THE ANATOMICAL RECORD 249:117–127 (1997) Golgi–Canalicular Reticulum System in Ion Transporting Fibrocytes and Outer Sulcus Epithelium of Gerbil Cochlea SAMUEL S. SPICER* AND BRADLEY A. SCHULTE Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina ABSTRACT Background: Five types of highly specialized fibrocytes have been identified in the spiral ligament of the gerbil cochlea. Type I, II, and IV fibrocytes function in cycling back to the stria vascularis K1 effluxed from outer hair cells and nerves during auditory transduction. Thus, evidence exists for a transcellular path of K1 movement from outer sulcus cells through fibrocytes to the strial interstitial space, but a mechanism for facilitating such ion flow within the cells has not been elucidated. Methods: The spiral ligament of glutaraldehyde–osmium tetroxide-fixed and Epon-embedded gerbil cochlea was examined by transmission electron microscopy. Results: Ultrastructural examination disclosed an extensive membrane limited reticulum in the cytoplasm of type I, II, IV, and V fibrocytes of the lateral wall and in outer sulcus cells and their root processes. This system resembled the tubulocisternal endoplasmic reticulum present in some ion-transporting epithelia but appeared more to constitute a network of canaliculi and is referred to as the canalicular reticulum (CR). Many typical small Golgi complexes invariably accompanied the CR in the fibrocytes and sulcus cells, as we have found to be true of other epithelia known to contain CR and function in ion transport. Numerous mitochondria populated cytosol-containing CR. Conclusions: The data support the concept of transcellular K1 flux in type I, II, IV, and V fibrocytes and outer sulcus cells in the cochlea and lend credence to the view of CR as functioning in the movement of ions through cells. The constant and precise association of Golgi complexes with CR in the different cell types implies a functional relationship possibly concerned with biosynthesis of CR by Golgi elements, and the abundance of mitochondria near CR indicates an energy requirement for function of the reticulum or its biosynthesis. Anat. Rec. 249:117–127, 1997. r 1997 Wiley-Liss, Inc. Key words: inner ear; spiral ligament; ultrastructure; electrolytes; ion transport Maintenance of the K1 gradient between endolymph and perilymph is essential for normal hearing and depends primarily on activity of the stria vascularis (for review, see Marcus, 1986; Wangemann et al., 1995). Abundant Na,K-ATPase in the stria (Inuma, 1967; Kuijpers and Bonting, 1969) and more precisely the basolateral plasmalemma of strial marginal cells (Kerr et al., 1982; Schulte and Adams, 1989; Nakazawa et al., 1995) provides a pumping mechanism for preserving the 150 mM K1 level unique to endolymph among extracellular body fluids. Fibrocytes in the spiral ligament of the cochlea appear to function in supplying K1 to the strial pump by cycling back to the stria (Schulte and Steel, 1994; Spicer and Schulte, 1994, 1996) ions that efflux from scala media during auditory transducr 1997 WILEY-LISS, INC. tion (Konishi et al., 1978; Wada et al., 1979; Marcus, 1986; Salt and Konishi, 1986; Sterkers et al., 1988). The lateral wall of the cochlea encloses five types of fibrocytes that have been differentiated on the basis of location, ultrastructural features, and content of enzymes that mediate or energize ion transport, including Na,K-ATPase, carbonic anhydrase, and creatine kinase Contract grant sponsor: National Institute on Deafness and Other Communication Disorders, National Institutes of Health; Contract grant number: DC00713; contract grant number: DC00422. *Correspondence to: Dr. Samuel S. Spicer, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. Received 25 November 1996; accepted 14 March 1997. 118 S.S. SPICER AND B.A. SCHULTE (Spicer and Schulte, 1991, 1996) (Fig. 1). Evidence indicates that plasmalemmal Na,K-ATPase of type II fibrocytes functions to lower K1 in fluid bathing outer sulcus cells and thereby promote K1 efflux from these cells, while also creating an elevated intracellular K1 level from which the ion diffuses down gradient to type I fibrocytes enroute to strial basal and marginal cells. Extensive plasmalemmal foldings that increase the ATPase-rich surfaces of type II fibrocytes bordering outer sulcus cells and the contact of branching narrow expansions from the inferior pole of type II fibrocytes with type Ib fibrocytes fit with the view of K1 flow from sulcus cells through polarized type II fibrocytes and downgradient to type Ia fibrocytes. Broad and narrow contacts (Spicer and Schulte, 1996) and gap junctions (Iurato et al., 1976; Nadol, 1978; Santos-Sacchi and Dallos, 1983; Kikuchi et al., 1995) between fibrocytes and between these cells and strial basal cells further testify to the possibility of diffusion of K1 through the fibrocytes to the stria vascularis. The available data thus point to K1 return to the stria vascularis in the lateral wall via a transcellular rather than an extracellular route. How structural and biochemical characteristics favor an intracellular rather than extracellular path has not been explained. Further inquiry into the fine structure of lateral wall cells reported in the present study delineates a previously undetected system of membrane-limited cisternal elements that apparently constitute a reticulum and possibly relate to ion circulation in these cells. MATERIALS AND METHODS The Mongolian gerbils (Meriones unguiculatis) used in the present study were housed in a low-noise room from birth to death at 3–6 months of age. Similarly aged animals from the gerbil colony maintained in this facility have consistently shown normal hearing as judged by evoked potentials of brainstem and auditory nerve (Schmiedt, 1989; Mills et al., 1990; Boettcher et al., 1993). Procedures for the care and use of animals were approved by the Animal Use Committee of the Medical University of South Carolina under NIH grant DC 00713. The gerbils under urethane anesthesia (1.5 gm/kg, i.p.) were perfused with minimal pressure via a cardiac catheter employing 10 ml of near-body-temperature 0.9% saline solution that contained 0.1% NaNO2. After exsanguination, the bodies were perfused with 50 ml of room-temperature fixative fluid consisting of freshly depolymerized 4.0% paraformaldehyde and 2.0% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The bullae were then opened, the stapes was removed, the round window was perforated, and 1.0 ml of fixative was injected gently into the oval window until it escaped through the round window. The cochleas were dissected free and immersed in fixative overnight in the refrigerator. The scalae were subsequently flushed gently with phosphate buffer, and the inner ears were trimmed of excess bone and decalcified by perfusion with and then immersion in 0.12 M EDTA, pH 7.0, containing 0.2% glutaraldehyde. The EDTA solution was changed every 48 hr until decalcification was complete (about 2 weeks). The specimens were bisected along the plane of the modiolus with a razor blade. To reduce compressive distortion of the organ of Corti during bisection, the Fig. 1. Distribution of type Ia, Ib, II, III, IV, and V fibrocytes differs in gerbil cochlea at the 500-Hz place, providing three routes for ion transport toward the stria vascularis. Na,K-ATPase-rich type II, IV, and V fibrocytes serve in the context of a pumping station to draw electrolytes from the organ of Corti, Hensen, Claudius, and outer sulcus cells (large arrows), from scala tympani (small arrows), and from scala vestibuli (arrowheads) and to move the ions into type Ib fibrocytes enroute to the stria (Spicer and Schulte, 1996). Inset shows suprastrial type V fibrocytes overlying type Ib fibrocytes. CC, Claudius cells; OSC, outer sulcus cells and roots; SP, spiral prominence; StV, stria vascularis; SM, scala media; ST, scala tympani; SV, scala vestibuli. Toluidine blue-stained epoxy thick section. 3470. Inset, 3225. scalae were perfused with warm (45°C) 10% gelatin (300 bloom) and then chilled on an ice bath until the gelatin congealed. Following bisection, the gelatin was 119 ION TRANSPORT BY COCHLEAR FIBROCYTES removed from the half cochleas by agitation in phosphate buffer warmed to approximately 45°C. The tissue was immersed in 0.8% orcein in acetic acid/alcohol for 3 min (Clark, 1960), rinsed, and placed in 1% osmium tetroxide in buffer for 30 min, washed in distilled water, then dehydrated and embedded in Epon resin. Prior to complete polymerization of the resin, the cochleas were sliced into individual half-turns and reembedded in resin in a slide mold to facilitate subsequent viewing of the tissue as a surface preparation. The cochleas were mapped, and radial slices were cut with a razor blade from cochlear regions encoding 0.5–20 kHz (Tarnowski et al., 1991). These slices were reembedded in Epon. Thick sections and adjacent thin sections were taken at several levels, 25 µm apart from each epoxy block, and the thick sections were stained with toluidine blue. Ultrathin sections were selected for electron microscopic examination and stained with uranyl acetate and lead citrate when the adjacent thick section displayed a full-length view of well-preserved spiral ligament and stria vascularis. Sections were examined ultrastructurally from three cochleas at the 2-, 10-, and 20-kHz regions. RESULTS Type Ia fibrocytes contacted strial basal cells extensively (Fig. 1) at gap junctionlike connections (Fig. 2). The Ia fibrocytes made similar but narrower contact with one another throughout the underlying spiral ligament (Fig. 3). The fibrocytes bordering basal cells enclosed membrane-limited profiles that appeared to constitute a network of canalicular structures (Fig. 2). In some Ia fibrocytes more removed from the stria, this canalicular reticulum (CR) occupied an axial core in the cell (Fig. 3). Numerous mitochondria infiltrated CRrich areas (Fig. 2, inset at right), and in longitudinal fibrocyte profiles, the mitochondria showed moderate to extreme elongation in the direction of the long axis of the fusiform cell (Fig. 3). Some Ia cell profiles exhibited Golgi complexes possessing a distinctive fine structure. These complexes commonly consisted of one or two neighboring stacks of about five short cisternae (Fig. 2, inset at left). Occasional wider complexes formed a shallow arch. One face of the Golgi complex often consisted of an uninterrupted cisterna and the other of a segmented cisterna or adjacent aligned vesicles. The Golgi zones occupied cytosolic areas near profiles of CR. Type Ib fibrocytes located inferior to Ia and superior and deep to IIb fibrocytes (Fig. 1) differed from the Ia cells in their lesser asymmetry and more abundant perinuclear cytosol and mitochondria (Figs. 4 & 5) and in their contact by slender processes from type II fibrocytes (Fig. 6). A reticulum of canalicular profiles filled much of the cytosol of Ib fibrocytes (Fig. 4) most densely as a rule in a perinuclear location (Fig. 5). Distribution of the CR corresponded closely with that of numerous mitochondria (Figs. 4 & 5). Golgi zones of Ib resembled those of Ia fibrocytes in structure and in their abundance in cell locations endowed with CR (Fig. 4). Type IIb fibrocytes occurred in the spiral prominence area. These cells showed structural polarity by their plasmalemmal outfoldings from the superior and lateral surfaces of the elongated cell body, where it closely neighbored outer sulcus cells (Fig. 7). In contrast to their upper segments, type IIb fibrocytes extended from their inferior pole slender processes that contacted Ib fibrocytes (Fig. 6). Prominent profiles of CR spread throughout the cytosol (Figs. 7, 8, & 10), extending in lesser abundance into small cell branches but not into the plentiful villuslike protrusions of plasmalemma (Figs. 6–10). Some profiles of type II fibrocytes enclosing widespread CR (Fig. 7) differed from others in displaying less prevalent canalicular and more abundant vesicular structures. Type IV and V fibrocytes are thought to function like type II fibrocytes but to serve in facilitating electrolyte flow to type I fibrocytes from scala tympani and scala vestibuli, respectively, rather than from organ of Corti (Spicer and Schulte, 1996; Fig. 1). The type IV and V fibrocytes also enclosed profiles of CR. Numerous mitochondria infiltrated in greater density the CR-rich region bordering one pole of the nucleus in longitudinally sectioned type II fibrocytes (Figs. 7 & 8). Mitochondria located in paranuclear cytosol containing a dense concentration of CR and prominent Golgi elements appeared smaller and less asymmetric than mitochondria in the distal slender processes of IIb fibrocytes (Figs. 7 & 10). An exceptional abundance of distinctive Golgi zones that otherwise resembled those in type I cells in their small size, organization, and distribution with the CR characterized type II fibrocytes. A cell displaying greater prevalence of vesicular than cisternal membranous elements showed especially numerous Golgi zones (Fig. 7). The Golgi complexes appeared confined to a region of the cell opposite one end of the nucleus and enriched with the greatest concentration of mitochondria (Figs. 7 & 8). Root bundles composed of multiple outer sulcus cell processes extended into the spiral prominence region populated by type IIb fibrocytes and dense stromal strands. The cytologic structure of the root processes differed widely within a bundle (Galic and Giebel, 1989). Content of CR differed comparably among the root processes. Some of the processes enclosed abundant profiles of CR, other processes enclosed only sparse cisternae generally oriented across the process, and still others did not enclose this organelle (Figs. 11 & 12). Mitochondria infiltrated in greater numbers cytoplasmic regions exhibiting greater volume density of CR (Figs. 11 & 12). Typical small Golgi complexes occurred frequently in regions of roots containing prominent CR and mitochondria but not elsewhere (Fig. 11). DISCUSSION A system of membrane-limited tubules and cisternae referred to as the tubulocisternal endoplasmic reticulum (TCER) has been demonstrated in certain epithelia by en bloc staining with an uranyl acetate/PbNO2/ CuSO4/OsO4 sequence (Møllgård and Rostgaard, 1978). Although the TCER in these cells in general resembled the system observed in the present study in routinely processed tissue, the membranous profiles in the cochlear fibrocytes and sulcus cells appeared to consist mainly of branching and interconnected canals. As indicative of the system’s structure and perceived function, the term ‘‘canalicular reticulum’’ seems preferable 120 S.S. SPICER AND B.A. SCHULTE Fig. 2. A type Ia fibrocyte apposed at gap junctions (arrowhead) to strial basal cells (BC) encloses profiles of canalicular reticulum (CR; arrows) and fairly numerous interspersed mitochondria. Inset at lower left shows a typical Golgi zone from a Ia fibrocyte and that at lower right shows more detail of branching elements of CR and many associated mitochondria. 38,000. Left inset, 318,750. Right inset, 317,500. Fig. 3. A longitudinally sectioned type Ia fibrocyte in the midregion of spiral ligament joins other Ia fibrocytes via gap junctions (arrowheads) and contains a central core of CR, with neighboring mitochondria elongated parallel to the long axis of the cell. 310,000. at least for the cochlear cells and is employed in the present study. TCER has been interpreted as promoting transcellular flow of fluid and ions. The organelle has been implicated in Na1 transport in frog skin (Voûte et al., 1975) and fluid and ion movement across ileal epithelium (Møllgård and Rostgaard, 1981). It has been visualized in hepatocytes (Møller et al., 1983) and in ion-transporting epithelium of the gallbladder, choroid plexus, proximal renal tubule (Møllgård and Rost- ION TRANSPORT BY COCHLEAR FIBROCYTES Fig. 4. A type Ib fibrocyte discloses CR densely and evenly distributed throughout the cell body and approaching close to the plasmalemma. Golgi complexes (arrows) consist of compact stacks of a few short cisternae. Inset shows a fibrocyte’s Golgi complex at higher magnification. 38,500. Inset, 337,500. Fig. 5. In a transversely sectioned type Ib fibrocyte, CR and interspersed mitochondria encircle the central nucleus. 35,500. 121 Fig. 6. Slender processes of type IIb fibrocytes identified in part by their large dark mitochondria extend between and make gap junctionlike contact (arrowheads) with Ib fibrocytes. Elements of CR populate the type II fibrocytes but not the plasmalemmal foldings from these cells. The inset shows processes from a IIb fibrocyte abutting on a Ib fibrocyte below. 38,000. Inset, 313,750. 122 S.S. SPICER AND B.A. SCHULTE Fig. 7. A longitudinally sectioned IIb fibrocyte, closely bordering the dense stromal fibers that envelope a root bundle (RB), possesses extensive membranous elements. These take the form of cytoplasmic vesicles or cross-sectioned tubules more than canaliculi except at the poles of the cell. Numerous short, compact Golgi stacks (arrows) intermingle with densely populated mitochondria opposite one pole of the nucleus. Evaginations of the plasmalemma amplify the cell surface. Insets show details of Golgi complexes of type II fibrocytes. 310,500. Upper inset, 334,000. Lower inset, 330,000. gaard, 1978, 1981; Rostgaard and Møllgård, 1980; Bergeron and Thiéry, 1981), and human eccrine sweat gland (Baron et al., 1984). In the cochlea, strial marginal cells (Forge, 1982) and Reissner’s epithelium (Qvortrup and Rostgaard, 1990) contain TCER. The CR shown in cochlear fibrocytes and sulcus cells resembles TCER and presumably functions similarly in mediating ion and fluid movement. TCER has been described only in polarized epithlelial cells, where it presumably directs ion translocation between luminal and abluminal surfaces. Because fibrocytes lack the polarity that is established in epithelia by tight junctions, they would seem not to require or be adapted to utilize TCER. However, the abundant canalicular network demonstrated in the present study in the specialized fibrocytes of the cochlear spiral ligament can be assumed to perform like that in epithelia and to provide thereby an indication of polarized transport activity in these cells. Support for this perception stems from the evidence (Spicer and Schulte, 1996) in type IIb fibrocytes of a structural polarity thought to underlie a capacity for directing ion flow. The position and fine structure of IIb fibrocytes point to such a possibility. Thus, the upper end of these asymmetric cells, where they border outer sulcus cell roots, exhibited marked surface expansion in the form of plasmalemmal evaginations in contrast to the lower pole of the cell, which contacted type I fibrocytes at the end of cell process arborizations (Fig. 6). Type II fibrocytes apparently resorb at their superior pole K1 effluxing from root bundles and release the ion through gap junctions with Ib fibrocytes at the opposite end of the cell. Such a prominent system of CR in spiral ligament cells must contribute importantly to their bioactivity. Whether the rate of extracellular K1 diffusion to the stria through a tissue densely populated with cells and interstitial fibers would suffice for the strial Na,KATPase pump to maintain the 150 mM K1 of endolymph is open to question, as is the supposition that the rate of diffusion through cytosol would contribute a significant advantage over extracellular flow. A network such as the CR offers a means of channeling ION TRANSPORT BY COCHLEAR FIBROCYTES Fig. 8. A longitudinally sectioned IIb fibrocyte exhibits an elaborate array of branching, interconnected canaliculi, and numerous vesicles. These elements of CR spread throughout the cytosol. Frequent, relatively short Golgi stacks (arrows) intermingle with CR on one side 123 of the nucleus in an area containing CR and the densest collection of mitochondria. Plasmalemmal outfoldings protrude from the cells. Inset shows an abundance of Golgi complexes (arrows) in a CR-rich area containing many mitochondria. 318,000. Inset, 316,000. 124 S.S. SPICER AND B.A. SCHULTE Fig. 9. CR occurs in processes of type IIb fibrocytes but not the plasmalemmal outfoldings (arrowheads) from these cells. 39,500. Fig. 10. Numerous profiles of CR border the poles of the nucleus in a IIb fibrocyte. Mitochondrial profiles near this CR (arrows) appear smaller than those in more narrow and distal cell processes (arrowheads). 39,000. ION TRANSPORT BY COCHLEAR FIBROCYTES 125 Fig. 11. One root process (arrow) in a root bundle differs from others in containing numerous mitochondria and profiles of CR with associated Golgi zones (arrowheads). Prevalence of elements of CR among the root processes parallels that of mitochondria and of Golgi elements. A IIb fibrocyte adjacent to the root bundle at lower left contains abundant CR and mitochondria. Inset shows an enlargement of a Golgi zone from the root bundle. 35,500. Inset, 337,500. unimpeded K1 diffusion in the cell and influencing the rate and direction of ion translocation. In addition, the reticulum provides a mechanism for sequestering electrolytes from cytosol and avoiding a cytotoxic effect of elevated ion concentration on cell metabolism. In accord with the latter concept is the observation that Hensen, Claudius, and inner sulcus cells, which lack CR but conduct electrolyte flow in the cochlea, possess a lucent cytosol with essentially none of the cytologic organelles that might suffer from ion toxity (Spicer and Schulte, 1997). The finding that some outer sulcus cells and types Ia, Ib, and II fibrocytes contain CR supports the view that together these cells constitute a path for the recycling of K1 back to the stria (Spicer and Schulte, 1996). If this thesis is correct, the presence of CR in these cell types provides further evidence for the role of this organelle in electrolyte transport by cells. Subsurface cytosol generally separated profiles of canalicular structures from the plasmalemma. Type II fibrocytes disclosed little evidence for ion resorption or secretion through direct fusion of CR elements with plasmalemma. Therefore, participation of the CR in moving transcellularly the K1 resorbed by plasmalemmal ATPase in type II fibrocytes should theoretically require K1 permeability in the membrane delimiting this system. K1 conductance in the CR membrane could allow the CR to resorb K1 from cytosol possessing a high K1 level at the superior pole of the fibrocyte and to release the ion into cytosol with lower K1 at the inferior pole. By way of affirming this concept, the plasmalemmal Na,K-ATPase more abundant at the superior end of the type II fibrocytes functions to maintain there a relatively high cytosolic K1, whereas gap junctions lower cytosolic K1 at the inferior pole of these cells by permitting the ion to escape into Ib fibrocytes. Whereas identification of TCER required special en bloc staining, the organelle was visualized in the cochlear fibrocytes and outer sulcus cells in specimens prepared routinely for electron microscopy. The membra- Fig. 12. A root from an outer sulcus cell displays profiles of CR, mitochondria, and microtubules (arrowheads). 330,000. 126 S.S. SPICER AND B.A. SCHULTE nous reticulum in the cochlear fibrocytes appeared labile to tissue processing, however, as it was encountered in abundance in some but only sparsely or not at all in other specimens. An increased volume density of mitochondria accompanied the CR in the region of fibrocytes and outer sulcus cells populated by the reticulum. The codistribution of mitochondria and CR directs attention to transport processes performed by the reticulum, possibly requiring a source of energy in lateral wall cells. Extremely asymmetric mitochondria bordered CR profiles closely over a considerable distance in an arrangement consistent with their serving as an energy source for the membranous system (Fig. 3). However, there is presently no knowledge that CR contains an ATPase or a protein kinase, either of which would utilize high energy phosphate for ion transport, and it is accordingly uncertain as to whether this system demands energy for its presumed function in facilitating ion flow. Moreover, the elaborate display of CR in other sites such as Deiters cells of gerbil cochlea and the human eccrine sweat gland lack an abundance of closely associated mitochondria (Spicer and Schulte, 1993; Baron et al., 1984). If mitochondria associated with CR energize ion movement in type II fibrocytes, the transport mechanism involved in this site must differ from that in Deiters cells and sweat glands. Alternatively, mitochondria populating CR-rich areas of cytosol could provide an energy source for biosynthesis of the membranous complex. The abundance of mitochondria showing a distinctive rounded profile in paranuclear regions rich in CR and Golgi zones (Figs. 3, 7, & 8) is consistent with such an interpretation. In this case, information concerning possible differences in function or rate of turnover is needed for explaining the more impressive codistribution of mitochondria with Golgi cisternae in the fibrocytes than in Deiters cells. Cytoplasmic areas with dense profiles of CR and mitochondria in all lateral wall cells invariably possessed a few to many Golgi complexes. A similar association of CR with Golgi elements has been encountered in other sites including human eccrine sweat glands (Baron et al., 1984) and cochlear Deiters cells (Spicer and Schulte, 1993). Moreover, a striking development of Golgi cisternae takes place in Deiters cells and pillar cells of neonatal gerbils 14–15 days after birth, at the precise time when CR appears in these cells (Ito et al., 1995). The hyperabundance of both systems occurred transiently, as they disappeared together from pillar cells during further postnatal development but in Deiters cells persisted into adulthood with a somewhat diminished volume of Golgi complexes and associated mitochondria. The codistribution of Golgi zones and CR in many cell types of the cochlear lateral wall and elsewhere, the concurrence of Golgi zones and CR only in a paranuclear area rich in mitochondria in type II fibrocytes, and the simultaneous timing of the appearance and disappearance of Golgi zones and CR during postnatal development all point to a possible role for Golgi cisternae in biosynthesis of the membranous reticulum. 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