ABR/DPOAE publications

Noise-induced hearing loss (NIHL) is one of the most prevalent forms of acquired hearing loss, and it is associated with aberrant microglial status and reduced hippocampal neurogenesis; however, the nature of these associations is far from being elucidated. Beyond its direct effects on the auditory system, exposure to intense noise has previously been shown to acutely activate the stress response, which has increasingly been linked to both microglial activity and adult hippocampal neurogenesis in recent years. Given the pervasiveness of noise pollution in modern society and the important implications of either microglial activity or hippocampal neurogenesis for cognitive and emotional function, this study was designed to investigate how microglial status and hippocampal neurogenesis change over time following acoustic exposure and to analyze the possible roles of the noise exposure-induced stress response and hearing loss in these changes. To accomplish this, adult male C57BL/6J mice were randomly assigned to either a control or noise exposure (NE) group. Auditory function was assessed by measuring ABR thresholds at 20 days post noise exposure. The time-course profile of serum corticosterone levels, microglial status, and hippocampal neurogenesis during the 28 days following noise exposure were quantified by ELISA or immunofluorescence staining. Our results illustrated a permanent moderate-to-severe degree of hearing loss, an early but transient increase in serum corticosterone levels, and time-dependent dynamic alterations in microglial activation status and hippocampal neurogenesis, which both present an early but transient change and a late but enduring change. These findings provide evidence that both the stress response and hearing loss contribute to the dynamic alterations of microglia and hippocampal neurogenesis following noise exposure; moreover, noise-induced permanent hearing loss rather than noise-induced transient stress is more likely to be responsible for perpetuating the neurodegenerative process associated with many neurological diseases.

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.

Although fear memory formation is essential for survival and fear-related mental disorders, the neural circuitry and mechanism are incompletely understood. Here, we utilized trace fear conditioning to study the formation of trace fear memory in mice. We identified the entorhinal cortex (EC) as a critical component of sensory signaling to the amygdala. We adopted both loss-of-function and gain-of-function experiments to demonstrate that release of the cholecystokinin (CCK) from the EC is required for trace fear memory formation. We discovered that CCK-positive neurons project from the EC to the lateral nuclei of the amygdala (LA), and inhibition of CCK-dependent signaling in the EC prevented long-term potentiation of the auditory response in the LA and formation of trace fear memory. In summary, high-frequency activation of EC neurons triggers the release of CCK in their projection terminals in the LA, potentiating auditory response in LA neurons. The neural plasticity in the LA leads to trace fear memory formation.

Recent studies have identified sex-differences in auditory physiology and in the susceptibility to noise-induced hearing loss (NIHL). We hypothesize that 17β-estradiol (E2), a known modulator of auditory physiology, may underpin sex-differences in the response to noise trauma. Here, we gonadectomized B6CBAF1/J mice and used a combination of electrophysiological and histological techniques to study the effects of estrogen replacement on peripheral auditory physiology in the absence of noise exposure and on protection from NIHL. Functional analysis of auditory physiology in gonadectomized female mice revealed that E2-treatment modulated the peripheral response to sound in the absence of changes to the endocochlear potential compared to vehicle-treatment. E2-replacement in gonadectomized female mice protected against hearing loss following permanent threshold shift (PTS)- and temporary threshold shift (TTS)-inducing noise exposures. Histological analysis of the cochlear tissue revealed that E2-replacement mitigated outer hair cell loss and cochlear synaptopathy following noise exposure compared to vehicle-treatment. Lastly, using fluorescent in situ hybridization, we demonstrate co-localization of estrogen receptor-2 with type-1C, high threshold spiral ganglion neurons, suggesting that the observed protection from cochlear synaptopathy may occur through E2-mediated preservation of these neurons. Taken together, these data indicate the estrogen signaling pathways may be harnessed for the prevention and treatment of NIHL.

Usher syndrome type I (USH1) is characterized by deafness, vestibular areflexia, and progressive retinal degeneration. The protein-truncating p.Arg245* founder variant of PCDH15 (USH1F) has an ~2% carrier frequency amongst Ashkenazi Jews accounts for ~60% of their USH1 cases. Here, longitudinal phenotyping in 13 USH1F individuals revealed progressive retinal degeneration, leading to severe vision loss with macular atrophy by the sixth decade. Half of the affected individuals were legally blind by their mid-50s. The mouse Pcdh15 R250X variant is equivalent to human p.Arg245*. Homozygous Pcdh15 R250X mice also have visual deficits and aberrant light-dependent translocation of the phototransduction cascade proteins, arrestin, and transducin. Retinal pigment epithelium (RPE)-specific retinoid cycle proteins, RPE65 and CRALBP, were also reduced in Pcdh15 R250X mice, indicating a dual role for protocadherin-15 in photoreceptors and RPE. Exogenous 9- cis retinal improved ERG amplitudes in Pcdh15 R250X mice, suggesting a basis for a clinical trial of FDA-approved retinoids to preserve vision in USH1F patients.

Auditory brainstem response (ABR) serves as an objective indication of auditory perception at a given sound level and is nowadays widely used in hearing function assessment. Despite efforts for automation over decades, ABR threshold determination by machine algorithms remains unreliable and thereby one still relies on visual identification by trained personnel. Here, we described a procedure for automatic threshold determination that can be used in both animal and human ABR tests. The method terminates level averaging of ABR recordings upon detection of time-locked waveform through cross-correlation analysis. The threshold level was then indicated by a dramatic increase in the sweep numbers required to produce "qualified" level averaging. A good match was obtained between the algorithm outcome and the human readouts. Moreover, the method varies the level averaging based on the cross-correlation, thereby adapting to the signal-to-noise ratio of sweep recordings. These features empower a robust and fully automated ABR test.

Little is known about the ontogeny of lingual echolocation. We examined the echolocation development of Rousettus aegyptiacus , the Egyptian fruit bat, which uses rapid tongue movements to produce hyper-short clicks and steer the beam's direction. We recorded from day 0 to day 35 postbirth and assessed hearing and beam-steering abilities. On day 0, R. aegyptiacus pups emit isolation calls and hyper-short clicks in response to acoustic stimuli, demonstrating hearing. Auditory brainstem response recordings show that pups are sensitive to pure tones of the main hearing range of adult Rousettus and to brief clicks. Newborn pups produced clicks in the adult paired pattern and were able to use their tongues to steer the sonar beam. As they aged, pups produced click pairs faster, converging with adult intervals by age of first flights (7–8 weeks). In contrast with laryngeal bats, Rousettus echolocation frequency and duration are stable through to day 35, but shift by the time pups begin to fly, possibly owing to tongue-diet maturation effects. Furthermore, frequency and duration shift in the opposite direction of mammalian laryngeal vocalizations. Rousettus lingual echolocation thus appears to be a highly functional sensory system from birth and follows a different ontogeny from that of laryngeal bats.

Spiral ganglion neurons (SGNs) can be injured by a wide variety of insults. However, there still is a lack of degeneration models to specifically damage the SGNs without disturbing other types of cells in the inner ear. This study aims to generate an SGN-specific damage model using the Cre-LoxP transgenic mouse strains. The Cre-inducible diphtheria toxin receptor ( iDTR +/+ ) knock-in mouse strain was crossed with a mouse strain with Cre activity specific to neurons ( Nefl CreER/CreER ). Expression of the Cre-recombinase activity was evaluated using the reporter mouse strain Ai9 at pre-hearing, hearing onset, and post-hearing stages. Accordingly, heterozygous Nefl CreER/ + ;iDTR +/– mice were treated with tamoxifen on postnatal days 1–5 (P1–5), followed by diphtheria toxin (DT) or vehicle injection on P7, P14, and P21 to evaluate the SGN loss. Robust tamoxifen-induced Cre-mediated Ai9 tdTomato fluorescence was observed in the SGN area of heterozygous Nefl CreER/ + ;Ai9 +/– mice treated with tamoxifen, whereas vehicle-treated heterozygote mice did not show tdTomato fluorescence. Compared to vehicle-treated Nefl CreER/ + ;iDTR +/– mice, DT-treated Nefl CreER/ + ;iDTR +/– mice showed significant auditory brainstem response (ABR) threshold shifts and SGN cell loss. Hair cell count and functional study did not show significant changes. These results demonstrate that the Nefl CreER/CreER mouse strain exhibits inducible SGN-specific Cre activity in the inner ear, which may serve as a valuable SGN damage model for regeneration research of the inner ear.

Age-related hidden hearing loss is often described as a cochlear synaptopathy that results from a progressive degeneration of the inner hair cell (IHC) ribbon synapses. The functional changes occurring at these synapses during aging are not fully understood. Here, we characterized this aging process in IHCs of C57BL/6J mice, a strain which is known to carry a cadherin-23 mutation and experiences early hearing loss with age. These mice, while displaying a large increase in auditory brainstem thresholds due to 50% loss of IHC synaptic ribbons at middle age (postnatal day 365), paradoxically showed enhanced acoustic startle reflex suggesting a hyperacusis-like response. The auditory defect was associated with a large shrinkage of the IHCs' cell body and a drastic enlargement of their remaining presynaptic ribbons which were facing enlarged postsynaptic AMPAR clusters. Presynaptic Ca 2+ microdomains and the capacity of IHCs to sustain high rates of exocytosis were largely increased, while on the contrary the expression of the fast-repolarizing BK channels, known to negatively control transmitter release, was decreased. This age-related synaptic plasticity in IHCs suggested a functional potentiation of synaptic transmission at the surviving synapses, a process that could partially compensate the decrease in synapse number and underlie hyperacusis.

Objectives: This study was designed to determine the morphology changes of noise-induced hidden hearing loss (NIHHL). Method: Fifteen guinea pigs were divided into three groups: noise-induced hidden hearing loss (NIHHL) group, noise-induced hearing loss (NIHL) group, and normal control group. For the noise-induced hidden hearing loss group, the guinea pigs were exposed to 15 times of impulse noise at one time. For the noise-induced hearing loss group, the animals were exposed to a total of 200 times of impulse noise in two times, and the time interval is 24 hours. Auditory brain response (ABR) was tested before, immediately, 24h, 1week, and one month after noise exposure to evaluate cochlear physiology changes. One month after noise exposure, all guinea pigs in three groups were sacrificed, and basement membranes were carefully dissected immediately after ABR tests. The cochlea samples were observed by transmission electron microscopy (TEM) to found out the monograph changes. Result: The ABR results showed that 15 times of impulse noise exposure could cause NIHHL in guinea pigs and 200 times could cause completely hearing loss. Impulse noise exposure could cause a dramatic increase in chondriosome in the inner hair cell. The structures of ribbon synapses and heminodes were also obviously impaired compared to the normal group. The nerve fibers and myelin sheaths remained intact after impulse noise exposure. Conclusion: This research revealed for the first time that impulse noise could cause hidden hearing loss, and the changes in inner hair cells, ribbon synapse, and heminode all played a vital role in the pathogenesis of hidden hearing loss.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters’ cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation ( Klhl18 lowf ) and a targeted mutation ( Klhl18 tm1a(KOMP)Wtsi ). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18 lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18 lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

Hearing loss is a common disability affecting the world’s population today. While several studies have shown that inner ear gene therapy can be successfully applied to mouse models of hereditary hearing loss to improve hearing, most of these studies rely on inner ear gene delivery in the neonatal age, when mouse inner ear has not fully developed. However, the human inner ear is fully developed at birth. Therefore, in order for inner ear gene therapy to be successfully applied in patients with hearing loss, one must demonstrate that gene delivery can be safely and reliably performed in the mature mammalian inner ear. In this study, we examine the steps involved in posterior semicircular canal gene delivery in the adult mouse inner ear. We find that the duration of perilymphatic leakage and injection rate have a significant effect on the post-surgical hearing outcome. Our results show that although AAV2.7m8 has a lower hair cell transduction rate in adult mice compared to neonatal mice at equivalent viral load, AAV2.7m8 is capable of transducing the adult mouse inner and outer hair cells with high efficiency in a dose-dependent manner.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1−/− mice. RNA sequencing analysis (GSE176114) indicated that Dusp1−/− cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1−/− mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1−/− mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation.

Objective: To investigate the hearing protection outcomes of different drug-eluting analog electrode arrays implanted into guinea pig cochleae. Methods: Sixty guinea pigs were randomly divided into a negative control group and five experimental groups implanted separately with blank (drug carrier), dexamethasone (DXM), aracytine (Ara-C), Ara-C+DXM, and nicotinamide adenine dinucleotide (NAD+) eluting analog electrode arrays. Micro CT was used to supervise the surgical procedure. Auditory brainstem response (ABR) thresholds of the guinea pigs were measured and analyzed. Results and conclusions: Compared with the negative control, all other groups showed a significant increase in ABR threshold (p<0.001) after surgery. Among them, there was no obvious difference between the blank (0 vs 90 days: 59.70±10.57 vs 64.60±9.47 dB SPL) and the NAD+ group (0 vs 90 days: 59.90±9.87 vs 64.70±8.65 dB SPL). On the other hand, the ABR thresholds in the DXM (0 days: 58.10±10.73 dB SPL; 90 days: 51.70±9.07 dB SPL) and the Ara-C group (0 days: 59.00±10.05 dB SPL; 90 days: 51.60±8.48 dB SPL) decreased significantly compared with the former two groups (p<0.001). However, the Ara-C+DXM group showed no further benefit (p>0.05). In addition, a significantly higher survival rate of spiral ganglion neurons in cochleae was observed in the Ara-C and/or DXM groups.

The protective effect of the efferent system against acoustic trauma (AT) has been shown by several experimental approaches, including damage to one ear, sectioning of the olivocochlear bundle (OCB) in the floor of the IV ventricle, and knock-in mice overexpressing outer hair cell (OHC) cholinergic receptors, among others. Such effects have been related to changes in the regulation of the cholinergic efferent system and in cochlear amplification, which ultimately reverse upon protective hearing suppression. In addition to well-known circuits of the brainstem, the descending corticofugal pathway also regulates efferent neurons of the olivary complex. In this study, we applied our recently developed experimental paradigm of multiple sessions of electrical stimulation (ES) to activate the efferent system in combination with noise overstimulation. ABR thresholds increased 1 and 2 days after AT (8–16 kHz bandpass noise at 107 dB for 90 min) recovering at AT + 14 days. However, after multiple sessions of epidural anodal stimulation, no changes in thresholds were observed following AT. Although an inflammatory response was also observed 1 day after AT in both groups, the counts of reactive macrophages in both experimental conditions suggest decreased inflammation in the epidural stimulation group. Quantitative immunocytochemistry for choline acetyltransferase (ChAT) showed a significant decrease in the size and optical density of the efferent terminals 1 day after AT and a rebound at 14 days, suggesting depletion of the terminals followed by a long-term compensatory response. Such a synthesis recovery was significantly higher upon cortical stimulation. No significant correlation was found between ChAT optical density and size of the buttons in sham controls (SC) and ES/AT + 1day animals; however, significant negative correlations were shown in all other experimental conditions. Therefore, our comparative analysis suggests that cochleotopic cholinergic neurotransmission is also better preserved after multisession epidural stimulation.

Significance Some animals have evolved an adaptive ability for vocalizing and hearing ultrasonic frequencies that are inaudible to humans (>20 kHz). For decades, it has been postulated that animals hear ultrasonic frequencies with cochlear hair cells using an identical set of mechanotransduction molecules in the hair bundles for hearing audible frequencies. Here, we show that mice lacking mechanosensitive PIEZO2 ion channels have difficulty hearing at ultrasonic frequencies but remain remarkably sensitive to audible frequencies. Thus, animals may use a partially different mechanism for sensing ultrasonic sound emissions.

Neural hyperactivity induced by sound exposure often correlates with the development of hyperacusis and/or tinnitus. In laboratory animals, hyperactivity is typically induced by unilateral sound exposure to preserve one ear for further testing of hearing performance. Most ascending fibers in the auditory system cross into the superior olivary complex and then ascend contralaterally. Therefore, unilateral exposure should be expected to mostly affect the contralateral side above the auditory brain stem. On the other hand, it is well known that a significant number of neurons have crossing fibers at every level of the auditory pathway, which may spread the effect of unilateral exposure onto the ipsilateral side. Here we demonstrate that unilateral sound exposure causes development of hyperactivity in both the contra and ipsilateral inferior colliculus in mice. We found that both the spontaneous firing rate and bursting activity were increased significantly compared to unexposed mice. The neurons with characteristic frequencies at or above the center frequency of exposure showed the greatest increase. Surprisingly, this increase was more pronounced in the ipsilateral inferior colliculus. This study highlights the importance of considering both ipsi- and contralateral effects in future studies utilizing unilateral sound exposure.

Objective To describe and study the population statistics, hearing phenotype, and pathological changes of a porcine congenital single-sided deafness (CSSD) pedigree. Methods Click auditory brainstem response (ABR), full-frequency ABR, and distortion product otoacoustic emission (DPOAE) were used to assess the hearing phenotype of the strain. Tympanogram was used to assess the middle ear function since birth. Celloidin embedding–hematoxylin–eosin (CE-HE) stain and scanning electron microscopy (SEM) were used to study the pathological changes of cochlear microstructures. Chi-square analysis was used to analyze the relation between hearing loss and other phenotypes. Results The mating mood of CSSD with CSSD was most efficient in breeding-targeted CSSD phenotype (47.62%), and the prevalence of CSSD reached 46.67% till the fifth generation, where 42.22% were bilateral hearing loss (BHL) and 9.00% were normal hearing (NH) individuals. Hearing loss was proved to have no relation with coat color ( P = 0.0841 > 0.05) and gender ( P = 0.4621 > 0.05) by chi-square analysis. The deaf side of CSSD offspring in the fifth generation had no relation with that of their maternal parent ( P = 0.2387 > 0.05). All individuals in this strain exhibited congenital severe to profound sensorineural hearing loss with no malformation and dysfunction of the middle ear. The good hearing ear of CSSD stayed stable over age. The deaf side of CSSD and BHL presented cochlear and saccular degeneration, and the hair cell exhibited malformation since birth and degenerated from the apex to base turn through time. The pathology in BHL cochlea progressed more rapidly than CSSD and till P30, the hair cell had been totally gone. The stria vascularis (SV) was normal since birth and degenerated through time and finally exhibited disorganization of three layers of cells. Conclusion This inbred porcine strain exhibited high and stable prevalence of CSSD, which highly resembled human non-syndromic CSSD disease. This porcine model could be used to further explore the etiology of CSSD and serve as an ideal tool for the studies of the effects of single-sided hearing deprivation on neural, cognitive, and behavioral developments and the benefits brought by CI in CSSD individuals.