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.     

Bar synapses in the end buds of lamprey skin

Summary The end buds of lamprey epidermis have been considered to be similar to taste buds, but the synapses on the receptor cells are of the type with a dense mass surrounded by lucent vesicles, which is not found in gustatory cells. It is suggested that the end buds may belong to the lateralis sensory system and be involved in the light sensitivity of lamprey skin.     

Rat gustatory neurons in the geniculate ganglion express glutamate receptor subunits

Taste receptor cells are innervated by primary gustatory neurons that relay sensory information to the central nervous system. The transmitter(s) at synapses between taste receptor cells and primary afferent fibers is (are) not yet known. By analogy with other sensory organs, glutamate might a transmitter in taste buds. We examined the presence of AMPA and NMDA receptor subunits in rat gustatory primary neurons in the ganglion that innervates the anterior tongue (geniculate ganglion). AMPA and NMDA type subunits were immunohistochemically detected with antibodies against GluR1, GluR2, GluR2/3, GluR4 and NR1 subunits. Gustatory neurons were specifically identified by retrograde tracing with fluorogold from injections made into the anterior portion of the tongue. Most gustatory neurons in the geniculate ganglion were strongly immunoreactive for GluR2/3 (68%), GluR4 (78%) or NR1 (71%). GluR1 was seen in few cells (16%). We further examined if glutamate receptors were present in the peripheral terminals of primary gustatory neurons in taste buds. Many axonal varicosities in fungiform and vallate taste buds were immunoreactive for GluR2/3 but not for NR1. We conclude that gustatory neurons express glutamate receptors and that glutamate receptors of the AMPA type are likely targeted to synapses within taste buds.     

Synaptophysin as a probable component of neurotransmission occurring in taste receptor cells

Taste signal is received in taste buds and transmitted via sensory afferent nerves to the brainstem. Although a signaling pathway involving phospholipase C-β2 has been shown to transduce taste signals of bitterness, sweetness and umami in taste receptor cells (Type II cells), these taste receptor cells appear to be different from the presynaptic cells (Type III cells) containing afferent synapses associated with nerve processes. To elucidate the neurotransmission system in the taste receptor cells expressing phospholipase C-β2, we searched for candidate molecules involved in the neurotransmission, and identified synaptophysin. Synaptophysin was expressed in the taste receptor cells expressing phospholipase C-β2, as well as in the presynaptic cells harboring synaptic structures with taste nerves and containing serotonin. Synaptophysin-immunoreactive signals were not limited to gustducin-positive bitter taste receptor cells, and sweet/umami taste receptor cells were indicated to also express synaptophysin. Expression of synaptophysin was already initiated 6 days after cell division, almost in synchrony with the initiation of phospholipase C-β2 expression. Synaptophysin-containing cells co-expressed vesicular-associated membrane protein 2, a v-SNARE molecule which is important for exocytosis. In addition, majority of the synaptophysin-expressing cells also expressed cholecystokinin, a neuropeptide expressed in taste buds. These results suggest that the taste receptor cells have a neurotransmission system involving synaptophysin, which occurs alternatively or additionally to a recently shown hemichannel system.     

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.     

Behavioral, cellular, and molecular analysis of memory in aplysia I: intermediate-term memory

Serotonin (5HT) induces short-term and long-term synaptic facilitation(STF and LTF, respectively) at sensory neuron to motor neuron(SN-MN) synapses in Aplysia, and these forms of plasticity arethought to contribute to short-term and long-term memory forbehavioral sensitization. Recent evidence in Aplysia has identifieda third phase of synaptic facilitation—intermediate-termfacilitation (ITF)—that is temporally and mechanisticallydistinct from STF and LTF. Here, we review the findings of recentstudies that have examined this unique intermediate-term phaseat molecular, cellular, and behavioral levels. The results indicatethat, at tail SN-MN synapses, multiple forms of ITF can be distinguished;they are induced via distinct mechanisms and use parallel molecularpathways for their expression. Moreover, we have incorporatedthe temporal and molecular features of these different formsof ITF at tail SN-MN synapses into behavioral analyses, andfound that they accurately predict distinct forms of intermediate-termmemory for sensitization of the tail-elicited siphon withdrawalreflex. These findings indicate that different types of experiencesengage distinct molecular pathways in the service of memoryretention over the same time domain.     

Immunohistochemical Localization of Neuropeptides and Neurotransmitters in the Nucleus Solitarius

The nucleus tractus solitarii (NTS), which receives visceralafferent information from the cardiovascular, respiratory, gastrointestinaland taste systems, contains multiple neurotrasmitters and neuropeptidesthroughout its rostral to caudal extent. The neurotransmittersand neuropeptides immunoreactivity is located predominatelyin varicose fibers and small puncta throughout the neuropil.In addition, immunoreactive NTS neurons for a variety of neurotransmittersand neuropeptides are present in subnuclear regions. The neuroactive substances localized immunohistochemically inthe NTS include acetylcholine, the neuropeptides, substanceP, methionine- and leucine-enkephalin, ß-endorphin,cholecystokinin, neurotensin, galanin, calcitonin gene-relatedpeptide, somatostatin, FMRMamide, neuropeptide Y, angiotensinII, vasoactive intestinal polypeptide, vasopressin, oxytocin,thyrotropin-releasing hormone, luteinizing hormone-releasinghormone, atrial natriuretic peptide, the catecholamines, dopamine,norepinephrine, epinephrine, serotonin, histamine and the aminoacids, GABA and glutamate. The pattern of innervation for eachneurotransmitter and neuropeptide is not homogeneously distributedthroughout the NTS. Each substance has a unique pattern withinthe NTS as each subnuclear region contains different immunohistochemicalstaining patterns and densities of fibers. At the ultrastructural level both neurotransmitters and neuropeptidesare present in synaptic terminals that are in contact with differentparts of the neuronal membranes. Typically, the labeled terminalscontain both small, clear vesicles and large, dense core vesicleswith the exception of synaptic terminals containing acetylcholine,GABA and glutamate which do not typically have the large, densecore vesicles. The most frequent post-synaptic target are dendritesand spinous processes. Less frequently, synaptic contacts arepresent on the cell soma. Chem. Senses 21: 367–376, 1996.     

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.     

The effects of beta-bungarotoxin on the morphogenesis of taste papillae and taste buds in the mouse

Although it has been long accepted that innervation by a tastenerve is essential for maintenance of taste buds, it is notclear what role, if any, innervation plays in the morphogenesis oftaste papillae and taste bud development. The following studywas undertaken to determine what effects lack of sensory innervationhave on the development of taste papillae and the formationof taste buds in the mouse. Timed-pregnant female mice (n =3) at gestational day 12 (gd12) were anesthetized and a 1 µlsolution (1 µg/µl) of ß-bungarotoxin (ß-BTX),a neurotoxin that disrupts sensory and motor neuron development,was injected into the amniotic cavity of two embryos per dam.Two shams were injected with PBS. Fetuses were harvested atgd18, 1 day before birth, and four ß-BTX-injected embryos,two shams and two controls were fixed in buffered paraformaldehyde.Serial sections were examined for the presence and morphologyof taste papillae and taste buds. No nerve profiles were observedin ß-BTX-injected tongues. Although circumvallate papillaewere present on ß-BTX tongues, only five fungiform papillaecould be identified. Taste buds were present on a large percentageof fungiform papillae profiles (24% and on circumvallate papillaein sham and control fetuses; in contrast, no taste buds wereassociated with taste papillae in ß-BTX fetuses. Theseresults implicate a significant role for innervation in tastepapillae and taste bud morphogenesis.     

Immunohistochemical distribution of CD44 and some of its isoforms during human taste bud development

 Taste buds are accumulations of elongated bipolar cells situated on lingual papillae. The factors that determine the sites where a taste bud may develop are largely obscure, although it is known that the early invasion of nerve fibers plays one of the key roles in taste bud development and maturation. The conditions under which taste bud primordium cells develop are influenced by the interaction between epithelial cells and extracellular matrix molecules of the mesenchyma, such as hyaluronan. Thus, we investigated immunohistochemically the distribution pattern of the receptor for hyaluronan, CD44s, and its epithelial variant isoforms CD44v6 and CD44v9, in taste buds of human embryonic, fetal, perinatal, and adult tongues. Furthermore, we wanted to determine the temporal and spatial relationships of CD44 to sensory innervation of taste bud primordia. In early gestational stages (weeks 7–9), CD44 and its isoforms are expressed on membranes of apical perigemmal (marginal) cells covering taste bud primordia. It seems that CD44 serves as a marker for marginal cells (perigemmal cells) in early developmental stages. The expression of CD44 follows rather than precedes the invasion of sensory nerve fibers and the development of taste bud primordia (weeks 7–8). In new-born and adult taste bud cells, only the standard molecule, CD44s, is expressed; the variant isoforms, CD44v6 and CD44v9, occur only in the adjacent epithelium. From these results it is likely that marginal cells are of the utmost importance for the development and maturation of taste buds. We presume that CD44 is involved in local binding, reuptake, and degradation of hyaluronan in the early stages of taste bud formation. CD44 probably does not induce the transformation of epithelial cells into taste bud primordial cells. What is more, CD44 may change its function in the course of developmental events. Accepted: 13 January 1998     

Phagocytic Cells in the Taste Buds of Rat Circumvallate Papillae after Denervation

Phagocytic cells in the taste buds of rat circumvallate papillaeafter the sectioning of bilateral glossopharyngeal nerves wereexamined by electron microscopy and immunohistochemistry. Electronmicrographs taken 1 day after denervation revealed that flat-shapedcells were present just beneath the taste buds and that theircellular processes extended toward the debris from the degeneratingtaste buds. At 2–6 days after denervation, long and thinprocesses of the flat cells surrounded the debris and appearedto have taken them up into the cytoplasm as small vesicles.Evidence for phagocytosis by the flat cells was seen up to 9days after denervation and again at 24 and 40 days, in correlationto the degeneration and regeneration of the taste buds. Pre-embeddingimmunohistochemistry using anti-vimentin antibody showed thatflat cells strongly reacted with vimentin. Light microscopicimmunohistochemistry using anti-macrophage antibodies (ED1,ED2) showed that throughout the post-operative days macrophageswere not present underneath or within the taste buds. Most ofthe ED2-immunoreactive resident macrophages were located inthe deep layer of connective tissue, and a few were found inthe nerve bundle. ED1-immunoreactive cells were seen in theduct cells of von Ebner's glands and a few were in the trenchwall of circumvallate papillae; however, they were also immunoreactivefor anti-OX62 antibody, which recognizes dendritic cells. Theresults indicate that the phagocytic cells of the taste budsare fibroblasts, not macrophages. Moreover, resident macrophagesparticipate in phagocytosis of degenerated nerves together withSchwann cells. Chem. Senses. 21: 467–476, 1996.     

The innervation of frog's taste organ “A histochemical study”

Morphological investigations on the fungiform papilla of the frog (Rana pipiens) have shown that this taste organ contains two distinct populations of cells: associate and sensory. Messages received by the sensory cells are believed to be transmitted through the mediation of an adrenergic transmitter. This chemical was shown by fluorescence microscopy and electron histochemistry to be stored in synaptic granular vesicles which accumulate at the membrane of the cytoplasmic processes of the sensory cells in typical chemical synaptic complexes. The sensory cell cytoplasmic processes form the presynaptic component of these complexes whose post synaptic components are the nerve fibres supplying the taste buds. These sensory nerve fibres contain agranular vesticles and are probably cholinergic, since they show positive cholinesterase activity at the light and electron microscopical levels.     

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.     

The pit organs of elasmobranchs: a review

Elasmobranchs have hundreds of tiny sensory organs, called pit organs, scattered over the skin surface. The pit organs were noted in many early studies of the lateral line, but their exact nature has long remained a mystery. Although pit organs were known to be innervated by the lateral line nerves, and light micrographs suggested that they were free neuromasts, speculation that they may be external taste buds or chemoreceptors has persisted until recently. Electron micrographs have now revealed that the pit organs are indeed free neuromasts. Their functional and behavioural role(s), however, are yet to be investigated.     

Ultrastructure of the skin mechanoreceptors of the Chinese giant salamander, Andrias davidianus

The ultrastructure of two kinds of mechanoreceptive organs, pit organs and neuromasts, in the skin of adult giant salamanders (Andrias davidianus) was studied by transmission electron microscopy. Neuromasts and pit organs differ in their types of synapses, the spatial distribution of kinocilia on sensory cells, and in the degree to which sensory cells are separated by processes of the supporting cells; the two organs probably serve complementary functions. The neuromasts in A. davidianus differ from those of other salamanders in the orientation of kinocilia, in the extent of intrusion of supporting cells into the sensory layer, in the degree of thickening of the synaptic membranes, in the distribution of synaptic spheres, and by the absence of a cupula.     

Evolutionary origins of taste buds: phylogenetic analysis of purinergic neurotransmission in epithelial chemosensors

Taste buds are gustatory endorgans which use an uncommon purinergic signalling system to transmit information to afferent gustatory nerve fibres. In mammals, ATP is a crucial neurotransmitter released by the taste cells to activate the afferent nerve fibres. Taste buds in mammals display a characteristic, highly specific ecto-ATPase (NTPDase2) activity, suggesting a role in inactivation of the neurotransmitter. The purpose of this study was to test whether the presence of markers of purinergic signalling characterize taste buds in anamniote vertebrates and to test whether similar purinergic systems are employed by other exteroceptive chemosensory systems. The species examined include several teleosts, elasmobranchs, lampreys and hagfish, the last of which lacks vertebrate-type taste buds. For comparison, Schreiner organs of hagfish and solitary chemosensory cells (SCCs) of teleosts, both of which are epidermal chemosensory end organs, were also examined because they might be evolutionarily related to taste buds. Ecto-ATPase activity was evident in elongate cells in all fish taste buds, including teleosts, elasmobranchs and lampreys. Neither SCCs nor Schreiner organs show specific ecto-ATPase activity, suggesting that purinergic signalling is not crucial in those systems as it is for taste buds. These findings suggest that the taste system did not originate from SCCs but arose independently in early vertebrates.     

Apoptosis in mouse taste buds after denervation

Apoptotic cells in the taste buds of mouse circumvallate papillae after the sectioning of bilateral glossopharyngeal nerves were examined by the method of DNA nick-end labeling (TUNEL), together with standard electron microscopy. The taste buds decreased in number and size 3–11 days after denervation and disappeared at 11 days. The TUNEL method revealed only a few positively stained nuclei in normal taste buds but, in those of mice 1–5 days after denervation, the number of positive nuclei had increased to 3–5 times that of taste buds from normal mice. Electron-microscopic observation after denervation demonstrated taste bud cells containing condensed and fragmentary nuclei in a cytoplasm with increased density. The results show that taste bud cells under normal conditions die by apoptosis at the end of their life span, and that gustatory nerve sectioning causes apoptosis of taste bud cells with taste buds decreasing in number and ultimately disappearing. Received: 20 November 1995 / Accepted: 15 May 1996     

Peptide regulators of peripheral taste function

The peripheral sensory organ of the gustatory system, the taste bud, contains a heterogeneous collection of sensory cells. These taste cells can differ in the stimuli to which they respond and the receptors and other signaling molecules they employ to transduce and encode those stimuli. This molecular diversity extends to the expression of a varied repertoire of bioactive peptides that appear to play important functional roles in signaling taste information between the taste cells and afferent sensory nerves and/or in processing sensory signals within the taste bud itself. Here, we review studies that examine the expression of bioactive peptides in the taste bud and the impact of those peptides on taste functions. Many of these peptides produced in taste buds are known to affect appetite, satiety or metabolism through their actions in the brain, pancreas and other organs, suggesting a functional link between the gustatory system and the neural and endocrine systems that regulate feeding and nutrient utilization.     

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.