Localization of the cochlear amplifier in living sensitive ears

Background

To detect soft sounds, the mammalian cochlea increases its sensitivity by amplifying incoming sounds up to one thousand times. Although the cochlear amplifier is thought to be a local cellular process at an area basal to the response peak on the spiral basilar membrane, its location has not been demonstrated experimentally.

Methodology and Principal Findings

Using a sensitive laser interferometer to measure sub-nanometer vibrations at two locations along the basilar membrane in sensitive gerbil cochleae, here we show that the cochlea can boost soft sound-induced vibrations as much as 50 dB/mm at an area proximal to the response peak on the basilar membrane. The observed amplification works maximally at low sound levels and at frequencies immediately below the peak-response frequency of the measured apical location. The amplification decreases more than 65 dB/mm as sound levels increases.

Conclusions and Significance

We conclude that the cochlea amplifier resides at a small longitudinal region basal to the response peak in the sensitive cochlea. These data provides critical information for advancing our knowledge on cochlear mechanisms responsible for the remarkable hearing sensitivity, frequency selectivity and dynamic range.     

Waves on Reissner's membrane: a mechanism for the propagation of otoacoustic emissions from the cochlea

Sound is detected and converted into electrical signals within the ear. The cochlea not only acts as a passive detector of sound, however, but can also produce tones itself. These otoacoustic emissions are a striking manifestation of the cochlea's mechanical active process. A controversy remains of how these mechanical signals propagate back to the middle ear, from which they are emitted as sound. Here, we combine theoretical and experimental studies to show that mechanical signals can be transmitted by waves on Reissner's membrane, an elastic structure within the cochlea. We develop a theory for wave propagation on Reissner's membrane and its role in otoacoustic emissions. Employing a scanning laser interferometer, we measure traveling waves on Reissner's membrane in the gerbil, guinea pig, and chinchilla. The results are in accord with the theory and thus support a role for Reissner's membrane in otoacoustic emissions.     

Manipulation of the Endocochlear Potential Reveals Two Distinct Types of Cochlear Nonlinearity

The mammalian hearing organ, the cochlea, contains an active amplifier to boost the vibrational response to low level sounds. Hallmarks of this active process are sharp location-dependent frequency tuning and compressive nonlinearity over a wide stimulus range. The amplifier relies on outer hair cell (OHC)-generated forces driven in part by the endocochlear potential, the ∼+80 mV potential maintained in scala media, generated by the stria vascularis. We transiently eliminated the endocochlear potential in vivo by an intravenous injection of furosemide and measured the vibrations of different layers in the cochlea’s organ of Corti using optical coherence tomography. Distortion product otoacoustic emissions were also monitored. After furosemide injection, the vibrations of the basilar membrane lost the best frequency (BF) peak and showed broad tuning similar to a passive cochlea. The intra-organ of Corti vibrations measured in the region of the OHCs lost the BF peak and showed low-pass responses but retained nonlinearity. This strongly suggests that OHC electromotility was operating and being driven by nonlinear OHC current. Thus, although electromotility is presumably necessary to produce a healthy BF peak, the mere presence of electromotility is not sufficient. The BF peak recovered nearly fully within 2 h, along with the recovery of odd-order distortion product otoacoustic emissions. The recovery pattern suggests that physical shifts in operating condition are a critical step in the recovery process.     

Power amplification in the mammalian cochlea

It was first suggested by Gold in 1948 [1] that the exquisite sensitivity and frequency selectivity of the mammalian cochlea is due to an active process referred to as the cochlear amplifier. It is thought that this process works by pumping energy to augment the otherwise damped sound-induced vibrations of the basilar membrane [2-4], a mechanism known as negative damping. The existence of the cochlear amplifier has been inferred from comparing responses of sensitive and compromised cochleae [5] and observations of acoustic emissions [6, 7] and through mathematical modeling [8, 9]. However, power amplification has yet to be demonstrated directly. Here, we prove that energy is indeed produced in the cochlea on a cycle-by-cycle basis. By using laser interferometry [10], we show that the nonlinear component of basilar-membrane responses to sound stimulation leads the forces acting on the membrane. This is possible only in active systems with negative damping [11]. Our finding provides the first direct evidence for power amplification in the mammalian cochlea. The finding also makes redundant current hypotheses of cochlear frequency sharpening and sensitization that are not based on negative damping.     

The Spatial Origins of Cochlear Amplification Assessed by Stimulus-Frequency Otoacoustic Emissions

Cochlear amplification of basilar membrane traveling waves is thought to occur between a tone’s characteristic frequency (CF) place and within one octave basal of the CF. Evidence for this view comes only from the cochlear base. Stimulus-frequency otoacoustic emissions (SFOAEs) provide a noninvasive alternative to direct measurements of cochlear motion that can be measured across a wide range of CF regions. Coherent reflection theory indicates that SFOAEs arise mostly from the peak region of the traveling wave, but several studies using far-basal suppressor tones claimed that SFOAE components originate many octaves basal of CF. We measured SFOAEs while perfusing guinea pig cochleas from apex to base with salicylate or KCl solutions that reduced outer-hair-cell function and SFOAE amplification. Solution effects on inner hair cells reduced auditory nerve compound action potentials (CAPs) and provided reference times for when solutions reached the SFOAE-frequency CF region. As solution flowed from apex to base, SFOAE reductions generally occurred later than CAP reductions and showed that the effects of cochlear amplification usually peaked ∼1/2 octave basal of the CF region. For tones ≥2 kHz, cochlear amplification typically extended ∼1.5 octaves basal of CF, and the data are consistent with coherent reflection theory. SFOAE amplification did not extend to the basal end of the cochlea, even though reticular lamina motion is amplified in this region, which indicates that reticular lamina motion is not directly coupled to basilar membrane traveling waves. Previous reports of SFOAE-frequency residuals produced by suppressor frequencies far above the SFOAE frequency are most likely due to additional sources created by the suppressor. For some tones <2 kHz, SFOAE amplification extended two octaves apical of CF, which highlights that different vibratory motions produce SFOAEs and CAPs, and that the amplification region depends on the cochlear mode of motion considered. The concept that there is a single “cochlear amplification region” needs to be revised.     

Using Drosophila for studying fundamental processes in hearing

Apart from detecting sounds, vertebrate ears occasionally produce sounds. These spontaneous otoacoustic emissions are the most compelling evidence for the existence of the cochlear amplifier, an active force-generating process within the cochlea that resides in the motility of the hair cells. Insects have neither a cochlea nor hair cells, yet recent studies demonstrate that an active process that is equivalent to the cochlear amplifier occurs in at least some insect ears; like hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila actively generate forces that augment the minute vibrations they transduce. This neuron-based force-generation, its impact on the ear's macroscopic performance, and the underlying molecular mechanism are the topics of this article, which summarizes some of the recent findings on how the Drosophila organ of hearing works. Functional parallels with vertebrate auditory systems are described that recommend the fly for the study of fundamental processes in hearing.     

The group delay and suppression pattern of the cochlear microphonic potential recorded at the round window

Background

It is commonly assumed that the cochlear microphonic potential (CM) recorded from the round window (RW) is generated at the cochlear base. Based on this assumption, the low-frequency RW CM has been measured for evaluating the integrity of mechanoelectrical transduction of outer hair cells at the cochlear base and for studying sound propagation inside the cochlea. However, the group delay and the origin of the low-frequency RW CM have not been demonstrated experimentally.

Methodology/Principal Findings

This study quantified the intra-cochlear group delay of the RW CM by measuring RW CM and vibrations at the stapes and basilar membrane in gerbils. At low sound levels, the RW CM showed a significant group delay and a nonlinear growth at frequencies below 2 kHz. However, at high sound levels or at frequencies above 2 kHz, the RW CM magnitude increased proportionally with sound pressure, and the CM phase in respect to the stapes showed no significant group delay. After the local application of tetrodotoxin the RW CM below 2 kHz became linear and showed a negligible group delay. In contrast to RW CM phase, the BM vibration measured at location ∼2.5 mm from the base showed high sensitivity, sharp tuning, and nonlinearity with a frequency-dependent group delay. At low or intermediate sound levels, low-frequency RW CMs were suppressed by an additional tone near the probe-tone frequency while, at high sound levels, they were partially suppressed only at high frequencies.

Conclusions/Significance

We conclude that the group delay of the RW CM provides no temporal information on the wave propagation inside the cochlea, and that significant group delay of low-frequency CMs results from the auditory nerve neurophonic potential. Suppression data demonstrate that the generation site of the low-frequency RW CM shifts from apex to base as the probe-tone level increases.     

In Vivo Impedance of the Gerbil Cochlear Partition at Auditory Frequencies

The specific acoustic impedance of the cochlear partition was measured from 4 to 20 kHz in the basal turn of the gerbil cochlea, where the best frequency is ∼40 kHz. The acoustic impedance was found as the ratio of driving pressure to velocity response. It is the physical attribute that governs cochlear mechanics and has never before been directly measured, to our knowledge. The basilar membrane velocity was measured through the transparent round window membrane. Simultaneously, the intracochlear pressure was measured close to the stapes and quite close to the cochlear partition. The impedance phase was close to −90° and the magnitude decreased with frequency, consistent with stiffness-dominated impedance. The resistive component of the impedance was relatively small. Usually the resistance was negative at frequencies below 8 kHz; this unexpected finding might be due to other vibration modes within the cochlear partition.     

The cochlear frequency map of the mustache bat,Pteronotus parnellii

The frequency-place map of the cochlea of mustache bats was constructed by the analysis of HRP-transport patterns in spiral ganglion cells following iontophoretic tracer injections into cochlear nucleus regions responsive to different frequencies. The cochlea consists of 5 half turns (total length 14.3 mm) and the representation of certain frequency bands can be assigned to specific cochlear regions: The broad high frequency range between 70 and 111 kHz is represented in the most basal half turn within only 3.2 mm. This region is terminated apically by a distinct narrowing of the scala vestibuli that coincides with a pronounced increase in basilar membrane (BM) thickness. The narrow intermediate frequency range between 54 and 70 kHz is expanded onto 50% of cochlear length between 4.0 and 11.1 mm distance from apex. The frequency range around 60 kHz, where the tuning characteristics of the auditory system are exceptionally sharp, is located in the center of this expanded BM-region in the second half turn within a maximum of innervation density. These data can account for the vast overrepresentation of neurons sharply tuned to about 60 kHz at central stations of the auditory pathway. In the cochlear region just basal to the innervation maximum, where label from injections at 66 and 70 kHz was found, a number of morphological specializations coincide: the BM is maximally thickened, innervation density is low, the spiral ligament is locally enlarged, and the 'thick lining', a dense covering of the scala tympani throughout the basal halfturn, suddenly disappears. Low frequencies up to 54 kHz are represented within the apical half turns over a 4 mm span of the basilar membrane. The data are compared to the cochlea of horseshoe bats and the possible functional role of the morphological discontinuities for sharp tuning and the generation of otoacoustic emissions is discussed.     

Consequences of Location-Dependent Organ of Corti Micro-Mechanics

The cochlea performs frequency analysis and amplification of sounds. The graded stiffness of the basilar membrane along the cochlear length underlies the frequency-location relationship of the mammalian cochlea. The somatic motility of outer hair cell is central for cochlear amplification. Despite two to three orders of magnitude change in the basilar membrane stiffness, the force capacity of the outer hair cell’s somatic motility, is nearly invariant over the cochlear length. It is puzzling how actuators with a constant force capacity can operate under such a wide stiffness range. We hypothesize that the organ of Corti sets the mechanical conditions so that the outer hair cell’s somatic motility effectively interacts with the media of traveling waves—the basilar membrane and the tectorial membrane. To test this hypothesis, a computational model of the gerbil cochlea was developed that incorporates organ of Corti structural mechanics, cochlear fluid dynamics, and hair cell electro-physiology. The model simulations showed that the micro-mechanical responses of the organ of Corti are different along the cochlear length. For example, the top surface of the organ of Corti vibrated more than the bottom surface at the basal (high frequency) location, but the amplitude ratio was reversed at the apical (low frequency) location. Unlike the basilar membrane stiffness varying by a factor of 1700 along the cochlear length, the stiffness of the organ of Corti complex felt by the outer hair cell remained between 1.5 and 0.4 times the outer hair cell stiffness. The Y-shaped structure in the organ of Corti formed by outer hair cell, Deiters cell and its phalange was the primary determinant of the elastic reactance imposed on the outer hair cells. The stiffness and geometry of the Deiters cell and its phalange affected cochlear amplification differently depending on the location.     

An Active Oscillator Model Describes the Statistics of Spontaneous Otoacoustic Emissions

Even in the absence of external stimulation, the cochleas of most humans emit very faint sounds below the threshold of hearing, sounds that are known as spontaneous otoacoustic emissions. They are a signature of the active amplification mechanism in the cochlea. Emissions occur at frequencies that are unique for an individual and change little over time. The statistics of a population of ears exhibit characteristic features such as a preferred relative frequency distance between emissions (interemission intervals). We propose a simplified cochlea model comprising an array of active nonlinear oscillators coupled both hydrodynamically and viscoelastically. The oscillators are subject to a weak spatial disorder that lends individuality to the simulated cochlea. Our model captures basic statistical features of the emissions: distributions of 1), emission frequencies; 2), number of emissions per ear; and 3), interemission intervals. In addition, the model reproduces systematic changes of the interemission intervals with frequency. We show that the mechanism for the preferred interemission interval in our model is the occurrence of synchronized clusters of oscillators.     

An Active Oscillator Model Describes the Statistics of Spontaneous Otoacoustic Emissions

Even in the absence of external stimulation, the cochleas of most humans emit very faint sounds below the threshold of hearing, sounds that are known as spontaneous otoacoustic emissions. They are a signature of the active amplification mechanism in the cochlea. Emissions occur at frequencies that are unique for an individual and change little over time. The statistics of a population of ears exhibit characteristic features such as a preferred relative frequency distance between emissions (interemission intervals). We propose a simplified cochlea model comprising an array of active nonlinear oscillators coupled both hydrodynamically and viscoelastically. The oscillators are subject to a weak spatial disorder that lends individuality to the simulated cochlea. Our model captures basic statistical features of the emissions: distributions of 1), emission frequencies; 2), number of emissions per ear; and 3), interemission intervals. In addition, the model reproduces systematic changes of the interemission intervals with frequency. We show that the mechanism for the preferred interemission interval in our model is the occurrence of synchronized clusters of oscillators.     

Persistence of past stimulations: storing sounds within the inner ear

Tones cause vibrations within the hearing organ. Conventionally, these vibrations are thought to reflect the input and therefore end with the stimulus. However, previous recordings of otoacoustic emissions and cochlear microphonic potentials suggest that the organ of Corti does continue to move after the end of a tone. These after-vibrations are characterized here through recordings of basilar membrane motion and hair cell extracellular receptor potentials in living anesthetized guinea pigs. We show that after-vibrations depend on the level and frequency of the stimulus, as well as on the sensitivity of the ear. Even a minor loss of hearing sensitivity caused a sharp reduction in after-vibration amplitude and duration. Mathematical models suggest that after-vibrations are driven by energy added into organ of Corti motion after the end of an acoustic stimulus. The possible importance of after-vibrations for psychophysical phenomena such as forward masking and gap detection are discussed.     

Developmental gene expression profiling along the tonotopic axis of the mouse cochlea

The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. Tnc (tenacin C) and Nov (nephroblastoma overexpressed gene) are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, Fst (Follistatin), an antagonist of TGF-β/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of Fst is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy.     

Mechanical basis of otoacoustic emissions in tympanal hearing organs

Tympanal hearing organs of insects emit distortion–product otoacoustic emissions (DPOAEs), which in mammals are used as indicator for nonlinear cochlear amplification, and which are highly vulnerable to manipulations interfering with the animal’s physiological state. Although in previous studies, evidence was provided for the involvement of auditory mechanoreceptors, the source of DPOAE generation and possible active mechanisms in tympanal organs remained unknown. Using laser Doppler vibrometry in the locust ear, we show that DPOAEs mechanically emerge at the tympanum region where the auditory mechanoreceptors are attached. Those emission-coupled vibrations differed remarkably from tympanum waves evoked by external pure tones of the same frequency, in terms of wave propagation, energy distribution, and location of amplitude maxima. Selective inactivation of the auditory receptor cells by mechanical lesions did not affect the tympanum’s response to external pure tones, but abolished the emission’s displacement amplitude peak. These findings provide evidence that tympanal auditory receptors, comparable to the situation in mammals, comprise the required nonlinear response characteristics, which during two-tone stimulation lead to additional, highly localized deflections of the tympanum.     

Mechanical filtering of sound in the inner ear.

We have studied the distortion generated by the cochlea to gain insight into the mechanisms responsible for the sharp tuning or 'frequency selectivity' of the inner ear. We used two stimulating tones of moderate intensity which are progressively separated in frequency, and measured the ear canal cubic distortion components which are generated as a consequence of the stimulus interaction in the cochlea. We inferred that the distortion is generated from the frequency region of the higher of the two stimulus tones and that it is then band-pass filtered by a structure which is tuned to a frequency just over half an octave below that of the high-frequency tone. We suggest that the structure responsible for this band-pass filtering is the tectorial membrane, and we conclude that our results support theories of cochlear mechanics in which resonances due to the tectorial membrane interact with those of the basilar membrane to enhance the frequency selectivity of the inner ear.     

Ear morphology of the frog-eating bat (Trachops cirrhosus,family: Phyllostomidae): Apparent specializations for low-freqency hearing

The frog-eating bat (Trachops cirrhosus) is unusual among bats studied because of its reliance on low-frequency (<5 kHz) sounds emitted by frogs for prey localization. We investigated the ear of this bat in order to identify anatomical features that might serve as adaptations for low-frequency hearing. Trachops cirrhosus has a variety of anatomical features that might enhance low-frequency hearing, either by increasing sensitivity to low-frequency sounds or expanding the total frequency range to include lower frequencies. These bats have long pinnae, and a long and wide basilar membrane. The basal portion of the basilar membrane is much stiffer than the apical portion, and the basal portion of the tectorial membrane is more massive than the apical portion. There is also a concentration of mass in the apical portion of the cochlea. T. cirrhosus possesses the largest number of cochlear neurons reported for any mammal, the second highest density of cochlear neurons innervation known among mammals, and three peaks of cochlear neuron density. Other bats have two peaks of cochlear neuron density, lacking the apical concentration, while other mammals usually have only one. T. cirrhosus differs from most other small mammals and bats in characteristics of the apical portion of the cochlea, i.e., that area where the place theory of hearing predicts that low frequencies are detected.     

Basilar membrane responses to two-tone and broadband stimuli.

The responses to sound of mammalian cochlear neurons exhibit many nonlinearities, some of which (such as two-tone rate suppression and intermodulation distortion) are highly frequency specific, being strongly tuned to the characteristic frequency (CF) of the neuron. With the goal of establishing the cochlear origin of these auditory-nerve nonlinearities, mechanical responses to clicks and to pairs of tones were studied in relatively healthy chinchilla cochleae at a basal site of the basilar membrane with CF of 8-10 kHz. Responses were also obtained in cochleae in which hair cell receptor potentials were reduced by systemic furosemide injection. Vibrations were recorded using either the M?ssbauer technique or laser Doppler-shift velocimetry. Responses to tone pairs contained intermodulation distortion products whose magnitudes as a function of stimulus frequency and intensity were comparable to those of distortion products in cochlear afferent responses. Responses to CF tones could be selectively suppressed by tones with frequency either higher or lower than CF; in most respects, mechanical two-tone suppression resembled rate suppression in cochlear afferents. Responses to clicks displayed a CF-specific compressive nonlinearity, similar to that present in responses to single tones, which could be profoundly and selectively reduced by furosemide. The present findings firmly support the hypothesis that all CF-specific nonlinearities present in the auditory nerve originate in analogous phenomena of basilar membrane vibration. However, because of their lability, it is almost certain that the mechanical nonlinearities themselves originate in outer hair cells.     

Prestin's role in cochlear frequency tuning and transmission of mechanical responses to neural excitation

The remarkable power amplifier [1] of the cochlea boosts low-level and compresses high-level vibrations of the basilar membrane (BM) [2]. By contributing maximally at the characteristic frequency (CF) of each point along its length, the amplifier ensures the exquisite sensitivity, narrow frequency tuning, and enormous dynamic range of the mammalian cochlea. The motor protein prestin in the outer hair cell (OHC) lateral membrane is a prime candidate for the cochlear power amplifier [3]. The other contender for this role is the ubiquitous calcium-mediated motility of the hair cell stereocilia, which has been demonstrated in vitro and is based on fast adaptation of the mechanoelectrical transduction channels [4, 5]. Absence of prestin [6] from OHCs results in a 40-60 dB reduction in cochlear neural sensitivity [7]. Here we show that sound-evoked BM vibrations in the high-frequency region of prestin(-/-) mice cochleae are, surprisingly, as sensitive as those of their prestin(+/+) siblings. The BM vibrations of prestin(-/-) mice are, however, broadly tuned to a frequency approximately a half octave below the CF of prestin(+/+) mice at similar BM locations. The peak sensitivity of prestin(+/+) BM tuning curves matches the neural thresholds. In contrast, prestin(-/-) BM tuning curves at their best frequency are >50 dB more sensitive than the neural responses. We propose that the absence of prestin from OHCs, and consequent reduction in stiffness of the cochlea partition, changes the passive impedance of the BM at high frequencies, including the CF. We conclude that prestin influences the cochlear partition's dynamic properties that permit transmission of its vibrations into neural excitation. Prestin is crucial for defining sharp and sensitive cochlear frequency tuning by reducing the sensitivity of the low-frequency tail of the tuning curve, although this necessitates a cochlear amplifier to determine the narrowly tuned tip.