % after a 1 min exposure to 12,000 Ix 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 Ix 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.