Auditory Research publications

The contactin-associated protein-like 2 gene, CNTNAP2, is a highly penetrant risk gene thought to play a role in the genetic etiology of language-related disorders, such as autism spectrum disorder and developmental language disorder. Despite its candidacy for influencing language development, few preclinical studies have examined the role of CNTNAP2 in auditory processing. Using in vivo and in vitro electrophysiological recordings in a rat model with translational validity, we report that a loss of the Cntnap2 gene function caused immature-like cortical evoked potentials, delayed multiunit response latencies to acoustic stimuli, impaired temporal processing, and led to a pattern of hyperexcitability in both multiunit and single cell recordings in adulthood. These collective results provide direct evidence that a constitutive loss of Cntnap2 gene function in rats can cause auditory processing impairments similar to those seen in language-related human disorders, indicating that its contribution in maintaining cortical neuron excitability may underlie the cortical activity alterations observed in Cntnap2−/− rats.

Mammalian hearing involves the mechanoelectrical transduction (MET) of sound-induced fluid waves in the cochlea. Essential to this process are the specialised sensory cochlear cells, the inner (IHCs) and outer hair cells (OHCs). While genetic hearing loss is highly heterogeneous, understanding the requirement of each gene will lead to a better understanding of the molecular basis of hearing and also to therapeutic opportunities for deafness. The Neuroplastin ( Nptn ) gene, which encodes two protein isoforms Np55 and Np65, is required for hearing, and homozygous loss-of-function mutations that affect both isoforms lead to profound deafness in mice. Here we have utilised several distinct mouse models to elaborate upon the spatial, temporal, and functional requirement of Nptn for hearing. While we demonstrate that both Np55 and Np65 are present in cochlear cells, characterisation of a Np65-specific mouse knockout shows normal hearing thresholds indicating that Np65 is functionally redundant for hearing. In contrast, we find that Nptn -knockout mice have significantly reduced maximal MET currents and MET channel open probabilities in mature OHCs, with both OHCs and IHCs also failing to develop fully mature basolateral currents. Furthermore, comparing the hearing thresholds and IHC synapse structure of Nptn- knockout mice with those of mice that lack Nptn only in IHCs and OHCs shows that the majority of the auditory deficit is explained by hair cell dysfunction, with abnormal afferent synapses contributing only a small proportion of the hearing loss. Finally, we show that continued expression of Neuroplastin in OHCs of adult mice is required for membrane localisation of Plasma Membrane Ca 2+ ATPase 2 (PMCA2), which is essential for hearing function. Moreover, Nptn haploinsufficiency phenocopies Atp2b2 (encodes PMCA2) mutations, with heterozygous Nptn -knockout mice exhibiting hearing loss through genetic interaction with the Cdh23 ahl allele. Together, our findings provide further insight to the functional requirement of Neuroplastin for mammalian hearing.

Sensory hypersensitivity, especially in the auditory system, is a common symptom in Fragile X syndrome (FXS), the most common monogenic form of intellectual disability. However, linking phenotypes across genetic background strains of mouse models has been a challenge and could underly some of the issues with translatability of drug studies to the human condition. This study is the first to characterize the auditory brain stem response (ABR), a minimally invasive physiological readout of early auditory processing that is also used in humans, in a commonly used mouse background strain model of FXS, C57BL/6J. We measured morphological features of pinna and head and used ABR to measure the hearing range, and monaural and binaural auditory responses in hemizygous males, homozygous females, and heterozygous females compared with those in wild-type mice. Consistent with previous study, we showed no difference in morphological parameters across genotypes or sexes. There was no significant difference in hearing range between the sexes or genotypes, however there was a trend towards high frequency hearing loss in male FXS mice. In contrast, female mice with homozygous FXS had a decreased amplitude of wave IV of the monaural ABR, while there was no difference in males for amplitudes and no change in latency of ABR waveforms across sexes and genotypes. Finally, males with FXS had an increased latency of the binaural interaction component (BIC) at 0 interaural timing difference compared with that in wild-type males. These findings further clarify auditory brain stem processing in FXS by adding more information across genetic background strains allowing for a better understanding of shared phenotypes.

Outer hair cell (OHC) degeneration is a major cause of progressive hearing loss and presbycusis. Despite the high prevalence of these disorders, targeted therapy is currently not available. Methods: We generated a mouse model harboring Kcnq4W276S/+ to recapitulate DFNA2, a common genetic form of progressive hearing loss accompanied by OHC degeneration. After comprehensive optimization of guide RNAs, Cas9s, vehicles, and delivery routes, we applied in vivo gene editing strategy to disrupt the dominant-negative allele in Kcnq4 and prevent progressive hearing loss. Results:In vivo gene editing using a dual adeno-associated virus package targeting OHCs significantly improved auditory thresholds in auditory brainstem response and distortion-product otoacoustic emission. In addition, we developed a new live-cell imaging technique using thallium ions to investigate the membrane potential of OHCs and successfully demonstrated that mutant allele disruption resulted in more hyperpolarized OHCs, indicating elevated KCNQ4 channel activity. Conclusion: These findings can facilitate the development of targeted therapies for DFNA2 and support the use of CRISPR-based gene therapy to rectify defects in OHCs.

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo . In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

Offset responses in auditory processing appear after a sound terminates. They arise in neuronal circuits within the peripheral auditory system, but their role in the central auditory system remains unknown. Here, we ask what the behavioral relevance of cortical offset responses is and what circuit mechanisms drive them. At the perceptual level, our results reveal that experimentally minimizing auditory cortical offset responses decreases the mouse performance to detect sound termination, assigning a behavioral role to offset responses. By combining in vivo electrophysiology in the auditory cortex and thalamus of awake mice, we also demonstrate that cortical offset responses are not only inherited from the periphery but also amplified and generated de novo. Finally, we show that offset responses code more than silence, including relevant changes in sound trajectories. Together, our results reveal the importance of cortical offset responses in encoding sound termination and detecting changes within temporally discontinuous sounds crucial for speech and vocalization.

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.

Biological and mechanical systems, whether by their overuse or their aging, will inevitably fail. Hearing provides a poignant example of this with noise-induced and age-related hearing loss. Hearing loss is not unique to humans, however, and is experienced by all animals in the face of wild and eclectic differences in ear morphology and operation. Here we exploited the high throughput and accessible tympanal ear of the desert locust, Schistocerca gregaria (mixed sex) to rigorously quantify changes in the auditory system due to noise exposure (3 kHz pure tone at 126 dB SPL) and age. We analysed tympanal dispalcements, morphology of the auditory Müller’s organ and measured activity of the auditory nerve, the transduction current and electrophysiological properties of individual auditory receptors. We found that noise mildly and transiently changes tympanal displacements, decreases both the width of the auditory nerve and the transduction current recorded from individual auditory neurons. Whereas age – but not noise - decreases the number of auditory neurons and increases their resting potential. Multiple other properties of Müller’s organ were unaffected by either age or noise including: the number of supporting cells in Müller’s organ or the nerve, membrane resistance and capacitance of the auditory neurons. The sound-evoked activity of the auditory nerve decreased as a function of age and this decrease was exacerbated by noise, with the largest difference during the middle of their life span. This ‘middle-aged deafness’ pattern of hearing loss mirrors that found for humans exposed to noise early in their life. Key point summary Age and noise lead to hearing loss. Tympanal displacements have a transient and delayed reduction after noise. The number of auditory neurons decreases with age. The width of the auditory nerve is reduced with noise exposure. Locusts repeatedly exposed to noise lost their hearing compared to silent controls, but their hearing became similar to old silent control locusts due to age-related hearing loss dominating. The electrophysiological properties of the auditory neurons and the transduction current remained unchanged for aged locusts but repeated noise exposure led to a cumulative decrease in the transduction current.

Binaural hearing is critically important for the perception of sound spatial locations. The primary auditory cortex (AI) has been demonstrated to be necessary for sound localization. However, after hearing onset, how the processing of binaural cues by AI neurons develops, and how the binaural processing of AI neurons is affected by reversible unilateral conductive hearing loss (RUCHL), are not fully elucidated. Here, we determined the binaural processing of AI neurons in four groups of rats: postnatal day (P) 14–18 rats, P19–30 rats, P57–70 adult rats, and RUCHL rats (P57–70) with RUCHL during P14–30. We recorded the responses of AI neurons to both monaural and binaural stimuli with variations in interaural level differences (ILDs) and average binaural levels. We found that the monaural response types, the binaural interaction types, and the distributions of the best ILDs of AI neurons in P14–18 rats are already adult-like. However, after hearing onset, there exist developmental refinements in the binaural processing of AI neurons, which are exhibited by the increase in the degree of binaural interaction, and the increase in the sensitivity and selectivity to ILDs. RUCHL during early hearing development affects monaural response types, decreases the degree of binaural interactions, and decreases both the selectivity and sensitivity to ILDs of AI neurons in adulthood. These new evidences help us to understand the refinements and plasticity in the binaural processing of AI neurons during hearing development, and might enhance our understanding in the neuronal mechanism of developmental changes in auditory spatial perception.

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.

The medial olivocochlear (MOC) efferent feedback circuit projecting to the cochlear outer hair cells (OHCs) confers protection from noise-induced hearing loss and is generally thought to be driven by inner hair cell (IHC) - type I spiral ganglion afferent (SGN) input. Knockout of the Prph gene ( Prph KO) encoding the peripherin type III intermediate filament disrupted the OHC - type II SGN innervation and virtually eliminated MOC – mediated contralateral suppression from noise delivered to the opposite ear, measured as a reduction in cubic distortion product otoacoustic emissions. Electrical stimulation of the MOC pathway elicited contralateral suppression indistinguishable between wildtype (WT) and Prph KO mice, indicating that the loss of contralateral suppression was not due to disruption of the efferent arm of the circuit; IHC – type I SGN input was also normal, based on auditory brainstem responses. High-intensity, broadband noise (108 dB SPL, 1 hour) produced permanent hearing loss in Prph KO mice, but not in WT littermates. These findings associate OHC-type II input with MOC efferent - based otoprotection at loud sound levels.

The binaural interaction component (BIC) is a sound-evoked electrophysiological signature of binaural processing in the auditory brainstem that has received attention as a potential biomarker for spatial hearing deficits. Yet the number of trials necessary to evoke the BIC, or its measurability, seems to vary across species: while it is easily measured in small rodents, it has proven to be highly variable and less reliably measured in humans. This has hindered its potential use as a diagnostic tool. Further measurements of the BIC across a wide range of species could help us better understand its origin and the possible reasons for the variation in its measurability. Statistical analysis on the function relating BIC DN1 amplitude and the interaural time difference has been performed in only a few small rodent species, thus it remains to be shown how the results apply to more taxonomically diverse mammals, and those with larger heads. To fill this gap, we measured BICs in rhesus macaque. We show the overall behavior of the BIC is the same as in smaller rodents, suggesting that the brainstem circuit responsible for the BIC is conserved across a wider range of mammals. We suggest that differences in measurability are likely because of differences in head size.

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.

Unlike olfaction, taste, touch, vision, and proprioception, which are widespread across animal phyla, hearing is found only in vertebrates and some arthropods. The vast majority of invertebrate species are thus considered insensitive to sound. Here, we challenge this conventional view by showing that the earless nematode C. elegans senses airborne sound at frequencies reaching the kHz range. Sound vibrates C. elegans skin, which acts as a pressure-to-displacement transducer similar to vertebrate eardrum, activates sound-sensitive FLP/PVD neurons attached to the skin, and evokes phonotaxis behavior. We identified two nAChRs that transduce sound signals independently of ACh, revealing an unexpected function of nAChRs in mechanosensation. Thus, the ability to sense airborne sound is not restricted to vertebrates and arthropods as previously thought, and might have evolved multiple times independently in the animal kingdom, suggesting convergent evolution. Our studies also demonstrate that animals without ears may not be presumed to be sound insensitive.

Hearing loss is the most common human sensory deficit. Hearing relies on stereocilia, inserted into the cuticular plate of hair cells (HCs), where they play an important role in the perception of sound and its transmission. Although numerous genes have been associated with hearing loss, the function of many hair cell genes has yet to be elucidated. Herein, we focused on nonerythroid spectrin αII (SPTAN1), abundant in the cuticular plate, surrounding the rootlets of stereocilia and along the plasma membrane. Interestingly, mice with HC-specific Sptan1 knockout exhibited rapid deafness, abnormal formation of stereocilia and cuticular plates, and loss of HCs from middle and apical turns of the cochlea during early postnatal stages. Additionally, Sptan1 deficiency led to the decreased spreading of House Ear Institute-Organ of Corti 1 cells, and induced abnormal formation of focal adhesions and integrin signaling in mouse HCs. Altogether, our findings highlight SPTAN1 as a critical molecule for HC stereocilia morphology and auditory function via regulation of focal adhesion signaling.

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.

Background Individuals with Fragile X syndrome (FXS) and autism spectrum disorder (ASD) exhibit an array of symptoms, including sociability deficits, increased anxiety, hyperactivity, and sensory hyperexcitability. It is unclear how endocannabinoid (eCB) modulation can be targeted to alleviate neurophysiological abnormalities in FXS as behavioral research reveals benefits to inhibiting cannabinoid (CB) receptor activation and increasing endocannabinoid ligand levels. Here, we hypothesize that enhancement of 2-arachidonoyl-sn-glycerol (2-AG) in Fragile X mental retardation 1 gene knock-out ( Fmr1 KO) mice may reduce cortical hyperexcitability and behavioral abnormalities observed in FXS. Methods To test whether an increase in 2-AG levels normalized cortical responses in a mouse model of FXS, animals were subjected to electroencephalography (EEG) recording and behavioral assessment following treatment with JZL-184, an irreversible inhibitor of monoacylglycerol lipase (MAGL). Assessment of 2-AG was performed using lipidomic analysis in conjunction with various doses and time points post-administration of JZL-184. Baseline electrocortical activity and evoked responses to sound stimuli were measured using a 30-channel multielectrode array (MEA) in adult male mice before, 4 h, and 1 day post-intraperitoneal injection of JZL-184 or vehicle. Behavior assessment was done using the open field and elevated plus maze 4 h post-treatment. Results Lipidomic analysis showed that 8 mg/kg JZL-184 significantly increased the levels of 2-AG in the auditory cortex of both Fmr1 KO and WT mice 4 h post-treatment compared to vehicle controls. EEG recordings revealed a reduction in the abnormally enhanced baseline gamma-band power in Fmr1 KO mice and significantly improved evoked synchronization to auditory stimuli in the gamma-band range post-JZL-184 treatment. JZL-184 treatment also ameliorated anxiety-like and hyperactivity phenotypes in Fmr1 KO mice. Conclusions Overall, these results indicate that increasing 2-AG levels may serve as a potential therapeutic approach to normalize cortical responses and improve behavioral outcomes in FXS and possibly other ASDs.

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.

Slow waves and cognitive output have been modulated in humans by phase-targeted auditory stimulation. However, to advance its technical development and further our understanding, implementation of the method in animal models is indispensable. Here, we report the successful employment of slow waves’ phase-targeted closed-loop auditory stimulation (CLAS) in rats. To validate this new tool both conceptually and functionally, we tested the effects of up- and down-phase CLAS on proportions and spectral characteristics of sleep, and on learning performance in the single-pellet reaching task, respectively. Without affecting 24 hr sleep-wake behavior, CLAS specifically altered delta (slow waves) and sigma (sleep spindles) power persistently over chronic periods of stimulation. While up-phase CLAS does not elicit a significant change in behavioral performance, down-phase CLAS exerted a detrimental effect on overall engagement and success rate in the behavioral test. Overall CLAS-dependent spectral changes were positively correlated with learning performance. Altogether, our results provide proof-of-principle evidence that phase-targeted CLAS of slow waves in rodents is efficient, safe, and stable over chronic experimental periods, enabling the use of this high-specificity tool for basic and preclinical translational sleep research.

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.