Cochlear outer hair cell electromotility enhances organ of Corti motion on a cycle-by-cycle basis at high frequencies in vivo.

Mammalian hearing depends on an amplification process involving prestin, a voltage-sensitive motor protein that enables cochlear outer hair cells (OHCs) to change length and generate force. However, it has been questioned whether this prestin-based somatic electromotility can operate fast enough in vivo to amplify cochlear vibrations at the high frequencies that mammals hear. In this study, we measured sound-evoked vibrations from within the living mouse cochlea and found that the top and bottom of the OHCs move in opposite directions at frequencies exceeding 20 kHz, consistent with fast somatic length changes. These motions are physiologically vulnerable, depend on prestin, and dominate the cochlea's vibratory response to high-frequency sound. This dominance was observed despite mechanisms that clearly low-pass filter the in vivo electromotile response. Low-pass filtering therefore does not critically limit the OHC's ability to move the organ of Corti on a cycle-by-cycle basis. Our data argue that electromotility serves as the primary high-frequency amplifying mechanism within the mammalian cochlea.

Amplification and Suppression of Traveling Waves along the Mouse Organ of Corti: Evidence for Spatial Variation in the Longitudinal Coupling of Outer Hair Cell-Generated Forces.

Mammalian hearing sensitivity and frequency selectivity depend on a mechanical amplification process mediated by outer hair cells (OHCs). OHCs are situated within the organ of Corti atop the basilar membrane (BM), which supports sound-evoked traveling waves. It is well established that OHCs generate force to selectively amplify BM traveling waves where they peak, and that amplification accumulates from one location to the next over this narrow cochlear region. However, recent measurements demonstrate that traveling waves along the apical surface of the organ of Corti, the reticular lamina (RL), are amplified over a much broader region. Whether OHC forces accumulate along the length of the RL traveling wave to provide a form of "global" cochlear amplification is unclear. Here we examined the spatial accumulation of RL amplification. In mice of either sex, we used tones to suppress amplification from different cochlear regions and examined the effect on RL vibrations near and far from the traveling-wave peak. We found that although OHC forces amplify the entire RL traveling wave, amplification only accumulates near the peak, over the same region where BM motion is amplified. This contradicts the notion that RL motion is involved in a global amplification mechanism and reveals that the mechanical properties of the BM and organ of Corti tune how OHC forces accumulate spatially. Restricting the spatial buildup of amplification enhances frequency selectivity by sharpening the peaks of cochlear traveling waves and constrains the number of OHCs responsible for mechanical sensitivity at each location.SIGNIFICANCE STATEMENT Outer hair cells generate force to amplify traveling waves within the mammalian cochlea. This force generation is critical to the ability to detect and discriminate sounds. Nevertheless, how these forces couple to the motions of the surrounding structures and integrate along the cochlear length remains poorly understood. Here we demonstrate that outer hair cell-generated forces amplify traveling-wave motion on the organ of Corti throughout the wave's extent, but that these forces only accumulate longitudinally over a region near the wave's peak. The longitudinal coupling of outer hair cell-generated forces is therefore spatially tuned, likely by the mechanical properties of the basilar membrane and organ of Corti. Our findings provide new insight into the mechanical processes that underlie sensitive hearing.

Hair cell force generation does not amplify or tune vibrations within the chicken basilar papilla.

Frequency tuning within the auditory papilla of most non-mammalian species is electrical, deriving from ion-channel resonance within their sensory hair cells. In contrast, tuning within the mammalian cochlea is mechanical, stemming from active mechanisms within outer hair cells that amplify the basilar membrane travelling wave. Interestingly, hair cells in the avian basilar papilla demonstrate both electrical resonance and force-generation, making it unclear which mechanism creates sharp frequency tuning. Here, we measured sound-induced vibrations within the apical half of the chicken basilar papilla in vivo and found broadly-tuned travelling waves that were not amplified. However, distortion products were found in live but not dead chickens. These findings support the idea that avian hair cells do produce force, but that their effects on vibration are small and do not sharpen tuning. Therefore, frequency tuning within the apical avian basilar papilla is not mechanical, and likely derives from hair cell electrical resonance.

Minimal basilar membrane motion in low-frequency hearing.

Low-frequency hearing is critically important for speech and music perception, but no mechanical measurements have previously been available from inner ears with intact low-frequency parts. These regions of the cochlea may function in ways different from the extensively studied high-frequency regions, where the sensory outer hair cells produce force that greatly increases the sound-evoked vibrations of the basilar membrane. We used laser interferometry in vitro and optical coherence tomography in vivo to study the low-frequency part of the guinea pig cochlea, and found that sound stimulation caused motion of a minimal portion of the basilar membrane. Outside the region of peak movement, an exponential decline in motion amplitude occurred across the basilar membrane. The moving region had different dependence on stimulus frequency than the vibrations measured near the mechanosensitive stereocilia. This behavior differs substantially from the behavior found in the extensively studied high-frequency regions of the cochlea.

Optical Coherence Tomography to Measure Sound-Induced Motions Within the Mouse Organ of Corti In Vivo.

The measurement of mechanical vibrations within the living cochlea is critical to understanding the first nonlinear steps in auditory processing, hair cell stimulation, and cochlear amplification. However, it has proven to be a challenging endeavor. This chapter describes how optical coherence tomography (OCT) can be used to measure vibrations within the tissues of the organ of Corti. These experimental measurements can be performed within the unopened cochlea of living mice routinely and reliably.

Perspectives for the treatment of sensorineural hearing loss by cellular regeneration of the inner ear.

Sensorineural hearing loss is a caused by the loss of the cochlear hair cells with the consequent deafferentation of spiral ganglion neurons. Humans do not show endogenous cellular regeneration in the inner ear and there is no exogenous therapy that allows the replacement of the damaged hair cells. Currently, treatment is based on the use of hearing aids and cochlear implants that present different outcomes, some difficulties in auditory discrimination and a limited useful life. More advanced technology is hindered by the functional capacity of the remaining spiral ganglion neurons. The latest advances with stem cell therapy and cellular reprogramming have developed several possibilities to induce endogenous regeneration or stem cell transplantation to replace damaged inner ear hair cells and restore hearing function. With further knowledge of the cellular and molecular biology of the inner ear and its embryonic development, it will be possible to use induced stem cells as in vitro models of disease and as replacement cellular therapy. Investigation in this area is focused on generating cellular therapy with clinical use for the treatment of profound sensorineural hearing loss.

Synaptic calcium regulation in hair cells of the chicken basilar papilla.

Cholinergic inhibition of hair cells occurs by activation of calcium-dependent potassium channels. A near-membrane postsynaptic cistern has been proposed to serve as a store from which calcium is released to supplement influx through the ionotropic ACh receptor. However, the time and voltage dependence of acetylcholine (ACh)-evoked potassium currents reveal a more complex relationship between calcium entry and release from stores. The present work uses voltage steps to regulate calcium influx during the application of ACh to hair cells in the chicken basilar papilla. When calcium influx was terminated at positive membrane potential, the ACh-evoked potassium current decayed exponentially over ∼100 ms. However, at negative membrane potentials, this current exhibited a secondary rise in amplitude that could be eliminated by dihydropyridine block of the voltage-gated calcium channels of the hair cell. Calcium entering through voltage-gated channels may transit through the postsynaptic cistern, since ryanodine and sarcoendoplasmic reticulum calcium-ATPase blockers altered the time course and magnitude of this secondary, voltage-dependent contribution to ACh-evoked potassium current. Serial section electron microscopy showed that efferent and afferent synaptic structures are juxtaposed, supporting the possibility that voltage-gated influx at afferent ribbon synapses influences calcium homeostasis during long-lasting cholinergic inhibition. In contrast, spontaneous postsynaptic currents ("minis") resulting from stochastic efferent release of ACh were made briefer by ryanodine, supporting the hypothesis that the synaptic cistern serves primarily as a calcium barrier and sink during low-level synaptic activity. Hypolemmal cisterns such as that at the efferent synapse of the hair cell can play a dynamic role in segregating near-membrane calcium for short-term and long-term signaling.

Instrumentation for studies of cochlear mechanics: from von Békésy forward.

Georg von Békésy designed the instruments needed for his research. He also created physical models of the cochlea allowing him to manipulate the parameters (such as volume elasticity) that could be involved in controlling traveling waves. This review is about the specific devices that he used to study the motion of the basilar membrane thus allowing the analysis that lead to his Nobel Prize Award. The review moves forward in time mentioning the subsequent use of von Békésy's methods and later technologies important for motion studies of the organ of Corti. Some of the seminal findings and the controversies of cochlear mechanics are mentioned in relation to the technical developments.

In vivo outer hair cell length changes expose the active process in the cochlea.

BACKGROUND: Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification. METHODOLOGY/PRINCIPAL FINDINGS: Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea. CONCLUSIONS/SIGNIFICANCE: The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.

Expression analysis of prestin and selected transcription factors in newborn rats.

Transcription factors (TFs) have a central role to play in regulating gene expression. To analyze the co-expression patterns of selected TFs with the motor protein prestin of the outer hair cells, we applied an real-time PCR approach combining several kinds of information: (i) expression changes during postnatal development, (ii) expression changes by exposure of organotypic cultures of the organ of Corti to factors which significantly affect prestin expression [thyroid hormone (T4), retinoic acid (RA), butyric acid (BA), increased KCl concentration] and (iii) changes along the apical-basal gradient. We found that the mRNA levels of the TF Brn-3c (Pou4f3), a member of the POU family, are significantly associated with the regulation of prestin during postnatal development and in cultures supplemented with T4 (0.5 µM), BA (0.5-2.0 mM), and high KCl (50 mM) concentration. The mRNA level of the constitutively active TF C/ebpb (CCAAT/enhancer binding protein beta) correlates positively with the prestin expression during postnatal development and in cultures exposed to T4 and RA (50-100 µM). The mRNA levels of the calcium-dependent TF CaRF correlates significantly with the prestin expression in cultures exposed to T4 and high KCl concentration. The observed coexpression patterns may suggest that the TFs Brn-3c, C/ebpb, and Carf contribute to regulating the expression of prestin under the investigated conditions.

Neuronal circuits involved in the middle-ear acoustic reflex.

Human and animal studies have shown that certain aromatic solvents such as toluene can cause hearing loss and can exacerbate the effects of noise. The latter effects might be due to a modification of responses of motoneurons controlling the middle-ear acoustic reflex. In the present investigation, the audition of Long-Evans rats was evaluated by measuring cubic (2f1 - f2) distortion otoacoustic emissions (f1 = 8000 Hz; f2 = 9600 Hz; f1/f2 = 1.2) prior to, during, and after activation of the middle-ear acoustic reflex. A noise suppressor was used to modify the amplitude of the 2f1 - f2 distortion otoacoustic emissions. It was delivered either contralaterally (band noise centered at 4 kHz), or ipsilaterally (3.5 kHz sine wave) to test the role played by the central auditory nuclei. This audiometric approach was used to study the physiological efficiency of the middle-ear acoustic reflex during an injection of a bolus of Intralipid (as a vehicle) containing 58.4, 87.4, or 116.2mM toluene via the carotid artery. The results showed that toluene could either increase or decrease middle-ear acoustic reflex efficiency, depending on the toluene concentration and the ear receiving noise suppressor. A new neuronal circuit of the middle-ear acoustic reflex has been proposed to explain findings obtained in this investigation. Finally, the depressing action of toluene on the central auditory nuclei driving the middle-ear acoustic reflex might explain the synergistic effects of a co-exposure to noise and aromatic solvents.

Brightness-compensated 3-D optical flow algorithm for monitoring cochlear motion patterns.

A method for three-dimensional motion analysis designed for live cell imaging by fluorescence confocal microscopy is described. The approach is based on optical flow computation and takes into account brightness variations in the image scene that are not due to motion, such as photobleaching or fluorescence variations that may reflect changes in cellular physiology. The 3-D optical flow algorithm allowed almost perfect motion estimation on noise-free artificial sequences, and performed with a relative error of <10% on noisy images typical of real experiments. The method was applied to a series of 3-D confocal image stacks from an in vitro preparation of the guinea pig cochlea. The complex motions caused by slow pressure changes in the cochlear compartments were quantified. At the surface of the hearing organ, the largest motion component was the transverse one (normal to the surface), but significant radial and longitudinal displacements were also present. The outer hair cell displayed larger radial motion at their basolateral membrane than at their apical surface. These movements reflect mechanical interactions between different cellular structures, which may be important for communicating sound-evoked vibrations to the sensory cells. A better understanding of these interactions is important for testing realistic models of cochlear mechanics.

A digital heterodyne laser interferometer for studying cochlear mechanics.

Laser interferometry is the technique of choice for studying the smallest displacements of the hearing organ. For low intensity sound stimulation, these displacements may be below 1 nm. This cannot be reliably measured with other presently available techniques in an intact organ of Corti. In a heterodyne interferometer, light is projected against an object of study and motion of the target along the optical axis causes phase and frequency modulations of the back-reflected light. To recover object motion, the reflected light is made to interfere with a reference beam of artificially altered frequency, producing a beating signal. In conventional interferometers, this carrier signal is demodulated with analog electronics. In this paper, we describe a digital implementation of the technique, using direct carrier sampling. In order to obtain the necessary reference signal for demodulation we introduce an additional third light path. Together, this results in lower noise and reduces the cost of the system. Within the hearing organ, different structures may move in different directions. It is therefore necessary to precisely measure the angle of incidence of the laser light, and to precisely localize the anatomical structure where the measurement is performed. Therefore, the interferometer is integrated with a laser scanning confocal microscope that permits us to map crucial morphometric parameters in each experiment. We provide key construction parameters and a detailed performance characterization. We also show that the system accurately measures the diminutive vibrations present in the apical turn of the cochlea during low-level sound stimulation.

The role of organ of Corti mass in passive cochlear tuning.

The mechanism for passive cochlear tuning remains unsettled. Early models considered the organ of Corti complex (OCC) as a succession of spring-mass resonators. Later, traveling wave models showed that passive tuning could arise through the interaction of cochlear fluid mass and OCC stiffness without local resonators. However, including enough OCC mass to produce local resonance enhanced the tuning by slowing and thereby growing the traveling wave as it approached its resonant segment. To decide whether the OCC mass plays a role in tuning, the frequency variation of the wavenumber of the cochlear traveling wave was measured (in vivo, passive cochleae) and compared to theoretical predictions. The experimental wavenumber was found by taking the phase difference of basilar membrane motion between two longitudinally spaced locations and dividing by the distance between them. The theoretical wavenumber was a solution of the dispersion relation of a three-dimensional cochlear model with OCC mass and stiffness as the free parameters. The experimental data were only well fit by a model that included OCC mass. However, as the measurement position moved from a best-frequency place of 40 to 12 kHz, the role of mass was diminished. The notion of local resonance seems to only apply in the very high-frequency region of the cochlea.

In vivo imaging and low-coherence interferometry of organ of Corti vibration.

An optical coherence tomography (OCT) system is built to acquire in vivo both images and vibration measurements of the organ of Corti of the guinea pig. The organ of Corti is viewed through a approximately 300-microm-diam hole in the bony wall of the cochlea at the scala tympani of the first cochlear turn. In imaging mode, the image is acquired as reflectance R(x,z). In vibration mode, the basilar membrane (BM) or reticular lamina (RL) are selected by the investigator interactively from the R(x,z) image. Under software control, the system moves the scanning mirrors to bring the sensing volume of the measurement to the desired membrane location. In vivo images of the organ of Corti are presented, indicating reflectance signals from the BM, RL, tectorial membrane, and Reissner's membrane. The tunnel of Corti and the inner sulcus are also visible in the images. Vibrations of +/-2 and +/-22 nm are recorded in the BM in response to low and high sound levels at 14 kHz above a noise floor of 0.2 nm.

Rapid confocal imaging for measuring sound-induced motion of the hearing organ in the apical region.

We describe a novel confocal image acquisition system capable of measuring the sound-evoked motion of the organ of Corti. The hearing organ is imaged with a standard laser scanning confocal microscope during sound stimulation. The exact temporal relation between each image pixel and the sound stimulus is quantified. The motion of the structures under study is obtained by fitting a Fourier series to the time dimension of a continuous sequence of acquired images. Previous versions of this acquisition system used a simple search to find pixels with similar phase values. The Fourier series approach permits substantially faster image acquisition with reduced noise levels and improved motion estimation. The system is validated by imaging various vibrating samples attached to a feedback-controlled piezoelectric translator. When using a rigid sample attached to the translator, the system is capable of measuring motion with peak-to-peak amplitudes smaller than 50 nm with an error below 20% at frequencies between 50 and 600 Hz. Examples of image sequences from the inner ear are given, along with detailed performance characteristics of the method.

Cochlear traveling-wave amplification, suppression, and beamforming probed using noninvasive calibration of intracochlear distortion sources.

Originally developed to estimate the power gain of the cochlear amplifier, so-called "Allen-Fahey" and related experiments have proved invaluable for probing the mechanisms of wave generation and propagation within the cochlea. The experimental protocol requires simultaneous measurement of intracochlear distortion products (DPs) and ear-canal otoacoustic emissions (DPOAEs) under tightly controlled conditions. To calibrate the intracochlear response to the DP, Allen-Fahey experiments traditionally employ invasive procedures such as recording from auditory-nerve fibers or measuring basilar-membrane velocity. This paper describes an alternative method that allows the intracochlear distortion source to be calibrated noninvasively. In addition to the standard pair of primary tones used to generate the principal DP the noninvasive method employs a third, fixed tone to create a secondary DPOAE whose amplitude and phase provide a sensitive assay of the intracochlear value of the principal DP near its characteristic place. The method is used to perform noninvasive Allen-Fahey experiments in cat and shown to yield results in quantitative agreement with the original, auditory-nerve-based paradigm performed in the same animal. Data obtained using a suppression-compensated variation of the noninvasive method demonstrate that neither traveling-wave amplification nor two-tone suppression constitutes the controlling influence in DPOAE generation at close frequency ratios. Rather, the dominant factor governing the emission magnitude appears to be the variable directionality of the waves radiated by the distortion-source region, which acts as a distortion beamformer tuned by the primary frequency ratio.

Serotonergic innervation of the inner ear: is it involved in the general physiological control of the auditory receptor?

The auditory pathway of mammals is composed of two complementary ascending afferent and descending efferent independent systems. The brainstem nuclei and cochlear projections for these systems are now well-known. In addition, a highly conspicuous distribution for serotonergic fibers was recently reported. This study focused on these serotonergic fibers and their neurons of origin. We identified several different types of serotonergic brainstem neurons surrounding the superior olivary complex and around the periolivary nuclei. Even though the 5-hydroxytryptamine (5-HT) efferent cochlear innervation originates in the periolivary area of the superior olivary complex system projecting to the cochlea, it is not involved in the transduction of pure tones during auditory processing. However, recent findings, after cochlear blockade of serotonin transporters, strongly suggested that this neuroactive substance has an important turnover within the auditory receptor. The presence of a conspicuous peripheral nerve distribution together with a particular brainstem origin could define a complex role for this innervation. Therefore, 5-HT fibers projecting to the cochlea might be involved, as in other parts of the auditory pathway, in alertness, attention, control of sleep or wakefulness cycles, and state of urgency prior to the transduction processing at the auditory receptor. A lack, or reduction, of the function of these fibers could result in pathological alterations.

The cochlear amplifier: augmentation of the traveling wave within the inner ear.

PURPOSE OF REVIEW: There have been many recent advancements in our understanding of cochlear function within the past ten years. In particular, several mechanisms that underlie the sensitivity and sharpness of mammalian tuning have been discovered. This review focuses on these issues. RECENT FINDINGS: The cochlear amplifier is essentially a positive feedback loop within the cochlea that amplifies the traveling wave. Thus, vibrations within the organ of Corti are sensed and then force is generated in synchrony to increase the vibrations. Mechanisms that generate force within the cochlea include outer hair cell electromotility and stereociliary active bundle movements. These processes can be modulated by the intracellular ionic composition, the lipid constituents of the outer hair cell plasma membrane, and the structure of the outer hair cell cytoskeleton. SUMMARY: A thorough understanding of the cochlear amplifier has tremendous implications to improve human hearing. Sensorineural hearing loss is a common clinical problem and a common site of initial pathology is the outer hair cell. Loss of outer hair cells causes loss of the cochlear amplifier, resulting in progressive sensorineural hearing loss.