Time course of morphological alterations of fungiform papillae and taste buds following chorda tympani transection in neonatal rats

The time course of structural changes in fungiform papillae was analyzed in rats that received unilateral chorda tympani nerve transection at 10 days of age. Morphological differences between intact and denervated sides of the tongue were first observed at 8 days postsection, with an increase in the number of fungiform papillae that did not have a pore. In addition, the first papilla with a filiform-like appearance was noted on the denervated side at 8 days postsectioning. By 11 days after surgery, the total number of papillae and the number of papillae with a pore were significantly lower on the transected side of the tongue as compared to the intact side. At 50 days postsection, there was an average of 70.5 fungiform papillae on the intact side and a mean of only 20.8 fungiform papillae the denervated side. Of those few remaining papillae on the cut side, an average of 13.5 papillae were categorized as filiform-like, while no filiform-like papillae occurred on the intact side. Significant reduction in taste bud volume was noted at 4 days posttransection and further decrements in taste bud volume were noted at 8 and 30 days postsection. Electron microscopy of the lingual branch of the trigeminal nerve from adult rats that received neonatal chorda tympani transection showed normal numbers of both myelinated and unmyelinated fibers. Thus, in addition to the well-characterized dependence of taste bud maintenance on the chorda tympani nerve, the present study shows an additional role of the chorda tympani nerve in papilla maintenance during early postnatal development.     

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     

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.     

The time course of taste bud regeneration after glossopharyngeal or greater superficial petrosal nerve transection in rats

We previously have published data detailing the time course of taste bud regeneration in the anterior tongue following transection of the chorda tympani (CT) nerve in the rat. This study extends the prior work by determining the time course of taste bud regeneration in the vallate papilla, soft palate and nasoincisor ducts (NID) following transection of either the glossopharyngeal (GL) or greater superficial petrosal (GSP) nerve. Following GL transection in rats (n = 6 per time point), taste buds reappeared in the vallate papilla between 15 and 28 days after surgery, and returned to 80.3% of control levels (n = 12) of taste buds by 70 days postsurgery. The first appearance and the final percentage of the normal complement of regenerated vallate taste buds after GL transection resembled that seen previously in the anterior tongue after CT transection. However, in the latter case, regenerated taste buds reached asymptotic levels by 42 days after surgery, whereas within the time frame of the present study, a clear asymptotic return of vallate taste buds was not observed. In contrast to the posterior (and anterior) tongue, only 25% of the normal complement of palatal taste buds regenerated by 112 days and 224 days after GSP transection (n = 9). The difference in regenerative capacity might relate to the surgical approach used to transect the GSP. These experiments provide useful parametric data for investigators studying the functional consequences of gustatory nerve transection and regeneration.     

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.     

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.     

Epithelial overexpression of BDNF and NT4 produces distinct gustatory axon morphologies that disrupt initial targeting

Most fungiform taste buds fail to become innervated when BDNF or NT4 is overexpressed in the basal layer of tongue epithelium. Here, we examined when and how overexpression of BDNF and NT4 disrupt innervation to fungiform papillae. Overexpression of either factor disrupted chorda tympani innervation patterns either before or during the initial innervation of fungiform papillae. NT4 and BDNF overexpression each disrupted initial innervation by producing different gustatory axon morphologies that emerge at distinct times (E12.5 and E14.5, respectively). Chorda tympani nerve branching was reduced in NT4 overexpressing mice, and neuronal fibers in these mice were fasciculated and remained below the epithelial surface, as if repelled by NT4 overexpression. In contrast, many chorda tympani nerve branches were observed near the epithelial surface in mice overexpressing BDNF, and most were attracted to and invaded non-taste filiform papillae instead of gustatory papillae. These results suggest that BDNF, but not NT4, normally functions as a chemoattractant that allows chorda tympani fibers to distinguish their fungiform papillae targets from non-gustatory epithelium. Since BDNF and NT4 both signal through the p75 and TrkB receptors, trophin-specific activation of different internal signaling pathways must regulate the development of the distinct gustatory axon morphologies in neurotrophin-overexpressing mice.     

Regeneration ability of fungiform papillae and taste-buds in rats

We have earlier shown that the taste-bud-bearing fungiform papillaeform a stable pattern on the tongue of rats. In this study theeffect of removal of the fungiform papillae in rats was investigated.The fungiform papillae on a 10 x 5-mm area on one side of thetongue were removed after mapping of both sides under an operatingmicroscope. Serial sections of five rat tongues within 1 dayof biopsy showed that all but one papilla were gone. After 4,6 and 12 months an average of seven papillae with taste-budswere found in the operated area, compared to 20, 26 and 23 inthe controls. Comparison of tongue maps before and after theseperiods showed that papillae had not migrated from areas outsidethe area of the biopsies. To test the assumption that the extentof biopsy determined the amount of regeneration, only the upperpart of the papillae with their taste buds were removed in 15rats. Complete regeneration of papillae and taste buds was obtainedwithin 14 days. The function of the regenerated taste buds wastested by bilateral recording from the chorda tympani propernerves. No difference in response amplitudes was observed betweenthe sides. When, however, the whole papilla including its basewas removed, neither the papilla nor the taste-bud regenerated.The results show that the ability of the fungiform papilla andthe taste-bud to regenerate after removal of the papilla isrelated to the extent of the biopsy. If the entire papilla includingits base is removed, it will not regenerate. If only the upperpart is removed, complete regeneration of both papilla and itstaste-bud will occur.     

RECEPTIVE FIELDS OF CAT TASTE FIBERS

The receptive fields of 25 cat chorda tympani taste fibers weremapped by the application of taste solutions to individual fungiformpapillae on the tongue. Chemical stimulation of other siteson the tongue was ineffective. The receptive fields were ingeneral small and discrete, but could extend for distances ofmore than 5 mm with one or more unresponsive papillae interposedbetween responsive papillae. Since most fibers branched andinnervated at least 2 fungiform papillae (mean 2.7), it waspossible to stimulate 2 papillae within a fiber's receptivefield independently. The two sets of taste receptors innervatedby a given fiber had similar relative chemical sensitivity.Therefore, branches of a taste fiber are not connected at randomto the various kinds of taste receptor cells.     

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.     

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.     

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

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

Location of taste buds in intact taste papillae by a selective staining method.

Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.     

Location of taste buds in intact taste papillae by a selective staining method

Summary Taste buds were found to stain strongly and selectively in intact papillae with highly acidic dyes such as ponceau S. In intact tongues the taste buds in the fungiform, circumvallate and foliate papillae of the cynomolgus monkey and in the fungiform papillae of the rat as well as the taste discs in the fungiform papillae of the frog could be visualized. This method enables a rapid location and counting of taste buds in taste papillae without preparing histological sections. In cynomolgus tongue material fixed in formalin, the dyes penetrate into the buds. In fresh tongues only the taste pore region of the buds stains, which suggests that in vivo taste buds are impenetrable underneath the pore.     

Peripheral Interactions among Single Papilla Inputs to Gustatory Nerve Fibers

Recordings were made from single fibers of the rat chorda tympani nerve while the peripheral receptor fields were mapped using a stimulator developed to stimulate single fungiform papillae which in the rat contain a solitary taste bud. The results indicate that several fungiform papillae may supply input to a single fiber, and the most sensitive papilla of these provided, on the average, about one-half of the response of that fiber to stimulation of the entire tongue. The magnitude of the response to each concentration of stimulus and the shape of the concentration-response curves differ among papillae innervated by the same fiber. If one of the papillae supplying input to the fiber was stimulated individually with NaCl solution, application of this stimulus to the tongue surface surrounding the isolated papilla resulted in enhancement of the fiber response. If the papilla was stimulated with NaCl and potassium benzoate solution was applied to the surround, a depression of the response occurred. The excitatory input of the cationic stimuli and the depressing influence of the anionic stimuli interacted to determine the resultant steady-state impulse frequency of the single afferent fiber. A hypothetical model involving the summation of generator currents along the unmyelinated terminals of the single afferent neuron is presented as a speculative explanation of the integration of inputs from several receptors innervated by the same single fiber.     

Taste placodes are primary targets of geniculate but not trigeminal sensory axons in mouse developing tongue

Tongue embryonic taste buds begin to differentiate before the onset of gustatory papilla formation in murine. In light of this previous finding, we sought to reexamine the developing sensory innervation as it extends toward the lingual epithelium between E 11.5 and 14.5. Nerve tracings with fluorescent lipophilic dyes followed by confocal microscope examination were used to study the terminal branching of chorda tympani and lingual nerves. At E11.5, we confirmed that the chorda tympani nerve provided for most of the nerve branching in the tongue swellings. At E12.5, we show that the lingual nerve contribution to the overall innervation of the lingual swellings increased to the extent that its ramifications matched those of the chorda tympani nerve. At E13.0, the chorda tympani nerve terminal arborizations appeared more complex than those of the lingual nerve. While the chorda tympani nerve terminal branching appeared close to the lingual epithelium that of the trigeminal nerve remained rather confined to the subepithelial mesenchymal tissue. At E13.5, chorda tympani nerve terminals projected specifically to an ordered set of loci on the tongue dorsum corresponding to the epithelial placodes. In contrast, the lingual nerve terminals remained subepithelial with no branches directed towards the placodes. At E14.5, chorda tympani nerve filopodia first entered the apical epithelium of the developing fungiform papilla. The results suggest that there may be no significant delay between the differentiation of embryonic taste buds and their initial innervation.     

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.     

Scanning Electron Microscopical Studies of Developing Gustatory Papillae in Humans

Development and morphological changes of human gustatory papillaeduring postovulatory weeks 6–15 have been studied usingscanning and transmission electron microscopy. The first papillaof the tongue appears around postovulatory week 6 in its caudalmidline near the foramen caecum. In contrast, the dorsal epitheliumof the anterior part of the tongue shows only small hillock-or papilla-like elevations from week 6 on, which comprise anaggregation of 5–20 epithelial cells. From week 7 on,most prominent fungiform papillae develop near the median sulcusand at the margins of the anterior part of the tongue. At theirtops, the first primitive taste pores are found around week10; these are often covered with processes of adjacent epithelialcells. Most pores, however, develop around weeks 14–15.The maturation of taste buds does not coincide with the appearanceof taste pores, since taste bud cells are not fully differentiatedin the observed period of time. Fungiform papillae are developedbefore filiform papillae, which do not occur within the first15 weeks of gestation. Fungiform papillae tend to grow betweenweeks 8 and 15 of gestation, whereas the size of vallate papillaeseems to be constant during this period. Chem. Senses 22: 601–612,1997.     

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.