Taste bud density in circumvallate and fungiform papillae of the bovine tongue

Taste bud quantitation may provide useful parameters for interspecies comparisons of the gustatory system. The present study is a morphometric analysis of bovine taste papillae. Circumvallate and fungiform papillae from six bovine tongues were serially sectioned and, following staining, analyzed. Circumvallate papillae were found to have a mean volume of 3.66 +/- 2.82 mm3, a mean number of taste buds per papilla of 445 +/- 279, and a mean taste bud density of 155 +/- 112 buds/mm3. Values for lateral fungiform papillae for the same three parameters were 0.384 +/- 0.184 mm3, 13.2 +/- 13.4, and 40.8 +/- 46.6 buds/mm3, respectively. Values for dorsal fungiform papillae were 0.438 +/- 0.246 mm3, 4.39 +/- 4.78, and 14.0 +/- 17.1 buds/mm3, respectively. Circumvallate papillae were found to have a significantly greater volume, number of taste buds per papilla, and taste bud density than either type of fungiform papilla. These data should serve as background for biochemical, endocrinological, or neurological studies involving the bovine tongue.     

Multiple Shh signaling centers participate in fungiform papilla and taste bud formation and maintenance

The adult fungiform taste papilla is a complex of specialized cell types residing in the stratified squamous tongue epithelium. This unique sensory organ includes taste buds, papilla epithelium and lateral walls that extend into underlying connective tissue to surround a core of lamina propria cells. Fungiform papillae must contain long-lived, sustaining or stem cells and short-lived, maintaining or transit amplifying cells that support the papilla and specialized taste buds. Shh signaling has established roles in supporting fungiform induction, development and patterning. However, for a full understanding of how Shh transduced signals act in tongue, papilla and taste bud formation and maintenance, it is necessary to know where and when the Shh ligand and pathway components are positioned. We used immunostaining, in situ hybridization and mouse reporter strains for Shh, Ptch1, Gli1 and Gli2-expression and proliferation markers to identify cells that participate in hedgehog signaling. Whereas there is a progressive restriction in location of Shh ligand-expressing cells, from placode and apical papilla cells to taste bud cells only, a surrounding population of Ptch1 and Gli1 responding cells is maintained in signaling centers throughout papilla and taste bud development and differentiation. The Shh signaling targets are in regions of active cell proliferation. Using genetic-inducible lineage tracing for Gli1-expression, we found that Shh-responding cells contribute not only to maintenance of filiform and fungiform papillae, but also to taste buds. A requirement for normal Shh signaling in fungiform papilla, taste bud and filiform papilla maintenance was shown by Gli2 constitutive activation. We identified proliferation niches where Shh signaling is active and suggest that epithelial and mesenchymal compartments harbor potential stem and/or progenitor cell zones. In all, we report a set of hedgehog signaling centers that regulate development and maintenance of taste organs, the fungiform papilla and taste bud, and surrounding lingual cells. Shh signaling has roles in forming and maintaining fungiform papillae and taste buds, most likely via stage-specific autocrine and/or paracrine mechanisms, and by engaging epithelial/mesenchymal interactions.     

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in pig taste papillae.

Immunoreactivity to neuron-specific enolase (NSE), a specific neuronal marker, and calcitonin gene-related peptide (CGRP) was localized in lingual taste papillae in the pigs. Sequential staining for NSE and CGRP by an elution technique allowed the identification of neuronal subpopulations. NSE-staining revealed a large neuronal network within the subepithelial layer of all taste papillae. NSE-positive fibers then penetrated the epithelium as isolated fibers, primarily in the foliate and circumvallate papillae, or as brush-shaped units formed by a multitude of fibers, especially in the fungiform papillae and in the apical epithelium of the circumvallate papilla. Taste buds of any type of taste papillae were found to express a dense subgemmal/intragemmal NSE-positive neuronal network. CGRP-positive nerve fibers were numerous in the subepithelial layer of all three types of taste papillae. In the foliate and circumvallate papillae, these fibers penetrated the epithelium to form extragemmal and intragemmal fibers, while in the fungiforms, they concentrated almost exclusively in the taste buds as intragemmal nerve fibers. Intragemmal NSE- and CGRP-positive fiber populations were not readily distinguishable by typical neural swellings as previously observed in the rat. The NSE-positive neuronal extragemmal brushes never expressed any CGRP-like immunoreactivity. Even more surprising, fungiform taste buds, whether richly innervated by or devoid of NSE-positive intragemmal fibers, always harboured numerous intragemmal CGRP-positive fibers. Consequently, NSE is not a general neuronal marker in porcine taste papillae. Our observations also suggest that subgemmal/intragemmal NSE-positive fibers are actively involved in synaptogenesis within taste buds. NSE-positive taste bud cells were found in all three types of taste papillae. CGRP-positive taste bud cells were never observed.     

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.     

Expression of Mash1 in basal cells of rat circumvallate taste buds is dependent upon gustatory innervation

Mash1, a mammalian homologue of the Drosophila achaete-scute proneural gene complex, plays an essential role in differentiation of subsets of peripheral neurons. In this study, using RT-PCR and in situ RT-PCR, we investigated if Mash1 gene expression occurs in rat taste buds. Further, we examined dynamics of Mash1 expression in the process of degeneration and regeneration in denervated rat taste buds. In rat tongue epithelium, Mash1 gene expression is confined to circumvallate, foliate, and fungiform papilla epithelia that include taste buds. In taste buds, Mash1-expressing cells are round cells in the basal compartment. In contrast, the mature taste bud cells do not express the Mash1 gene. Denervation and regeneration experiments show that the expression of Mash1 requires gustatory innervation. We conclude that Mash1 is expressed in cells of the taste bud lineage, and that the expression of Mash1 in rat taste buds is dependent upon gustatory innervation.     

NT4/5 mutant mice have deficiency in gustatory papillae and taste bud formation.

Neurotrophins are key determinants for controlling the survival of peripheral neurons during development. Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT4/5) exert their action through a common trkB receptor but independently support gustatory sensory neurons. To assess the role of NT4/5 during development, we examined the postnatal development and maintenance of fungiform taste buds in mice carrying a deletion of NT4/5. The absence of NT4/5 results in embryonic deficits in gustatory innervation and a reduced number of fungiform papillae at birth. No degenerative deficits of fungiform papillae were observed for the first 3 weeks of postnatal development. However, these remaining fungiform papillae were smaller in appearance and many did not contain taste pores. By postnatal day 60, there was 63% decrease in the number of fungiform papillae, and remaining papillae were smaller in size or modified into filiform-like spines. These papillae had either no taste bud or a taste bud with a reduced number of taste cells compared to controls. These findings demonstrate that the NT4/5 gene functions in the maintenance of fungiform gustatory papillae and raises the possibility for an earlier role in development.     

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.     

Keratin 19-like immunoreactivity in receptor cells of mammalian taste buds

Three monoclonal antibodies, 4.62, LPZK and 170.2.14, were usedto evaluate keratin 19-like immunoreactivity in gustatory epithelia.Keratin 19-like immunoreactivity was restricted to the intragemmalcells for all types of mammalian taste buds examined. Thesetaste buds included fungiform, foliate and vallate taste budsin rat, gerbil and rabbit, and nasopalatine, epiglottal andpalatine taste buds in rat. There was no keratin 19-like immunoreactivityin basal cells or in perigemmal cells lateral to the immunoreactivetaste receptor cells. Denervation of the rat vallate papillaeliminated all taste buds, as well as all immunoreactive tastecells. That the immunoreactive material in the taste cells waskeratin 19 was supported by the comparable staining of rat tastebuds with each of three monoclonal antibodies specific for keratin19. Furthermore, as predicted, these antibodies selectivelystained luminal cells of ral bile ducts, bladder, salivary ducts,trachea, ureter and uterus. It was concluded that monoclonalantibodies against keratin 19 can usefully distinguish intragemmaltaste receptor cells from keratinocytes, and from the perigemmaland basal cells of gustatory epithelia. Anti-keratin 19 antibodiesmay serve to identify differentiated taste cells in gustatoryepithelia undergoing taste bud development, renewal, degenerationor regeneration.     

Early dietary sodium restriction disrupts the peripheral anatomical development of the gustatory system

Dietary sodium restriction has profound effects on the development of peripheral taste function and central taste system anatomy. This study examined whether early dietary sodium restriction also affects innervation of taste buds. The number of geniculate ganglion cells that innervate single fungiform taste buds were quantified for the midregion of the tongue in two groups of rats: those fed either a low-sodium diet and those fed a sodium replete diet (control rats) from early prenatal development through adulthood. The same mean number of ganglion cells in developmentally sodium-restricted and control adult rats innervated taste buds on the midregion of the tongue. However, the characteristic relationship of the larger the taste bud, the more neurons that innervate it did not develop in sodium-restricted rats. The failure to form such a relationship in experimental rats was likely due to a substantially smaller mean taste bud volume than controls and probably not to changes in innervation. Further experiments demonstrated that the altered association between number of innervating neurons and taste bud size in restricted rats was reversible. Feeding developmentally sodium-restricted rats a sodium replete diet at adulthood resulted in an increase in taste bud size. Accordingly, the high correlation between taste bud volume and innervation was established in sodium-replete rats. Findings from the current study reveal that early dietary manipulations influence neuron-target interactions; however, the effects of dietary sodium restriction on peripheral gustatory anatomy can be completely restored, even in adult animals.     

Morphometry and cellular dynamics of denervated fungiform taste buds in the hamster

For most species and gustatory papillae denervation resultsin a virtual disappearance of taste buds. This is not the casefor hamster fungiform papillae, which contain taste buds thatsurvive denervation. To characterize these taste buds, in thisstudy, counts and measurements were made of all buds on theanterior 3 mm of the hamster tongue at 36 or 91 days after resectingthe chorda/lingual nerve. Taste bud numbers were, at both timeperiods, unaffected by denervation. However, bud dimensionswere affected with denervated buds 25–30% smaller thancontrol ones. Counts of taste bud cells indicated that decreasesin bud size may result from shrinkage, but not a loss of cells.Tritiated thymidine autoradiography was used to evaluate whetherdenervation influences the mitotic activity or the migratorypattern of bud cells. For every animal, the average number oflabelled cells per bud was slightly lower on the denervatedthan the control side of the tongue. However, when labelledcell positions were evaluated at 0.25, 3 and 6 days after thymidine,the distances from the sides of the bud increased at increasingtimes after injection for both the innervated and the denervatedbuds. Stem cells were located laterally or basally in the bud.Labelled cells that migrated into the centers of the buds werefew and seen only at 6 days post-injection time in both controland experimental buds. The moderate effects of denervation ontaste bud sizes and mitotic activities may indicate a generalizedatrophy. Remarkably intact were taste bud numbers and the migratorypatterns of cells, features of anterior tongue taste buds inthe hamster that are relatively invulnerable to resection ofthe chorda /lingual nerve.     

Stimulus-secretion coupling of glucose-induced insulin release. LI. Divalent cations and ATPase activity in pancreatic islets

Pancreatic islets can be viewed as a fuel-sensor organ. The amount of ATP used by the islet cells for the maintenance of adequate Ca2+ gradients across membranes is not known. An indirect approach to this issue consists in the measurement of Ca-ATPase activity. The kinetics of Ca-ATPase in islet homogenates yielded a Km for ATP close to 0.1 mM and two Km values for Ca2+ close to 0.13 and 4-6 microM, respectively. Within limits, the Ca-ATPase appeared as a distinct entity from Mg-ATPase. Several divalent cations, including Mg2+, inhibited the Ca-ATPase activity. Calmodulin also inhibited, significantly albeit modestly Ca-ATPase. The activity of the enzyme was increased at high pH or in the presence of bicarbonate. The reaction velocity at close-to-physiological concentrations of ATP, Ca2+ and H+ suggests that the consumption of ATP by the Ca-ATPase may account for a major fraction of the overall rate of ATP breakdown in intact islets.     

Zinc localization in taste bud membranes

Zinc was measured by flame aspiration atomic absorption spectrophotometry in homogenates and in enriched fractions and subfractions from bovine taste bud membranes and from surrounding control tissues that contained no taste buds. Zinc was found in significantly higher concentrations in tissues containing taste buds and increased in concentration as biochemical and electron microscopic purity increased. The role of zinc in taste bud membranes could relate to its role in membrane stabilization or to its activity in alkaline phosphatase, a zinc-dependent enzyme whose specific activity increased in taste bud membranes in the same manner as did zinc concentration.     

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.     

Cell-to-Cell Communication in the Taste Bud: ATP and Acetylcholine as Primary Mediators

It was shown that physiological processes in taste buds (peripheral sensory gustatory organs in vertebrates) are realized with the involvement of several signal systems. In these structures, a number of “classical” neurotransmitters, including glutamate, serotonin, GABA, ATP, noradrenaline, and others, as well as receptors to these agents, were identified. The physiological roles of the above systems (separate ones and all as a whole) remain, however, far from final elucidation. We studied purinergic and cholinergic systems in the taste buds. Based on the data obtained in behavioral experiments using knockout animals, which indicated that ATP is an afferent neurotransmitter, we found stimulation-induced secretion of ATP by type-II cells. The release of ATP does not require the entry of external calcium and is mediated by ion channels permeable for ATP. The obtained data allowed us to explain the fact that classical synaptic structures are absent in the type-II cells. The type-I cells coat other elements including type-II cells; they provide formation of compartments in the intercellular space of the taste buds (this limits ATP diffusion). We showed that taste cells of just type I mostly generate calcium signals in response to the action of ATP and acetylcholine. These cell responses are generated with the involvement of metabotropic purine receptors (isoforms P2Y1, P2Y2, and P2Y4) and muscarinic receptors (isoforms M1, M3, and M5), respectively. Functioning of these receptors is combined with a phosphoinositide cascade, mobilization of intracellular Ca²⁺, and subsequent activation of calcium-activated Cl^– channels. It seems probable that purinergic and cholinergic signal systems in type-I cells are elements of negative feedback in the taste buds, which promote the process of adaptation to the action of gustatory stimuli.     

Development of anterior gustatory epithelia in the palate and tongue requires epidermal growth factor receptor.

We characterized the gustatory phenotypes of neonatal mice having null mutations for epidermal growth factor receptor (egfr(-/-)), brain-derived neurotrophic factor (bdnf(-/-)), or both. We counted the number and diameter of fungiform taste buds, the prevalence of poorly differentiated or missing taste cells, and the incidence of ectopic filiform-like spines, each as a function of postnatal age and anterior/posterior location. Egfr(-/-) mice and bdnf(-/-) mice had similar reductions in the total number of taste buds on the anterior portions of the tongue and palate. Nonetheless, there were significant differences in their gustatory phenotypes. EGFR deficiency selectively impaired the development of anterior gustatory epithelia in the mouth. Only bdnf(-/-) mice had numerous taste buds missing from the foliate, vallate, and posterior fungiform papillae. Only egfr(-/-) fungiform taste papillae had robust gustatory innervation, markedly reduced cytokeratin 8 expression in taste cells, and a high incidence of a filiform-like spine. Egfr/bdnf double-null mutant mice had a higher frequency of failed fungiform taste bud differentiation. In bdnf(-/-) mice taste cell development failed because of sparse gustatory innervation. In contrast, in young egfr(-/-) mice the abundance of axons innervating fungiform papillae and the normal numbers of geniculate ganglion neurons implicate gustatory epithelial defects rather than neural defects.