Fine structure of the taste bud in guinea pigs. II. Localization of spot 35 protein, a cerebellar Purkinje cell-specific protein, as revealed by electron-microscopic immunocytochemistry

The localization of spot 35 protein, a cerebellar Purkinje cell-specific protein, was studied in guinea pig taste buds by means of electron-microscopic immunocytochemistry. The immunoreactivity was localized in the cytoplasmic matrix of discrete bud cells. The ultrastructural features of the reactive cells indicated that they corresponded to the Type III or gustatory cells making a synaptic contact with the intragemmal nerves. All other cells specified as basal, Type I, and Type II were immunonegative for spot 35 protein. This finding indicates a method for specifically demonstrating the gustatory cells in the guinea pig taste bud and, further, gives new evidence that paraneurons may share neuron-specific substances with neurons.     

Three-dimensional structure of the gustatory cell in the mouse fungiform taste buds: a computer-assisted reconstruction from serial ultrathin sections

Taste buds in fungiform papillae of the mouse were examined with transmission electron microscopy and computer-assisted, three-dimensional reconstructions from serial ultrathin sections. In accord with observation by Murray (1971), four distinct cell types, type I, II, III and basal cells, were identified. Of these, only the type III cell made synaptic contacts with nerve terminals and contained both small, clear vesicles and dense-cored granules. The former vesicles were synaptic-type and accumulated in the cytoplasm just below the synaptic in membrane thickenings. This finding clearly indicates a sensory function for the type III cell. One to three type III cells were identified within a taste bud. The type III cell had at most eight synapses with nerve terminals. One nerve fiber making two synapses with the type III cell was occasionally observed in its terminal region.     

Intermediate filaments in mouse taste bud cells

The intermediate filaments in mouse taste bud cells were studied by immunocytochemistry using antikeratin antibodies, and by conventional electron microscopy. Taste bud cells (types I, II, and III) possessed less densely aggregated bundles of intermediate filaments than the surrounding epithelial cells. Type III cells, however, contained more densely aggregated bundles than type I or II cells. Basal cells in the taste buds showed aggregations of filaments as dense as those seen in the epithelial cells, although their bundles were more slender than those of the epithelial cells. The antibodies to keratins from the bovine muzzle and human stratum corneum stained all types of the taste bud cells as well as the surrounding epithelial cells. PKK2 antibody reacted with the surrounding epithelial cells, but did not react with the taste bud cells. These results show that keratins are present in both taste bud and surrounding epithelial cells, although the keratin subtype differs between those cells. This finding has led us to the supposition that all cell types comprising the taste buds--including type III (receptor) cells--originate from the epithelial cells surrounding the taste buds. It is also suggested that both keratin subtypes and aggregation patterns of intermediate filament bundles change during differentiation from surrounding epithelial cells to taste bud cells, and from basal cells in the taste buds to types I, II, or III cells.     

Response of the taste receptor cell to the umami-substance stimulus. An electron-microscopic study.

The chemosensory gustatory cells in taste buds form chemical synapses with intragemmal neurites. We investigated the ultrastructure of the guinea pig gustatory cells after stimulation with a mixture of monosodium L-glutamate and guanosine 5'-monophosphate. The gustatory cells responded to the stimulus. The dense-cored vesicles localized in the presynaptic regions discharged the contents into the synaptic cleft by means of exocytosis, which resulted in a marked decrease of their population. These findings strongly suggest that the transmitter or transmitters contained in the vesicles are released in response to the taste stimulation at the cell apex, to conduct the excitement of the cell to the nerves.     

Taste cells in the gut and on the tongue. Their common, paraneuronal features

Chemoreception of foodstuff in the gut is performed by endocrine cells dispersed in the gut epithelium. They are bipolar cells extending an apical process to the gut lumen and releasing their messenger substances from the cell base in response to the apical stimuli. The cells share cell-biological features with neurons and are classified as paraneurons. Noteworthy, the gut paraneurons do not seem to be stimulated by monosodium glutamate (MSG) as our dog experiment indicates. Administration of 50 mM MSG into the duodenal loop of anesthetized dogs did not cause changes in the volume and protein output in the pancreatic juice. The gustatory cells in the taste bud show essentially the same structure and function as the gut endocrine cells. A single gustatory cell type (type III) seems to receive different chemical stimuli, whereas different endocrine cell types in the gut react to different stimuli. The gustatory cells possess numerous peptidergic-type granules besides synapse-associated small clear vesicles. The former granules, in the guinea pig and dog, are abundant in the cytoplasm, giving an endocrine-like appearance to the cell. Peptidic signal substances contained in the granules remain to be identified. Comparison of the gustatory cells in the taste bud with the endocrine cells in the gut and with other paraneurons may put into front certain hitherto unexplored structures and functions of the cells.     

Cell lineage and differentiation in taste buds

Mammalian taste buds are maintained through continuous cell renewal so that taste bud cells are constantly generated from progenitor cells throughout life. Taste bud cells are composed of basal cells and elongated cells. Elongated cells are derived from basal cells and contain taste receptor cells (TRC). Morphologically, elongated cells consist of three distinct types of cells: Types I, II and III. In contrast to the remarkable progress in understanding of the molecular basis for taste reception, the mechanisms of taste bud maintenance have remained a major area of inquiry. In this article, we review the expression of regulatory genes in taste buds and their involvement in taste bud cell differentiation. Three major topics include: 1) the Sonic hedgehog (Shh)-expressing cell in the basal cell in taste buds as a transient precursor of elongated cells and as a signal center for the proliferation of progenitor cells; 2) the Mash1-expressing cell as an immature cell state of both Type II and Type III cells and as a mature cell state of Type III cell; and 3) the nerve dependency of gene expression in taste buds. Problems in the application of NCAM for the type III cell marker are also discussed.     

Fine structure of the taste bud in the mud puppy,Necturus maculosus

The taste receptor of Necturus was studied because of its potential usefulness in electrophysiological intracellular recording. The taste bud has about twice the length and twice the diameter of buds in most other vertebrates. The large size can be attributed to both a greater number of cells and a greater size of the individual cells. The cells of Necturus taste buds appear to be homologous to those in other vertebrates. Three distinct cell types were found: dark cells (60%), light cells (30%), and basal cells (10%). Dark cells contained granular endoplasmic reticulum, Golgi bodies, and many clusters of characteristic granules that reacted positively when treated with the periodic acid-silver methenamine technique. They are probably secretory cells. Light cells, found in the center of the taste bud, were virtually filled with agranular endoplasmic reticulum, mitochondria and Golgi bodies. Their morphology is similar to that of chloride cells in fish gills, and they may have an ion transport function important in the gustatory response. Basal cells, located at the periphery in the basal half of the bud, contained characteristic dense-core vesicles about 70–90 mμ in diameter. The lower half of the taste bud contained many intraepithelial nerve processes adjacent to the cells.     

Taste buds and nerve fibers in the rat larynx: an ultrastructural and immunohistochemical study

We investigated the rat laryngeal taste buds and their innervation by electron microscopy and immunohistochemical methods. Taste buds were densely arranged in the surface facing the laryngeal cavity of the epiglottis, the aryepiglottic fold, and the cuneiform process of the arytenoid cartilages. The cells of the buds were classified into types I, II, III, and basal cells, the ultrastucture of which was almost the same as that previously reported in lingual taste buds. The type III cells that had synaptic contacts with nerve fibers were considered to be sensory cells. Immunohistochemical analysis revealed thick calbindin D28k-immunoreactive fibers and thin varicose fibers immunoreactive for calcitonin gene-related peptide or substance P in and around the taste bud. Serotonin-immunoreactive cells were also observed here. The results revealed the innervation pattern of laryngeal taste buds to be the same as that in lingual taste buds. Carbonic anhydrase (CA) is known to catalyze the hydration of CO2 and dehydration of H2CO3, and seems to be essential in CO2 reception. Immunoreactivity for CAI was detected in slender cells and that for CAIII was observed in barrel-like cells in the laryngeal taste buds. The pH-sensitive inward rectifier K+ (Kir) channel in the cell membrane may be involved in CO2 reception as well. CAII-reactive cells were also reactive to Kir4.1, PGP 9.5 and serotonin. Our results indicated that CAII and Kir4.1 are located in type III cells of the laryngeal taste buds, and supported the idea that the buds may be involved in the recognition of CO2.     

Fine structure of taste buds located on the lamb epiglottis

Background: Taste buds located on the aryepiglottal folds and laryngeal surface of the epiglottis are the principal receptors responsible for the initiation of the laryngeal chemoreflex. In contrast to the wealth of information available concerning the ultrastructure of oral taste buds, little comparable data exists for taste buds located at the entrance to the larynx. Therefore, the present study was designed to investigate the fine structure of taste buds located on the lamb epiglottis. Materials: Stained thick and semi-serial thin sections from taste buds located on the lamb epiglottis were examined with light and electron microscopy. Results: Based on morphological criteria, three types of cells could be identified in the taste bud: Type I, Type II, and basal cells. Both Type I and Type II cells extended into the apical taste pore, but there were differences between these two cell types with regard to nuclear profiles, electron density, and the relative density of ribosomes, apical mitochondria, and rough and smooth endoplasmic reticulum. Basal cells did not extend a process into the taste pore. Nerve processes were observed throughout the taste bud. Synapses were observed between both Type I and Type II cells and nerve fibers. These synapses exhibited membrane thickenings and accumulations of clear and dense-cored vesicles of varying proportions in the taste cell cytoplasm adjacent to membrane specializations. Conclusions: The taste buds located on the lamb epiglottis share several structural similarities to taste buds located in the oral cavity and other regions of the pharynx and larynx of many mammalian species. The presence of synapses on both Type I and Type II cells of the lamb epiglottal taste bud suggests that both cell types are involved in laryngeal chemorecption. © 1994 Wiley-Liss, Inc.     

Structure of taste buds in foliate papillae of the rhesus monkey,Macaca mulatta

Taste buds in foliate papillae of the rhesus monkey were examined by electron microscopy. Three distinct cell types were identified. Type I cells were narrow elongated cells containing an oval nucleus, bundles of intermediate filaments, several Golgi bodies, and characteristic apical membrane-bounded dense granules. These cells exhibited morphological variations: some had a moderately dense cytoplasm, perinuclear free ribosomes, and flattened sacs of rough endoplasmic reticulum; others had a more lucent cytoplasm, dilated irregular rough endoplasmic reticulum, lysosome-like dense bodies, and lipid droplets. Type II cells typically contained a spherical, pale nucleus, a prominent nucleolus, supranuclear and infranuclear Golgi bodies, mitochondria with tubular cristae, and one or two centrioles. This cell type, too, showed some variation in the relative amounts of ribosomes and smooth endoplasmic reticulum, which varied inversely with each other. Type III cells were characterized by a clear apical cytoplasm essentially devoid of ribosomes and containing microtubules. In a few type III cells, the peri- and infranuclear regions contained many ribosomes and some rough endoplasmic reticulum. In most Type III cells, there were large numbers of dense and clear vesicles in the peri- and infranuclear regions; some of the vesicles were grouped in synapse-like arrangements with adjacent nerves. The morphological variations exhibited by all three cell types could be accounted for by age differences in each of the cells. This would be consistent with the notion that cell renewal occurs in each of the three cell populations.     

Possible role of serotonin in Merkel-like basal cells of the taste buds of the frog, Rana nigromaculata

Merkel-like basal cells in the taste buds of the frog were examined by fluorescence histochemistry, immunohistochemistry and electron microscopy. There were about 16–20 basal cells arranged in a radial fashion at the base of each taste bud. These cells were strongly immunopositive for serotonin antiserum. They were characterised by the presence of numerous dense-cored granules in the cytoplasm ranging from 80 to 120 nm in diameter, and of microvilli protruding from the cell surface. For 4 mo after sensory denervation by cutting the gustatory nerves, all cell types of the taste bud were well preserved and maintained their fine structure. Even at 4 mo after denervation, the basal cells exhibited a strong immunoreaction with serotonin antiserum. To investigate the function of serotonin in the basal cells in taste bud function, serotonin deficiency was induced by administration of p -chlorophenylalanine (PCPA), an inhibitor of tryptophan hydroxylase, and of p -chloroamphetamine (PCA), a depletor of serotonin. After administration of these agents to normal and denervated frogs for 2 wk, a marked decrease, or complete absence, of immunoreactivity for serotonin was observed in the basal cells. Ultrastructurally, degenerative changes were observed in both types of frog; numerous lysosome-like myelin bodies were found in all cell types of the taste buds. The number of dense-cored granules in the basal cells also was greatly decreased by treatment with these drugs. Serotonin in Merkel-like basal cells appears to have a trophic role in maintenance of the morphological integrity of frog taste bud cells.     

Mash1-expressing cells could differentiate to type III cells in adult mouse taste buds

The gustatory cells in taste buds have been identified as paraneuronal; they possess characteristics of both neuronal and epithelial cells. Like neurons, they form synapses, store and release transmitters, and are capable of generating an action potential. Like epithelial cells, taste cells have a limited life span and are regularly replaced throughout life. However, little is known about the molecular mechanisms that regulate taste cell genesis and differentiation. In the present study, to begin to understand these mechanisms, we investigated the role of Mash1-positive cells in regulating adult taste bud cell differentiation through the loss of Mash1-positive cells using the Cre-loxP system. We found that the cells expressing type III cell markers—aromatic L-amino acid decarboxylase (AADC), carbonic anhydrase 4 (CA4), glutamate decarboxylase 67 (GAD67), neural cell adhesion molecule (NCAM), and synaptosomal-associated protein 25 (SNAP25)—were significantly reduced in the circumvallate taste buds after the administration of tamoxifen. However, gustducin and phospholipase C beta2 (PLC beta2)—markers of type II taste bud cells—were not significantly changed in the circumvallate taste buds after the administration of tamoxifen. These results suggest that Mash1-positive cells could be differentiated to type III cells, not type II cells in the taste buds.     

Fine structure of degeneration and regeneration in denervated rabbit vallate taste buds

Taste buds of rabbit circumvallate papillae were studied with the electron microscope at intervals from six hours to 11 weeks after section of the glossopharyngeal nerve distal to the petrosal ganglion. Nerve endings were first affected, showing degeneration as early as 12 hours and disappearing by 48 hours. Rapid loss of cells and of all buds by ten days followed. Numerous inclusion bodies within type I and type II cells were interpreted as autophagic activity in type II cells and both phagocytic and autophagic activity in type I cells. Type III cells were lost primarily by pyknosis, and phagocytized by type I cells. No clear evidence of dedifferentiation, or extrusion of dead cells, was observed. Regenerated nerves appeared beneath the epithelium at 21 days but new buds first appeared at 25 days, after nerves had penetrated the basement membrane. Intimate contact of nerves with epithelial cells appears to be a precondition for taste bud renewal. Early appearance of cells resembling basal cells (type IV) followed by relatively simultaneous appearance of type I, II and III suggest independent origins for these three types. The data support a humoral hypothesis of trophic action but do not rule out a role for impulse transmission.     

Organization of geniculate and trigeminal ganglion cells innervating single fungiform taste papillae: a study with tetramethylrhodamine dextran amine labeling.

Single gustatory nerve fibers branch and innervate several taste buds. In turn, individual taste buds may receive innervation from numerous gustatory nerve fibers. To evaluate the pattern of sensory innervation of fungiform papilla-bearing taste buds, we used iontophoretic fluorescent injection to retrogradely label the fibers that innervate single taste papillae in the hamster. For each animal, a single taste papilla was injected through the gemmal pore with 3.3% tetramethylrhodamine dextran amine. Fungiform papillae either at the tongue tip (0.5-1.5 mm from the tip) or more posteriorly (1.5-3.0 mm from the tip) were injected. After one to seven days survival, the geniculate and trigeminal ganglia and the tongue were sectioned and examined for labeled cells and fibers, respectively. Analysis of the number and topographic distribution of geniculate cells innervating single taste papillae, shows that: (i) 15 +/- 4 (S.D.) ganglion cells converge to innervate a single fungiform taste bud; (ii) more ganglion cells innervate anterior- (range: 13-35 cells) than posterior-lying buds (range: five to 12 cells), which, in part, may be related to bud volume (microm3); and (iii) ganglion somata innervating a single taste bud are scattered widely within the geniculate ganglion. Analysis of labeled fibers in the tongue demonstrated that two to eight taste buds located within 2 mm of the injected taste bud share collateral innervation with the injected taste bud. Since all buds with labeled fibers were located in close proximity (within a 2-mm radius), widely dispersed geniculate ganglion cells converge to innervate closely spaced fungiform taste buds. Trigeminal ganglion (mandibular division) cells were also labeled in every case and, as with the geniculate ganglion, a dispersed cell body location and collateralization pattern among papillae were observed. This study shows that iontophoresis of tetramethylrhodamine dextran amine, selectively applied to individual peripheral receptor end-organs, effectively locates sensory ganglion cells in two different ganglia that project to these sites. Moreover, the marker demonstrates collateral branches of sensory afferents associated with the labeled fibers and the nearby receptor areas innervated by these collaterals. The labeling of single or clusters of receptor cells, as well as identified sensory afferents, affords future possibilities for combining this technique with immunocytochemistry to establish the relationships of innervation patterns with neurotransmitters and neurotropic substances within identified cells.     

Oral sensory papillae, chemo- and mechano-receptors, in the snake, Elaphe quadrivirgata. A light and electron microscopic study

The oral sensory papillae of the snake (Elaphe quadrivirgata), comprising a compound sensory system located along the tooth rows, were studied by light microscopy, immunohistochemistry for neuron specific enolase and S 100 protein, and scanning and transmission electron microscopy. Each sensory papilla exhibited a single taste bud and free nerve endings in the epithelium, and Meissner-like corpuscles, branched coiled terminals, and lamellated corpuscles in the connective tissue. The taste buds consisted of four types of cells; the type III cells, exclusively synapsing onto intragemmal nerves, were identified as gustatory in function. The gustatory cells included dense-cored and clear vesicles in the cytoplasm. These vesicles were accumulated both in the presynaptic and infranuclear regions, suggesting dual functions: the synaptocrine and paracrine/endocrine release of signal substances. The free nerve endings constantly contained mitochondria and frequent clear vesicles. The Meissner-like corpuscles were located in the uppermost zone of the connective tissue. These corpuscles consisted of nerve fibers and lamellar cells. The nerve fibers, rich in mitochondria, were folded and layered on each other. The branched coiled terminals were localized in the connective tissue along the side wall of the papillae. Nerve fibers, free from a Schwann-cell covering, swelled up to make terminals which accumulated mitochondria and glycogen particles. The lamellated corpuscles were associated with the nerve-fiber bundles in the connective tissue. Consisting of a central nerve axon and lamellar cells encircling it, these corpuscles resembled mammalian Vater-Pacini corpuscles, except that they lacked a capsule. These findings demonstrated that the snake sensory papilla represents one of the most specialized, compound sensory systems among vertebrates, which may play an important role in receiving chemical and mechanical information on prey.     

Taste cell formation does not require gustatory and somatosensory innervation

Dependency of taste buds and taste papillae on innervation has been debated for a long time. Previous research showed neurotrophins, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), play an important role for the establishment of the lingual gustatory and somatosensory innervation. BDNF null mutant mice showed severe deficits in gustatory innervation and loss of taste buds while NT-3 null mutation reduced lingual somatosensory innervation to tongue papillae. These results proved BDNF or NT-3 null mutations affected different sensory modalities (i.e. gustatory and somatosensory, respectively). In this study, we analyzed taste bud development in BDNF × NT-3 double knockout mice to examine the relationship between taste bud development and gustatory/somatosensory innervation. Our results demonstrate that, at the initial stage, before nerve fibers reached the appropriate areas in the papilla, taste bud formation did not require innervation. However, at the synaptogenic stage, after nerve fibers ramified into the apical epithelium, innervation was required and played an essential role in the development of taste buds/papillae.     

Ultrastructure of monoaminergic terminals in the intermediolateral nucleus of the cat spinal cord

Monoaminergic innervation of the intermediolateral nucleus of the cat spinal cord was investigated by fluorescence histochemistry and electron microscopy. Large numbers of monoaminergic terminals were labeled by prior administration of the false neurotransmitter 5-hydroxydopamine (5-OHDA). Ultrastructurally, 5-OHDA-labeled terminals fell into three types. Type I, which made up 55% of the labeled terminals, contained abundant, large and densely labeled vesicles and only a few small and unlabeled vesicles. This type was "bouton de passage". Type II, which made up 40% of the terminals, made asymmetrical synaptic contacts with typical postsynaptic structures. This type contained many small vesicles, some of which were labeled, and a few large dense-core vesicles. Type III, which made up 5% of the terminals, made close contact with presynaptic nerve endings containing abundant small unlabeled clear vesicles. The type III terminals contained many large and densely labeled vesicles and a few small flattened vesicles, most of which were unlabeled.     

Confocal imaging of Merkel-like basal cells in the taste buds of zebrafish

The oropharyngeal cavity in fish supports a range of sensory modalities, including detection of chemical and mechanical stimuli. Taste buds are found throughout this tissue and may participate in both processes. We used confocal microscopy and immunohistochemistry to characterize the morphology of Merkel-like cells and their association with other cell types and nerve fibers of the taste bud in the vertebrate model, the zebrafish. In addition, we document procedures for the observation of these structures in whole-tissue preparations from larvae and adults using zebrafish-specific and monoclonal antibodies. A single microvillus Merkel-like cell was found in each taste bud regardless of age or location. Merkel-like cells were neurosecretory, as indicated by labelling with the styryl dye, FM1-43, and the synaptic vesicle marker, SV2. Merkel-like cells were associated with SV2- and calretinin-positive taste receptor cells, received innervation from discoid aggregations of nerve fibers, and retained serotonin-filled synaptic vesicles oriented within the cytoplasm toward adjacent innervation. Moreover, a ring-like formation of nerve endings was identified with the neuronal marker, zn-12 that circumscribed the taste receptor area, surrounding calretinin-immunoreactive taste cell microvilli, and appeared to associate with the nerve plexus adjacent to Merkel-like cells. We suggest that these nerve fibers are somatosensory, perhaps associated with mechanoreception or the common chemical sense.     

Ultrastructure of palatal taste buds in the perihatching chick

Palatal taste buds of perihatching chicks were examined by electron microscopy. Four intragemmal cell types were characterized. (1) Light: with voluminous, electron-lucent cytoplasm containing scattered free ribosomes, rough and smooth endoplasmic reticulum, plump mitochondria, sparse perinuclear filaments, occasional Golgi bodies, and numerous clear and dense-cored vesicles. Clear vesicles sometimes aggregate in a presynaptic-like configuration apposed to an axonal profile. These cells contained large, spherical, uniformly granular nuclei with one nucleolus. (2) Dark: with dense cytoplasm containing filamentous bundles surrounding the nucleus, occasional clear vesicles, centrioles, rough endoplasmic reticulum, and compact mitochrondria. The apical cytoplasm noticeably lacks dense secretory granules. Irregular to lobulated nuclei are densely granular, and contain scattered clumps of chromatin, adhering especially to the inner leaflet of the nuclear membrane, and at least one nucleolus. Cytoplasmic extensions of dark cells envelop other intragemmal cell types and nerve fibers. Light and dark cells project microvilli into the taste pore. (3) Intermediate: contain gradations of features of light and dark cells. (4) Basal: darker than the other intragemmal cell types and confined to the ventral bud region. Putative afferent synapses in relation to light cells, and axo-axonal contacts are described. While the appearance of axo-axonal contacts may be a transient developmental event, other bud features are consonant with observations in adult chickens and suggest that the peripheral gustatory apparatus is mature at hatching in this precocial avian species.     

Bovine circumvallate taste buds: taste cell structure and immunoreactivity to alpha-gustducin

The taste buds of bovine circumvallate papillae were investigated under light and electron microscopy both by histological and immunohistochemical methods. Taste buds existed in the inner epithelium of the trench of the papillae. Under electron microscopy, two types of taste cells, type I and type II, could be classified according to the existence of dense-cored vesicles and cytoplasmic density. Type I had electron-lucent cytoplasm and possessed many electron-dense cored vesicles in the apical cytoplasm. It was considered that the electron-dense materials of the vesicles were released and constituted the pore substance. This type of cell possessed long and thick apical processes in the taste pore. Type II had denser electron cytoplasm compared with that of type I and possessed many electron-lucent vesicles in the apical cytoplasm. This type of cell possessed microvilli in the taste pore. To know the immunoreactivity to alpha-gustducin in bovine circumvallate taste buds, we used the immunoblotting method and the immunohistochemical method. The alpha-gustducin reaction band at 40 kDa was displayed in the specimen of Western blots. The immunohistochemical property of the antiserum to alpha-gustducin was investigated by using the avidin-biotin complex (ABC) method and the 1.4-nm gold and silver enhancement methods. A subset of taste cells showed the immunoreactivity under light microscopy. The electron microscopic specimens with the 1.4-nm gold and silver enhancement method revealed that only type II cells exhibited the alpha-gustducin immunoreactivity.