Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in pig taste papillae.

Immunoreactivity to neuron-specific enolase (NSE), a specific neuronal marker, and calcitonin gene-related peptide (CGRP) was localized in lingual taste papillae in the pigs. Sequential staining for NSE and CGRP by an elution technique allowed the identification of neuronal subpopulations. NSE-staining revealed a large neuronal network within the subepithelial layer of all taste papillae. NSE-positive fibers then penetrated the epithelium as isolated fibers, primarily in the foliate and circumvallate papillae, or as brush-shaped units formed by a multitude of fibers, especially in the fungiform papillae and in the apical epithelium of the circumvallate papilla. Taste buds of any type of taste papillae were found to express a dense subgemmal/intragemmal NSE-positive neuronal network. CGRP-positive nerve fibers were numerous in the subepithelial layer of all three types of taste papillae. In the foliate and circumvallate papillae, these fibers penetrated the epithelium to form extragemmal and intragemmal fibers, while in the fungiforms, they concentrated almost exclusively in the taste buds as intragemmal nerve fibers. Intragemmal NSE- and CGRP-positive fiber populations were not readily distinguishable by typical neural swellings as previously observed in the rat. The NSE-positive neuronal extragemmal brushes never expressed any CGRP-like immunoreactivity. Even more surprising, fungiform taste buds, whether richly innervated by or devoid of NSE-positive intragemmal fibers, always harboured numerous intragemmal CGRP-positive fibers. Consequently, NSE is not a general neuronal marker in porcine taste papillae. Our observations also suggest that subgemmal/intragemmal NSE-positive fibers are actively involved in synaptogenesis within taste buds. NSE-positive taste bud cells were found in all three types of taste papillae. CGRP-positive taste bud cells were never observed.     

The Formation of Endoderm-Derived Taste Sensory Organs Requires a Pax9-Dependent Expansion of Embryonic Taste Bud Progenitor Cells

In mammals, taste buds develop in different regions of the oral cavity. Small epithelial protrusions form fungiform papillae on the ectoderm-derived dorsum of the tongue and contain one or few taste buds, while taste buds in the soft palate develop without distinct papilla structures. In contrast, the endoderm-derived circumvallate and foliate papillae located at the back of the tongue contain a large number of taste buds. These taste buds cluster in deep epithelial trenches, which are generated by intercalating a period of epithelial growth between initial placode formation and conversion of epithelial cells into sensory cells. How epithelial trench formation is genetically regulated during development is largely unknown. Here we show that Pax9 acts upstream of Pax1 and Sox9 in the expanding taste progenitor field of the mouse circumvallate papilla. While a reduced number of taste buds develop in a growth-retarded circumvallate papilla of Pax1 mutant mice, its development arrests completely in Pax9-deficient mice. In addition, the Pax9 mutant circumvallate papilla trenches lack expression of K8 and Prox1 in the taste bud progenitor cells, and gradually differentiate into an epidermal-like epithelium. We also demonstrate that taste placodes of the soft palate develop through a Pax9-dependent induction. Unexpectedly, Pax9 is dispensable for patterning, morphogenesis and maintenance of taste buds that develop in ectoderm-derived fungiform papillae. Collectively, our data reveal an endoderm-specific developmental program for the formation of taste buds and their associated papilla structures. In this pathway, Pax9 is essential to generate a pool of taste bud progenitors and to maintain their competence towards prosensory cell fate induction.     

Immunohistochemical localization of neuron-specific enolase and calcitonin gene-related peptide in rat taste papillae.

Calcitonin gene-related peptide-like and neuron-specific enolase-like immunoreactivity (CGRP-IR and NSE-IR) were surveyed immunohistochemically in the fungi-form, foliate and circumvallate papillae in rats. A dense CGRP-IR network (subgemmal and extragemmal) in the taste papillae is linked to the presence of taste buds, even though CGRP-IR fibers are rarely present in the taste buds. Three typical fiber populations were detected with these two markers. (a) A population of coarse NSE-IR intragemmal fibers characterized by thick neural swellings, never expressing CGRP-immunoreactivity. (b) A population of thin varicose intragemmal NSE/CGRP-IR fibers. (c) A population of subgemmal and extragemmal NSE-/CGRP-IR fibers that partly penetrated the epithelium. The common distribution of CGRP-IR and NSE-IR fibers at the base of taste buds, their differential distribution and morphology within taste buds, added to their restricted nature (gustatory or somatosensory) suggest that a population of CGRP-IR fibers undergoes a target-induced inhibition of its CGRP phenotype while entering the taste buds. The combined use of NSE and CGRP allowed a better characterization of nerve fibers within and between all three types of taste papillae. NSE was also a very good marker for a subtype of taste bud cells in the foliate and in the circumvallate papillae, but no such cells could be observed in the fungiform papillae.     

Induction of apoptosis by colchicine in taste bud and epithelial cells of the mouse circumvallate papillae

Apoptotic cells in the taste buds and epithelia of mouse circumvallate papillae after colchicine treatment were examined by the methods of in situ DNA nick-end labeling, immunocytochemistry, and electron microscopy. After colchicine treatment, numerous positive cells appeared in the taste buds by DNA nick-end labeling, and some epithelial cells in the basal and suprabasal layers in and around the circumvallate papillae also revealed positive staining. Condensed and fragmented nuclei with a high density were occasionally found in the taste bud cells and in the basal and suprabasal layer epithelial cells by electron-microscopic observation. An immunocytochemical reaction for tubulin revealed weak staining in taste bud cells, because of the depolymerization of microtubules, and a decrease of the microtubules in the taste bud cells was observed by electron microscopy. These results indicate that colchicine treatment of mice induces the apoptosis of taste bud and epithelial cells in the circumvallate papillae and dorsal epithelial cells around the circumvallate papillae.     

Immunohistochemical observation of growth-associated protein 43 (GAP-43) in the developing circumvallate papilla of the rat

The distribution and development of growth-associated protein 43 (GAP-43)-like immunoreactivity (-LI) in the rat circumvallate papilla (CVP) were compared to those of protein gene product 9.5 (PGP 9.5)-LI. In the adult, thick GAP-43-like immunoreactive (-IR) structures gathered densely in the subgemmal region. Some of these further penetrated the apical epithelium and trench wall epithelium. At least two types of GAP-43-IR structures were recognized; taste bud-related and non-gustatory GAP-43-IR neural elements. Immunoelectron microscopy revealed that GAP-43-LI was localized predominantly in the Schwann cells, and a few axons displayed GAP-43-LI in the lamina propria. In the trench epithelium, GAP-43-LI was detected in the cytoplasmic side of the axonal membrane. Some intragemmal GAP-43-IR axons made synaptic-like contacts with taste bud cells. At least four developmental stages were defined on the basis of the changes in distribution of GAP-43-LI. In stage I [embryonic day (E) 16–17] GAP-43-IR structures accumulated at the lamina propria just beneath the newly-formed circumvallate papilla. In stage II (E18–19) GAP-43-IR nerve fibers began to penetrate the apical epithelium. In stage III [E20-postnatal day (P) 0] GAP-43-IR nerve fibers first appeared in the trench wall epithelium. Penetration of GAP-IR nerve fibers occurred in the inner trench wall epithelium first, and then in the outer trench wall epithelium. In stage IV (P1-) the distribution of GAP-43-LI was similar to that observed in the adult; but the density of GAP-43-LI was much higher than in adults. PGP 9.5-LI showed a similar distribution pattern to that of GAP-43-LI, except for round-shaped cells in the apical epithelium at the late embryonic stages, and in taste bud cells and intralingual ganglionic cells which lacked GAP-43-LI. The similarities in distribution patterns of GAP-43-LI and PGP 9.5-LI during the development and mature circumvallate papilla suggest that GAP-43 may be a key neuronal molecule for induction and maintenance of the taste buds.     

Immunohistochemical localization of carbonic anhydrase isozyme II in the gustatory epithelium of the adult rat.

The distribution of carbonic anhydrase isozyme II (CA II)-like immunoreactivity (-LI) in the gustatory epithelium was examined in the adult rat. In the circumvallate and foliate papillae, CA II-LI was observed in the cytoplasm of the spindle-shaped taste bud cells, with weak immunoreaction in the surface of the gustatory epithelium. No neuronal elements displayed CA II-LI in these papillae. There was no apparent difference in the distribution pattern between the anterior and posterior portions of the foliate papillae. In immunoelectron microscopy, immunoreaction products for CA II were diffusely distributed in the entire cytoplasm of the taste bud cells having dense round granules at the periphery of the cells. No taste bud cells displaying CA II-LI were detected in the fungiform papillae, but a few thick nerve fibers displayed CA II-LI. In the taste buds of the palatal epithelium, neither taste bud cells nor neuronal elements exhibited CA II-LI. The present results indicate that CA II was localized in the type I cells designated as supporting cells in the taste buds located in the posterior lingual papillae of the adult animal.     

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 cyclin-dependent kinase inhibitors in taste buds of mouse and hamster

Taste buds are specialized epithelial cell clusters in the oral squamous cell epithelium. Although taste buds have been reported to renew rapidly, the mechanism of cell cycle control in these specialized structures remains unresolved. To clarify the cell cycle status and role of cyclin-dependent kinase inhibitors (CDKI) for cell cycle control in the taste buds, we analyzed cell proliferation activity using bromodeoxyuridine (BrdU) and Ki-67 immunostainings and the expression of the Cip/Kip family of CDKI (p21Cip1, p27Kip1, and p57Kip2) in the circumvallate papillae of mouse and hamster. BrdU-positive cells were detected in the basal layer of the oral epithelium. In the taste buds, Ki-67-positive cells were seen in the basal area, with only a very few positive cells in the taste buds. Both p21Cip1 and p27Kip1 positive cells were seen in the suprabasal layer of the non-gustatory oral epithelium. In the taste buds, stronger p27Kip1 staining was detected than in the non-gustatory epithelium. Western blotting analysis revealed that p27Kip1 was abundant in the mucosal tissues from circumvallate papillae. Thus, our study suggests that the taste bud cells except for basal cells are post-mitotic cells and that the cell cycle arrest associated with taste bud cell differentiation could be regulated predominantly by p27Kip1.     

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.     

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.     

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     

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.     

Chronic hypoxia alters calbindin D-28k immunoreactivity in lingual and laryngeal taste buds in the rat

The distribution and abundance of the calcium binding protein, calbindin D-28k (CB) immunoreactivity in the taste buds of the circumvallate papillae and larynx were compared between normoxic and chronically hypoxic rats (10% O2 for 8 weeks). In the normoxic rats, CB immunoreactivity was observed in some cells and fibers of the intragemmal region of the taste buds in the circumvallate papillae. In contrast, in the subgemmal region of the laryngeal taste buds, fibers but not cells were immunoreactive for CB. In chronically hypoxic rats, CB immunoreactive cells and fibers in the taste buds were decreased in the circumvallate papillae. In the laryngeal taste buds, the density of the subgemmal CB immunoreactive fibers in chronically hypoxic rats was greater than in normoxic rats. It is considered that function of the laryngeal taste buds is different from that of the lingual taste buds, so that laryngeal taste buds may be involved in chemosensation other than taste. The altered density of CB immunoreactive cells and fibers in the lingual and laryngeal taste buds is a predominant feature of hypoxic adaptation, and chronic hypoxic exposure might change the chemical sensitivity of the circumvallate papillae and larynx through the regulation of intracellular Ca2+.     

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.