Further studies on the mechanics of the cochlear partition in the mustached bat. I. Ultrastructural observations on the tectorial membrane and its attachments

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HWRIIIC, RCH Hearing Research 94 (1996) 63-77

Further studies on the mechanics of the cochlear partition in the mustached bat. I. Ultrastructural observations on the tectorial membrane and its attachments M a r i a n n e V a t e r a, *, M a n f r e d K~Sssl b lnstitut fiir Zoologie, Universit~it Regensburg, Unicersitiitsstrasse 31, 93040 Regensburg, Germany b Zoologisches lnstitut der Unilersitiit Miinchen, Luisenstrasse 14, 80333 Miinchen. Germany

Received 31 March 1995; accepted 8 December 1995

Abstract From semithin and ultrathin sections of the mustached bat cochlea, baso-apical gradients in ultrastructural composition, shape and attachment site of the tectorial membrane (TM) were determined in relation to gradients in hair cell size and stereocilia size. These provide a data base for estimates of the mechanical properties of the organ of Corti as they relate to specialized aspects of the cochlear frequency map (KiSssl and Vater, 1996). As in other mammals, the TM is composed of type A and type B protofibrils. Measurements of the packing density of type A protofibrils reveal gradients in both the radial and longitudinal direction. Distinct variations in packing density of type A protofibrils across the radial extent of the TM allow the definition of more subregions than in other mammals. Throughout the cochlea, packing density is highest in the 'stripe' region located close to the spiral limbus. The centrally located 'core' region of the middle zone contains distinctly fewer type A protofibrils than the laterally located 'mantle' region of the middle zone. The TM in the specialized basal turn (first and second half-turns) features a higher packing density of type A protofibrils in the 'mantle' than the TM in the apical cochlea (upper third to fifth half-turns), and an incorporation of longitudinally directed type A protofibrils in the marginal zone. Among cochlear turns, there are pronounced changes in cross-sectional area of the TM and the extent of its limbal attachment site. Within the densely innervated second half-turn that contains an expanded representation of the 60 kHz constant frequency (CF) component of the echolocation signal, both the cross-sectional area (see also Henson and Henson, 1991) and the attachment site of the TM are enlarged. An extended limbal attachment site is also observed in the densely innervated region of the lower first half-turn that represents the upper harmonics of the call. Within the sparsely innervated region of the upper first half-turn, the limbal attachment site of the TM is significantly diminished. Size of outer hair cells (OHC) ranges between 12 and 13 /xm throughout the basal 80% of cochlear length and reaches maximal values of 20 /xm in the apex. Size of OHC stereocilia ranges between 0.7 and 0.8 /xm throughout the basal 60% of cochlear length and reaches a maximal size of 2.2 /xm in the apex. These data corroborate and extend previous notions that morphological specializations of the TM in concert with specializations of the basilar membrane and perilymphatic spaces play an integral role in creating specialized cochlear tuning in the mustached bat. Keywords: Audition; Echolocation; Cochlear tuning; Hair cell

1. I n t r o d u c t i o n The tectorial m e m b r a n e ( T M ) plays an integral role in stimulus transduction in the m a m m a l i a n cochlea. In the classical m o d e l it is thought to act as a stiff anchor for the

* Corresponding author. Tel.: (94-1) 943-3058; Fax: (94-1) 943-3304; E-mail: marianne.vater @biologie.uni-regensburg.de. 0378-5955/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S 0 3 7 8 - 5 9 5 5 ( 9 6 ) 0 0 0 0 5 - 6

tips o f hair cell stereocilia (e.g., Davis, 1956) so that shearing motion b e t w e e n the T M and the reticular lamina leads to a radial deflection o f the stereocilia and production o f receptor potentials. In an alternative view, the T M is thought to act as a mass load on the stereocilia with a resonant f r e q u e n c y distinctly b e l o w the characteristic freq u e n c y o f the respective c o c h l e a r place (Zwislocki and Kletzky, 1979; Allen, 1980; Z w i s l o c k i , 1988; Z w i s l o c k i et al., 1988; Z w i s l o c k i and Cefaratti, 1989; Allen and Fahey, 1993). The resonant T M is p r o p o s e d to act as a second

64

M. Vclter. M. KiJssl/ Hearing Research 94 ¢1996) 63-77

filter superimposed on basilar membrane (BM) mechanics (e.g., Brown et al., 1992; Allen and Fahey, 1993) The structural composition of the TM is thought to govern its mechanical properties and to play a role in ion balance of the subtectorial space above the hair cells (review Steel, 1983). Longitudinal gradients in TM mass are thought to influence cochlear tuning (Strelioff et al., 1985). The TM is an acellular structure that is secreted into the scala media by the action of several non-sensory cell types of the cochlear duct during ontogeny (e.g., Lira, 1972, 1987). Biochemical investigations reveal the presence of at least three collagen types and several non-collagenous glycosylated polypetides (Richardson et al., 1987). Ultrastructurally, the TM is composed of two types of protofibrils. Type A protofibrils are straight and unbranched (diameter: 10 nm) and form distinct bundles. Type B protofibrils form the matrix in which type A protofibrils can be embedded; they are coiled, branched and can exist in weakly and strongly hydrated states (Kronester-Frei, 1978; Hasko and Richardson, 1988). Immunocytochemical data (Slepecky et al.. 1992) suggest that type II and type IX collagen form the type A protofibrils. The matrix of type B protofibrils is thought to be composed of type V collagen. Type II collagen, as the major fibrillar component of the TM, likely contributes to structural stabilization and tensile strength of the TM (Slepecky et al., 1992), and the density of type A protofibrils has been used to estimate TM stiffness (Weaver and Schweizer, 1994). Type IX and type V collagen and associated proteoglycans contribute to the hydration of the matrix, its cation composition and cross-linking (Slepecky et al., 1992). Regional variations in the relative proportions of type A protofibrils distinguish a number of different zones within the TM (guinea pig, squirrel monkey, rhesus monkey: Lira, 1972; mouse; guinea pig; squirrel monkey: Kronester-Frei, 1978; mouse: Hasko and Richardson, 1988; gerbil: Weaver and Schweitzer, 1994). Together with type B protofibrils, they form the limbal and middle zones. The marginal band, the Hensen's stripe, the subsurface of the TM facing the organ of Corti, and the cover net are mainly composed of weakly hydrated type B protofibrils. Although there are reports on the pattern of changes in TM shape and cross-sectional area along the cochlear coils in certain bats ( H i p p o s i d e r o s : Dannhof and Bruns, 1991: mustached bat: Henson and Henson, 1991), the ultrastructure of the TM of echolocating bats has not been investigated. The mustached bat emits multiharmonic CF-FM calls (CF: constant frequency component; FM: frequency modulated component) with the dominant second harmonic CF frequency at about 60 kHz (review: Schnitzler and Henson, 1980). Its cochlea contains an expanded representation of a narrow frequency band around 60 kHz (K/Sssl and Vater, 1985a; Zook and Leake, 1989) and is exceptionally sharply tuned to frequency bands around 60 kHz and 90 kHz (Pollak et al., 1972; Suga et al., 1975; Henson et al., 1985). In addition to morphological special-

izations of the basilar membrane (Henson, 1978; K~Sssl and Vater, 1985a) and the perilymphatic spaces (Henson et al., 1977), the gradients in size of the TM reveal species characteristic specializations which have been proposed to play important roles in creating the unusually sharp tuning capabilities of the cochlea (Henson and Henson, 1991). The present study is designed to extend the observations on the TM to the ultrastructural level in order to provide a database that allows a comparison between the TM of a cochlea designed for hearing in the ultrasonic range up to frequencies of 120 kHz and the TM in the cochleae of non-echolocating mammals. Furthermore. we were particularly interested in defining longitudinal gradients in the shape and ultrastructural composition of the TM of mustached bats in order to relate them to gradients in hair cell size and stereocilia size. These data can provide estimates of the mechanical properties of the organ of Corti in relation to specialized aspects of the cochlear frequency map (K/Sssl and Vater, 1996).

2. Materials and m e t h o d s

Results are based on five cochleae from five adult mustached bats, P t e r o n o t u s p a r n e l l i i captured in Jamaica. Under deep pentobarbital anaesthesia (60 rag/100 g), the bats were decapitated and the cochleae were quickly removed from the skull and immersed in fixative (2.5% glutaraldehyde in 0.1 M phosphate buffer). The round and oval windows were immediately opened and a small hole was drilled in the apical turn. The fixative was peffused through the cochlea via the oval window. Specimens were postfixed in the same fixative for 3 h to several days. The fixed cochleae were decalcified in 7.5% EDTA (pH 7.3) for 48 h. Using a thin razor blade, the cochleae were split into two halves by midmodiolar sections in planes about 30 ° apart and referenced to the plane passing through the apex and a point between the round and oval windows. The individual half-turns were separated by sections parallel to the basilar membrane. After rinsing in buffer, the half-turns were treated with 1% OsO 4 (1 h), rinsed again, dehydrated in increasing series of alcohol (30-100%) and embedded in epoxy resin Durcopan. For assessment of cochlear length, the preparations were photographed at × 2 0 magnification (Wild stereoscope). Serial semithin sections (2 /,m) in the radial direction were cut from all half-turns by consecutively adjusting the plane of section parallel to the course of peripheral dendrites of spiral ganglion cells. The sections were mounted on non-coated glass slides and stained with Richardson blue. They were used to assess longitudinal gradients in the morphology of the organ of Corti with the light microscope ( × 100 to × I000 magnification). Selected sections were remounted in Durcopan and ultrathin sections were cut. The ultrathin sections were observed with a Zeiss EMIO at magnifica-

M. Vater, M. Kgssl / Hearing Research 94 (1996) 63-77

tions ranging from X 1700 to X 30000. Photographs were printed at 3 times the original magnification. The fixation, decalcification and dehydration protocols used here and in other studies of the TM (Lira, 1972; Hasko and Richardson, 1988; Henson and Henson, 1991; Weaver and Schweitzer, 1994) provide valuable information on subregional differences in ultrastructure of the TM and baso-apical gradients in TM shape and structure but only provide estimates of the normal dimensions and appearance of the TM in situ. It is known that the ionic composition of the fixative (Kronester-Frei, 1979; Freeman et al., 1994; Shah et al., 1995), and its exact components (Hasko and Richardson, 1988) critically influence TM morphology. The most prominent shrinkage appears to be produced by dehydration procedures (e.g., Zwicker, 1971; Lim, 1977, 1980). In the mustached bat, comparisons of the width of fixed, non-dehydrated TMs with specimens that were dehydrated and embedded showed similar regional specific changes (Henson and Henson, 1991). Therefore, it is likely that the relative sizes are preserved and that they are proportional to those in the living animal. The shape and cross-sectional area of the TM in different turns of three cochleae was analysed from drawings of semithin sections at magnifications of X 70. The drawings were digitized with a graphical tablet. The TM cross-sectional area and the extent of its attachment site with the spiral limbus was calculated with the program SigmaScan. Photomontages of ultrathin sections of the TM were constructed at X5100 final magnifications. From these, the individual subdivisions of the TM were defined according to criteria of packing density and course of type A protofibrils and the extent of the matrix. At selected positions spanning the radial and vertical extent of the TM, high power photomicrographs were taken (final magnification X 30000) in which the number of type A protofibrils crossing a 2 /~m line was counted. From adjacent semithin sections of the same cochleae, hair cell length (inner hair cells: IHC; outer hair cells: OHC), BM width and thickness, and the length of IHC stereocilia were measured with an ocular micrometer at X 1000 magnification. The length of OHC stereocilia was measured from photomicrographs of ultrathin sections taken at X5100 to X60000 final magnification.

3. Results 3.1. Shape of the tectorial membrane in different turns The mustached bat cochlea is coiled into five half-turns which are numbered starting from the base. According to the frequency map (K~Sssl and Vater, 1985a) and regional variations in innervation density (Henson, 1973; Zook and Leake, 1989), it can be sudivided into four regions (Fig. 1). (1) The apical cochlear regions (fifth to third half-turn) represent frequencies below the dominant second harmonic

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Fig. 1. The subdivisions of the mustached bat cochlea. A: Whole mount preparations of the basal turn show maxima in innervation density within the hook and the CF2 region and a minimum within the SI zone. The horizontal projection of the complete BM coil is superimposed. The asteriscs indicates a local enlargement of the spiral ligament (redrawn after Henson and Henson, 1991 and K~ssl and Vater, 1985). B. The cochlear frequency map of the mustached bat (redrawn after KSssl and Vater, 1985).

CF component of the echolocation signal and have an unspecialized innervation pattern. (2) The second half-turn contains an expanded representation of the dominant CF2 portion of the echolocation signal and is densely innervated. (3) The apical portion of the first half-turn responds to frequencies of about 62-72 kHz and is sparsely innervated (SI zone). (4) The basal portion of the first half-turn (hook) represents the third and fourth harmonics of the echolocation signal (H3 and H4) and is characterized by high innervation density. The morphology of the organ of Corti in these regions is illustrated in Fig. 2. The distinct apical to basal gradients in shape and cross-sectional area of the TM are further illustrated schematically in Fig. 3A. The TM of the apical cochlea is a thin elongated structure. Only one third of its area is attached to the spiral limbus (SL). In the apical portion of the second half-turn, a change in TM shape and a pronounced increase in TM cross-sectional area has occurred which is mainly due to a prominent thickening of the TM over the hair cell region. The TM is a massive structure that has a predominantly vertical orientation relative to the organ of Corti. Most of the underside of the TM is attached to the SL. At the transition between the second and first half-turn, a further change in TM shape takes place. The most prominent thickening of the TM is now located medial to the hair cell level and forms a bulbous extension above the attachment site to the SL. In addition,

66

M. Vater, M. Ki)ssl / Hearing Research 94 (1996) 63 77

the attachment area to the SL is considerably decreased. The bulbous thickening reaches its maximal extent at the basal end of the SI zone and is attached to a tongue-like extension of the SL. In the hook region, the shape of the TM again resembles that observed in the CF2 region; however, its area is distinctly smaller. The cross-sectional area of the TM and the extent of its limbal attachment site are quantified in Fig. 3B and C. The basic pattern of change in cross-sectional area of the TM corroborates previous findings (Henson and Henson, 1991). The cross-sectional area of the TM (Fig. 3B) increases from the basal end of the hook to the basal end of the SI zone. Within the SI zone, a plateau including a shallow maximum is reached. At the transition of the SI zone and CF2 region, TM area distinctly increases to maximal values within the CF2 region. Since the scatter of values derived from different cochleae is quite large, we could not reproduce the presence of a focal maximum of TM crosssectional area at the apical end of the CF2 region (see Henson and Henson, 1991). Our data, however, closely match the gradients obtained for TM width and size of the spiral limbus by the same authors. Within the third halfturn, TM area decreases towards the apex. Note that the size of the TM in the apex is within the range observed in the hook. The baso-apical gradient in the extent of the limbal attachment site (Fig. 3C) reveals a broad minimum corresponding to the SI zone, two maxima within the hook and the CF2 region, respectively; and a decrease at about 65% distance from the base towards the apex. These changes in cross-sectional area and shape of the TM and in the degree of attachment to the SL lead to a redistribution of mass relative to the organ of Corti and thus may change the resonant properties of the cochlear partition. 3.2. Subdivisions o f the T M

The TM is generally subdivided into a limbal, middle and marginal zone (Lim, 1972; Kronester-Frei, 1978). This basic nomenclature will be adopted, however, for an accurate description of the TM of mustached bats it is necessary to extend the subdivisions further. Fig. 4 schematically illustrates the subdivisions based on ultrastructural observations. The limbal zone of the TM is composed of an inner and an outer part. The inner part is separated from the middle zone by the 'stripe'. This stripe is continuous with the basal layer of the middle zone. The middle zone of the TM in CF2, SI and H 3 / 4 is distinctly subdivided into a core and a mantle. The outermost part of the mantle is continuous with the outer part of the limbal zone. This distinction in mantle and core is less apparent in the apex. The marginal zone of the TM is subdivided into a homogenous part (black) and a heterogenous part (dotted). The heterogenous zone is only present in the C F 2 , SI and H 3 / 4 regions.

Fig. 2. Low-power photomicrographs of the organ of Corti (radial 2 /xm sections). A: Third half-turn (70% distance from base). B: CF2 region (second half-turn; 55% distance from base) C: SI zone (first half-turn; 30% distance from base). D: Hook (14% distance from base). Note changes in TM shape, TM cross-sectional area and attachment of the TM to the spiral limbus. Cal. bar: 50 txm.

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Fig. 3. A: Schematic drawings of the TM and spiral limbus at different baso apical positions. Arrowheads denote the extent of the limbal attachment site of the TM. B: Cross-sectional area of the TM is plotted vs. normalized BM length for three specimens (different symbols). C: The extent of the limbal attachment site of the TM is plotted vs. normalized BM length for the same specimens.

3.3. Ultrastructure of the TM As in other studies (Kronester-Frei, 1978; Hasko and Richardson, 1988), two types of protofibrils can be distinguished: type A protofibrils are thick (10 nm) and unbranched; type B protofibrils are thinner and branched, and they exist in weakly or strongly hydrated states. The relative proportions of these protofibrils and their arrangements are characteristic of different zones of the TM. 3.3.1. Limbal zone The inner part of the limbal zone is composed of loosly packed type A protofibrils which form irregularly arranged bundles separated by electronlucent zones. It is attached to the processes of the interdental cells of the SL by a band of homogenous matrix (Fig. 5A). The bundles of protofibril A typically form criss-crossing patterns and are embedded in islands of matrix. The outer part of the limbal zone is composed of dense type A protofibrils arranged in parallel, and colocalized with type B protofibrils (Fig. 5A). These arrangements are found in all cochlear regions. The main difference in TM shape between the SI zone and other regions is created by a vast increase of the inner part of the limbal zone (Fig. 4). 3.3.2. Stripe The stripe of the TM in the basal turn is distinctly narrower than in the apical cochlea (Figs. 4 and 6A,B) but

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68

M. Vater, M. Ki~ssl/Hearing Research 94 (1996) 63 77

has the s a m e u l t r a s t r u c t u r a l c o m p o s i t i o n . Its m a i n feature is the d e n s e parallel a r r a n g e m e n t o f type A p r o t o f i b r i l s (Fig. 5C). T h e trajectory o f type A p r o t o f i b r i l s is at a slightly s t e e p e r a n g l e t h a n the l i m b a l a t t a c h m e n t o f the

T M . T h e stripe is c o n t i n u o u s with the f i l a m e n t o u s z o n e o f the basal l a y e r o f the m i d d l e z o n e w h e r e the p r o t o f i b r i l s are o r i e n t e d parallel to the s u b s u r f a c e o f the T M (Figs. 4 and 6A,B).

Fig. 5. Electron micrographs of the limbal zone (A, B) and stripe (C) of the TM. A: Low-power electron micrograph of the attachment of the limbal zone of the TM to the spiral limbus in the third half-turn (70% distance from base). The arrowheads indicate the processes of the interdental cells (ID) of the spiral limbus. OL, outer limbal zone; iL, inner limbal zone. Cal. bar: 5 /zm. B: High-power electron micrograph of the inner limbal zone. Note the irregular arrangement of protofibrils A (arrows) and their colocalization with electron dense areas of matrix. C: High-power photomicrograph of the stripe (CF2 region 55% distance from base). Note dense parallel arrangement of protofibrils A interconnected by crossbridges. Cal. bar B,C: 0.2 /zm.

M. Vater, M. KOssl/ Hearing Research 94 (1996) 63-77 3.3.3. M i d d l e zone

The middle zone of the TM in the apical cochlea is of rather homogenous composition. Type A protofibrils accompanied by type B protofibrils form distinct bundles separated by large electronlucent zones (Fig. 6C and 7A).

69

The bundles are between 0.2 and 1.26 /xm thick. They arise from the basal layer of the middle zone with an inclination angle of 4 5 - 5 0 ° and arch medially in a course parallel to the scala media face of the TM (Fig. 7C). There is no distinct borderline between core and mantle, although

Fig. 6. Low-power electron micrographs of the differentzones of the TM in the apical cochlea (third half-turn;A,B) and the hook (C,D). A,C: transitions between limbal zone, stripe and middle region of the TM. B,D: the outer part of the middle zone (core and mantle). Cal. bar A-D: 5 /xm.

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M. Vater, M. Ki~ssl / Hearing Research 94 (1996) 63-77

M. Vater, M. K6ssl/Hearing Research 94 (1996) 63-77

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Fig. 8. Electron micrographs of the marginal band and subsurface of the TM in the apical cochlea (third half-turn; A,B) and the CF2 region (second half-turn; C-D). A: The marginal band (MB) in the apical cochlea is composed of weakly hydrated type B protofibrils. Arrows indicate imprints of tallest stereocilia of innermost (1) to outermost (3) row of OHCs in Hardesties membrane of the subsurface of the TM. B: Hensen's stripe in the apical cochlea. C: The marginal band in the basal turn (shown here for CF2 region) contains a heterogeneous region composed of Type A protofibrils. The arrows indicate imprints of OHC stereocilia. D: Hensen's stripe in the basal cochlea. E: High-power electronmicrograph of the marginal band in the basal turn. E,F: Stereocilia of OHC1 in the basal turn (E) and the apical turn (F); Cal. bars: A - D : 5 # m ; E,F: 0.5 /xm; G: 0.2 /xm.

Fig. 7. High-power electron micrographs of the middle region of the TM in the apical cochlea (third half-turn; left column A,C,E) and the CF2 region (second half-turn; right column, B,D,F). A,B: Central part of the core region of the TM. C,D: Core region of the TM close to the subsurface of the BM; basal layer (bl), and Hardesties membrane (hm); asterix in D indicates the dense weakly hydrated protofibrils B that interface the protofibril A bundles with the basal layer. E,F: Mantle region of the TM. Note that the cover net (arrows) is interrupted in the apical cochlea whereas it forms a continuous sheet in the basal cochlea. Cal. bar A - F : 0.2 /xm.

M. Vater, M. K6ssl/Hearing Research 94 (1996) 63 77

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rils (Fig. 7B). There are only a few small electron lucent zones. In the CF2 region and the hook (Fig. 6D and 7D), the filament bundles of the core arise from the basal layer above the OHCs with a steep inclination angle of 80-85 °. In the SI zone, their angle of inclination amounts to 45-50 °. In contrast to the apical cochlea, the type A protofibril bundles in the middle zone of the basal turn are interfaced with the basal layer of the TM by a dense weakly hydrated matrix (compare Fig. 7C and D). The mantle is composed of densely packed parallel sheets of type A protofibrils whose trajectory follows the scala media surface of the TM. There is little or no electronlu~ cent material between the sheets of protofibrils (Fig. 7F).

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3.3.4. Marginal zone, Hardesties membrane and Hensen's

the mantle may be distinguished by a higher packing of filament bundles and their course parallel to the scala media surface of the TM (Figs. 4 and 6C and 7E). In the CF2, SI and H 3 / 4 zones of the cochlea, there is a clear division into core and mantle regions (Fig. 6D). The core region is characterized by the presence of thin bundles of type A protofibrils (diameter: 0.-0.4 /xm) separated from each other by a dense matrix of hydrated type B protofib40

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Fig. 10. Measurements of hair cell length (A), stereocilia length (B), diameter of IHCs (C) and BM thickness and width (D) are plotted vs. normalized BM length for three different cochleae (different symbols).

M. Vater, M. K6ssl/Hearing Research 94 (1996) 63-77

of weakly hydrated type B protofibrils. In the marginal zone of the CF2, SI and H 3 / 4 regions, a prominent heterogenous zone is characterized by bundles of crosssectioned profiles of type A protofibrils within electron lucent zones (Fig. 8B,E). This pattern suggests a longitudinal trajectory of the protofibrils. Hardesties membrane and Hensen's stripe are present throughout the cochlea, but their dimensions vary (Fig. 8A-D). In the basal turn both are continuous with each other whereas in more apical regions, Hensen's stripe is separate from Hardesties membrane. Hensen's stripe in the apex is less distinct than in more basal cochlea (Fig. 8B,D). The maximum size of Hensen's stripe is observed in the SI zone. Except for the apex, Hardesties membrane over the innermost row of OHCs (OHC1) forms a distinct protrusion that carries an indentation for the tallest stereocilia of OHC1 (Fig. 8A,C). The tips of the tallest stereocilia of OHC2 and OHC3 form imprints in the smooth Hardesties membrane below the marginal zone (Fig. 8A,C).

3.4. Gradients in densit3, of ~pe A protofibrils Medio-lateral gradients in the density of type A protofibrils have been quantified in the gerbil and were used to estimate TM stiffness (Weaver and Schweizer, 1994). We also counted the number of type A protofibrils crossing a 2 /xm line for several medio-lateral positions of the TM of the mustached bat at four different longitudinal positions. While the minimal spacing distance of type A protofbrils is invariant, the spaces among the protofibril bundles can differ among turns. The density profiles measured across the limbal zone, the stripe, the core and the mantle exhibit a clear minimum within the core region (Fig. 9). In the apex, this minimum is created by the presence of pronounced electronlucent zones separating filament bundles. At other locations, however, this minimum is created by the separation of filament bundles by matrix. There are no significant differences in filament densities of limbal zone, stripe, and core region among the turns (Fig. 9). However, the values obtained for the mantle zone in the apex are distinctly lower than in other cochlear locations.

3.5. Hair cell stereocilia, hair cell length and BM dimensions In order to determine how the specialized TM morphology relates to gradients in hair cell morphology, the length of the cell bodies of the IHCs and OHCs and the length of their tallest stereocilia was measured (Fig. 10A,B). Additionally, we determined the diameter of the IHCs (Fig. 10C) and the thickness and width of the BM from the same sections (Fig. 10D). Within the basal 60% of cochlear length (hook, SI zone and CF2), there was no systematic change in OHC length (12-13 /xm) and the stereocilia length ranged between 0.7 and 0.8 /xm. In the apical 40% of the cochlea there was a

73

slight increase in OHC length to values of 15-16 /xm and an increase in stereocilia height to values of 1.5 /xm. Throughout the basal turn, the IHC stereocilia had a length of 2 /xm. Beyond 60% distance from base their length increased to maximum values of 4 /xm in the apex. IHC length varied between 26 and 32 /xm with no clear trend for an increase towards the apex. There was however a region of minimum length corresponding to the SI zone. Measurements of IHC diameter revealed a clear minimum within the SI zone. BM thickness is maximal within the SI zone, reaches a plateau within the CF2 region and only systematically decreases as is common in mammals within the apicalmost 30% of cochlear length (see also K/Sssl and Vater, 1985a). BM width exhibits a broad plateau between about 25 and 70% distance from the cochlear base thus encompassing the SI zone and the CF2 zone.

4. Discussion

4.1. General.features The basic ultrastructural composition of the TM of the mustached bat is similar to other mammals (Lira, 1972; Ross, 1974; Kronester Frei, 1978; Hasko and Richardson, 1988; Weaver and Schweitzer, 1994). It is composed of type A and type B protofibrils. The latter form the matrix and appear to exist in two different hydration states. This similarity further suggests that protofibrils A may be composed of collagen type II and IX as in other species (Slepecky et al., 1992) and that they may crucially determine the structural stability and tensile strength of the TM (Thalmann et al., 1987). Significantly, the regional increases in TM cross-sectional area observed here and in a previous study (Henson and Henson, 1991) are created by addition of protofibrils and not by differences in overall hydration of the TM. Distinct variations in packing density of type A protofibrils and relative amount of matrix in the TM of the mustached bat produce more subregions than described in other species especially within the middle zone and the marginal band of the TM. These regional differences within cross-sectional profiles of the TM are less prominent in the apical cochlea than within the functionally specialized upper and lower basal turns. Baso-apical differences in the ultrastructural organization of the TM as observed here have not been reported in other species. These differences and the subregional differences in protofibril A packing density in cross-sections of the TM of all species studied suggest that the mechanical properties of the TM are inhomogeneous as also proposed by direct stiffness measurements in situ (Zwislocki and Cefaratti, 1989). In the gerbil, a radially directed gradient in density of protofibrils A in the middle zone of the TM was observed throughout the cochlea that was most pronounced in high-frequency regions (Weaver and Schweitzer, 1994). The decrease in protofibril A density

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from more limbal towards more external regions of the TM was taken as evidence for a higher compliance of external zones of the TM and thought to contribute to the characteristics of motion of the cochlear partition. The use of a similar measurement paradigm in the mustached bat yielded different results. In all cochlear regions, there was a distinct minimum in the packing density of protofibrils A within the core region of the middle zone. The organization into core and mantle may endow the bat TM with specialized internal resonant properties and generate complicated vibration modes. Neither the data obtained in gerbil nor the data obtained here match the in vivo stiffness measurements (Zwislocki et al., 1988; Zwislocki and Cefaratti, 1989) which suggest that the TM is more compliant near the limbus than over the organ of Corti. This is not surprising given the fact that direct stiffness measurements will be affected by the geometry of the TM and its attachment site, a factor not taken into account in measurements of protofibril density. A further interesting parameter in TM architecture is the course of protofibril A bundles of the middle zone relative to the subsurface of the TM. In the guinea pig it was noted that protofibrils A form distinct bundles which ascend obliquely from the subsurface of the TM in an arched course (Kronester-Frei, 1978). In whole mounts of the guinea pig TM it was further noted that fibril bundles are arranged radially with a slight slant in the longitudinal direction (Lim, 1972). In the mustached bat, there are differences among cochlear regions in the angle at which protofibril bundles ascend from the lower surface of the TM. This angle was shallow ( 4 5 - 5 0 °) in the apical cochlea and in the SI zone, and steep ( 8 0 - 8 5 °) in the CF2 and the hook region of the cochlea. A general property of collagen bundles in matrix is that they tend to be aligned in the direction of maximum stress thus forming trajectories indicating the direction of force. Using this line of argument for the TM of the mustached bat it follows that the major force component is directed more vertically in the CF2 region and the hook than in the apical cochlea and the SI zone. Together with differences in TM shape and extent of limbal attachment site among cochlear regions this feature may be indicative of differences in vibration modes of the TM (see below). During development, differences in the extent of particular subregions of the TM may be generated by different timing of secretory activity of particular cell types. However, it is an open problem how the orderly and regional specific arrangements of type A protofibril bundles are generated during development and maintained during adult life given the absence of active fibroblasts within the body of the TM. The subsurface of the TM of the mustached bat also reveals some specialized features. Except at the extreme apex, a distinct Hensen's stripe is present which represents the putative attachment site for the tallest stereocilia of IHCs (e.g., Lira, 1986; Vater and Lenoir, 1992; Vater et al., 1992). As in the horseshoe bat (Vater et al., 1992) the

Hensen's stripe is especially prominent in the basal turn and is continuous with Hardesties membrane, a structure located above the stereocilia of the OHCs. In contrast to the horseshoe bat, however, there were no clear indications of direct coupling of IHC stereocilia to the TM. Similar to the horseshoe bat, a reinforced mechanical coupling of the innermost row of OHCs to the TM is suggested by the presence of a protrusion of the subsurface of the TM above this row which carries the imprints of the tallest OHC stereocilia. These patterns differ from the TM of mammals with more generalized hearing capabilities like the rat where Hensen's stripe is only observed in the basal cochlea (e.g., Lenoir et al., 1987). The dimensions of OHC stereocilia stay remarkably constant throughout the basal turn of the mustached bat cochlea. Similar patterns were observed in Hipposideros bats (Dannhof and Bruns, 1991) and in the horseshoe bat (Vater et al., 1992). All these species possess an expanded frequency mapping of a narrow frequency band around the dominant harmonic of the CF-echolocation call component (mustached bat cochlea: K~Sssl and Vater, 1985a; horseshoe bat cochlea: Vater et al., 1985; Hipposideros central auditory system: Riibsamen et al., 1988). Although it is tempting to correlate 'constant' stereocilia size with expanded frequency mapping, the findings in the mustached bat argue against this interpretation. Stereocilia size in the hook ( 9 0 - 1 2 0 kHz) is not significantly different from that in the CF2 region (60 kHz). This finding may be related to the limited precision in measurement of stereocilia length given their small absolute length (0.7-0.8 ~m). Alternatively, it may be that there is a lower limit in dimensions that guarantees function at high frequencies. If there is a lower limit in stereocilia size then other micromechanical factors must govern the regular tonotopic order in the high-frequency range of the mammalian audiogram. One such factor may be the mass of the TM that is forming a resonant system with the OHC stereocilia (Strelioff et al., 1985). Another parameter related to tonotopic organization is the length of the OHC body (e.g., Brundin et al., 1989; Dannhof et al., 1991, Pujol et al., 1992), but there is no clear gradient in OHC length throughout the basal turn of the mustached bat cochlea. Absolute size of OHCs in the SI zone and CF2 region of the mustached bat cochlea (12-13 /~m) closely agrees with the value derived from a function relating OHC length to frequency in different mammals (62 kHz corresponds to a hair cell length of 12.7 /~m; Dannhof et al., 1991). According to this relation, one should expect a length difference of at least 4 / ~ m between the CF2 region and the hook region which is tuned to frequencies about one octave higher, and we were not able to demonstrate such a difference in the mustached bat. Measurements in the Hipposideros bat on OHCs tuned to frequencies of 128 kHz (the highest lYequency available for the calculations) give a value of 11 /~m which closely fits our data in the mustached bat. This value may thus represent the minimum value for OHC length in mammals.

M. Vater, M. KSssl/Hearing Research 94 (1996) 63 77

Furthermore, the maximum OHC length observed in the apex of the mustached bat cochlea (16-17 /~m) would correlate with a frequency of about 32 kHz which certainly does not represent the lower cut-off frequency of hearing in this species. Thus calculations of the cochlear frequency map based on OHC length may be valuable for positions in middle cochlear regions and the general location of best hearing but not for estimations of lower and upper limits of the hearing range at extreme positions of the cochlea. In contrast to other C F - F M bats (Dannhof and Bruns, 1991; Vater et al., 1992), there is no prominent discontinuity in size of the IHC stereocilia at the basal end of the expanded frequency region in the mustached bat. However, IHCs of the SI zone of the mustached bat cochlea are clearly smaller in size than in other cochlear regions (this study). This feature may not represent a mechanically important factor as it is the case for OHC size (see above). Rather, the reduced dimensions of IHCs correlate with the drastically diminished innervation density of the SI zone (Henson, 1973; Zook and Leake, 1989). This opens the interesting possibility that during early cochlear development, a trophic influence of afferent terminals and their number will determine IHC size.

4.2. TM specializations for sharp tuning ? Comparative analyses of the TM cross-sectional area in several mammalian species show an overall increase in size from base to apex (domestic pig: Zwicker, 1971; guinea pig: Lim, 1986; rat: Roth and Bruns, 1992; frog eating bat, mole rat, gerbil: Bruns et al., 1989; Hipposideros bats: Dannhof and Bruns, 1991). There are, however, only very few studies that systematically measured TM cross-sectional area throughout the cochlea. Bruns et al. (1989) suggested that the baso-apical gradients are species specific and that some species possess local maxima in TM cross-sectional area which appear to correspond to regions of highest cochlear sensitivity. The mustached bat also presents maximal TM cross-sectional area in the cochlear region of highest sensitivity, i.e., the CF2 region, but the baso-apical gradients in TM morphology are highly specialized and paralleled by changes in size of the spiral limbus (Henson and Henson, 1991). These findings prompted the suggestion that the discontinuous change in TM cross-sectional area is correlated with enhanced tuning capacities to the CF2 frequency (Henson and Henson, 1991) and interpreted as support for the theory that the TM acts as a second resonator superimposed on BM motion (Zwislocki et al., 1988). Data from the present study are in agreement with the previous report (Henson and Henson, 1991) except that we did not find a very focal maximum of TM cross-sectional area in the apical second half-turn that was present in some of their specimens. The present findings do corroborate the notion that the TM in addition to specialized gradients of BM morphology (K~ssl and Vater, 1985a; this study) and the volume of perilymphatic spaces (Henson et al., 1977)

75

plays an integral role in cochlear tuning in the mustached bat (Henson and Henson, 1991) but suggest that its action may be considerably more complex than previously supposed. As a significant finding of the present study, the distinct changes in cross-sectional area of the TM are accompanied by profound changes in TM shape and geometry of the limbal attachment site. In the CF2 region, TM mass is increased and positioned directly above the organ of Corti; furthermore the enlarged attachment to the limbus suggests that it is rigidly anchored. A similar general appearance is found in the hook, although the TM area is smaller. Significantly, in both cochlear regions, tuning sharpness is increased beyond standard mammalian values (Pollak et al., 1972; Suga et al., 1975; KiSssl and Vater, 1985b) and the low-frequency slopes of the tuning curves of many single units tuned to the CF2 and CF3 signal ranges are steeper than the high-frequency slopes (Krssl and Vater, 1990). In contrast, the TM in the SI zone is characterized by an elongated shape, and a redistribution of the mass to more medial locations. It is less rigidly anchored to the spiral limbus and may thus be capable of larger displacements than in other regions of the basal turn. Interpretations of morphological data are inspired by previous models of the TM that incorporate an elastic element in series with a mass above the organ of Corti (Allen, 1980; Allen and Fahey, 1993). The resonance frequency of this complex is given by the square root of the quotient of stiffness of the elastic element and the mass above the hair cells (Zwislocki and Cefaratti, 1989; Allen and Fahey, 1993) and is about half an octave lower than the characteristic frequency at the given cochlear location. This arrangement produces a relative null in hair cell excitation at the series resonance of the TM (Allen and Fahey, 1993). Measurements of the TM frequency response by Gummer et al. (1995) support this model whereas Ulfendahl et al. (1995) provide conflicting evidence. In accordance with these models the inferred greater stiffness of the TM attachment at strategic cochlear positions in the mustached bat (representation sites of CF2 and CF3 frequencies) may produce a relatively higher frequency of the series resonance of the TM that is closer to the maximum of BM vibration than in other cochlear locations and in the cochlea of other mammals. This may contribute to a sharpening of the cochlear filter function beyond values encountered in non-specialized cochleae. Indirect measurements of the TM frequency map (K~ssl and Vater, 1996) support this idea. The increased mass of the TM, in particular within the CF2 region, may decrease the damping and thus further increase filter sharpness. The sharply tuned cochlear regions are separated by the SI zone which features characteristic specializations of the BM and TM and represents frequencies between 62 and 70 kHz (KiSssl and Vater, 1985a; this study). Measurements of distortion product otoacoustic emissions (K~Sssl, 1994) and measurements of BM vibration (KiSssl and Russell, pers. comm.) indicate the presence of significant BM

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M. Vater. M. KiJssl / Hearing Research 94 (1996) 63 77

vibration a m p l i t u d e s within the SI z o n e to the f r e q u e n c i e s o f the e m i t t e d C F 2 c o m p o n e n t s w h i c h are not r e f l e c t e d in n e u r o n a l tuning at t h e s e locations. A p o s s i b l e e x p l a n a t i o n w o u l d be a m o t i o n o f the T M in p h a s e with the B M m o t i o n with the net e f f e c t o f a r e d u c e d s h e a r o f stereocilia a n d c o n s e q u e n t l y hair cell excitation. If o n e a s s u m e s that the e x t e n t o f the limbal a t t a c h m e n t o f the T M crucially c o n t r i b u t e s to its stiffness it f o l l o w s that the r e s o n a n c e f r e q u e n c y o f the T M in the S1 z o n e m a y not be h i g h e r than m o r e apically d e s p i t e its m a s s b e i n g smaller. Instead, it m a y b e t u n e d to f r e q u e n c i e s similar to or l o w e r than the T M in the CF2 r e g i o n as i n d i c a t e d in the c o m p a n i o n p a p e r (K~Sssl and Vater, 1996). F o r i n t e r p r e t a t i o n o f the f u n c t i o n o f the s p e c i a l i z e d T M a r r a n g e m e n t s in the basal turn o f the m u s t a c h e d bat c o c h l e a it is not s u f f i c i e n t to solely c o n s i d e r i n f e r r e d stiffness and m a s s o f the TM. Rather, the specialized g e o m e t r y o f the T M a t t a c h m e n t , the s p e c i a l i z e d T M s h a p e and its o r i e n t a t i o n relative to the reticular l a m i n a and B M s h o u l d b e taken into account. T h e s e p a r a m e t e r s are likely to a f f e c t the e x t e n t and d e g r e e o f f r e e d o m o f T M d i s p l a c e m e n t . W e tentatively p r o p o s e that the T M in the h o o k and C F 2 r e g i o n is relatively i m m o b i l e in transversal and radial d i r e c t i o n w h e r e a s the s p e c i a l i z e d g e o m e t r y o f the T M in the SI z o n e a l l o w s strong oscillations that m a y p r o v i d e m e c h a n i c a l e n e r g y w h i c h s p r e a d s to the m o r e apical r e p r e s e n t a t i o n p l a c e o f the C F 2 f r e q u e n c i e s . Its action w o u l d thus be a p a s s i v e e n h a n c e m e n t o f m e c h a n i c a l sensitivity a n d tuning at m o r e apical c o c h l e a r p o s i t i o n s . T h e s e s u g g e s t i o n s are o f h i g h l y s p e c u l a t i v e nature and m u s t be s u b s t a n t i a t e d by direct m e a s u r e m e n t s o f T M stiffness and T M m o t i o n relative to BM. A n u n d e r s t a n d i n g o f the f u n c t i o n o f the h i g h l y s p e c i a l i z e d c o c h l e a o f the m u s t a c h e d bat will only be g a i n e d by integrating the m u l t i p l e s p e c i a l i z a t i o n s o f the h y d r o m e c h a n i c a l s y s t e m into 3 - d i m e n s i o n a l m o d e l s that also a c c o u n t for active o u t e r hair cell f u n c t i o n a n d for the p h a s e relations a m o n g different motion components.

Acknowledgements W e t h a n k the J a m a i c a n Natural R e s o u r c e s C o n s e r v a t i o n A u t h o r i t y for their p e r m i s s i o n to e x p o r t bats to G e r m a n y . W e t h a n k lan Russell for m a n y helpful d i s c u s s i o n s on this topic. W e also thank him, Ellen C o v e y and G e r h a r d N e u w e i l e r for critically r e a d i n g the first v e r s i o n s o f the m a n u s c r i p t . W e t h a n k G e s a T h i e s for e x c e l l e n t technical assistant and H i l d e g a r d H a l l m e r for p h o t o g r a p h i c work. This w o r k was s u p p o r t e d by S F B 204.

References Allen, J.B. (1980)Cochlear micromechanics: a physical model of transduction. J. Acoust. Soc. Am. 68, 1660-1670. Allen, J.B. and Fahey, P.F. (1993) A second cochlear-frequency map that

correlates distortion product and neural measurements. J. Acoust. Soc. Am. 94. 809-816. Brown, A.M., Gaskill, S.A. and Williams, D.M. (1992) Mechanical filtering of sound in the inner ear. Proc. R. Soc. Lond. 250, 29-34. Brundin, L., Flock, A. and Canlon, B. (1989) Sound-induced motility of isolated cochlear hair cells is frequency-specific. Nature 342, 814-816. Bruns, B., Burda, H. and Ryan, M.J. (1989) Ear morphok)gy in the frog-eating bat (Trachops cirrhosus, Family Phylostonuaidae): apparent specializations for low frequency hearing. J. Morphol. 199, 103118. Dannhof, B.J. and Bruns, V. (1991) The organ of Corti in the bat Hipposidetvs bicolor. Hear. Res. 53, 253-268. Dannhof, B.J., Roth, B. and Bruns, V. (1991) Length of hair cells as a measure of frequency representation in the mammalian inner ear. Naturwissenschaften 78, 570 573. Davis, H. (1956) Initiation of nerve impulses in the cochlea and other mechanoreceptors. In: T.H,Bullock (Ed.), Physiological Triggers and Discontinuous Rate Processes. Am. Physiol. Soc., Washington, DC, pp. 60-71. Freeman, D.M.. Cotanche, D.A., Ehsani, F. and Weiss, T.F. (1994) The osmotic response of the isolated tectorial membrane of the chick to isnosmotic solutions: effect of Na +, K*, and Ca2+ concentration. Hear. Res. 79, 197-215. Gummer, A.W., Hemmert, W. and Zenner. H.P. (1995) The tectorial membrane is mechanically resonant at a frequency 0.5 oct below the characteristic frequency of the cochlear partition, Abstracts of the 181h Midwinter Research Meeting of the ARO, St. Petersburg Beach. FL, p, 188. Hasko, J.A. and Richardson, G.P. (1988) The ultrastructural organization and properties of the mouse tectorial membrane matrix. Hear. Res. 35, 21-38. Henson. M.M. (1973) Unusual nerve-fiber distribution in the cochlea of the bat, Pleronolus p. parnellii (Gray), J. Acoust. Soc. Am. 32, 380 384. Henson, M.M. (1978) The basilar membrane of the hat. Pteronotus p. partwllii. Am. J. Anat. 153, 143-158. Henson, M.M., Henson, O.W. Jr. and Goldman, L.J. (1977) The perilymphatic spaces in the cochlea of the bat, Pwronotus p. parnellii (Gray). Anat. Rec. 187. 767. Henson, M.M. and Henson, O.W. Jr. (1991) Specializations for sharp tuning in the mustached bat: the tectorial membrane and spiral limbus. Hear. Res. 56. 122 132. Henson, O.W. Jr., Schuller G. and Vater, M. (1985) A comparative study of the physiological properties of the inner ear in Doppler shift compensating bats (Rhinolol)hus rouxi and Pteronotus parnellii). J. Comp. Physiol. A157. 587-597. K/Sssl, M. (1994) Evidence l\)r a mechanical filter in the cochlea of the 'constant fl-equency' bats Rhinolophns rouM and Pteronotus parnellii. Hear. Res. 72. 73 80. Ki:issl, M. and Vater, M. (1985a) The cochlear frequency map of the mustache bat, Pteronotus parnellii. J. Comp. Physiol. A 157,687 697. K(5,ssl, M. and Vater. M. (1985b) Ew)ked acoustic emissions and cochlear microphonics in the mustache bat, Pteronotus partlellii, Hear. Res. 19. 157-170. K~ssl, M. and Vater, M. (1990) Resonance phenomena in the cochlea of the mustache bat and their contribution to neuronal response characteristics in the cochlear nucleus. J. Comp. Physiol. A166. 711 720. K/3ssl, M.and Vater. M. (1996) Further studies on the mechanics of the cochlear partition in the mustached bat. II. A second cochlear frequency map derived from acoustic distortion products. Hear. Res. 94, 76 86. Kronester Frei, A. (1978) Ultrastructure of different zones of the rectorial membrane. Cell Tiss. Res. 193, I 1-23. Kronester-Frei, A. (1979) The effect of changes in endolymphatic ion concentrations on the tectorial membrane. Hear. Res. I. 81 94. Lenoir, M.. Puel, J.-L. and Pujol, R. (1987) Stereocilia and tectorial

M. Vater, M. Ki~ssl/ Hearing Research 94 (1996) 63-77 membrane development in the rat cochlea. A SEM study. Anat. Embryol. 175, 477-487. Lim, DJ. (1972) Fine morphology of the tectorial membrane. Arch. Otolaryngol. 96, 199-215. Lim, D.J. (1986) Functional structure of the organ of Corti: a review. Hear. Res. 22, 117-146. Lira, D.J. (1987) Development of the tectorial membrane. Hear. Res. 28, 9-21. Pollak, G.D., Henson, O.W. Jr. and Novick, A. (1972) Cochlear microphonic audiograms in the pure tone bat, Chilonycteris parnellii. Science 176, 66-68. Pujol, R., Lenoir, M., Ladrech, S., Tribillac, F. and Rebillard, G. (1992) Correlation between the length of outer hair cells and the frequency coding of the cochlea. In: Cazals, Demany and Horner (Eds.), Advances in Biosciences. Auditory Physiology and perception, Pergamon Press. Richardson, G.P., Russell, I.J., Duance, V.C. and Bailey, A.J. (1987) Polypeptide composition of the tectorial membrane. Hear. Res. 25, 454-460. Ross, M. (1974) The tectorial membrane of the rat. Am. J. Anat. 139, 449-482. Roth, B. and Bruns, V. (1992) Postnatal development of the rat organ of Corti. I. General morphology, basilar membrane, tectorial membrane and border cells. Anat. Embryol. 185, 559-569. Riibsamen, R., Neuweiler, G. and Sripathi, K. (1988) Comparative collicular tonotopy in two bat species adapted to movement detection, Hipposideros speoris and Megaderma lyra. J. Comp. Physiol. A163, 271-285. Schnitzler, H.U. and Henson, O.W. Jr. (1980) Performance of airborne animal sonar systems: echolocation in microchiropteran bats. In: R.G. Busnel and J.F. Fish (Eds.), Animal Sonar Systems, Plenum Press, New York, pp. 109-182. Shah, D.M., Freeman, D.M., and Weiss, T.F. (1995) The osmotic response of the isolated, unfixed mouse tectorial membrane to isoosmotic solutions: effect of Na +, K + and Ca 2+ concentration. Hear. Res. 87, 187-208. Slepecky, N.B., Savage, J.E., Cefaratti, L.K. and Yoo, T.C. (1992) Electron-microscopic localization of type II, IX, and V collagen in the organ of Corti of the gerbil. Cell Tiss. Res. 267, 413-418. Steel, K.P. (1983) The tectorial membrane of mammals. Hear. Res. 9, 327-359.

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Strelioff, D., Flock, A. and Minser, K.E. (1985) Role of inner and outer hair cells in mechanical frequency selectivity of the cochlea. Hear Res. 18, 169-175. Suga, N., Simmons, J.A., and Jen, P.H.-S. (1975) Peripheral specializations for fine analysis of Doppler-shifted echoes in the auditory system of the ' C F - F M ' bat Pteronotus parnellii. J. Exp. Biol. 63, 161-192. Thalmann, I., Thallinger, G., Crouch, E.C., Comegys, T.H., Barrett, N. and Thalmann, R. (1987) Composition and supramolecular organization of the tectorial membrane. Laryngoscope 97, 357-367. Ulfendahl, M., Khanna, S.M. and Heneghan, C. (1995) Shearing motion between the tectorial membrane and the reticular lamina in the isolated temporal bone preparation. Abstracts of the 18th Midwinter Research Meeting of the ARO, St. Petersburg Beach, FL, p. 188. Vater, M. and Lenoir, M. (1992) Ultrastructure of the horseshoe bat's organ of Corti. I. Scanning electron microscopy. J. Comp. Neurol. 318, 367-369. Vater, M., Feng, A.S. and Betz, M. (1985) An HRP-study of the frequency-place map of the horseshoe bat cochlea: morphological correlates of the sharp tuning to a narrow frequency band. J. Comp. Physiol. A157, 671-686. Vater, M., Lenoir, M. and Pujol, R. (1992) UItrastructure of the horseshoe bat's organ of Corti. II. Transmission electron microcopy. J. Comp. Neurol. 318, 380-391. Weaver, S.P. and Schweitzer, L. (1994) A radial gradient of fibril density in the gerbil tectorial membrane. Hear. Res. 76, 1-6. Zook, J.M. and Leake, P.A. (1989) Connections and frequency representation in the auditory brainstem of the mustache bat, Pteronotus parnellii. J. Comp. Neurol. 290, 243-261. Zwicker, E. (1971) Die Abmessungen des lnnenohrs des Hausschweins. Acustica 25, 232-239. Zwislocki, J.J. (1988) Mechanical properties of the tectorial membrane in situ. Acta Otolaryngol. 105, 450-456. Zwislocki, J.J. and Kletzky, E.J. (1979) Tectorial membrane: a possible effect on frequency analysis in the cochlea. Science 204, 639-641. Zwislocki, J.J., Chamberlain, S.C. and Slepecky, N.B. (1988) Tectorial membrane. I. Static mechanical properties in vivo. Hear. Res. 33, 207-222. Zwislocki, J.J. and Cefaratti, L.K. (1989) Tectorial membrane. II. Stiffness measurements in vivo. Hear. Res. 42, 211-228.