Radixin modulates the function of outer hair cell stereocilia.

The stereocilia of the inner ear sensory cells contain the actin-binding protein radixin, encoded by RDX. Radixin is important for hearing but remains functionally obscure. To determine how radixin influences hearing sensitivity, we used a custom rapid imaging technique to visualize stereocilia motion while measuring electrical potential amplitudes during acoustic stimulation. Radixin inhibition decreased sound-evoked electrical potentials. Other functional measures, including electrically induced sensory cell motility and sound-evoked stereocilia deflections, showed a minor amplitude increase. These unique functional alterations demonstrate radixin as necessary for conversion of sound into electrical signals at acoustic rates. We identified patients with RDX variants with normal hearing at birth who showed rapidly deteriorating hearing during the first months of life. This may be overlooked by newborn hearing screening and explained by multiple disturbances in postnatal sensory cells. We conclude radixin is necessary for ensuring normal conversion of sound to electrical signals in the inner ear.

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.

Effects of salicylate on sound-evoked outer hair cell stereocilia deflections.

Hearing depends on sound-evoked deflections of the stereocilia that protrude from the sensory hair cells in the inner ear. Although sound provides an important force driving stereocilia, forces generated through mechanically sensitive ion channels and through the motor protein prestin have been shown to influence stereocilia motion in solitary hair cells. While a possible influence of prestin on mechanically sensitive ion channels has not been systematically investigated, a decrease in transducer currents is evident in solitary hair cells when prestin is blocked with salicylate, raising the question of whether a reduced prestin activity or salicylate itself affected the mechanotransduction apparatus. We used two- and three-dimensional time-resolved confocal imaging to visualize outer hair cell stereocilia during sound stimulation in the apical turn of cochlear explant preparations from the guinea pig. Surprisingly, following application of salicylate, outer hair cell stereocilia deflections increased, while cochlear microphonic potentials decreased. However, when prestin activity was altered with the chloride ionophore tributyltin, both the cochlear microphonic potential and the stereocilia deflection amplitude decreased. Neither positive nor negative current stimulation abolished the bundle movements in the presence of salicylate, indicating that the observed effects did not depend on the endocochlear potential. These data suggest that salicylate may alter the mechanical properties of stereocilia, decreasing their bending stiffness.

Filtering of acoustic signals within the hearing organ.

The detection of sound by the mammalian hearing organ involves a complex mechanical interplay among different cell types. The inner hair cells, which are the primary sensory receptors, are stimulated by the structural vibrations of the entire organ of Corti. The outer hair cells are thought to modulate these sound-evoked vibrations to enhance hearing sensitivity and frequency resolution, but it remains unclear whether other structures also contribute to frequency tuning. In the current study, sound-evoked vibrations were measured at the stereociliary side of inner and outer hair cells and their surrounding supporting cells, using optical coherence tomography interferometry in living anesthetized guinea pigs. Our measurements demonstrate the presence of multiple vibration modes as well as significant differences in frequency tuning and response phase among different cell types. In particular, the frequency tuning at the inner hair cells differs from other cell types, causing the locus of maximum inner hair cell activation to be shifted toward the apex of the cochlea compared with the outer hair cells. These observations show that additional processing and filtering of acoustic signals occur within the organ of Corti before inner hair cell excitation, representing a departure from established theories.

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.

Sound rhythms are encoded by postinhibitory rebound spiking in the superior paraolivary nucleus.

The superior paraolivary nucleus (SPON) is a prominent structure in the auditory brainstem. In contrast to the principal superior olivary nuclei with identified roles in processing binaural sound localization cues, the role of the SPON in hearing is not well understood. A combined in vitro and in vivo approach was used to investigate the cellular properties of SPON neurons in the mouse. Patch-clamp recordings in brain slices revealed that brief and well timed postinhibitory rebound spiking, generated by the interaction of two subthreshold-activated ion currents, is a hallmark of SPON neurons. The I(h) current determines the timing of the rebound, whereas the T-type Ca(2+) current boosts the rebound to spike threshold. This precisely timed rebound spiking provides a physiological explanation for the sensitivity of SPON neurons to sinusoidally amplitude-modulated (SAM) tones in vivo, where peaks in the sound envelope drive inhibitory inputs and SPON neurons fire action potentials during the waveform troughs. Consistent with this notion, SPON neurons display intrinsic tuning to frequency-modulated sinusoidal currents (1-15Hz) in vitro and discharge with strong synchrony to SAMs with modulation frequencies between 1 and 20 Hz in vivo. The results of this study suggest that the SPON is particularly well suited to encode rhythmic sound patterns. Such temporal periodicity information is likely important for detection of communication cues, such as the acoustic envelopes of animal vocalizations and speech signals.

A differentially amplified motion in the ear for near-threshold sound detection.

The ear is a remarkably sensitive pressure fluctuation detector. In guinea pigs, behavioral measurements indicate a minimum detectable sound pressure of ∼20 µPa at 16 kHz. Such faint sounds produce 0.1-nm basilar membrane displacements, a distance smaller than conformational transitions in ion channels. It seems that noise within the auditory system would swamp such tiny motions, making weak sounds imperceptible. Here we propose a new mechanism contributing to a resolution of this problem and validate it through direct measurement. We hypothesized that vibration at the apical side of hair cells is enhanced compared with that at the commonly measured basilar membrane side. Using in vivo optical coherence tomography, we demonstrated that apical-side vibrations peaked at a higher frequency, had different timing and were enhanced compared with those at the basilar membrane. These effects depend nonlinearly on the stimulus sound pressure level. The timing difference and enhancement of vibrations are important for explaining how the noise problem is circumvented.

Membrane cholesterol modulates cochlear electromechanics.

Changing the concentration of cholesterol in the plasma membrane of isolated outer hair cells modulates electromotility and prestin-associated charge movement, suggesting that a similar manipulation would alter cochlear mechanics. We examined cochlear function before and after depletion of membrane cholesterol with methyl-ß-cyclodextrin (MßCD) in an excised guinea pig temporal bone preparation. The mechanical response of the cochlear partition to acoustic and/or electrical stimulation was monitored using laser interferometry and time-resolved confocal microscopy. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents. Exposure to MßCD increased the magnitude and asymmetry of the response, without changing the frequency tuning of sound-evoked mechanical responses or cochlear microphonic potentials. Sodium salicylate reversibly blocked the enhanced electromechanical response in cholesterol depleted preparations. The increase of sound-evoked vibrations during positive current injection was enhanced following MßCD in some preparations. Imaging was used to assess cellular integrity which remained unchanged after several hours of exposure to MßCD in several preparations. The enhanced electromechanical response reflects an increase in outer hair cell electromotility and may reveal features of cholesterol distribution and trafficking in outer hair cells.

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.

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.

Imaging the living inner ear using intravital confocal microscopy.

Confocal laser scanning microscopy permits detailed visualization of structures deep within thick fluorescently labeled specimen. This makes it possible to investigate living cells inside intact tissue without prior chemical sample fixation and sectioning. Isolated guinea pig temporal bones have previously been used for confocal experiments in vitro, but tissue deterioration limits their use to a few hours after the death of the animal. In order to preserve the cochlea in an optimal functional and physiological condition, we have developed an in vivo model based on a confocal microscopy approach. Using a ventral surgical approach, the inner ear is exposed in deeply anaesthetized, tracheotomized, living guinea pigs. To label the inner ear structures, scala tympani is perfused via an opening in the basal turn, delivering tissue culture medium with fluorescent vital dyes (RH 795 and calcein AM). An apical opening is made in the bony shell of cochlea to enable visualization using a custom-built objective lens. Intravital confocal microscopy, with preserved blood and nerve supply, may offer an important tool for studying auditory physiology and the pathology of hearing loss. After acoustic overstimulation, shortening and swelling of the sensory hair cells were observed.

Imaging of the guinea pig cochlea following round window gadolinium application.

Precise, non-invasive determination of the aetiology and site of pathology of inner ear disorders is difficult. The aim of this study was to describe an alternative method for inner ear visualization, based on local application of the paramagnetic contrast agent gadolinium. Using a 4.7 T MRI scanner, high contrast images of all four cochlear turns were obtained 3.5 h after placing gadolinium on the round window membrane. Gadolinium cleared from the cochlea within 96 h. Auditory brainstem response measurements performed on a separate group of animals showed no significant threshold shifts after the application, indicating that gadolinium is non-toxic to the guinea pig cochlea.

Measuring hearing organ vibration patterns with confocal microscopy and optical flow.

A new method for visualizing vibrating structures is described. The system provides a means to capture very fast repeating events by relatively minor modifications to a standard confocal microscope. An acousto-optic modulator was inserted in the beam path, generating brief pulses of laser light. Images were formed by summing consecutive frames until every pixel of the resulting image had been exposed to a laser pulse. Images were analyzed using a new method for optical flow computation; it was validated through introducing artificial displacements in confocal images. Displacements in the range of 0.8 to 4 pixels were measured with 5% error or better. The lower limit for reliable motion detection was 20% of the pixel size. These methods were used for investigating the motion pattern of the vibrating hearing organ. In contrast to standard theory, we show that the organ of Corti possesses several degrees of freedom during sound-evoked vibration. Outer hair cells showed motion indicative of deformation. After acoustic overstimulation, supporting cells contracted. This slowly developing structural change was visualized during simultaneous intense sound stimulation and its speed measured with the optical flow technique.

Alterations of basilar membrane response phase and velocity after acoustic overstimulation.

To investigate the physiology of noise-induced hearing loss, the sound-induced vibrations of the basilar membrane (BM) of the inner ear were measured in living anesthetized guinea pigs before and after intense sound exposure. The vibrations were measured using a laser Doppler velocimeter after placing reflective glass beads on the BM. Pseudo-random noise waveforms containing frequencies between 4 and 24 kHz were used to generate velocity tuning curves. Before overstimulation, sharp response peaks were seen at stimulus frequencies between 15 and 17 kHz, consistent with the expected best frequency of the recording location. The response to low level stimuli lagged the high level ones by up to 90 degrees at the characteristic frequency. Following exposure to loud sound, the BM vibrations showed a pronounced reduction in amplitude, primarily at low stimulus levels, and the best frequency moved to approximately 12 kHz. At higher levels, the reduction was either absent or much smaller. In addition to the amplitude changes, increased phase lags were seen at frequencies near the characteristic frequency. In animals with more severe exposures, response phases were altered also at frequencies showing no change of the amplitude. The phase was independent of stimulus level after severe exposures.