Auditory Research publications

Cisplatin-induced ototoxicity is one of the major adverse effects in cisplatin chemotherapy, and hearing protective approaches are unavailable in clinical practice. Recent work unveiled a critical role of autophagy in cell survival in various types of hearing loss. Since the excessive activation of autophagy can contribute to apoptotic cell death, whether the activation of autophagy increases or decreases the rate of cell death in CDDP ototoxicity is still being debated. In this study, we showed that CDDP induced activation of autophagy in the auditory cell HEI-OC1 at the early stage. We then used rapamycin, an autophagy activator, to increase the autophagy activity, and found that the cell death significantly decreased after CDDP injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly increased cell death. In accordance with in vitro results, rapamycin alleviated CDDP-induced death of hair cells in zebrafish lateral line and cochlear hair cells in mice. Notably, we found that CDDP-induced increase of Sirtuin 1 (SIRT1) in the HEI-OC1 cells modulated the autophagy function. The specific SIRT1 activator SRT1720 could successfully protect against CDDP-induced cell loss in HEI-OC1 cells, zebrafish lateral line, and mice cochlea. These findings suggest that SIRT1 and autophagy activation can be suggested as potential therapeutic strategies for the treatment of CDDP-induced ototoxicity.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

Evidence shows that selective attention to visual stimuli modulates the gain of cochlear responses, probably through auditory-cortex descending pathways. At the cerebral cortex level, amplitude and phase changes of neural oscillations have been proposed as a correlate of selective attention. However, whether sensory receptors are also influenced by the oscillatory network during attention tasks remains unknown. Here, we searched for oscillatory attention-related activity at the cochlear receptor level in humans. We used an alternating visual/auditory selective attention task and measured electroencephalographic activity simultaneously to distortion product otoacoustic emissions (a measure of cochlear receptor-cell activity). In order to search for cochlear oscillatory activity, the otoacoustic emission signal, was included as an additional channel in the electroencephalogram analyses. This method allowed us to evaluate dynamic changes in cochlear oscillations within the same range of frequencies (1-35 Hz) in which cognitive effects are commonly observed in electroencephalogram works. We found the presence of low frequency (<10 Hz) brain and cochlear amplifier oscillations during selective attention to visual and auditory stimuli. Notably, switching between auditory and visual attention modulates the amplitude and the temporal order of brain and inner ear oscillations. These results extend the role of the oscillatory activity network during cognition in neural systems to the receptor level.

Interaural time differences (ITD) are one of several principle cues for localizing sounds. However, ITD are in the sub-millisecond range for most animals. Because the neural processing of such small ITDs pushes the limit of temporal resolution, the precise ITD-range for a given species and its usefulness - relative to other localization cues - was a powerful selective force in the evolution of the neural circuits involved. Birds and other non-mammals have internally coupled middle ears working as pressure-difference receivers that may significantly enhance ITD, depending on the precise properties of the interaural connection. Here, the extent of this internal coupling was investigated in chickens, specifically under the same experimental conditions as typically used in neurophysiology of ITD-coding circuits, i.e. with headphone stimulation. Cochlear microphonics (CM) were recorded simultaneously from both ears of anesthetized chickens under monaural and binaural stimulation, using pure tones from 0.1 to 3 kHz. Interaural transmission peaked at 1.5 kHz at a loss of only −5.5 dB; the mean interaural delay was 264 μs. CM amplitude strongly modulated as a function of ITD, confirming significant interaural coupling. The “ITD heard” derived from the CM phases in both ears showed enhancement, compared to the acoustic stimuli, by a factor of up to 1.8. However, the closed sound delivery systems impaired interaural transmission at low frequencies (< 1 kHz). We identify factors that need to be considered when interpreting neurophysiological data obtained under these conditions, and relating them to the natural free-field condition. Summary statement The interaural time differences that chickens can use for sound localization are significantly greater than their small head size suggests. Closed-system sound stimulation can, however, produce complex artefacts.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

The caudomedial nidopallium (NCM) is a higher level region of auditory cortex in songbirds that has been implicated in encoding learned vocalizations and mediating perception of complex sounds. We made cell-attached recordings in awake adult male zebra finches ( Taeniopygia guttata) to characterize responses of single NCM neurons to playback of tones and songs. Neurons fell into two broad classes: narrow fast-spiking cells and broad sparsely firing cells. Virtually all narrow-spiking cells responded to playback of pure tones, compared with approximately half of broad-spiking cells. In addition, narrow-spiking cells tended to have lower thresholds and faster, less variable spike onset latencies than did broad-spiking cells, as well as higher firing rates. Tonal responses of narrow-spiking cells also showed broader ranges for both frequency and amplitude compared with broad-spiking neurons and were more apt to have V-shaped tuning curves compared with broad-spiking neurons, which tended to have complex (discontinuous), columnar, or O-shaped frequency response areas. In response to playback of conspecific songs, narrow-spiking neurons showed high firing rates and low levels of selectivity whereas broad-spiking neurons responded sparsely and selectively. Broad-spiking neurons in which tones failed to evoke a response showed greater song selectivity compared with those with a clear tuning curve. These results are consistent with the idea that narrow-spiking neurons represent putative fast-spiking interneurons, which may provide a source of intrinsic inhibition that contributes to the more selective tuning in broad-spiking cells. NEW & NOTEWORTHY The response properties of neurons in higher level regions of auditory cortex in songbirds are of fundamental interest because processing in such regions is essential for vocal learning and plasticity and for auditory perception of complex sounds. Within a region of secondary auditory cortex, neurons with narrow spikes exhibited high firing rates to playback of both tones and multiple conspecific songs, whereas broad-spiking neurons responded sparsely and selectively to both tones and songs.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major NADPH-producing enzyme which is essential for maintaining the mitochondrial redox balance in cells. We sought to determine whether IDH2 deficiency induces mitochondrial dysfunction and modulates auditory function, and investigated the protective potential of an antioxidant agent against reactive oxygen species (ROS)-induced cochlear damage in Idh2 knockout (Idh2-/-) mice. Idh2 deficiency leads to damages to hair cells and spiral ganglion neurons (SGNs) in the cochlea and ultimately to apoptotic cell death and progressive sensorineural hearing loss in Idh2-/- mice. Loss of IDH2 activity led to decreased levels of NADPH and glutathione causing abnormal ROS accumulation and oxidative damage, which might trigger apoptosis signal in hair cells and SGNs in Idh2-/- mice. We performed ex vivo experiments to determine whether administration of mitochondria-targeted antioxidants might protect or induce recovery of cells from ROS-induced apoptosis in Idh2-deficient mouse cochlea. MitoQ almost completely neutralized the H2O2-induced ototoxicity, as the survival rate of Idh2-/- hair cells were restored to normal levels. In addition, the lack of IDH2 led to the accumulation of mitochondrial ROS and the depolarization of ΔΨm, resulting in hair cell loss. In the present study, we identified that IDH2 is indispensable for the functional maintenance and survival of hair cells and SGNs. Moreover, the hair cell degeneration caused by IDH2 deficiency can be prevented by MitoQ, which suggests that Idh2-/- mice could be a valuable animal model for evaluating the therapeutic effects of various antioxidant candidates to overcome ROS-induced hearing loss.

Much of our understanding about how acetylcholine modulates prefrontal cortical (PFC) networks comes from behavioral experiments that examine cortical dynamics during highly attentive states. However, much less is known about how PFC is recruited during passive sensory processing and how acetylcholine may regulate connectivity between cortical areas outside of task performance. To investigate the involvement of PFC and cholinergic neuromodulation in passive auditory processing, we performed simultaneous recordings in the auditory cortex (AC) and PFC in awake head fixed mice presented with a white noise auditory stimulus in the presence or absence of local cholinergic antagonists in AC. We found that a subset of PFC neurons were strongly driven by auditory stimuli even when the stimulus had no associative meaning, suggesting PFC monitors stimuli under passive conditions. We also found that cholinergic signaling in AC shapes the strength of auditory driven responses in PFC, by modulating the intra-cortical sensory response through muscarinic interactions in AC. Taken together, these findings provide novel evidence that cholinergic mechanisms have a continuous role in cortical gating through muscarinic receptors during passive processing and expand traditional views of prefrontal cortical function and the contributions of cholinergic modulation in cortical communication.

Hearing impairment in older people is thought to arise from impaired temporal processing in auditory circuits. We used a systems-level (scalp recordings) and a microcircuit-level (extracellular recordings) approach to investigate how aging affects the sensitivity to temporal envelopes of speech-like sounds in rats. Scalp-recorded potentials suggest an age-related increase in sensitivity to temporal regularity along the ascending auditory pathway. The underlying cellular changes in the midbrain were examined using extracellular recordings from inferior colliculus neurons. We observed an age-related increase in sensitivity to the sound's onset and temporal regularity (i.e., periodicity envelope) in the spiking output of inferior colliculus neurons, relative to their synaptic inputs (local field potentials). This relative enhancement for aged animals was most prominent for multi-unit (relative to single-unit) spiking activity. Spontaneous multi-unit, but not single-unit, activity was also enhanced in aged compared with young animals. Our results suggest that aging is associated with altered sensitivity to a sound's temporal regularities, and that these effects may be due to increased gain of neural network activity in the midbrain.

Contemporary cochlear implants (CI) are generally very effective for remediation of severe to profound sensorineural hearing loss, but outcomes are still highly variable. Auditory nerve survival is likely one of the major factors underlying this variability. Neurotrophin therapy therefore has been proposed for CI recipients, with the goal of improving outcomes by promoting improved survival of cochlear spiral ganglion neurons (SGN) and/or residual hair cells. Previous studies have shown that glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor, and neurotrophin-3 can rescue SGNs following insult. The current study was designed to determine whether adeno-associated virus vector serotype 5 (AAV-5) encoding either green fluorescent protein or GDNF can transduce cells in the mouse cochlea to express useful levels of neurotrophin and to approximate the optimum therapeutic dose(s) for transducing hair cells and SGN. The findings demonstrate that AAV-5 is a potentially useful gene therapy vector for the cochlea, resulting in extremely high levels of transgene expression in the cochlear inner hair cells and SGN. However, overexpression of human GDNF in newborn mice caused severe neurological symptoms and hearing loss, likely due to Purkinje cell loss and cochlear nucleus pathology. Thus, extremely high levels of transgene protein expression should be avoided, particularly for proteins that have neurological function in neonatal subjects.

This paper investigates the safety of a novel 'magnetic injection' method of delivering therapy to the cochlea, in a rodent model. In this method of administration, a magnetic field is employed to actively transport drug-eluting superparamagnetic iron-oxide core nanoparticles into the cochlea, where they then release their drug payload (we delivered the steroid prednisolone). Our study design and selection of control groups was based on published regulatory guidance for safety studies that involve local drug delivery. We tested for both single and multiple delivery doses to the cochlea, and found that magnetic delivery did not harm hearing. There was no statistical difference in hearing between magnetically treated ears versus ears that received intra-tympanic steroid (a mimic of a standard-of-care for sudden sensorineural hearing loss), both 2 and 30 days after treatment. Since our treatment is local to the ear, the levels of steroid and iron circulating systemically after our treatment were low, below mass-spectrometry detection limits for the steroid and no different from normal for iron. No adverse findings were observed in ear tissue histopathology or in animal gross behavior. At 2 and 30 days after treatment, inflammatory changes examined in the ear were limited to the middle ear, were very mild in severity, and by day 90 there was ongoing and almost complete reversibility of these changes. There were no ear tissue scarring or hemorrhage trends associated with magnetic delivery. In summary, after conducting a pre-clinical safety study, no adverse safety issues were observed.

auditory function. However, the differences between the patterns of endocytosis in cochlear inner hair cells (IHCs) and outer hair cells (OHCs) remain unclear. Both IHCs and OHCs were obtained from adult C57 mice. Patterns of endocytosis in cells were estimated by analyzing the uptake of FM1-43, a fluorescent. The observations were made using live confocal imaging, fluorescence intensities were calculated statistically. Results revealed the details about following phenomenon, i) sites of entry: the FM1-43 dye was found to enter IHC at the apical area initially, the additional sites of entry were then found at basolateral membrane of the cells, The entry of the dye into OHCs initially appeared to be occurring around whole apical membranes area, which then diffused towards the other membrane surface of the cells, ii) capacity of endocytosis: fluorescence intensity in IHCs showed significantly higher than that of OHCs (P<0.01). We have found different patterns of endocytosis between IHCs and OHCs, this indicated functional distinctions between them. Moreover, FM1-43 dye can be potentially used as an indicator of the functional loss or repair of cochlear hair cells.

A 'pattern alternation paradigm' has been previously used in human ERP recordings to investigate the brain encoding of complex auditory regularities, but prior studies on regularity encoding in animal models to examine mechanisms of adaptation of auditory neuronal responses have used primarily oddball stimulus sequences to study stimulus-specific adaptation alone. In order to examine the sensitivity of neuronal adaptation to expected and unexpected events embedded in a complex sound sequence, we used a similar patterned sequence of sounds. We recorded single unit activity and compared neuronal responses in the rat inferior colliculus (IC) to sound stimuli conforming to pattern alternation regularity with those to stimuli in which occasional sound repetitions violated that alternation. Results show that some neurons in the rat inferior colliculus are sensitive to the history of patterned stimulation and to violations of patterned regularity, demonstrating that there is a population of subcortical neurons, located as early as the level of the midbrain, that can detect more complex stimulus regularities than previously supposed and that are as sensitive to complex statistics as some neurons in primary auditory cortex. Our findings indicate that these pattern-sensitive neurons can extract temporal and spectral regularities between successive acoustic stimuli. This is important because the extraction of regularities from the sound sequences will result in the development of expectancies for future sounds and hence, the present results are compatible with predictive coding models. Our results demonstrate that some collicular neurons, located as early as in the midbrain level, are involved in the generation and shaping of prediction errors in ways not previously considered and thus, the present findings challenge the prevailing view that perceptual organization of sound only emerges at the auditory cortex level.

It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear spiral ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

Speech audiometry is an essential part of audiological diagnostics and clinical measurements. Development times of speech recognition tests are rather long, depending on the size of speech corpus and optimization necessity. The aim of this study was to examine whether this development effort could be reduced by using synthetic speech in speech audiometry, especially in a matrix test for speech recognition. For this purpose, the speech material of the German matrix test was replicated using a preselected commercial system to generate the synthetic speech files. In contrast to the conventional matrix test, no level adjustments or optimization tests were performed while producing the synthetic speech material. Evaluation measurements were conducted by presenting both versions of the German matrix test (with natural or synthetic speech), alternately and at three different signal-to-noise ratios, to 48 young, normal-hearing participants. Psychometric functions were fitted to the empirical data. Speech recognition thresholds were 0.5 dB signal-to-noise ratio higher (worse) for the synthetic speech, while slopes were equal for both speech types. Nevertheless, speech recognition scores were comparable with the literature and the threshold difference lay within the same range as recordings of two different natural speakers. Although no optimization was applied, the synthetic-speech signals led to equivalent recognition of the different test lists and word categories. The outcomes of this study indicate that the application of synthetic speech in speech recognition tests could considerably reduce the development costs and evaluation time. This offers the opportunity to increase the speech corpus for speech recognition tests with acceptable effort.

Bilateral cochlear-implant (CI) users and single-sided deaf listeners with a CI are less effective at localizing sounds than normal-hearing (NH) listeners. This performance gap is due to the degradation of binaural and monaural sound localization cues, caused by a combination of device-related and patient-related issues. In this study, we targeted the device-related issues by measuring sound localization performance of 11 NH listeners, listening to free-field stimuli processed by a real-time CI vocoder. The use of a real-time vocoder is a new approach, which enables testing in a free-field environment. For the NH listening condition, all listeners accurately and precisely localized sounds according to a linear stimulus–response relationship with an optimal gain and a minimal bias both in the azimuth and in the elevation directions. In contrast, when listening with bilateral real-time vocoders, listeners tended to orient either to the left or to the right in azimuth and were unable to determine sound source elevation. When listening with an NH ear and a unilateral vocoder, localization was impoverished on the vocoder side but improved toward the NH side. Localization performance was also reflected by systematic variations in reaction times across listening conditions. We conclude that perturbation of interaural temporal cues, reduction of interaural level cues, and removal of spectral pinna cues by the vocoder impairs sound localization. Listeners seem to ignore cues that were made unreliable by the vocoder, leading to acute reweighting of available localization cues. We discuss how current CI processors prevent CI users from localizing sounds in everyday environments.