The reticular lamina and basilar membrane vibrations in the transverse direction in the basal turn of the living gerbil cochlea.

The prevailing theory of cochlear function states that outer hair cells amplify sound-induced vibration to improve hearing sensitivity and frequency specificity. Recent micromechanical measurements in the basal turn of gerbil cochleae through the round window have demonstrated that the reticular lamina vibration lags the basilar membrane vibration, and it is physiologically vulnerable not only at the best frequency but also at the low frequencies. These results suggest that outer hair cells from a broad cochlear region enhance hearing sensitivity through a global hydromechanical mechanism. However, the time difference between the reticular lamina and basilar membrane vibration has been thought to result from a systematic measurement error caused by the optical axis non-perpendicular to the cochlear partition. To address this concern, we measured the reticular lamina and basilar membrane vibrations in the transverse direction through an opening in the cochlear lateral wall in this study. Present results show that the phase difference between the reticular lamina and basilar membrane vibration decreases with frequency by ~ 180 degrees from low frequencies to the best frequency, consistent with those measured through the round window. Together with the round-window measurement, the low-coherence interferometry through the cochlear lateral wall demonstrates that the time difference between the reticular lamina and basilar membrane vibration results from the cochlear active processing rather than a measurement error.

Best frequencies and temporal delays are similar across the low-frequency regions of the guinea pig cochlea.

The cochlea maps tones with different frequencies to distinct anatomical locations. For instance, a faint 5000-hertz tone produces brisk responses at a place approximately 8 millimeters into the 18-millimeter-long guinea pig cochlea, but little response elsewhere. This place code pervades the auditory pathways, where neurons have "best frequencies" determined by their connections to the sensory cells in the hearing organ. However, frequency selectivity in cochlear regions encoding low-frequency sounds has not been systematically studied. Here, we show that low-frequency hearing works according to a unique principle that does not involve a place code. Instead, sound-evoked responses and temporal delays are similar across the low-frequency regions of the cochlea. These findings are a break from theories considered proven for 100 years and have broad implications for understanding information processing in the brainstem and cortex and for optimizing the stimulus delivery in auditory implants.

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points.

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

An outer hair cell-powered global hydromechanical mechanism for cochlear amplification.

It is a common belief that the mammalian cochlea achieves its exquisite sensitivity, frequency selectivity, and dynamic range through an outer hair cell-based active process, or cochlear amplification. As a sound-induced traveling wave propagates from the cochlear base toward the apex, outer hair cells at a narrow region amplify the low level sound-induced vibration through a local feedback mechanism. This widely accepted theory has been tested by measuring sound-induced sub-nanometer vibrations within the organ of Corti in the sensitive living cochleae using heterodyne low-coherence interferometry and optical coherence tomography. The aim of this short review is to summarize experimental findings on the cochlear active process by the authors' group. Our data show that outer hair cells are able to generate substantial forces for driving the cochlear partition at all audible frequencies in vivo. The acoustically induced reticular lamina vibration is larger and more broadly tuned than the basilar membrane vibration. The reticular lamina and basilar membrane vibrate approximately in opposite directions at low frequencies and in the same direction at the best frequency. The group delay of the reticular lamina is larger than that of the basilar membrane. The magnitude and phase differences between the reticular lamina and basilar membrane vibration are physiologically vulnerable. These results contradict predictions based on the local feedback mechanism but suggest a global hydromechanical mechanism for cochlear amplification. This article is part of the Special Issue Outer hair cell Edited by Joseph Santos-Sacchi and Kumar Navaratnam.

The mechanoelectrical transducer channel is not required for regulation of cochlear blood flow during loud sound exposure in mice.

The mammalian cochlea possesses unique acoustic sensitivity due to a mechanoelectrical 'amplifier', which requires the metabolic support of the cochlear lateral wall. Loud sound exposure sufficient to induce permanent hearing damage causes cochlear blood flow reduction, which may contribute to hearing loss. However, sensory epithelium involvement in the cochlear blood flow regulation pathway is not fully described. We hypothesize that genetic manipulation of the mechanoelectrical transducer complex will abolish sound induced cochlear blood flow regulation. We used salsa mice, a Chd23 mutant with no mechanoelectrical transduction, and deafness before p56. Using optical coherence tomography angiography, we measured the cochlear blood flow of salsa and wild-type mice in response to loud sound (120 dB SPL, 30 minutes low-pass filtered noise). An expected sound induced decrease in cochlear blood flow occurred in CBA/CaJ mice, but surprisingly the same sound protocol induced cochlear blood flow increases in salsa mice. Blood flow did not change in the contralateral ear. Disruption of the sympathetic nervous system partially abolished the observed wild-type blood flow decrease but not the salsa increase. Therefore sympathetic activation contributes to sound induced reduction of cochlear blood flow. Additionally a local, non-sensory pathway, potentially therapeutically targetable, must exist for cochlear blood flow regulation.

Revealing the morphology and function of the cochlea and middle ear with optical coherence tomography.

Optical coherence tomography (OCT) has revolutionized physiological studies of the hearing organ, the vibration and morphology of which can now be measured without opening the surrounding bone. In this review, we provide an overview of OCT as used in the otological research, describing advances and different techniques in vibrometry, angiography, and structural imaging.

A mechanoelectrical mechanism for detection of sound envelopes in the hearing organ.

To understand speech, the slowly varying outline, or envelope, of the acoustic stimulus is used to distinguish words. A small amount of information about the envelope is sufficient for speech recognition, but the mechanism used by the auditory system to extract the envelope is not known. Several different theories have been proposed, including envelope detection by auditory nerve dendrites as well as various mechanisms involving the sensory hair cells. We used recordings from human and animal inner ears to show that the dominant mechanism for envelope detection is distortion introduced by mechanoelectrical transduction channels. This electrical distortion, which is not apparent in the sound-evoked vibrations of the basilar membrane, tracks the envelope, excites the auditory nerve, and transmits information about the shape of the envelope to the brain.

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.

Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function.

The phospholipid- and Ca(2+)-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear's sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5(-/-) mice. Annexins have been proposed to mediate Ca(2+)-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5(-/-) mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle's key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

Minimally invasive surgical method to detect sound processing in the cochlear apex by optical coherence tomography.

Sound processing in the inner ear involves separation of the constituent frequencies along the length of the cochlea. Frequencies relevant to human speech (100 to 500 Hz) are processed in the apex region. Among mammals, the guinea pig cochlear apex processes similar frequencies and is thus relevant for the study of speech processing in the cochlea. However, the requirement for extensive surgery has challenged the optical accessibility of this area to investigate cochlear processing of signals without significant intrusion. A simple method is developed to provide optical access to the guinea pig cochlear apex in two directions with minimal surgery. Furthermore, all prior vibration measurements in the guinea pig apex involved opening an observation hole in the otic capsule, which has been questioned on the basis of the resulting changes to cochlear hydrodynamics. Here, this limitation is overcome by measuring the vibrations through the unopened otic capsule using phase-sensitive Fourier domain optical coherence tomography. The optically and surgically advanced method described here lays the foundation to perform minimally invasive investigation of speech-related signal processing in the cochlea.

Optogenetic Control of Mouse Outer Hair Cells.

Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects.

Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir-based regenerative shifts between two RP states could form a critical mechanism for conduction/spread of vasomotion along the arteriole axis.

JAK2/STAT3 inhibition attenuates noise-induced hearing loss.

Signal transducers and activators of transcription 3 (STAT3) is a stress responsive transcription factor that plays a key role in oxidative stress-mediated tissue injury. As reactive oxygen species (ROS) are a known source of damage to tissues of the inner ear following loud sound exposure, we examined the role of the Janus kinase 2 (JAK2)/STAT3 signaling pathway in noise induce hearing loss using the pathway specific inhibitor, JSI-124. Mice were exposed to a moderately damaging level of loud sound revealing the phosphorylation of STAT3 tyrosine 705 residues and nuclear localization in many cell types in the inner ear including the marginal cells of the stria vascularis, type II, III, and IV fibrocytes, spiral ganglion cells, and in the inner hair cells. Treatment of the mice with the JAK2/STAT3 inhibitor before noise exposure reduced levels of phosphorylated STAT3 Y705. We performed auditory brain stem response and distortion product otoacoustic emission measurements and found increased recovery of hearing sensitivity at two weeks after noise exposure with JAK2/STAT3 inhibition. Performance of cytocochleograms revealed improved outer hair cell survival in JSI-124 treated mice relative to control. Finally, JAK2/STAT3 inhibition reduced levels of ROS detected in outer hair cells at two hours post noise exposure. Together, these findings demonstrate that inhibiting the JAK2/STAT3 signaling pathway is protective against noise-induced cochlear tissue damage and loss of hearing sensitivity.

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.

Thin and open vessel windows for intra-vital fluorescence imaging of murine cochlear blood flow.

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions.

Non-uniform distribution of outer hair cell transmembrane potential induced by extracellular electric field.

Intracochlear electric fields arising out of sound-induced receptor currents, silent currents, or electrical current injected into the cochlea induce transmembrane potential along the outer hair cell (OHC) but its distribution along the cells is unknown. In this study, we investigated the distribution of OHC transmembrane potential induced along the cell perimeter and its sensitivity to the direction of the extracellular electric field (EEF) on isolated OHCs at a low frequency using the fast voltage-sensitive dye ANNINE-6plus. We calibrated the potentiometric sensitivity of the dye by applying known voltage steps to cells by simultaneous whole-cell voltage clamp. The OHC transmembrane potential induced by the EEF is shown to be highly nonuniform along the cell perimeter and strongly dependent on the direction of the electrical field. Unlike in many other cells, the EEF induces a field-direction-dependent intracellular potential in the cylindrical OHC. We predict that without this induced intracellular potential, EEF would not generate somatic electromotility in OHCs. In conjunction with the known heterogeneity of OHC membrane microdomains, voltage-gated ion channels, charge, and capacitance, the EEF-induced nonuniform transmembrane potential measured in this study suggests that the EEF would impact the cochlear amplification and electropermeability of molecules across the cell.

Changes in cochlear blood flow in mice due to loud sound exposure measured with Doppler optical microangiography and laser Doppler flowmetry.

In this work we determined the contributions of loud sound exposure (LSE) on cochlear blood flow (CoBF) in an in vivo anesthetized mouse model. A broadband noise system (20 kHz bandwidth) with an intensity of 119 dB SPL, was used for a period of one hour to produce a loud sound stimulus. Two techniques were used to study the changes in blood flow, a Doppler optical microangiography (DOMAG) system; which can measure the blood flow within individual cochlear vessels, and a laser Doppler flowmetry (LDF) system; which averages the blood flow within a volume (a hemisphere of ~1.5 mm radius) of tissue. Both systems determined that the blood flow within the cochlea is reduced due to the LSE stimulation.

Depth-resolved dual-beamlet vibrometry based on Fourier domain low coherence interferometry.

We present an optical vibrometer based on delay-encoded, dual-beamlet phase-sensitive Fourier domain interferometric system to provide depth-resolved subnanometer scale vibration information from scattering biological specimens. System characterization, calibration, and preliminary vibrometry with biological specimens were performed. The proposed system has the potential to provide both amplitude and direction of vibration of tissue microstructures on a single two-dimensional plane.

MEASUREMENT OF AMPLITUDE AND DELAY OF STIMULUS FREQUENCY OTOACOUSTIC EMISSIONS.

Although stimulus frequency otoacoustic emissions (SFOAEs) have been used as a non-invasive measure of cochlear mechanics, clinical and experimental application of SFOAEs has been limited by difficulties in accurately deriving quantitative information from sound pressure measured in the ear canal. In this study, a novel signal processing method for multicomponent analysis (MCA) was used to measure the amplitude and delay of the SFOAE. This report shows the delay-frequency distribution of the SFOAE measured from the human ear. A low level acoustical suppressor near the probe tone significantly suppressed the SFOAE, strongly indicating that the SFOAE was generated at characteristic frequency locations. Information derived from this method may reveal more details of cochlear mechanics in the human ear.

Effects of hypoxia on cochlear blood flow in mice evaluated using Doppler optical microangiography.

Reduced cochlear blood flow (CoBF) is a main contributor to hearing loss. Studying CoBF has remained a challenge due to the lack of available tools. Doppler optical microangiography (DOMAG), a method to quantify single-vessel absolute blood flow, and laser Doppler flowmetry (LDF), a method for measuring the relative blood flow within a large volume of tissue, were used for determining the changes in CoBF due to systemic hypoxia in mice. DOMAG determined the change in blood flow in the apical turn (AT) with single-vessel resolution, while LDF averaged the change in the blood flow within a large volume of the cochlea (hemisphere with ∼1 to 1.5 mm radius). Hypoxia was induced by decreasing the concentration of oxygen-inspired gas, so that the oxygen saturation was reduced from >95% to ∼80%. DOMAG determined that during hypoxia the blood flow in two areas of the AT near and far from the helicotrema were increased and decreased, respectively. The LDF detected a decrease in blood flow within a larger volume of the cochlea (several turns averaged together). Therefore, the use of DOMAG as a tool for studying cochlear blood flow due to its ability to determine absolute flow values with single-vessel resolution was proposed.