A Transmission and Scanning Electron Microscopic Study of the Saccule in Five Species of Catfishes DAVID B. J E N K I N S Department of Anatomy, T h e Milton S. Hershey Medical Center, T h e Pennsylvania State University, Hershey, Pennsylvania I7033

ABSTRACT The sacculi of five species of catfishes were studied by transmission and scanning electron microscopy. In four species, the sagitta exhibited a multifluted anterior part and a tapered posterior part; in Corydoras aeneus, however, the fluted part was absent, and a vertical component extended dorsally to terminate near the opening of the transverse canal. In all species, the otoliths had a laminar structure. An otolithic membrane was present, and hair cell bundles projected into cavities on the macular surface of the membrane. Attachments of the otolithic membrane to the neuroepithelium included short extensions of the membrane to the tallest components of the hair cell bundles of the peripheral cells and more delicate connections to the kinocilium and taller stereocilia of central cells; in addition, attachments to the microvilli of supporting cells were present. In both hair cells and supporting cells single microtubules and bundles of microtubules were present; the bundles had an orderly arrangement and were associated with cytoplasmic densities surrounding the desmosomes. The hair cells were innervated by both afferent and efferent nerve endings. Studies of the polarization of the hair cells in all species (except C. aeneus) showed that there was a single longitudinal axis that divided dorsally polarized cells from those oriented ventrally. In Doras spinosissimus and Bunocephalus bicolor, an additional line of polarization was evident in a small area in the anterior part of the macula; therefore, in these forms there was a double bipolar orientation

The inner ears of ostariophysans (e.g., catfish, goldfish, carp, minnows, loaches) are of special interest due to the auditory capabilities of these fish (Manning, '24; Stetter, '29; von Frisch, '38; Weiss et al., '69);these capabilities include broad band sensitivity and low thresholds, and they appear to be correlated with the Weberian ossicles (Weber, 1820) which link the swim bladder to the membranous labyrinth. Although many early reports were centered upon the Weberian apparatus, investigations of the membranous labyrinth have been limited and only a few studies have used electron microscopy. The first report concerning ultrastructure was that of Hama ('69) on the saccular macula of the common Japanese goldfish, Carassius auratus, and that was followed by studies on the lagenae of the Crussian carp, Carassius carassius (Terui and Saito, '71; Saito, '73), and a loach (Saito, '73), and the synaptic relationAM.

J. ANAT. (1979)154: 81-102

ships in the saccular macula of goldfish (Nakajima and Wang, '74; Hama and Saito, '77). Platt ('77) has recently reported on the inner ear of goldfish utilizing scanning electron microscopy. The basic morphology and histology of the inferior division of the membranous labyrinth (saccule and lagena) has been described in five species of catfishes by Jenkins ('77); that study recognized specific patterns of phylogenetic progression in the inferior division, especially in the saccule. The present study was undertaken to provide information on the ultrastructure of the sagitta, otolithic membrane and saccular macula and to determine whether there are ultrastructural changes Received No". 7 , '77. Accepted June 27, '78. This study was supported in part by USPHS Research Grant NS07860. 'Present address: Department of Anatomy, The University of North Carolina at Chapel Hill, 102 Building "D' 331H, Chapel Hill, North Carolina 27514.

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which parallel the phylogenetic progressions observed a t the light microscopic level. MATERIALS AND METHODS

were cut, oriented, and affixed to blank plastic stubs with epoxy cement. Thin sections (45-80 nm) were cut with a diamond knife, collected on 200-, or 75 by 300-mesh copper grids, stained with saturated aqueous uranyl acetate followed with lead citrate (Venable and Coggeshall, '651, and examined with a Philips 300 transmission electron microscope. One group of labyrinths fixed and decalcified as described above, as well as undecalcified specimens (post-fixation in osmium tetroxide omitted in both cases), were prepared for scanning electron microscopy. A second series of specimens was prepared for scanning electron microscopy utilizing the osmium-thiocarbohydrazide-osmium(OTO) procedure of Kelley et al. ('73). The tissues in both groups were then dehydrated (see below) and dried in a critical-point dryer. Some specimens were dehydrated in increasing concentrations of ethanol and then transferred through graded mixtures of ethanol and Freon 113 and two changes of Freon 113; the tissue remained in each solution for ten minutes. These tissues were then dried by the recommended procedure in a Bomar SPC900 critical-point dryer using Freon 13 as the transitional fluid. Other specimens were dehydrated in increasing concentrations of acetone and then placed in the chamber of a Samdri PVT-3 critical-point dryer. The chamber was immediately filled with liquid CO, and the tissue remained immersed in this fluid for five minutes; a continuous exchange of liquid CO, was maintained throughout this period. No difference in specimen quality was observed between the two processes. All specimens were oriented and affixed to aluminum stubs within two hours after drying. Any unwanted tissue, and remnants of otolith or otolithic membrane were carefully removed with fine forceps or a jet of Freon gas directed through a small-caliber (0.5 mm ID) plastic tube. The stubs and tissue were coated with a thin layer of palladium-gold alloy (40:60) in an Edwards evaporator or Hummer sputtering unit. The OTO-treated material was coated thinly, but, to avoid charging, other specimens were more thickly coated. The specimens were examined with an AMR900 or a JSM-35 scanning electron microscope.

One species from each of five families was used as a sampling of the three major taxonomic groups of catfishes described by Chardon ('68). The species selected were Kryptopterus bicirrhis (glass catfish - siluroid group), Bunocephalus bicolor (banjo catfish - primitive loricaroid), Corydoras aeneus (bronze catfish - advanced loricaroid), Zctalurus nebulosus (brown bullhead - primitive bagroid) and Dorm spinosissimus (talking catfish - advanced bagroid). The inner ears from adult (22-30 cm) specimens of Zctalurus nebulosus showed no marked differences, other than size, from the ears of smaller (5-8 cm) animals. For convenience, therefore, small specimens of all species were used. Each animal was sacrificed by decapitation. The head was then immersed in a cold (0-4 C") 4.0%solution of glutaraldehyde in 0.2 M s-collidine buffer (pH 7.3-7.4) containing 4.0% sucrose, and the ear was dissected partially to insure adequate exposure to the fixative. Blocks of tissue containing the otic capsules were dissected from the head and fixed for one hour. The membranous labyrinth was then dissected free and allowed to remain in fresh cold fixative overnight. Except for those specimens to be used for scanning electron microscopy of the sagitta, each was placed in a chelating agent, 0.1 M tetrasodium ethylenediamine tetraacetic acid (Na,EDTA) containing 4.0%glutaraldehyde (Baird et al., '67). The decalcifying solution was changed every second day until the otoliths were clear (usually 1 week). Specimens to be used for transmission electron microscopy were next rinsed in 0.2 M s collidine buffer for one hour, and then postfixed for one hour in a solution of 2.0%osmium tetroxide in similar buffer. The tissue was then placed in 3.0% neutral buffered formalin for one hour, dehydrated in 15-minute changes of ethyl alcohol G O % , 95%, loo%),and cleared in propylene oxide for 40 minutes. The specimens were transferred to a 50:50 mixture of propylene oxide and Epon 812 for one hour and then to undiluted resin in a vacuum chamber for an additional hour. The tissues were then placed in fresh resin in flat molds RESULTS and polymerization was completed during a 1. General morphology sequence of temperature increases (37'C- 16 hours, 45OC-8 hours, 80°C-2 days). Blocks The inferior division of the membranous containing the desired parts of the labyrinth labyrinth in catfishes consists of a saccule and

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lagena. In four of the species (except C. aeneus) the large ovoid lagena lies lateral to the elongate saccule. In K. bicirrhis, I. nebulosus and D. spinosissimus the lagena is positioned posteriorly, but in B. bicolor it lies adjacent to the anterior part of the saccule. In C. aeneus the lagena is positioned posterolateral to a spheroidal saccule. The inferior divisions of the right and left membranous labyrinths are united by a transverse canal. In all forms except C. aeneus the canal joins the dorsomedial aspect of the saccule, continues anteriorly on the saccular roof and communicates ventromedially with the saccular lumen through a narrow elongate opening. In C. aeneus the transverse canal crosses dorsal to the saccular roof and opens into the dorsolateral surface of the anterior part of the saccule by a small rounded foramen. 2. Sagitta In all species the saccule contains a solid otolith, the sagitta, which is positioned adjacent to an otolithic membrane overlying the saccular macula. In all forms except C. aeneus, described separately below, the sagitt a consists of a fluted anterior and a rod-like posterior part (fig. 1).Four flutes are usually present anteriorly, radiating from the body of the sagitta (Jenkins, '77); a fifth flute is exhibited in I. nebulosus. The delicate superior flute (figs. 1 , 2 ) extends dorsomedially from the sagittal body. At the level of the posterior extremity of the opening of the transverse canal into the saccule, the superior flute is inclined obliquely and attaches to the saccular wall ventral to the lumen of the canal. Anteriorly, the flute has a vertical orientation and extends dorsally into the anterior extremity of the lumen of the transverse canal. In I. nebulosus, lateral to the oblique part of the superior flute, a short vertical flute projects dorsally from the body of the sagitta into the saccular lumen (fig. 2); between the two flutes, a small elongate cavity is formed. The medial flute has a smooth medial edge (fig. 11, along which the otolithic membrane presumably attaches; it must be noted, however, that no remnants of the membrane were seen in any of the specimens examined. The broad, flat, lateral flute projects ventromedially; it is widest anteriorly, narrows gradually posteriorly, and extends for a short distance along the posterior part of the body of the sagitta (fig. 2). The inferior flute, the longest of the otolithic processes, extends ventromedially from the sagit-

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tal body; it is widest anteriorly and is absent only from the posterior tip of the sagitta. Anteriorly, its ventral edge is thickened, and the surface which attaches to perimacular epithelium ventral t o the macula is rough. In I. nebulosus, D. spinosissimus and B. bicolor the orientation of the inferior flute changes posteriorly in relation to the shift of the posterior part of the saccular macula from the medial wall toward the saccular floor. The change is most marked in B. bicolor, where the flute swings abruptly from a ventromedial to a ventrolateral position (fig. 31, reflecting the abrupt transition of the macula from a vertical to a horizontal orientation. On the medial surface of the anterior part of the sagitta, in the junctional area of the superior, medial and inferior flutes, large calcareous accumulations are present (figs. 1, 3). These irregular masses extend from the body of the sagitta, are attached to the adjacent surfaces of the flutes, and apparently act as supports to strengthen the delicate processes. Where the supports are present, the diameter of the body of the sagitta is also significantly increased. In B. bicolor, the superior and medial flutes, together with their supporting masses of otolithic material, blend posteriorly with a ridgelike enlargement of the sagitta (fig. 3). The ridge occurs a t the junction of the anterior and posterior parts of the otolith; the major part of the ridge projects dorsally, but it extends ventrally on both the medial and lateral surfaces of the sagitta. An anterior extension of the sagitta is evident (fig. l) and appears t o be a continuation of the body or central axis of the otolith. Its medial surface is rough and is associated with a small anteroventral projection of the saccula macula. Unlike the sagitta in the other forms, that in C. aeneus is massive and lacks an anterior fluted part (fig. 4).The horizontal component, however, shows a basic similarity to the posterior tapered part of the sagitta in the other species; in the area occupied by the posterior part of the inferior flute in species where the posterior part of the macula occupies the saccular floor, there is a ventrolateral ridge. In addition, a ventromedial ridge is evident along the length of the horizontal process. The ridges exhibit some similarities to the flutes of the sagittae in the other forms studied and are reinforced by globular accumulations of otolithic material. The concave area between the ventromedial and ventrolateral processes of

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the otolith is associated with the otolithic membrane overlying the saccular floor (fig. 4). Anteriorly, a columnar vertical component extends dorsally into close association with the orifice of the transverse canal. In all five forms, the matrix of the otolith is composed of arrays of layers of granular or fine filamentous material (fig. 5). The thickness of the layers varies, but each is demarcated from surrounding material by dense lines a t its junctions with adjacent layers. Centrally, near the axis of the sagitta and its buttresses, the internal structure becomes irregular, and the matrix material may be arranged in several different patterns. 3. Otolithic membrane

The sacular macula is covered by an otolithic membrane which is thickened centrally and thinner a t the periphery. The entire macular surface of the membrane is perforated, but on the otolithic surface, holes are present only peripherally. The otolithic surface of the central area is relatively smooth and, therefore, the holes are incomplete, being capped superiorly by otolithic membrane material but open on the macular surface. Generally, a single hair cell bundle protrudes into each cavity or hole (figs. 6, 7) and the tallest components of the bundles are attached to the membrane. Between adjacent bundles the membrane is affixed to the apical surface of the supporting cells (fig. 8). In addition, short processes of the membrane attach to the tallest components of the hair cell bundles of the marginal sensory cells.

4. Saccular macula Underlying the otolithic membrane is the saccular macula; it is bordered by cells with numerous microvilli and a single, short, central cilium. The macula is a continuous strip of neuroepithelium that shows positional differences among the species studied (Jenkins, '77). In C. aeneus, the macula lies primarily on the saccular floor, but also extends onto the posterior wall and the anterior wall and adjacent anterior part of the saccular roof. In the other four species, two distinct parts of the macula are evident (fig. 9) and the sensory epithelium is confined to the medial wall and floor of the saccule. The posterior part is large and oval shaped; in K. bicirrhis, it lies on the medial wall, but, in I. nebulosus and D. spinosissimus, some representation of the macula is evident on the saccular floor. In B.bicolor, the

entire posterior part of the macula is positioned horizontally. The posterior region is continuous with a narrow anterior part oriented vertically on the medial wall in all four forms. A short anterior projection (fig. 9) is continuous with the anteroventral extremity of the main macular area by a narrow neck; beyond the constriction, the extension expands dorsally. In all five forms, the macula is composed of both hair (sensory) cells and supporting (sustentacular) cells (fig. 10).The tall, columnar, hair cells have centrally located nuclei and are rounded basally (fig. 11);unlike the supporting cells, they do not reach the basal lamina. The nuclei of the supporting cells are located basally, and the apical part of each cell extends toward the saccular lumen between adjacent hair cells. Myelinated nerve fibers penetrate the basal lamina, pass between the basal parts of adjacent supporting cells, and make synaptic contact with the hair cells. Below the basal lamina a highly vascularized stroma is present. Each tall columnar supporting cell exhibits a slightly constricted supranuclear part which, a t its apical extremity, is somewhat expanded; these expansions frequently indent or overlap adjacent hair cells. The supranuclear parts of three to five supporting cells surround each hair cell, and a t the subapical level, long irregular processes interdigitate between adjacent supporting cells (fig. 12). In C. aeneus, intervening cells may be absent and the membranes of two hair cells are juxtaposed (fig. 13). On the luminal surface of each supporting cell are numerous microvilli and a short (0.410.46 pm) central cilium; only peripherally positioned tubules are evident in each cilium, and these extend into the apical portion of the cell. A centriole usually lies immediately below these tubules, parallel to the surface of the macula. The cytoplasm of the supporting cell is more electron-opaque than that of the hair cell but, except for its microtubular content (discussed below), the cytoplasmic organization is not remarkable. Beneath the centriole is a prominent Golgi complex which is generally confined to the compressed supranuclear region. In addition, numerous profiles of granular endoplasmic reticulum, mitochondria, vesicles and multivesicular bodies are scattered throughout the cytoplasm; these are most abundant in the basal part of the cell. Junctional complexes unite adjacent supporting cells and supporting cells and hair

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cells (figs. 14, 15). Immediately below the sac- often found in close association with the latcular lumen, the apposing membranes are sep- ter. Large numbers of vesicles are scattered arated by an intercellular space of 6-8nm; in throughout the cytoplasm of the hair cell, but the cytoplasm adjacent to this region of the these are most numerous in the basal region. Microtubules constitute a prominent feamembranes, no coalescence of cytoplasmic material was found. Beneath this area the in- ture in the cytoplasm of both the sensory and tercellular spaces are wider, the juxtaposed supporting cells. Single tubules, 20-22.5 nm in membranes are more electron-opaque, and diameter, lie primarily parallel to the longituthere is an accumulation of a fine filamentous dinal axis of the cell, but some do course material within the cytoplasm adjacent to the obliquely (fig. 19). These tubules have fremembranes. Still further basally, desmosomes quent curving profiles which suggests that they may be flexible. Other microtubules, in are present. The hair cells are basically columnar (27- bundled arrays, are more prominent and nu32 pm x 3.5-7 pm) but are slightly narrowed merous than the single tubules. Apically, in apically, possibly due to the expansion of the both hair cells and supporting cells, bundles of adjacent supporting cells a t this level. Pro- microtubules are present in planes both paraljecting from the luminal surface of each hair lel and perpendicular to the surface of the cell is a bundle of sensory hairs composed of macula (fig. 12). In the hair cell, the bundles numerous stereocilia and a single kinocilium. are limited to the peripheral part of the cytoThere are usually 30 to 40 stereocilia in each plasm by the cuticular plate. Between the apibundle (fig. 16),but in C. aeneus some bundles cal and basal portions of both hair cells and may have as many as 50 stereocilia. In tangen- supporting cells, the aggregations of tubules tial section (fig. 17) i t is evident that the course predominantly vertically, and their stereocilia are arranged in parallel rows and number is greater in supporting cells than in taper gradually toward the surface of the cell. hair cells. Basally in both cell types only a few The height of the stereocilia is ordered, in bundles are present and these lie in a plane steplike fashion, from the shortest on the side parallel to the basal lamina (fig. 20). The numof the cell opposite the kinocilium, to the ber of bundles in the basal regions of both cell longest adjacent to the kinocilium. Each types is fewer than that found apically. The number of microtubules in each bundle stereocilium contains a dense central core continuous basally with a conical rootlet. In varies considerably; as few as five and as longitudinal section, the rootlet appears as a many as thirty have been noted. Each tubule pair of diverging processes extending into but is usually pentagonal (some may be hexnot passing through a granular cuticular plate agonal) in cross section, with an outside diam(fig. 14). Numerous microtubules extend from eter of 19.5-22.5 nm and a n inside diameter of the underlying cytoplasm into the cuticular 8-10 nm (fig. 21). Adjacent tubules share a plate, but no continuity between these and the common wall and, frequently, the walls of peripheral tubules of the bundle are incomplete. rootlets of the stereocilia is evident. Unlike the stereocilia, the kinocilium does The bundles terminate in the cytoplasmic dennot taper near the surface of the cell (fig. 181, sities of desmosomes (fig. 151, and have been and it is generally as tall as the longest observed traversing the cell between two stereocilia (3.1-5.3 pm). Paired central tu- opposite desmosomes (fig. 22). bules terminate a t the junction of the luminal In C. aeneus there are numerous microtubuand basal parts of the kinocilium; peripheral lar bundles in the lateral perimacular cells tubules, however, extend into a basal body underlying the ventrolateral ridge of the which is embedded in the cytoplasm occupying sagitta (fig. 231. In the apical parts of these a notch in the cuticular plate. No basal foot cells, bundles lie predominantly parallel to the cell surface, although some enter the area was found in any of the forms examined. Generally, the hair cells can be said to con- obliquely. All aggregations of microtubules tain a typical complement of cytoplasmic or- appear to terminate in and contribute to denganelles. Throughout the cytoplasm are nu- sities associated with desmosomes. merous free ribosomes, multivesicular bodies 5. Nerve endings of diverse sizes, and elongate mitochondria. In the supranuclear area, scattered profiles of Nerve fibers innervating the macula are granular endoplasmic reticulum and a promi- characteristically myelinated beneath the banent Golgi complex are present; vesicles are sal lamina and unmyelinated within the neu-

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roepithelium, but myelinated fibers are occasionally present within the macula. Fibers penetrating the thin, filamentous, basal lamina (fig. 10) course between adjacent supporting cells and make synaptic contacts with the basal regions of hair cells. Many fibers have multiple synaptic endings on one or two sensor y cells. Two types of nerve endings are present in catfishes, and both types can be observed in synaptic relationship with the same hair cell. The first type (fig. 20) is evident in all parts of the saccular macula. It is variable in size and essentially devoid of vesicles. Bordering a synaptic cleft of 19-21nm, both the membrane of the hair cell and that of the nerve ending are thickened; the membrane of the nerve terminal is more electron-opaque than that of the hair cell (fig. 20). Associated with the synaptic site is a spheroidal synaptic body, a dense, osmiophilic, granular structure situated in the hair cell immediately adjacent to the synaptic membrane. Small, round vesicles (40-48 nm) surround the synaptic body (figs. 20, 2426). Vesicles lying between the synaptic body and synaptic membrane are usually separated by projections of the synaptic body, but profiles of two vesicles are sometimes evident between adjacent processes (fig. 20). The stalk of each projection extends toward the synaptic membrane and terminates in a bulbous enlargement separated from the membrane by a distance of 3-4.5 nm. The membrane of the hair cell adjacent to that enlargement bulges slightly into the synaptic cleft, but due to a corresponding depression i n t h e opposing membrane of the nerve ending, a uniform width of the cleft is maintained. In some cells clusters of synaptic bodies occur, and as many as fourteen have been seen in a single cluster (fig. 24). Multiple synaptic bodies are frequently associated with synapses of two hair cells with a single nerve ending (fig. 25). When close alignment of synaptic bodies does occur, only a single layer of vesicles is usually evident between adjacent bodies. The second type of nerve ending is also present in all areas of the saccular macula but is less numerous than the first type. It is characterized by large numbers of round vesicles (4055 nm) distributed throughout a bulbous terminal (figs. 26, 27); in some cases vesicles are closely aligned along the synaptic membrane. Few organelles are evident in the ending although mitochondria are occasionally present. A synaptic space of 15-20nm separates the ap-

posing synaptic membranes, which are only slightly thicker and more electron-opaque than the adjacent membranes of the hair cell and nerve terminal. A flattened channel, the subsynaptic cistern, is positioned parallel to the synaptic cleft in the hair cell. The cistern varies in length and width, but its extremities and central region are usually enlarged. Dense granular accumulations resembling ribosomes are positioned on the side of the cistern opposite the synaptic site. 6. Hair cell polarization The eccentric position of the kinocilium in the hair cell bundle (adjacent to the tallest stereocilia) provides each hair cell with a morphological and physiological polarization. The morphological polarization of hair cells was studied in four species (excluding C. aeneus). The general pattern in the saccular macula is basically a bipolar orientation along a single longitudinal axis (fig. 28A); the hair cells dorsal to the axis are polarized dorsally while those on the ventral side are polarized in the opposite direction. In B. bicolor, however, the polarization in the posterior part of the macula is in a medial-lateral direction due to the horizontal position of that region of the macula. In all forms the axis of polarization curves dorsally from the midline in the anterior part of the macula. The most peripheral hair cells are frequently positioned so that their vector of polarization is perpendicular to the curving edge of the macula. In two species, D. spinosissimus and B. bicolor, a significant variation occurs in a small anterior area of the macula. The hair cells on the dorsal and ventral edges of the macula are polarized dorsally and ventrally, respectively, as is found in the other forms; in the central region of the area, however, the cells are polarized toward the midline. This arrangement creates three axes of polarization and a double bipolar orientation pattern (fig. 28B). DISCUSSION

The complex internal matrix of the sagitta has not been observed previously a t the transmission electron microscopic level. The internal structure is basically similar in all five species of catfishes, and it resembles the laminated appearance of individual otoconia in the mammalian ear (Lim, ’73a). The arrangement of electron-opaque lines and intervening, wide, relatively electron-lucent bands may be interpreted as the “growth rings” described by

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Pannella ('71) in fish a t the light microscopic level. The light bands presumably correspond to the fast-growth zones and the opaque lines to the slow-growth zones. The gelatinous layer (slow-growth band) described by Lim ('74) in an otolith of a goldfish was not apparent in the catfishes. The attachments and position of the sagitta and its flutes are of comparative and functional significance. Although variations in the shape of the sagittae and the fluting pattern occur, similarities in the function of various parts of the otolith can be proposed. Several of the processes of the sagitta provide attachment to the perimacular epithelium and, therefore, maintain the relationship of the sagitta to the adjacent otolithic membrane and saccular macula. In four of the species (except C. aeneus), the widened margin of the inferior flute, for example, i s roughened where it is associated with the perimacular epithelium. This provides the basis for a firm attachment of the extracellular material that binds the otolith to the perimacular cells, and tends to support the concept (Jenkins, '77) that this attachment establishes the pivotal axis of the sagitta. A similar function is shared by the ventrolateral ridge of the horizontal component of the sagitta in C. aeneus. This process anchors the otolith to the lateral perimacular epithelium and establishes an axis of rotation a t that attachment. The delicate superior flute, which is not present in C. aeneus, provides an additional attachment of the sagitta ventral to the macula; such an attachment may limit torsion around the axis at the foot of the inferior flute. The oblique part of the superior flute is affixed immediately below the orifice of the transverse canal and thus lies in the expected path of fluid displacement. The lateral flute in K. bicirrhis, I. nebulosus, D. spinosissimus and B. bicolor and the vertical flute in 1. nebulosus project directly into the path of expected fluid disturbances and should provide additional areas for impingement of fluid on the sagitta to augment rotational movement. In C. aeneus, where the anterior fluted part of the sagitta is absent, the dorsal end of the vertical component of the otolith lies close to the opening of the transverse canal and is in position to be directly influenced by fluid displacements. This component, therefore, functionally replaces the superior, lateral and vertical flutes in the other species. Forces applied to the vertical arm should produce rotation at

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the line of attachment of the horizontal component to the perimacular cells and result in a "rocking" of the entire otolith. In light of the accepted view of the function of the otolith/otolithic membrane complex (van Bergeijk, '671, i t is important to determine the sites of attachment of the sagitta to the otolithic membrane; however, due in part to shrinkage during tissue preparation, which has been reported in the inner ear to range from 10-215: (Hunter-Duvar, '77; Platt, '771, some of the areas of attachment were difficult to determine. In the posterior region of the saccule in K. bicirrhis, 1. nebulosus, D. spinosissimus and B. bicolor, the membrane is affixed to the nonfluted surface of the otolith; the same relationship is present between the membrane and the horizontal component of the sagitta in C. aeneus. Anteriorly (except in C. aeneus where the flutes are absent), light microscopy (Jenkins, '77) suggests attachment of the membrane to the medial flute; in tissues examined with the scanning electron microscope, however, no remnants of the otolithic membrane were observed along the smooth edge of the flute. If such an attachment of the membrane in this region is present, it is more delicate than that found posteriorly and was detached during tissue preparation. Attachment of the otolithic membrane to the periphery of the macula by fingerlike projections to the marginal hair cell bundles has not been described previously, but it appears to constitute a very stable connection. On the surface of the otolithic membrane, the cavities into which single hair cell bundles project are similar to those described in the utricular otolithic membrane of the cod (Dale, '76, '77) and the tectorial membrane of the bullfrog's basilar papilla (Frishkopf and Flock, '74). Contact of the distal ends of the kinocilium and stereocilia of each bundle with the otolithic membrane provides additional attachments of the membrane to the macula. Similar attachments, a t least with the kinocilia, have been reported in reptiles and birds (Dohlman, '71; Bagger-Sjoback and Wersall, '73; BaggerSjoback, '74; Baird, '74a; Tanaka and Smith, '75). In the organ of Corti of mammals the kinocilium is absent but contact of the tectorial membrane with the stereocilia of the outer hair cells (Kimura, '66; Lim, '72, '77; Ross, '74; Hoshino, '74, '771, and to some extent the inner hair cells (Ross, '74; Hoshino, '74, '761, has been described.

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Transmission electron microscopy has shown that in catfishes the otolithic membrane extends to the apical surface of the supporting cells; such extensions (which form the walls of the cavities housing the hair cell bundles) resemble the "veils" of membrane described in birds and mammals by Dohlman ('71) and Lim ('73b). Except for the lateral perimacular cells in C. aeneus, no contact of the membrane with peripheral supporting cells, similar to that described in the basilar papilla of the bullfrog (Frishkopf and Flock, '741, has been observed. The attachment of the otolithic membrane to the macula provides a mechanical link between the membrane and hair cell bundles; this, coupled with attachment of the membrane to the sagitta, establishes an arrangement whereby rotational movements of the otolith during vibratory stimulation could be transmitted as shearing forces to the ciliary bundles. The relationships are such that the hair cell processes should be bent in planes essentially transverse to the long axis of the receptor; such movement would generally correspond to the polarization patterns observed in the macula and, according to the studies of Flock ('711, should constitute the most effective stimulus for the sensory cells. In the five forms investigated, the basic characteristics of the neuroepithelium of the saccular macula conform to the description of a "primitive" sensory epithelium (Baird, '74a,b). Within the cytoplasm of both the hair cells and supporting cells, however, both single microtubules and bundles of microtubules are evident. Although Hama ('69) described structures in the saccular macula of the goldfish which he considered to be aggregates of either filaments or tubules, no measurements or descriptions of orientation were provided. In this study, the presence of single tubules, their similarity in size to individual units of the bundles, and the appearance of the tubular units in cross section, all suggest that the bundles are specialized aggregates of microtubules. Baird ('67, '69, '70a,b, '74a,b) has suggested that groups of single microtubules serve a cytoskeletal function and contribute structural stability to the auditory receptors in various reptiles. In catfishes, it seems that the role has been assumed by the bundles of microtubules since they course considerable distances through the cells and terminate in the densities adjacent to desmosomes; some bundles are evident traversing

the full width of cells between desmosomes. Such attachments would appear to stiffen individual cells and contribute to the overall strength of the neuroepithelium. In addition, in C. aeneus, large populations of bundles are evident a t the apical ends of the perimacular cells to which the massive sagitta is affixed. Their arrangement, predominantly parallel to the luminal surface, suggests that the tubules act in strengthening this area to assist in support of the sagitta. Hillman ('77) has recently reported microtubules in peripheral supporting cells to which the cupula attaches in Rana catesbeiana; there the tubules terminate in association with junctional complexes, but, unlike those in C. aeneus, are arranged parallel to the longitudinal axis of the cell. With regard to the two types of nerve endings, the first, similar to Hama's ('69) type I ending, resembles synapses commonly present and considered to be afferent in the labyrinthine neuroepithelia of lower vertebrates (Flock, '71; von During et al., '74; Nakajima and Wang, '74; Hoshino, '75; Hama and Saito, '77). The most prominent feature a t the synaptic site is a synaptic body surrounded by vesicles similar in size to those reported in the saccular hair cells of the goldfish (Nakajima and Wang, '74; Hama and Saito, '77). No consideration has previously been given to the relation of the presynaptic membrane to the small dense bodies which are situated between the synaptic body and synaptic membrane and are connected to the body by a narrow stalk. In catfishes, these processes are separated from the membrane by a space of 34.5 nm and, therefore, do not lie directly upon the presynaptic membrane. The second type of nerve terminal, equivalent to Hama's ('69) type I1 ending, has the structural characteristics of efferent endings identified in the ears of other vertebrates (Smith and Takasaka, '71; von During et al., '74; Nakajima and Wang, '74). Notable are the accumulation of vesicles in the terminal, absence of vesicles in the related area of the hair cell, and the presence of a subsynaptic cistern within the hair cell. The size of the vesicles (40-50 nm) contained in these terminals is intermediate to those in the goldfish; Hama ('69) reported a range of 60-70 nm, while Nakajima and Wang ('74) found notably smaller vesicles (31-36 nm). The general pattern of polarization of hair cells of the saccular macula in the goldfish, has been described (Hama, '69; Platt, '77) as

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an orientation a t right angles to a central, longitudinal axis; only a slight dorsal deviation of the axis occurs in the anterior part of the macula (Platt, '77). This generalized pattern corresponds to studies of the saccule in other vertebrates (Lowenstein et al., '64; Jargensen, '70, '74; Jargensen and Andersen, '73) but differs greatly from the patterns reported in flatfish (Jargensen, '761, cod (Dale, '76) and other non-ostariophysans (Popper, '76, '77). In the present study the predominant pattern is a dorsal-ventral orientation along a longitudinal axis similar to that in the goldfish; however, the axis curves dorsally in the anterior part of the macula so that more cells are polarized ventrally than dorsally. In addition, a t the periphery of the macula of catfishes the cells deviate from the axis as noted in the previous section. The functional significance of such variation is not known. There are differences in the polarization patterns of hair cells in the saccular maculae not only among ostariophysans but also among non-ostariophysans; the double bipolar orientation in D. spinosissimus and B. bicolor, and the single bipolar orientation in the other species of catfishes, demonstrate that significant variations occur among closely related fishes. The double bipolar pattern has not been reported in other fishes, but does occur in the basilar papillae of certain lizards (Baird, '74a; Miller, '74). The presence of this rather complex polarization pattern in catfishes, where the ossicular chain is closely linked to the saccule, suggests that the orientation may be related to specialized hearing capabilities. It has been reported (Jenkins, '77) that, in catfishes, the structure of the membranous labyrinth exhibits variations which are generally in accord with the phylogenetic progression proposed by Chardon ('68); those changes in gross morphology observed a t the light microscopic level, and supported by scanning electron microscopic results of the present study, include: variation in the position of the lagena relative to the saccule, change in the shape of the saccule and sagitta, and variation in the position of the saccular macula. The most notable changes have occurred in the advanced loricaroid, C. ueneus. Although ultrastructural variations have been noted in saccular structures among the five species, none of these variations Le., double bipolar orientation observed in the advanced bagroid, D. spinosissimus, and primitive loricaroid, B. bicolor) follow t h e proposed phylogenetic

lines. Such findings are in agreement with the premise that ultrastructural characteristics of sensory epithelia of the membranous labyrinth show only relatively minor modifications in fish and amphibians (Baird, '74a,b). ACKNOWLEDGMENTS

The author wishes to thank Doctor I. L. Baird of The M. S. Hershey Medical Center of The Pennsylvania State University for support and advice during the major part of this study and Doctor 0. W. Henson, Jr., of The University of North Carolina a t Chapel Hill for critical review of the final manuscript. LITERATURE CITED Bagger-Sjoback, D. 1974 The sensory hairs and their a t tachments in the lizard basilar papilla. Brain, Behav. and Evol., 10: 88-94. Bagger-Sjoback, D., and J. Wersall 1973 The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calotes uersicolor. J. Neurocytol., 2: 329-350. Baird, I. L. 1967 Some histological and cytological features of t h e basilar papilla in the lizard,Anolis carolinensis. Anat. Rec., 157: 208-209. 1969 Some findings of comparative fine structural studies of the basilar papilla in certain reptiles. Anat. Rec., 163: 149. 1970a The anatomy of the reptilian ear. In: Biology of the Reptilia. Vol. 2. C. Gans and T. S. Parsons, eds. Academic Press, London-New York, pp. 193-275. _ _ 1970b A preliminary report on light and electron microscopic studies of a crocodilian basilar papilla. Anat. Rec., 166: 274. - 1974a Anatomical features of the inner ear in submammalian vertebrates. In: Handbook of Sensory Physiology. Vol. V/1. W. D. Keidel and W. D. Neff, eds. Springer-Verlag, Berlin-Heidelberg-New York, pp, 159-212. 1974b Some aspects of t h e comparative anatomy and evolution of the inner ear in submammalian vertebrates. Brain, Behav. and Evol., 10: 11-36. Baird, I. L., W. B. Winborn and D. E. Bockman 1967 A technique of decalcification suited to electron microscopy of tissues closely associated with bone. Anat. Rec., 159: 281-289. Bergeijk, W. A. van 1967 The evolution of vertebrate hearing. In: Contributions to Sensory Physiology. Vol. 2. W. D. Neff, ed. Academic Press, New York-London, pp. 1-49. Chardon, M. 1968 Anatomie compark de l'appareil de Weber e t des structures connexes chez les Siluriformes. M u s k Royal de 1'Afrique Centrale-Terouren, Belgique Annales-Serie In-8". Sciences Zoologiques, 169: 1-273. Dale, T. 1976 The labyrinthine mechanoreceptor organs of the cod Gadus morhua (Teleostei: Gadidae). Norw. J. Zool., 24: 85-128. - 1977 Functional-morphological correlations in acoustico-lateralis sensory organs. IITRUSEM, IZ: 445-452. Dohlman, G. F. 1971 The attachment of t h e cupulae, otolith and tectorial membranes to the sensory cell areas. Acta Otolaryng., 71: 89-105. During, M. von, A. Karduck and H.-G. Richter 1974 The fine structure of the inner ear in Caiman crocodilus. 2. Anat. Entwick1.-Gesch., 145: 41-65.

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Flock, A. 1971 Sensory transduction in hair cells. In: Handbook of Sensory Physiology. Vol. I. W. R. Loewenstein, ed. Springer-Verlag, Berlin-Heidelberg-New York, pp. 396-441. Frisch, K. von 1938 Uber die Bedeutung des Sacculus und der Lagena fur den Gehorsinn der Fische. Z. vergl. Physiol., 25: 703-747. Frischkopf, L. S., and A. Flock 1974 Ultrastructure of t he basilar papilla, an auditory organ in t he bullfrog. Acta Otolaryng., 77: 176-184. Hama, K. 1969 A study on the fine structure of the saccular macula of the goldfish. Z. Zellforsch., 94: 155-171. Hama, K., and K. Saito 1977 Fine structure of the afferent synapse of the hair cells in the saccular macula of the goldfish, with special reference to t he anastomosing tubules. J. Neurocytol., 6: 361-373. Hillman, D. E. 1977 Relationship of the sensory cell cilia to the cupula. IITRI/SEM, 11: 415-420. Hoshino, T. 1974 Relationship of the tectorial membrane to the organ of Corti. A scanning electron microscope study of cats and guinea pigs. Arch. Histol. Jap.,37: 25-39. 1975 An electron microscopic study of t h e otolithic maculae of the lamprey (Entosphenus japonicusi. Acta Otolaryng., 80: 43-53. 1976 Attachment of t he inner sensory cell hairs to the tectorial membrane. ORL, 38: 11-18. 1977 Contact between the tectorial membrane and the cochlear sensory hairs in t he human and t he monkey. Arch. Oto-Rhino-Laryng., 21 7: 53-60. Hunter-Duvar, I. M. 1977 Morphology of thenormal and the acoustically damaged cochlea. IITRIBEM, 11: 421-428. Jenkins, D. B. 1977 A light microscopic study of the saccule and lagena in certain catfishes. Am. J. Anat., 150: 605-630. Jmgensen, J. M. 1970 On the structure of t he macula lagenae in birds with some notes on the avian maculae utriculi and sacculi. Vidensk. Meddr. dansk naturh. Foren., 133: 121-147. 1974 The sensory epithelia of t he inner ear of two turtles, Testudo graeca L. and Pseudemys scripta (Schoepff). Acta Zool., 55: 289-298. 1976 Hair cell polarization in the flatfish inner ear. Acta Zool., 57: 37-39. Jmgensen, J. M., and T. Andersen 1973 On the structureof the avian maculae. Acta Zool., 54: 121~130. Kelley, R. O., R. A. F. Dekker and J. G. Bluemink 1973 Ligand-mediated osmium binding: Its application in coating biological specimens for scanning electron microscopy. J. Ultrastruct. Res., 45: 254-258. Kimura, R. S. 1966 Hairs of the cochlear sensory cells and their attachment t o t he tectorial membrane. Acta Otolaryng., 61: 55-72. Lim, D. J. 1972 Fine morphology of the tectorial membrane: Its relationship to the organ of Corti. Arch. Otolaryng., 96: 199-215.

1973a Formation and fate of the otoconia: Scanning and transmission electron microscopy. Ann. Otol. Rhinol. Laryngol., 82: 23-35. 1973b Ultrastructure of the otolithic membrane and the cupula: A scanning electron microscopic observation. Adv. Oto-Rhino-Laryng., 19: 35-49. 1974 The statoconia of the non-mammalian species. Brain, Behav. and Evol., 10: 37-51. 1977 Current review of SEM techniques for inner ear sensory organs. IITRUSEM, II: 401-408. Lowenstein, O., M. P. Osborne and J. Wersall 1964 Structure and innervation of the sensory epithelia of the labyrinth in the thornback ray (Raja clauatat. Proc. Roy. SOC.B., 160: 1-12. Manning, F. B. 1924 Hearing in the goldfish in relation t o the structure of its ear. J. Exp. Zool., 41: 5-20. Miller, M. R. 1974 Scanning electron microscopy of the lizard papilla basilaris. Brain, Behav. and Evol., 10: 95-112. Nakajima, Y., and D. W. Wang 1974 Morphology of afferent and efferent synapses in hearing organ of the goldfish. J. Comp. Neur., 156: 403-416. Pannella, G. 1971 Fish otoliths: Daily growth layers and periodical patterns. Science, 173: 1124-1127. Platt, C. 1977 Hair cell distribution and orientation in goldfish otolith organs. J. Comp. Neur., 172: 283-298. Popper, A. N. 1976 Ultrastructure of the auditory regions in the inner ear of the lake whitefish. Science, 192: 1020-1023. 1977 A scanning electron microscopic study of the sacculus and lagena in the ears of fifteen species of teleost fishes. J. Morph., 153: 397-418. Ross, M. D. 1974 The tectorial membrane of the rat. Am. J. Anat., 139: 449-482. Saito, K. 1973 Fine structure of macula of lagena in the teleost inner ear. Acta Anat. Nip., 48: 1-18. Smith, C. A,, and T. Takasaka 1971 Auditory receptor organs of reptiles, birds, and mammals. In: Contributions to Sensory Physiology. Vol. 5. W. D. Neff, ed. Academic Press, New York-London, pp. 129-178. Stetter, H. 1929 Untersuchungen uber den Gehorsinne des Fische besonders vonPhorinus laeuis L. undAmiurus nebulosus RAF. Z. vergl. Physiol., 9: 339.477. Tanaka, K., and C. A. Smith 1975 Structure of the avian tectorial membrane. Ann. Otol. Rhinol. Laryngol., 84: 287-296. Terui, S.,and K. Saito 1971 Endoplasmic reticulum accompanied with vesicles observed in hair cells of the inner ear of the lagena of the Crussian carp. Acta Anat. Nip., 46: 359-367. Venable, J. H., and R. Coggeshall 1965 A simplified lead citrate stain for use in electron microscopy. J. Cell Biol., 25: 407-408. Weber, E. H. 1820 De aure et auditu hominis e t animalium. Pars I. De aure animalium aquatilium. Lipsiae. Weiss, B. A., W. Strother and G. Hartig 1969 Auditory sen. sitivity in the bullhead catfish (Ictalurus nebulosus). Proc. Natl. Acad. Sci., 64: 552-556.

PLATES

Abbreviations bl, basal lamina cp, cuticular plate h, horizontal component hc, hair cell if, inferior flute k, kinocilium

If, lateral flute mf, medial flute ne, nerve ending nf, nerve fiber om, otolithic membrane r, rootlets sag, sagitta

sb, synaptic body sc, supporting cell sf, superior flute ssc, subsynaptic cistern v, vertical component vf, vertical flute

PLATE 1 EXPLANATION OF FIGURES

1 Medial view (anterior to the right) of the sagitta in I. nebulosus. Note the superior (sf), medial imf), inferior (if) and lateral (If) flutes and t h e anterior extension (arrow) projecting from the central axis of the otolith. X 21.

2 Dorsolateral view (anterior to the bottom right) of the sagitta in I. nebulosus showing the lateral (If),vertical (vf) and superior (sf) flutes. Note the continuation of the lateral flute (large arrowhead) onto the posterior part of sagitta and the oblique (small arrowhead) and vertical (small double arrowhead) parts of the superior flute. X 40. 3 Medial view (anterior to the right) of the junction of the anterior and posterior parts of the sagitta in B. bicolor. Superior isf) and inferior (if) flutes and buttresses (solid star) are related to the ridgelike accumulation of calcareous material (arrows). Note the shift of t h e inferior flute from a ventromedial to a ventrolateral position (open star) and the position of the medial (mf) and lateral (If) flutes. x 140. 4 Medial surface (anterior to the right) of the sagitta in C. aeneus showing the vertical (v) and horizontal (h) components. A ventromedial (star) and ventrolateral process (double star) are evident along the horizontal component; note the remnants (arrow) of otolithic membrane between t h e processes. x 90.

5 Transmission electron micrograph of a decalcified sagittal flute in D. sprnosissirnus showing the laminated appearance and electron-opaque lines (arrowheads) t h a t separate the bands of delicate fibrillar material of the otolithic matrix. X 2,690. 6 Scanning electron micrograph of the otolithic membrane overlying the anterior part of the saccular macula in B. bicolor. The central thickened part of the membrane has a smooth luminal surface but ciliary bundles (arrowhead) project into perforations in the thin peripheral region. Note the attachments (arrows) of the membrane to peripheral ciliary bundles. X 2,080,

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PLATE 1

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PLATE 2 EXPLANATION OF FIGURES

7 Peripheral part of t h e otolithic membrane in I. nebulosus. Note the projection of single ciliary bundles (arrowheads) into perforations in the membrane. X 5,600. 8 Transmission electron micrograph of a longitudinal section through t h e apical parts of two hair cells and interposed supporting cell (sc) in K. bicirrhis. Note the cavities in t h e otolithic membrane (om) into which the ciliary bundles extend and attachments of the membrane to components of the ciliary bundle of the hair cell (arrowheads) and microvilli of the supporting cell (arrow). X 6,610.

9 Scanning electron micrograph of the saccular macula in K . bicirrhis; the anterior end is to the upper left and the dorsal margin is toward the top of the micrograph. Note the large oval posterior part (double arrowhead), elongate anterior part (arrowhead) and short anterior projection (star). X 73. 10 Fractured preparation of the saccular macula in I. nebulosus. Shown are the columnar hair cells (hc), nerve fibers (nf), nerve ending h e ) , areas of synaptic clefts (arrowheads) bases of supporting cells (4, and the shelf-like basal lamina (bl). Vessels lie immediately below the basal lamina. X 2,610. 11 Fractured preparation of the saccular macula in I. nebulosus. Evident are two hair cells (hc) and the apical part of an intervening supporting cell (sc). X 3,000.

12 Transverse section through the apical region of a hair cell (hc) and surrounding supporting cells (sc) in I. nebulosus showing t h e interdigitating processes of the supporting cells (arrowheads). Cross-sections of peripheral bundles of microtubules in the hair cell (arrows) are not numerous a t this level. X 8,420.

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PLATE 3 EXPLANATION OF FIGURES

13 Transverse section showing the central regions of the hair cells (hc) and supporting cells (sc) in the saccular macula of C. aeneus. Intervening supporting cells are absent and t h e membranes of t h e hair cells (arrowheads) a r e closely apposed: this arrangement is not typical but does occur in the macula. X 6.700. 14 Micrograph of a longitudinal section through the apical region of a hair cell in K. bicirrhis. Rootlets (r) of the stereocilia are evident in the cuticular plate (cp). Note the junctional complex showing zones of closely apposed (star) and widely separated (double star) membranes. x 20,690.

15 Longitudinal section of the macula in L nebulosus illustrating the microtubular bundles and their association with t h e desmosomes (star). Also evident is the closely apposed region (arrowhead) of the junctional complex immediately below the macular surface. X 20,400. 16 Ciliary bundle of a hair cell in the saccular macula of C. aeneus. Note the step-like progression of the stereocilia and the remnants of otolithic membrane attached to the taller components of the bundle (arrowhead). X 15,000. 17 Oblique section through the surface of the saccular macula in I. nebulosus. Stereocilia with dense central cores (arrowheads), a kinocilium (k)and a supporting cell (sc) are evident. The stereocilia taper near the cell surface and their dense cores form rootlets (r) which penetrate the cuticular plate. X 12,850.

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PLATE 3

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PLATE 4 EXPLANATION OF FIGURES

18 Longitudinal section of a kinocilium projecting from the apex of a hair cell in K. bicirrhis. An adjacent supporting cell (sc) is joined to a second supporting cell by a prominent junctional complex. The central tubules of the kinocilium (arrow) terminate above t h e cell surface. X 9,920. 19 Apical region of a hair cell in 1. nebulosus. Evident are t h e cuticular plate (cp) and single microtubules (arrowhead). X 36,450. 20 Longitudinal section of t h e basal region of a hair cell in 1. nebufosus. Microtubular bundles lie approximately parallel to the base of the cell. A synaptic body (sb) is related to a thickened presynaptic membrane. Note the profiles of two vesicles (arrowheads) between adjacent processes and the continuity of t h e processes with t h e synaptic body and their separation from t h e presynaptic membrane. X 37,620. 21 Longitudinal section of a lateral perimacular cell in C. aeneus demonstrating microtubular bundles. Note the absence of t h e outer wall of a peripheral tubule (arrowhead). X 39,660. 22 Transverse section through the subapical region of a hair cell and supporting cell (sc) in I. nebulosus. A microtubular bundle traverses t h e hair cell and terminates (arrowheads) i n densities adjacent to desmosomes. Large bundles of microtubules are present in the supporting cell (arrow). x 9,070. 23 Longitudinal section of t h e apical region of the lateral perimacular cells in C. aeneus. Microtubular bundles are oriented both obliquely (at bottom of micrograph) and parallel to the cell surface (arrowheads). Densities surrounding the desmosomes are prominent (stars). Note t h e attachment of the sagitta (sag) to the surface of t h e cell by a n intervening extension of t h e otolithic membrane (om). x 10,120.

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PLATE 4

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PLATE 5 EXPLANATION OF FIGURES

24 Multiple synaptic bodies in t h e basal portion of a hair cell in I. nebulosus. Although the section is tangential to t h e synaptic bodies in the lower left of the micrograph (arrowheads), the vesicles maintain a circular arrangement around each body. x 39,000.

25 Longitudinal section of a synapse of a single nerve ending with two hair cells (hc) in I. nebulosus. Evident are the thickened membranes of both the nerve terminal and hair cells and a single row of vesicles between adjacent synaptic bodies. X 39,900. 26 Oblique section of the basal region of a hair cell in K. bicirrhis. Vesiculated nerve endings (stars) are typically associated with subsynaptic cisterns (ssc) showing densities (arrowhead) aligned on one membrane. The adjacent synapse a t the left shows a synaptic body and t h e different structure of t h e nerve ending. x 30,320.

27 Vesiculated nerve ending in I. nebulosus. Note the subsynaptic cistern (arrowhead) within the hair cell. X 71,400. 28A Schematic representation of the polarization pattern of the hair cells of the saccular macula in K. bicirrhis and I. nebulosus. Note the orientation away from the longitudinal axis and the dorsal curve of the axis in the anterior part of the macula. B Polarization pattern of the anterior part of the macula in D.spinosissirnus and B. bicolor. Aside from the double bipolar organization anteriorly t h e remainder of t h e saccular macula in these forms has the same pattern depicted in figure 28A.

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dor. lant.

A

B

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