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

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.

Action Potentials↗

Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue.

The role of amiloride-sensitive Na+ channels (ASSCs) in the transduction of salty taste stimuli in rat fungiform taste buds has been well established. Evidence for the involvement of ASSCs in salt transduction in circumvallate and foliate taste buds is, at best, contradictory. In an attempt to resolve this apparent controversy, we have begun to look for functional ASSCs in taste buds isolated from fungiform, foliate, and circumvallate papillae of male Sprague-Dawley rats. By use of a combination of whole-cell and nystatin-perforated patch-clamp recording, cells within the taste bud that exhibited voltage-dependent currents, reflective of taste receptor cells (TRCs), were subsequently tested for amiloride sensitivity. TRCs were held at -70 mV, and steady-state current and input resistance were monitored during superfusion of Na(+)-free saline and salines containing amiloride (0.1 microM to 1 mM). Greater than 90% of all TRCs from each of the papillae responded to Na+ replacement with a decrease in current and an increase in input resistance, reflective of a reduction in electrogenic Na+ movement into the cell. ASSCs were found in two thirds of fungiform and in one third of foliate TRCs, whereas none of the circumvallate TRCs was amiloride sensitive. These findings indicate that the mechanism for Na+ influx differs among taste bud types. All amiloride-sensitive currents had apparent inhibition constants in the submicromolar range. These results agree with afferent nerve recordings and raise the possibility that the extensive labeling of the ASSC protein and mRNA in the circumvallate papillae may reflect a pool of nonfunctional channels or a pool of channels that lacks sensitivity to amiloride.

Amiloride↗

Substance-P-containing fibers in the incisive papillae of the rat hard palate. Light- and electron-microscopic immunohistochemical study.

Substance P (SP)-containing fibers in the incisive papillae of rat hard palates, which include various components of sensory receptors, i.e. mechanoreceptors, free nerve endings and chemosensory corpuscles (taste buds), were examined using immunoperoxidase techniques and light and electron microscopes. Immunolabeled fibers were consistently distributed in the medial part of the orifice of the incisive canals, i.e. in the taste-bud-enriched region. Dense immunolabeled fibers were found in subgemmal regions and in the lamina propria papillae. Some fine fibers entered and ascended the taste buds or occasionally the epithelium outside the taste buds. In addition, a rich innervation by SP-containing fibers close to blood capillaries was clearly identified. Electron microscopy revealed no specialized synaptic contact between the immunolabeled fibers and taste bud cells. Synaptic-like images could be found only between nonimmunolabeled nerve endings and the underlying taste bud cells. In the lamina propria papillae, mechanoreceptors observed in the present study contained no immunoperoxidase end products, whereas free nerve endings with an immunolabeled small-diameter axon (630-730 nm in diameter) were frequent. Similar axons were located at the adventitia of the blood capillaries. The possible functional role of SP-containing fibers in the incisive papillae was given attention.

Animals↗

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.

Animals↗

The cell biology of vertebrate taste receptors.

New technologies in neurophysiology and ultrastructural research are bringing about rapid advances in our understanding of taste, particularly at the cellular level. The model of chemosensory processing in the taste bud presented here can now be explored in great detail. The synaptic organization of the taste bud indicates a potential for intriguing peripheral integrative mechanisms, including cross-talk between taste cells, summation of chemoreceptor responses by interneurons (basal cells) in the taste bud, and centrifugal control of taste buds via efferent input from the CNS. Figure 2 summarizes these findings. The existence of voltage-gated ionic channels on taste cells and their unequal distribution in apical and basolateral membrane suggests mechanisms for chemosensory transduction: A primary event in the transduction process for many taste stimuli is likely to be the closure of apical potassium channels, thus leading to a depolarizing receptor potential. The closure of these apical potassium channels is probably mediated via cyclic nucleotides or intracellular Ca2+.

Animals↗

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide.

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12-13)-a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14-16) - thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17-18) - thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0-3] - many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5-10) - a small number of PGP 9.5-IR cells in the taste buds appeared, and their number increased gradually. Stage VI (P14-adult) - the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.

Age Factors↗

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.

Animals↗

Binding of [35S]saccharin to a protein fraction of rat tongue epithelia.

The binding of [35S]saccharin to ammonium sulfate fractions from homogenates of rat tongue epithelia was measured by equilibrium dialysis. The 40--60% saturated ammonium sulfate fraction from the buffer-soluble fraction had the highest saccharin-binding activity. Binding of [35S]saccharin to the 40--60% ammonium sulfate fraction was inhibited by unlabeled saccharin sodium salt. The inhibition increased with increasing unlabeled saccharin concentration and was nearly complete above 10 mM. [35S]Saccharin binding to the 40--60% ammonium sulfate fraction extracted from the tongue epithelia was inhibited by glucose, lactose and sucrose, while binding to similar fractions from tongue muscle was not affected by these sugars. The inhibition of binding of labeled saccharin to the epithelial fraction increased with increasing glucose concentrations. About 35% of the binding was inhibited by 1 M glucose. No significant difference in the amount of inhibition was seen among the three sugars at 0.1 M. The 40--60% ammonium sulfate fraction from tongue epithelium devoid of taste buds bound much less [35S]saccharin than did a similar fraction from epithelium with taste buds. Binding of [35S]saccharin by the preparation from epithelium devoid of taste buds was not inhibited by glucose. The results provide evidence that the 40--60% ammonium sulfate fraction from tongue epithelia with taste buds contains a protein which binds saccharin and sugars. We hypothesize that it is a sweet taste receptor protein.

Animals↗

Efficacy of exogenous oral zinc in treatment of patients with carbonic anhydrase VI deficiency.

BACKGROUND: We previously described a disorder in 18 patients with decreased parotid saliva gustin/carbonic anhydrase (CA) VI secretion associated with loss of taste (hypogeusia) and smell (hyposmia) and distorted taste (dysgeusia) and smell (dysosmia). Because gustin/CAVI is a zinc-dependent enzyme we instituted a study of treatment with exogenous zinc to attempt to stimulate synthesis/secretion of gustin/CAVI and thereby attempt to correct the symptoms of this disorder. METHODS: Fourteen of the 18 patients with this disorder completed the study. They were treated with 100 mg of exogenous zinc daily for 4 to 6 months, in an open clinical trial. Both before and after treatment, measurements were obtained of parotid saliva gustin/CAVI, parotid saliva, serum and urine zinc, taste and smell function, and, in some patients, examination of circumvallate taste buds by electron microscopy. RESULTS: Treatment success was predicated upon significant increases in parotid saliva gustin/CAVI. This occurred in 10 of the 14 patients who were labeled responders; they also exhibited improvement in taste and smell acuity, a diminution in dysgeusia and dysosmia and increased zinc concentrations in parotid saliva, serum, and urine. Taste bud morphology returned to normal in each responder in whom it was measured. No increase in gustin/CAVI occurred in 4 patients who were labeled nonresponders; they exhibited no improvement in taste or smell acuity and no increases in parotid saliva zinc. However, serum and urine zinc increased to levels similar to those measured in the 10 responders. Two of 4 nonresponders reported diminution in dysgeusia and dysosmia. Taste bud morphology did not change from the abnormal state in the 1 nonresponder in whom it was measured. CONCLUSIONS: Zinc treatment is effective in patients in whom this trace metal increases synthesis/secretion of gustin/CAVI and ineffective in those in whom it does not. Increased gustin/CAVI in this disorder is probably associated with zinc stimulation of the gene responsible for the synthesis/secretion of gustin/CAVI. Among nonresponders, zinc was ineffective for several possible reasons, including resistance to zinc and possible sialylation of gustin/CAVI, which may render it functionally ineffective. Results suggest the hypothesis that gustin/CAVI is a trophic factor that promotes growth and development of taste buds through its action on taste bud stem cells.

Administration, Oral↗

Specification of pharyngeal endoderm is dependent on early signals from axial mesoderm.

The development of taste buds is an autonomous property of the pharyngeal endoderm, and this inherent capacity is acquired by the time gastrulation is complete. These results are surprising, given the general view that taste bud development is nerve dependent, and occurs at the end of embryogenesis. The pharyngeal endoderm sits at the dorsal lip of the blastopore at the onset of gastrulation, and because this taste bud-bearing endoderm is specified to make taste buds by the end of gastrulation, signals that this tissue encounters during gastrulation might be responsible for its specification. To test this idea, tissue contacts during gastrulation were manipulated systematically in axolotl embryos, and the subsequent ability of the pharyngeal endoderm to generate taste buds was assessed. Disruption of both putative planar and vertical signals from neurectoderm failed to prevent the differentiation of taste buds in endoderm. However, manipulations of contact between presumptive pharyngeal endoderm and axial mesoderm during gastrulation indicate that signals from axial mesoderm (the notochord and prechordal mesoderm) specify the pharyngeal endoderm, conferring upon the endoderm the ability to autonomously differentiate taste buds. These findings further emphasize that despite the late differentiation of taste buds, the tissue-intrinsic mechanisms that generate these chemoreceptive organs are set in motion very early in embryonic development.

Ambystoma↗

Immunohistochemical localization of pleiotrophin and midkine in the lingual epithelium of the adult rat.

Distribution of two heparin-binding molecules, pleiotrophin (PTN) and midkine (MK), was investigated by immunohistochemistry in the lingual epithelium of the adult rat. In the lingual epithelium, both PTN- and MK-like immunoreactivities were observed in its basal cell layers, showing a mesh-like appearance. These molecules were also found along the surface of the taste bud cells; an intense immunoreaction was detected at the base of the taste buds in the circumvallate and foliate papillae. At the electron microscope level, the immunoreactive products were localized on the cell surface and basement membrane at the base of the taste buds. The immunoreactivity for PTN was distributed more diffusely than that for MK. It was suggested that these molecules may be involved in the differentiation and maintenance of taste bud cells, possible via their trophic effect upon approaching nerves.

Animals↗

Evolution of taste and solitary chemoreceptor cell systems.

Vertebrates possess four distinct chemosensory systems distinguishable on the basis of structure, innervation and utilization: olfaction, taste, solitary chemoreceptor cells (SCC) and the common chemical sense (free nerve endings). Of these, taste and the SCC sense rely on secondary receptor cells situated in the epidermis and synapsing on sensory nerve fibers innervating them near their base. The SCC sense occurs in anamniote aquatic craniates, including hagfish, and may be used for feeding or predator avoidance. The sense of taste occurs only in vertebrates and is always utilized for feeding. The SCC system achieves a high degree of specialization in two teleosts: sea robins (Prionotus) and rocklings (Ciliata). In sea robins, SCCs are abundant on the three anterior fin rays of the pectoral fin which are free of fin webbing and are used in active exploration of the substrate. Behavioral and physiological studies show that this SCC system responds to feeding cues and drives feeding behavior. It is connected centrally like a somatosensory system. In contrast, the specialized SCC system of rocklings occurs on the anterior dorsal fin which actively samples the surrounding water. This system responds to mucus substances and may serve as a predator detector. The SCC system in rocklings is connected centrally like a gustatory system. Taste buds contain multiple receptor cell types, including a serotonergic Merkel-like cell. Taste receptor cells respond to nutritionally relevant substances. Due to similarities between SCCs and one type of taste receptor cell, the suggestion is made that taste buds may be compound sensory organs that include some cells related to SCCs and others related to cutaneous Merkel cells. The lack of taste buds in hagfish and their presence in all vertebrates may indicate that the phylogenetic development of taste buds coincided with the elaboration of head structures at the craniate-vertebrate transition.

Animals↗

Ultrastructure of premetamorphic taste organs of the Bombina variegata.

Ultrastructural studies of taste organs in tadpoles of Bombina variegata indicate that these are taste buds (TBs). They occur in the apical parts of papillae of the mouth cavity lining. Each taste bud consists of taste cells and supporting cells; no basal cells were found. In the sensory area of taste bud there are two types of sensory cells: cells with a tuft of short (approximately 1.5 m) microvilli and cells with a single, longer (up to 3.7 microns) process, definitely longer than in TBs of other studied species of Anura. Fairly often, twin TBs were observed.

Animals↗

A histomorphometric study of the effect of chronic vitamin A deficiency on the circumvallate papilla of the rat.

Although a deterioration of taste sensitivity is known to occur during severe vitamin A depletion, the structural changes in the taste buds themselves have not been described. Circumvallate papillae from rats deprived of vitamin A were examined histomorphometrically at intervals, and changes in the gustatory apparatus were quantitated. The cross-sectional area of the circumvallate trenches increases 30-fold because of keratin accumulation when vitamin A deficiency is extreme. After 190 days on a vitamin A-free diet, 38% of rats had no detectable taste buds and the remaining taste buds appeared atrophied. The involution of the taste buds may be the result of keratin buildup in the adjacent trench or a direct consequence of vitamin A deficiency. The atrophy of the gustatory apparatus during vitamin A deficiency and the consequent presumed loss of taste sensation may contribute to a diminished interest in food and the subsequent protein-calorie malnutrition seen in vitamin A-deficient populations.

Animals↗

Keratins as markers of differentiated taste cells of the rat.

Cytokeratins in taste buds were immunocytochemically evaluated with monoclonal antibodies. In each of six different epithelial sites in the rat oral cavity, intragemmal cells of taste buds were immunoreactive for keratin polypeptides 8, 18, and 19, as well as for keratin 7, which has not been previously reported in taste buds. Keratin-18-like immunoreactivity was present in fewer than half of the intragemmal cells, whereas all intragemmal cells were immunopositive for keratins 7, 8, and 19. Apart from some salivary duct cells, no other cells in the tongue were immunoreactive for any of these four keratins. Morphological and immunocytochemical profiles indicate that taste buds are islets of simple epithelium embedded in an expanse of stratified squamous epithelium. These simple epithelial cells and their keratins are nerve-dependent, since denervation eliminated all four keratins and replaced elongated taste cells of the vallate papilla with stratified squamous epithelium. We conclude that antibodies against keratins 7, 8, or 19 are useful markers for intragemmal cells in studies of taste bud development, degeneration, regeneration, turnover and tissue culture.

Animals↗

Functional redundancy and gustatory development in bdnf null mutant mice.

In the mouse nasopalate papilla and in the trenches of the foliate and vallate papillae, taste buds accumulated primarily during the first 2 weeks after birth. Null mutation for brain-derived neurotrophic factor caused extensive death of embryonic taste neurons, with the secondary outcome that most taste buds failed to form. However not all taste neurons died; functional redundancy rescued a variable number. The primary research objective was to identify the likely site of the taste neuron rescue factor that substituted for BDNF. In this quest taste bud abundance served as a useful gauge of taste neuron abundance. The proportion of taste buds that developed was variable and uncorrelated among the nasopalate, vallate, and foliate gustatory papillae within each bdnf null mutant mouse. Thus, in spite of shared IXth nerve innervation, the vallate and foliate papillae independently varied in residual gustatory innervation. This variation rules against the rescue of gustatory neurons by system-wide factors or by factors acting on the IXth ganglion or nerve trunk. Therefore it is likely that surviving BDNF-deprived taste neurons were stochastically rescued by a redundant neurotrophic factor at the level of the local gustatory epithelium. These findings broaden the classic expectation that target tissue supplies only a single neurotrophic factor that can sustain sensory (taste) neurons.

Accessory Nerve↗

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush.

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.

Amiloride↗

Gustatory trophic action of arterial chemosensory neurones in the cat.

The proximal stump of the carotid sinus nerve (c.s.n.) branch of the IXth cranial nerve was surgically cross-anastomosed to the distal stump of the lingual nerve (l.n.) branch of the same cranial nerve to test the ability of regenerating c.s.n. axons to reinnervate and induce taste buds on cat circumvallate papillae. The l.n. branch of the IXth nerve, which normally innervates circumvallate papillae, was directed away from the tongue by suturing it to the distal stump of the c.s.n. Animals with normal and re-anastomosed (cut, then resutured) l.n. served as control preparations. 2-19 months following cross-anastomosis, circumvallate papillae contained 59.8 +/- 9.1% (mean +/- S.E. of mean) of the normal incidence of taste buds, indicating that axons possessing specific gustatory trophic properties had reinnervated the epithelium. Further experimentation was concerned with confirming the identity of the putative foreign axons which reinnervated the taste buds. Radiolabelled axoplasmic transport and autoradiography showed that 89 +/- 4.4% of the taste buds in cross-anastomosed, and 97 +/- 2.1% in normal preparations were innervated by axons from the principal sensory ganglion (petrosal) of the IXth nerve (P greater than 0.05). Whole-nerve recordings in cross-anastomosed preparations demonstrated gustatory activity in the transposed c.s.n., which was similar to responses present in normal and re-anastomosed l.n. preparations. The largest response was evoked by 4 M-NaCl, followed by 1 M-NH4Cl and 0.02 M-quinine HCl. Sucrose (0.5 M) elicited insignificant nerve activity. In normal animals, gustatory or mechanical stimulation did not alter cardiopulmonary function, but in bilaterally cross-anastomosed preparations, gustatory stimuli produced respiratory excitation while mechanical stimulation resulted in reductions in blood pressure and respiratory inhibition. These data suggest that following cross-anastomosis, arterial chemosensory axons innervate taste buds and barosensory axons innervate tongue mechanoreceptors (which normally evoke cardiopulmonary reflexes from the carotid body and carotid sinus, respectively). We conclude that arterial chemosensory neurones share with gustatory chemosensory neurones a trophic function essential for the development and maintenance of taste buds.

Action Potentials↗