ABR/DPOAE publications

The ability to discriminate modulation frequencies is important for speech intelligibility because speech has amplitude and frequency modulations. Neurophysiological responses assessed by envelope following responses (EFRs) significantly decline at faster amplitude modulation frequencies (AMF) in older subjects. A typical assumption is that a decline in EFRs will necessarily result in corresponding perceptual deficits. To test this assumption, we investigated young and aged Fischer-344 rats’ behavioral AMF discrimination abilities and compared to their EFRs. A modified version of prepulse inhibition (PPI) of acoustic startle reflex (ASR) was used to obtain behavioral performance. A PPI trial contains pulses of sinusoidal AM (SAM) at 128 Hz presented sequentially, a SAM prepulse with different AMF and a startle-eliciting-stimulus. To account for hearing threshold shift or age-related synaptopathy, stimulus levels were presented at 10-dB lower or match to the aged peripheral neural activation (using auditory brainstem response wave I amplitude). When AMF differences and modulation depths were large, young and aged animals’ behavioral performances were comparable. Aged animals’ AMF discrimination abilities declined as the AMF difference or the modulation depth reduced, even compared to the young with peripheral matching. Young animals showed smaller relative decreases in EFRs with reduced modulation depths. The correlation of EFRs and AM perception was identified to be more consistent in young animals. The overall results revealed larger age-related deficits in behavioral perception compared to EFRs, suggesting additional factors that affect perception despite smaller degradation in neural responses. Hence, behavioral and physiological measurements are critical in unveiling a more complete picture on the auditory function.

Gene therapy for genetic deafness is a promising approach by which to prevent hearing loss or to restore hearing after loss has occurred. Although a variety of direct approaches to introduce viral particles into the inner ear have been described, presumed physiological barriers have heretofore precluded investigation of systemic gene delivery to the cochlea. In this study, we sought to characterize systemic delivery of a rAAV2/9 vector as a non-invasive means of cochlear transduction. In wild-type neonatal mice (postnatal day 0–1), we show that intravenous injection of rAAV2/9 carrying an eGFP-reporter gene results in binaural transduction of inner hair cells, spiral ganglion neurons and vestibular hair cells. Transduction efficiency increases in a dose-dependent manner. Inner hair cells are transduced in an apex-to-base gradient, with transduction reaching 96% in the apical turn. Hearing acuity in treated animals is unaltered at postnatal day 30. Transduction is influenced by viral serotype and age at injection, with less efficient cochlear transduction observed with systemic delivery of rAAV2/1 and in juvenile mice with rAAV2/9. Collectively, these data validate intravenous delivery of rAAV2/9 as a novel and atraumatic technique for inner ear transgene delivery in early postnatal mice.

Plasticity of myelination represents a mechanism to tune the flow of information by balancing functional requirements with metabolic and spatial constraints. The auditory system is heavily myelinated and operates at the upper limits of action potential generation frequency and speed observed in the mammalian CNS. This study aimed to characterize the development of myelin within the trapezoid body, a central auditory fiber tract, and determine the influence sensory experience has on this process in mice of both sexes. We find that in vitro conduction speed doubles following hearing onset and the ability to support high-frequency firing increases concurrently. Also in this time, the diameter of trapezoid body axons and the thickness of myelin double, reaching mature-like thickness between 25 and 35 d of age. Earplugs were used to induce ∼50 dB elevation in auditory thresholds. If introduced at hearing onset, trapezoid body fibers developed thinner axons and myelin than age-matched controls. If plugged during adulthood, the thickest trapezoid body fibers also showed a decrease in myelin. These data demonstrate the need for sensory activity in both development and maintenance of myelin and have important implications in the study of myelin plasticity and how this could relate to sensorineural hearing loss following peripheral impairment.SIGNIFICANCE STATEMENT The auditory system has many mechanisms to maximize the dynamic range of its afferent fibers, which operate at the physiological limit of action potential generation, precision, and speed. In this study we demonstrate for the first time that changes in peripheral activity modifies the thickness of myelin in sensory neurons, not only in development but also in mature animals. The current study suggests that changes in CNS myelination occur as a downstream mechanism following peripheral deficit. Given the required submillisecond temporal precision for binaural auditory processing, reduced myelination might augment sensorineural hearing impairment.

The spectral analysis of the spontaneous activity recorded with an electrode positioned near the round window of the guinea pig cochlea shows a broad energy peak between 800 and 1,000 Hz. This spontaneous electric activity is called round window noise or ensemble background activity. In guinea pigs, the proposed origin of this peak is the random sum of the extracellular field potentials generated by action potentials of auditory nerve neurons. In this study, we used a non-invasive method to record the tympanic electric noise (TEN) in humans by means of a tympanic wick electrode. We recorded a total of 24 volunteers, under silent conditions or in response to stimuli of different modalities, including auditory, vestibular, and motor activity. Our results show a reliable peak of spontaneous activity at ~1,000 Hz in all studied subjects. In addition, we found stimulus-driven responses with broad-band noise that in most subjects produced an increase in the magnitude of the energy band around 1,000 Hz (between 650 and 1,200 Hz). Our results with the vestibular stimulation were not conclusive, as we found responses with all caloric stimuli, including 37°C. No responses were observed with motor tasks, like eye movements or blinking. We demonstrate the feasibility of recording neural activity from the electric noise of the tympanic membrane with a non-invasive method. From our results, we suggest that the 1,000 Hz component of the TEN has a mixed origin including peripheral and central auditory pathways. This research opens up the possibility of future clinical non-invasive techniques for the functional study of auditory and vestibular nerves in humans.

Inner ear hair cells detect sound through deflection of stereocilia, the microvilli-like projections that are arranged in rows of graded heights. Calcium and integrin-binding protein 2 is essential for hearing and localizes to stereocilia, but its exact function is unknown. Here, we have characterized two mutant mouse lines, one lacking calcium and integrin-binding protein 2 and one carrying a human deafness-related Cib2 mutation, and show that both are deaf and exhibit no mechanotransduction in auditory hair cells, despite the presence of tip links that gate the mechanotransducer channels. In addition, mechanotransducing shorter row stereocilia overgrow in hair cell bundles of both Cib2 mutants. Furthermore, we report that calcium and integrin-binding protein 2 binds to the components of the hair cell mechanotransduction complex, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 mutations. We conclude that calcium and integrin-binding protein 2 is required for normal operation of the mechanotransducer channels and is involved in limiting the growth of transducing stereocilia.

Glucocorticoids have been used to treat hearing loss and vestibular dysfunction for many years. However, some reports have indicated that a subset of patients with these disorders exhibit glucocorticoid insensitivity or resistance. A reduction in histone deacetylase 2 (HDAC2) activity and expression has been reported to play a critical role in glucocorticoid resistance. Here, we investigated the protective effects of aminophylline on HDAC2 expression and glucocorticoid sensitivity in lipopolysaccharide (LPS)-induced sudden sensorineural hearing loss in guinea pigs. We assessed hearing recovery in LPS-applied guinea pigs, which were either left untreated or were systemically treated with either dexamethasone, aminophylline, or a combination of the two. We utilized fluorescence microscopy and enzyme-linked immunosorbent assay to analyze the distribution patterns of HDAC2 and detect its levels in the cochlea. We used hematoxylin-eosin staining to examine cochlear histopathological changes. In the absence of treatment, significant hearing loss was detected in LPS-exposed animals. A synergistic effect was observed between aminophylline and dexamethasone in maintaining HDAC2 expression levels, preventing hearing loss in LPS-exposed animals and reducing cochlear damage. This study indicates that aminophylline can restore glucocorticoid sensitivity, which provides a new approach to treating patients with hearing disorders who are refractory to glucocorticoids.

Extensive research on humans has improved our understanding of how the brain integrates information from our different senses, and has begun to uncover the brain regions and large-scale neural activity that contributes to an observer's ability to perceive the relative timing of auditory and visual stimuli. In the present study, we developed the first behavioral tasks to assess the perception of audiovisual temporal synchrony in rats. Modeled after the parameters used in human studies, separate groups of rats were trained to perform: (1) a simultaneity judgment task in which they reported whether audiovisual stimuli at various stimulus onset asynchronies (SOAs) were presented simultaneously or not; and (2) a temporal order judgment task in which they reported whether they perceived the auditory or visual stimulus to have been presented first. Furthermore, using in vivo electrophysiological recordings in the lateral extrastriate visual (V2L) cortex of anesthetized rats, we performed the first investigation of how neurons in the rat multisensory cortex integrate audiovisual stimuli presented at different SOAs. As predicted, rats (n = 7) trained to perform the simultaneity judgment task could accurately (~80%) identify synchronous vs. asynchronous (200 ms SOA) trials. Moreover, the rats judged trials at 10 ms SOA to be synchronous, whereas the majority (~70%) of trials at 100 ms SOA were perceived to be asynchronous. During the temporal order judgment task, rats (n = 7) perceived the synchronous audiovisual stimuli to be "visual first" for ~52% of the trials, and calculation of the smallest timing interval between the auditory and visual stimuli that could be detected in each rat (i.e., the just noticeable difference (JND)) ranged from 77 ms to 122 ms. Neurons in the rat V2L cortex were sensitive to the timing of audiovisual stimuli, such that spiking activity was greatest during trials when the visual stimulus preceded the auditory by 20-40 ms. Ultimately, given that our behavioral and electrophysiological results were consistent with studies conducted on human participants and previous recordings made in multisensory brain regions of different species, we suggest that the rat represents an effective model for studying audiovisual temporal synchrony at both the neuronal and perceptual level.

The purpose of this study was to test the hypothesis that listeners with frequent exposure to loud music exhibit deficits in suprathreshold auditory performance consistent with cochlear synaptopathy. Young adults with normal audiograms were recruited who either did ( n = 31) or did not ( n = 30) have a history of frequent attendance at loud music venues where the typical sound levels could be expected to result in temporary threshold shifts. A test battery was administered that comprised three sets of procedures: (a) electrophysiological tests including distortion product otoacoustic emissions, auditory brainstem responses, envelope following responses, and the acoustic change complex evoked by an interaural phase inversion; (b) psychoacoustic tests including temporal modulation detection, spectral modulation detection, and sensitivity to interaural phase; and (c) speech tests including filtered phoneme recognition and speech-in-noise recognition. The results demonstrated that a history of loud music exposure can lead to a profile of peripheral auditory function that is consistent with an interpretation of cochlear synaptopathy in humans, namely, modestly abnormal auditory brainstem response Wave I/Wave V ratios in the presence of normal distortion product otoacoustic emissions and normal audiometric thresholds. However, there were no other electrophysiological, psychophysical, or speech perception effects. The absence of any behavioral effects in suprathreshold sound processing indicated that, even if cochlear synaptopathy is a valid pathophysiological condition in humans, its perceptual sequelae are either too diffuse or too inconsequential to permit a simple differential diagnosis of hidden hearing loss.

Ca2+ signaling has been implicated in the initial pathophysiologic mechanisms underlying the cochlea's response to acoustic overstimulation. Intracellular Ca2+ signaling (ICS) waves, which occur in glia and retinal cells in response to injury to activate cell regulatory pathways, have been proposed as an early event in cochlear injury. Disruption of ICS activity is thought to underlie Connexin 26-associated hearing loss, the most common genetic form of deafness, and downstream sequelae of ICS wave activity, such as MAP kinase pathway activation, have been implicated in noise-induced hearing loss. However, ICS waves have only been observed in neonatal cochlear cultures and are thought to be quiescent after the onset of hearing. In this study, we employ an acute explant model of an adult, hearing cochlea that retains many in vivo physiologic features to investigate Ca2+ changes in response to sound. We find that both slow monotonic changes in intracellular Ca2+ concentration as well as discrete ICS waves occur with acoustic overstimulation. The ICS waves share many intrinsic features with their better-described neonatal counterparts, including ATP and gap-junction dependence, and propagation velocity and distance. This identification of ICS wave activity in the adult, hearing cochlea thus confirms and characterizes an important early detection mechanism for cochlear trauma and provides a target for interventions for noise-induced and Connexin 26-associated hearing loss.

Globally, a body of comparative case-control studies suggests that rheumatoid arthritis (RA) patients are more prone to developing hearing loss (HL). However, experimental evidence that supports this hypothesis is still lacking because the human auditory organ is not readily accessible. The aim of this study was to determine the association between bone damage to the ossicles of the middle ear and HL, using a widely accepted murine model of collagen-induced arthritis (RA mice). Diarthrodial joints in the middle ear were examined with microcomputer tomography (microCT), and hearing function was assessed by auditory brainstem response (ABR). RA mice exhibited significantly decreased hearing sensitivity compared to age-matched controls. Additionally, a significant narrowing of the incudostapedial joint space and an increase in the porosity of the stapes were observed. The absolute latencies of all ABR waves were prolonged, but mean interpeak latencies were not statistically different. The observed bone defects in the middle ear that were accompanied by changes in ABR responses were consistent with conductive HL. This combination suggests that conductive impairment is at least part of the etiology of RA-induced HL in a murine model. Whether the inner ear sustains bone erosion or other pathology, and whether the cochlear nerve sustains pathology await subsequent studies. Considering the fact that certain anti-inflammatories are ototoxic in high doses, monitoring RA patients' auditory function is advisable as part of the effort to ensure their well-being.

Sorting nexins are a large family of evolutionarily conserved proteins that play fundamental roles in endocytosis, endosomal sorting and signaling. As an important member of sorting nexin family, sorting nexin 9 (SNX9) has been shown to participate in coordinating actin polymerization with membrane tubulation and vesicle formation. We previously showed that SNX9 is expressed in mouse auditory hair cells and might regulate actin polymerization in those cells. To further examine the physiological role of SNX9, we generated Snx9 knockout mice using homologous recombination method. Unexpectedly, Snx9 knockout mice have normal viability and fertility, and are morphologically and behaviorally indistinguishable from control mice. Further investigation revealed that the morphology and function of auditory hair cells are not affected by Snx9 inactivation, and Snx9 knockout mice have normal hearing threshold. In conclusion, our data revealed that Snx9-deficient mice do not show defects in development as well as auditory function, suggesting that SNX9 is not essential for mice development and hearing.

Hidden hearing refers to the functional deficits in hearing without deterioration in hearing sensitivity. This concept is proposed based upon recent finding of massive noise-induced damage on ribbon synapse between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in the cochlea without significant permanent threshold shifts (PTS). Presumably, such damage may cause coding deficits in auditory nerve fibers (ANFs). However, such deficits had not been detailed except that a selective loss of ANFs with low spontaneous rate (SR) was reported. In the present study, we investigated the dynamic changes of ribbon synapses and the coding function of ANF single units in one month after a brief noise exposure that caused a massive damage of ribbon synapses but no PTS. The synapse count and functional response measures indicates a large portion of the disrupted synapses were re-connected. This is consistent with the fact that the change of SR distribution due to the initial loss of low SR units is recovered quickly. However, ANF coding deficits were developed later with the re-establishment of the synapses. The deficits were found in both intensity and temporal processing, revealing the nature of synaptopathy in hidden hearing loss.

Connexin26 (Cx26, encoded by GJB2) mutations are the most common cause of non-syndromic deafness. GJB2 is thought to be involved in noise-induced hearing loss (NIHL). However, the role of Cx26 in NIHL is still obscure. To explore the association between Cx26 and NIHL, we established a Cx26 knockdown (KD) mouse model by conditional knockdown of Cx26 at postnatal day 18 (P18), and then we observed the auditory threshold and morphologic changes in these mice with or without noise exposure. The Cx26 KD mice did not exhibit substantial hearing loss and hair cell degeneration, while the Cx26 KD mice with acoustic trauma experienced higher hearing loss than simple noise exposure siblings and nearly had no recovery. Additionally, extensive outer hair cell loss and more severe destruction of the basal organ of Corti were observed in Cx26 KD mice after noise exposure. These data indicate that reduced Cx26 expression in the mature mouse cochlea may increase susceptibility to noise-induced hearing loss and facilitate the cell degeneration in the organ of Corti.

WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.

The patients with DiGeorge syndrome (DGS), caused by deletion containing dozens of genes in chromosome 22, often carry cardiovascular problem and hearing loss associated with chronic otitis media. Inside the deletion region, a transcription factor TBX1 was highly suspected. Furthermore, similar DGS phenotypes were found in the Tbx1 heterozygous knockout mice. Using ENU-induced mutagenesis and G1 dominant screening strategy, here we identified a nonsynonymous mutation p.W118R in T-box of TBX1, the DNA binding domain for transcription activity. The mutant mice showed deficiency of inner ear functions, including head tossing and circling, plus increased hearing threshold determined by audiometry. Therefore, our result further confirms the pathogenic basis of Tbx1 in DGS, points out the crucial role of DNA binding activity of TBX1 for the ear function, and provides additional animal model for studying the DGS disease mechanisms.

We proposed a piezoelectric artificial basilar membrane (ABM) composed of a microelectromechanical system cantilever array. The ABM mimics the tonotopy of the cochlea: frequency selectivity and mechanoelectric transduction. The fabricated ABM exhibits a clear tonotopy in an audible frequency range (2.92–12.6 kHz). Also, an animal model was used to verify the characteristics of the ABM as a front end for potential cochlear implant applications. For this, a signal processor was used to convert the piezoelectric output from the ABM to an electrical stimulus for auditory neurons. The electrical stimulus for auditory neurons was delivered through an implanted intra-cochlear electrode array. The amplitude of the electrical stimulus was modulated in the range of 0.15 to 3.5 V with incoming sound pressure levels (SPL) of 70.1 to 94.8 dB SPL. The electrical stimulus was used to elicit an electrically evoked auditory brainstem response (EABR) from deafened guinea pigs. EABRs were successfully measured and their magnitude increased upon application of acoustic stimuli from 75 to 95 dB SPL. The frequency selectivity of the ABM was estimated by measuring the magnitude of EABRs while applying sound pressure at the resonance and off-resonance frequencies of the corresponding cantilever of the selected channel. In this study, we demonstrated a novel piezoelectric ABM and verified its characteristics by measuring EABRs.

Spiral ganglion neuron (SGN) damage and apoptosis can lead to noise-induced hearing loss, age-associated hearing loss and, in certain cases, auditory neuropathy. The apoptosis-inducing factor (AIF)-associated pathway may be important in this process. The present study aimed to investigate the expression levels of AIF and calpain in damaged SGNs. Glutamate (Glu) perfusion and cell culture in different concentrations of Glu were performed to damage the SGNs of Sprague-Dawley (SD) rats, with saline water used as a control Different concentrations (5, 10, 20 and 40 mM) of Glu were injected into the cochlear tympanic canal of 18 SD rats, and 10, 20 and 40 mM Glu were added to SGN cultures. Auditory brainstem responses (ABR) were measured prior to and 2 days following the injection of Glu. Immunofluorescent staining was used to detect the SGN damage and the expression levels of AIF and calpain in vivo and in in vitro. Transmission electron microscopy (TEM) was used to measure cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to analyse the gene expression levels of AIF and calpain in the damaged SGNs. The TEM identified mitochondrial vacuolisation, swelling of the SGN and heterochromatin formation. Injection of Glu reduced the number of SGNs and induced apoptosis. AIF was observed to translocate into the nuclei of the SGNs in the 20 and 40 mM Glu groups, and the expression levels of AIF and calpain were markedly upregulated in the modiolus of the Glu-damaged SGNs. The upregulation of AIF and calpain may be important in the process of SGN damage and apoptosis.

Chronic tinnitus, or "ringing of the ears", affects upwards of 15% of the adult population. Identifying a cost-effective and objective measure of tinnitus is needed due to legal concerns and disability issues, as well as for facilitating the effort to assess neural biomarkers. We developed a modified gap-in-noise (GIN) paradigm to assess tinnitus in mice using the auditory brainstem response (ABR). We then compared the commonly used acoustic startle reflex gap-prepulse inhibition (gap-PPI) and the ABR GIN paradigm in young adult CBA/CaJ mice before and after administrating sodium salicylate (SS), which is known to reliably induce a 16 kHz tinnitus percept in rodents. Post-SS, gap-PPI was significantly reduced at 12 and 16 kHz, consistent with previous studies demonstrating a tinnitus-induced gap-PPI reduction in this frequency range. ABR audiograms indicated thresholds were significantly elevated post-SS, also consistent with previous studies. There was a significant increase in the peak 2 (P2) to peak 1 (P1) and peak 4 (P4) to P1 amplitude ratios in the mid-frequency range, along with decreased latency of P4 at higher intensities. For the ABR GIN, peak amplitudes of the response to the second noise burst were calculated as a percentage of the first noise burst response amplitudes to quantify neural gap processing. A significant decrease in this ratio (i.e. recovery) was seen only at 16 kHz for P1, indicating the presence of tinnitus near this frequency. Thus, this study demonstrates that GIN ABRs can be used as an efficient, non-invasive, and objective method of identifying the approximate pitch and presence of tinnitus in a mouse model. This technique has the potential for application in human subjects and also indicates significant, albeit different, deficits in temporal processing in peripheral and brainstem circuits following drug induced tinnitus.

Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1 −/− mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1 −/− mice compared with that in wild-type controls both in vivo and in vitro , indicating Bmi1 −/− hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1 −/− mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1 −/− hair cells due to the aggravated disequilibrium of antioxidant–prooxidant balance. Furthermore, the antioxidant N -acetylcysteine rescued Bmi1 −/− hair cells from neomycin injury both in vitro and in vivo , suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1 −/− hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.

MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream affecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46, XY sex reversal. Until recently, the only phenotype observed in Map3k1tm1Yxia mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1tm1Yxia mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1tm1Yxia mutant mice have supernumerary functional OHCs and Deiters' cells. Loss of MAP3K1 function resulted in the down-regulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in the otic placode induction or in otic epithelium development in mice and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the critical role of MAP3K1 in the development and function of the mouse inner ear and hearing.