Vibration responses of the organ of Corti and the tectorial membrane to electrical stimulation

Vibration responses of the organ of Corti and the tectorialmembrane to electrical stimulation

Manuela Nowotnya) and Anthony W. Gummerb)

Faculty of Medicine, Section of Physiological Acoustics and Communication, Eberhard Karls UniversityTubingen, Elfriede-Aulhorn-Straße 5, 72076 Tubingen, Germany

(Received 17 June 2011; revised 13 September 2011; accepted 14 September 2011)

Coupling of somatic electromechanical force from the outer hair cells (OHCs) into the organ of

Corti is investigated by measuring transverse vibration patterns of the organ of Cori and tectorial

membrane (TM) in response to intracochlear electrical stimulation. Measurement places at the

organ of Corti extend from the inner sulcus cells to Hensen’s cells and at the lower (and upper) sur-

face of the TM from the inner sulcus to the OHC region. These locations are in the neighborhood of

where electromechanical force is coupled into (1) the mechanoelectrical transducers of the stereoci-

lia and (2) fluids of the organ of Corti. Experiments are conducted in the first, second, and third

cochlear turns of an in vitro preparation of the adult guinea pig cochlea. Vibration measurements

are made at functionally relevant stimulus frequencies (0.48–68 kHz) and response amplitudes

(<15 nm). The experiments provide phase relations between the different structures, which, de-

pendent on frequency range and longitudinal cochlear position, include in-phase transverse motions

of the TM, counterphasic transverse motions between the inner hair cell and OHCs, as well as

traveling-wave motion of Hensen’s cells in the radial direction. Mechanics of sound processing in

the cochlea are discussed based on these phase relationships. VC 2011 Acoustical Society of America.

[DOI: 10.1121/1.3651822]

PACS number(s): 43.64.Bt, 43.64.Kc [BLM] Pages: 3852–3872

I. INTRODUCTION

The mammalian cochlea exhibits two clearly separated

hair-cell types: inner hair cells (IHCs) and outer hair cells

(OHCs). Harboring 90%–95% of the afferent innervation

(Spoendlin, 1969), the IHCs are the true sensory cells of the

cochlea. Although also afferently innervated, the OHCs have

mainly a motor function: In response to a change of mem-

brane potential (Dallos et al., 1991), the soma is motile

(Brownell et al., 1985), with synchronous motion up to high

frequencies (Dallos and Evans, 1995; Gale and Ashmore,

1997), up to at least 70 kHz (Frank et al., 1999). The electro-

mechanical force produced by the OHC soma appears to be

the basis for the exquisite sensitivity (Liberman et al., 2002;

Cheatham et al., 2004; Mellado Lagarde et al., 2008) and

frequency selectivity (Cheatham et al., 2004; Mellado

Lagarde et al., 2008) of the cochlea, acting on a cycle-by-

cycle basis (Gao et al., 2007) to amplify the traveling wave

on the basilar membrane (BM) over a narrow spatial region

(Russell and Nilsen, 1997). Traveling-wave motion longitu-

dinally along the tectorial membrane (TM) appears to be

instrumental for cooperative injection of the electromechani-

cal forces from neighboring OHCs to enable both high gain

and wide bandwidth (Ghaffari et al., 2007; Ghaffari et al.,2010; Meaud and Grosh, 2010). Nevertheless, the amplifica-

tion mechanisms are not yet understood, although several

mechanical feedback pathways are probably involved (Lu

et al., 2006; Meaud and Grosh, 2010). It is the purpose of

this study to use intracochlear electrical stimulation to under-

stand how electromechanical force from the OHCs might be

coupled to cochlear structures, concentrating on the phase

relationships between the transverse components at the TM

and apical surface of the organ of Corti.

Direct electrical stimulation of the cochlea, both in vivo(Xue et al., 1995; Nuttall et al., 1999) and in vitro (Reuter etal., 1992; Mammano and Ashmore, 1993), provides informa-

tion on the role of the OHCs in cochlear amplification,

including the possibilities of piezoelectric resonance of the

OHC soma (Grosh et al., 2004; Scherer and Gummer,

2004b; Zheng et al., 2007), stereociliary motility (Chan and

Hudspeth, 2005a,b), inducing resonant TM motion in the ra-

dial direction (Mammano and Ashmore, 1993; Gummer etal., 1996), generating fluid motion within the organ of Corti

(Karavitaki and Mountain, 2007a,b), and stimulating coun-

terphasic transversal motion of the reticular lamina (RL) and

TM at the IHC (Nowotny and Gummer, 2006). Specifically,

by stimulating electromechanical feedback pathways

directly, in the absence of acoustical stimulation, the cou-

pling of OHC electromechanical forces into the organ of

Corti and TM can be investigated directly.

In vitro preparations cannot (yet) reproduce all salient

mechanical properties of the in vivo cochlea (Robles and

Ruggero, 2001), but nevertheless they have the advantage of

allowing vibration measurements both within the organ of

Corti and also on the surfaces of the TM. Although in vivomeasurement of RL vibration in the high-frequency sensitive

region of the cochlea is now technically possible (Chen

et al., 2011), it is still not possible to measure TM and RL

a)Present address: Institute of Cell Biology and Neuroscience, AK Neuro-

biology und Biosensors, Siesmayerstraße 70 A, 60323 Frankfurt/Main,

Germany.b)Author to whom correspondence should be addressed. Electronic mail:

[email protected]

3852 J. Acoust. Soc. Am. 130 (6), December 2011 0001-4966/2011/130(6)/3852/21/$30.00 VC 2011 Acoustical Society of America

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motion differentially in such preparations—because of the

lower optical reflectivity of the TM relative to the RL and

the small distance between TM and RL compared with the

depth of focus. Nevertheless, these exquisite experiments

demonstrate that RL motion is not simply a replica of BM

motion, but is tuned to a slightly higher frequency (�440 Hz

in 16 kHz) and leads BM motion (up to 86�) around the best

frequency at low intensities (Chen et al., 2011). These results

highlight the importance of understanding how OHC electro-

mechanical force is coupled into the organ of Corti and TM

to overcome limitations incurred by viscosity, capacitance,

and Brownian motion.

Unfortunately, to date, most in vitro preparations suffer

from the problem that the vibration detection systems do not

have adequate sensitivity at all functionally relevant frequen-

cies. Detection problems are commonly circumvented by (1)

introducing reflecting materials into the cochlea, (2) using

large-amplitude stimulation, (3) using low-frequency stimula-

tion, and/or (4) restricting recordings to low-frequency coch-

lear regions.

Using a sensitive confocal laser interferometric system

(Scherer and Gummer, 2004b), it has been shown that it is

possible to make direct measurements of the transversal

motion not only of the RL, but also of the lower surface of the

TM bordering the subtectorial space (Nowotny and Gummer,

2006). Those experiments, conducted in an in vitro prepara-

tion, show that somatic electromechanical force from the

OHC induces counterphasic motion of the TM and RL at the

IHC for stimulus frequencies up to 3 kHz in the first, second,

and third turns of the guinea pig cochlea. The experiments

uncovered a mechanism for stereocilia deflection, in addition

to the classical shearing mechanism between RL and TM

(Davis, 1958; Rhode and Geisler, 1967).

Here, in response to intracochlear electrical stimulation,

the transversal motion of the organ of Corti and TM is

described in greater detail than hitherto, making measure-

ments at 22 different radial positions. The data provide fur-

ther insights into the complex behavior of the TM and entireupper surface of the organ of Corti under the influence of

OHC electromechanical force for both low- and high-

frequency regions of the cochlea.

II. METHOD

Preparation, stimulation, and vibration measurement

protocols have been described in Nowotny and Gummer

(2006). This information is also presented here and extended

for completeness.

A. Preparation

In situ preparations were made from the first three turns

of the excised cochlea of the mature, pigmented guinea pig.

Under CO2 anesthesia, animals of weight 250–500 g and with

positive Preyer’s reflexes were sacrificed by rapid cervical

dislocation. The bulla was rapidly removed (�1 min postmortem) and placed in ice-cooled Hanks’ balanced salt solu-

tion (HBSS; Sigma-Aldrich Chemie GmbH, Steinheim, Ger-

many). The HBSS had been supplemented with 4.1 mM

NaHCO3 and 10 mM HEPES buffer and its osmolarity (Osm)

adjusted with glucose to 320 mOsm for the first cochlear turn

and 300 mOsm for the other turns (pH 7.35). Using a higher

osmolarity for experiments from the first cochlear turn

improved the lifetime of the cells and TM, and is a protocol

based on the higher osmolarity of the first turn in vivo(Sterkers et al., 1984) and on similar experiences with the via-

bility of isolated OHCs (Preyer et al., 1996). Within the prep-

aration time of �15 min, the solution warmed up to room

temperature, which in turn was controlled to 20–22 �C. The

bulla was opened and the lateral bony walls of the other coch-

lear turns were removed. For better preservation of the TM

and organ of Corti, Reissner’s membrane in the remaining

turn was kept intact, as ascertained visually. The cochlear was

then placed in a fluid-filled experimental chamber (�27 mL)

and mounted on a free-moving platform to position the RL

approximately perpendicular to the laser beam of the interfer-

ometer. The tympanic surface of the BM was also bathed in

the HBSS, enabling the BM to vibrate.

The length of the BM in the excised cochlear section

was �7–8 mm for the first and second turns and �5 mm for

the third turn. For the first, second, and third turns, respec-

tively, the excised BM began at 0, 4, and 10 mm from the ba-

sal end of the BM. The apical end of the excised cochlea

section was open; the basal end was open for the second-

and third-turn preparations and closed for the first-turn

preparation.

The morphological condition of the preparation was visu-

ally checked throughout the experiment. Using a total magni-

fication of 400, the preparation was considered viable when

the following conditions were maintained: (1) Apical surface

of the organ of Corti aligned along its entire length within the

focal plane of the microscope, (2) cylindrically shaped OHCs

and no blebbing, and (3) constant position and shape of the

TM. The first condition was always satisfied. Typically,

OHCs began to swell, becoming noncylindrical, at �60 min

post mortem in the first cochlear turn, and no earlier than

90 min post mortem in the other two turns. The position and

shape of the TM is extremely sensitive to its ionic environ-

ment (Kronester-Frei, 1979; Edge et al., 1998; Freeman et al.,2003). Therefore, the TM was considered patent when the

tallest OHC stereocilia remained in the same focal plane as

the protofibrils in the lower surface of the TM. Typically, this

condition was maintained for 90 min post mortem for all

cochlear turns, after which the TM began to retract medially.

Damage to Reissner’s membrane also caused TM retraction,

presumably because of mixing of the endolymph with the arti-

ficial perilymph from Scala vestibuli. The experiment was ter-

minated if these viability conditions were not satisfied.

B. Stimulation

For intracochlear electrical stimulation, two platinum

electrodes (diameter of 0.3 mm) were placed in Scala vesti-buli and a gold reference electrode in Scala tympani (width

of 1.1 mm); the transversal distance between the electrodes

was 4 mm. Using two electrodes rather than one in Scalavestibuli enabled better “focusing” of the extracellular elec-

tric field along the principal axis of the OHC (Scherer and

Gummer, 2004b); typically, the field strength was a factor of

J. Acoust. Soc. Am., Vol. 130, No. 6, December 2011 M. Nowotny and A. W. Gummer: Cochlea electromechanical responses 3853


9 smaller in the longitudinal cochlear direction (Nowotny

and Gummer, 2006). The gold electrode also served as a mir-

ror for intracochlear illumination from a white-light source

coupled via a glass fiber (diameter of 1 mm).

There was no means of measuring the frequency response

of the electric field within the organ of Corti. Such measure-

ments would require small-diameter electrodes to avoid

mechanically disrupting the organ and such electrodes are

notoriously difficult to calibrate reliably at high frequencies.

Therefore, as a compromise, in a series of control experiments,

the frequency response was measured with a soda-glass capil-

lary of large tip-diameter (40–50 lm) placed above the organ

of Corti and TM. Reissner’s membrane was disrupted to place

the capillary. The capillary was connected directly to an oscil-

loscope via a platinum wire. The voltage responses of the cap-

illary and oscilloscope were corrected. The voltage response

in the HBSS was slightly dependent on frequency (<2.4 dB

and 5� up to 68 kHz). In the presence of the preparation, a

(small) high-frequency roll-off was introduced above 20 kHz

which amounted to an amplitude slope of �4 dB/octave and a

phase decrease of 20� up to 68 kHz. The 3 dB frequency was

31–34 kHz depending on the exact measurement position

above the organ of Corti and TM. The vibration data were cor-

rected for the frequency response of the voltage in HBSS in

the absence of the preparation, but not for the high-frequency

loss due to the presence of the preparation.

Likewise, the transmembrane potential and its frequency

response are unknown for this preparation. However, it is

possible to estimate some bounds. Assuming the cell to be a

voltage divider with a ratio of 1:1, the largest low-frequency

transmembrane potential change is estimated to be 0.6 mV

for the shortest (30 lm) and 1.7 mV for the longest (90 lm)

OHCs, where the maximum electric field gradient between

the stimulus electrodes in Scalae vestibuli and tympani was

measured to be 37 V/m per frequency point (Nowotny and

Gummer, 2006). Therefore, the 1 mV reference potential is

approximately equal to the transmembrane potential driving

the electromotility. Clearly, an upper bound for the trans-

membrane potential provides a lower bound for the small

signal gain (nm/mV). In any case, no attempt was made to

correct the vibration data for a possible dependence of trans-

membrane potential on cell length along the cochlea; thus,

the true low-frequency gain might be larger for the basal

location [say, 20 log(1/0.6)¼ 4 dB] and smaller for the api-

cal location (5 dB). Such differences are found to be within

the interanimal variation of the vibration data (Sec. III).

Finally, as the radial distance between adjacent OHCs is

much smaller than the distance between the stimulating elec-

trodes and any radial variation of cell length is much smaller

than the longitudinal variation, one can assume that all three

rows of OHCs experience the same extracellular voltage

and, presumably, the same transmembrane potential.

To maintain the viability of the preparation for as long as

possible, the stimulus amplitudes were kept as small as possi-

ble. Measured low-frequency displacement amplitudes were

typically between 2 and 15 nm. At these levels, the second

and third harmonic components were more than 30 dB below

the fundamental component. Therefore, the vibration response

is assumed to be a linear function of the stimulus voltage and

a multitone signal can be safely used as stimulus. The multi-

tone signal contained 81 frequency components of equal am-

plitude and random phase uniformly distributed on the

interval [0, 2p]. The frequency range extended from 480 Hz

to 67.848 kHz, with an almost logarithmic frequency spacing

(ratio� 1.07) between adjacent frequencies. To further reduce

harmonic distortion products in the measured velocity signal,

care was taken that no stimulus frequency was within 1% of

the first four harmonics of a lower frequency.

C. Vibration measurements

Vibration measurements were made with a Polytec

(Waldbronn, Germany) laser Doppler vibrometer (LDV;

OFV-302, wavelength 633 nm, power 1 mW) fitted with a ve-

locity decoder (OFV-3000, bandwidth 100 kHz). Although

vibration was measured as velocity, data are presented here as

displacement. The laser beam was coupled via a shortwave-

path dichroic beam splitter (AHF Analysentechnik, Tubingen,

Germany) into the optical path of an upright microscope (Axi-

oskop 2FS, Zeiss, Jena, Germany). The transition wavelength

of the beam splitter was 590 nm.

The microscope objective was a water-immersion objec-

tive with magnification 40�, numerical aperture 0.8 and

working distance 3.61 mm (Zeiss Achroplan, Jena, Germany).

The laser spot had an approximately Gaussian profile and a

full-width at 1/e2 of maximum power of 0.63 lm, as quanti-

fied with a knife-edge method. The measured velocity

response was corrected for the (measured) transfer function of

the LDV. Phase is defined as positive for motion toward the

microscope objective; this phase convention corresponds to

motion toward Scala vestibuli. A velocity response was

derived by averaging 100–200 velocity spectra; the effective

averaging time was 25–50 s. For this amount of averaging, the

displacement noise floor typically decreased from 100 pm at

480 Hz to 1 pm at 68 kHz.

Confidence in being able to measure transverse velocities

selectively from the lower surface of the TM and its opposing

point on the organ of Corti is based on two experimental

observations. First, the standard deviation of the vibration

phase of different points at approximately the same anatomi-

cal location was less than �5�. A systematic error was

unlikely because the relative interferometric reflection phase

of an unwanted contribution, from an optically rough surface,

is uniformly distributed over 2p and would induce an error

with standard deviation related to the amplitude of the

unwanted contribution (e.g., Dalhoff et al., 2001; de La

Rochefoucauld et al., 2005). Second, any LDV system is

inherently confocal, especially if reference and object light of

the interferometer is focused onto the detector. In that case,

the reference light spot can be thought of to “act as a synthetic

pinhole” (Wilson, 1990, p. 399). In the case of the Polytec

LDV, the situation is more complicated, because object and

reference beams are collimated at the detector plane. How-

ever, the mixing efficiency of the interferometer will also

decrease in this case, if the measuring beam is not perfectly

focused onto the object. For a suitable glass–air interface at

normal incidence, we measured the attenuation of the hetero-

dyne signal of our setup as a function of backfocal distance

3854 J. Acoust. Soc. Am., Vol. 130, No. 6, December 2011 M. Nowotny and A. W. Gummer: Cochlea electromechanical responses


and found a resolution depth of 61.8 lm, measured as attenu-

ation of the heterodyne signal of 10 dB with respect to its

peak value. Using a numerical aperture of 0.8, this value is

approximately 2� the theoretically achievable resolution

depth for an interferometer focusing the object and the refer-

ence beam onto the detector. This depth is smaller than the

depth of the subtectorial space, which amounts to 4–8 lm

from first to third turn, respectively. Taken together, these two

experimental observations strongly suggest that the contribu-

tion of reflected light from outside the focal plane is negligi-

ble in the present experiments.

D. Confocal laser scanning microscopy

In a separate set of experiments, the condition of the

TM was investigated with a confocal laser scanning micro-

scope (LSM510, Zeiss, Jena, Germany). A small rupture was

made in Reissner’s membrane to apply a fluorescent dye,

dextran conjugated with Oregon green 514 (D-7174, Molec-

ular Probes, Invitrogen GmbH, Germany; 10 000 MW), into

Scala media (Ulfendahl et al., 2001). For better orientation,

hair cells were stained with FM 1-43 (T3163, Molecular

Probes, Invitrogen GmbH, Germany). Both dyes were

excited with a wavelength of 514 nm. The collected absorp-

tion spectrum for the dextran dye was at 505–560 nm and for

the FM 1-43 dye at 610–720 nm.

E. Curve fitting and statistics

Amplitude responses were fitted numerically with the

amplitude responses of second-order low-pass or resonant

filters, using the Levenberg–Marquardt algorithm in

SigmaPlotVR

8 (Systat Software, Inc., Richmond, CA). Pa-

rameters are given as mean 6 standard deviation (SD). For

collated data, mean amplitudes and frequencies were esti-

mated on logarithmic axes (i.e., decibel and octave axes,

respectively). Test of statistical significance (Student’s t)was performed at the 95% confidence level.

F. Animal welfare

Care and maintenance of the animals was in accordance

with institutional guidelines at the University of Tubingen

III. RESULTS

The results derive from in situ preparations of 77 mature

guinea pig cochleae. Vibration measurements began at

�20 min post mortem and were made from the first turn

(characteristic frequency, CF¼ 24 kHz, n¼ 21), second turn

(CF¼ 3.0 kHz, n¼ 30), and third turn (CF¼ 0.8 kHz,

n¼ 26) of the cochlea. The preparation was not successful in

the fourth, most apical turn, because of anatomical con-

straints. The CF values were calculated using the neuronal

tonotopic map of Tsuji and Liberman (1997), and refer to

in vivo CF.

At a given longitudinal position along the cochlea,

vibration measurements were made usually at ten positions

on the organ of Corti, extending from the inner sulcus cells

(ISCs) to Hensen’s cells (HeCs), and at six positions on the

upper and lower surfaces of the TM [Fig. 1(B)], the exact

number mainly depending on the time available for acquir-

ing (reliable) data. When presenting average data across

preparations, the number of samples at each radial location

is given in brackets after the mean and SD. The measure-

ment sequence was randomized to avoid time-dependent

phenomena. However, to ascertain the physiological stability

of the preparation, the first and last measurements were

always made on the RL at the second row of OHCs. With

increasing duration of the experiment, the response ampli-

tudes tend to decrease—by < 4 dB below 4 kHz and < 10 dB

above 4 kHz. The absolute phase changed by no more than

25�. Measurements with low-frequency amplitude loss

>8 dB were rejected. For mean values of a given parameter,

the SD is mainly due to a variation in the physiological

FIG. 1. Measurement positions on the organ of Corti and tectorial membrane.

(A) Schematic drawing of the guinea pig cochlea in the first turn. (B) Simpli-

fied representation of A showing the positions (circles) at which vibration

measurements were made. For easier reading, the circles are alternately black

and white. Measurement points on the upper surface of the tectorial membrane

(TM) are labeled according to the structures located directly below on the

organ of Corti. The reticular lamina (RL; exaggerated thickest black line in A)

comprises the apical surfaces of the hair cells, inner pillar cell, and Deiter’s

cells. The RL, as well as the Hensen’s cells and the measured region of the

inner sulcus cells, was orientated approximately perpendicular to the laser

beam (error < 10%), as indicated by the arrow; vibration data are not cor-

rected for this (relatively small) angle. Abbreviations: basilar membrane

(BM), Hensen’s cells (HeCs), Hensen’s cell area (HA), Hensen’s stripe (HS),

inner hair cell (IHC), inner sulcus (IS), IS cells (ISCs), outer hair cells

(OHCs), outer tunnel (OT), pillar cells (PCs), Scala media (SM), and Scalatympani (ST).



condition across preparations. Uncertainty in the longitudi-

nal recording position within a given cochlear turn was no

more than 0.3 mm, so that this interpreparation variation is

unlikely to contribute significantly to statistical variability.

A. Vibration responses of the organ of Corti

Some general properties of the vibration pattern of the

organ of Corti can be gleaned from the envelope patterns in

Fig. 2. Highest amplitudes are found on the OHCs, the

region where the OHC electromechanical force is coupled

directly to the RL and, thus, appear to drive the other regions

of the organ of Corti. The amplitudes tend to be largest at

the second row of OHCs, denoted by OHC2 [Figs. 2, 3(A),

and 3(C)]. The middle region of Hensen’s cell area (HA)

presents amplitudes comparable to those of OHCs in the first

and third rows. At in vivo CF, amplitudes of the IHC region

are �3� smaller than those of OHCs. Smallest amplitudes

are found on the pillar cells (PCs). Moreover, the HeCs near

the third row of OHCs, denoted by HA1, and which cover

the outer tunnel, also tend to show relatively low amplitudes.

In the second and third turns, the IHC moves in an opposite

phase to the OHC region [Figs. 2(A) and 2(B)], at least up to

�24 kHz. In the first turn, this counterphasic motion is found

up to �5 kHz; the phase roll-off of the IHC becomes increas-

ingly larger at higher frequencies, so that eventually IHC

and OHC appear to move in phase [Fig. 2(C)].

Therefore, consistent with the lowest amplitudes being

found at the PC and HAl, the vibration patterns suggest that,

depending on frequency and cochlear turn, the RL can appear

to act as a stiff element (plate) between IHC and OHCs, pivot-

ing about the PC; lateral to OHCs in the HeC region, the

surface of the organ of Corti appears less stiff, allowing coun-

terphasic motion between OHCs and the HeCs.

1. OHC vibration response on the RL

In all three turns, the OHC response on the RL can be

described as that of a low-pass filter (Fig. 3), apart from a

high-frequency antiresonance in the first turn [e.g., at

15.7 kHz in Figs. 3(E) and 3(F)]. The filter shape is usually of

second order, exhibiting high-frequency amplitude roll-off of

about �12 dB/octave and total phase delay of �180�. The

amplitude responses are best described by two first-order low-

FIG. 2. Envelope of the transversal dis-

placement pattern of the organ of Corti (OC)

and lower surface of the TM (TMLS) in the

third (A), second (B), and first (C) cochlear

turns. The stimulus was a single sinusoid

with frequency equal to the in vivo charac-

teristic frequency for the given recording

location: 0.8, 3, and 24 kHz, respectively.

The response envelope is designated by the

shaded area, which connects the maxima

and minima of the response at each measure-

ment point. In each panel, the response is

plotted relative to the largest response in that

panel; i.e., data are only comparable at a

given surface (OC or TMLS). Relative phase

is indicated by the black lines. Notice that

the envelope for the TMLS terminates at

OHC3 because the HeCs are not covered by

the TM. MS X¼ guinea pig preparation

numbers.



pass filters (in series), as ascertained by least-mean-square fit-

ting with the Levenberg-Marquardt algorithm (Sec. II E). The

two 3 dB frequencies of the filters, estimated from the fitting

procedure and denoted by fc1 and fc2, are illustrated with the

crossed lines for the example in Fig. 3(A). On average,

for the OHC2 these frequencies are in the third turn

fc1¼ 4.3 6 1.8 kHz and fc2¼ 35.1 6 14.1 kHz (n¼ 20), in

the second turn fc1¼ 3.8 6 1.4 kHz and fc2¼ 26.1 6 14.2 kHz

(n¼ 27), and in the first turn fc1¼ 10.0 6 4.6 kHz and

fc2¼ 33.0 6 14.5 kHz (n¼ 11 of 12). The values of fc1 in the

second and third turns are not significantly different; the value

in the first turn is significantly larger than in the other two

turns. Statistically, fc2 is the same in all three turns, and has a

mean value of 31.2 6 14.2 kHz. Finally, for any given longi-

tudinal position along the cochlea, there is usually no signifi-

cant difference between the shapes of the frequency

responses from the three rows of OHCs (Fig. 3); an example

of the only type of exception is given in Figs. 3(A) and 3(B)

and is discussed in the following.

Average amplitudes at in vivo CF are given in Table I

and are lowest in the first turn, where the in vivo CF

(24 kHz) is higher than the first 3 dB frequency (10 kHz).

The low-frequency OHC2 amplitudes—evaluated at 504 Hz

(Fig. 4)—are, on average, for the third turn 4.8 6 2.4 nm/mV

(n¼ 20), for the second turn 7.8 6 6.4 nm/mV (n¼ 27) and

for the first turn 4.9 6 3.8 nm/mV (n¼ 12). These values are

not significantly different. There is a tendency for the low-

frequency OHC2 amplitudes to be slightly larger (�4 dB

across all turns) than that for the other two rows. However,

the mean difference across cochleae is not statistically sig-

nificant (for OHC2 re. OHC1: 4.1 6 2.1, 3.4 6 2.8, and

2.9 6 3.2 dB for turns 3, 2, and 1, respectively; for OHC2 re.

OHC3: 5.9 6 4.3, 5.5 6 4.2, and� 0.5 6 4.0 dB for turns 3,

2, and 1, respectively).

In the first turn, half of the amplitude responses exhibit

an obvious antiresonance below in vivo CF [Figs. 3(E) and

3(F)]. On average, it is located at 16.4 6 1.0 kHz for OHC2,

and 15.8 6 1.1 kHz for IHC (n¼ 6); or 0.55 6 0.09 and

0.60 6 0.10 octaves below in vivo CF, respectively. That is,

the antiresonance is located at the same frequency for IHC

and OHC2; indeed, the antiresonance is located at the same

frequency for all OHC rows. For the other half of the first-

turn recordings, the antiresonance is not as pronounced,

appearing simply as a change of high-frequency slope. Thus,

at �1 octave below in vivo CF, there is either the beginnings

of a prominent amplitude roll-off or a more-or-less pro-

nounced resonance in the OHC and IHC responses. On aver-

age, this frequency is located at 12.1 6 1.1 kHz for OHC2

and 12.8 6 1.4 kHz for IHC (n¼ 6); or 0.97 6 0.12 and

0.91 6 0.15 octaves below in vivo CF, respectively.

Low-frequency phase responses of OHCs on the RL usu-

ally asymptote to about �180� [Figs. 3(B), 3(D), 3(F), and 5],

meaning that the RL of OHCs moves toward Scala tympanifor positive potential in Scala vestibuli. On average, the low-

frequency phase response—evaluated at 504 Hz for OHC2—

is for the third turn �171 6 33� (n¼ 20), for the second turn

�165 6 19� (n¼ 27) and for the first turn �152 6 18�

(n¼ 12). These values are not significantly different from

�180�.In all three turns, the phases of OHC1 and OHC2 exhibit

a phase roll-off of �180� from 0.48 kHz up to 68 kHz. How-

ever, OHC3 in the third turn exhibits a high-frequency phase

FIG. 3. Displacement amplitude (A), (C), (E) and phase (B), (D), (F) of the first to third row of OHCs on the RL at different positions along the cochlea. (A),

(B) Third turn, MS 124; (C), (D) second turn, MS 101; (E), (F) first turn, MS 149. Arrows indicate the in vivo characteristic frequency (CF) of the measured

region, calculated from the neuronal tonotopic map of Tsuji and Liberman (1997). The two sets of crossed lines in (A) mark the position of the 3 dB frequencies,

which define the low-pass filter shape of the responses; the slopes of their constituent lines are 0, �6, and �12 dB/octave. Negative phase means that the OHCs

contract for positive voltage in Scala vestibuli. Red diamonds¼OHC1, black circles¼OHC2, and purple triangles¼OHC3. MS X¼ guinea pig preparation

numbers.



roll-off of �360� in most cases [82% of n¼ 20, as illustrated

in Fig. 3(B)], and the usual 180� in the remainder of the

recordings. In the second turn, OHC3 moves in some cases

(16% of n¼ 27) in counterphase to OHC1 and OHC2 at all

frequencies (not illustrated); otherwise, they move in-phase

with OHC1 and OHC2. In the first turn, all OHCs are in

phase and always show a phase roll-off of �180�. In other

words, in the second and third turns, the OHC3 could move

counterphasic to OHC1 and OHC2, either at all frequencies

(16% of cases in the second turn) or at very high frequencies

(>20 kHz in 82% of cases in the third turn).

2. IHC vibration response on the RL

As with the OHCs, the IHC responses exhibit high-

frequency amplitude slopes of about �12 dB/octave and total

phase roll-off of �180� (Fig. 6), indicative of responses of

second order.

However, in contrast to the situation with the OHCs, in

only 40% of IHCs can the amplitude responses be described

by two first-order low-pass filters [Fig. 6(A)], with two dis-

tinct 3 dB frequencies. On average, the frequencies are in

the third turn fc1¼ 5.5 6 2.1 kHz and fc2¼ 20.2 6 6.3 kHz

TABLE I. Mean displacement amplitude (A) and phase (u) at the in vivo

characteristic frequency (CF) for IHC and second-row OHC on the reticular

lamina (RL) at a mean distance (x) from the BM basal end.

Position

on RL x (mm) na

CF

(kHz) A (nm/mV) u (�)u (�) OHC

RL re. IHC RLb

OHC2 2.5 12 24 1.3 6 0.4 �284 6 25 67 6 45

OHC2 9 27 3 6.6 6 5.3 �230 6 20 211 6 26

OHC2 13 20 0.8 6.7 6 4.7 �177 6 23 176 6 37

IHC 2.5 12 24 0.4 6 0.3 � 340 6 76 —

IHC 9 25 3 2.2 6 1.4 �17 6 26 —

IHC 13 20 0.8 2.8 6 1.8 1 6 32 —

aNumber of preparations.bThe mean of the relative phase is calculated by averaging the relative phase

differences from the individual preparations.

FIG. 4. Displacement amplitudes at 504 Hz averaged across preparations for

different radial and longitudinal positions along the cochlea. (A) Third turn; (B)

second turn; (C) first turn. Error bars are SDs (Sec. II E). Measurement locations

are shown in Fig. 1(B). Closed circles¼ organ of Corti, opened diamonds¼TM

lower surface, and opened triangles¼TM upper surface. Clearly, the HeCs are

not covered by the TM. ISC¼ inner sulcus cell, IHC¼ inner hair cell,

PC¼ pillar cell, OHC¼ outer hair cell, and HA¼Hensen’s cell area.

FIG. 5. Displacement phases at 504 Hz averaged across preparations for dif-

ferent radial and longitudinal positions along the cochlea. (A) Third turn;

(B) second turn. (C) first turn. Error bars are SDs (Sec. II E). Measurement

locations are shown in Fig. 1(B). Closed circles¼ organ of Corti, opened

diamonds¼TM lower surface, and opened triangles¼TM upper surface.

ISC¼ inner sulcus cell, IHC¼ inner hair cell, PC¼ pillar cell, OHC¼ outer

hair cell, and HA¼Hensen’s cell area.



(n¼ 12 of 20), in the second turn fc1¼ 5.8 6 2.5 kHz and

fc2¼ 20.3 6 10.5 kHz (n¼ 9 of 25) and in the first turn

fc1¼ 10.3 6 1.6 kHz and fc2¼ 29.7 6 17.4 kHz (n¼ 2 of

12). In these cases, no significant differences to the shapes

of OHC amplitude responses are found.

In the second and third turns, the balance of amplitude

responses on the IHC RL exhibit either a broad resonance

response between� 2 and 10 kHz (13 of 45), as illustrated

in Fig. 6(C), or an over-damped second-order resonance

response (11 of 45). The former cases can be best described

by high-pass filtering of the second-order low-pass filter

response, where the 3 dB frequency is located at low fre-

quencies. For example, for the amplitude response presented

in Fig. 6(C), the low-frequency amplitude response has a

slope of 6.4 dB below 1 kHz and 3 dB frequency of 1 kHz.

This slope value is close to the value of 6 dB/octave for a

first-order high-pass filter. For the cases of an over-damped

second-order resonance, on average, the 3 dB frequency is in

the third turn fc1¼ 11.8 6 3.0 kHz (n¼ 4 of 20), and in the

second turn fc1¼ 9.0 6 2.6 kHz (n¼ 4 of 25). Although this

group can also be fitted with two first-order low-pass filters

with two distinct 3 dB frequencies, statistically the fits are

not as good as with the over-damped second-order resonance

response. In the first turn, the balance of amplitude responses

(10 of 12) exhibits the wide-band response and the high-

frequency antiresonance found for the OHCs.

On average, the low-frequency amplitude response of the

IHCs, evaluated at 504 Hz, is for the third turn 2.6 6 1.8 nm/

mV (n¼ 20), for the second turn 1.7 6 0.6 nm/mV (n¼ 25)

and for the first turn 1.3 6 0.5 nm/mV (n¼ 12). These values

are not significantly different. On average, low-frequency

IHC amplitude responses are �10 dB smaller than those of

the OHCs; namely, in the third turn 7.8 6 5.7 dB (n¼ 19), in

the second turn 13.5 6 6.4 dB (n¼ 25) and in the first turn

11.5 6 5.8 dB (n¼ 6).

Low-frequency phase responses of IHCs on the RL usu-

ally asymptote to �0� [Figs. 5, 6(B), and 6(D)]. This means

that at low frequencies the RL of the IHC is counterphasic to

the RL of OHC1 and OHC2, and usually to OHC3 (see Sec.

III A 1). Those cases exhibiting a high-pass filter response at

low frequencies [Figs. 6(C) and 6(D)] exhibit a low-frequency

phase lead, as expected for a high-pass filter, which typically

amounts to �40� down to 480 Hz; that is, in these cases, the

phase difference between IHC and OHCs is typically 220�.On average, the low-frequency phase response, evaluated at

504 Hz, is 29�6 38� (n¼ 20) in the third turn, 14�6 21�

(n¼ 25) in the second turn and 14�6 32� (n¼ 12) in the first

turn. These values are not significantly different from 0�.

FIG. 6. Two types of IHC response on the RL. Displacement amplitude (A), (C) and phase (B), (D) of IHCs (closed triangles) and, for comparison, second-

row OHCs (closed circles), also recorded on the RL. Arrows indicate the in vivo CF. Lines in (A) and (C) are mean-square regression curves derived by fitting

the amplitude responses with two first-order low-pass filters, with 3 dB frequencies fc1 and fc2; these frequencies are delineated for OHC2 by the crossed lines

in (A). Notice that the IHC amplitude response in (C) cannot be represented by this filter response because it exhibits amplitude attenuation below� 3 kHz. In

(A): for OHC2, fc1¼ 6.0 6 0.1 kHz, and fc2¼ 32.1 6 1.0 kHz; for IHC, fc1¼ 4.4 6 0.3 kHz, and fc2¼ 20.3 6 1.7 kHz. In (C): for OHC2, fc1¼ 2.4 6 0.1 kHz,

and fc2¼ 30.7 6 3.0 kHz. MS X¼ guinea pig preparation numbers.



The IHC phase response exhibits a high-frequency phase

roll-off of �180� in the third and second turns; the roll-off is

significantly larger in the first turn. Specifically, the average

roll-offs are 201�6 43� (n¼ 20) and 178�6 38� (n¼ 25) for

the third and second turns, respectively. This means that

in the third and second turns, the phase shift of �180�

between the IHCs and OHCs on the RL is maintained over

the entire functionally relevant frequency range for this coch-

lear region. Mean data relative to OHC2 are given in Table I

for stimulus frequencies at in vivo CF. In the first turn, how-

ever, the phase difference of 180� is maintained to only

�5 kHz; the total phase roll-off is, on average, 413�6 40�

(n¼ 12) and the phase difference between OHC and IHC at

in vivo CF of 24 kHz amounts, on average, to only 67�6 45�

(n¼ 12). In other words, IHC and OHC move approximately

in phase at the in vivo CF of the first turn. Importantly, this is

due to an additional 180� high-frequency phase roll-off, rela-

tive to that for the other two turns, found on the IHC RL, but

not on the OHC RL. Interestingly, the phase shift of 180�

between IHC and OHC in the third and second turns is main-

tained at frequencies as high as the 24 kHz in vivo CF of the

first turn; the average values at that frequency are 188�6 23�

(n¼ 18) and 176�6 26� (n¼ 23), respectively.

3. Supporting-cell vibration responses

Vibration responses were measured on three regions of

supporting cells: (1) Head plate of the inner pillar cell,1

referred to as pillar-cell (PC) recordings, (2) lateral to

OHC3, referred to as Hensen’s cell area (HA), and (3) ISCs

[Fig. (1B)]. Vibration measurements were made at three or

four, approximately equidistant locations on the HA. In the

third turn, the HeCs were clearly visible because of their

highly reflecting lipid droplets and were, typically, located at

HA3 and HA4 [Fig. 1(B)].

a. Pillar cell. Lowest amplitudes are measured on the

PC [Figs. 4 and 7(A)]. At 504 Hz, the PC amplitudes are

smaller than OHC amplitudes by �16 dB; specifically,

16.2 6 4.2 dB (n¼ 15) in the third turn, 19.0 6 7.6 dB

(n¼ 24) in the second turn, and 14.2 6 5.6 dB (n¼ 11) in the

first turn. The amplitudes at 504 Hz are 0.8 6 0.7 nm/mV

(n¼ 16) in the third turn, 0.9 6 1.1 nm/mV (n¼ 23) in the

second turn, and 1.0 6 0.9 nm/mV (n¼ 13) in the first turn.

Consistent with the amplitude data, the scatter in the phase

data in the PC region is much larger than at the neighboring

locations of IHC and OHC1, the standard deviation of the

phases at 504 Hz being at least a factor of 2 and as much as

8 greater than at the IHC or OHC1 (Fig. 5). Together with

the observation that the IHC can vibrate in opposite phases

to the OHCs [Sec. III A 2, Figs. 5, 6(B), and 6(D)], the find-

ing of least vibration amplitude at the PC allows the conclu-

sion that, depending on stimulus frequency, the RL acts as a

stiff element pivoting about the PC. The frequency range for

such motion in the second and third turns extends to at least

24 kHz, and in the first turn to �5 kHz. The exact position of

the pivot point at the PC can be extracted from the amplitude

and phase data in Fig. 7, where one observes that the ampli-

tudes are smallest at �5 lm medial to the midpoint between

IHC and OHC1. Indeed, according to observations with

the confocal microscope (Sec. III C), this point is located

approximately over the apex of the triangle formed by the

inner surfaces of the pillar cells. The radial location of the

apex is a factor of �2.5 closer to the IHC than to the OHC1.

This eccentricity can account for most of the observation

(Sec. III A 2) that, on average, IHC amplitudes are �10 dB

smaller than OHC amplitudes (20 log 2.5¼ 8 dB).

b. Hensen’s cells. There is a tendency for the HeC

amplitudes to eventually decrease radially with position to-

ward Stria vascularis, the total amplitude decrease amount-

ing to �10 dB in the second and first turns [Figs. 4(B) and

4(C)]. Moreover, the HeC area near OHC3, denoted by HA1

[Fig. 1(B)], tends to have smaller amplitudes than at HA2,

particularly in the third turn [Figs. 4(A) and 8(A)]. However,

for a given turn, averaging across the three OHC and the

four HeC recording locations, the interanimal scatter is such

that there is no detectable difference between the low-

frequency amplitude responses: Evaluated at 504 Hz (Fig. 4),

FIG. 7. Displacement amplitude (A) and phase (B) of the PC region. The

responses at the RL of OHC2 and IHC are included for comparison. Arrows

indicate the in vivo CF. Black circles¼OHC2, black triangles¼ IHC, black

squares¼PC (midway between OHC1 and IHC), blue circles¼PC-OHC

(� 5 lm from PC toward OHC1), and red triangles¼PC-IHC (� 5 lm from

PC toward IHC). Notice that for most frequencies the relatively small ampli-

tudes at PC and PC-IHC, together with the near 180� phase difference

between the region extending laterally and medially from these points

implies that a near frequency-independent pivot point lies in the region of

PC to PC-IHC. MS X¼ guinea pig preparation number.



the HA responses relative to OHC responses are 3.8 6 5.4 dB

(n¼ 14) for the third turn, 9.3 6 9.1 dB (n¼ 18) for the sec-

ond turn and 5.5 6 8.6 dB (n¼ 10) for the first turn, all of

which are not statistically different from 0 dB. At 504 Hz,

amplitudes at HA2 are 2.3 6 2.8 nm/mV (n¼ 15) for the

third turn, 3.9 6 3.7 nm/mV (n¼ 22) for the second turn, and

4.0 6 2.3 nm/mV (n¼ 11) for the first turn. However, the

slope of the high-frequency amplitude response for the HA is

greater than that for OHCs [Figs. 8(A) and 8(C)], being typi-

cally �16 dB/octave as opposed to �12 dB/octave. A value

of �16 dB/octave implies a low-pass filter of order greater

than two (�12 dB/octave), and close to three (�18 dB/

octave).

The phase response of the HA presents different roll-offs

in the different cochlear turns. In the first turn, the roll-off is

�180� [Fig. 8(D)], whereas in the third turn it can be as much

as 360� or even 450� [Fig. 8(B)]. Based on the asymptotic

high-frequency amplitude slopes, such phase roll-offs are

larger than those of a minimum-phase system.2 As, in the

third turn, the phase roll-off is much greater than the 180� pre-

sented by the OHCs, the HA phase responses cross the OHC

phase response curve [Fig. 8(B)]. In fact, the crossover fre-

quency decreases with increasing radial distance from the

OHCs toward Stria vascularis. Expressed another way, for a

given stimulus frequency, the phase delay increases with ra-

dial distance toward Stria vascularis—this is reminiscent of a

radial traveling wave in the HA. For example, the wavelength

at 5.68 kHz (the crossover frequency for OHC2 and HA4) is

113 lm. However, here it should be emphasized that this type

of wave motion is only significant well above in vivo CF, and

is only prominent in the third turn—it is much less distinctive

in the first and second turns.

At low frequencies, the HA tends to vibrate in counter-

phase to the OHCs [Figs. 5, 8(B), and 8(D)]; specifically, at

504 Hz the difference is 224�6 43� (n¼ 14) in the third turn,

196�6 19� (n¼ 19) in the second turn, and 175�6 22�

(n¼ 9) in the first turn. The HA phase at 504 Hz is 31�6 44�

(n¼ 16) in the third turn, 33�6 23� (n¼ 22) in the second

turn, and 14�6 54� (n¼ 11) in the first turn. Although these

mean values are not statistically different from 180� and 0�,respectively, throughout the HA one observes a large scatter

in the mean phases, as well as in the standard deviations,

compared with the IHC and OHC phase data (Fig. 5).

In summary, lateral to OHC3, the surface of the organ

of Corti appears to be much less stiff than in the region from

IHC to OHCs: Although the HA moves counterphasic to the

OHCs at low frequencies, it exhibits wave-like motion at

higher frequencies, particularly in the third turn.

c. Inner sulcus cells. The shapes of the amplitude and

phase responses of the ISCs are similar to those of the IHCs

up to at least 10 kHz [Figs. 9(A) and 9(B)]. The amplitudes

FIG. 8. Displacement amplitude (A), (C) and phase (B), (D) of the Hensen’s cell area (HA). The response at the RL of OHC2 is included for comparison.

Arrows indicate the in vivo CF. Closed circles¼OHC2, dashed line¼HA1 located� 20 lm from the lateral edge of OHC3, dash-dot-dot-dash line¼HA2,

dash-dot-dash line¼HA3, and solid line¼HA4. The distance between adjacent HA-recording locations is� 20 lm. Notice the larger high-frequency ampli-

tude slope and phase roll-off for the HA compared with the OHC. Also, for a given stimulus frequency, the phase delay increases with lateral position from

OHC3; this is reminiscent of a radial traveling wave in the HA. This traveling-wave signature is not as pronounced for the first-turn responses (D). MS

X¼ guinea pig preparation numbers.



decrease along the ISC by �20 dB over a distance of 15 lm

from the IHC. ISC vibration is below background noise by

�40 lm distant from the IHC. At 504 Hz, the ISC ampli-

tudes are smaller than OHC2 amplitudes by �14 dB; specifi-

cally, 11.2 6 4.8 dB (n¼ 15) in the third turn, 15.4 6 5.0 dB

(n¼ 14) in the second turn, and 15.1 6 5.6 dB (n¼ 10) in the

first turn. The ISCs move in-phase with the IHC (Fig. 5) up

to at least 10 kHz [Fig. 9(B)]. Therefore, contrary to the sit-

uation for the HA in the third turn, there is no evidence of ra-

dial traveling wave motion on the ISC.

B. TM vibration responses

TM vibration measurements were made directly oppo-

site those on the underlying RL and inner sulcus [Fig. 1(B)]

and, therefore, the same nomenclature is used as in previous

sections to denote their radial recording locations. Clearly,

there are no vibration measurements above the HeCs

because they are not covered by the TM (Lim, 1972).

For a given radial location, responses at the lower and

upper surfaces of the TM are not significantly different. Spe-

cifically, for a given radial location, the phase responses of

the two TM surfaces well-nigh superimpose at all stimulus

frequencies [Fig. 10(B)] and the amplitude responses are of

similar shape [Fig. 10(A)]. The amplitude responses at the

upper surface tend to be smaller than at the overlying lower

surface, but the difference of typically 2–6 dB across animals

and stimulus frequencies is within the limits of reproducibil-

ity. Therefore, in the remainder of this section the findings

are presented for the lower surface of the TM.

1. TM vibration response over the OHC

Over the entire frequency range, TM responses over the

OHCs faithfully follow the responses of the underlying

OHCs [Figs. 4, 5, and 10; Nowotny and Gummer (2006),

their Fig. 2], the phase responses well-nigh superimposing

and the amplitude difference being no more than 2 dB, which

is within the limits of reproducibility.

Averaged across animals, at 504 Hz the TM amplitude

relative to RL amplitude at the OHCs is �1.5 6 2.5 dB

(n¼ 17) in the third turn, 0.8 6 1.5 dB (n¼ 15) in the second

turn, and �1.7 6 1.4 dB (n¼ 10) in the first turn; these val-

ues are not significantly different from 0 dB.

Clearly, superposition of these amplitude and phase

responses indicates that the TM is strongly coupled to the

OHCs by means of their stereocilia. Indeed, superposition of

the TM and RL responses at the OHCs is an indicator of the

patency of the preparation—retraction or lifting of the TM

results in significantly smaller TM responses compared with

RL responses; such experiments are not included in the data

set.

FIG. 9. Displacement responses on the organ of Corti (A), (B) and lower surface of the tectorial membrane (C), (D) extending from OHC to inner sulcus. Am-

plitude (A), (C) and phase (B), (D) responses measured at the second-row OHC (closed circles), IHC (closed triangles), and inner sulcus cells� 5 lm (closed

diamonds) and �15 lm (closed squares) distant from the IHC. The full lines in (C) and (D) are the responses of the IHC at the RL. Arrows indicate the in vivoCF. Notice that for the organ of Corti the low-frequency amplitudes decrease from the IHC region toward the inner sulcus by �20 dB/15 lm and for the lower

surface of the TM by �12 dB per 15 lm. MS X¼ guinea pig preparation numbers.



2. TM vibration response over the IHC

The situation is quite different in the IHC region com-

pared with the OHC region.

First, in all turns, the amplitudes of the TM and RL at the

IHC tend to be similar up to �3.5–5 kHz [Fig. 4; Nowotny

and Gummer (2006), their Fig. 2]. However, there are some

exceptions, as, e.g., in Fig. 9(C), where at low frequencies the

TM amplitudes can be as much as 6 dB smaller than those on

the RL. Averaged across preparations, at 504 Hz TM ampli-

tude relative to RL amplitude at the IHC is �3.5 6 5.1 dB

(n¼ 16) in the third turn, �1.6 6 8.5 dB (n¼ 23) in the sec-

ond turn, and �1.9 6 3.4 dB (n¼ 6) in the first turn; these val-

ues are not significantly different from 0 dB. Above 3 kHz,

TM amplitudes tend to be smaller than RL amplitudes, typi-

cally up to 12 dB. However, here again there are some excep-

tions, where in the first turn they can be similar [Nowotny and

Gummer (2006), their Fig. 2E].

Second, up to �3.5 kHz in all turns, there is a phase dif-

ference of 180� between the TM and the underlying RL at the

IHC [Figs. 5 and 9(D); Nowotny and Gummer (2006), their

Fig. 2]. Consistent with this observation, at all frequencies

below �3.5 kHz, the TM moves in phase along its length

between the IHC and OHCs [Figs. 5 and 9(D); Nowotny and

Gummer (2006), their Fig. 2]. At the TM, low-frequency

motion overlying the IHC is �11 dB smaller than over the

OHCs; specifically, at 504 Hz, 10.2 6 3.3 dB (n¼ 16) in the

third turn, 14.3 6 7.3 dB (n¼ 15) in the second turn, and

7.5 6 3.8 dB (n¼ 6) in the first turn. This average value of

11 dB at the TM is not significantly different from the average

value of 10 dB for the relative motions of the RL at the OHC

re. IHC (Sec. III A 2). Thus, for frequencies up to �3.5 kHz,

OHC RL and its overlying TM move with equal amplitude

and phase, and at the IHC with approximately equal ampli-

tude but opposite phase.

Third, at higher frequencies in all turns, the response of

the TM above the IHCs presents an additional phase roll-off

relative to the TM above the OHCs [Fig. 9(D); Nowotny and

Gummer (2006), their Fig. 2], asymptoting to 180� at high

frequencies (at least above 24 kHz). That is, at high frequen-

cies, the TM and RL at the IHC move in phase. [At 24 kHz,

the in vivo CF for the first-turn recording site, the IHC RL

phase relative to the overlying TM phase is 298�6 47�

(n¼ 23) and 271�6 44� (n¼ 16) for the second and third

turns, respectively.] This asymptotic in-phase motion

between RL and TM at the IHC derives from the additional

180� phase roll-off of the TM relative to its underlying IHC

at high frequencies, which in turn presents a total phase roll-

off of only 180�. The additional asymptotic phase roll-off of

180� for the TM implies a second-order low-pass filter. For

the second and third cochlear turns, the 90� frequency for

this filter is located, on average, at 19.4 kHz (n¼ 38). Reca-

pitulating, TM phase above the OHCs exhibits a high-

frequency roll-off of �180� and above the IHC �360�.

3. TM vibration response over the ISC

TM amplitudes decrease by �12 dB over a distance of

15 lm from the IHC toward the ISC [Fig. 9(C)]. That is,

for this most medial recording location, TM amplitudes are

�1/4� the amplitudes over the IHC (up to at least 10 kHz).

C. Condition of the hair cells and TM

As further documentation of the condition of the prepara-

tion, in a separate set of morphological experiments, fluores-

cent staining and confocal laser scanning microscopy (LSM

510, Zeiss, Germany) was used to examine microscopically

the organ of Corti and TM. Experiments were conducted in

the third (n¼ 10) and second (n¼ 6) turns.

OHC and IHC somae stain green with FM 1-43 and ster-

eocilia stain yellow using super-saturated FM 1-43 (Fig. 11).

Nonstained cellular material appears black. OHCs of all

three rows present their typical cylindrical shape (not illus-

trated). No anatomical or time-dependent differences

between OHCs in the three rows are found, which might oth-

erwise have explained the counterphasic motion between

OHC3 and OHC1, OHC2 in the second turn (16% of the

measurements, Sec. III A 1).

In a subset of these third-turn experiments (n¼ 7 of 10),

the TM, including Hensen’s stripe and trabeculae, were

made visible (black) with a negative contrast method. This

was achieved by staining the endolymph—surrounding

and within the TM—red with a dextran dye (Oregon green

FIG. 10. Displacement responses of the RL and TM surfaces at the OHC.

Amplitude (A) and phase (B) responses measured at the second-row OHC

on the RL (black circles), lower (blue circles), and upper (purple circles)

surfaces of the TM. Arrows indicate the in vivo CF. Notice that the phase

responses well-nigh superimpose and, correspondingly, that the amplitude

responses are of similar shape. MS X¼ guinea pig preparation number.



514-conjugated dextran). Thus, in Fig. 11, the black area

above the organ of Corti corresponds to the TM. The TM is

found to remain in contact with the stereocilia of all three

rows of OHCs for up to 120 min post mortem. This

“survival” period corresponds to that observed for the vibra-

tion experiments—using light microscopy, the TM is found

to extend out to the third row of OHCs, in the usual manner

(Lim, 1972), and the TM and the tip of the (tallest) stereoci-

lia are found in the same confocal plane for up to 120 min

post mortem in the second and third turns (and 90 min postmortem in the first turn).

Two TM structures, well-described elsewhere (e.g.,

Lim and Lane, 1969; Lim, 1972), are always visible when

using this negative contrast method: (1) Hensen’s stripe

(HS), a ridge running longitudinally along the lower surface

of the TM in the vicinity of the IHCs, and (2) trabeculae

(Tr), small bridges connecting the TM to the border cells

and/or inner phalangeal cells. These structures are visible

in the preparations (Fig. 11), for up to at least 90 min after

opening the cochlea. Clearly, because these morphological

experiments require staining, one cannot directly compare

the actual positions of the HS and Tr with those in the

vibration experiments. Nevertheless, their presence in the

morphological experiments provides strong evidence that

the condition of the hair cells and TM in the vibration

experiments is not seriously compromised by the in vitroconditions.

IV. DISCUSSION

Using intracochlear electrical stimulation in an in vitropreparation, the transversal motion of the upper surface of

the organ of Corti and the overlying TM was measured in

response to electromechanical force produced by the OHCs,

without the additional influence of acoustically induced

vibrations. That is, the mechanical feedback loops are effec-

tively opened to study the effect of electromechanical feed-

back, in a similar way that others have done for in vivorecordings at the BM (Xue et al., 1995; Nuttall et al., 1999;

Grosh et al., 2004). Here, however, feedback is being studied

at the RL and TM, in the immediate neighborhood of the for-

ward, mechanoelectrical pathway, and also at the apical

surfaces of the ISCs and HeCs. Although located further

way from the mechanoelectrical transducers of the hair-cell

stereocilia, the motion of these latter surfaces might influ-

ence longitudinal fluid flow and, therefore, mechanical cou-

pling within the organ of Corti (de Boer, 1993; Karavitaki

et al., 2007b). Thus, in contrast to the earlier report of Now-

otny and Gummer (2006), which concentrated on the vibra-

tion responses of the RL and TM at the surfaces of the

subtectorial space, this report extends to the ISCs and HeCs,

as well as to the upper surface of the TM.

The data were gathered in the first three cochlear turns

spanning an in vivo CF range from 0.8 to 24 kHz, using stim-

uli that cover the entire functionally relevant frequency range

and above. The nanometer range of displacement amplitudes

(2–15 nm for OHCs at low frequencies) corresponds to BM

displacement amplitudes near neural CF-threshold (Narayan

et al., 1998); the relative displacement amplitudes (on aver-

age, 6 nm/mV at low frequencies for OHC2) are consistent

with values expected from the small-signal gain of OHC elec-

tromotility and the stiffness of the RL (Scherer and Gummer,

2004b; Nowotny and Gummer, 2006).

Using a chloride channel blocker (anthracene-9-carbox-

ylic acid; 9-AC) that reversibly attenuates somatic electro-

motility (Scherer and Gummer, 2004b), Nowotny and

Gummer (2006) have shown that in this preparation the me-

chanical force driving the RL and, therefore, other cochlear

structures, derives mainly from the electromechanical action

of the OHC, rather than from the stereocilia. Moreover,

based on vibration measurements from fine fibers (Kleenex

tissue or cotton, with diameters of 8–40 lm), they demon-

strated that nonspecific electromechanical effects associated

with cochlear-fluid charge, TM charge (Weiss and Freeman,

1997), or glycocalyx charge at the apical surfaces of the hair

cells (Dolgobrodov et al., 2000) are negligible.

A. Wide-band responses

In the second and third turns, the displacement responses

at the OHCs (Sec. III A 1) and most IHCs (Sec. III A 2),

having the first 3 dB frequency no smaller than 4 kHz, are

wide-band relative to the in vivo CFs of 3 and 0.8 kHz,

respectively. In contrast, the first 3 dB frequency in the first

turn is �10 kHz and, therefore, below the in vivo CF of

24 kHz for that turn. The wide-band responses are observed at

both the RL and TM. Clearly, for all three turns, these band-

widths are much larger than the bandwidth of the receptor

FIG. 11. Cross sections of the subtectorial space in the region of an IHC

in the third turn. The picture was reconstructed from a z-stack. (A) Apical

end of an inner hair cell (green), its stereocilia bundle (yellow) and tecto-

rial membrane (black). (B) Hensen’s stripe and the trabeculae, which con-

nect the tectorial membrane with the reticular lamina. HS¼Hensen’s

stripe, IHC¼ inner hair cell, TM¼ tectorial membrane, Tr¼ trabeculae.

Green¼FM 1-43 dye (soma), yellow¼ super-saturated FM 1-43 dye (ster-

eocilia), red¼ dextran dye (endolymph), and black¼ nonstained cellular

material. Notice that as endolymph is also contained within the TM, the

TM does not appear as a purely black structure.



potential (Preyer et al., 1996) and electrical impedance

(Housley and Ashmore, 1992; Preyer et al., 1996) of isolated

OHCs, where the 3 dB frequency lies 3–6 octaves below

in vivo CF, with the 6 octave value being for OHCs from the

first turn (Preyer et al., 1996). They are also larger than the

electrical bandwidths for OHCs in an in situ preparation of

the rat or gerbil cochlea (Johnson et al., 2011). Although the

current pathways were not investigated, the wide bandwidth

is almost certainly due to the mode of extracellular stimula-

tion: In a similar fashion to that described for electrical stimu-

lation of an OHC held in an electrically sealed micropipette

(Dallos et al., 1991), the OHC probably acts as a voltage di-

vider with the input-voltage pathway contributing a high-pass

filter (HPF) to the ratio of the transmembrane and Scala vesti-buli source voltages. It is, of course, the transmembrane volt-

age that drives somatic electromotility (Dallos et al., 1991).

The transmembrane voltage is almost certainly responsible

for the observed vibration responses because: (1) At low

frequencies (<3 kHz), positive voltage in Scala vestibuliresults in motion of the OHC RL toward Scala tympani (Sec.

III A 1), which corresponds to OHC contraction in response

to a depolarizing transmembrane voltage, as also demon-

strated by Scherer and Gummer (2004b) and Karavitaki and

Mountain (2007b), and (2) 9-AC reversibly attenuates the

vibration responses independent of stimulus frequency

[20 6 2 dB; Nowotny and Gummer (2006)].

The finding that some IHC responses in the second and

third turns (13 of 45) exhibit a broad resonance response

between �2 and 10 kHz [Fig. 6(C); Sec. III A 2], concurs

with similar findings in the whole-mount experiments of

Scherer and Gummer (2004b). The broad resonance can also

be described as high-pass filtering of the usual second-order

low-pass filter response of the other IHCs [e.g., Figs. 6(A)

and 6(C)], where the 3 dB frequency of the HPF is located at

low frequencies. As the TM was removed in the experiments

of Scherer and Gummer (2004b), this type of response

appears to be a fundamental electrical and/or mechanical

property of the organ of Corti. Electrically, for example, one

might propose that it is due to the 3 dB frequency of the OHC

basolateral electrical impedance being higher than in most

preparations, such that the ratio of the transmembrane and

Scala vestibuli source voltages continues to increase with fre-

quency into the low-frequency measurement range

(>480 Hz). For the example illustrated in Figs. 6(C) and 6(D),

the appearance of the 3 dB frequency of the HPF at 1 kHz

would place the basolateral 3 dB frequency near 1 kHz, which

is well above that observed for isolated OHCs (Housley and

Ashmore, 1992; Preyer et al., 1996). Moreover, up to

�3 kHz, the OHC vibration responses are frequency inde-

pendent, so that this mechanism appears unlikely to explain

the observed HPF response of these IHCs. Perhaps the most

parsimonious explanation is that as stimulus frequency is

reduced, the electromechanical force produced by the OHCs

is increasingly coupled longitudinally through the fluid rather

than to the RL of the IHC, as suggested by Scherer and

Gummer (2004b) and evident in fluid-flow data of Karavitaki

and Mountain [2007a, their Fig. 3(A)], the exact frequency

below which this effect becomes evident in the vibration data

being dependent on the preparation.

The observed bandwidths, as well as the appearance of a

high-frequency antiresonance in first-turn responses at and

above the OHCs and IHC [Figs. 3(E) and 3(F); Nowotny and

Gummer (2006), their Figs. 2(E) and 2(F)], emphasize the im-

portance of the presence of the TM and BM mobility on band-

width. Thus, in the whole-mount experiments of Scherer and

Gummer (2004b), where the TM was removed and the BM

mechanically clamped, the 3 dB frequencies were higher than

in the present preparation (0.8–1.3 octave); in that prepara-

tion, they were around 10 kHz in the second and third turns

and 17 kHz in the first turn. Moreover, in those experiments,

there was never any evidence for high-frequency antireso-

nance. Here, it should be emphasized that an antiresonance

has been measured on the BM in vivo for bipolar electrical

stimulation (Grosh et al., 2004; Zheng et al., 2007), so that

the antiresonance is probably not an anomaly of our in vitropreparation. Although this antiresonant response might par-

tially result from piezoelectric resonance of the OHC soma

(Spector et al., 2003; Weitzel et al., 2003; Zheng et al.,2007), the absence of an antiresonant response of the organ of

Corti in the absence of the TM (Scherer and Gummer, 2004b)

led Nowotny and Gummer (2006) to propose that the antire-

sonance derives from a mechanical TM resonance associated

with inertia of the TM for acceleration in the radial direction

(Zwislocki, 1980; Allen, 1980; Mammano and Nobili, 1993).

Here, it should be noted that TM resonance: (1) has been

demonstrated in other in vitro preparations (Gummer et al.,1996; Hemmert et al., 2000a,b), (2) has evolved from coch-

lear models (Cai et al., 2004; Meaud and Grosh, 2010), (3)

has been inferred from otoacoustic emission responses

(Brown et al., 1992; Allen and Fahey, 1993; Lukashkin and

Russell, 2003) and when also combined with simultaneous

recordings of masking neural tuning curves (Lukashkin et al.,2007), and (4) has been evidenced in vivo using a-tectorin

mouse mutants (Legan et al., 2000; Lukashkin et al., 2004;

Legan et al., 2005).

Compared with the whole-mount preparation without

TM (Scherer and Gummer, 2004b), it is therefore tempting

to speculate that the bandwidth of the electromechanical

response of the organ of Corti and TM in the present prepa-

ration is reduced by the inertia of the TM. However, one

cannot discount the possibility that the electrical bandwidth

of the preparation is lower than that in Scherer and Gummer

(2004b). After all, except for a narrow region under the

OHCs, the BM was mechanically clamped and electrically

isolated in their preparation to allow greater transversal fo-

cusing of current to the OHCs, the current then exiting the

organ of Corti through the narrow BM region to the refer-

ence electrode in Scala tympani. Although the present vibra-

tion data are corrected for the frequency response of the

voltage in HBSS in the absence of the preparation (<2.4 dB

up to 68 kHz), there was no experimental means of meas-

uring the frequency response of the electric field within the

organ of Corti (Sec. II B). Clearly, a smaller electrical band-

width might yield a lower mechanical 3 dB frequency. In

any case, based on frequency response measurements of the

voltage above the organ of Corti and TM (Sec. II B), it is

suspected that the second 3 dB frequency, with mean value

of 31 kHz for all three turns (Sec. III A 1), is probably due to



electrical properties of the preparation. Summa summarum,

although responses can be interpreted using physical princi-

ples, the unknown spatial and temporal characteristics of the

applied electric field within the organ of Corti and TM do

remain limiting factors.

B. Counterphasic motion of the IHC relative to OHC1and OHC2

OHC1 and OHC2 move in opposite phase to the IHC, for

measurement frequencies at least up to 24 kHz in the second

and third turns [Figs. 5, 6(B), 6(D), 7(B), 9(B), and 9(D)] and

up to �5 kHz in the first turn [Nowotny and Gummer (2006),

their Fig. 2]. Counterphasic motion is an extremely robust

property, occurring in all preparations. Thus, in response to an

electromechanical force applied to the RL by the OHCs, the

RL appears to simply rotate like a stiff element (plate) about a

point located between OHC1 and IHC (Figs. 4, 5, and 7). A

similar conclusion was reached by Karavitaki and Mountain

(2007b) for electrically induced motion measured in the radial

and longitudinal planes within the organ of Corti of the mid-

dle and apical turns of the gerbil cochlea; their optical mea-

surement technique was limited to very low frequencies

(30–120 Hz). The present vibration measurements on the head

plate of the inner pillar cell (Fig. 7), together with the 10 dB

smaller IHC than OHC amplitudes (Fig. 4), place the pivot

point directly above the apex of the triangle formed by the

medial surfaces of the PCs. Using optical flow analysis of

electrically induced vibrations, Chan and Hudspeth (2005b)

report a point of inflection at the PC, with counterphasic radial

motion on either side. Counterphasic motion of OHCs and

IHC in response to OHC electromechanical force was origi-

nally predicted by Geisler (1986) based on the geometry of

the RL and pillar cells. It has also been reported in the ab-

sence of the TM and with mechanically clamped BM (Scherer

and Gummer, 2004b). Therefore, counterphasic motion

appears to be an inherent mechanical property of the organ of

Corti in response to electromechanical somatic force from the

OHC, determined by the RL and PCs, independent of the

presence of TM or BM.

Other groups, who have stimulated electrically and made

vibration measurements of the organ of Corti and/or TM

in vitro, have not measured the transverse motion of the RL.

Thus, Mammano and Ashmore (1993) measured the vibration

of a bead on a HeC. Chan and Hudspeth (2005b) measured

the transverse vibration of beads on the upper surface of the

TM.

Here it should be emphasized that for low-frequency

acoustical (Hemmert et al., 2000a,b; Fridberger and Boutet de

Monvel, 2003; Fridberger et al., 2004; Chan and Hudspeth,

2005b; Fridberger et al., 2006;) or hydrodynamical (Hu et al.,1999; Cai et al., 2003; Fridberger et al., 2002) stimulation,

the RL also appears to move as a stiff element, with the

exception of one report showing complex two-dimensional

motion of the RL (Ulfendahl et al., 1995). In contrast to the

situation for electrical stimulation, those former reports sug-

gest that the pivot point is located medial to the IHC. Under

in vivo conditions, with acoustical stimulation, the exact

position of the pivot point is probably determined by an

intensity-dependent interplay of two sources of force—fluid

pressure and OHC electromechanical force.

In contrast to the situation in the second and third turns,

above 5 kHz in the first turn, the phase difference between RL

motion at the OHC and IHC has been shown to decrease grad-

ually, so that above �10 kHz the RL moves in phase along its

length (Nowotny and Gummer, 2006). This gradual loss of

counterphasic motion results from an additional 180� rotation

of the IHC phase response. As the high-frequency antireso-

nance was found on both sides of the subtectorial space at the

IHC and OHCs, the antiresonant mechanism is probably not

directly responsible for this high-frequency phase response.

The phase shift is consistent with the observation that the real

part of the point impedance—i.e., the viscous component—at

the IHC is largest in the first turn (Scherer and Gummer,

2004a); specifically, both coefficients of the 1/f2 and exponen-

tial frequency dependence of the real part, denoted by c1 and

c3 in that publication, are largest in the first turn.

C. Motion of OHC3 relative to OHC1 and OHC2

OHC3 moves in phase with OHC1 and OHC2 for all

first-turn and most (84%) second-turn recordings [Figs. 3(D)

and 3(F)] and, therefore, counterphasic to the IHC. In the

third turn, OHC3 also moves in phase with OHC1 and

OHC2, but only within its functionally relevant frequency

range [Fig. 3(B)]—above in vivo CF, in most cases (82%)

the phase response exhibits additional roll-off, yielding a

total phase roll-off of 360� at high frequencies [Fig. 3(B)].

Moreover, with few exceptions and within experimental

error or reproducibility (<4 dB), there is no evidence of dif-

ferences in the amplitude responses between the three rows.

The exceptions are of the type found for OHC3 in the third

turn [Fig. (3A)], where there is additional phase roll-off.

Therefore, contrary to suggestions based on the greatest sus-

ceptibility of OHC1 to noise damage (e.g., Robertson, 1982;

Liberman, 1987) and anatomical observations of the stereo-

cilia bundle and overlying TM (Glueckert et al., 2005), there

is usually no evidence for OHC3 behaving micromechani-

cally any differently to OHC1 or OHC2, at least under these

electromechanical stimulus conditions. This observation sup-

ports the notion that strong radial coupling of the three

OHCs at their apical surface—through both the RL and also

the tips of the tallest stereocilia being connected to the

TM—enhances the cooperativity of the OHCs and, therefore,

cochlear amplification (Gavara and Chadwick, 2009).

In a small proportion of preparations from the second

turn (16%), OHC3 exhibited anomalous phase behavior:

OHC3 moved in opposite phase to OHC1 and OHC2 and at

all stimulus frequencies. Although low-frequency counterpha-

sic motion has been reported for swollen OHCs from the

same row (Karavitaki and Mountain, 2007b), there was no

sign of cell swelling at the light microscopic level in the prep-

arations reported here. As the present study finds a similar

proportion of recordings (9%) exhibiting counterphasic

motion to that for vibration measurements in the absence of

the TM (Scherer and Gummer, 2004b), this behavior is

unlikely to be due directly to some pathological decoupling of

OHC3 stereocilia from the TM. Moreover, at the light



microscopic level, the patency of the TM was always

checked—that the TM was not lifting or retracting. As the

HeC area also moves in opposite phase to OHC1 and OHC2

[Figs. 5, 8(B), and 8(D)], it is as if this relatively small pro-

portion of OHC3 are more strongly coupled to the motion of

the HA than to the RL. Interestingly, counterphasic motion

between OHC3 and the other two rows was the rule in the

preparation of Karavitaki and Mountain (2007b), rather than

the exception reported here. However, it is difficult to make

comparisons with their preparation because their vibration

amplitudes and stimulus voltages were much larger, the esti-

mated cell-length changes amounting to 280–1440 nm

(0.7%–3.6% of a 40 lm OHC), compared with the low-

frequency transverse displacement amplitudes of 2–15 nm

presented here. More exactly, taking the oblique angle

between the OHC longitudinal axis and RL into account;

namely, 100�–120� for turns 1–3, respectively (Y. Yarin, per-

sonal communication), the maximum low-frequency cell-

length change in the present experiments is estimated to be

17 nm, which is 1–2 orders of magnitude smaller than for the

experiments of Karavitaki and Mountain (2007b). Their stim-

ulus voltages were correspondingly larger. At least for the

present preparation, it is presumed that this counterphasic

behavior, in an albeit small proportion of preparations, derives

from a pathological condition at the level of the lateral end of

the RL, which was not detectable at the light microscopic

level. For example, using laser scanning confocal microscopy

in fluorescent stained organ of Corti in a temporal-bone prepa-

ration, there is suggestion of a reversible disassembly–-

assembly mechanism in the apical part of the cytoskeleton in

the Deiter’s cells in response to acoustic trauma (Flock et al.,1999). The result could be that the RL of OHC3 loses its cou-

pling to OHC2 and becomes more tightly coupled to the

motion of the HA—at low frequencies, the HA is found con-

sistently to vibrate counterphasic to OHC1 and OHC2 [Figs.

8(B) and 8(D)].

For most third-turn recordings (82%), there is an addi-

tional phase roll-off in the OHC3 response [Fig. (3B)], which

begins no less than an octave above in vivo CF and asymp-

totes to 180� by �20 kHz. That is, at asymptotically high fre-

quencies, OHC3 moves in the opposite direction to OHC1

and OHC2 in this turn. Clearly, being 4–5 octaves above

in vivo CF, this counterphasic motion is of no functional sig-

nificance for the third turn. Instead of being a pathological

condition, it is suggested that this motion is related to unique

anatomical and vibrational features of the HA in the third

turn; this point is discussed in Sec. IV F.

D. In-phase motion of the TM

The TM moves in phase along its radial extent from the

region covering the OHCs to the region covering the ISCs—

for each cochlear turn and for frequencies up to 3 kHz

[Figs. 5, 9(D), and 10(B)]. In-phase motion has also been

reported for beads on the upper surface of the TM (Chan and

Hudspeth, 2005b). However, here it should be mentioned

that (transversal) vibration measurements at the TM upper

surface, over the OHCs, provide an accurate picture of the

vibration pattern at the TM lower surface overlying the

OHCs, at all frequencies, but at the TM lower surface over-

lying the IHC, above �1 kHz, there can be frequency-

dependent attenuation and phase shift relative to the TM

upper surface over the IHC.

This in-phase motion implies that the TM is behaving as

a stiff element for motion in the transverse direction. This

interpretation is consistent with the morphological feature

that the TM is composed of a dense meshwork of radially

orientated collagen fibrils, which are supposed to impart

large stiffness and small compressibility to the matrix within

which they are found (Hasko and Richardson, 1988; Weaver

and Schweitzer, 1994; Goodyear and Richardson, 2002). It is

also consistent with point impedance (Abnet and Freeman,

2000; Gu et al., 2008) and shear modulus (Shoelson et al.,2004) measurements of isolated TM; namely, that the me-

chanical space constant for a point load is �50 lm (Gu

et al., 2008) and, based on the data of Shoelson et al. (2004),

that TM shear modulus is relatively constant radially over

the measurement region of the present experiments. The

results also support the hypothesis that the TM must be stiff

in the radial direction for efficient transmission of force to

and from the OHC stereocilia (de Boer, 1993; Goodyear and

Richardson, 2002; Gavara and Chadwick, 2009) and, indeed,

that TM and stereocilia appear to be compliance matched

(Shoelson et al., 2004).

As, in this low-frequency range, TM amplitude monot-

onically decreases, but phase remains constant, with radial

position toward the limbus (Sec. III B 3), the TM appears to

rotate as a stiff element about some point in the limbal zone.

Preliminary evidence for rotation about the limbal zone has

also been reported by Karavitaki and Mountain (2007b).

Clearly, an amplitude decrease could also be caused by

increasing TM material stiffness toward the limbus, a gradi-

ent that has been proposed based on the larger collagen fibril

density in the limbal zone (Weaver and Schweitzer, 1994;

Vater and Kossl, 1996), and found experimentally using

atomic force microscopy (Shoelson et al., 2004; Gueta et al.,2006) and osmotic stress (Masaki et al., 2006). However, the

stiffness gradient across the entire TM is only a factor of

somewhere between 1.2 and 2 (Shoelson et al., 2004; Gueta

et al., 2006; Masaki et al., 2006), depending on the measure-

ment technique, and the present vibration measurements

were made lateral to the limbal zone [Fig. 1(B)]. Moreover,

being a viscoelastic material (Abnet and Freeman, 2000; Gu

et al., 2008), for a significant stiffness effect one would also

expect a phase change with radial position. As the phases

measured in the present experiments are independent of ra-

dial position, one can rule out material anisotropy as the

main reason for the reduced amplitude with radial position.

Rotation of the TM as a stiff element (for frequencies up to

3 kHz) supports the classical view of TM motion (Davis,

1958; Rhode and Geisler, 1967). Clearly, it does not support

the two-degrees of freedom model of de Boer (1993), in

which the TM is composed of two stiff elements connected

by a flexible hinge over the apex of the PCs.

At frequencies above 3 kHz, the TM over the IHC

exhibits an additional phase roll-off relative to its motion

over the OHCs [Fig. 9(D)]; the phase difference accumulates

to �180� at high frequencies. This relative phase response is



presumably due to inertial viscoelastic impedance of the TM

(Allen, 1980) between its driving point at the OHC stereoci-

lia and its termination at the limbus.

E. Motion of the TM relative to the RL at the IHC

The counterphasic motion between the TM and the RL

at the IHC for stimulus frequencies below �3 kHz is found

in all experiments independent of cochlear turn. This obser-

vation was originally reported in Nowotny and Gummer

(2006) and as explained in that article is due to: (1) The TM

moving in phase along its lower surface, rotating like a stiff

element about a point in the limbal zone (Sec. IV D), and (2)

the IHC moving in opposite phase to the OHCs, the RL

rotating like a stiff element about the apex of the PCs

(Sec. IV B). The finding that the ratios of IHC amplitude to

OHC amplitude are approximately equal on either side of

the subtectorial space (difference< 10 dB) suggests that,

apart from considerations of geometric lever ratios, the trans-

verse stiffness of the TM and RL are of the same order. As

the TM and RL are firmly coupled at the OHCs through the

(tallest) stereocilia (Takasaka et al., 1983), rotation of these

two (relatively) stiff elements causes the TM and RL at the

IHC to move apart for OHC elongation, and in apposition

for OHC contraction. In other words, OHC electromotility

causes cycle-by-cycle extension and squeezing of the subtec-

torial space in the region of the IHC.

Using an hydrodynamical analysis of small-amplitude

fluid motion between two closely separated elastic plates

(Hassan and Nagy, 1997), it has been shown theoretically

(Nowotny and Gummer, 2006)3 that this counterphasic trans-

verse motion of the RL and TM is capable of producing radial

fluid motion in the subtectorial space, which in turn cannot

only deflect IHC stereocilia but also amplify their motion rel-

ative to OHC motion. In other words, this mechanism presents

a means for directly coupling OHC electromechanical force

to the IHC stereocilia, without involving the BM. This mecha-

nism could be loosely described as a second cochlear-

amplifier mechanism. Importantly, this mechanism is active

at all functionally relevant frequencies in the second and third

turns and on the low-frequency tail of neural responses in the

first turn. Thus, in contrast to the conventional—first—coch-

lear amplifier, it is not tuned to the CF place. Coupling OHC

electromechanical force directly to the IHC stereocilia, this

mechanism readily explains the observation of Guinan et al.(2005), based on electrical stimulation of the medial olivoco-

chlear efferents, that there appears to exist a mechanism for

directly deflecting IHC stereocilia, which is independent of

the classical BM traveling-wave mechanism.

Independent of our work, using an elastic shell model for

the organ of Corti, Steele and Puria (2005) actually predicted

modulation of the width of the subtectorial space, whereas

based on lubrication theory and the large viscous forces

involved, Chadwick et al. (1996) found no theoretical evi-

dence for such an effect. The model of Steele and Puria

(2005) requires the TM to be sufficiently stiff so that pressure

changes within the inner sulcus cannot be accommodated by

transversal vibration of the overlying TM. The data in Fig.

9(C) support their assumption, there being a progressive

attenuation of TM amplitude with distance into the inner sul-

cus, which at low frequencies amounts to �12 dB/20 lm

from a point over the IHC.

Here, it must be emphasized that counterphasic motion

between TM and RL at the IHC is not expected to be found

for acoustical or hydrodynamical stimulation in those

in vitro preparations for which the somatic electromechani-

cal force was relatively insignificant because the change of

the transmembrane potential was too small. Thus, for electri-

cal stimulation the change of transmembrane potential is

produced artificially by the applied electric field, whereas for

acoustical or hydrodynamical stimulation a change of trans-

membrane potential requires bidirectional transduction; i.e.,

in addition to somatic electromechanical force, it also

requires functional mechanoelectrical transduction, a non-

zero resting membrane potential and, possibly, an endoco-

chlear potential.

With increasing frequency above 3 kHz in all turns,

the additional 180� phase roll-off of the TM over the IHC

(Sec. IV D) means that at above 3 kHz the counterphasic

motion of the TM and RL surfaces at the IHC is gradually lost,

so that at high frequencies the TM and RL eventually move in

phase. In addition to inertial viscoelastic impedance in the TM

(Allen, 1980) being responsible for the high-frequency phase

roll-off (Sec. IV D), there is also the possibility of a contribu-

tion from the fluid of the subtectorial space. Thus, this fluid

mode is expected to vanish if slip at the TM and RL becomes

excessively large at high frequencies, as found by Lloyd and

Redwood (1965) for the case of a narrow fluid layer between

elastic plates. This interpretation concurs, of course, with the

customary notion that TM and RL cannot move independently

at high frequencies because of viscous coupling between their

surfaces (de Boer, 1993; Zwislocki, 2002).

F. Motion of Hensen’s cell area

The finding of low-frequency counterphasic (approxi-

mately equal amplitude) motion between the OHC RL and

the middle region of the HA (Figs. 4, 5, and 8) supports a sug-

gestion by de Boer (1993) that the HA should be relatively

“flexible” to allow fluid motion in and around the OHC

region—otherwise, the fluid regions of the organ of Corti

would act as narrow channels, mechanically impeding even

OHC motion. Indeed, point impedance measurements of the

upper surface of the organ of Corti have shown that the HA is

much less stiff than the PC, namely, a factor of 12–14 in the

first and second turns and a factor of 36 in the third turn

(Gummer and Scherer, 2004a). Robust longitudinal fluid

motion in the tunnel of Corti in response to OHC electromo-

tility has been demonstrated in the gerbil cochlea, the space

constant of the longitudinal extent being as much as 1.3 mm

at CF in the 4 kHz cochlear region (Karavitaki and Mountain,

2007a). The counterphasic motion between OHCs and HA

observed in the present experiments, and between OHC1, 2

and HA by Karavitaki and Mountain (2007b), suggests that

OHC contraction causes fluid flow from the spaces of Nuel

not only into the tunnel of Corti (Karavitaki and Mountain,

2007a), but also into the outer tunnel,4 formed by the lateral

surface of OHC3 and the medial surface of the HA



[Fig. 1(A)].5 That is, it appears that the fluid pressure could be

sufficiently large and the HA sufficiently flexible that electro-

mechanically induced OHC contraction causes counterphasic

motion of the middle of the HA toward Scala vestibuli. This

type of motion is predicted by the three-fluid-compartment

model of de Boer (1991), as well as by the elastic shell model

for the organ of Corti by Steele and Puria (2005). Clearly, the

amplitudes and phases of the HA relative to the RL depend

on the relative impedances of Deiter’s cells, BM, OHCs, PCs,

and HA. A similar conclusion was reached by Hu et al.(1999) based on hydrodynamical stimulation in a hemico-

chlear preparation.

Here it should be added that such counterphasic motion

is not found, at least in the third and fourth turns, for acousti-cal stimulation in temporal-bone preparations (Hemmert

et al., 2000a,b), where electromechanical action of the

OHCs was practically absent because of loss of the positive

endocochlear potential post mortem (Bosher, 1979). In that

case, the upper surface of the organ of Corti—including the

HA—rotated as a stiff element for stimulus frequencies up

to approximately one octave above in vivo CF. Under in vivoconditions in the healthy cochlea, where the OHCs are elec-

tromechanically functioning, it is expected that the motion

of the HA is a superposition of the two types of vibration

response.

The situation changes at frequencies above �3 kHz,

mainly in the third turn [Figs. 8(A) and 8(B)], with the HA

no longer following the OHCs, but presenting larger ampli-

tude and phase slopes than those of the OHC responses. Con-

sistent with this result, decoupling of the Hensen’s cell

vibration response from the rest of the organ of Corti has

been reported in an hemicochlear preparation for intraco-

chlear hydrodynamical stimulation for frequencies “close

and above the best frequency of the location” (Richter and

Dallos, 2003). It is tempting to speculate that the additional

180� phase roll-off found for most (82%) OHC3 recordings

in the third turn (Sec. IV C) is probably associated with these

HA responses, the OHC3 being more strongly coupled to the

HA than to OHC2 at these high frequencies.

At frequencies well above in vivo CF, the finding that the

phase roll-off increases with radial position toward Stria vas-cularis is reminiscent of a traveling wave propagating in that

direction. This type of motion, found several octaves above

in vivo CF, is most obvious in the third turn [Fig. 8(B)]. The

HA in the third turn has several salient anatomical features

that might be responsible for its unique vibration pattern: (1)

Hensen’s cells increase in size and contain increasing

amounts of lipid inclusions toward the cochlear apex (Lim,

1980; Santi, 1988); (2) Hensen’s cells in the high-frequency

region of the cochlea rest on Boettcher cells, whereas in the

low-frequency region they rest on the BM (Roth and Bruns,

1992a; Spicer and Schulte, 1994a,b); and (3) the lumen of the

outer tunnel is at least 2� as wide and 2� as high in the third

turn compared with the first and second turns.6 Thus, having a

cross-sectional area at least 4� larger than in the first and sec-

ond turns, the outer tunnel in the third turn should present less

resistance to fluid flow. Moreover, point impedance measure-

ments (Scherer and Gummer, 2004a) indicate that the HA is

�3� more flexible in the third turn than in the first and sec-

ond turns. Thus, the smaller stiffness of Hensen’s cells in the

apical region is indicative of traveling-wave motion of shorter

wavelength than in the basal region. For example, for the

third-turn recording illustrated in Fig. 8(B), at 5.68 kHz one

observes 0.53 cycles of the traveling wave on the 60 lm

region from HA1 to HA4. Wavelength is approximately pro-

portional to stiffness for the dispersive traveling-wave motion

(Currie, 1974) suggested by the phase response in Fig. 8(B).

Therefore, if the stiffness were to increase by a factor of 3,

then only 0.18 cycles would fit onto this 60 lm region. If

much smaller than this value, the HA would appear to move

in phase across its width; i.e., traveling-wave motion would

not be detected. Finally, it should be emphasized that as

traveling-wave motion is found well above in vivo CF, the

present authors do not attach any functional significance to it.

G. Condition of the preparation with main focus onthe TM

Two important precautions were taken to reduce the rate

of mixing of endolymph with the perilymph-like bathing me-

dium and, therefore, to prolong the viability of the prepara-

tion: (1) Reissner’s membrane was left intact and (2) the

longest possible section of a cochlear turn was dissected

(�1/2). Ultimately, mixing cannot be prevented because the

ends of Scala media were open—both ends in the second

and third turns, and the distal end in the first turn (Sec. II A).

It was particularly important that the amount of mixing be

reduced as much as possible because the TM is extremely

sensitive to changes in Ca2þ, Kþ, or Naþ concentration

(Kronester-Frei, 1979; Shah et al., 1995; Edge et al., 1998)

and pH value (Kronester-Frei, 1979; Freeman et al., 1993;

Freeman et al., 2003). Therefore, it was imperative to moni-

tor the condition of the preparation during the course of the

vibration experiments. Using light microscopy with total

magnification of 400, the tips of the OHC stereocilia were

regularly examined to ascertain that they remained in the

same focal plane as the lower surface of the TM; this pro-

vides confidence that the TM did not lift from the OHC ster-

eocilia. Moreover, using the marginal edge of the TM as a

landmark, regular checks were made to ensure that the TM

had not retracted radially. Finally, the superposition of the

phase responses of the TM and RL at the OHCs [Fig. 10(B)]

provides the strongest evidence for the (tallest) stereocilia of

the OHCs being firmly connected to the TM.

Apart from checking that the RL remained in the same

focal plane along its entire length, it was hardly possible to

monitor TM conditions in the region of the IHC using light

microscopy. Therefore, in a separate series of experiments

(Sec. III C), this region was examined using confocal laser

scanning microscopy of fluorescent stained organ of Corti

and TM. The protocol was similar to that of Ulfendahl et al.(2001). In all preparations, up to at least 90 min after opening

the cochlea, HS was observed near the IHC stereocilia and

trabeculae were found projecting orthogonally from HS to

the head of the inner phalangeal cell (Fig. 11). These struc-

tures are well-described elsewhere (e.g., Lim and Lane,

1969; Lim, 1972). There is still no definitive evidence as to

whether the IHC stereocilia are attached to these structures.



Although there is some preliminary evidence from an

in vitro preparation that the HS might overlie the IHC stereo-

cilia (Edge et al., 1998), the present morphological data does

not support this observation, but instead concur with obser-

vations by Kronester-Frei (1978), Ulfendahl et al. (2001),

and Fridberger et al. (2006).

In conclusion, phase responses at and above the OHCs,

light-microscopic monitoring of the in vitro preparation, to-

gether with the results of confocal laser scanning microscopy

of stained organ of Corti and TM, suggest that the condition

of the organ of Corti and TM was not unduly compromised

by the in vitro conditions of the vibration experiments.

H. Hensen’s stripe and trabeculae

It has been proposed that HS might be involved in

deflection of IHC stereocilia (Crane, 1983; Steele and Puria,

2005). However, recently it has been shown that HS is not

essential at high CFs (20 kHz in mouse)—this structure is

absent in b-tectorin mutant mice and yet there is an increase

of cochlear tuning without significant loss of sensitivity

(Russell et al., 2007).7 Nevertheless, it might be required at

frequencies on the low-frequency tail of the neural tuning

curve (Russell et al., 2007). Moreover, as originally pointed

out by Nowotny and Gummer (2006), HS might be involved

in deflection of IHC stereocilia by means of counterphasic,

transverse motion of the TM and RL at the IHC, found up to

�3 kHz in each cochlear turn (Sec. IV E). Further, if the

present fluorescent images (Fig. 11) provide an accurate in-

dication of morphology during the vibration experiments,

then the counterphasic motion exists in the presence of the

trabeculae. As the low-frequency (<3 kHz) amplitudes and

phases of this motion can be explained by the geometry of

the organ of Corti and TM, with the RL and lower surface of

the TM acting as stiff elements, it is tempting to speculate

that the trabeculae are very elastic, as was also concluded by

Orman and Geisler (1986), and that they contribute little to

the measured transversal vibration responses.

V. CONCLUSION

Using intracochlear electrical stimulation in an in vitropreparation, the transverse velocity responses of the apical

surface of the organ of Corti and the upper and lower surfa-

ces of the TM to somatic electromechanical force from the

OHCs is described. Salient features include (1) relatively

wide-band responses, (2) equal amplitude and phase

responses of TM and RL at the OHCs, (3) up to �3 kHz,

counterphasic motion between TM and RL at the IHC, (4)

up to �3 kHz, on average, counterphasic, equal amplitude

motion between the OHC RL and middle region of the

HeCs, (5) radial traveling-wave motion on the HeCs, mainly

in the third turn several octaves above in vivo CF, (6) up to

�3 kHz, in-phase motion of the TM from the OHC region to

the ISC region, (7) in-phase motion of the upper surface of

the organ of Corti from the IHC to the ISCs, (8) up to

�3 kHz, IHC amplitude �10 dB smaller than OHC ampli-

tude, on both the RL and overlying TM, and (9) no evidence

of radial traveling-wave motion of the TM. The data are

explained in terms of geometry, morphology, and material

properties of the organ of Corti and TM, and elucidate some

underlying mechanical principles involved in the response of

these structures to somatic electromechanical force from the

OHCs. When the technology is available, the next step will

be to conduct such experiments in vivo, enabling electrome-

chanical feedback to be studied in the presence of fully func-

tional homeostatic systems.

ACKNOWLEDGMENTS

For helpful discussion and suggestions we thank Dr. C.

Chiaradia, Dr. E. Dalhoff, Dr. C. Harasztosi, Dr. M. P.

Scherer, and Dr. A. Vetesnık, and for technical assistance A.

Seeger and K. Vollmer. We are also grateful for the collegial

comments and suggestions of the two reviewers. This work

was supported by the Deutsche Forschungsgemeinschaft

(Grant No. DFG Gu 194/5-1, 2).

1The head plate of the outer pillar cell lies below the head plate of the inner

pillar cell and, therefore, is not accessible to the laser beam for vibration

measurements.2For a minimum phase system, an asymptotic high-frequency phase roll-off

of 90� is associated with each 6 dB/octave asymptotic high-frequency am-

plitude roll-off (Bode, 1945).3See supplementary material at http://www.pnas.org/content/suppl/2006/

02/06/0511125103.DC1/11125SuppText.pdf, entitled “Estimation of IHC

stereocilia deflection for counterphasic motion of the TM and RL.”4The outer tunnel is often referred to as the fourth space of Nuel (e.g., Roth

and Bruns, 1992b).5Here it should be noted that Karavitaki and Mountain (2007a) used motion

of the medial olivocochlear fibers crossing the tunnel of Corti to calculate,

indirectly, fluid motion in that region, and therefore, their experimental

protocol would not have allowed detection of fluid motion in the outer

tunnel.6According to dimensional measurements of paraformaldehyde-fixed mate-

rial (4%, pH 7.4), the outer tunnel in the first, second, and third cochlear

turns has widths of 10, 12, and 21 lm and heights of 21, 22, and 43 lm,

respectively, all of which are uncorrected for shrinkage (Y. Yarin, perso-

nal communication).7 In the meantime, there is strong experimental (Ghaffari et al., 2010) and

theoretical (Meaud and Grosh, 2010) evidence for the enhanced tuning in

these mutants being due to reduction of longitudinal coupling of OHC ster-

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Vibration responses of the organ of Corti and the tectorial membrane to electrical stimulation

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