Publications using the RZ6

Noise-induced hearing loss (NIHL) is a common health concern with significant social, psychological, and cognitive implications. Moderate levels of acoustic overstimulation associated with tinnitus and impaired speech perception cause cochlear synaptopathy, characterized physiologically by reduction in wave I of the suprathreshold auditory brainstem response (ABR) and reduced number of synapses between sensory hair cells and auditory neurons. The unfolded protein response (UPR), an endoplasmic reticulum stress response pathway, has been implicated in the pathogenesis and treatment of NIHL as well as neurodegeneration and synaptic damage in the brain. In this study, we used the small molecule UPR modulator Integrated Stress Response InhiBitor (ISRIB) to treat noise-induced cochlear synaptopathy in a mouse model. Mice pretreated with ISRIB prior to noise-exposure were protected against noise-induced synapse loss. Male, but not female, mice also exhibited ISRIB-mediated protection against noise-induced suprathreshold ABR wave-I amplitude reduction. Female mice had higher baseline wave-I amplitudes but greater sensitivity to noise-induced wave-I reduction. Our results suggest that the UPR is implicated in noise-induced cochlear synaptopathy, and can be targeted for treatment.

Large-scale epidemiological surveys suggest that hearing loss (HL) is a significant risk factor for dementia. We previously showed that noise-induced HL (NIHL) impairs hippocampal cognitive function and decreases hippocampal neurogenesis and neuronal complexity, suggesting a causal role of HL in dementia. To further investigate the influence of acquired peripheral HL on hippocampal neurogenesis with the aging process as well as the underlying mechanism, we produced NIHL in male CBA/J mice and assessed hippocampal neurogenesis and microglial morphology in the auditory brain and hippocampus at 4 days post-noise exposure (DPN) or 1, 3, 6, or 12 months post-noise exposure (MPN) by immunofluorescence labeling. We found that the age-related decline in hippocampal neurogenesis was accelerated in mice with NIHL. Furthermore, in mice with NIHL, prolonged microglial activation occurred from 1 MPN to 12 MPN across multiple auditory nuclei, while aggravated microglial deterioration occurred in the hippocampus and correlated with the age-related decline in hippocampal neurogenesis. These results suggest that acquired peripheral HL accelerates the age-related decline in hippocampal neurogenesis and that hippocampal microglial degeneration may contribute to the development of neurodegeneration following acquired peripheral HL.

Background In addition to the direct effects of irradiation, the induced inflammatory response may play an important role in the damage to the inner ear caused by radiotherapy for the treatment of head and neck cancers. Resolvin E1 (RvE1) has anti-inflammatory activity, acting by reducing neutrophil infiltration and proinflammatory cytokine expression. Therefore, in this study we sought to confirm whether the inflammation induced by irradiation was involved in damage to the inner ear after radiotherapy and to investigate the protective effect and underlying mechanism of RvE1 using mouse models. Methods A dose of RvE1 was delivered by intraperitoneal injection to mice before irradiation. Changes in the auditory brainstem response (ABR), relative balance ability, inner ear morphology and the expression levels of inflammatory factors in the inner ear were analyzed on days 7 and 14 after irradiation and compared among different experimental groups. Results Changes of ABR and relative balance ability showed the inner functions of experimental mice presented severe damage after irradiation, but the damage was significantly alleviated after RvE1 pretreatment compared to irradiation alone. Morphological analysis of the inner ear showed severe damage to the cochlea and vestibule after irradiation. In contrast, damage to the cochlea and vestibule was significantly reduced in the RvE1-pretreated group compared to that in the irradiation alone group. Along with these functional and morphological changes, the mRNA expression level of anti-inflammatory factors interleukin-2 was significantly increased, while those of proinflammatory factors interleukin-6 and tumor necrosis factor-α were significantly decreased in the inner ear of mice after RvE1 pretreatment compared to irradiation alone. Conclusions We believe that inflammation induced by irradiation is involved in the damage to the inner ear caused by radiotherapy, and that RvE1 reduces the damage caused by irradiation to the inner ear by regulating the induced inflammatory response.

Age-related hearing loss (ARHL) is the most common sensory disorder among the elderly, associated with aging and auditory hair cell death due to oxidative-stress-induced mitochondrial dysfunction. Although transgenic mice and long-term aging induction cultures have been used to study ARHL, there are currently no ARHL animal models that can be stimulated by intermittent environmental changes. In this study, an ARHL animal model was established by inducing continuous oxidative stress to promote short-term aging of cells, determined on the basis of expression of hearing-loss-induced phenotypes and aging-related factors. The incidence of hearing loss was significantly higher in dual- and triple-exposure conditions than in intermittent hypoxic conditions, high-fat diet (HFD), or d-galactose injection alone. Continuous oxidative stress and HFD accelerated cellular aging. An increase in Ucp2, usually expressed during mitochondrial dysfunction, was observed. Expression of Cdh23, Slc26a4, Kcnq4, Myo7a, and Myo6, which are ARHL-related factors, were modified by oxidative stress in the cells of the hearing organ. We found that intermittent hypoxia, HFD, and galactose injection accelerated cellular aging in the short term. Thus, we anticipate that the development of this hearing loss animal model, which reflects the effects of intermittent environmental changes, will benefit future research on ARHL.

Human pathogenic variants of TBC1D24 are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human TBC1D24 associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of TBC1D24 c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, Tbc1d24 expression was detected only in spiral ganglion neurons. We engineered mouse models of DFNB86 p.(Asp70Tyr) and DFNA65 p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human TBC1D24. Unexpectedly, no auditory dysfunction was detected in Tbc1d24 mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse Tbc1d24 and human TBC1D24.

Cells of the immune system are present in the adult cochlea and respond to damage caused by noise exposure. However, the types of immune cells involved and their locations within the cochlea are unclear. We used flow cytometry and immunostaining to reveal the heterogeneity of the immune cells in the cochlea and validated the presence of immune cell gene expression by analyzing existing single-cell RNA-sequencing (scRNAseq) data. We demonstrate that cell types of both the innate and adaptive immune system are present in the cochlea. In response to noise damage, immune cells increase in number. B, T, NK, and myeloid cells (macrophages and neutrophils) are the predominant immune cells present. Interestingly, immune cells appear to respond to noise damage by infiltrating the organ of Corti. Our studies highlight the need to further understand the role of these immune cells within the cochlea after noise exposure.

This protocol was used to collect data now published in the Journal of Neural Engineering, Sources of off-target effects of vagus nerve stimulation using the helical clinical lead in domestic pigs, see Metadata for citation. Additionally, the dataset is publicly available on Pennsieve 10.26275/qcuk-a8ty.

The ability to acquire a behavior can be facilitated by exposure to a conspecific demonstrator. Such social learning occurs under a range of conditions in nature. Here, we tested the idea that social learning can benefit from any available sensory cue, thereby permitting learning under different natural conditions. The ability of naïve gerbils to learn a sound discrimination task following 5 days of exposure adjacent to a demonstrator gerbil was tested in the presence or absence of visual cues. Naïve gerbils acquired the task significantly faster in either condition, as compared to controls. We also found that exposure to a demonstrator was more potent in facilitating learning, as compared to exposure to the sounds used to perform the discrimination task. Therefore, social learning was found to be flexible and equally efficient in the auditory or visual domains.

A GWAS study recently demonstrated single nucleotide polymorphisms (SNPs) in the human GLRB gene of individuals with a prevalence for agoraphobia. GLRB encodes the glycine receptor (GlyRs) β subunit. The identified SNPs are localized within the gene flanking regions (3' and 5' UTRs) and intronic regions. It was suggested that these nucleotide polymorphisms modify GlyRs expression and phenotypic behavior in humans contributing to an anxiety phenotype as a mild form of hyperekplexia. Hyperekplexia is a human neuromotor disorder with massive startle phenotypes due to mutations in genes encoding GlyRs subunits. GLRA1 mutations have been more commonly observed than GLRB mutations. If an anxiety phenotype contributes to the hyperekplexia disease pattern has not been investigated yet. Here, we compared two mouse models harboring either a mutation in the murine Glra1 or Glrb gene with regard to anxiety and startle phenotypes. Homozygous spasmodic animals carrying a Glra1 point mutation (alanine 52 to serine) displayed abnormally enhanced startle responses. Moreover, spasmodic mice exhibited significant changes in fear-related behaviors (freezing, rearing and time spent on back) analyzed during the startle paradigm, even in a neutral context. Spastic mice exhibit reduced expression levels of the full-length GlyRs β subunit due to aberrant splicing of the Glrb gene. Heterozygous animals appear normal without an obvious behavioral phenotype and thus might reflect the human situation analyzed in the GWAS study on agoraphobia and startle. In contrast to spasmodic mice, heterozygous spastic animals revealed no startle phenotype in a neutral as well as a conditioning context. Other mechanisms such as a modulatory function of the GlyRs β subunit within glycinergic circuits in neuronal networks important for fear and fear-related behavior may exist. Possibly, in human additional changes in fear and fear-related circuits either due to gene-gene interactions e.g., with GLRA1 genes or epigenetic factors are necessary to create the agoraphobia and in particular the startle phenotype.

Fragile X Syndrome (FXS) is a leading known genetic cause of intellectual disability. Many symptoms of FXS overlap with those in autism including repetitive behaviors, language delays, anxiety, social impairments and sensory processing deficits. Electroencephalogram (EEG) recordings from humans with FXS and an animal model, the Fmr1 knockout (KO) mouse, show remarkably similar phenotypes suggesting that EEG phenotypes can serve as biomarkers for developing treatments. This includes enhanced resting gamma band power and sound evoked total power, and reduced fidelity of temporal processing and habituation of responses to repeated sounds. Given the therapeutic potential of the antibiotic minocycline in humans with FXS and animal models, it is important to determine sensitivity and selectivity of EEG responses to minocycline. Therefore, in this study, we examined if a 10-day treatment of adult Fmr1 KO mice with minocycline (oral gavage, 30 mg/kg per day) would reduce EEG abnormalities. We tested if minocycline treatment has specific effects based on the EEG measurement type (e.g., resting versus sound-evoked) from the frontal and auditory cortex of the Fmr1 KO mice. We show increased resting EEG gamma power and reduced phase locking to time varying stimuli as well as the 40 Hz auditory steady state response in the Fmr1 KO mice in the pre-drug condition. Minocycline treatment increased gamma band phase locking in response to auditory stimuli, and reduced sound-evoked power of auditory event related potentials (ERP) in Fmr1 KO mice compared to vehicle treatment. Minocycline reduced resting EEG gamma power in Fmr1 KO mice, but this effect was similar to vehicle treatment. We also report frequency band-specific effects on EEG responses. Taken together, these data indicate that sound-evoked EEG responses may serve as more sensitive measures, compared to resting EEG measures, to isolate minocycline effects from placebo in humans with FXS. Given the use of minocycline and EEG recordings in a number of neurodegenerative and neurodevelopmental conditions, these findings may be more broadly applicable in translational neuroscience.

The neural mechanisms underlying forward suppression in the auditory cortex remain a puzzle. Little attention is paid to thalamic contribution despite the important fact that the thalamus gates upstreaming information to the auditory cortex. This study compared the time courses of forward suppression in the auditory thalamus, thalamocortical inputs and cortex using the two-tone stimulus paradigm. The preceding and succeeding tones were 20-ms long. Their frequency and amplitude were set at the characteristic frequency and 20 dB above the minimum threshold of given neurons, respectively. In the ventral division of the medial geniculate body of the thalamus, we found that the duration of complete forward suppression was about 75 ms and the duration of partial suppression was from 75 ms to about 300 ms after the onset of the preceding tone. We also found that during the partial suppression period, the responses to the succeeding tone were further suppressed in the primary auditory cortex. The forward suppression of thalamocortical field excitatory postsynaptic potentials was between those of thalamic and cortical neurons but much closer to that of thalamic ones. Our results indicate that early suppression in the cortex could result from complete suppression in the thalamus whereas later suppression may involve thalamocortical and intracortical circuitry. This suggests that the complete suppression that occurs in the thalamus provides the cortex with a "silence" window that could potentially benefit cortical processing and/or perception of the information carried by the preceding sound.

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2 , encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

Introduction: In utero, exposure to sevoflurane (a commonly used inhalation anesthetic) can lead to hearing impairment in offspring mice, but the underlying impairment mechanism is not known. Materials and methods: Day-15 pregnant mice were treated with 2.5% sevoflurane for 2 h to investigate sevoflurane ototoxicity. Cochleae from offspring mice were harvested for hair-cell and ribbon-synapse assessments. Hearing in offspring mice was assessed at postnatal day 30 using an auditory brainstem-response (ABR) test. Cochlear-explant cultures from offspring mice were exposed to 2.5% sevoflurane for 6 h. Immediately after treatment, explants were assessed for hair-cell morphology, mitochondrial oxidative stress, and autophagy. Results: In utero, sevoflurane exposure impaired hearing in the offspring is demonstrated by a decrease in ABR wave I amplitudes, a marker for ribbon-synapse functionality. Sevoflurane exposure caused no obvious damage to hair cells, but cochlear ribbon synapses were reduced in postnatal day 15 offspring, and partially recovered by postnatal day 30. Sevoflurane treatment also increased mitochondrial reactive-oxygen species stress and decreased autophagy in the cochlear explants. Conclusion: These results suggest that oxidative stress and reduced autophagy may underly ribbon-synapse involvement in sevoflurane-induced hearing loss.

Declining auditory spatial processing is hypothesized to contribute to the difficulty older adults have detecting, locating, and selecting a talker from among others in noisy listening environments. Though auditory spatial processing has been associated with several cortical structures, little is known regarding the underlying white matter architecture or how age-related changes in white matter microstructure may affect it. The arcuate fasciculus is a target for understanding age-related differences in auditory spatial attention based on normative spatial attention findings in humans. Similarly, animal and human clinical studies suggest that the corpus callosum plays a role in the cross-hemispheric integration of auditory spatial information important for spatial localization and attention. The current investigation used diffusion imaging to examine the extent to which age-group differences in the identification of spatially cued speech were accounted for by individual differences in the white matter microstructure of the right arcuate fasciculus and the corpus callosum. Higher right arcuate and callosal fractional anisotropy (FA) predicted better segregation and identification of spatially cued speech across younger and older listeners. Further, individual differences in callosal microstructure mediated age-group differences in auditory spatial processing. Follow-up analyses suggested that callosal tracts connecting left and right pre-frontal and posterior parietal cortex are particularly important for auditory spatial processing. The results are consistent with previous work in animals and clinical human samples and provide a cortical mechanism to account for age-related deficits in auditory spatial processing. Further, the results suggest that both intrahemispheric and interhemispheric mechanisms are involved in auditory spatial processing.

Auditory dysfunction is the most prevalent injury associated with blast overpressure exposure (BOP) in Warfighters and civilians, yet little is known about the underlying pathophysiological mechanisms. To gain insights into these injuries, an advanced blast simulator was used to expose rats to BOP and assessments were made to identify structural and molecular changes in the middle/inner ears utilizing otoscopy, RNA sequencing (RNA-seq), and histopathological analysis. Deficits persisting up to 1 month after blast exposure were observed in the distortion product otoacoustic emissions (DPOAEs) and the auditory brainstem responses (ABRs) across the entire range of tested frequencies (4–40 kHz). During the recovery phase at sub-acute time points, low frequency (e.g. 4–8 kHz) hearing improved relatively earlier than for high frequency (e.g. 32–40 kHz). Perforation of tympanic membranes and middle ear hemorrhage were observed at 1 and 7 days, and were restored by day 28 post-blast. A total of 1,158 differentially expressed genes (DEGs) were significantly altered in the cochlea on day 1 (40% up-regulated and 60% down-regulated), whereas only 49 DEGs were identified on day 28 (63% up-regulated and 37% down-regulated). Seven common DEGs were identified at both days 1 and 28 following blast, and are associated with inner ear mechanotransduction, cytoskeletal reorganization, myelin development and axon survival. Further studies on altered gene expression in the blast-injured rat cochlea may provide insights into new therapeutic targets and approaches to prevent or treat similar cases of blast-induced auditory damage in human subjects.

Dopamine guides behavior and learning through pleasure, according to classic understanding. Dopaminergic neurons are traditionally thought to signal positive or negative prediction errors (PEs) when reward expectations are, respectively, exceeded or not matched. These signed PEs are quite different from the unsigned PEs, which report surprise during sensory processing. But mounting theoretical accounts from the predictive processing framework postulate that dopamine, as a neuromodulator, could potentially regulate the postsynaptic gain of sensory neurons, thereby scaling unsigned PEs according to their expected precision or confidence. Despite ample modeling work, the physiological effects of dopamine on the processing of surprising sensory information are yet to be addressed experimentally. In this study, we tested how dopamine modulates midbrain processing of unexpected tones. We recorded extracellular responses from the rat inferior colliculus to oddball and cascade sequences, before, during, and after the microiontophoretic application of dopamine or eticlopride (a D2-like receptor antagonist). Results demonstrate that dopamine reduces the net neuronal responsiveness exclusively to unexpected sensory input without significantly altering the processing of expected input. We conclude that dopaminergic projections from the thalamic subparafascicular nucleus to the inferior colliculus could encode the expected precision of unsigned PEs, attenuating via D2-like receptors the postsynaptic gain of sensory inputs forwarded by the auditory midbrain neurons. This direct dopaminergic modulation of sensory PE signaling has profound implications for both the predictive coding framework and the understanding of dopamine function.

Primary cortical areas contain maps of sensory features, including sound frequency in primary auditory cortex (A1). Two-photon calcium imaging in mice has confirmed the presence of these global tonotopic maps, while uncovering an unexpected local variability in the stimulus preferences of individual neurons in A1 and other primary regions. Here we show that local heterogeneity of frequency preferences is not unique to rodents. Using two-photon calcium imaging in layers 2/3, we found that local variance in frequency preferences is equivalent in ferrets and mice. Neurons with multipeaked frequency tuning are less spatially organized than those tuned to a single frequency in both species. Furthermore, we show that microelectrode recordings may describe a smoother tonotopic arrangement due to a sampling bias towards neurons with simple frequency tuning. These results help explain previous inconsistencies in cortical topography across species and recording techniques.

Language development builds upon a complex network of interacting subservient systems. It therefore follows that variations in, and subclinical disruptions of, these systems may have secondary effects on emergent language. In this paper, we consider the relationship between genetic variants, hearing, auditory processing and language development. We employ whole genome sequencing in a discovery family to target association and gene x environment interaction analyses in two large population cohorts; the Avon Longitudinal Study of Parents and Children (ALSPAC) and UK10K. These investigations indicate that USH2A variants are associated with altered low-frequency sound perception which, in turn, increases the risk of developmental language disorder. We further show that Ush2a heterozygote mice have low-level hearing impairments, persistent higher-order acoustic processing deficits and altered vocalizations. These findings provide new insights into the complexity of genetic mechanisms serving language development and disorders and the relationships between developmental auditory and neural systems.

There are several technical challenges to obtaining high-quality recordings of cochlear potentials in human electrocochleography (ECochG). These challenges include electrical artifacts from devices such as acoustic transducers, biological artifacts from excessive myogenic and electroencephalographic potentials, and issues associated with the placement of a tympanic membrane (TM) electrode on the eardrum. This article presents approaches for dealing with these challenges for ECochG measurement using a TM electrode. Emphasis is placed on eliminating stimulus artifact, optimizing the placement of the electrode, and comparing a custom-made electrode with a commercially-available electrode. This comparison revealed that the custom-made electrode results in greater subject comfort, superior ease of placing the electrode on the eardrum, and larger compound action potential (CAP) amplitudes.

AMP-activated protein kinase (AMPK) integrates the regulation of cell growth and metabolism. AMPK activation occurs in response to cellular energy decline and mitochondrial dysfunction triggered by reactive oxygen species (ROS). In aged Tg-mtTFB1 mice, a mitochondrial deafness mouse model, hearing loss is accompanied with cochlear pathology including reduced endocochlear potential (EP) and loss of spiral ganglion neurons (SGN), inner hair cell (IHC) synapses and outer hair cells (OHC). Accumulated ROS and increased apoptosis signaling were also detected in cochlear tissues, accompanied by activation of AMPK. To further explore the role of AMPK signaling in the auditory phenotype, we used genetically knocked out AMPKα1 as a rescue to Tg-mtTFB1 mice and observed: improved ABR wave I, EP and IHC function, normal SGNs, IHC synapses morphology and OHC survivals, with decreased ROS, reduced pro-apoptotic signaling (Bax) and increased anti-apoptotic signaling (Bcl-2) in the cochlear tissues, indicating that reduced AMPK attenuated apoptosis via ROS-AMPK-Bcl2 pathway in the cochlea. To conclude, AMPK hyperactivation causes accelerated presbycusis in Tg-mtTFB1 mice by redox imbalance and dysregulation of the apoptosis pathway. The effects of AMPK downregulation on pro-survival function and reduction of oxidative stress indicate AMPK serves as a target to rescue or relieve mitochondrial hearing loss.