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.