哺乳动物舌面味蕾的发育

根据近年来有关大鼠、小鼠味觉发育方面的大量研究,对哺乳动物味蕾(taste buds)发育的情况进行了综述和讨论.哺乳动物舌面上的味蕾分布在菌状乳头(fungiform papillae,FF)、叶状乳头(foliate papillae,FL)、轮廓状乳头(circumvallate papillae,CV)之中,味蕾细胞(taste bud cells)不断地进行着周期性的更新,味蕾的形态、数量和功能随动物随年龄而变化.有关味孔头的研究表明,味乳头(gustatory papillae)在味蕾形成和维持味蕾存在及正常发育方面有着独特的功能.味乳头和味蕾的发育过程与细胞信号分子(signaling molecules)、味觉神经(gustatory nerve fibers)等许多因素有着密切的关系,其中有些作用机理至今尚无定论.     

Expression of gustducin overlaps with that of type III IP3 receptor in taste buds of the rat soft palate

Type III IP3 receptor (IP3R3) is one of the common critical calcium-signaling molecules for sweet, umami, and bitter signal transduction in taste cells, and the total IP3R3-expressing cell population represents all cells mediating these taste modalities in the taste buds. Although gustducin, a taste cell-specific G-protein, is also involved in sweet, umami, and bitter signal transduction, the expression of gustducin is restricted to different subsets of IP3R3-expressing cells by location in the tongue. Based on the expression patterns of gustducin and taste receptors in the tongue, the function of gustducin has been implicated primarily in bitter taste in the circumvallate (CV) papillae and in sweet taste in the fungiform (FF) papillae. However, in the soft palate (SP), the expression pattern of gustducin remains unclear and little is known about its function. In the present paper, the expression patterns of gustducin and IP3R3 in taste buds of the SP and tongue papillae in the rat were examined by double-color whole-mount immunohistochemistry. Gustducin was expressed in almost all (96.7%) IP3R3-expressing cells in taste buds of the SP, whereas gustducin-positive cells were 42.4% and 60.1% of IP3R3-expressing cells in FF and CV, respectively. Our data suggest that gustducin is involved in signal transduction of all the tastes of sweet, umami, and bitter in the SP, in contrast to its limited function in the tongue.     

β-Catenin Signaling Biases Multipotent Lingual Epithelial Progenitors to Differentiate and Acquire Specific Taste Cell Fates

Continuous taste bud cell renewal is essential to maintain taste function in adults; however, the molecular mechanisms that regulate taste cell turnover are unknown. Using inducible Cre-lox technology, we show that activation of β-catenin signaling in multipotent lingual epithelial progenitors outside of taste buds diverts daughter cells from a general epithelial to a taste bud fate. Moreover, while taste buds comprise 3 morphological types, β-catenin activation drives overproduction of primarily glial-like Type I taste cells in both anterior fungiform (FF) and posterior circumvallate (CV) taste buds, with a small increase in Type II receptor cells for sweet, bitter and umami, but does not alter Type III sour detector cells. Beta-catenin activation in post-mitotic taste bud precursors likewise regulates cell differentiation; forced activation of β-catenin in these Shh⁺ cells promotes Type I cell fate in both FF and CV taste buds, but likely does so non-cell autonomously. Our data are consistent with a model where β-catenin signaling levels within lingual epithelial progenitors dictate cell fate prior to or during entry of new cells into taste buds; high signaling induces Type I cells, intermediate levels drive Type II cell differentiation, while low levels may drive differentiation of Type III cells.     

Gα-gustducin is extensively coexpressed with sweet and bitter taste receptors in both the soft palate and fungiform papillae but has a different functional significance

To clarify the regional differences in the expression and functional significance of Gα-gustducin in soft palate (SP) and fungiform (FF) taste buds, we examined the coexpression of Gα-gustducin with taste receptors and the impact of Gα-gustducin knockout (gKO) on neural responses to several sweet and bitter compounds. Sweet responses from both the greater superficial petrosal (GSP) and chorda tympani (CT) nerves in gKO mice were markedly depleted, reflecting overlapping expression of Gα-gustducin and Tas1r2. However, although Gα-gustducin was expressed in 87% and 88% of Tas2rs cells in the SP and FF, respectively, there were no statistically significant differences in the CT responses to quinine-HCl (QHCl) and denatonium (Den) between gKO and wild-type (WT) mice. In contrast, GSP responses to these compounds were markedly reduced in gKO mice with an apparent elevation of thresholds (>10-fold). These results suggest that 1) Gα-gustducin plays a critical role in sweet transduction in both the SP and the FF, 2) other Gα subunits coexpressed with Gα-gustducin in the FF are sufficient for responses to QHCl and Den, and 3) robust GSP responses to QHCl and Den occur in the SP by a Gα-gustducin-dependent mechanism, which is absent in the FF.     

Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chordalingual nerve

In normal rats there is one taste bud on the apical surfaceof each fungiform papilla. These taste buds are innervated bythe chorda tympani proper nerve (CT). According to general consensus,after cutting the nerve the taste buds should disappear. Inthis study, performed on 24 rats divided in six groups, theCT nerve on the left side (singly denervated) and the combinedchorda-lingual (CT-L) nerve on the other side (doubly denervatedwere permanently interrupted. The animals were sacrificed after5, 10, 20, 35,60 and 100 days and their tongues serially sectionedfor light microscope examiation. Some papillae were examinedunder an electron microscope. The papillae were categorizedinto three groups: papillae with a normal looking taste bud,with an abnormal looking taste bud and without a taste bud.The results showed a substantial number of papillae with a normallooking taste bud present at all time intervals in all animals.More specifically, on the singly denervated side the proportionof normal looking taste buds stayed below 10% until day 60,when it increased to 15% and to 23% on day 100. The proportionof abnormal looking taste buds decreased from above 92% by day5 to 49% on day 100. The percentage of fungiform papillae withoutsigns of a taste bud was relatively low on the singly denervatedside at times (1, 5, 16, 29, 34 and 28%). On the doubly denervatedside fewer than than 4% normal looking taste buds were foundthroughout the time period. The proportion of abnormal lookingtaste buds decreased from {small tilde} 96% by day 5 to 35%on day 100. A significantly higher proportion of papillae withno taste bud was observed on this side from day 10 onwards.(1, 29, 32, 52, 60 and 63%). The reasons for the differencein tast bud number between the doubly and singly denervatedsides are unknown, but it is possible that collaterals fromother (non-gustatory) nerves have an ability, although limited,to induce and maintain fungiform taste buds. In other words,the capacity to induce taste bud formation is not limited exclusivelyto gustatory nerves.     

Developmental changing of rat taste responses from chorda tympani nerve

In order to clarify developmental changing of gustatory system, histological and electrophysiological experiments were performed in the rat. Histological examination on the anterior tongue innervated by chorda tympani nerve showed that the ratio of matured taste buds which possess a taste pore were only 9% of all taste buds observed at 1 week of postnatal age, and 81.3% at 3 weeks of age. Recording integrated responses from the chorda tympani nerve reveals that taste buds with a pore at 1 week of age responded to NaCl, HCl, and quinine-HCl as well as in adult rats, which suggests that these relatively young taste buds are matured functionally for these three stimuli. However, the response magnitudes for various sugars at 1 week of age were smaller compared to those in the adult rat, reached to the maximum at 3 weeks of age, then decreased gradually with age. Also, results from the experiment of cross-adaptation among different sugars, effects of pronase-E treatment of the tongue, analysis of correlation between on- and off-responses to sugars, showed that qualitative changes for sugar responses continues after 3 weeks of age. These results suggest that functional changes occur in the gustatory processing of sugars during postnatal development in the rat chorda tympani nerve.     

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     

The effect of β-bungarotoxin, or geniculate ganglion lesion on taste bud development in the chick embryo

Chick taste bud (gemmal) primordia normally appear on embryonic day (E) 16 and incipient immature, spherical-shaped buds at E17. In ovo injection of β-bungarotoxin at E12 resulted in a complete absence of taste buds in lower beak and palatal epithelium at developmental ages E17 and E21. However, putative gemmal primordia (solitary clear cells; small, cell groupings) remained, lying adjacent to salivary gland duct openings as seen in normal chick gemmal development. Oral epithelium was immunonegative to neural cell adhesion molecule (NCAM) suggesting gemmal primordia are nerve-independent. Some NCAM immunoreactivity was evident in autonomic ganglion-like cells and nerve fibers in connective tissue. After unilateral geniculate ganglion/otocyst excision on E2.5, at developmental ages E18 and posthatching day 1, ∼12% of surviving ipsilateral geniculate ganglion cells sustained ∼54% of the unoperated gemmal counts. After E18, proportional stages of differentiation in surviving developing buds probably reflect their degree of innervation, as well as rate of differentiation. Irrespective of the degree of geniculate ganglion damage, the proportion of surviving buds can be sustained at the same differentiated bud stage as on the unoperated side, or may differentiate to a later bud stage, consistent with the thesis that bud maturation, maintenance, and survival are nerve-dependent.     

Changes in the immunoreactivity of substance P and calcitonin gene-related peptide in the laryngeal taste buds of chronically hypoxic rats

The distribution of substance P (SP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers in the taste buds of the epiglottis and aryepiglottic folds was compared between normoxic control and chronically isocapnic hypoxic rats (10% O2 and 3-4% CO2 for 3 months). In the normoxic laryngeal taste buds, SP- and CGRP-immunoreactive fibers were detected within the taste buds, where they appeared as thin processes with many varicosities. Most CGRP fibers showed coexistence with SP, but a few fibers showed the immunoreactivity of CGRP only. The density of intra- and subgemmal SP and CGRP fibers penetrating into the laryngeal taste buds was significantly higher in chronically hypoxic rats than in normoxic control rats. Water intake in the hypoxic rats was significantly lower than in the normoxic rats. These results indicate that the increased density of SP- and CGRP-containing nerve fibers within the laryngeal taste buds is a predominant feature of hypoxic adaptation. The altered peptidergic innervation and reduced water intake support the hypothesis that the laryngeal taste buds are involved in water reception, and that the water reception may be under the control of peptidergic innervation.     

The chemosensory support of feeding behavior in precocial and altricial mammals during ontogeny]

The development of the structure and function of chemosensory apparatus of the tongue of mature and immature mammals was studied by scanning electron microscopy and in behavioral tests. Heterochronic development of receptors structures of dorsal surface of the tongue was established. At birth the chemoreceptors of body and root of the tongue were relatively mature in morphological aspects (the number of taste buds with pore). Taste pores in buds of anterior part of the tongue were found immediately at birth in mature and at the third week of postnatal period in immature animals. Behavioral tests found functional immaturity of chemosensory structures. Immature pups were able to recognize flavoured solutions only on the 7-10 day of age, and preference-aversion reactions of mature pups appeared to the third day of age. The data obtained are discussed in the respect of different ways of adaptation to food contacts with environment of the pups of mature and immature animals.     

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.     

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

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

Neuron/target matching between chorda tympani neurons and taste buds during postnatal rat development

During postnatal development, a relationship is established between the size of individual taste buds and number of innervating neurons. To determine whether rearrangement of neurons that innervate taste buds establishes this relationship, we labeled single taste buds at postnatal day 10 (P10) and again at either P15, P20, or P40 with retrograde fluorescent neuronal tracers. The number of single- and double-labeled geniculate ganglion cells was counted, and the respective taste bud volumes were measured for the three groups of rats. The current study replicates findings from an earlier report demonstrating that the larger the taste bud, the more geniculate ganglion cells that innervate it. This relationship between taste bud size and number of innervating neurons is not apparent until P40, when taste bud size reaches maturity. These findings are extended here by demonstrating that the number of neurons that innervate taste buds at P10, when taste bud size is small and relatively homogeneous, predicts the size that the respective taste bud will become at maturity. Moreover, while there is some neural rearrangement of taste bud innervation from P10 to P40, rearrangement does not impact the relationship between taste bud size and innervating neurons. That is, the neurons that maintain contact with taste buds from P10 through P40 accurately predict the mature taste bud size. Therefore, the size of the mature taste bud is determined by P10 and relates to the number of sensory neurons that innervate it at that age and the number of neurons that maintain contact with it throughout the first 40 days of postnatal development.     

Cytokeratin 14 is expressed in immature cells in rat taste buds

We analyzed the differentiation of taste bud cells, by precisely describing expression profiles of cytokeratins (CKs) 8 and 14 in relation to those of marker molecules including label of 5-bromo-2′-deoxy uridine (BrdU) injected. In rat circumvallate papillae, cell division was observed at the basal layer of the epithelium expressing CK14 and located outside taste buds. The progenitor cells began to migrate toward the apical surface and maintained CK14 expression at 1 day after BrdU injection (day 1). On the other hand, a minor population of newly divided cells was infrequently incorporated into taste buds and also maintained CK14 expression at day 1. In taste buds, the conversion of CK subtypes occurred from CK14 to cytokeratin 8 (CK8) at day 2–3, showing the differentiation from immature cells expressing CK14 into mature or maturing cells expressing CK8. Functionally matured cells such as taste receptor cells expressing inositol triphospate receptor type 3 (IP₃R3) never expressed CK14, suggesting that CK14 would be expressed only in immature cells. On the other hand, a small but distinct population of BrdU-positive cells still showed CK14 immunoreactivity in taste buds even at day 12, which might correspond to the cells that remain undifferentiated for a long period within taste buds.     

Comparative lectin histochemistry on taste buds in foliate, circumvallate and fungiform papillae of the rabbit tongue.

Taste buds (TB) in the foliate, circumvallate and fungiform papillae of the rabbit tongue were examined with lectin histochemistry by means of light (LM) and electron (EM) microscopy. Biotin- and gold-labeled lectins were used for the detection of carbohydrate residues in TB cells and subcutaneous salivary glands. At the LM level, the lectins of soybean (SBA) and peanut (PNA) react with material of the foliate and circumvallate taste pores only after pretreatment of the section with neuraminidase. This indicates that the terminal trisaccharide sequences are as follows: Sialic acid-Gal-GalNAc in O-glycosylated glycoproteins or Sialic acid-Gal-GlcNAc in N-glycosylated glycoproteins. In fungi-form taste buds the lectins of Dolichos biflorus (DBA) and Helix pomatia (HPA), also specific to GalNAc residues, are reactive without preincubation with neuraminidase. Wheat germ agglutinin (WGA), specific to GlcNAc, reacts with TBs of all papillae; and the lectin from Ulex europaeus (UEA I), specific to fucose, binds to individual TB cells. The presence of sialic acid may protect mucus or other glycoproteins in TB cells and inside the taste pore from premature enzymatic degradation. In a post-embedding EM procedure on LR-White-embedded tissue sections, only gold-labeled HPA was found to bind especially on membrane surfaces of the microvilli which protrude into the taste pore; however HPA did not bind to the electron-dense mucus inside the taste pore. The mucus situated in the trough and at the top of the adjacent epithelial cells also is strongly HPA-positive, but is of different origin and composition than that found in the taste pore. These results demonstrate distinct carbohydrate histochemical differences between fungiform and circumvallate/foliate taste buds. The different configuration of galactosyl residues and the occurrence of mannose in circumvallate and foliate TBs leads to the suggestion that the lectin reactivities of TBs are not only due to the presence of mucins, but also to N-linked glycoproteins, possibly with a hormone-like paraneuronal function. A possible relationship to v. Ebner glands in these papillae is discussed.     

The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance

Taste buds on the dorsal tongue surface are continually bathedin saliva rich in epidermal growth factor (EGF). In the followingexperiment, taste bud number and morphology were monitored followingsubmandibular and sublingual salivary gland removal (sialoadenectomy),to determine if EGF plays a role in the maintenance and formationof taste buds. Adult male rats were divided into four groups:sialoadenectomized (SX, n = 4); sialoadenectomized with EGFreplacement (SX + EGF, n = 5); sham-operated (SH, n = 4); andsham-operated with exogenous EGF (SH + EGF, n = 5). After a3 week recovery, SX + EGF and SH + EGF animals were given 50µg/day EGF in their drinking water for 14 days. At day14, saliva was collected, the animals were killed and the presenceof EGF determined by radioligand-binding assay. Tongues wereremoved and histologically examined for the presence and morphologyof taste buds on fungiform and circumvallate papillae, or immunostainedfor the presence of EGF, TGF     

Tachykinins stimulate a subset of mouse taste cells

The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+)-imaging on isolated taste cells, it was observed that SP induces Ca(2+) -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+)-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+)-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+)-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+) responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.     

Developmental time course of peripheral cross‐modal sensory interaction of the trigeminal and gustatory systems

Few sensory modalities appear to engage in cross‐modal interactions within the peripheral nervous system, making the integrated relationship between the peripheral gustatory and trigeminal systems an ideal model for investigating cross‐sensory support. The present study examined taste system anatomy following unilateral transection of the trigeminal lingual nerve (LX) while leaving the gustatory chorda tympani intact. At 10, 25, or 65 days of age, rats underwent LX with outcomes assessed following various survival times. Fungiform papillae were classified by morphological feature using surface analysis. Taste bud volumes were calculated from histological sections of the anterior tongue. Differences in papillae morphology were evident by 2 days post‐transection of P10 rats and by 8 days post in P25 rats. When transected at P65, animals never exhibited statistically significant morphological changes. After LX at P10, fewer taste buds were present on the transected side following 16 and 24 days survival time and remaining taste buds were smaller than on the intact side. In P25 and P65 animals, taste bud volumes were reduced on the denervated side by 8 and 16 days postsurgery, respectively. By 50 days post‐transection, taste buds of P10 animals had not recovered in size; however, all observed changes in papillae morphology and taste buds subsided in P25 and P65 rats. Results indicate that LX impacts taste receptor cells and alters epithelial morphology of fungiform papillae, particularly during early development. These findings highlight dual roles for the lingual nerve in the maintenance of both gustatory and non‐gustatory tissues on the anterior tongue. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 626–641, 2016