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The fine structure 0f the sacculus and lagena of a teleost fish
Hearing Research, 5 (1981) 245-263 El~vier~Nox~-Ho~and Biomedical Press
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THE FINE STRUCTURE OF THE SACCULUS AND LAGENA OF A TELEOST FISH
ARTHUR N. POPPER and BECKY HOKTER Department of Anatomy. Georgetown UniversitySchools of Medicine and Dentistry, 3900 Reservoir Road N. W., Washington,DC 20007, U.S.A. (Received 18 December 1980; accepted 21 April 1981)
The ultrastructure of the sensory epithelia in the auditory re8ions of the ear, the sacculus and lagena, were investigated in the blue gourami, 7Wkoguster trichopterus, using transmission and scanning electron microscopy. The sensory epi~elium consists of sensory hair cefls surrounded by sup porting cells, both of which are quite similar to comparable cells found in other fishes. The apical surface of each sensory cell contains a ciliary bundle which varies in length in different epithelial regions. Tight junctions and one or more levels of desmosomes are located between supporting cells, and between sensory and supporting cells, just below the apical cell membrane. Peripheral to the actual sensory epi~eBum is a region of epicene cells that resemble the ~pporti~ cells on the sensory epithelium itself. However, interspersed among these cells are other cells containing huge numbers of mitochondria, extensive smooth endoplasmic reticulum, and large vacuoles. Investigations of the orientation patterns of the ciliary bundles on the sensory hair cells demonstrate that the lagena is typical of other Perciform fishes while the position of two of the four orientation groups normally found in the Perciform sacculus are quite different in lzichogasrer from that found in other species. Comparisons of the ultrastructure of the sensory and supporting epithelia of Trichogaster and other fishes shows that, with the exception of the mitochondria-fiied cells, there are no apparent signiircant interspecific differences with regard to ultrast.ructure of the. sensory and supporting cells themselves, althou&~ there are differences in hair cell orientation patterns among the same fish groups. Key words: sacculus; lagena; fish; hair cell orientation;
ciliary bundles.
A number of studies have demonstrated that there is considerable inter-specific variation in the gross morphology of the vertebrate ear (e.g. [31,35]), a fact that is especially evident in the auditory portions of the ear in the teleost fishes, the saccuhrs and lagena [6,22,23,31]. Inter-specific differences in the sacculus and lagena are seen in a number of features of these organs, and include the shape and size of the otoliths and the area of the sensory epithelium that lies directly in contact with the otohth. ~~~t~ctur~ variations include the lengths of the cilia extending from the hair cells on different regions of the, sensory epithelium and the orientations of these cilia on the sensory cells at different regions of the epithelium [4,5,17,18,22,23,25,27]. While the scanning electron microscope (SEM) studies demonstrate differences in surface features in the teleost sacculus and lagena, both on different parts of a particular sensory organ of a single species and in the same organ of different species, few comparable data are available derived from the transmission electron microscopy (TEM) of the 0378~5955/81/0000-0000/$02.50
0 1981 Elsevier/North-Holland
Biomedical Press
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cell bodies. In only one of the several TEM studies on the teleost ear [4,7,9,11,25], that of the moray eel, Gymnothorax [2.5], were data sought regarding correlations between variation in surface features, such as ciliary bundle length, and variation in cell body structure. Furthermore, only the study of Gymnothorax considered differences in cell body ultrastructure at different regions of the sensory epithelium. In order to develop a general intra-specific and inter-specific understanding of the teleost auditory sensory epithelia, it will be necessary to obtain a broader data base with regards to comparative cell structure. Due to the extended time needed to gather TEM data, however, we cannot expect to sample as extensive a group of fishes with TEM as has been done with SEM (e.g. [23,24,27]). Still, by comparison of species showing variation in SEM studies of the sensory epithelia we should be able to get an indication of cell structure in different species. The present investigation extends the comparative data available regarding the structure of the ear in fishes by presenting TEM and SEM data on the sacculus and lagena in the blue gourami, Trichogaster trichopterus. Data on this species is of particular interest since it is a member of the order Perciformes, the largest and one of the most diverse of the teleost groups [15] and one for which no TEM data on the ear are currently available. Trichogaster was also chosen because as one of the labyrinth fishes and a member of the suborder Anabantoidei (family Belontiidae), it has an air chamber near the sacculus in the buccal cavity that may be involved in hearing as a peripheral auditory structure [I]. This buccal air cavity is likely to enhance detection of sound [37], much in the same way as the swimbladder in other species [6,29]. Studies of other species having swimbladder, or swimbladder-like, specializations for sound detection [23,26], including another belontiid [35], have demonstrated specializations in the shape of the sensory epithelia and the orientation patterns of the sensory hair cells that are rarely found in species without such swimbladder adaptations. However, we have no information as to whether such specializations in surface features of the sensory epithelia, as determined with SEM, extend to other features of the sensory epithelia at the level studied with TEM. MATERIALSANDMETHODS
Six specimens of blue gourami were obtained from local pet distributors. Specimens were prepared using standard techniques of fixation and preparation described earlier [23,25]. Briefly, the fish were anesthetized, the crania opened, and the brains removed by suction to expose the ears. Cold 2.5% gluteraldehyde in 0.2 M S-Collidine buffer with 4% sucrose was poured into the cranial opening and used as the initial fixative. The ears were exposed as much as possible and then immersed in a large volume of cold fixative for 6 to 24 h to complete fixation. If necessary, excess tissue was dissected away and the ears were rapidly washed in cold buffer (containing 13% sucrose) and post-fixed in 1% osmium tetroxide in 0.2 M S-Collidine buffer for one hour at 4°C. The tissue was then run through graded alcohols and stored in 70% alcohol. Tissues for SEM were trimmed and treated for several seconds in an ultrasonic cleaner [3,23] to remove any debris which could mar viewing of the epithelial surfaces. The maculae were dehydrated to absolute alcohol and then critical point dryed in a Samdri critical point dryer, mounted on an aluminum stub, and viewed with an ETEC Autoscan SEM.
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Tissues for transmission electron microscopy were embedded in Araldite 812, sectioned with an MT-2 ultramicrotome and picked up on 0.3 mm 200 mesh or slotted copper grids coated with formvar. The grids were stained with 1% potassium permanganate in 2% aqueous uranyl acetate, post stained with lead citrate, and viewed with an AEI-801 or a Jeol 100s TEM. RESULTS
The ear in Trichogaster (Fig. 1) lies in the cranial cavity at about the level of the medulla. The ear is typical of that found in other fishes [23,24,31] in having three mutually perpendicular semi-circular canals and associated ampullary regions and three sac-like otolithic organs, the sacculus, lagena and utricle. Each of the otolithic chambers contains a single dense calcareous otolith which is separated from the sensory epithelium (Fig. 1) by a thin gelatinous otolithic membrane. The sensory epithelium attaches to the inner surface of the wall of the otolithic chamber. The ear is oriented so that the saccular and lagenar sensory epithelia lie on the animals’ vertical plane. The sacculus, the largest of the three otolithic organs, and the lagena, the smallest of the organs (Fig. 1) are connected by a small opening in the wall of the otolithic chambers. The saccular and lagenar otoliths each fill about 75% of the volume of their respective chambers. The saccular otolith has a medial groove, or sulcus, in which the sensory epithelium lies, while no such sulcus is found in the smaller lagenar otolith. The saccular otolith does not contact the dorsal edge of the wide rostral saccular epithelium, while the lagenar otolith does not contact the very dorsal tip of the lagenar sensory epithelium. The saccular epithelium or.macula, in Trichogaster, is oblong shaped with a bulbous rostral portion that tapers into a substantially thinner caudal region (Fig. 1). The lagenar
Mdiil
SM’
Fig. 1. Drawingsof the right ear of Trichogaster(medial view on the left, lateral on the right). The medial view shows the sensory epithelia of the sacculus (SM) and portions of the innervating eighth nerve (AN, LN). The outlines of the otoliths are denoted with dashed lines. The otoliths lie close to the sensory epithelia, and are separated from them by the thin otolithic membrane. Note the large bulbous rostral end of the saccular macula. A, anterior semi-circular canal; CC, crus commune; H, horizontal semi-circular canal; L, lagena; LO, lagenar otolith; P, posterior semicircular canal; S, sacculus; SO, saccular otolith; U, utriculus; UO, utricular otolith.
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sensory epithelium is almost crescent shaped (Fig. 1). The sensory epithelium in both organs sits on the inner side of the wall of their respective connective tissue chambers. A general surface examination of the sensory epithelia (e.g. Fig. 2) shows that they are covered with bundles of cilia in some areas and microvilli in others. The entire sensory epithelium is covered by a thin otolithic membrane which contains holes through which the ciliary bundles project, and which separates the macula from its otolith. Surrounding the sensory epithelium itself is non-sensory epithelial tissue which is solely covered by
Fig. 2. View of a region of the surface of the saccular macula in the middle of its long axis showing the ciliary bundles from the apical ends of the sensory hair cells. The sensory region is surrounded by non-sensory epithelial cells, a portion of which is illustrated by the region at the top of this SEM. Different types of ciliary bundles are found at various regions of the sensory epithelium (see text). A few rows of F2 ciliary bundles are located around the whole macula and can be seen at the edges of the tissue at the top of the figure. Several rows of F3 bundles are just inside of the marginal region, while the bulk of the bundless on the macula are type Fl.
249 microvilli. A cross-sectional view of both saccular (Fig. 3) and lagenar sensory epithelia shows that the sensory region contains two distinct types of cells: sensory and supporting. Each of the sensory cells is surrounded by several supporting cells. The non-sensory, or extramacular, area extends out from the sensory regions to a distance greater than the width of each sensory region and is composed of cells that, for the most part (see below), closely resemble the supporting cells of the sensory region. The sensory region of both organs is two layers thick, in contrast to the single layer of the surrounding non-sensory epithelial surface. The sensory and extramacular regions he on a thin basement membrane which contacts the connective tissue wall of the otolithic chambers (Fig. 3).
Surface features
The apical surface of the sensory epithelium, as examined with SEM, is covered with numerous ciliary bundles, each of which are the projections from one sensory cell (Fig. 2). The surfaces of the supporting cells are covered with short microvilli (Figs. 4-6) and similar microvilli are found on the epithehal cells surround~g the sensory region. As is typical of ciliary bundles in other vertebrate ears [36], those in Trichogaster contain a single eccentrically positioned kinocilium and a larger number of stereocilia which are graded in size, the longest being closest to the kinocilium (Fig, 4). The ciliary bundles in
Fig, 3. Low power TEM showing the cross-section of an edge of the saccular sensory epithelium leading into the thinner non-sensory extramacular epithelial region. This TEM represents a region at the edge of the sensory epithelium, such as at the top of Fig. 2. Scauning electron microscopic study shows that the sensory hair cells in this marginal region of the sensory epithehum have type F2 ciliary bundles (see Fig. 2). BM, basement membrane; Hc, sensory hair cell; NV, microvilli on extramacular supporting cells; SC, supporting cell.
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Fig. 4. Type Fl ciliary bundles from the saccular macula. Fl ciliary bundles have kinocilia one or two times longer than the longest of the graded stereocilia. Note the microvilli on the surfaces of the supporting cells that separate the sensory cells. K, kinoclium; Mv, microvilli on supporting cells;; ST,
stereocilia.
Trichoguster vary in absolute lengths, and in the relative lengths of the kinocilia and stereocilia. There are three distinct types of bundles in this species. An Fl bundle [23] has a kinocilium that is one to two times the length of the longest of the graded stereocilia (Fig. 4). This bundle is the predominant type on both the saccular and lagenar maculae, and it covers the entire sensory epithelial surface except for the edges of the sensory regions (Fig. 2). The F2 bundles (Fig. 5) [23] are found at the very edges of both maculae (Fig. 2) and have very short stereocilia (Fig. 5) and a substantially longer kinocilium. The third type of ciliary bundle (type F3 [23]) is generally located just inside the rows of F2 ciliary bundles and extends for several rows before the more ubiquitous Fl bundles (Fig. 2). The F3 bundle (Fig. 6) resembles the Fl, except that the entire F3 bundle is longer than the Fl and has more sharply graded stereocilia.
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Fig. 5. Type F2 ciliary bundles, such as those found at the edges of both saccular and lagenar maculae. The kinocilium (K) in F2 ciliary bundles are substantially longer than the stereocilia (ST).
Hair cell orientation In examining surface features of both the saccular and lagenar sensory epithelia it is apparent that they are divided into several regions, with all of the cihary bundles in any one region having their kinocilia on the same side of the ciliary bundle (see Fig. 4). The saccular macula is divided into four hair cell orientation groups, two of which are located on the wide rostral region of the macula and two on the thin caudal region (Fig. 7). One of the groups in the caudal region of the macula is oriented so that the kinociha of the ciliary bundles are pointed towards the dorsal side of the fish, while the ciliary bundles ventral to this group have all of the cells with their kinocilia oriented towards the ventral
Fig. 6. Type F3 ciliary bundles. Note that these bundles are longer than the type Fl (Fig. 4) bundles and that the stereocilia (ST) are more sharply graded in length. K, kinocilia.
side of the animal. The groups on the rostra1 macula region are oriented along the animal’s horizontal axis. The cells at the rostra1 end of the macula are oriented so that their kinocilia are directed towards the caudal end of the animal while the ciliary bundles just behind this are oriented so that their kinocilia are directed towards the rostral end of the animal. The transition Bnes dividing the various cell groups (dashed line in Fig. 7) are not discrete, and cells of one group may impinge slightly into the region of another group. Further, there is no evidence of there being a distinct striola region [3.53 separating the various orientation groups. It is important to note that in going from one orientation group
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D
L
Trichogaster
A
trichopterus
Fig. 7. Schematic drawing of the hair cell orientation patterns on the saccular (right) and lagenar (left) maculae in Trichogmrer. The arrows point from the stereocilia towards the side of the sensory cells on which the kinocilium is located. The dashed lines indicate the approximate transition lines between opposing hair cell orientation groups. Anterior (A) and dorsal (D) sides of the animals are indicated.
the cihary bundles on both the saccular and lagenar maculae are oriented in distinct directions, as opposed to their ‘grading’ into one another through gradual changes in orientation. The crescent-shaped lagena macula has two hair cell orientation groups. The cells on the anterior side of the macular are oriented dorsally, while those on the posterior side of the macula are oriented ventrally (Fig. 7). There is also a gradual shift in orientation within the confines of the major orientation groups as the macula curves, but this is never as great as the diametric opposition of cells across the transition line. to another,
Cell ultrastructure
Transmission electron microscopic examination of the sensory cells in the sacculus and lagena in Trichogaster show them to be quite similar to those in other fishes examined to date [4,9,17,25] and to the Type 2 sensory cells of mammals [36]. The cells are rectangular in shape and are slightly narrower at the apical than basal end (Figs. 3 and 8). A large nucleus is centrally located in the cells while mitochondria and vacuoles are distributed throughout the cytoplasm. No differences were observed between the sensory cells in the sacculus and lagena, nor were there any noteworthy differences in the supporting cells. Cells were examined at all points along the saccular and lagenar maculae and specifically in macula regions known, through SEM examination, to have cells with different ciliary bundle types (e.g. regions illustrated in Fig. 2). No differences in cells with regard to their size, shape, or organelle content were found that could in any way be correlated with the position of the cells on the sensory epithelium or the lengths of the ciliary bundles. The sensory cells are separated from one another by at least one, and usually several,
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Fig. 8. TEM of sensory (Hc) and supporting cells (SC) of the saccular macula. Note that several supporting cells a_refound between sensory cells. C, cuticle; D, desmosome; M, mitochondria; N, nucleus of sensory cell; St, stereocilia.
Fig. 9, High power TEM showing a series of supporting cells near a single sensory cell. Tight junctions (T) and associated desmosomes (D) connect supporting cells to one another and to the sensory cell. C, cuticle; M, mitochondria.
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Fig. 10. This high-power TEM demonstrates the fact that the tight junctions (T) and the desmosome connections (D) are found at and below the apical border of the sensory epithelium.
supporting cells (Figs. 3, 8 and 9). The cuboidal-shaped supporting cells are lined up along the basement membrane and send slender projections up between the sensory cells (Fig. 8). At the ~cro~~i~overed apical surface, tight junctions and desmosomes provide connections between supporting cells and between supporting and sensory cells (Figs. 9 and 10). The desmosomal connections are found near the surface of the epithelium and at one or more points further down, towards the basement membrane (Figs. 9 and 10). The region surrounding the sensory epithehum consists of non-sensory epitheliai cells (Fig. 3) which vary from rectangular shaped near the sensory region to cuboidal shaped at the extremes of this extramacular area. The non-sensory epithelial cells make up a single layer of cells lying on a basement membrane. Their apical surfaces contain numerous microvillous projections of varying lengths.
Fig. 11. SEM showing the extramacular epithelial region containing the more common type of extramacular epithelial cells (EC) as well as cells with a raised ‘bump-like’ appearance (Lc).
Fig. 12. TEM of the region just outside of the sensory epithehal region showing a cross section of simBar cells to those shown in Fig. 11. These huge cells (Lc) are separated by more typical extramacular epithelial cells. These large cells have an extraordinarily high concentration of mitochondria and smooth endoplasmic reticulum. BM, basement membrane; Mv, microviih.
Fig. 13. High-power TEM showing some of the mitochondria, smooth endoplasmic reticulum (Ser), and large, presumably Iipid containing, vacuoles (Li) in one of the ‘bump-like’ cells. The large membrane bound spaces may have contained lipid that was leached out during some stage of the fvcation process, or dehydration.
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Interspersed among the more common cells of this non-sensory region are other cells that differ in that they contain an inordinate number of mitochondria. On examination with SEM these cells appear on the surface of the extramacular region as raised bumps with a microvillar surface (Fig. 11). TEM shows these cells to have a large number of mitochondria around the nucleus and throughout the cytoplasm (Fig. 12) as well as numerous coated vesicles in the cytoplasm and some large vacuoles which may contain lipid (Fig. 13). DISCUSSION
The gross structure of the ear in Trichoguster is similar to the ears in representatives of other major teleost taxa [23,25-281 with the exception of one taxonomic group, the Ostariophysi [9,13,21]. The similarities between the gourami and non-ostariophysines (species in taxonomic groups other than the Ostariophysi) also extend to most inner ear features including the general shape and size relationships of the otolithic organs, the shape of the otoliths and their relationship to the sensory epithelia, the presence of sensory and supporting cells on the macula, and the division of the maculae into orientation groups [4,5,13,14,22-271. In addition, the ciliary bundles and the bodies of the sensory cells in Trichoguster closely resemble the same structures in the other teleosts that have been studied with both SEM and TEM including the goldfish, Carassiusauratus [8,21], a moray eel, Gymnothorax sp. [25], a cod, Gadus morhua [4] and several species of catfish [13]. While Trichoguster has many inner ear features in common with those found in other teleost fishes, several aspects of the ear in the gourami are of considerable significance and important to note. These include the lack of variation in the cell bodies, the cells containing numerous mitochondria found outside of the sensory region, and the orientation patterns of the ciliary bundles on the saccular macula. Cell bodies An important question relates to the nature of any differences in sensory cell bodies
at different regions of the sensory epithelium or that may be associated with the various types of ciliary bundles. Studies of a Carcharhinid shark have demonstrated the presence of dark and light sensory cells in the macula neglecta [3] and a similar observation has been made for the lagena of one teleost, a loach of unidentified species [32]. Investigations of a lamprey, Entosephenus japonicus, have shown that cells with different length ciliary bundles in that species have different types of organelles [ 111. The present study on i’khoguster, and an earlier investigation of Gymnothorux [23], have not, however, revealed the presence of dark and light sensory cells, nor is there any evidence for cytological differences in cells with different ciliary bundles. In fact, there is no evidence for any cellular differences in any region of either otolithic macula in Trichogaster. Thus, it now appears that in spite of differences in ciliary bundle lengths, all of the sensory cells in a number of different teleost species have similar cell bodies. Surface features
One of the most striking features of the ear in Tkichogaster is the orientation pattern
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of the sensory hairs on the rostra1 end of the macula. The saccuiar sensory region in most fishes, other than the Ostariophysi, is elongate with a moderately enlarged anterior region containing the horizontally oriented hair cells [23,27]. The horizontally oriented cells in most of these species are organized so that the caudally oriented cell group is located on the dorsal quadrant of the macula, while the rostrally oriented group is on the ventral quadrant. The region containing horizontally oriented hair cells in Trichogaster, in contrast, is bulbous (Figs. 1 and 7), while the caudal half of the macula is very thin. The gourami-like hair cell o~entation pattern, invol~ng one group of rostral sensory cells in front of, rather than dorsal to, the other, is uncommon among fishes. It has only been reported in one other perciform, Colisa labiosa [34], another genus of the same subfamily as Trichogaster. Even more significant is the finding of a somewhat similar pattern of saccular hair cell o~entation in several species that are t~ono~ca~y distinct from the anabantids including the clown knife-fish (Notoptems chitda) [26] and several deep-sea myctophids, or lantern-fishes [23]. While data are severely limited, it is tempting to speculate that the specialized saecular hair cell orientation patterns found in the anabantids and the other species are, in some way, adapted for s~cialized,hearing abilities. In fact, the special hair cell orientation pattern may potentially be correlated with other specializations in auditory structures peripheral to the ear. For example, Trichogaster has a small bubble of air in the buccal cavity close to the ear that is thought to enhance auditory sensitivity [ 1,371. While structurally different, Noto~te~s has a swimbladder that terminates on the auditory bulla, a condition often associated with enhanced hearing capabilities [22]. Although hearing abilities are not known for the myctophids, the third group having a hair cell orientation pattern similar to that in ~c~ogaster, it has been suggested that members of the myctophid family use sound in communication {17f. The specific functional significance of the specializations in the horizontally oriented hair cells in Trichogaster and other species, as opposed to the more ‘standard’ pattern found in many other fishes, is unclear. The presence of ho~zont~y, as well as vertically, oriented sensory hair cells have been implicated in the detection of sound source direction (e.g. sound localization) [3-6,22,23,28] and it is possible that the differences seen among these specialized species represents a particular adaptation for extracting certain types of directional information, or directional information under certain types of acoustic conditions encountered in the environments of these species. It is also possible that the adaptions seen in Trichogaster have nothing to do with directional detection, but instead have bearing on basic mechanisms of acoustic analysis by the fish. It has been suggested ]22,28] that the sound processing in the teleost ear is complex and significantly affected by the mechanisms by which sound reaches the ear and the specific relationship between the otolith and the ciliary bundles. Any factors affecting these structures, such as the shape of the otolith or the sensory epithelium, the lengths of the ciliary bundles, or whether the ciliary bundles are directly under the otolith or in regions not covered by the otolith, could markedly affect the way that the fish detects and/or analyzes sounds [ 221. Consequently, it is possible that the variation seen in the morphology of the teleost ear reflects interspecific variation in the way that fishes use sound. However, little is known about sound detection and processing in any teleost species, and the differences in the use of sound by Trichogaster and Notoptems, as opposed to other species, are totally unknown.
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Supporting cells While the supporting cells within the sensory maculae in Trichogaster, and the epithe-
lial cells in the extramacular region, are typical of those seen in other fishes, the large mitochondria-filed cells in the extramacular region are not ubiquitous among teleosts. Cells of this type were not seen in the moray eel, nor were they reported in the sacculus of Carassius [9], or any of the otolithic organs of Gadus [4] or a lamprey [ll]. They have, however, been seen in a sunfish (Lepomis) (Popper, unpublished) and in several species of catfish (Jenkins, personal communication; Popper, unpublished). Furthermore, SEM studies of several other species, including the squirrelfish Myripristis, have demonstrated the presence of cells with similar surface features in extramacular regions
[231. The functional significance of these large cells is not known, and cells of similar types have not been reported in the mammalian ear (e.g. [12]) or in the teleost lateral line [9, 10,201. However, cells have been reported in the teleost lateral line [20], in the supporting region of the shark macula neglecta [3], and in the mammalian ear [ 12,16,30] which contain large numbers of mitochondria and highly invaginated basal ends. These cells in mammals have been associated with otoconia formation or reabsorption [ 16,301 or with the production or reabsorption of endolymph [18]. The role of such cells in fishes and sharks is also not known, but it has been suggested that they may have a secretory or reabsorptive role involved with cupula formation or roles in maintenance of the metabolic activity of the sensory cells [9,20]. It is reasonable to suggest, based upon the presence of extensive mitochondria and smooth endoplasmic reticulum, that the extramacular cells in Zlichogaster have high metabolic activity and are probably associated with secretion or absorption of materials. Their precise role, however, is not clear even after considering data from secretory cells in the ears of other vertebrates, and it will be necessary to await experimental studies on the teleost ear to determine the function of these cells. However, one potential area that should be considered when studying these cells is the likelihood that they may be involved in secretion of calcium carbonate for otolith formation or in the metabolism of endolymph. Virtually no data are available regarding both functions in the teleost ear, but we would expect such activity to involve cells with high metabolic activity and extensive secretory abilities. Finally, while it is clear that such cells are found in a number of taxonomically diverse species, they have not been reported in all species. Although it is possible that these cells were not noticed by earlier workers, it is more likely that they may only be present in fishes under certain conditions, such as during periods of rapid growth or perhaps during development of certain ear structures, such as the otoliths. Taxonomic significance The present TEM and SEM studies of the ear in Wchogaster are of considerable inter-
est and importance when we consider that these are the first TEM data for any member of one of the major advanced teleost orders [ 151, the Perciformes. With these data, we now have some information on the ultrastructure of the sensory epithelia of four diverse teleost taxonomic orders, the Gadiformes [4,7], the Ostariophysi [9,13], the Anguilliformes [25] and Perciformes. It is clear that there is substantial similarity in the cytology
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of the sensory and supporting cells among all of these groups. This is encountered in spite of the extensive variation found in the ears of the same species as determined with the SEM (e.g. [4,13,21,25]) and the considerable differences in the gross structure of their ears and more peripheral auditory structures. The significance of the similarities in the TEM observations of the sensory and supporting cells in these species may indicate that the major adaptive changes encountered in the ear are associated only with the regions of the ear actually involved in mechanical responses to acoustic energy, such as the ciliary bundles on the sensory cells and the orientation patterns of the hair cells, as well as in the structures more peripheral to these, such as swimbladder structure or the morphological relationship between the swimbladder and the inner ear. In contrast, the function of the cell bodies for the support of the ciliary bundles may be sufficient to operate in a wide range of different acoustic conditions. Thus, similarities in the cell bodies of the sensory hair cells may represent a conservative arrangement among fishes, as well as in the ears of most other vertebrates, that is not affected by selective pressures acting upon the sound detection and processing system of the ear. ACKNOWLEDGEMENTS
This work was supported by NSF grants BNS 79-17920 and BNS 80-09343, and by a U.S. Public Health Service Research grant NS-15090 and Career Development Award (NS 003 12) to A.N.P. REFERENCES [l] Bader, R. (1937): Bau, Entwicklung und Funktion des akzessorischen Atmungsorgans der Labyrinthflsche. Z. Zool. 149,323-401. [ 21 Buwalda, R.J.A. and van der Steen, J. (1979): The sensitivity of the cod sacculus to directional and nondirectional sound stimuli. Comp. Biochem. Physiol. 64A, 467-471. [3] Corwin, J.T. (1977): Morphology of the macula neglecta in sharks of the genus Carcharhinus. J. Morphol. 152,341-362. [4] Dale, T. (1976): The labyrinthine mechanoreceptor organs of the cod Gadus morhua L. (Teleostei: Gadidae). Norw. J. Zool. 24,85-128. [5] Enger, P.S. (1976): On the orientation of hair cells in the labyiinth of perch (Perca fluviarilis). In: Sound Reception in Fish, pp. 396-411. Editors: A Schuijf and A.D. Hawkins. Elsevier, Amsterdam. [6] Fay, R.R. and Popper, A.N. (1980): Structure and function in teleost auditory systems. In: Comparative Studies of Hearing in Vertebrates, pp. l-42. Editors: A.N. Popper and R.R. Fay. Springer-Verlag, New York. [7] Flock, A. (1964): Structure of the macula utriculi with special reference to directional interplay of sensory responses as revealed by morphological polarization. J. Cell Biol. 22,413-431. [ 81 Hama, K. (1965): Some observations on the fine structure of the lateral line organ in the Japanese sea eel, Lyncozmba nystromi. J. Cell Biol. 24,193-210. [ 91 Hama, K. (1969): A study on the tine structure of the saccular macula of the goldfish. Z. Zellforsch. 94,155-171. [lo] Hama, K. and Y. Yamada (1977): Fine structure of the ordinary lateral line organ II. The lateral line canal organ of the spotted shark,Mustelus manazo. Cell Tissue Res. 176,23-36. [11] Hoshino, T. (1975): An electron microscopic study of the otolithic maculae of the lamprey (Entosephenusjaponicus). Acta OtoIaryngol. 80,43-53. [ 121 Iurato, S. (1967): Submicroscopic Structure of the Ear. Pergamon Press, New York.
263 [ 131 Jenkins, D.B. (1979): A transmission and scanning electron microscopic study of the saccule in five species of catfishes. Am. I. Anat. 154,81-101. [ 141Jdrgensen, J.M. (1976): Hair cell polarization in the flatfish inner ear. Acta Zool. 57,37-39. [ 15) Liem, K.F. and Lauder, G.V. (1982): The evolution and interrelationships of the Actinopterygian fishes. In: Neurobiology and Behavior of Fishes. Editors: R.G. Northcutt and R.E. Davis. University of Michigan Press, Ann Arbor (in press). [l6] Lim, D. (1973): Formation and fate of the otoconia. AM. Otol. Rhinol. Laryngol. 82,23-35. [ 171 Marshall, N.B. (1967): Sound-producing mechanisms and the biology of deep-sea fishes. In: Marine Bio-Acoustics, pp. 123-133. Editor: W.N. Tavolga. Pergamon Press, Oxford. 1181 Nakai, Y. and Hilding, D. (1968): Vestibular endolymph-producing epithelium. Acta Otolaryngal. 66,120-128. [ 191 Nakajima, Y. and Wang, D.W. (1974): Morphology of afferent and efferent synapses in hearing organ of the goldfish. J. Comp. Neural. 156,403-416. [ 201 Petraitis, R. (1966): Fine structure of supporting cells in the lateral-line canal organ of Fundulus. J. Morphol. 118, 367-378. [21] Platt, C. (1977): Hair cell distribution and orientation in goldfish otolith organs. J. Comp. Neurol. 172,283-298. [22] Platt, C. and Popper, A.N. (1981): Fine structure and function of the ear. In: Hearing and Sound Communication in Fishes. Editors: W.N. Tavolga, A.N. Popper and R.R. Fay. Springer-Verlag, New York (in press). [ 231 Popper, A.N. (1977) A scanning electron microscopic study of the sacculus and lagena in the ears of fifteen species of teleost fishes. J. Morphol. 153,397-418. [ 241 Popper, A.N. (1978): A comparative study of the otolithic organs in fishes. Scanning Electron Microscopyll978, pp. 405-416. (251 Popper, A.N. (1979): The ultrastructure of the sacculus and lagena in a moray eel (Gymnorhorax sp.). J. Morphol. 161,241-256. [26] Popper, A.N. (1979): Inner ear auditory receptors in Osteoglossomorph fishes. Sot. Neurosci. Abstr. 5, 29. [ 271 Popper, A.N. (1980): Scanning electron microscopic study of the sacculus and lagena In several deep sea fishes. Am. J. Anat. 157,115-136. [28] Popper, A.N. (1982): Organization of the inner ear and auditory processing. In: Fish Neurobiology and Behavior. Editors: R.G. Northcutt and R.E. Davis. University of Michigan Press, AM Arbor, Mich. (in press). [29] Popper, A.N. and Fay, R.R. (1973): Sound detection and processing by teleost fishes: a critical review. J. Acoust. Sot. Am. 53, 1515-l 529. [30] Preston, R.E., Johnsson, L.-G. and Schacht, J. (1975): Incorporation of radioactive calcium into otoBthic membrane and middle ear ossicles. Acta Otolaryngol. 80, 269-275. [ 313 Retzius, G. (1881): Das Gehororgan der Wirbeltiere. Vol. I. Samson and Wallin, Stockholm. [32] Saito, K. (1975): Fine structure of the macula of the lagena in the teleost ear. Acta Anat.Nippon. 48, 1-18. [ 331 Schneider, H. (1941): Die Bedeutung der Atemhiihle der Labyrhrthflsche fur ihr Horvermogen. Z. Vergl. Physiol. 29.172-194. [ 341 Wegner, N.T. (1979): The orientation of hair cells in the otolithic organs and the papilla neglecta in the Inner ear of the anabantide fish, Colisa Zubiosu (Day). Acta Zool. 60,205-216. [35] Werner, C.F. (1960): Das Gehororgan der Wirbeltiere und des Menschen. G. Thieme, Leipzig. [36] Wersiill, J., Flock, A. and Lundquist, P.-G. (1965): Structural basis for directional sensitivity in cochlear and vestibular sensory receptors. Cold Spring Harbor Symp. Quant. Biol. 30, 115-132.
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THE FINE STRUCTURE OF THE SACCULUS AND LAGENA OF A TELEOST FISH
ARTHUR N. POPPER and BECKY HOKTER Department of Anatomy. Georgetown UniversitySchools of Medicine and Dentistry, 3900 Reservoir Road N. W., Washington,DC 20007, U.S.A. (Received 18 December 1980; accepted 21 April 1981)
The ultrastructure of the sensory epithelia in the auditory re8ions of the ear, the sacculus and lagena, were investigated in the blue gourami, 7Wkoguster trichopterus, using transmission and scanning electron microscopy. The sensory epi~elium consists of sensory hair cefls surrounded by sup porting cells, both of which are quite similar to comparable cells found in other fishes. The apical surface of each sensory cell contains a ciliary bundle which varies in length in different epithelial regions. Tight junctions and one or more levels of desmosomes are located between supporting cells, and between sensory and supporting cells, just below the apical cell membrane. Peripheral to the actual sensory epi~eBum is a region of epicene cells that resemble the ~pporti~ cells on the sensory epithelium itself. However, interspersed among these cells are other cells containing huge numbers of mitochondria, extensive smooth endoplasmic reticulum, and large vacuoles. Investigations of the orientation patterns of the ciliary bundles on the sensory hair cells demonstrate that the lagena is typical of other Perciform fishes while the position of two of the four orientation groups normally found in the Perciform sacculus are quite different in lzichogasrer from that found in other species. Comparisons of the ultrastructure of the sensory and supporting epithelia of Trichogaster and other fishes shows that, with the exception of the mitochondria-fiied cells, there are no apparent signiircant interspecific differences with regard to ultrast.ructure of the. sensory and supporting cells themselves, althou&~ there are differences in hair cell orientation patterns among the same fish groups. Key words: sacculus; lagena; fish; hair cell orientation;
ciliary bundles.
A number of studies have demonstrated that there is considerable inter-specific variation in the gross morphology of the vertebrate ear (e.g. [31,35]), a fact that is especially evident in the auditory portions of the ear in the teleost fishes, the saccuhrs and lagena [6,22,23,31]. Inter-specific differences in the sacculus and lagena are seen in a number of features of these organs, and include the shape and size of the otoliths and the area of the sensory epithelium that lies directly in contact with the otohth. ~~~t~ctur~ variations include the lengths of the cilia extending from the hair cells on different regions of the, sensory epithelium and the orientations of these cilia on the sensory cells at different regions of the epithelium [4,5,17,18,22,23,25,27]. While the scanning electron microscope (SEM) studies demonstrate differences in surface features in the teleost sacculus and lagena, both on different parts of a particular sensory organ of a single species and in the same organ of different species, few comparable data are available derived from the transmission electron microscopy (TEM) of the 0378~5955/81/0000-0000/$02.50
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Biomedical Press
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cell bodies. In only one of the several TEM studies on the teleost ear [4,7,9,11,25], that of the moray eel, Gymnothorax [2.5], were data sought regarding correlations between variation in surface features, such as ciliary bundle length, and variation in cell body structure. Furthermore, only the study of Gymnothorax considered differences in cell body ultrastructure at different regions of the sensory epithelium. In order to develop a general intra-specific and inter-specific understanding of the teleost auditory sensory epithelia, it will be necessary to obtain a broader data base with regards to comparative cell structure. Due to the extended time needed to gather TEM data, however, we cannot expect to sample as extensive a group of fishes with TEM as has been done with SEM (e.g. [23,24,27]). Still, by comparison of species showing variation in SEM studies of the sensory epithelia we should be able to get an indication of cell structure in different species. The present investigation extends the comparative data available regarding the structure of the ear in fishes by presenting TEM and SEM data on the sacculus and lagena in the blue gourami, Trichogaster trichopterus. Data on this species is of particular interest since it is a member of the order Perciformes, the largest and one of the most diverse of the teleost groups [15] and one for which no TEM data on the ear are currently available. Trichogaster was also chosen because as one of the labyrinth fishes and a member of the suborder Anabantoidei (family Belontiidae), it has an air chamber near the sacculus in the buccal cavity that may be involved in hearing as a peripheral auditory structure [I]. This buccal air cavity is likely to enhance detection of sound [37], much in the same way as the swimbladder in other species [6,29]. Studies of other species having swimbladder, or swimbladder-like, specializations for sound detection [23,26], including another belontiid [35], have demonstrated specializations in the shape of the sensory epithelia and the orientation patterns of the sensory hair cells that are rarely found in species without such swimbladder adaptations. However, we have no information as to whether such specializations in surface features of the sensory epithelia, as determined with SEM, extend to other features of the sensory epithelia at the level studied with TEM. MATERIALSANDMETHODS
Six specimens of blue gourami were obtained from local pet distributors. Specimens were prepared using standard techniques of fixation and preparation described earlier [23,25]. Briefly, the fish were anesthetized, the crania opened, and the brains removed by suction to expose the ears. Cold 2.5% gluteraldehyde in 0.2 M S-Collidine buffer with 4% sucrose was poured into the cranial opening and used as the initial fixative. The ears were exposed as much as possible and then immersed in a large volume of cold fixative for 6 to 24 h to complete fixation. If necessary, excess tissue was dissected away and the ears were rapidly washed in cold buffer (containing 13% sucrose) and post-fixed in 1% osmium tetroxide in 0.2 M S-Collidine buffer for one hour at 4°C. The tissue was then run through graded alcohols and stored in 70% alcohol. Tissues for SEM were trimmed and treated for several seconds in an ultrasonic cleaner [3,23] to remove any debris which could mar viewing of the epithelial surfaces. The maculae were dehydrated to absolute alcohol and then critical point dryed in a Samdri critical point dryer, mounted on an aluminum stub, and viewed with an ETEC Autoscan SEM.
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Tissues for transmission electron microscopy were embedded in Araldite 812, sectioned with an MT-2 ultramicrotome and picked up on 0.3 mm 200 mesh or slotted copper grids coated with formvar. The grids were stained with 1% potassium permanganate in 2% aqueous uranyl acetate, post stained with lead citrate, and viewed with an AEI-801 or a Jeol 100s TEM. RESULTS
The ear in Trichogaster (Fig. 1) lies in the cranial cavity at about the level of the medulla. The ear is typical of that found in other fishes [23,24,31] in having three mutually perpendicular semi-circular canals and associated ampullary regions and three sac-like otolithic organs, the sacculus, lagena and utricle. Each of the otolithic chambers contains a single dense calcareous otolith which is separated from the sensory epithelium (Fig. 1) by a thin gelatinous otolithic membrane. The sensory epithelium attaches to the inner surface of the wall of the otolithic chamber. The ear is oriented so that the saccular and lagenar sensory epithelia lie on the animals’ vertical plane. The sacculus, the largest of the three otolithic organs, and the lagena, the smallest of the organs (Fig. 1) are connected by a small opening in the wall of the otolithic chambers. The saccular and lagenar otoliths each fill about 75% of the volume of their respective chambers. The saccular otolith has a medial groove, or sulcus, in which the sensory epithelium lies, while no such sulcus is found in the smaller lagenar otolith. The saccular otolith does not contact the dorsal edge of the wide rostral saccular epithelium, while the lagenar otolith does not contact the very dorsal tip of the lagenar sensory epithelium. The saccular epithelium or.macula, in Trichogaster, is oblong shaped with a bulbous rostral portion that tapers into a substantially thinner caudal region (Fig. 1). The lagenar
Mdiil
SM’
Fig. 1. Drawingsof the right ear of Trichogaster(medial view on the left, lateral on the right). The medial view shows the sensory epithelia of the sacculus (SM) and portions of the innervating eighth nerve (AN, LN). The outlines of the otoliths are denoted with dashed lines. The otoliths lie close to the sensory epithelia, and are separated from them by the thin otolithic membrane. Note the large bulbous rostral end of the saccular macula. A, anterior semi-circular canal; CC, crus commune; H, horizontal semi-circular canal; L, lagena; LO, lagenar otolith; P, posterior semicircular canal; S, sacculus; SO, saccular otolith; U, utriculus; UO, utricular otolith.
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sensory epithelium is almost crescent shaped (Fig. 1). The sensory epithelium in both organs sits on the inner side of the wall of their respective connective tissue chambers. A general surface examination of the sensory epithelia (e.g. Fig. 2) shows that they are covered with bundles of cilia in some areas and microvilli in others. The entire sensory epithelium is covered by a thin otolithic membrane which contains holes through which the ciliary bundles project, and which separates the macula from its otolith. Surrounding the sensory epithelium itself is non-sensory epithelial tissue which is solely covered by
Fig. 2. View of a region of the surface of the saccular macula in the middle of its long axis showing the ciliary bundles from the apical ends of the sensory hair cells. The sensory region is surrounded by non-sensory epithelial cells, a portion of which is illustrated by the region at the top of this SEM. Different types of ciliary bundles are found at various regions of the sensory epithelium (see text). A few rows of F2 ciliary bundles are located around the whole macula and can be seen at the edges of the tissue at the top of the figure. Several rows of F3 bundles are just inside of the marginal region, while the bulk of the bundless on the macula are type Fl.
249 microvilli. A cross-sectional view of both saccular (Fig. 3) and lagenar sensory epithelia shows that the sensory region contains two distinct types of cells: sensory and supporting. Each of the sensory cells is surrounded by several supporting cells. The non-sensory, or extramacular, area extends out from the sensory regions to a distance greater than the width of each sensory region and is composed of cells that, for the most part (see below), closely resemble the supporting cells of the sensory region. The sensory region of both organs is two layers thick, in contrast to the single layer of the surrounding non-sensory epithelial surface. The sensory and extramacular regions he on a thin basement membrane which contacts the connective tissue wall of the otolithic chambers (Fig. 3).
Surface features
The apical surface of the sensory epithelium, as examined with SEM, is covered with numerous ciliary bundles, each of which are the projections from one sensory cell (Fig. 2). The surfaces of the supporting cells are covered with short microvilli (Figs. 4-6) and similar microvilli are found on the epithehal cells surround~g the sensory region. As is typical of ciliary bundles in other vertebrate ears [36], those in Trichogaster contain a single eccentrically positioned kinocilium and a larger number of stereocilia which are graded in size, the longest being closest to the kinocilium (Fig, 4). The ciliary bundles in
Fig, 3. Low power TEM showing the cross-section of an edge of the saccular sensory epithelium leading into the thinner non-sensory extramacular epithelial region. This TEM represents a region at the edge of the sensory epithelium, such as at the top of Fig. 2. Scauning electron microscopic study shows that the sensory hair cells in this marginal region of the sensory epithehum have type F2 ciliary bundles (see Fig. 2). BM, basement membrane; Hc, sensory hair cell; NV, microvilli on extramacular supporting cells; SC, supporting cell.
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Fig. 4. Type Fl ciliary bundles from the saccular macula. Fl ciliary bundles have kinocilia one or two times longer than the longest of the graded stereocilia. Note the microvilli on the surfaces of the supporting cells that separate the sensory cells. K, kinoclium; Mv, microvilli on supporting cells;; ST,
stereocilia.
Trichoguster vary in absolute lengths, and in the relative lengths of the kinocilia and stereocilia. There are three distinct types of bundles in this species. An Fl bundle [23] has a kinocilium that is one to two times the length of the longest of the graded stereocilia (Fig. 4). This bundle is the predominant type on both the saccular and lagenar maculae, and it covers the entire sensory epithelial surface except for the edges of the sensory regions (Fig. 2). The F2 bundles (Fig. 5) [23] are found at the very edges of both maculae (Fig. 2) and have very short stereocilia (Fig. 5) and a substantially longer kinocilium. The third type of ciliary bundle (type F3 [23]) is generally located just inside the rows of F2 ciliary bundles and extends for several rows before the more ubiquitous Fl bundles (Fig. 2). The F3 bundle (Fig. 6) resembles the Fl, except that the entire F3 bundle is longer than the Fl and has more sharply graded stereocilia.
251
Fig. 5. Type F2 ciliary bundles, such as those found at the edges of both saccular and lagenar maculae. The kinocilium (K) in F2 ciliary bundles are substantially longer than the stereocilia (ST).
Hair cell orientation In examining surface features of both the saccular and lagenar sensory epithelia it is apparent that they are divided into several regions, with all of the cihary bundles in any one region having their kinocilia on the same side of the ciliary bundle (see Fig. 4). The saccular macula is divided into four hair cell orientation groups, two of which are located on the wide rostral region of the macula and two on the thin caudal region (Fig. 7). One of the groups in the caudal region of the macula is oriented so that the kinociha of the ciliary bundles are pointed towards the dorsal side of the fish, while the ciliary bundles ventral to this group have all of the cells with their kinocilia oriented towards the ventral
Fig. 6. Type F3 ciliary bundles. Note that these bundles are longer than the type Fl (Fig. 4) bundles and that the stereocilia (ST) are more sharply graded in length. K, kinocilia.
side of the animal. The groups on the rostra1 macula region are oriented along the animal’s horizontal axis. The cells at the rostra1 end of the macula are oriented so that their kinocilia are directed towards the caudal end of the animal while the ciliary bundles just behind this are oriented so that their kinocilia are directed towards the rostral end of the animal. The transition Bnes dividing the various cell groups (dashed line in Fig. 7) are not discrete, and cells of one group may impinge slightly into the region of another group. Further, there is no evidence of there being a distinct striola region [3.53 separating the various orientation groups. It is important to note that in going from one orientation group
253
D
L
Trichogaster
A
trichopterus
Fig. 7. Schematic drawing of the hair cell orientation patterns on the saccular (right) and lagenar (left) maculae in Trichogmrer. The arrows point from the stereocilia towards the side of the sensory cells on which the kinocilium is located. The dashed lines indicate the approximate transition lines between opposing hair cell orientation groups. Anterior (A) and dorsal (D) sides of the animals are indicated.
the cihary bundles on both the saccular and lagenar maculae are oriented in distinct directions, as opposed to their ‘grading’ into one another through gradual changes in orientation. The crescent-shaped lagena macula has two hair cell orientation groups. The cells on the anterior side of the macular are oriented dorsally, while those on the posterior side of the macula are oriented ventrally (Fig. 7). There is also a gradual shift in orientation within the confines of the major orientation groups as the macula curves, but this is never as great as the diametric opposition of cells across the transition line. to another,
Cell ultrastructure
Transmission electron microscopic examination of the sensory cells in the sacculus and lagena in Trichogaster show them to be quite similar to those in other fishes examined to date [4,9,17,25] and to the Type 2 sensory cells of mammals [36]. The cells are rectangular in shape and are slightly narrower at the apical than basal end (Figs. 3 and 8). A large nucleus is centrally located in the cells while mitochondria and vacuoles are distributed throughout the cytoplasm. No differences were observed between the sensory cells in the sacculus and lagena, nor were there any noteworthy differences in the supporting cells. Cells were examined at all points along the saccular and lagenar maculae and specifically in macula regions known, through SEM examination, to have cells with different ciliary bundle types (e.g. regions illustrated in Fig. 2). No differences in cells with regard to their size, shape, or organelle content were found that could in any way be correlated with the position of the cells on the sensory epithelium or the lengths of the ciliary bundles. The sensory cells are separated from one another by at least one, and usually several,
254
Fig. 8. TEM of sensory (Hc) and supporting cells (SC) of the saccular macula. Note that several supporting cells a_refound between sensory cells. C, cuticle; D, desmosome; M, mitochondria; N, nucleus of sensory cell; St, stereocilia.
Fig. 9, High power TEM showing a series of supporting cells near a single sensory cell. Tight junctions (T) and associated desmosomes (D) connect supporting cells to one another and to the sensory cell. C, cuticle; M, mitochondria.
256
Fig. 10. This high-power TEM demonstrates the fact that the tight junctions (T) and the desmosome connections (D) are found at and below the apical border of the sensory epithelium.
supporting cells (Figs. 3, 8 and 9). The cuboidal-shaped supporting cells are lined up along the basement membrane and send slender projections up between the sensory cells (Fig. 8). At the ~cro~~i~overed apical surface, tight junctions and desmosomes provide connections between supporting cells and between supporting and sensory cells (Figs. 9 and 10). The desmosomal connections are found near the surface of the epithelium and at one or more points further down, towards the basement membrane (Figs. 9 and 10). The region surrounding the sensory epithehum consists of non-sensory epitheliai cells (Fig. 3) which vary from rectangular shaped near the sensory region to cuboidal shaped at the extremes of this extramacular area. The non-sensory epithelial cells make up a single layer of cells lying on a basement membrane. Their apical surfaces contain numerous microvillous projections of varying lengths.
Fig. 11. SEM showing the extramacular epithelial region containing the more common type of extramacular epithelial cells (EC) as well as cells with a raised ‘bump-like’ appearance (Lc).
Fig. 12. TEM of the region just outside of the sensory epithehal region showing a cross section of simBar cells to those shown in Fig. 11. These huge cells (Lc) are separated by more typical extramacular epithelial cells. These large cells have an extraordinarily high concentration of mitochondria and smooth endoplasmic reticulum. BM, basement membrane; Mv, microviih.
Fig. 13. High-power TEM showing some of the mitochondria, smooth endoplasmic reticulum (Ser), and large, presumably Iipid containing, vacuoles (Li) in one of the ‘bump-like’ cells. The large membrane bound spaces may have contained lipid that was leached out during some stage of the fvcation process, or dehydration.
259
Interspersed among the more common cells of this non-sensory region are other cells that differ in that they contain an inordinate number of mitochondria. On examination with SEM these cells appear on the surface of the extramacular region as raised bumps with a microvillar surface (Fig. 11). TEM shows these cells to have a large number of mitochondria around the nucleus and throughout the cytoplasm (Fig. 12) as well as numerous coated vesicles in the cytoplasm and some large vacuoles which may contain lipid (Fig. 13). DISCUSSION
The gross structure of the ear in Trichoguster is similar to the ears in representatives of other major teleost taxa [23,25-281 with the exception of one taxonomic group, the Ostariophysi [9,13,21]. The similarities between the gourami and non-ostariophysines (species in taxonomic groups other than the Ostariophysi) also extend to most inner ear features including the general shape and size relationships of the otolithic organs, the shape of the otoliths and their relationship to the sensory epithelia, the presence of sensory and supporting cells on the macula, and the division of the maculae into orientation groups [4,5,13,14,22-271. In addition, the ciliary bundles and the bodies of the sensory cells in Trichoguster closely resemble the same structures in the other teleosts that have been studied with both SEM and TEM including the goldfish, Carassiusauratus [8,21], a moray eel, Gymnothorax sp. [25], a cod, Gadus morhua [4] and several species of catfish [13]. While Trichoguster has many inner ear features in common with those found in other teleost fishes, several aspects of the ear in the gourami are of considerable significance and important to note. These include the lack of variation in the cell bodies, the cells containing numerous mitochondria found outside of the sensory region, and the orientation patterns of the ciliary bundles on the saccular macula. Cell bodies An important question relates to the nature of any differences in sensory cell bodies
at different regions of the sensory epithelium or that may be associated with the various types of ciliary bundles. Studies of a Carcharhinid shark have demonstrated the presence of dark and light sensory cells in the macula neglecta [3] and a similar observation has been made for the lagena of one teleost, a loach of unidentified species [32]. Investigations of a lamprey, Entosephenus japonicus, have shown that cells with different length ciliary bundles in that species have different types of organelles [ 111. The present study on i’khoguster, and an earlier investigation of Gymnothorux [23], have not, however, revealed the presence of dark and light sensory cells, nor is there any evidence for cytological differences in cells with different ciliary bundles. In fact, there is no evidence for any cellular differences in any region of either otolithic macula in Trichogaster. Thus, it now appears that in spite of differences in ciliary bundle lengths, all of the sensory cells in a number of different teleost species have similar cell bodies. Surface features
One of the most striking features of the ear in Tkichogaster is the orientation pattern
260
of the sensory hairs on the rostra1 end of the macula. The saccuiar sensory region in most fishes, other than the Ostariophysi, is elongate with a moderately enlarged anterior region containing the horizontally oriented hair cells [23,27]. The horizontally oriented cells in most of these species are organized so that the caudally oriented cell group is located on the dorsal quadrant of the macula, while the rostrally oriented group is on the ventral quadrant. The region containing horizontally oriented hair cells in Trichogaster, in contrast, is bulbous (Figs. 1 and 7), while the caudal half of the macula is very thin. The gourami-like hair cell o~entation pattern, invol~ng one group of rostral sensory cells in front of, rather than dorsal to, the other, is uncommon among fishes. It has only been reported in one other perciform, Colisa labiosa [34], another genus of the same subfamily as Trichogaster. Even more significant is the finding of a somewhat similar pattern of saccular hair cell o~entation in several species that are t~ono~ca~y distinct from the anabantids including the clown knife-fish (Notoptems chitda) [26] and several deep-sea myctophids, or lantern-fishes [23]. While data are severely limited, it is tempting to speculate that the specialized saecular hair cell orientation patterns found in the anabantids and the other species are, in some way, adapted for s~cialized,hearing abilities. In fact, the special hair cell orientation pattern may potentially be correlated with other specializations in auditory structures peripheral to the ear. For example, Trichogaster has a small bubble of air in the buccal cavity close to the ear that is thought to enhance auditory sensitivity [ 1,371. While structurally different, Noto~te~s has a swimbladder that terminates on the auditory bulla, a condition often associated with enhanced hearing capabilities [22]. Although hearing abilities are not known for the myctophids, the third group having a hair cell orientation pattern similar to that in ~c~ogaster, it has been suggested that members of the myctophid family use sound in communication {17f. The specific functional significance of the specializations in the horizontally oriented hair cells in Trichogaster and other species, as opposed to the more ‘standard’ pattern found in many other fishes, is unclear. The presence of ho~zont~y, as well as vertically, oriented sensory hair cells have been implicated in the detection of sound source direction (e.g. sound localization) [3-6,22,23,28] and it is possible that the differences seen among these specialized species represents a particular adaptation for extracting certain types of directional information, or directional information under certain types of acoustic conditions encountered in the environments of these species. It is also possible that the adaptions seen in Trichogaster have nothing to do with directional detection, but instead have bearing on basic mechanisms of acoustic analysis by the fish. It has been suggested ]22,28] that the sound processing in the teleost ear is complex and significantly affected by the mechanisms by which sound reaches the ear and the specific relationship between the otolith and the ciliary bundles. Any factors affecting these structures, such as the shape of the otolith or the sensory epithelium, the lengths of the ciliary bundles, or whether the ciliary bundles are directly under the otolith or in regions not covered by the otolith, could markedly affect the way that the fish detects and/or analyzes sounds [ 221. Consequently, it is possible that the variation seen in the morphology of the teleost ear reflects interspecific variation in the way that fishes use sound. However, little is known about sound detection and processing in any teleost species, and the differences in the use of sound by Trichogaster and Notoptems, as opposed to other species, are totally unknown.
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Supporting cells While the supporting cells within the sensory maculae in Trichogaster, and the epithe-
lial cells in the extramacular region, are typical of those seen in other fishes, the large mitochondria-filed cells in the extramacular region are not ubiquitous among teleosts. Cells of this type were not seen in the moray eel, nor were they reported in the sacculus of Carassius [9], or any of the otolithic organs of Gadus [4] or a lamprey [ll]. They have, however, been seen in a sunfish (Lepomis) (Popper, unpublished) and in several species of catfish (Jenkins, personal communication; Popper, unpublished). Furthermore, SEM studies of several other species, including the squirrelfish Myripristis, have demonstrated the presence of cells with similar surface features in extramacular regions
[231. The functional significance of these large cells is not known, and cells of similar types have not been reported in the mammalian ear (e.g. [12]) or in the teleost lateral line [9, 10,201. However, cells have been reported in the teleost lateral line [20], in the supporting region of the shark macula neglecta [3], and in the mammalian ear [ 12,16,30] which contain large numbers of mitochondria and highly invaginated basal ends. These cells in mammals have been associated with otoconia formation or reabsorption [ 16,301 or with the production or reabsorption of endolymph [18]. The role of such cells in fishes and sharks is also not known, but it has been suggested that they may have a secretory or reabsorptive role involved with cupula formation or roles in maintenance of the metabolic activity of the sensory cells [9,20]. It is reasonable to suggest, based upon the presence of extensive mitochondria and smooth endoplasmic reticulum, that the extramacular cells in Zlichogaster have high metabolic activity and are probably associated with secretion or absorption of materials. Their precise role, however, is not clear even after considering data from secretory cells in the ears of other vertebrates, and it will be necessary to await experimental studies on the teleost ear to determine the function of these cells. However, one potential area that should be considered when studying these cells is the likelihood that they may be involved in secretion of calcium carbonate for otolith formation or in the metabolism of endolymph. Virtually no data are available regarding both functions in the teleost ear, but we would expect such activity to involve cells with high metabolic activity and extensive secretory abilities. Finally, while it is clear that such cells are found in a number of taxonomically diverse species, they have not been reported in all species. Although it is possible that these cells were not noticed by earlier workers, it is more likely that they may only be present in fishes under certain conditions, such as during periods of rapid growth or perhaps during development of certain ear structures, such as the otoliths. Taxonomic significance The present TEM and SEM studies of the ear in Wchogaster are of considerable inter-
est and importance when we consider that these are the first TEM data for any member of one of the major advanced teleost orders [ 151, the Perciformes. With these data, we now have some information on the ultrastructure of the sensory epithelia of four diverse teleost taxonomic orders, the Gadiformes [4,7], the Ostariophysi [9,13], the Anguilliformes [25] and Perciformes. It is clear that there is substantial similarity in the cytology
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of the sensory and supporting cells among all of these groups. This is encountered in spite of the extensive variation found in the ears of the same species as determined with the SEM (e.g. [4,13,21,25]) and the considerable differences in the gross structure of their ears and more peripheral auditory structures. The significance of the similarities in the TEM observations of the sensory and supporting cells in these species may indicate that the major adaptive changes encountered in the ear are associated only with the regions of the ear actually involved in mechanical responses to acoustic energy, such as the ciliary bundles on the sensory cells and the orientation patterns of the hair cells, as well as in the structures more peripheral to these, such as swimbladder structure or the morphological relationship between the swimbladder and the inner ear. In contrast, the function of the cell bodies for the support of the ciliary bundles may be sufficient to operate in a wide range of different acoustic conditions. Thus, similarities in the cell bodies of the sensory hair cells may represent a conservative arrangement among fishes, as well as in the ears of most other vertebrates, that is not affected by selective pressures acting upon the sound detection and processing system of the ear. ACKNOWLEDGEMENTS
This work was supported by NSF grants BNS 79-17920 and BNS 80-09343, and by a U.S. Public Health Service Research grant NS-15090 and Career Development Award (NS 003 12) to A.N.P. REFERENCES [l] Bader, R. (1937): Bau, Entwicklung und Funktion des akzessorischen Atmungsorgans der Labyrinthflsche. Z. Zool. 149,323-401. [ 21 Buwalda, R.J.A. and van der Steen, J. (1979): The sensitivity of the cod sacculus to directional and nondirectional sound stimuli. Comp. Biochem. Physiol. 64A, 467-471. [3] Corwin, J.T. (1977): Morphology of the macula neglecta in sharks of the genus Carcharhinus. J. Morphol. 152,341-362. [4] Dale, T. (1976): The labyrinthine mechanoreceptor organs of the cod Gadus morhua L. (Teleostei: Gadidae). Norw. J. Zool. 24,85-128. [5] Enger, P.S. (1976): On the orientation of hair cells in the labyiinth of perch (Perca fluviarilis). In: Sound Reception in Fish, pp. 396-411. Editors: A Schuijf and A.D. Hawkins. Elsevier, Amsterdam. [6] Fay, R.R. and Popper, A.N. (1980): Structure and function in teleost auditory systems. In: Comparative Studies of Hearing in Vertebrates, pp. l-42. Editors: A.N. Popper and R.R. Fay. Springer-Verlag, New York. [7] Flock, A. (1964): Structure of the macula utriculi with special reference to directional interplay of sensory responses as revealed by morphological polarization. J. Cell Biol. 22,413-431. [ 81 Hama, K. (1965): Some observations on the fine structure of the lateral line organ in the Japanese sea eel, Lyncozmba nystromi. J. Cell Biol. 24,193-210. [ 91 Hama, K. (1969): A study on the tine structure of the saccular macula of the goldfish. Z. Zellforsch. 94,155-171. [lo] Hama, K. and Y. Yamada (1977): Fine structure of the ordinary lateral line organ II. The lateral line canal organ of the spotted shark,Mustelus manazo. Cell Tissue Res. 176,23-36. [11] Hoshino, T. (1975): An electron microscopic study of the otolithic maculae of the lamprey (Entosephenusjaponicus). Acta OtoIaryngol. 80,43-53. [ 121 Iurato, S. (1967): Submicroscopic Structure of the Ear. Pergamon Press, New York.
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