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.     

Epithelial overexpression of BDNF or NT4 disrupts targeting of taste neurons that innervate the anterior tongue

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are essential for the survival of geniculate ganglion neurons, which provide the sensory afferents for taste buds of the anterior tongue and palate. To determine how these target-derived growth factors regulate gustatory development, the taste system was examined in transgenic mice that overexpress BDNF (BDNF-OE) or NT4 (NT4-OE) in basal epithelial cells of the tongue. Overexpression of BDNF or NT4 caused a 93 and 140% increase, respectively, in the number of geniculate ganglion neurons. Surprisingly, both transgenic lines had severe reduction in fungiform papillae and taste bud number, primarily in the dorsal midregion and ventral tip of the tongue. No alterations were observed in taste buds of circumvallate or incisal papillae. Fungiform papillae were initially present on tongues of newborn BDNF-OE animals, but many were small, poorly innervated, and lost postnatally. To explain the loss of nerve innervation to fungiform papillae, the facial nerve of developing animals was labeled with the lipophilic tracer DiI. In contrast to control mice, in which taste neurons innervated only fungiform papillae, taste neurons in BDNF-OE and NT4-OE mice innervated few fungiform papillae. Instead, some fibers approached but did not penetrate the epithelium and aberrant innervation to filiform papillae was observed. In addition, some papillae that formed in transgenic mice had two taste buds (instead of one) and were frequently arranged in clusters of two or three papillae. These results indicate that target-derived BDNF and NT4 are not only survival factors for geniculate ganglion neurons, but also have important roles in regulating the development and spatial patterning of fungiform papilla and targeting of taste neurons to these sensory structures.     

Lack of functional and morphological susceptibility of the greater superficial petrosal nerve to developmental dietary sodium restriction

Restriction of dietary sodium during gestation has major effects on taste function and anatomy in the offspring. The chorda tympani nerve of offspring that are maintained on sodium-reduced chow throughout life (NaDep) has reduced neurophysiological responses to sodium and altered morphology of its terminal field in the nucleus of the solitary tract. There are many anatomical and physiological similarities between the chorda tympani nerve that innervates taste buds on the anterior tongue and the greater superficial petrosal nerve (GSP) that innervates taste buds on the palate. To determine if the GSP is similarly susceptible to the effects of dietary sodium restriction, the present study examined neurophysiological responses and the terminal field of the GSP in NaDep and control rats. Neurophysiological responses of the GSP to a variety of sodium and non-sodium stimuli did not differ between NaDep and control rats. Furthermore, the volume and shape of the GSP terminal field in the nucleus of the solitary tract did not differ between the groups. Therefore, despite the high degree of functional and anatomical correspondence between the chorda tympani nerve and the GSP, the GSP does not appear to be susceptible to the effects of lifelong dietary sodium restriction.     

Sensitive periods for the effect of dietary sodium restriction on intact and denervated taste receptor cells

Unilateral chorda tympani nerve (CT) section combined with dietary sodium restriction leads to striking alterations in sodium taste function. The regenerated rat CT exhibits deficits in sodium sensitivity, and surprisingly, there are also functional alterations in the intact, contralateral nerve. The studies presented here describe the functional "sensitive periods" for these aberrations and the number of taste buds present during corresponding stages. The regenerated CT is sensitive to dietary sodium restriction during the first 2 wk after denervation, whereas the intact CT is sensitive to dietary manipulation during the first week postsection. Therefore, distinct mechanisms are responsible for the effects of sodium restriction combined with denervation, because separate sensitive periods exist for the regenerated and intact CT nerves. Identification of mature taste buds with an antibody directed at anti-keratin 19 revealed that there is a loss of ~85% of taste buds on the denervated side of the tongue under control and low-sodium diets within the first week postsection. Thus, sodium restriction does not differentially affect the loss of taste buds following denervation.     

Effect of sensory and sympathetic denervation of the frog tongue on the catecholamine containing cells of the taste buds]

The structure of catecholamine-containing dumb-bell shaped cells of the taste buds was studied by luminescent microscopy in the epithelial layer of the frog's tongue (Rana temporaria). On the unilateral section of the lingual nerve, a maintained adrenergic innervation of vessels and of the epithelium was observed, a decreased number of dumb-bell shaped cells in the taste bud, and their significant enlargement, and increased cathecholamine luminescence. With desympathization, no adrenergic nerves were observed on the vessels and the epithelium of the tongue. The size of the taste buds in desympathized cells of the tongue is sharply decreased and their number is increased. There is a tendency to grouping of the dumbbell shaped cells into 3--4 taste buds in one fungiform papillina. The experiments with sensory and sympathetic denervation of the frog tongue distinctly showed the trophic action of sensory and sympathetic nerves on the taste organ of the frog.     

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.     

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.     

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     

Insulin-Like Growth Factors Are Expressed in the Taste System,but Do Not Maintain Adult Taste Buds

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1r^lox/lox). While K14-Cre::Igf1r^lox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1r^lox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C β2- (PLCβ2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1r^lox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1r^lox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.     

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.     

Effect of polaprezinc on taste disorders in zinc-deficient rats

The effect of polaprezinc, a chelate compound consisting of zinc ion and L-carnosine, on abnormalities of taste sensation induced by feeding a zinc-deficient diet to rats was examined by using the two-bottle preference test (quinine hydrochloride as a bitter taste and sodium chloride as a salty taste). Rats were fed either a zinc-deficient or a zinc-sufficient diet. The zinc-deficient diet increased the preference for both taste solutions, while polaprezinc (at doses of 3 and 10 mg/kg) restored the altered taste preferences. We also evaluated the proliferation of taste bud cells using 5-bromo-2'-deoxyuridine (BrdU). The BrdU incorporation into taste bud cells was significantly reduced in rats fed a zinc-deficient diet compared with rats fed a zinc-sufficient diet (from 50.8% to 45.0%, p<0.05) and this reduction was reversed by polaprezinc at doses of 1, 3, and 10 mg/kg, increasing to 50.2%, 53.5%, and 52.5%, respectively. These findings indicate that zinc deficiency induces the delayed of proliferation of taste bud cells, while polaprezinc improves cell proliferation. In conclusion, polaprezinc had a therapeutic effect in a rat model of abnormal taste sensation. Its mechanism of action was suggested to involve improvement of the decrease in taste bud cell proliferation caused by zinc deficiency.     

Neural crest contribution to lingual mesenchyme, epithelium and developing taste papillae and taste buds

The epithelium of mammalian tongue hosts most of the taste buds that transduce gustatory stimuli into neural signals. In the field of taste biology, taste bud cells have been described as arising from "local epithelium", in distinction from many other receptor organs that are derived from neurogenic ectoderm including neural crest (NC). In fact, contribution of NC to both epithelium and mesenchyme in the developing tongue is not fully understood. In the present study we used two independent, well-characterized mouse lines, Wnt1-Cre and P0-Cre that express Cre recombinase in a NC-specific manner, in combination with two Cre reporter mouse lines, R26R and ZEG, and demonstrate a contribution of NC-derived cells to both tongue mesenchyme and epithelium including taste papillae and taste buds. In tongue mesenchyme, distribution of NC-derived cells is in close association with taste papillae. In tongue epithelium, labeled cells are observed in an initial scattered distribution and progress to a clustered pattern between papillae, and within papillae and early taste buds. This provides evidence for a contribution of NC to lingual epithelium. Together with previous reports for the origin of taste bud cells from local epithelium in postnatal mouse, we propose that NC cells migrate into and reside in the epithelium of the tongue primordium at an early embryonic stage, acquire epithelial cell phenotypes, and undergo cell proliferation and differentiation that is involved in the development of taste papillae and taste buds. Our findings lead to a new concept about derivation of taste bud cells that include a NC origin.     

Functional morphology of the taste buds of Florida manatee,Trichechus manatus latirostris

The Florida manatee, Trichechus manatus latirostris, is a fully aquatic, threatened marine mammal for which increased understanding of their physiology, reproduction, and nutrition supports management decisions. Manatees may use taste to distinguish saltwater gradients, toxin detection, food assessment, and social interactions. This study sought to locate and characterize manatee taste buds comparing location, structure, number, and size to other species. Entire heads from manatees (6 males, 4 females) of various ages were obtained. The muzzle, tissue surrounding the nares, oral cavity, and epiglottis were examined grossly for pits and papillae. Tissues were examined using light and transmission electron microscopy. Within the predominant taste bud location, the tongue root, taste bud number was estimated using samples from four animals. The average number of taste buds within the tongue root was 11,534 (range 2,711–23,237) with sparse taste buds located on the soft palate and epiglottis. The location along the lateral surface of the tongue root and bordered by grooves, through which tastants could be easily transported, has functional significance. Large numbers of taste buds within the tongue root suggest that taste is an important component of manatee sensory systems and behavioral research would clarify this.     

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.     

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.     

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.     

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.