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.

CRISPR/Cas9 editing of the MYO7A gene in rhesus macaque embryos to generate a primate model of Usher syndrome type 1B.

Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.

Optimizing Auditory Brainstem Response Acquisition Using Interleaved Frequencies.

Auditory brainstem responses (ABRs) require averaging responses to hundreds or thousands of repetitions of a stimulus (e.g., tone pip) to obtain a measurable evoked response at the scalp. Fast repetition rates lead to changes in ABR amplitude and latency due to adaptation. To minimize the effect of adaptation, stimulus rates are sometimes as low as 10 to 13.3 stimuli per second, requiring long acquisition times. The trade-off between reducing acquisition time and minimizing the effect of adaptation on ABRs is an especially important consideration for studies of cochlear synaptopathy, which use the amplitude of short latency responses (wave 1) to assess auditory nerve survival. It has been proposed that adaptation during ABR acquisition can be reduced by interleaving tones at different frequencies, rather than testing each frequency serially. With careful ordering of frequencies and levels in the stimulus train, adaptation in the auditory nerve can be minimized, thereby permitting an increase in the rate at which tone bursts are presented. However, widespread adoption of this stimulus design has been hindered by lack of available software. Here, we develop and validate an interleaved stimulus design to optimize the rate of ABR measurement while minimizing adaptation. We implement this method in an open-source data acquisition software tool that permits either serial or interleaved ABR measurements. The open-source software library, psiexperiment, is compatible with widely used ABR hardware. Consistent with previous studies, careful design of an interleaved stimulus train can reduce ABR acquisition time by more than half, with minimal effect on ABR thresholds and wave 1 latency, while improving measures of wave 1 amplitude.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

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.

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.

Evaluating psychoacoustic measures for establishing presence of tinnitus.

The Department of Veterans Affairs (VA) considers tinnitus a disability. Veterans can claim tinnitus as a "service-connected" disability if the tinnitus is thought to be connected to military service. The VA adjudicates each claim and determines whether reasonable evidence exists to support it. Currently, determining the presence of tinnitus is based on subjective reporting-objective measures do not exist. The aim of this study was to develop and document a test for detecting the presence/absence of tinnitus with high confidence. Using our computer-automated, self-guided tinnitus evaluation system, we conducted three phases of testing to compare psychoacoustic measures of tinnitus between participants with versus without tinnitus. Phase 1 measures included loudness match, pitch match, minimum masking level, residual inhibition, Békésy, and forced-choice double staircase. Phases 2 and 3 measures were chosen based on results of the previous phase. The number of tests and time of testing decreased during each successive phase. Differences were seen between groups; most notably, higher low-frequency loudness matches and higher median pitch matches were observed for participants with tinnitus. Results of this study suggest that further efforts can produce a defined psychoacoustic test battery for identifying the presence/absence of tinnitus.

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.

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.

Fast reverse propagation of sound in the living cochlea.

The auditory sensory organ, the cochlea, not only detects but also generates sounds. Such sounds, otoacoustic emissions, are widely used for diagnosis of hearing disorders and to estimate cochlear nonlinearity. However, the fundamental question of how the otoacoustic emission exits the cochlea remains unanswered. In this study, emissions were provoked by two tones with a constant frequency ratio, and measured as vibrations at the basilar membrane and at the stapes, and as sound pressure in the ear canal. The propagation direction and delay of the emission were determined by measuring the phase difference between basilar membrane and stapes vibrations. These measurements show that cochlea-generated sound arrives at the stapes earlier than at the measured basilar membrane location. Data also show that basilar membrane vibration at the emission frequency is similar to that evoked by external tones. These results conflict with the backward-traveling-wave theory and suggest that at low and intermediate sound levels, the emission exits the cochlea predominantly through the cochlear fluids.

Auditory test result characteristics of subjects with and without tinnitus.

Tinnitus is the perception of sound that does not have an acoustic source in the environment. Ascertaining the presence of tinnitus in individuals who claim tinnitus for compensation purposes is very difficult and increasingly becoming a problem. This study examined the potential to observe differences in loudness and pitch matches between individuals who experience tinnitus versus those who do not. This study follows a previous pilot study we completed that included 12 subjects with and 12 subjects without tinnitus. The current study included 36 subjects with and 36 without tinnitus. Results of this study revealed no significant differences between groups with regard to decibel sensation level (SL) loudness matches and within-session loudness-match reliability. Between-group differences revealed that the tinnitus subjects had (1) greater decibel sound pressure level loudness matches, (2) better between-session loudness-match reliability, (3) better pitch-match reliability, and (4) higher frequency pitch matches. These findings support the data from our pilot study with the exception that decibel SL loudness matches were greater for the tinnitus subjects in the pilot study. Tinnitus loudness and pitch matching may have some value in an overall battery of tests for evaluating tinnitus claims.

Inverted direction of wave propagation (IDWP) in the cochlea.

The "classical" view on wave propagation is that propagating waves are possible in both directions along the length of the basilar membrane and that they have identical properties. Results of several recently executed experiments [T. Ren, Nat. Neurosci. 2, 333-334 (2004) and W. X. He, A. L. Nuttall, and T. Ren, Hear. Res., 228, 112-122 (2007)] appear to contradict this view. In the current work measurements were made of the velocity of the guinea-pig basilar membrane (BM). Distortion products (DPs) were produced by presenting two primary tones, with frequencies below the characteristic frequency f(0) of the BM location at which the BM measurements were made, with a constant frequency ratio. In each experiment the phase of the principal DP, with frequency 2f(1)-f(2), was recorded as a function of the DP frequency. The results indicate that the DP wave going from the two-tone interaction region toward the stapes is not everywhere traveling in the reverse direction, but also in the forward direction. The extent of the region in which the forward wave occurs appears larger than is accounted for by classical theory. This property has been termed "inverted direction of wave propagation." The results of this study confirm the wave propagation findings of other authors. The experimental data are compared to theoretical predictions for a classical three-dimensional model of the cochlea that is based on noise-response data of the same animal. Possible physical mechanisms underlying the findings are discussed.

Reverse wave propagation in the cochlea.

Otoacoustic emissions, sounds generated by the inner ear, are widely used for diagnosing hearing disorders and studying cochlear mechanics. However, it remains unclear how emissions travel from their generation sites to the cochlear base. The prevailing view is that emissions reach the cochlear base via a backward-traveling wave, a slow-propagating transverse wave, along the cochlear partition. A different view is that emissions propagate to the cochlear base via the cochlear fluids as a compressional wave, a fast longitudinal wave. These theories were experimentally tested in this study by measuring basilar membrane (BM) vibrations at the cubic distortion product (DP) frequency from two longitudinal locations with a laser interferometer. Generation sites of DPs were varied by changing frequencies of primary tones while keeping the frequency ratio constant. Here, we show that BM vibration amplitude and phase at the DP frequency are very similar to responses evoked by external tones. Importantly, the BM vibration phase at a basal location leads that at a more apical location, indicating a traveling wave that propagates in the forward direction. These data are in conflict with the backward- traveling-wave theory but are consistent with the idea that the emission comes out of the cochlea predominantly through compressional waves in the cochlear fluids.