Three-Dimensional Ultrastructure of Taste Bud Cells and Nerve Fibers in the Mouse

Jpn. J. Oral Biol., 27:672-678, 1985.

Three-dimensional ultrastructure of taste bud cells and nerve fibers in the mouse

Yuko Suzuki and Masako Takeda

Department of Oral Anatomy, Higashi-Nippon-Gakuen University, School of Dentistry, Tobetsu, Ishikarigun, Hokkaido 061-02, Japan (Chief: Prof. Masako Takeda)

[Accepted for publication : February 6, 1985;

Key words: three-demensional ultrastructure / taste bud cell / nerve fiber

Abstract: The three-dimensional ultrastructures of the taste bud cells and the nerve fibers in the mouse were investigated by means of electron microscopy using serial ultra-thin sections . Most of the taste bud cells were spindle-shaped and smooth in outline, but a few of type-II cells protruded in cytoplasmic processes from the basal portions. The nerve fibers entering the taste buds through the basal lamina, branched several times, the terminals were formed repeatedly, and then, they narrowed and back to slender, finally ending in the form of a teminal. The number of nerve fibers and terminals which innervate a single taste bud cell differed among the cell types. Each type of cell came in contact with the following per cell , type- I and basal cells with 1-2 nerve fibers, type- II cells with 1-4 nerve fibers, and type-DI (gustatory) cells with 2-4 nerve fibers. The number of nerve terminals in contact with each type of cell was 1-2 per cell in type- I and basal cells, 2-4 type-il and 3-6 in type-III. The nerve fibers which came in contact with type-III cells always formed several (mean number of 8 per cell) afferent synapses. A single nerve fiber innervated 4-14 (mean number of 9) taste bud cells which were composed of different cell types. Seven of nine nerve fibers innervated both type-DI cells and other types of cells . This suggests that most of the nerve fibers in the taste bud not only transmit gustatory stimulus but also have other functions, such as the trophic effect on the taste bud cells .

Introduction addition to type- I and type- II cells which have been distinguished before and probably Although very extensive literature has des- were sustentacular in nature, according to many cribed electron microscopic and histochemical investigators1-3,5). Furthermore, it has been observations pertaining to the mammalian assumed from the observation of degeneration taste buds1-9), there has been only a few data and regeneration in denervated taste bud cells on the detailed relation between the nerves that type- I, II and III cells originated sep- and the taste bud cells. On the basis of ele- arately from the basal cells13). Subsequent ctrophysiological studies, it has been suggest- studies have supported the belief that the taste ed that several taste bud cells are innervated buds consist of these four types of cells14-16). by a single nerve fiber10). Beidler11) has ob- It is not yet known whether each type of served in the rat fungiform papillae that about cell in the taste bud is innervated by different fifty nerve fibers in a cross section entered kinds of nerve fibers, or whether a single within a taste bud and branched several times, multiple branching nerve fiber innervates and that one taste bud cell might be in con- both type-Ill cells and other types of cells. In tact with many nerve branches. addition, there have been no reports describ- Murray et al.12) have reported type-M (gas- ing the three-dimensional reconstructions of tatory or receptor) cells in rabbit foliate papil- entire taste bud cells. lae which have been characterized by both the In the present study, a three-dimensional presence of dense-cored vesicles and afferent analysis by the reconstruction of serial thin synaptic contacts with nerve terminals, in sections for electron microscopy was used to Y. Suzuki et al.: Taste bud cell and nerve fiber 673

acquire accurate data on the relation between A B the nerve fibers and the taste bud cells.

Materials and Methods

Adult dd-mice were fixed by perfusion

through the left ventricle with a phosphate

buffered ice-cold mixture of 2% paraformal-

dehyde and 2% glutalaldehyde. The cir-

cumvallate papillae, the soft palate and the

pharynx were removed, immersed in the same fixative for 1 hr and postfixed in 1% 0504 for

1 hr. The tissues were dehydrated with eth-

anol series and embedded in Epok 812. Sev-

eral sets of serial ultra-thin sections (approx-

imately 100 nm in thickness) were cut . One series consisted of 250 consecutive sections.

They were mounted on one-hole grids with

formvar coats, stained with uranyl acetate and

lead citrate and observed by electron micro-

scopy. The electron micrographs of a final

magnification of •~5000 were used to recons-

truct the cells and the nerves. The outlines

of nerves and the cells were traced on trans- Fig. 1 Three-dimensional reconstructions of

parent sheets of paper and superimposed to the type- I cells which are in cont- make a three-dimensional model. Some fig- act with the nerve fibers (N). A is

ures were transferred to the cardboard papers, located in periphery of the taste bud while B is in central region of the cut out and superimposed. taste bud. The nerve terminals are indicated by the arrows. BL: basal Results lamina.

A total of 27 cells and 9 nerve fibers in

several taste buds were reconstructed. The were localized in the basal part of the taste

three-dimensional shapes of type- I, type- II bud. Most of the nerve fibers branched sev-

and type-III cells were spindle, extending eral times within the taste bud, repeated en.-

from the basal lamina to the taste pore sur- largement and narrowing and terminated to

face (Figs. 1, 2, 3 and 4). The outlines of form a swollen terminal. The nerve ter-

the cells were relatively smooth, but there minals were classified as two types :- the broad

were small irregular cytoplasmic processes terminals with extensive contacts to the cell

like interdigitations which were not reconst- cytoplasm, and the small bulbous terminals

ructed by the use of magnification of X 5000. which penetrated the cytoplasm. Both termi-

Three of eight type- II cells protruded large nals were 1.5-3ƒÊm in diameter and contained mitochondria, dense-cored vesicles (100 nm in cytoplasmic processes from the basal part of

the cytoplasm (Fig. 3). The basal cells were diameter) and small clear vesicles (40-60 nm

oval-shaped and did not extend to the taste in diameter). In the serial sections, agg- regations of vesicles were observed at some pores (Fig. 5). The nerve fibers entering through the places in a nerve terminal. However no thic- kening of the membrane of nerves were found basal lamina into the taste bud branched, there (Fig. 6). formed swollen terminals and again narrowed

to slender fibers. The course of nerve fibers Innervation of a single taste bud cell was varied; some fibers extended upward and Type- I and type- II cells received innerva- terminated near the taste pore, while others tion of similar pattern in nerve branching. 674 Jpn. J. Oral Biol., 27:672-678, 1985

A B

Fig. 2 Three-dimensional reconstructions of the type- II cells which are in contact with the nerve fibers (N). A is loca- Fig. 3 Three-dimensional reconstruction of ted in periphery of the taste bud a type- II cell with a cytoplasmic while B is in the central region. process extending toward the basal The nerve terminals are indicated lamina. N: nerve fiber, BL: basal by the arrows. BL: basal lamina. lamina. The nerve terminals are indicated by the arrows. A nerve fiber entering through the basal lamina branched at the basal or middle por- nerve fibers extended to the upper portions of tion of the taste bud and came very close to type-Ill cells and formed larger terminals than the cells to form one or two terminals and those of type- I and type- II cells (Fig. 4B). extended farther toward other taste bud cells. The basal cells received simple innervation in However, those two types of cells made no which one or two nerve fibers terminated to synaptic contacts with the nerves. During form small bulbous endings with the cyto- the course of the nerve fiber, the cells were plasm (Fig. 5). partly surrounded by the nerve fibers (Figs. 1B The number of nerve fibers and terminals and 2B). At the periphery of the taste bud, which were in contact with each type of cell the nerve branchings occurred before the is summarized in Fig. 7. Each type of cell contacts with the cells and the nerve fiber was innervated by the following per cell: terminated to form one or two terminals with -type- I and basal cells by 1-2 nerve fibers the cells (Figs. 1A and 2A). The basal (mean number of 1.2), type- II cells by 1-4 cytoplasmic processes of the type- ft cells also nerve fibers (mean number of 2.3), and type- made the contacts with nerve terminals (Fig. In cells by 2-4 nerve fibers (mean number of 3). Afferent synaptic contacts between the 2.6, the largest number of the four types of nerve terminals and type-III cells were mainly cells). The mean number of nerve fibers, localized in the basal portions of the taste buds which innervated four types of cells, was 2 (Fig. 4A). Also in the basal portion, the per cell. The number of nerve terminals in nerve fibers and terminals were found to form contact with each type of cell was 1-2 per cell a plexus in touch with the underlying basal in type- I and basal cells, 2-4 in type- II and lamina (Fig. 4A, inset). In a few cases, the 3-6 in type-III. Each nerve fiber which made Y. Suzuki et al.: Taste bud cell and nerve fiber 675

A B

C D

Fig. 6 Four serial sections with 0.3ƒÊm interval of a nerve terminal showing the large Fig. 4 Three-dimensional reconstructions of extensive contact with a type- II cell. the type-DI cells which are in cont- The increase of small clear and large act with the nerve fibers (N). Both cored vesicles is seen in C. Subsurfaced A and B cells are located in the cisterns of smooth endoplasmic reticulum central region of the taste buds obta- are observed in the cytoplasm along ined from the different specimens. the nerve terminal. This specimen is

The inset figure represents the base obtained from Fig. 2 A. •~ 10,000. of A which is viewed from the basal lamina (BL). The nerve terminals are indicated by the arrows.

Fig. 5 Three-dimensional reconstructions of basal cells which are in contact with the nerve fibers (N). The nerve terminals are indicated by the arrows. BL: basal lamina. contacts with type-III cells, was sure to have Fig. 7 The number of the nerve fibers (upper several afferent synapses, and a maximum of graph) and the nerve terminals (lower 7 synapses was counted in one terminal, and graph), which come in contact with each the maximum of 15 synapses in one type-III type of cells (Type I , II , III and Basal cell (Table 1). cells) per cell in the taste bud. 676 Jpn. J. Oral Biol., 27:672-678, 1985

Table 2 The number of different four types of cells (Type- I, II, III and basal cells) innervated by a single nerve fiber

and made two afferent synaptic contacts with a type-III (3) cell. The fiber divided into two branches ; one soon terminated to form a bulbous ending in the cytoplasm of type- I (la) cell, and the other branch extended up- Fig. 8 Three-dimensional reconstruction of the ward, branched further three times and ter- course of a single nerve fiber (N) in the taste bud which comes in contact with minated to form the broad endings which the different types of cells. la, lb: type- partly surrounded the type- II (2a, 2b) and I cells. 2a, 2b: type-II cells. 3: type- type- I (1b) cells. On the other hand, only III cell. BL: basal lamina. The arrows two nerve fibers (No. 6 and 8 in Table 2) had indicate the afferent synaptic sites. no relation to type-III cells, but made contact with type-I, type- II or basal cells. Innervation of each type of cells by a single The number of taste bud cells innervated by nerve fiber a single nerve fiber ranged from 4 to 14 The types and the numbers of the taste bud (Table 2). A single fiber supplied many cells innervated by a single nerve fiber are branches to different types of cells. Also a shown in Fig. 8 and Table 2. Seven of nine single fiber tended to innervate a greater nerve fibers (No. 1, 2, 3, 4, 5, 7 and 9 in Table number of type- I cells than other cell types, 2) formed a contact with both type-III cells and because type- I cells showed the largest num- other types of cells. Figure 8 indicates a part ber in the taste bud cells. The number of of the course of No. 5 nerve fiber, which en- type-M cells innervated by a single nerve tered through the basal part of the taste bud fiber was varied ; the maximum was seen in No. 1 fiber which innervated 5 type-III cells, Table 1 The number of afferent synapses per while No. 2, 5, 9 fibers innervated only one nerve fiber which innervates the type-III cells type-III cell. In addition, it was never seen that a single nerve fiber innervated only particular cell types such as type-III (gustatory) cells.

Discussion

Most of the taste bud cells showed smooth outlines in the three-dimensional reconstructions and only a few of type- II cells had cytoplasmic processes extending from the Y. Suzuki et al.: Taste bud cell and nerve fiber 677 basal portions of the cell, although their cell types, and afferent synaptic contacts were function has not been clarified. Type- II and always observed between the type-III cells and type-Ill cells received relatively numerous the nerve terminals. This suggests that most nerve supply and the basal processes of type- of the nerve fibers in the taste buds transmit II cells also made contact with nerve termi- gustatory stimulus and simultaneously secrete nals. This may be related to the dependency a trophic factor. In addition, the present of the taste bud cells on the nerves, since study also demonstrated that a few nerve type- II and type-Ill cells disappeared more fibers did not form the synaptic contacts with rapidly than type- I cells in the denervated type-III cells. Electrophysiological data has taste buds17). indicated that the nerve fibers branched before Our data showed that the average number their penetration of the basal part of the taste of nerve fibers, which innervated a single bud20). Therefore, it is quite possible that taste bud cell was 2 ranging from 1 to 4. these fibers innervate type-III cells in the Beidler11) reported that an average of 9 nerve other adjacent taste buds. fibers with a range of 0 to 22 innervated a Recent immunohistological study have de- single taste bud cell in the rat fungiform monstrated the substance-P activity in the papillae. Our study also revealed that the nerve fibers which do not form synaptic con- number of the taste bud cells which a single tacts with type-III cells in the rat taste buds21). nerve fiber innervated was relatively smaller Therefore, it is undetermined whether these than that of Beidler's. A possible explanation substance-P immunoreactive fibers are sensory for the difference is that Beidler's data has in nature. So far there have been only a not distinguished the terminal ' from the small number of sympathetic nerves in the fiber: -therefore, many profiles of the fiber taste buds22). Thus, it is reasonable to cons- with attachments to the cells ought to be ider that most of the nerve fibers entering the counted. On the other hand, our study was taste buds are afferent (sensory) in nature, and carried out by counting only nerve fibers that there are only a few efferent fibers. El- which possessed nerve terminals (swellings) ectrophysiologically, taste responses have not coming in contact with the cells. been considered to be the simple summation Numerous vesicles similar in size to the of passively transducted stimulus, and the synaptic vesicles were occasionally observed in nerve fibers in the taste buds might be con- the nerve terminals. In our previous study the cerned with modifying the sensitivity or re- nerve terminals during early postnatal period, sponsiveness of the taste receptor23). Namely, when the taste bud cells rapidly differentiated sensory fibers not only transmit the impulse and developed, contained more vesicles than to the central nervous system but also bring those of adult mice18). Torrey19 has speculat- about a trophic effect on the various types of ed from the studies of degeneration and re- taste bud cells. Additionally, they might mod- generation in denervated taste buds that the ify the taste stimulus through the type- I cells nerve terminals might secrete a substance for or type- II cells which possess subsurfaced maintaining the taste bud cells or promoting cisterns of the endoplasmic reticulum such as cell differentiation. Thus, it seems reasonable observed by many investigators14,16). that the vesicles in the nerve terminals con- Acknowledgement tain a trophic factor for maintaining the taste buds rather than a neurotransmitter substance. This work was supported in part by a Grant-in- Furthermore, we demonstrated that most of Aid (No. 58771245)for Encouragement of Young the single nerve fibers with multiple branches Scientist from the Japan Ministry of Education, innervated both type-III cells and other several Science and Culture.

抄録:マウス味蕾の超薄連続切片を作製し,味蕾細胞と神経線維の三次元的構築について調べた。大部分の味蕾細胞は紡錘形をしており,表面は平滑であったが一部のII型細胞では胞体の基底側が不規則に分岐していた。神経線維は基底膜から味蕾内に侵入し,数回分岐し,ところどころに膨大部を形成し, 678 Jpn. J. Oral Biol., 27:672-678, 1985

次いで神経線維に戻ることをくり返し,最後には膨大した終末で終っていた。1個の細胞に接触する神経線維の数は,I型と基底細胞が最も少なく1～2本(平均1.2本)で,II型細胞で1～4本(平均2.3 本)であり最も多く神経支配を受けているのはIII型(味)細胞で,2～4本(平均2.6本)であった。また1個の細胞に接触する神経終末の数はI型と基底細胞ではそれぞれ1～2個でII型細胞では2～4個で,III型細胞では3～6個であった。III型細胞に接触する神経線維は,必らず求心性シナプスを形成しおり,その数は細胞1個につき平均8個であった。一本の神経線維は4個から14個(平均9個)の味蕾細胞を支配しており,通常異なった型の細胞を同時に支配していた。9本のうち7本の線維がIII型(味) 細胞と他の細胞型とを同時に支配していた。このことから,大部分の味蕾内の神経線維は,味覚刺激を伝導するだけではなく,他の何らかの作用(たとえば味蕾細胞に対する栄養的効果など)を及ぼすのではないかと思われる。

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