Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit.

The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with alpha-MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla. From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.     

Pharmaco-histochemical studies on a specific monoamine in the gustatory epithelia of the rabbit

Summary The foliate, vallate and fungiform papillae of the rabbit's tongue were studied fluorescence-histochemically under normal and experimental conditions. In normal animals a yellow fluorescence suggesting the presence of a serotonin-like monoamine was demonstrated only in taste bud cells of the foliate papilla, though its intensity was very weak. The fluorescence disappeared completely following reserpine treatment, while it was significantly enhanced by the treatment with nialamide. The fluorescence of taste bud cells could be clearly distinguished from that of catecholamines by the treatment with -MMT followed by nialamide. When 5-HTP, 5-HT and 5,6-DHT were administered separately, each of these drugs was selectively taken up in taste bud cells of the foliate and vallate papillae, but no fluorescent cells were observed in the fungiform papilla.From the present results, it seems reasonable to conclude that the fluorigenic amine of taste bud cells may be 5-HT (serotonin), or at least an indoleamine derivative. Also, it is suggested that the taste bud of the vallate papilla contains a cell type which can potentially synthesize a biogenic amine in situ, or is actually synthesizing it in a very small amount just like in the case of the taste bud of the foliate one.     

The occurrence of the cell type containing a specific monoamine in the taste bud of the rabbit's foliate papila.

The fluorescence histochemical examination on biogenic amines of the rabbit's foliate papilla revealed that a specific monoamine exhibiting an yellow fluorescence was present in a certain cell type of taste buds. The fluorescence had the emission maximum at 520 mmu and faded rapidly under the influence of the UV-irradiation. The green fluorescence of adrenergic nerve had the emission maximum at 480 mmu and was fairly stable upon the UV-irradiation. The yellow fluorescence disappeared completely following reserpine treatment, while it was markedly enhanced by nialamide treatment. From the observations, it is suggested that certain taste bud cells of the foliate papilla contain a biogenic monoamine, probably 5-hydroxytryptamine (serotonin).     

Effect of monoamines on the taste buds in the mouse

Summary Mouse taste buds were investigated following administration of monoamines and their precursors by fluorescence and electron microscopy. The appearance of fluorescent cells within the taste bud and the ultrastructural changes of vesicles in the gustatory cells were due to the treatment of 5-hydroxytryptophan. Small dense-cored vesicles (30–60 nm in diameter) appeared throughout the cytoplasm and accumulated especially at the presynaptic membranes of afferent synapses. Large dense-cored vesicles (80–100 nm) increased twice in number, and electron densities of their cores became more dense as compared with untreated mice. Fluorescent cells appeared in the taste bud of l-DOPA treated mice, whereas no ultrastructural changes were observed. These results suggest that the gustatory cells of the taste bud are capable of taking up and storing monoamines, which might act as neurotransmitters from the gustatory cells to the nerves.     

Measurement of membrane potential and [Ca2+]i in cell ensembles: application to the study of glutamate taste in mice.

We have studied the spectral properties of the voltage-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca(2+)-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+]i). There is a small spillover of di-8-ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8-ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+]i estimate is < 1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+]i can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express both metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.     

Possible nature of the biogenic amine in the dumbbell-shaped cells of frog taste buds]

An attempt was made to identify specific monoamines contained in the dumb-bell shape cells of the frog taste bud by means of histochemical analysis. It was shown by fluorescent microscopy that preliminary administration of exogenous serotonin into the blood channel of frog tongue resulted in a sharp increase of specific fluorescence of the dumb-bell shape cells, whereas serotonin synthesis inhibition with p-chlorphenylalanine led to reduction and elimination of specific fluorescence. It was concluded that-specific monoamine of the dumb-bell shape cells was possibly of serotonin-like nature.     

Basal cells of the frog's taste organ: fluorescence histochemistry with the serotonin analogue 5,7-dihydroxytryptamine in supravital conditions

We utilized the fluorescent serotonin analogue 5,7-dihydroxytryptamine (5,7DHT) to visualize basal cells in the frog's taste organ in supravital conditions. In whole mounts of lingual mucosa, specifical and detailed morphological visualization of fluorescent basal cells was obtained in the peripheral and central region of the intact taste organ; similar results were obtained after mechanical dissociation. Preincubation with serotonin prevented any fluorescence in basal cells. Electron microscopy showed good preservation of the ultrastructural morphology of the taste disk after exposure to 5,7DHT. The advantages of the current method as compared with conventional ones are discussed. This simple, reliable procedure will be useful to further define the biology of neuroendocrine cells in taste as well as in other organs.     

Glutamate may be an efferent transmitter that elicits inhibition in mouse taste buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (～50%) respond to 100 μM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 μM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors.     

Identification of TRPM5 Ion Channels in Type-II Taste Cells of Mice

TRPM5 are ion channels belonging to the TRP family, which demonstrate a nonselective permeability for monovalent cations and are activated by an increase in the intracellular calcium level. TRPM5 are present in taste receptor cells of type II responsible for reception of bitter, sweet, and umami taste sensations. Knockout of the trpm5 gene in mice results in a nearly complete loss of sensitivity to taste stimuli of the above-mentioned modalities (taste blindness). The physiological activity of TRPM5 in taste receptive cells has practically not been studied. Using a patch-clamp technique, we carried out a comparative analysis of the properties of recombinant TRPM5 and Ca²⁺-activated membrane channels in type-II taste cells in mice. Dialysis of the studied cells with a high-Ca²⁺ solution and application of a calcium ionophore, ionomycin, caused activation of outward-rectification ion channels permeable for Na⁺, Cs⁺, and K⁺ in CHO-strain cells with exogenous TRPM5. These channels were blocked by 100 μM triphenylphosphine oxide (TPPO). Calcium-activated channels in type-II taste cells also possessed analogous properties. Application of the calcium ionophore ionomycin or a stepwise increase in the intracellular Ca²⁺ level using photolysis (uncaging) caused activation of channels nonselective with respect to Na⁺ and Cs⁺ and impermeable for N-methyl-D-glucamine (NMDG⁺). These channels had the current–voltage characteristics of outward rectification and a high thermosensitivity (Q₁₀ = 6.7 ± 0.5); they could be blocked by TPPO. It should be emphasized that TRPM5 were specific with respect to type-II cells. An increase in the intracellular calcium level induced the appearance of Cl– current in type-I cells and did not influence the basic current in type-III cells.     

A serotonin-sensitive sensor for investigation of taste cell-to-cell communication

Taste receptor cells are the taste sensation elements for sour, salty, sweet, bitter and umami sensations. It was demonstrated that there are cell-to-cell communications between type II (sour) and type III (sweet, bitter and umami) taste cells. Serotonin (5-HT) is released from type III cells, which is the only type of taste cells that has synaptic process with sensory afferent fibers. Then, taste information is transmitted via fibers to the brain. During this process, 5-HT plays important roles in taste information transmission. In order to explore a sensor to detect 5-HT released from taste cell or taste cell networks, we develop a 5-HT sensitive sensor based on LAPS chip. This sensor performs with a detection limit of 3.3 × 10(-13)M and a sensitivity of 19.1 mV per concentration decade. Upon the stimuli of sour and mix (bitter, sweet and umami) tastants, 5-HT released from taste cells could be detected flexibly, benefit from the addressability of LAPS chip. The experimental results show that the local concentration of 5-HT is around several nM, which is consistent with those from other methods. In addition, immunofluorescent imaging technique is utilized to confirm the functional existence of both type II and III cells in a cluster of isolated taste cells. Different types of taste cells are labeled with corresponding specific antibody. This 5-HT sensitive LAPS chip provides a potential and promising way to detect 5-HT and to investigate the taste coding and information communication mechanisms.     

Flavor Preference Learning Increases Olfactory and Gustatory Convergence onto Single Neurons in the Basolateral Amygdala but Not in the Insular Cortex in Rats

The basolateral amygdala (BLA) and the insular cortex (IC) represent two major areas for odor-taste associations, i.e. flavor integration. This learning may require the development of convergent odor and taste neuronal activation allowing the memory representation of such association. Yet identification of neurons that respond to such coincident input and the effect of flavor experience on odor-taste convergence remain unclear. In the present study we used the compartmental analysis of temporal activity using fluorescence in situ hybridization for Arc (catFISH) to visualize odor-taste convergence onto single neurons in the BLA and in the IC to assess the number of cells that were co-activated by both stimuli after odor-taste association. We used a sucrose conditioned odor preference as a flavor experience in rats, in which 9 odor-sucrose pairings induce a reliable odor-taste association. The results show that flavor experience induced a four-fold increase in the percentage of cells activated by both taste and odor stimulations in the BLA, but not in the IC. Because conditioned odor preference did not modify the number of cells responding selectively to one stimulus, this greater odor-taste convergence into individual BLA neurons suggests the recruitment of a neuronal population that can be activated by both odor and taste only after the association. We conclude that the development of convergent activation in amygdala neurons after odor-taste associative learning may provide a cellular basis of flavor memory.     

Making sense with TRP channels: store-operated calcium entry and the ion channel Trpm5 in taste receptor cells

The sense of taste plays a critical role in the life and nutritional status of organisms. During the last decade, several molecules involved in taste detection and transduction have been identified, providing a better understanding of the molecular physiology of taste receptor cells. However, a comprehensive catalogue of the taste receptor cell signaling machinery is still unavailable. We have recently described the occurrence of calcium signaling mechanisms in taste receptor cells via apparent store-operated channels and identified Trpm5, a novel candidate taste transduction element belonging to the mammalian family of transient receptor potential channels. Trpm5 is expressed in a tissue-restricted manner, with high levels in gustatory tissue. In taste cells, Trpm5 is co-expressed with taste-signaling molecules such as alpha-gustducin, Ggamma(13), phospholipase C beta(2) and inositol 1,4,5-trisphosphate receptor type III. Biophysical studies of Trpm5 heterologously expressed in Xenopus oocytes and mammalian CHO-K1 cells indicate that it functions as a store-operated channel that mediates capacitative calcium entry. The role of store-operated channels and Trpm5 in capacitative calcium entry in taste receptor cells in response to bitter compounds is discussed.     

Isolation of single taste cells from lingual epithelium

A method is described for obtaining large numbers of isolatedtaste cells with identified polarity from lingual epithelium.The procedure involves incubating lingual epithelium in collagenase,staining the apical surface with fluorescein-conjugated wheatgerm agglutinin (FTTC-WGA), peeling non-gustatory surface epitheliumfrom the underlying taste buds and connective tissue, and dissociatingisolated taste buds with Ca²⁺-free saline. Isolated taste cellsretain their characteristic morphology for at least 30 min afterdissociation, and the apical specialization can be identifiedas a single patch of fluorescence usually located at the tipof an elongate process. Isolated taste cells are amenable tostudy with the patch-clamp technique, and whole-cell patch-clamprecordings show that isolated taste cells have membrane propertiessimilar to taste cells of intact lingual epithelium. Evidenceis presented that FITC-WGA staining does not alter the voltage-dependentionic currents of the taste cell membrane.     

Calbindin D28k-like immunoreactivity in the developing and regenerating circumvallate papilla of the rat

The distribution of calbindin D28k (CB)-like immunoreactivity (-LI) in the circumvallate papilla (CVP) was examined during development and regeneration following bilateral crush injury to the glossopharyngeal nerve in the rat. In the adult CVP, CB-like immunoreactive (-IR) nerve fibers were observed in the subgemmal region and some penetrated into the taste buds. CB-LI was also detected in the cytoplasm of the spindle-shaped gustatory cells in the lower half of the trench epithelium, which contained numerous synaptic vesicles and bundles of intermediate filaments. These CB-IR gustatory cells made synapse-like contacts with CB-IR nerve terminals. Some CB-IR nerve terminals made contacts with the gustatory cells negative for CB-LI. At least three developmental stages were defined with regard to the developmental changes in the distribution of CB-LI: (1) Stage I (embryonic day (E) 18–postnatal day (P)5): CB-IR nerve fibers appeared in the lamina propria just beneath the newly-formed CVP at E18, but the gustatory epithelium of the CVP contained no CB-IR structures. Taste buds with taste pores appeared at P1. (2) Stage II (P5–10): thin CB-IR nerve fibers began entering the trench epithelium, but no CB-IR cells were observed. (3) Stage III (P10–adult): in addition to the intragemmal and perigemmal CB-IR nerve fibers, very few CB-IR cells appeared in the taste buds around P10, and their numbers increased progressively. The changes in the distribution of taste buds and CB-LI following glossopharyngeal nerve injury were similar to those observed during development. On post-operative day (PO) 4, the taste buds and CB-IR cells decreased markedly in number. These CB-IR cells became round in shape, and the number of CB-IR nerve fibers decreased markedly. On PO8, both taste buds and CB-IR cells disappeared completely. The regenerated taste buds were first observed on PO12, increased rapidly in number by PO20, and increased slowly thereafter. CB-IR nerve fibers accumulated at the subgemmal region and began penetrating into the trench wall epithelium around PO16. CB-IR cells appeared between PO20 and PO24, and their numbers increased progressively and reached the normal level on PO40. The topographical localizations of the taste buds and CB-IR cells during development and regeneration were comparable to those of normal animals. The delay of the time courses for appearance of CB-IR nerve fibers and CB-IR cells compared to the appearance of taste buds during development and regeneration suggests that CB in the gustatory epithelium may participate in the survival of the taste bud cells rather than in the induction of the taste buds.     

The selective serotonin reuptake inhibitor paroxetine does not alter consummatory concentration-dependent licking of prototypical taste stimuli by rats

Serotonin and the 5HT(1A) receptor are expressed in a subset of taste receptor cells, and the 5HT(3) receptor is expressed on afferent fibers innervating taste buds. Exogenous administration of the selective serotonin reuptake inhibitor, paroxetine, has been shown to increase taste sensitivity to stimuli described by humans as sweet and bitter. Serotonergic agonists also decrease food and fluid intake, and it is possible that modulations of serotonin may alter taste-based hedonic responsiveness; alternatively, or in combination, serotonin may interact with physiological state to impact ingestive behavior. In this study, the unconditioned licking of prototypical taste stimuli by rats in brief-access taste tests was assessed following paroxetine administration (0.3-10 mg/kg intraperitoneal). We also measured sucrose licking by rats in different deprivation states after paroxetine (5 mg/kg). In neither experiment did we find any evidence of an effect of paroxetine on licking relative to water to any of the taste stimuli in the brief-access test at doses that decreased food intake. However, in some conditions, paroxetine decreased trials initiated to tastants. Therefore, a systemic increase in serotonin via paroxetine administration can decrease appetitive behavior in brief-access tests but is insufficient to alter taste-guided consummatory behavior.     

Construction of a taste-blind medaka fish and quantitative assay of its preference-aversion behavior

In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference-aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference-aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.     

Uptake of l-Dopa by cells in the taste buds of the vallate papilla of the mouse

Summary Several precursor substances and biogenic amines were admistered intraperitoneally to mice and were examined by the histochemical formaldehyde induced fluorescence method. It was found that after treatment with l-Dopa a number of cells inside the taste buds showed fluorescence.     

Acid stimulation (sour taste) elicits GABA and serotonin release from mouse taste cells

Several transmitter candidates including serotonin (5-HT), ATP, and norepinephrine (NE) have been identified in taste buds. Recently, γ-aminobutyric acid (GABA) as well as the associated synthetic enzymes and receptors have also been identified in taste cells. GABA reduces taste-evoked ATP secretion from Receptor cells and is considered to be an inhibitory transmitter in taste buds. However, to date, the identity of GABAergic taste cells and the specific stimulus for GABA release are not well understood. In the present study, we used genetically-engineered Chinese hamster ovary (CHO) cells stably co-expressing GABA(B) receptors and Gαqo5 proteins to measure GABA release from isolated taste buds. We recorded robust responses from GABA biosensors when they were positioned against taste buds isolated from mouse circumvallate papillae and the buds were depolarized with KCl or a stimulated with an acid (sour) taste. In contrast, a mixture of sweet and bitter taste stimuli did not trigger GABA release. KCl- or acid-evoked GABA secretion from taste buds was Ca(2+)-dependent; removing Ca(2+) from the bathing medium eliminated GABA secretion. Finally, we isolated individual taste cells to identify the origin of GABA secretion. GABA was released only from Presynaptic (Type III) cells and not from Receptor (Type II) cells. Previously, we reported that 5-HT released from Presynaptic cells inhibits taste-evoked ATP secretion. Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the present results indicate that GABA and 5-HT are inhibitory transmitters in mouse taste buds and both likely play an important role in modulating taste responses.