Taste bud development in the zebrafish, Danio rerio.

Taste buds are chemosensory endorgans consisting of modified epithelial cells. Fish and other vertebrates use their taste bud cells to sample potential food, either selecting or rejecting substances according to their edibility. The adult gustatory system in fish has been studied thoroughly, including regeneration experiments. Taste buds occur in the epithelia of the lips, the mouth cavity, the oropharyngeal cavity, and also in the skin of the barbels, the head, and sometimes even all over the body surface. Despite its importance for feeding, little is known about the ontogeny of the fish taste system. We examined the development of taste buds in the zebrafish on the light microscopical and the scanning and transmission electron microscopical levels. Taste buds develop later than the olfactory organ and the solitary chemosensory cells, two other chemosensory systems in aquatic vertebrates. The first few taste bud primordia are visible within the epithelia of lips and gill arches 3 to 4 days after fertilization, and the first few taste buds with open receptor areas appear on the lips and simultaneously on the gill arches 4-5 days after fertilization, which coincides with the onset of feeding. Taste buds in the mouth cavity, on the head, and on the barbels are formed later in development. As seen in other fish, zebrafish taste buds contain elongate dark and light cells, termed according to their electron density. Dark cells with a cell apex of many short microvilli appear first, followed by the light cells with one large microvillus. In addition, the zebrafish has a third fusiform cell type, which appears last. This cell type is low in electron density and has a brush-like apical ending with several small microvilli. This cell type has not been described previously. Furthermore, in zebrafish, the ontogenetic processes of taste bud formation differ from regenerative processes described in the literature.     

Specificity and distribution of receptor cells in the olfactory mucosa of char (Salmo alpinus L.)

Olfactory receptor activity was studied in the char by two methods: (a) recording of the electro-olfactogram (EOG) with two electrodes simultaneously in the olfactory pit and (b) recordings from the olfactory bulb during olfactory stimulation and progressive removal of lamellae in the olfactory rosette. As stimuli were used methionine representing the amino acids and dilute char bile representing the bile salts. By cross-adaptation studies it was demonstrated that receptors sensitive to each of these two stimuli are functionally independent. The results show further that both types of receptors may be found on all lamellae, but differentially distributed within each lamella. Receptors sensitive to methionine are located closer to the raphe than receptors sensitive to bile. The spatial differentiation persists regardless of stimulus concentration. The results are discussed in relation to the projection and growth of primary nerve fibres into the olfactory bulb, and the existence of receptor cells with microvilli and with cilia.     

Solitary chemosensory cells in mammals?

In fish, solitary chemosensory cells (SCCs) occur in the oropharynx, gills and skin and have often been found in association with taste buds. Among amphibia, a diffuse chemosensory system has been described on the ventral skin of toads, and a structural resemblance of SCCs to taste bud cells has been reported in frogs. Putative solitary chemoreceptors have been described in mammals too, at specific sites in the digestive or respiratory apparatus. In newborn rodents, a specific set of SCCs (composed of elements positive for alpha-gustducin, a marker of chemosensory cells) is associated with the gustatory epithelium. In conclusion, the available data suggest that a SCC system is not restricted to fish but is present in amphibia and mammals as well. At our present level of knowledge, establishing a precise homology between different species is difficult. However, the data from mammals and amphibia fully confirm previous findings in fish, and the use of chemical markers to study the diffuse chemosensory systems of vertebrates seems promising.     

Heterogeneous distribution of taste cells in facial and vagal nerve-innervated taste buds

Input from the three gustatory nerves of vertebrates is used to evaluate the nutritional quality of food. In some species, these cranial nerves are modified to accomplish additional specific functions. For example, the facial nerve innervated taste buds distributed over the body surface of catfish aid food search. Physiological studies indicate that this extra-oral taste pathway is more sensitive to amino acids than either the glossopharyngeal or vagal systems of the oral cavity. The current investigation seeks to determine if differences in taste cell subtypes might contribute to the observed differences in sensitivity. The distributions of five low molecular weight metabolites, L-alanine, L-aspartate, L-glutamate, GABA, taurine and the tripeptide glutathione, were examined in 2118 individual taste cells innervated by either the facial or vagal nerve of the channel catfish, Ictalurus punctatus. The metabolite profiles of these cells were determined immunocytochemically and subjected to a k-means clustering algorithm. Fifteen cell classes with quantitatively different patterns of metabolite co-localization were identified. All but one small class of two cells were found in both facial and vagal nerve-innervated taste buds. Four classes (9% of the total cells) had high, two classes (17%) had intermediate and the remaining nine classes (74%) had low levels of GABA immunoreactivity. While the functional significance of differences in metabolite profile remains to be determined, taste cell classes were not uniformly distributed across vagal and facial nerve innervated taste buds and may provide an anatomical basis for previously reported differences in gustatory sensitivity.     

The gustatory sense in foetal sheep during the last third of gestation

1. Histological studies revealed that presumptive taste buds are present in the foetal sheep tongue at 50 days. By 100 days the taste buds appear morphologically mature.2. To determine if foetal taste buds are functional, electrophysiological recordings were made of activity in the chorda tympani nerve of sheep foetuses. Single and multi-fibre preparations were studied in foetuses aged 100 days to term.3. Responses were recorded to lingual stimulation with salts, acids, glycerol, glycine, sodium saccharin, quinine HCl and amniotic fluid. Responses of the foetal chorda tympani to lingual stimulation with a series of monochloride salts and with increasing concentrations of one salt were similar to responses recorded in lambs and adults.4. The peripheral gustatory system of the foetal sheep is functional for at least the last third of gestation. Foetal taste experiences may influence the formation of adult taste preferences or may aid the foetus in monitoring its environment.     

Are there efferent synapses in fish taste buds?

In fish, nerve fibers of taste buds are organized within the bud's nerve fiber plexus. It is located between the sensory epithelium consisting of light and dark elongated cells and the basal cells. It comprises the basal parts and processes of light and dark cells that intermingle with nerve fibers, which are the dendritic endings of the taste sensory neurons belonging to the cranial nerves VII, IX or X. Most of the synapses at the plexus are afferent; they have synaptic vesicles on the light (or dark) cells side, which is presynaptic. In contrast, the presumed efferent synapses may be rich in synaptic vesicles on the nerve fibers (presynaptic) side, whereas the cells (postsynaptic) side may contain a subsynaptic cistern; a flat compartment of the smooth endoplasmic reticulum. This structure is regarded as a prerequisite of a typical efferent synapse, as occurring in cochlear and vestibular hair cells. In fish taste buds, efferent synapses are rare and were found only in a few species that belong to different taxa. The significance of efferent synapses in fish taste buds is not well understood, because efferent connections between the gustatory nuclei of the medulla with taste buds are not yet proved.     

Artificial rearing of preweanling rats: the effectiveness of direct intragastric feeding

The stimulating effects of amino acids and related compounds on the gustatory receptors were studied in the Japanese minnow, Pseudorasbora parva, by recording electrical responses from the palatine nerve innervating the upper lip and the adjacent palate. All of the 21 amino acids and 6 related compounds elicited responses at a concentration of 10^?3 M. The order of the response magnitude to the 6 most effective of 18 L-amino acids was: proline > lysine-HCl > alanine > arginine-HCl > cysteine > serine. The threshold concentration for proline, the most potent among the amino acids was estimated to range between 10^?11 and 10^?10 M. The relationship between the log response magnitude and the log stimulus concentration for L-proline or L-alanine was linear in a relatively wide concentration range, showing a tendency for the response to be saturated at higher concentrations. The results of this study indicate that the amino acids are the most potent gustatory stimuli in the Japanese minnow among various chemicals so far tested including salts, sugars, quinine-HCl and ribonucleotides.     

Electrophysiological evidence for a chemotopy of biologically relevant odors in the olfactory bulb of the channel catfish

Extracellular electrophysiological recordings from single olfactory bulb (OB) neurons in the channel catfish, Ictalurus punctatus, indicated that the OB is divided into different functional zones, each processing a specific class of biologically relevant odor. Different OB regions responded preferentially at slightly above threshold to either a mixture of 1) bile salts (10(-7) to 10(-5) M Na(+) salts of taurocholic, lithocholic, and taurolithocholic acids), 2) nucleotides [10(-6) to 10(-4) M adenosine-5'-triphosphate (ATP), inosine-5'-monophosphate (IMP), and inosine-5'-triphosphate (ITP)], or 3) amino acids (10(-6) to 10(-4)M L-alanine, L-methionine, L-arginine, and L-glutamate). Excitatory responses to bile salts were observed primarily in a thin, medial strip in both the dorsal (100-450 microm) and ventral (900-1,200 microm) OB. Excitatory responses to nucleotides were obtained primarily from dorsal, caudolateral OB, whereas excitatory responses to amino acids occurred more rostrally in the dorsolateral OB, but continued more medially in the ventral OB. The chemotopy within the channel catfish OB is more comparable to that previously described by optical imaging studies in zebrafish than by field potential studies in salmonids. The present results are consistent with recent studies, suggesting that the specific spatial organization of output neurons in the OB is necessary for the quality coding/decoding of olfactory information.     

Functional anatomy of the peripheral olfactory system of the african lungfish Protopterus annectens owen: Macroscopic,microscopic, and morphometric aspects

An anatomical functional study of the olfactory system of Protopterus annectens along the course of the primary olfactory neuron was made by means of macroscopic and microscopic methods on samples of specimens weighing from 3 to 1.250 gm. The olfactory organ, opening by anterior and posterior nostrils into the oral cavity, may be considered a macrosmatic fish type according to the repartition of receptor areas along parallel grooves and on the basis of its active irrigation mechanism. A morphometric study of Protopterus, using a set of seven specimens weighing from 4 to 350 gm, is compared with two morphometric studies on Silurid fishes and reveals some original features concerning the increase of the surface area of the receptor epithelium. The olfactory organ is meant to function in water; its isolation from the aerial environment is carried out by closing the apertures with edema linked to the hypothyroid and cholinergic crisis during the starvation stage; it represents an adaptation to the life in a special environment. This study does not support the assimilation of the posterior intrabuccal opening to a choana, nor the presence of a functional vomeronasal organ. The olfactory nerve shows a definite degree of organization, but it was not possible to recognize any vomeronasal nerve linked with the former. The histological organization of the bulbar relay is of a primitive type; it was impossible to locate an accessory olfactory bulb corresponding to a functional vomeronasal organ, the presence and the innervation of which by the nervus terminalis is discussed.     

Responses of single facial taste fibers in the sea catfish, Arius felis, to amino acids

1. Taste buds in catfish are found not only within the oropharyngeal cavity, as in mammals, but are also located along the external body surface of the animal from the barbels and lips to the caudal fin. Because these taste buds are innervated by the facial (cranial VII) nerve, the extraoral taste system of catfish is analogous to the mammalian taste system of the anterior two-thirds of the tongue, which contains taste buds innervated by the chorda tympani nerve, and of the soft palate and nasoincisor ducts, which contain taste buds innervated by the greater superficial petrosal nerve. 2. The majority of information concerning the specificity of individual taste fibers in vertebrates has been obtained primarily in mammals to stimuli representing the four basic human taste qualities (i.e., salty, sweet, sour, and bitter). In the present report, we examine the evidence for gustatory fiber types within the stimulus class of amino acids, compounds known to be especially relevant gustatory stimuli for catfish and other teleosts. 3. Action potentials were recorded from 60 individual facial taste neurons obtained from 28 sea catfish (Arius felis). Stimuli were 10(-4) M concentrations of L-alanine, D-alanine, glycine, L-proline, L-histidine, and L-arginine, compounds selected from an original stimulus list of 28 amino acids. Responses were quantified as the number of action potentials evoked at various time intervals from the first 0.5 s up to 10 s of response time. 4. The spontaneous activity of 42 fully characterized neurons was 0.8 +/- 2.1 SD spikes/3 s. The average rate of spike discharge increased 50-fold during stimulation with the most effective amino acid (42 +/- 31 spikes/3 s, mean +/- SD). The majority of the sampled neurons were not narrowly tuned to the amino acid stimulants tested (mean breadth of responsiveness, H = 0.60; range 0-0.95). 5. Hierarchical cluster analysis of the fully characterized neurons identified two large and two small groups of cells. The largest group (n = 22) of neurons was stimulated most by L-alanine and glycine; the other large group (n = 17) was stimulated most by D-alanine. For this latter group, the response to glycine was relatively low, whereas the responses to L-alanine varied from 0 to nearly 100% of the D-alanine response.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solitary chemosensory cells in the developing chemoreceptorial epithelium of the vallate papilla

SummaryThe solitary chemosensory cells are considered typical of aquatic vertebrates and have never been described in the oral cavity of terrestrial vertebrates. We describe elements with ultrastructural characteristics of the solitary chemosensory cell in the gustatory epithelium of rats in the first week of extrauterine life. These elements appeared isolated, located among keratinocytes, and wrapped by glial-like elements. They showed a bipolar shape with a round cell body, a thin apical process, and a thicker basal one. Nerve fibers contacted the cell body and the processes. The cells showed small dense granules mainly located near nerve contacts. Small bundles of tonofilaments were visible in the perinuclear cytoplasm. Similar elements were not found in rats after the first week of extrauterine life. The present study demonstrates in a mammal that the development of taste buds is preceded by the presence of epithelial elements with ultrastructural characteristics of the solitary chemosensory cells described in lower vertebrates. This finding suggests that the oral chemoreception in the suckling rats may be mediated by three different patways (i.e., gustatory system, common chemical sense, and solitary chemosensory cell system).     

Expression of ATP-gated P2X3 receptors in rat gustatory papillae and taste buds

It has recently become evident that ATP and other extracellular nucleotides could play an important role in signal transductions. ATP mediates excitatory signaling by means of P2X receptors. P2X3, one of its subtypes, a membrane ligand-gated ion channel, is strongly expressed in peripheral sensory neurons. The aim of the present study was to examine the distribution of nerve fibers expressing P2X3 receptors in taste buds in the gustatory papillae and soft palate of rats by immunohistochemistry. We found that the fluorescence ATP marker quinacrine stained subsets of taste bud cells. Numerous nerve fibers innervating taste buds were intensely immunostained with the P2X3 receptor antibody. These nerve fibers ascended among intragemmal cells and terminated just below the taste pores. In order to examine whether P2X3 receptors are involved in signal modulation within taste buds, we used fluorescent double stainings to analyze the distribution of P2X3 receptors and their relationship to alpha-gustducin immunopositive taste receptor cells. Many varicose nerve fibers expressing P2X3 receptor-immunoreactivities were entangled with alpha-gustducin-immunopositive taste receptor cells and ended closely below the taste pores. In fungiform papillae, nerve fibers expressing both P2X3 receptors and PGP 9.5 were observed. In contrast, only PGP 9.5 immunoreactive nerve fibers were recognized in filiform papillae. These results suggest that P2X3 receptors might be involved in taste transmission pathways within taste buds. ATP may act as a neurotransmitter, co-transmitter, or neuromodulator at P2X3 receptors to generate activating gustatory nerve fibers.     

A metabotropic glutamate receptor variant functions as a taste receptor

Sensory transduction for many taste stimuli such as sugars, some bitter compounds and amino acids is thought to be mediated via G protein-coupled receptors (GPCRs), although no such receptors that respond to taste stimuli are yet identified. Monosodium L-glutamate (L-MSG), a natural component of many foods, is an important gustatory stimulus believed to signal dietary protein. We describe a GPCR cloned from rat taste buds and functionally expressed in CHO cells. The receptor couples negatively to a cAMP cascade and shows an unusual concentration-response relationship. The similarity of its properties to MSG taste suggests that this receptor is a taste receptor for glutamate.     

Ionic stimulation of the olfactory epithelium in the bullfrog and the carp.

The stimulating effects of mono- and divalent cations and anions were studied in the olfactory epithelia of the bullfrog and the carp. The rhythmic waves induced by these ions were recorded in the olfactory bulb. 1. Many mono- and divalent cations and anions showed stimulating actions in the bullfrog and the carp. 2. Microelectrode studies disclosed that the olfactory receptor cells respond to different ions differently. 3. When many ions were applied with various concentrations, responses appeared with long latencies while the concentration was very low (the "delayed responses"). The responses nearly disappeared at the intermediate concentrations, but then responses with short latencies appeared at the higher concentrations (the "initial responses"). Thus, many ions showed dual responses in the bullfrog, although some exceptional cases were found (choline+, Tl+, La3+, Cd2+). 4. Cd2+ and other heavy metal ions showed depressive actions upon the responses induced by other ions in the olfactory epithelium. 5. Tetrodotoxin of even 10 (-5) g/ml was found ineffective in depressing the rhythmic waves induced by ions. 6. Chemoreceptive activities of the olfactory epithelia of the bullfrog and the carp were compared with the activities of the gustatory receptors. They were also compared with the other chemoreceptors of the fish, namely the palatal organ, external chemoreceptors over the snout region and the lateral-line organ. Chemical senses of the fish were discussed.     

Postnatal development of the vallate papilla and taste buds in rats

The postnatal maturation of the vallate papilla and its taste buds was quantitatively investigated in rats by ligh microscopy. Specifically, we measured postnatal increases in the size of mature vallate taste buds and the vallate papilla, increases in the thickness of the gustatory epidermis, and increases in the number of mature taste buds and taste cells per bud. Mature taste buds, defined as those having a taste pore, are rare at birth but proliferate rapidly during the first postnatal month until an average of 610 mature taste buds has accumulated by 90 days. Throughout this postnatal period, mature taste buds adjust to the developmental thickening of the epidermis by continuously increasing in length. Mature taste buds also increase in width, in part due to a threefold increase from 10 and 45 days in the number of taste cells per bud. From 10 to 21 days there is an average daily net increase of three cells per mature taste bud. The maturational increase in taste buds and cells may contribute to the functional changes in taste nerve responses known to occur over the course of several generations of taste receptor cells. The dimensions of the vallate papilla and the surface area of the gustatory epithelium increase logarithmically with age. Although mature taste buds continue to increase in number until 90 days, both taste bud density (178/mm2) and the number of cells per mature taste bud (70-75 cells) reach ceilings by 45 days. Thus, density-dependent factors appear to control vallate taste bud maturation. The immaturity of lingual taste buds in newborn rats supports the view that odor, rather than taste, is the chemosensory signal that guides suckling in altricial rodents.     

The taste cell-related diffuse chemosensory system

Elements expressing the molecular mechanisms of gustatory transduction have been described in several organs in the digestive and respiratory apparatuses. These taste cell-related elements are isolated cells, which are not grouped in buds, and they have been interpreted as chemoreceptors. Their presence in epithelia of endodermal origin suggests the existence of a diffuse chemosensory system (DCS) sharing common signaling mechanisms with the "classic" taste organs. The elements of this taste cell-related DCS display a site-related morphologic polymorphism, and in the past they have been indicated with various names (e.g., brush, tuft, caveolated, fibrillo-vesicular or solitary chemosensory cells). It may be that the taste cell-related DCS is like an iceberg: the taste buds are probably only the most visible portion, with most of the iceberg more caudally located in the form of solitary chemosensory cells or chemosensory clusters. Comparative anatomical studies in lower vertebrates suggest that this 'submerged' portion may represent the most phylogenetically ancient component of the system, which is probably involved in defensive or digestive mechanisms. In the taste buds, the presence of several cell subtypes and of a wide range of molecular mechanisms permits precise food analysis. The larger, 'submerged' portion of the iceberg is composed of a polymorphic population of isolated elements or cell clusters in which the molecular cascade of cell signaling needs to be explored in detail. The little data we have strongly suggests a close relationship with taste cells. Morphological and biochemical considerations suggest that the DCS is a potential new drug target. Modulation of the respiratory and digestive apparatuses through substances, which act on the molecular receptors of this chemoreceptive system, could be a new frontier in drug discovery.     

Processing of bile salt odor information by single olfactory bulb neurons in the channel catfish

A chemotopic map of biologically relevant odorants (that include amino acids, bile salts, and nucleotides) exists in the olfactory bulb (OB) of channel catfish, Ictalurus punctatus. Neurons processing bile salt odorant information lie medially within this OB map; however, information as to how single neurons process bile salt odorant information is lacking. In the present report, recordings were obtained from 51 OB neurons from 30 channel catfish to determine the excitatory molecular receptive range (EMRR) of bile salt responsive neurons. All recordings were performed in vivo within the medial portions of the OB using extracellular electrophysiological techniques. Excitatory thresholds to bile salts typically ranged between 0.1 and 10 muM. The bile salt specificity of OB neurons were divided into three groups: neurons excited by taurine-conjugated bile salts only (group T), neurons excited by nonconjugated bile salts only (group N), and neurons excited by at least one member of each of the three classes of bile salts tested (group G). In addition to the conjugating group at C24 of the side-chain, OB neurons discriminated bile salts by the molecular features present at three other carbon positions (C3, C7, and C12) along the steroid backbone. These data suggest that OB neurons are selectively excited by combinations of molecular features found on the side-chain and along the steroid nucleus of bile salt molecules.     

The distribution of alkaline phosphatase activity in normal and cross-species regenerated rat and mouse taste buds

Alkaline phosphatase (ALK Pase) activity can be detected histochemically in the taste buds of rats but not mice. Since taste buds develop, regenerate and are maintained under the influence(s) of the sensory nerve it was decided to study cross-species regenerated buds of these two animals to determine whether the nerve also regulated ALK Pase development in taste cells. Grafts of rat sensory ganglion and mouse tongue or mouse ganglion and rat tongue were combined in the anterior chamber of the eyes of immunologically-deficient nude mice and the cross-species buds that developed at 35 days were examined histochemically for ALK Pase. The results revealed that the rat nerve did not cause ALK Pase to appear in any buds found in mouse tongue grafts and that mouse nerve could support buds containing ALK Pase in rat tongue tissue. Because the cross-species regenerated buds were histochemically characteristic of those normally found in rat or mouse tongue, there is no evidence that the foreign nerve altered gene expression for ALK Pase in the target organ, and the action of the nerve on gustatory epithelium appears to be that of activation and maintenance.     

The distribution of alkaline phosphatase activity in normal and cross-species regenerated rat and mouse taste buds.

Alkaline phosphatase (ALK Pase) activity can be detected histochemically in the taste buds of rats but not mice. Since taste buds develop, regenerate and are maintained under the influence(s) of the sensory nerve it was decided to study cross-species regenerated buds of these two animals to determine whether the nerve also regulated ALK Pase development in taste cells. Grafts of rats sensory ganglion and mouse tongue or mouse ganglion and rat tongue were combined in the anterior chamber of the eyes of immunologically-deficient nude mice and the cross-species buds that developed at 35 days were examined histochemically for ALK Pase. The results revealed that the rat nerve did not cause ALK Pase to appear in any buds found in mouse tongue grafts and that mouse nerve could support buds containing ALK Pase in rat tongue tissue. Because the cross-species regenerated buds were histochemically characteristic of those normally found in rat or mouse tongue, there is no evidence that the foreign nerve altered gene expression for ALK Pase in the target organ, and the action of the nerve on gustatory epithelium appears to be that of activation and maintenance.     

Building sensory receptors on the tongue

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds—the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.