The Tectorial Membrane of the Rat’

MURIEL D. ROSS Department of Anatomy, The University of Michigan, Ann Arbor, Michigan 48104

ABSTRACT Histochemical, x-ray analytical and scanning and transmission electron microscopical procedures have been utilized to determine the chemical nature, physical appearance and attachments of the tectorial membrane in nor- mal rats and to correlate these results with biochemical data on protein-carbo- hydrate complexes. Additionally, pertinent histochemical and ultrastructural findings in chemically sympathectomized rats are considered. The results indi- cate that the tectorial membrane is a viscous, complex, colloid of glycoprotein( s) possessing some oriented molecules and an ionic composition different from either endolymph or perilymph. It is attached to the reticular laminar surface of the organ of Corti and to the tips of the outer hair cells; it is attached to and encloses the hairs of the inner hair cells. A fluid compartment may exist within the limbs of the “W’formed by the hairs on each outer hair cell surface. Present biochemical concepts of viscous glycoproteins suggest that they are polyelectro- lytes interacting physically to form complex networks. They possess character- istics making them important in fluid and ion transport. Furthermore, the macro- molecular configuration assumed by such polyelectrolytes is unstable and subject to change from stress or shifts in pH or ions. Thus, the attachments of the tec- torial membrane to the hair cells may play an important role in the transduction process at the molecular level.

The present investigation is an out- of the tectorial membrane remain matters growth of a prior study of the effects of of dispute. As will be made evident in the chemical sympathectomy upon the struc- Review of the Literature which follows, tures of the inner ear. None of the findings one can find support for almost any inter- of this prior study were more interesting pretation of the structure of the tectorial than those concerning the internal struc- membrane or its degree of attachment to ture and the attachments of the tectorial the organ of Corti. Although the tectorial membrane. In chemically sympathectom- membrane is generally accepted to be rich ized rats, the filamentous organization of in acid mucopolysaccharide, this point is the membrane was often remarkably evi- also a matter of some contention. dent ultrastructurally, and the membrane It was decided, therefore, to undertake showed a proclivity for remaining attached histochemical and scanning electron micro- to the tips of the hairs of the outer hair scopical research which might provide new cells. These results raised questions con- information on certain of the controversial cerning the chemical nature and attach- aspects of the tectorial membrane (chemi- ments of the tectorial membrane in un- cal nature, structure, and attachments) in treated rats, and whether or not these rats with developed auditory systems (rats properties had been altered by chemical beyond 14 days of age). It was hoped that sympathec tomy. new insights into the functions of the A search of the literature showed that membrane would result from elucidation the answers to the questions posed were of these particular points. not to be found there for, although the It became apparent, however, that a membrane has been extensively studied clearer understanding of the functional from the time of Corti (1851) to the significance of the tectorial membrane de- present, the origin, attachments, chemical pended not only upon such chemical or nature, physical appearance and functions 1 Supported by grant NB-07306 USPHS. AM. J. ANAT., 139: 449482. 449 450 MURIEL D. ROSS structural evidence as could be obtained thickness from one species to another, by the methods chosen, but also upon cor- (40 A, guinea pig, Engstrom and Wersa, relation of this data with that obtained by '58; 100 A, rat, Iurato, '60; variable, cat, biochemists working in the field of pro- Spoendlin, '57). However, Spoendlin ('57) tein-carbohydrate complexes. Biochemical pointed out that the filaments could be knowledge of such complexes has advanced artifactitious and Engstrom and Wersa rapidly in recent years and has reached a ('58) indicated that, because of their size, . point where its application to structures they cannot be the same as those observed such as the inner ear membranes could be with the light microscope. The last-named most enlightening, not only with respect to authors suggested that the tectorial mem- understanding tectorial membrane func- brane might be jelly-like with micellar tions in the normal auditory organ, but structures interspersed in some substance also in perceiving possible mechanisms which dissolves away in preparation of underlying some degenerative diseases of the tissue. the inner ear. The integration of histo- Scanning electron microscopy has gen- chemical, ultrastructural and biochemical erally revealed the tectorial membraue to data is, then, a further important aim of be smooth on much of its under side but this report. to be grossly fibrillar on its upper side and in the interior (Kosaka et al., '71; Lim, Review of the Literature '72). In his diagram, Urn ('72) showed From the earliest descriptions of its mi- the smooth regions as separate sheets, cor- croscopic appearance, the tectorial mem- responding to Hensen's stripe and to brane has been considered to be a fibrillar Hardesty's membrane. structure (Boettcher, 1859; Kolliker, 1854, All of the early investigators thought 1861; Waldeyer, 1873; Henle, 1880; Ret- that the membrane was attached to the zius, 1884). Many observers have con- epithelial cells of the entire organ develop- sidered the membrane to be comprised of mentally, but differed in their opinion of fibrils embedded in a gelatinous or amor- its degree of attachment in the adult. Thus, phous matrix (Retzius, 1884; Kolmer, '07; many have stated that the membrane was Hardesty, '08, '15; Keith, '18; Wislocki free, or "floated" over the organ of Corti and Ladman, '55; Iurato, '60; Lim, '72); in the adult (Kolliker, 1861; Helmholtz, to be stratified, or composed of lamellae in 1863; Waldeyer, 1873; Retzius, 1884; toto or in part (Henle, 1880; Held, '02, '09; Rickenbacher, '01; Hardesty, '08; Held, Shambaugh, '07; Prentiss, '13); or to be a '09; Kolmer, '07, '27). Waldeyer com- reticulum (Coyne and Cannieu, 1895; mented, however, that he occasionally Prentiss, '13). Still others have thought found the hairs of the outer hair cells im- that the fibrils were either products of co- bedded in the membrane and, after hard- agulation of endolymph (Czinner and ening in alcohol, sometimes found the Hammerschlag, 1897) or were continua- lamina reticularis carried away with the tions of the hairs of the hair cells (Ayers, membrane as it contracted. Boettcher 1891; Borghesan, '49; '52; Mygind, '52). (1859) indicated that the membrane did Recently, Borghesan ('71) suggested that not float free but was connected to the sur- the hairs are deeply embedded in the mem- face of the organ of Corti, an opinion brane, and that they are artifactitiously shared by Coyne and Cannieu (1895), elongated as the shrinkage forces pull the Kishi ('07), Shambaugh ('07) and Prentiss membrane away from the hairs. ('13). Prentiss was emphatic in stating Research carried out by phase contrast that he meant that the attachments were or with polarized light has suggested that to the entire surface of the organ of Corti, the tectorial membrane is a filamentous as occurred in the fetal state. Corti (1851), structure, with the filaments coursing at Boettcher (1859) and Lowenberg (1864) 30"-40" angles in an apical direction (A. considered the tectorial membrane to ex- Hilding, '52; Iurato, '60). Transmission tend beyond the organ of Corti, even to electron microscopy demonstrates that the the spiral ligament; Henle (1880) found membrane appears to be comprised of it attached just beyond the last row of hair slender filaments which vary somewhat in cells. Keith ('18) mentioned an attachment TECTORIAL MEMBRANE OF THE RAT 45 1 of the tectorial membrane to inner support- vergent results. Biochemical studies have ing cells as well as a peripheral connection indicated that the fibrils of the tectorial to outermost Deiters cells. Among more membrane are not comprised of collagen recent investigators, Wittmack ('36) main. (Bairati and Iurato, '57; Iurato, '60; and tained that the tectorial membrane at- Naftalin et al., '64) and are not related to tached to the hairs of both inner and outer keratin (Belanger, '56a). Naftalin et al. hair cells; such connections were also ('64) found the tectorial membrane to be found by von BCkCsy ('60) and Hawkins a gel which binds calcium and magnesium, and Johnsson ('68). According to Witt- with calcium the more firmly bound, and mack ('36), other attachments existed to to contain potassium in higher concentra- border cells and to Hensen cells. These tion than in extracellular fluid but lower results were supported by A. Hilding ('52), than in endolymph. They and others (Ayers, who also observed attachments to outer, 1891; Shambaugh, '07; Wittmack, '36) but not inner, hair cells. The peripheral noted the great sensitivity of the tectorial attachment of the tectorial membrane to membrane to the state of hydration. In- the reticular lamina (von BekCsy, '60), vestigators have found the tectorial mem- or to Hensen cells (Tonndorf et al., '62), has been described as firm, effectively brane to be PAS-positive (Wislocki and Lad- forming a seal between the organ of Corti man, '55; Belanger, '56a; Friberg and and the endolymph. Ringertz, '56; Igarashi and Alford, '69; Scanning electron microscopists have Vinnekov and Titova, '64). Results with found attachments of the tectorial mem- toluidine blue are in dispute. Wislocki and brane to various parts of the organ of Corti. Ladman ('55) and Friberg and Ringertz Kosaka et al. ('71) reported indentations ('56) found the membrane to lack meta- of the tips of the hairs of outer hair cells chromasia, but BClanger ('53, '54, '56a,b), in the tectorial membrane; Lim ('72) Plotz and Perlman ('55) and Tonndorf et found connections of the membrane to the al. ('62) reported it to be metachromatic. outer hair cells and to the outermost row Additionally, Igarashi and Alford ('69) of Deiters cells; Tanaka et al. ('73) ob- found the inner ear membranes to stain served attachments of the membrane to moderately in alcian blue, and concluded both the inner and the outer hair cells. that the membranes contain neutral and According to Lim ('72), the outer margin acid mucopolysaccharides. This conclusion of the tectorial membrane is attached only is commonly accepted today. However, as far as the outermost row of hair cells in Iurato ('60, '67) showed that the tectorial some cases; thus, endolymph would have membrane consisted mostly of protein, free access to the surface of the organ of contained little carbohydrate and no hexu- Corti. ronic acid. He concluded, on these grounds, Transmission electron microscopists that the tectorial membrane cannot be com- have generally failed to find the tips of the posed chiefly of acid mucopolysaccharides. hairs connected to the tectorial membrane in mammals (Smith and Dempsey, '57; MATERIAL AND METHODS Spoendlin, '57; Engstrom and Wersslll, '58; The experiments reported upon here Iurato, '61). However, a few attachments were carried out on Sprague-Dawley rats were seen by Kimura ('66) in the case of ranging in weight from 30-385 gm. Some outer hair cells, and Iurato ('67) found of the animals were chemically sympa- the tips of the hairs of the inner hair cells thectomized by use of 6-hydroxydopamine in close contact with the membrane. In (6-OH DA) (Angeletti and Levi-Montal- birds, not only were hair cells connected cini, '70). Newborn animals were injected to the tectorial membrane, but also there subperitoneally with Bhydroxydopamine were veil-like attachments between the 50 pgm/gm body weight, in ascorbic acid tectorial membrane and the microvilli of or in physiological saline daily from day 1 the supporting cells (Dohlman, '71; Taka- to day 7. Controls were left untreated or saka and Smith, '71; Rosenhall, '71). were injected with the vehicle only. The Investigations using biochemical and experimental animals were then prepared histochemical techniques have led to di- in the following ways. 452 MURIEL D. ROSS

Histochemical procedures Ultrastructural procedures Twenty-four rats ranging from 25-75 Scanning electron microscopy. Cochleae gm and 15-35 days in age were used as from 34 normal, 13 6-hydroxydopamine normal controls. Five rats of the same treated, and five saline injected animals ranges of body weight and age were in- were prepared by a variety of procedures jected with 6-hydroxydopamine. After de- for scanning electron microscopy. Twenty capitation, the entire skull with inner ears rats were prepared by the following in situ was quickly frozen on dry ice method, referred to subsequently as the while being sprayed with Cryokwik (Inter- “ordinary procedure” for scanning electron microscopy : national Equipment co.). The frozen skull Animals were anesthetized with 3.5% was then mounted and sectioned in a cryo- chloral hydrate (1 m1/100 gm body weight), stat at -22°C. The 16 sections were and then fixed by intracardiac perfusion picked up on warm coverslips and placed with glutaraldehyde (3% )-paraformalde- in small covered coplin jars in the cryostat hyde (4% ) in Millonig buffer, pH 7.4. for drying. The sections were then ex- Cochleae were removed and opened in posed to paraformaldehyde vapor for fixa- fixative, and subsequently postfixed in 1% tion at either 37°C for two hours, or at osmic acid in Millonig buffer. The tissues 50°C for one hour. Tissues were well fixed were dehydrated in alcohol up to 70%, and by either procedure, but the 37°C fixation were stored in 70% alcohol until later dis- was routinely followed as it was considered section for freeze-drying. Cochleae were less drastic, Alternate sections were then broken into large pieces, freeze-dried, treated in the following ways : mounted on stubs, coated with gold and Periodic acid-Schiff (PAS). Sections observed in a JEOL JSM U3 scanning elec- were stained according to the method of tron microscope. Lillie (’65). Controls were exposed to the Four rats were perfused and cochleae Schiff reagent only. were removed and osmicated as outlined Toluidine blue. The toluidine blue above. After osmication the cochleae were method of Kramer and Windrum (’55) for broken into segments and pieces of the metachromasia was followed; pH of the tectorial membrane were removed from staining solution was 8.2. their attachments. These portions were Enzyme studies. Some sections were then dehydrated through absolute alcohol, pretreated with enzymes to determine immersed in amyl acetate (2 changes, 15 whether or not specific substrates were minutes), and dried in a critical-point dryer present in the tectorial membrane and con- (Denton). tributed to the staining reaction noted. The remaining 23 animals were not per- The enzymes were chosen for their useful- fused, but were decapitated. Cochleae were ness in distinguishing glycoproteins from removed and prepared according to the other substances which have, or might procedures outlined immediately below. have, similar histochemical reactivity. The These experiments were designed to reveal procedures followed were based upon data more about the physical appearance of the given by Thompson and Hunt (’66) and tectorial membrane, its attachments and in the Worthington Manual (’72): amy- ionic composition, under a variety of tech- lase (Sigma, Worthington), for hydrolysis nical conditions. of glycogen, 20 mg/lO ml in Millonig First, cochleae from two untreated and buffer, pH 6, at 37°C for one hour; neur- from three 6-hydroxydopamine treated rats aminidase ( Worthington), for hydrolysis were opened directly in the glutaraldehyde- of sialic acid, 0.2 mg/lO ml in acetate paraformaldehyde fixative. Except for this, buffer, pH 5, at 37” for from 30 minutes the ordinary procedure for scanning elec- to three hours; hyaluronidase (Worthing- tron microscopy was followed. ton), for hydrolysis of hyaluronic acid, 5 Second, in order to determine the conse- mg/lO ml in Sorenson buffer, pH 6, at quences of poor fixation and of freezing- 37°C for from 1-20 hours. Controls were thawing-refreezing upon inner ear struc- exposed to buffers only for the same periods tures, two animals were prepared as fol- of time. lows : after decapitation, one cochlea was TECTORIAL MEMBRANE OF THE RAT 453 allowed to remain unfixed for 15 minutes, were analyzed in an ARL EMX-SM electron whereupon it was opened in glutaralde- microprobe, utilizing a Multichannel ana- hyde-paraformaldehyde fixative and subse- lyzer, Model 710 (Northern Scientific Com- quently prepared according to the ordinary pany). For microprobe analyses, the sam- method given above. The other cochlea was ples were mounted on carbon stubs with allowed to remain unfixed for 30 minutes, carbon paint, and were carbon-coated. and then treated similarly. Cochleae from Transmission electron microscopy. Tis- the remaining animals were opened rap sues were obtained from seven untreated idly, fixed immediately in glutaraldehyde- and 13 Shydroxydopamine injected rats paraformaldehyde and prepared according which had been prepared by intracardiac to the usual procedure up to the quench- perfusion of glutaraldehyde-parafonnalde- ing. At this point, segments of these coch- hyde as given. After osmication, tissues for leae were quenched, then thawed, then transmission electron microscopy were de- quenched again and freeze-dried. hydrated and embedded in Epon. Thick Third, according to Hardesty ('15), fixa- sections (1-2 p) were used for orientation tion in Held's solution gives the best preser- purposes, then thin sections were cut and vation of the tectorial membrane. Cochleae mounted on 200-mesh grids covered with from two normal and from one 6-hydroxy- Formvar membranes. Sections were stained dopamine treated rats were opened in this with lead citrate (Reynolds, '63) and stud- fixative, left in it overnight, and then pre- ied on an Hitachi Hu 11A electron pared according to the ordinary procedure microscope. for scanning electron microscopy. RESULTS Fourth, various combinations of glyc- erin in glutaraldehyde were tried in an Histochemical findings attempt to prevent dehydration of the mem- The tectorial membrane is sometimes brane during the process of fixation, by torn away from the surface of the spiral replacing the water with glycerin. One limbus in cryostat sections, and may be cochlea was opened in lo%, and the other folded upon itself to some extent. These in 20% glycerin in 2% glutaraldehyde. are unavoidable artifacts in sections of un- These were left in the solutions overnight, decalcified cochleae. then dried briefly over silica gel, coated PAS stain. The tectorial membrane is and viewed in the scanning electron mi- highly PAS-positive in both normal and in croscope. Additionally, four cochleae were 6-hydroxydopamine treated rats (figs. 1,2). prepared by fixation in each of the above The dark, purplish-red color of the stained combinations of glycerin and glutaralde- membrane is not uniform throughout, but hyde, but were freeze-dried. this appears to be due to distortions (prin- Finally, nine cochleae were removed, cipally folding) of the membrane during quickly opened to varying degrees, and cutting procedures rather than to the pres- quenched in liquid Freon cooled in liquid ence of fibrils. nitrogen. These cochleae were immediately The PAS reactivity of the membrane is freeze-dried, No fixation or dehydration unaffected by pretreatment with amylase, was involved. Additionally, five cochleae hyaluronidase, or their buffers. The fact were removed quickly from decapitated that no detectable loss of staining capacity rats and immediately quenched in liquid occurs after amylase treatment indicates Freon cooled in liquid nitrogen. These that glycogen, which would stain with the cochleae were opened in the liquid Freon, PAS method, is not present in the tectorial according to the technique of Flock ('73). membrane. Analyses of fixed, dehydrated samples of There appears to be a slight decline in tectorial membrane and of freeze-dried the intensity of the staining reaction after tissue untouched by fluid fixative or dehy- exposure to neuraminidase. However, un- drating agents were carried out in the due emphasis cannot be placed upon this scanning electron microscope, utilizing an change at the present time. The slight shift x-ray analyzer and a Multichannel ana- in color may be due not to the hydrolysis lyzer, Model 710 (Northern Scientific Com- of sialic acid, but to the action of con- pany), Other samples of freeze-dried tissue taminant hexosidases, or to the blocking of 454 MURIEL D. ROSS otherwise reactive sites by their binding noted after treatment with neuraminidase with neuraminidase. Chemical analysis of indicates that some sialic acid is present the tectorial membrane should resolve this remains a question. This fmding is sub- issue. jective and must be considered tentative The tectorial membrane is an exceed- until definitive results are forthcoming ingly pale pink color in control sections from chemical analysis. exposed to the Schiff reagent only. This result demonstrates that the reactions noted Ulnastructural findings in membranes exposed to the entire stain- Scanning electron microscopy of the nor- ing procedure are not artifacts induced by mal organ of Corti and its relationship to the process of fixation in paraformalde- the tectorial membrane. The tectorial hyde vapor. membrane ordinarily pulls away from its Toluidine blue. The tectorial membrane attachments to the organ of Corti during is not metachromatic under any of the ex- preparation of the tissue for scanning elec- perimental conditions utilized here, indi- tron microscopy. If any part of it remains cating that it does not contain numerous extended over the organ, it is usually pur- free polyanions. The finding in the case of posely removed so that the entire surface the tectorial membrane was checked of the organ of Corti can be seen. Under against the staining reaction of mast cells, either of these circumstances, the true re- which were always metachromatic. The lationships of the membrane to the organ mast cells were present on every section in connective tissue remnants along blood of Corti are generally obliterated. However, vessels outside the skull, at times, shreds of the membrane remain The tectorial membrane stains dark blue attached accidentally, and adhering por- in both normal and in 6-hydroxydopamine tions can be left intact on purpose so that treated rats (figs. 3, 4). The membrane some concept of the attachments can be looks faintly striated, or even assumes a gained. feathery appearance, in toluidine blue When the membrane is completely re- stain. moved from the cochlea, the hairs of the The entire section stains less intensely outer hair cells are generally erect and with toluidine blue after exposure to an evenly spaced relative to one another enzyme-buffer solution or to a buffer alone. (fig. 7). Sometimes the hairs slope slightly In the case of the tectorial membrane, the inward, toward the limbus, but this could staining reaction remains orthochromatic, be artifactitious and related to manipula- but the color assumed by the membrane tion of the tissue during dissection. shifts to a more pale shade of blue. The The hairs are disposed in three rows of shift is slight in sections pretreated with decreasing height on the surfaces of the amylase or hyaluronidase (fig, 5) or their outer hair cells, in a “W configuration buffers, but is pronounced in the case of (fig. 7). The base of the “Wis broader in pretreatment with neuraminidase (fig. 6). the fist row of hair cells, with the “W’ Tectorial membranes pretreated with the of the third row being most narrow. The acetate buffer alone appear to be slightly tallest hairs are always outermost on any less affected than when neuraminidase is individual hair cell, but the hairs are of included in the solution in the present graded height from base to apex, being series. These results suggest that the shifts tallest at the apex. in staining color noted in the tectorial The tips of the tallest hairs of any given membrane are at least partly related to the set are smooth and bulbous when the tec- pH of the buffer solutions used (pH 6.0 in torial membrane is completely removed the case of the amylase or hyaluronidase; from them (fig. 7). However, if the mem- pH 5.0 for neuraminidase). However, as brane is left as undisturbed as possible, hyaluronidase had no remarkable effect these tips are covered over by the substance upon the staining reactivity of the tectorial of the tectorial membrane (fig. 8). More- membrane, it can be concluded that hyalu- over, the shafts of the hairs are intercon- ronic acid is not present in significant nected by similar substance in such cases amounts. Whether or not the shift in color (fig. 8). Wherever the hairs are acciden- TECTORIAL MEMBRANE OF THE RAT 455 tally slightly separated, the connections be- face of the organ of Corti, from inner tween them are evident. border cells to the phalangeal processes of In some cases, the attachments between the outermost row of Deiters cells (figs. the tectorial membrane and the hairs and 17-19). That these attachments are to mi- between the hairs themselves are so strong crovilli seems clear from electron micro- that the hair cells are lifted from their graphs taken at higher magnifications basal connections (fig. 9). In other cases, (fig. 20). the caps of the tips of the hairs strip away In the case of the outer hair cells, the and hang from the underside of the mem- presence of attachments to the tips of the brane (fig. lo), or the hairs may pull the tallest hairs, the roofing over of the hair attaching filaments loose, so that imprints region, and the attachments to the few of their arrangement (also in the form of microvilli present suggest that a fluid com- a “W) are visible in the tectorial mem- partment exists within the limbs of the brane (fig. 9). “W formed by the hairs. It would seem The hairs of the inner hair cells are that this small compartment may extend thick, somewhat club-shaped structures completely around the hairs on the basis forming what can sometimes be described of the distribution of the microvilli, al- as a shallow “W,” but more often a cres- though the present results can neither cent, on the cuticular surfaces (fig. 11). deny nor confirm this. The present findings The hairs are distributed in three main concerning the attachments of the tectorial rows on the surface of each cell, although membrane to the organ of Corti are shown extra hairs forming incomplete internal diagrammatically in figure 21. rows are common in the rat. The hairs of Scanning electron microscopy of the the outermost row on each hair cell are tectorial membrane. Various techniques the tallest, with the inner rows formed of have been followed in an attempt to leave hairs of decreasing height. The tips of the the tectorial membrane in as natural a tallest hairs are smooth and flat in ordinary condition as possible. This is a monu- scanning electron microscopical prepara- mental task, for the membrane is ex- tions, while the tips of the inner rows are tremely sensitive to any of the common smooth but angled in an upward and out- fixatives as well as to alcohol, so that it ward direction (fig. 11). The shafts of the always shrinks and pulls away from the hairs are rough in superficial appearance. organ of Corti during ordinary technical The tips of the tallest hairs of the inner procedures. Nor does freeze-drying of the hair cells are also attached to the tectorial membrane in situ in a freshly sacrificed membrane (figs, 12-14). In some prepa- animal surmount these difficulties, for this rations, when care is taken to leave por- procedure also results in shrinkage and tions of the tectorial membrane remaining detachment of the membrane as it is de- adherent to the organ of Corti undisturbed, hydrated. However, these and other studies the shreds of the membrane attach to all dealt with here are useful, for they demon- the hairs of the inner hair cells, as though strate unreservedly that the tectorial mem- enshrouding them. At times, hairs of the brane can assume any one of several physi- inner hair cells have been found hanging cal appearances, depending upon how it from the tectorial membrane. is treated. The hairs on each hair cell are inter- The tectorial membrane is gel-like in ap- connected, both between adjacent hairs of pearance in sections cut in a cryostat, fixed the same row and of neighboring rows over paraformaldehyde vapor, and kept dry (figs. 15, 16). Usually, the connections ap- over silica gel. Membranes fixed in situ, pear strand-like, but this is probably arti- osmicated, then removed from their attach- factitious and related to stretching of the ments prior to dehydration are not fibrillar substance as hairs are separated from one although they may appear to be thrown another either by accidental manipulation into folds and wrinkles (figs. 22-24). The or during the drying process. substance of the membrane is homogene- Finger-like shreds of the tectorial mem- ous and gel-like in appearance. Mem- brane have often been encountered at- branes which have been fixed, osmicated, tached to the microvilli of the entire sur- dehydrated up to 70% alcohol and allowed 456 MURIEL D. ROSS to enter the freeze-drying state in situ have cases, it is nearly impossible to define the their upper surfaces thrown into a series external limits of the membrane. of ridges which lend them a fibrillar ap Purposely induced artifact. After de- pearance (figs. 25-26). The ridges may be layed fixation, the hairs of both inner and curved, straight, or form broad longitudi- outer hair cells are often thread-like; ad- nal bands. The ridges are not of similar jacent hairs may fuse with one another, orientation even in adjacent, small por- and the tips of the hairs of inner hair cells tions of the membrane. The under side is are sometimes flattened and expanded. rough and sometimes has large superficial The reticular lamina of the supporting holes, as though the substance of the mem- cells is usually vacuolated and the micro- brane had pulled apart (fig, 27). villi are poorly preserved. The tectorial If the tectorial membrane is quenched in membrane unfixed for 30 minutes after liquid Freon cooled in liquid nitrogen and death of the animal lacks organization and immediately freeze-dried, the membrane is vacuolated. on the whole looks like a series of inter- Freezing-thawing of presumably well- connected ribbons of gelatinous material fixed cochleae results in artifacts which (fig. 28), although it may appear to be a are different from those just described. The gelatinous sheet at other sites (fig. 29). hairs of the outer hair cells have bizarre In cross section, the tectorial membrane, configurations. The hairs of all three rows freeze-dried and untouched by fluid ha- on the hair cell frequently merge together tives or dehydrating agents, is gel-like in in a solid line in a “V shape. At other appearance (fig. 30). The interior of the times, the limbs of the “V” are broken into membrane at first impression is striated. sections, as though hairs are missing at However, at higher magnifications the gela- intervals. On the other hand, the hairs of tinous nature of the interior is positively the inner hair cells often retain their in- revealed, with the striations being simply dividuality and form three rows on each thickenings of the constituent gelatinous hair cell surface. The reticular laminar material. surfaces of the supporting cells are some- Tectorial membranes fixed in 25% glu- times intact, although large holes are of taraldehyde and freeze-dried without al- common occurrence. Tectorial membranes cohol dehydration resemble greatly in ap- subjected to freezing-thawing generally pearance the freeze-dried membranes just have a reticulated or shredded appearance described (figs. 31, 32). In cross section over the proximal one-half to two thirds of the membrane is gelatinous, with thicken- their extent. The portion over the organ of ings of the gel-like substance radiating in Corti resembles a vacuolated, glassy sheet. a direction toward the under side of the The under side is usually smooth and tectorial membrane. glassy. Membranes prepared by the Held fixa- X-ray analysis of the tectorial membrane. tive appear rough and granular, and frag- X-ray analyses of tectorial membranes pre- ments of them commonly remain attached pared for scanning electron microscopy to the surface of the organ of Corti. How- proved to be inadequate, because the use ever, in those spots free of attaching tec- of aluminum stubs, of silver paint, and of torial membrane, the reticular lamina is gold coating interferes with the detection vacuolated as though poorly fixed. The of ions which are present in small quanti- hairs of the hair cells, in contrast, are often ties, The analyses are useful, however, for nearly typical or entirely normal in over- a comparison of results obtained from all appearance. freeze-dried membranes with fixed and de- When glycerol is used with the glutar- hydrated tissues shows that all ions are aldehyde in an attempt to replace the water greatly reduced after the membranes are of the membrane, the tectorial membrane subjected to various fluids. This emphasizes is gelatinous in appearance, The mem- the need for use of membranes untouched brane is also gelatinous-appearing if only by fixatives or dehydrating agents when dried over silica gel while left in situ or analyses are carried out. when entire cochleae are prepared by the The use of carbon paint and stubs in Flock (’73) method. However, in all these the procedure for microprobe analysis of TECTORIAL MEMBRANE OF THE RAT 457 the tectorial membrane introduces a dif- and the fine filaments are cross-linked ferent artifact, Phosphorous and silicon are (fig. 35). There are small areas within present in the paint, and silicon occurs in the membrane which seem to have under- the stub; these ions must be discounted. gone lysis, and detritus is entrapped in The analytical data given below are these spaces (fig. 35). based upon information obtained from The capping material from the tips of three samples of freeze-dried membrane 15 hairs remains attached to the under with the upper side (toward the endolymph) side of the tectorial membrane shown in and four samples with the lower side figure 35. The filaments of the tectorial (toward the organ of Corti) exposed in membrane in the immediate vicinity of the the microprobe. The samples represent tips of the hairs are more closely related three different cochleae and include por- to one another spatially than are those in tions of the tectorial membrane from base the interior of the membrane, and are fine to apex. (-40-50 A), In this part of the membrane The x-ray analyses described here are the fine filaments emerge from the coarse not quantitative, but give information con- ones directly nearest them, as though un- cerning the ions present and their relative winding much in the manner of fraying concentration in scans of equal duration rope. The fine filaments turn out almost and of approximately equal counts of emit- at right angles from the coarser ones, lie ted rays per second (1000). These analyses close to one another, and finally cross the show that the ions present in the tectorial halo-like region around the tips of the membrane are not distributed in similar hairs. As the frlaments cross to the hairs, proportions on its two sides (figs. 33, 34). they become fine and are spatially evenly On the upper side (fig. 33), K+ and C1- arranged (-30 A apart). as though held ions are present in almost equivalent in register. It is this even spacing of the amounts, although K+ is slightly more filaments which lends the halo-like appear- plentiful. Na+ is present in small and Mg++ ance to the zone of attachment. The fila- in trace quantities, but Ca++does not ap ments then course without break into the pear to exist in sufficient quantity to be caps of the hairs, into material which detected. On the under side of the mem- would seem to correspond to a cell coat. brane (fig. 34), C1- is the most plentiful, In all cases, adjacent filaments appear to followed by K' and then by Na+. Na+ is be cross-linked. abundant on the under side of the mem- brane. Mg++ and Ca++ are present only DISCUSSION in trace quantities. A small amount of sul- The chemical nature of the tectorial fur is found on both the upper and the membrane. One of the more important under sides and is likely incorporated into issues to resolve is whether the tectorial the protein of the membrane. membrane consists of acid mucopolysac- Transmission electron microscopy of the charide or of glycoprotein substances, Acid tectorial membrane. The portion of the mucopolysaccharides ( glycosaminoglycuro- tectorial membrane considered in detail noglycans) are widely distributed in con- here is the region of attachment to the nective tissues and are large molecular outer hair cells. Thus far, no essential dif- weight protein-carbohydrate complexes ferences have been found between tectorial with polysaccharide chains composed of membranes obtained from untreated and repeating disaccharide units. These repeat- from chemically sympathectomized rats. ing units contain uronic acids (glucuronic The interior of the membrane is com- or iduronic acid) and N-acetylhexosamine prised of numerous filaments which are (N-acetylglucosamine or N-acetylgalactos- either fine (-30 A wide), possessing nu- amine). With the exception of hyaluronic merous cross-hatchings so that they look acid most acid mucopolysaccharides con- feathered, and lacking apparent organiza- tain ester-sulfate bonds. Glycoproteins on tion; or else the filaments are coarse the other hand, are generally of lower mo- (-160 A wide), less branched and aggre- lecular weight and branched in structure, gated into groups having similar orienta- lack a serially repeating unit and are high tions (fig. 35) In these groups, the coarse in protein content. In general, they lack 458 MURIEL D. ROSS ester-sulfate bonds and contain a diversity ('65) produced experimental evidence of sugars or sugar derivatives, including N- which indicates that the tectorial mem- acetylglucosamine, mannose, galactose, brane is, in fact, electrically neutral. and/or fucose or sialic acid (Gottschalk, Attachments of the membrane to the '66). Glycoproteins are currently of great organ of Corti. The present scanning elec- interest for they are being shown to be tron microscopic findings show that the important constituents of cell coats and to tectorial membrane is attached to the tips participate in the immune reaction; in sur- of hairs of the outer hair cells, to the tips face charge, cell permeability and electri- and shafts of hairs of inner hair cells, and cal properties of cells; in cell division; and to the microvilli of the surface of the or- in determining cell to cell reactions (Ram- gan of Corti from the inner border cells to bourg, '71 ). the phalangeal processes of the outermost The present histochemical results show Deiters cells. Thus far, attachments to the that the tectorial membrane is highly PAS- Hensen cells have not been found. positive and is not metachromatic, in Although the connections to the micro- agreement with the original findings of villi could not have been observed with Wislocki and Ladman ('55). These obser- the light microscope, my present findings vations must be interpreted in the light of are in general agreement with those of recent histochemical and biochemical evi- Prentiss ('13) and the other few, early in- dence which shows that only glycoproteins vestigators who held that the tectorial are highly PAS-positive, while mucopoly- membrane was attached to the surface of saccharides react slowly and little, if at all the organ of Corti in adult mammals (Glegg et al., '52; Leblond et al., '57; Ram- (Boettcher, 1859; Coyne and Cannieu, bourg et al., '66; Rambourg, '71). On this 1895; Kishi, '07; Shambaugh, '07). My re- basis alone, the tectorial membrane would sults in juvenile and adult rats are strik- appear to contain glycoprotein( s). ingly similar to those obtained by Held A metachromatic reaction on the part of ('09) in fetal and newborn animals. Held a substance does not automatically cate- previously described not only the attach- gorize it as an acid mucopolysaccharide, ments of the tectorial membrane to the but only indicates that anions with which organ of Corti shown here, but also the the dye can react are present. The existing existence of a compartment around the discrepancies in the metachromatic reac- hairs of the outer hair cells. He indicated, tion of the tectorial membrane reported in however, that these relationships did not the previous studies are at least partially persist beyond the neonatal state. explainable on the grounds that much of Alternate viewpoints that the shreds of the work was carried out on inner ears material adhering to the microvilli are pre- after exposure to various fluids for fixation cipitated macromolecules formerly in solu- and/or dehydration, or even after decalci- tion in a subtectorial fluid, or are pieces fication, and often on embedded material. of membrane which have accidentally These manipulations could alter the ionic fallen into position, have been considered composition of the membrane. In the and discounted. A subtectorial fluid would present study, use of cryostat sections from be small in quantity; its macromolecular cochleae frozen in situ within the skull content would have to be very high (and and fixed in vapor rather than fluid avoids the fluid viscous) to account for the volume the possibility that metachromasia has of material I have observed clinging to the been artifactitiously conferred upon the microvilli. If endolymph were considered membrane. to be the subtectorial fluid, the chances of The lack of metachromasia in toluidine the shreds being precipitated macromole- blue sections indicates that the tectorial cules diminish remarkably. The viscosity membrane is not highly negatively charged. and the protein content of endolymph are Based on their studies of interdental cells low in mammals (in the human: viscosity, of the spiral limbus, Arnold and Vosteen 1.03-1.05 relative to water, 1.0; total pro- ('73) concluded that the tectorial mem- tein content, 20-30 mg/100 ml; Rauch, brane consists mostly of neutral glycopro- '64 ) . Moreover, the organization of the teins synthesized by these cells. Lawrence shreds and their resemblance to, and con- TECTORIAL MEMBRANE OF THE RAT 459 tinuity with, the tectorial membrane speak tion begins, but which shrinks and pulls for their being part of that membrane. Ac- away from its attachments to the organ of cidentally deposited debris is never organ- Corti as withdrawal of water continues. ized and never specifically related to par- Consistent curling back of the outer margin ticular structures. of the membrane suggests a greater effect The physical nature of the tectorial of dehydration upon the upper than on the membrane. My conclusion that the tec- under surface. torial membrane is a glycoprotein must be Thickenings of the gelatinous substance considered together with the findings of of the tectorial membrane radiate from the Naftalin et al. (’64) that the membrane region of the limbus toward the organ of is a gel containing approximately 90% Corti in cross sections of membranes pre- water in the adult guinea pig. This sug- pared by either freeze-drying or by fixation gests that the tectorial membrane is a sys- in 25% glutaraldehyde prior to freeze-dry- tem of glycoprotein molecules in close as- ing. They may correspond to the “fibrils” sociation with fluid, forming a colloidal or seen with phase-contrast or polarized light semisolid structure, and that no further microscopy (A. Hilding, ’52; Iurato, ’60) “amorphous” material is present. The and to the fibrillar structures depicted by amount of fluid entrained by the mem- Held (’09) as anchoring the tectorial mem- brane appears to change between the fetal brane to the reticular surfaces of the sup- and the functional states, for embryologists porting cells of the organ of Corti in the have consistently found the membrane to fetus. These thickenings would appear to be attached to the entire organ of Corti de- correspond to the groups of similarly ori- velopmentally but not in the adult. The ented filaments observed by transmission ease of preservation of the connections to electron microscopy (fig. 35). The aggre- the microvilli in birds implies that the gates of filaments do not correspond to membrane is less hydrated than in mam- “fibrils,” but possibly represent orientations mals, The extreme sensitivity of the mem- assumed by the glycoprotein macromole- brane to states of hydration must reflect cules due to the tensions placed upon the an important in vivo property which has membrane by its attachments. been little explored. Correlation of data obtained by trans- The conclusion that the tectorial mem- mission electron microscopy of the tec- brane is basically a gel has significant rami- torial membrane with biochemical and fications in considering the ultrastructural physical data. The filaments observed in results which have been interpreted to in- transmission electron microscopy of the dicate that the membrane is largely fibrillar tectorial membrane are the subunits of the or consists of fibrils in an amorphous ma- cohesive material seen with the scanning trix. As shown here, the grossly fibrillar electron microscope. They are presumed to appearance of the membrane described by be aggregates of macromolecules compris- many workers is artifactitious and related ing the glycoprotein of the membrane. The to the fixation and dehydration procedures arrangements of the macromolecules are followed, dependent upon electrostatic interactions Membranes dehydrated after being freed between or within the polyelectrolytes of their attachments are not fibrillar, but which act to attract or repel one another, are gelatinous in appearance, These results upon the steric configurations of the mole- demonstrate that great viscous forces exist cules themselves, and upon the effects of in the glycoprotein of the tectorial mem- tensions (stress) or of ions in the entrained brane which keep it a cohesive mass when fluid. it is dried free of attachments, The sur- The ’ electron microscopical observations faces of membranes simply dehydrated, or taken together with the well-known physi- fixed and dehydrated, in situ show various cal visco-elastic property of the membrane patterns in the residual, still cohesive ma- lead to the inference that the polyelectro- terial. The particular pattern seen reflects lyte glycoprotein molecular configuration the changing forces of stress placed upon is the extended flexible coil (Gibbons, ’66). the viscous membrane which is anchored One consequence of this configuration is at both ends as fixation and/or dehydra- that the molecules can interact physically 460 MURIEL -D. ROSS with one another to form a complex net- correspond to figures given for the ionic work which will possess properties making content of perilymph (in the human: Na+, it important in fluid and ion transport. 118-169; K+, 2.9-8.2; C1-, 102-135; When flexible polyelectrolyte molecules be- Rauch, '64). Thus, the electrolyte composi- come very extended, hydrodynamic inter- tion of the fluid in the colloid (tectorial action ceases to a point where appreciable membrane) is different from either endo- fluid can pass between and through the lymph or perilymph, as Naftalin et al. molecules. Such molecules are said to be ('64) have previously reported. partially or free draining, in contrast to Naftalin et al. ('64) have shown that more coiled or entwined molecules through the membrane binds some of the K+, and which less water can pass (Gibbons, '66). binds Ca++more tightly than Mg++.These Both the more closely entwined and the findings taken together with the present more extended-appearing, thread-like fda- results indicate that the tectorial mem- ments occur in the tectorial membrane, and brane is not simply a sponge for endo- in differing concentrations from one site to lymph, Rather, if the membrane is elec- another (fig. 35). Thus, fluid transport trically neutral as the present histochemi- capabilities are likely to differ from one cal and prior electrical evidence (Law- site to another within the membrane. How- rence, '65) suggests, it stands as a barrier ever, the amount of fluid entrained is pos- between regions of different electrolyte sibly in constant flux, for the configuration composition to retard diffusion of ions from of the molecules is unstable and can the one region to the other. The cells change (by further extending or recoiling) around the endolymphatic space must be due to solvent-solute interactions, slight discharging or actively removing ions shifts in pH or ions, stress, or even Brown- differently from those cells lying under ian movement (Gibbons, '66). the membrane. As the shifts noted are Ions can be moved through a complex, primarily in concentrations of Na+ and polyelectrolyte network by either of two K+, there are the ions which are most methods: free flow with the fluid solvent, concerned. or by ion-exchange. In either case, the hy- The fact that the ionic composition of drated radius of the ions (ionic size) and the tectorial membrane differs from that the charge on the polyelectrolyte molecules of endolymph means that a potential dif- must be considered pertinent to their ference will be produced, for potentials, movement . which can be quite high, are created where- The ionic composition of the tectorial ever charge separations exist. A series of membrane: its functional significance. finite charge separations probably occurs The present findings show that the tec- within the tectorial membrane itself. How- tonal membrane has some pore spaces that ever, if the entire membrane is considered are large (Ca++has a relative hydration as one liquid phase and endolymph as an- of 22 compared to 5.4 for K+ and 8.4 for other, then a charge separation exists at or Na+; Hober et al., '45), and that it will near the interface between the two. That admit both cations and anions. The find- this is the case has already been shown by ings also demonstrate that although the Lawrence ('65), who found a shift in po- tectorial membrane contains essentially the tential to occur from about 6-7 mv nega- same ions as endolymph, it does not tive (approximately base line) to about possess them in similar relative concen- 70 mv positive as his recording electrode trations on both sides of the membrane. left the tectorial membrane to enter endo- The relative concentrations found here for lymph. Other charge separations possibly the ions on the upper surface correspond exist between the tectorial membrane and well with figures expressed in milligram- the fluid compartments around the hairs equivalents per liter for endolymph (in of the outer hair cells, although there is the human: Na', 6-30; K+, 128-169; C1-, presently no electrical evidence for their 100-123; Rauch, '64). However, the rela- existence. Additionally, the tectorial mem- tive concentrations of the ions (Cl- > brane, by its anatomical position, partici- K+ > Na+) present on the underside of pates in the production of a charge sepa- the membrane are different; nor do they ration between the organ of Corti and the TECTORIAL MEMBRANE OF THE RAT 46 1 endolymph. Lawrence ('65) found a shift ('64, '65), although Mygind ('52) and Bor- from 40 mv negative in the organ of Corti ghesan ('71) have also supported it. to 70 mv positive in the endolymph, with One of the arguments against bending the shift toward zero taking place at the of the hairs, as postulated by BekCsy, has tectorial membrane. Thus, the tectorial been the fact that the hairs are stiff (Wal- membrane would appear to play a role in deyer, 1873) and will break before bend- the production of certain inner ear poten- ing (Engstrom et al., '62). In my prepara- tials, although the specific functions of tions for scanning electron microscopy, these potentials are currently unknown. hairs are sometimes broken at their bases Functional significance of the attach- and moved en masse from their positions ments of the tectorial membrane to the due to the fact that they are strongly tied hair cells. The concept that the extended, together by a binding material. The pres- flexible coil is an unstable molecular con- ence of this substance would preclude figuration would appear to be of extreme bending of the hairs, for the hairs would significance when considering the function be restrained in movement by their coup- of the attachment between the tectorial ling to one another, In my scanning prepa- membrane and the tips of the hairs of rations, both the hairs of outer and of inner outer hair cells. The filaments by which hair cells show this binding; previously, the structures are attached to one another Lindemann and Bredberg ('72) observed are thread-like, held in register with one similar connections in the case of hairs of another, and continuous with the cell coat inner hair cells in the cat. The binding of the tips of the hairs. They become inti- material seen by this method probably cor- mately related to or even a part of the cell responds to the amorphous or granular membranes of the hair cells on the one substance found along the surface of the hand, and are continuous with the sub- shafts of the hairs by all who have studied stance of the tectorial membrane on the the hair cells by transmission electron mi- other. Stimulation of these filaments by croscopy. It may be a cell coat; some have any of several means, (including stress or postulated the material contains acid muco- local shifts in pH or in ionic environment) polysaccharide (Kuttner and Geyer, '71). could result in changes in configuration of ACKNOWLEDGMENTS the molecules, leading to alteration in the permeability of the hair cell membrane to I thank Dr. Rita P. Liu for her techni- ions, and to production of a receptor po- cal assistance and Dr. Wilbur Bigelow, Di- tential. This would bring the transduction rector of the Scanning Electron Microscope of acoustical into electrical energy down Facility at the University of Michigan, for to a molecular level as Naftalin ('65) and access to the scanning electron microscope Lawrence ('65) have indicated is essential and the microprobe. I am also most grate- at lowermost thresholds of hearing. ful to Dr. Joseph E. Hawkins, Jr., Dr. Merle In a sense, the tips of the outer hairs, Lawrence and Dr. Edward Lauer for their the hairs of the inner hair cells, and the helpful remarks concerning this manu- tectorial membrane are continuous, for script and to Dr. George Jourdian, who they are physically bound together, and was kind enough to comment on the bio- their attachments may correspond to a chemical aspects of this report. binding of one cell coat to another. This I thank Merck, Sharp and Dohme Re- would suggest that the hairs and the tec- search Laboratories for their gift of a torial membrane are also functionally quantity of 6-hydroxydopamine, which was united, and that the tectorial membrane used in some of the experiments reported and not the basilar is the important struc- upon here. ture in delivering acoustical energy to the LITERATURE CITED hair cells. While this concept is not new Angeletti, P. U., and R. Levi-Montalcini 1970 (von Ebner, '02; Shambaugh, '07, '08; Har- Sympathetic nerve cell destruction in newborn desty, '08; Prentiss, '13) it has all but been mammals by GHydroxydopamine. Proc. Nat. abandoned since the work of Bekksy ('60). Acad. Sci. U.S.A., 65: 114-121. Arnold, W., and K.-H.Vosteen 1973 Zur sekre- Most noted among recent proponents of torischen Aktivitat der Interdental-zellen des this concept are Naftalin and his group Limbus spiralis. Acta Otolaryng., 75: 192-202. 462 MURIEL D. ROSS

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F. 1953 Autoradiographic detec- A. Gottschalk, ed. Elsevier, Amsterdam, Lon- tion of P5in the membranes of the inner ear don, and New York., pp. 29-95. of the rat. Science, 118: 52G521. Glegg, R. E., Y. Clermont and C. P. Leblond 1952 1954 Autoradiographic visualization of The use of lead tetraacetate, benzidine, O-dian- S35 incorporation and turnover by the mu- sidine and a “‘film test” in investigating the cous glands of the gastro-intestinal tract periodic acid-Schiff technic. Stain Tech., 27: and other soft tissues of rat and hamster. Anat. 277-305. Rec., 118: 755-771. Gottschalk, A. 1966 Definition of glycoproteins 1956a On the intimate composition of and their delineation from other carbohydrate- membranes of the inner ear. Science, 123: 1074. protein complexes. In: Glycoproteins - Their 1956b Observations on the develop- Composition, Structure and Function. B. B. A. ment, structure and composition of the cochlea Libraries, Vol. 5, Chapter 2. A. Gottschalk, ed. of the rat. Ann. 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Rambourg, A. 1971 Morphological and histo- Kolliker, A. 1854 Schnecke. In: Mikroskopische chemical aspects of glycoproteins at the sur- Anatomie. W. Engelmann, Leipzig, pp. 743- face of animal cells. Int. Rev. Cytol., 31: 57- 763. 114. 1861 Entwicklungsgeschichte des Rambourg, A., M. Neutra and C. P. Leblond Menschen und der hoheren Tiere. W. Engel- 1966 Presence of a “cell coat” rich in carbo- mann, Leipzig. hydrate at the surface of cells in the rat. Anat. Kolmer, W. 1907 Beitrage zur Kenntnis des Rec., 154: 41-52. feineren Baues des Gehororgans mit besonderer Rauch, S. 1964 Biochemie des Hororgans. Berucksichtigung der Haussaugetiere. Arch. George Thieme, Stuttgart. mikr. Anat., 70: 695-767. Retzius, G. 1884 Das Gehiirorgan der Wirbel- 1927 Gehororgan. In: Handbuch der thiere. Bd. 2. Samson and Wallin, Stockholm. mikroskopischen Anatomie des Menschen. W. Reynolds, E. S. 1963 The use of lead citrate at von Mollendorf and W. Bargmann, eds., high pH as an electron opaque stain in electron Springer, Berlin, pp. 25Ck478. microscopy. J. Cell Biol., 17: 208-212. Kosaka, N., T. Tanaka, T. Takiguchi, Y. Ozeki Rickenbacher, 0. 1901 Untersuchungen uber and S. Takahara 1971 Observation on the or- die Embryonale Membrana Tectoria des Meer- gan of Corti with scanning electron microscope. schweinchens. Anat. Hefte, Abth. 1, 16: 381- Acta Otolaryng., 72: 377-384. 412. Kramer, H., and G. M. Windrum 1955 The Rosenhall, U. 1971 Morphological patterns of metachromatic staining reaction. J. Histochem. the organ of Corti in birds. Arch. Ohr. Nas.- Cytochem., 3: 227-237. Kehlk. Heilk., 200: 42-63. Kuttner, K., and G. Geyer 1972 Elektronen- und Shambaugh, G. 1907 A restudy of the minute Lichtmfkroskopische Untersuchungen zur Dar- anatomy of structures in the cochlea with con- stellung der Glykokalyx im Bereich des Ductus clusions bearing on the solution of the problem Cochlearis des Meerschweinchens. Acta Oto- of tone perception. Am. J. Anat., 7: 245-257. laryng., 74: 183-190. 1908 The membrana tectoria and the Lawrence, M. 1965 Dynamic range of the coch- theory of tone perception. Arch. Otol., 37: 457- lear transducer. In: Symposia on Quantitative 467. Biology. Cold Spring Harbor Laboratory of Smith, C. A., and E. W. Dempsey 1957 Elec- Quantitative Biology, Cold Spring Harbor, 30: tron microscopy of the organ of Corti. Am. J. 159-167. Anat., 100: 337-368. Leblond, C. P., R. E. Glegg and D. Eidinger 1957 Spoendlin, H. 1957 Elektronenmikroskopische Presence of carbohydrates with free 1,2-glycol Untersuchungen am Corti’schen Organ des groups in sites stained by the periodic acid- Meerschweinchens. Pract. Oto-rhino-laryng., 19: Schiff technique. J. Hi’stochem. Cytochem., 5: 192-234. 445458. Takasaka, T., and C. A. Smith 1971 The struc- Lillie, R. D. 1965 Histopathologic Technic and ture and innervation of the pigeon’s basilar Practical Histochemistry. McGraw-Hill Book Co., papilla. J. Ultrastruct. Res., 35: 20-65. New York. Tanaka, T., N. Kosaka, T. Takiguchi, T. Aoki and Lim, D. J. 1972 Fine morphology of the tec- S. Takahara 1973 Observation on the cochlea torial membrane. Arch. Otolaryng., 96: 199- with SEM. In: Scanning Electron MicroscoPv/ 215. 1973. 0. Johari and I. Corvin, eds. IIT Research Lindeman, H. H., and G. Bredberg 1972 Scan- Inst., Chicago, pp. 427433. 464 MURIEL D. ROSS

Thompson, S. W., and R. D. Hunt 1966 Se- tive Histology. Vol. 3, Chapt. IV. S. Stricker, lected Histochemical and Histopathological ed. The New Sydenham Society, London. Methods. Charles C Thomas, Springfield. Wislocki, G. B., and A. J. Ladman 1955 Selec- Tonndorf, J., A. J. Duvall and J. P. Reneau tive and histochemical staining of the otolithic 1962 Permeability of intracochlear membranes membranes, cupulae and tectorial membrane to various vital stains. Ann. Otol. Rhinol. of the inner ear. J. Anat., 89: 3-12. Laryng., 71: 801-841. Vinnikov Ya. A., and L. K. Titova 1964 The Wittmaack, K. 1936 Uber Bau und Funktion Organ of Corti: Its Histophysiology and Histo- der Membrana tectoria. Acta Otolaryng., 24: chemistry. Translated by B. Haigh. Consultants 397-423. Bureau, New York. Worthington Biochemical Corporation 1972 Waldeyer, W. 1873 The auditory nerve and Worthington Enzyme Manual. Worthington cochlea. In: Manual of Human and Compara- Biochemical Corporation, Freehold.

PLATE 1

EXPLANATION OF FIGURES

1 The tectorial membrane (TM)is PAS-positive in the normal rat. SL, spiral limbus. Rat 21 days old. x 310. 2 After chemical sympathectomy with 6-hydroxydopamine, the tectorial membrane (TM) remains PAS-positive. SL, spiral limbus. Rat 22 days old. x 300. 3 The tectorial membrane (TM) stains orthochromatically in toluidine blue. The membrane often has a feathery appearance with this stain. SL, spiral limbus. Rat 24 days old. x 350. 4 After chemical sympathectomy, the tectorial membrane (TM) re- mains orthchromatic in toluidine blue. The intensity of the staining reaction is approximately the same as in untreated rats. SL, spiral limbus. Rat 22 days old. x 345. 5 In this specimen, exposed to hyaluronidase for three hours prior to staining with toluidine blue, the tectorial membrane (TM), is ortho- chromatic and shows little change in staining reaction from the membrane shown in figure 3. SL, spiral limbus. Rat 25 days old. X 325. 6 The tectorial membrane (TM) incubated in neuraminidase for 30 minutes prior to staining with toluidine blue stains a faint blue. SL, spiral limbus. Rat 25 days old. x 360. TECTORIAL MEMBRANE OF THE RAT PLATE 1 Muriel D. Ross PLATE 2

EXPLANATION OF FIGURES

7 The hairs of the outer hair cells of the normal rat are arranged in a “W configuration (indentation between limbs of the “W is marked with an arrow). The tips of the hairs are smooth and bulbous when entirely freed of tectorial membrane. Middle third of the cochlea. Rat 39 days old. x 18,000. 8 With care, the attachments of the tectorial membrane (TM) to the tips of the hairs of outer hair cells are preserved. Some of the con- nections between adjacent hairs are indicated by arrows. Note the few microvilli (M) on the cuticular surface of the hair cell. Some of the microvilli at upper right have bits of the substance of the tectorial membrane still adherent to them. Apical third of the coch- lea. Rat 32 days old. x 20,000. 9 The hairs of the outer hair cells are so firmly attached to the tec- torial membrane (TM) that they are sometimes torn loose from their basal relationships to the Deiters cells. Indentations, denoting the former positions of hairs of outer hair cells now pulled away, are obvious on the underside of the tectorial membrane (arrows). B, base of hair cell. Basal third of the cochlea. Adult rat, 320 gm. x 3800. 10 Shreds sometimes hang down from the tectorial membrane (TM), denoting positions of attachments (arrows) between the membrane and the tips of the hairs of outer hair cells. Middle third of the cochlea. Adult rat, 320 gm. x 3600.

466 TECTORIAL MEMBRANE OF THE RAT PLATE 2 Muriel D. Ross

467 PLATE 3

EXPLANATION OF FIGURES

11 The hairs of the inner hair cells are arranged in three rows, forming a shallow crescent, with the tallest hairs outermost. When the tec- torial membrane is completely removed from them, the hairs of the outermost row have blunt tips while those of the inner rows have tips which are flattened and sharply angled upward. IPC, inner pillar cell; IPhC, inner phalangeal cell. Middle third of the cochlea. Rat 39 days old. x 11,000. 12 The hairs of the inner hair cells appear to be enshrouded by the tec- torial membrane where it is left as intact as possible. At left, the tectorial membrane remains attached to the heads of the inner pillar cells (IPC). These attachments have been disrupted toward the center of the field. Toward the upper right, the attachments to the tips of the hairs of the inner hair cells are evident (arrow). Apical third of the cochlea. Rat 40 days old. x 3000. 13 The attachments of the tectorial membrane (TM) to the tips of the hairs of the inner hair cells are shown here at higher magnification than in figure 12. Most of the tectorial membrane appears fibrous, but this is artifactitious and related to fixation and dehydration of the tissue. However, the regular arrangement of elements of the membrane attaching to the hairs can be observed at upper right (arrows). Note that bits of the tectorial membrane cling to the shafts of the hairs. Apical third of the cochlea. Rat 40 days old. x 12,000. 14 Connections of the tectorial membrane (TM) to the tips of the hairs of inner hair cells are also demonstrated in this scanning electron micrograph. Connecting filaments (F) remain attached to the second hair from the left, although the hair was loosened from the mem- brane. Apical third of the cochlea. Rat 40 days old. x 21,000.

468 TECTORIAL MEMBRANE OF 'ME RAT PLATE 3 Muriel D. Ross

469 PLATE 4

EXPLANATION OF FIGURES

15 Filamentous strands connect adjacent hairs of the inner hair cells (arrows). Note that bits of the tectorial membrane (mow heads) still cling to microvilli of the inner pillar cell (IPC) and also to hairs of the inner hair cell. Middle third of the cochlea. Rat 39 days old. X 24,000. 16 Connections between adjacent hairs of the same row and of the neighboring row on an inner hair cell are evident here (arrows). A few of the sites where remnants of the tectorial membrane remain attached to microvilli of the inner phalangeal cell (IPhC) and the inner pillar cell (IPC) are indicated with arrow heads. Middle third of the cochlea. Rat 39 days old. X 12,700. 17 Shreds of the tectorial membrane (TM) attach to microvilli of the inner phalangeal cells (IPhC) and border cells (BC) in this speci- men. A few of the more obvious connections are indicated by arrows. Middle third of the cochlea. Rat 39 days old. X 8000. 18 Shreds of the tectorial membrane (TM) commonly attach to the microvilli of the inner pillar cells (IPC), outer pillar cells (OPC), and the Deiters cells (DC). Some shreds also adhere to microvilli of the cuticular plates of outer hair cells. A few of these attachments are indicated by arrows. Middle third of the cochlea. Rat 39 days old. x 8000.

470 TECTORIAL MEMBRANE OF THE RAT PLATE 4 Muriel D. Ross

471 PLATE 5

EXPLANATION OF FIGURES

19 Connections of the tectorial membrane to the microvilli on the pha- langeal processes of the second (single arrow) and third row (double arrows) of Deiters cells are indicated here. Rat 34 days old. Middle third of the cochlea. x 8000. 20 The shreds of tectorial membrane remaining adherent to the reticu- lar lamina of the organ of Corti attach to the microvilli (arrows), as demonstrated at high magnification here. The connections shown are to microvilli of inner phalangeal cells. HIHC, hairs of an inner hair cell. Rat 34 days old. X 22,000. 31 This drawing illustrates the anatomical position of the tectorid mem- brane according to the present findings. The membrane is closely ap- plied to the reticular laminar surface of the organ of Corti. It en- closes the hairs of the inner hair cells, but is attached only to the tips of the hairs of the outer hair cells. The relationship of the mem- brane to the hairs (H) of the outer hair cells is shown in the insert; a fluid compartment (C) may exist around and within the limbs of the “W” formed by the hairs on the cuticular surface of each outer hair cell. The tectorial membrane is depicted here as a gel; internal organization of the membrane has been omitted.

472 TECTORIAL MEMBRANE OF THE RAT PLATE 5 Muriel D. Ross

473 PLATE 6

EXPLANATION OF FIGURES

22 Tectorial membranes freed of their attachments prior to dehydration appear gelatinous. Upper surface of the tectorial membrane, apical third of the cochlea. Critical point dried. Rat 39 days old. x 750. 23 A portion of the upper surface of the tectorial membrane from the same specimen depicted in figure 22 is shown here at higher magni- fication. Note the band-like thickenings of the gelatinous substance near the outer margin of the membrane (arrows). X 5400. 24 This scanning electron micrograph shows the under side of the tec- torial membrane, as it appears when the membrane is dried free of its attachments. Critical point dried membrane. Rat 39 days old. x 6000. 25 Tectorial membranes fixed and dehydrated in situ commonly have their upper surfaces thrown into ridges, coursing in all directions. Near the peripheral margin of the membrane, at times, there are lon- gitudinally running bands. The bands (arrows) shown here corre- spond in position to those shown in figure 23. Middle third of the cochlea. Rat 40 days old. x 2400.

474 TECTORIAL MEMBRANE OF THE RAT PLATE 6 Muriel D. Ross

475 PLATE 7

EXPLANATION OF FIGURES

26 The fine ridges, disposed in all directions, which are found on the upper surface of the tectorial membrane fixed and dried in situ are shown here at higher magnification than in figure 25. This portion of the membrane lies proximal (toward the spiral limbus) to the part shown in figure 25. Middle third of the cochlea. Rat 40 days old. x 4800. 27 A small portion of the under surface of the tectorial membrane from the same specimen shown in figures 25 and 26 is shown here. The under side (UTM) is curled back over the upper surface (UpTM) at the peripheral margin of the tectorial membrane. The under surface is rough and irregular. Where tears occur, the interior of the mem- brane appears fibrous. Middle third of the cochlea. Rat 40 days old. x 3750. 28 In some places, the upper surface of tectorial membrane (UpTM) freeze-dried without prior fixation or dehydration exhibits a series of ribbon-like, gelatinous structures (arrows). Here, at the peripheral border of the membrane, the under side (UTM) is gelatinous and curled back upon the upper surface. Middle third of the cochlea. Rat 41 days old. x 3750. 29 The gelatinous substance of the upper side of the freeze-dried tec- torial membrane may be smooth, or show stubby protrusions. Middle third of the cochlea. Rat 41 days old. x 10,000.

476 TECTORIAL MEMBRANE OF THE RAT PLATE 7 Muriel D. Ross

477 PLATE 8

EXPLANATION OF FIGURES

30 A cross section of freeze-dried tectorial membrane (TM) is shown here. The interior of the membrane is gelatinous, not fibrillar. Thick- enings of the gelatinous material are indicated ty arrows. Basal third on the cochlea. Rat 41 days old. x 2300. 31 Compare this micrograph with figure 30. In cross section, the tec- torial membrane (TM) fixed in 25% glutaraldehyde prior to freeze- drying resembles greatly the tectorial membrane subjected to freeze- drying only. The organ of Corti (OC) has been disruFted during preparation of the tissue. Basal third of the cochka. Rat 71 days old. x 1270. 32 A portion of the interior of the tectorial membrane shown in fig- ure 31 is presented here at higher magnification. The gelatinous nature of the membrane and the presence of thickenings (arrows) of the gelatinous material are evident. Rat 71 days old. x 32,000.

478 TECTORIAL MEMBRANE OF THE RAT PLATE 8 Muriel D. Ross

479 PLATE 9

EXPLANATION OF FIGURES

33 This is an x-ray spectrum obtained upon the analysis of the upper surface of a segment of tectorial membrane from the basal third of the cochlea. The peaks given by Na+, C1- and K+ are indicated. The two major peaks between Na+ and C1- are those given by phos- phorus and sulfur, from left to right. Freeze-dried membrane. Adult rat, 385 gm. 34 This is an x-ray spectrum obtained upon analysis of the under side of a segment of tectorial membrane from the basal third of the coch- lea. Compare with figure 33. Freeze-dried membrane. Adult rat, 350 gm. 35 A portion of the tectorial membrane, with capping material from the tips of 15 hairs still attached, is shown in this transmission electron micrograph. Some of the filaments on the interior are thick (-160 A) and are aggregated into bundles (single arrow). Many of the remain- ing interior filaments are finer (+30 A) and lack regular arrange- ment. Near the under side of the membrane, thicker filaments are continuous with finer ones which are more closely spaced; fine fila- ments then cross the halo-like region to the caps of the hairs in regular array (see the series of 3 arrows, lower center). The fila- ments appear to be cross linked (arrow heads). Detritus (D) appears entrapped in the clear spaces in the membrane. Rat 22 days old, 6- hydroxydopamine treated. Basal third of the cochlea. x 28,500.

480 TECTORIAL MEMBRANE OF THE RAT PLATE 9 Muriel D. Ross

48 1