Although protective effects of the cochlea’s efferent feedback pathways have been well documented, prior work has focused on hair cell damage and cochlear threshold elevation and, correspondingly, on the high sound pressure levels (> 100 dB SPL) necessary to produce them. Here we explore the noise-induced loss of cochlear neurons that occurs with lower intensity exposures and in the absence of permanent threshold shifts. Using confocal microscopy to count synapses between hair cells and cochlear nerve fibers, and using measurement of auditory brainstem responses and otoacoustic emissions to assess cochlear pre- and post-synaptic function, we compare the damage from a weeklong exposure to moderate-level noise (84 dB SPL) in mice with varying degrees of cochlear de-efferentation induced by surgical lesion to the olivocochlear pathway. Such exposure causes minimal acute threshold shift and no chronic shifts in mice with normal efferent feedback. In de-efferented animals, there was up to 40% loss of cochlear nerve synapses and a corresponding decline in the amplitude of the auditory brainstem response. Quantitative analysis of the de-efferentation in inner vs. outer hair cell areas suggested that outer hair cell efferents are most important in minimizing this neuropathy, presumably by virtue of their sound-evoked feedback reduction of cochlear amplification. The moderate nature of this acoustic overexposure suggests that cochlear neurons are at risk even in everyday acoustic environments, and, thus, that the need for cochlear protection is plausible as a driving force in the design of this feedback pathway.
Auditory neuropathy; olivocochlear; hair cells; cochlea; noise; acoustic injury; feedback
Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To re-examine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 days) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that 1) IHCs are not necessary for neuronal survival, 2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and 3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.
Genetic tools available for the mouse make it a powerful model to study the modulation of cochlear function by descending control systems. Suppression of distortion product otoacoustic emission (DPOAE) amplitude by contralateral acoustic stimulation (CAS) provides a robust tool for noninvasively monitoring the strength of descending modulation, yet investigations in mice have been performed infrequently and only under anesthesia, a condition likely to reduce olivocochlear activation. Here, we characterize the contralateral olivocochlear reflex in the alert, unanesthetized mouse. Head-fixed mice were restrained between two closed acoustic systems, while an artifact rejection protocol minimized contamination from self-generated sounds and movements. In mice anesthetized with pentobarbital, ketamine or urethane, CAS at 80 dB SPL evoked, on average, a <1-dB change in DPOAE amplitude. In contrast, the mean CAS-induced DPOAE suppression in unanesthetized mice was nearly 8 dB. Experiments in mice with targeted deletion of the α9 subunit of the nicotinic acetylcholine receptor confirmed the contribution of the medial olivocochlear efferents to this phenomenon. These findings demonstrate the utility of the CAS assay in the unanesthetized mouse and highlight the adverse effects of anesthesia when probing the functional status of descending control pathways within the auditory system.
olivocochlear; corticofugal; arousal; attention; anesthesia; contralateral reflex; outer hair cell
The auditory system transduces sound-evoked vibrations over a range of input sound pressure levels spanning six orders of magnitude. An important component of the system mediating this impressive dynamic range is established in the cochlear sensory epithelium, where functional subtypes of cochlear nerve fibers differ in threshold sensitivity, and spontaneous discharge rate (SR), by more than a factor of 1000 (Liberman, 1978), even though, regardless of type, each fiber contacts only a single hair cell via a single ribbon synapse. To study the mechanisms underlying this remarkable heterogeneity in threshold sensitivity among the 5–30 primary sensory fibers innervating a single inner hair cell, we quantified the sizes of presynaptic ribbons and postsynaptic AMPA receptor patches in >1200 synapses, using high-power confocal imaging of mouse cochleas immunostained for CtBP2 (C-terminal binding protein 2, a major ribbon protein) and GluR2/3 (glutamate receptors 2 and 3). We document complementary gradients, most striking in mid-cochlear regions, whereby synapses from the modiolar face and/or basal pole of the inner hair cell have larger ribbons and smaller receptor patches than synapses located in opposite regions of the cell. The AMPA receptor expression gradient likely contributes to the differences in cochlear nerve threshold and SR seen on the two sides of the hair cell in vivo (Liberman, 1982a); the differences in ribbon size may contribute to the heterogeneity of EPSC waveforms seen in vitro (Grant et al., 2010).
Broadband transient sounds, such as clicks and consonants, activate a traveling wave in the cochlea. This wave evokes firing in auditory nerve fibers that are tuned to high frequencies several milliseconds earlier than in fibers tuned to low frequencies. Despite this substantial traveling wave delay, octopus cells in the brainstem receive broadband input and respond to clicks with submillisecond temporal precision. The dendrites of octopus cells lie perpendicular to the tonotopically organized array of auditory nerve fibers, placing the earliest arriving inputs most distally and the latest arriving closest to the soma. Here, we test the hypothesis that the topographic arrangement of synaptic inputs on dendrites of octopus cells allows octopus cells to compensate the traveling wave delay. We show that in mice the full cochlear traveling wave delay is 1.6 ms. Because the dendrites of each octopus cell spread across about one third of the tonotopic axis, a click evokes a soma directed sweep of synaptic input lasting 0.5 ms in individual octopus cells. Morphologically and biophysically realistic, computational models of octopus cells show that soma-directed sweeps with durations matching in vivo measurements result in the largest and sharpest somatic excitatory postsynaptic potentials (EPSPs). A low input resistance and activation of a low-voltage-activated potassium conductance that are characteristic of octopus cells are important determinants of sweep sensitivity. We conclude that octopus cells have dendritic morphologies and biophysics tailored to accomplish the precise encoding of broadband transient sounds.
ventral cochlear nucleus; octopus cell; cochlea; broadband transient sounds; cable analysis; compartmental modeling; traveling wave delay
In cases of acquired sensorineural hearing loss, death of cochlear neurons is thought to arise largely as a result of sensory-cell loss. However, recent studies of acoustic overexposure report massive degeneration of the cochlear nerve despite complete hair cell survival (Kujawa and Liberman, J Neurosci 29:14077–14085, 2009). To assess the primary loss of spiral ganglion cells (SGCs) in human ears, neuronal counts were performed in 100 temporal bones from 100 individuals, aged newborn to 100 years, selected to include only cases with a normal population of inner and outer hair cells. Ganglion cell counts declined at a mean rate of 100 cells per year of life. There were no significant gender or inter-aural differences, and a slight increase in degeneration in the basal turn re upper turns was not statistically significant. The age-related decline in SGCs was significantly less than that in prior studies that included ears with hair cell loss (Otte et al., Laryngoscope 88:1231–1246, 1978), but significantly more than for analogous data on vestibular ganglion cells in cases without vestibular hair cell loss (Velazquez-Villasenor et al., Ann Otol Rhinol Laryngol Suppl 181:14–19, 2000). The age-related decline in SGC counts may contribute to the well-known decline in hearing-in-noise performance, and the data will help in interpretation of histopathological findings from temporal bones with known otologic disease.
spiral ganglion; cochlear neurons; histopathology; otopathology
Lesion studies of the olivocochlear efferents have suggested that feedback via this neuronal pathway normally maintains an appropriate binaural balance in excitability of the two cochlear nerves (Darrow et al., 2006). If true, a decrease in cochlear nerve output from one ear, due to conductive or sensorineural hearing loss, should change cochlear nerve response in the opposite ear via modulation in olivocochlear feedback. To investigate this putative efferent-mediated interaural coupling, we measured cochlear responses repeatedly from both ears in groups of mice for several weeks before, and for up to 5 weeks after, a unilateral manipulation causing either conductive or sensorineural hearing loss. Response measures included amplitude-vs.-level functions for distortion product otoacoustic emissions (DPOAEs) and auditory brainstem responses (ABRs), evoked at 7 log-spaced frequencies. Ipsilateral manipulations included either tympanic membrane removal or an acoustic overstimulation designed to produce a reversible or irreversible threshold shift over a restricted frequency range. None of these ipsilateral manipulations produced systematic changes in contralateral cochlear responses, either at threshold or suprathreshold levels, either in ABRs or DPOAEs. Thus, we find no evidence for compensatory contralateral changes following ipsilateral hearing loss. We did, however, find evidence for age-related increases in DPOAE amplitudes as animals mature from 6 to 12 weeks and evidence for a slow apical spread of noise-induced threshold shifts, which continues for several days post-exposure.
hearing loss; feedback; acoustic injury
The strength of the medial olivocochlear (OC) reflex is routinely assayed by measuring suppression of ipsilateral responses such as otoacoustic emissions (OAEs) by a brief contralateral noise, e.g. (Berlin et al., 1995). Here, we show in anesthetized guinea pigs, that the magnitude of OC-mediated suppression of ipsilateral cochlear responses (i.e. compound actions potentials (CAPs), distortion product (DP) OAEs and round-window noise) slowly builds over 2–3 minutes during a sustained contralateral noise. The magnitude of this build-up suppression was largest at low ipsilateral stimulus intensities, as seen for suppression measured at contra-noise onset. However, as a function of stimulus frequency, build-up suppression magnitude was complementary to onset suppression, i.e. largest at the lowest and highest frequencies tested. Both build-up and onset suppression were eliminated by cutting the OC bundle. In contrast to “slow effects” of shock-evoked medial OC activity (Sridhar et al., 1995), which are mediated by slow intracellular changes in Ca concentration in OHCs, build-up effects of contralateral noise are immediately extinguished upon OC bundle transection and are likely mediated by central modulation of the response rates in MOC fibers due to the sustained noise. Results suggest that conventional tests of OC reflex strength may underestimate its magnitude in noisy environments.
feedback; inner ear; brainstem plasticity; olivocochlear reflex
Premature birth is a well-known risk factor for sensorineural hearing loss in general and auditory neuropathy in particular. However, relatively little is known about the underlying causes, in part because there are so few relevant histopathological studies. Here, we report on the analysis of hair cell loss patterns in 54 temporal bones from premature infants and a control group of 46 bones from full-term infants, all of whom spent time in the neonatal intensive care unit at the Hospital de Niños in San Jose, Costa Rica, between 1977 and 1993. The prevalence of significant hair cell loss was higher in the preterm group than the full-term group (41% vs. 28%, respectively). The most striking finding was the frequency of selective inner hair cell loss, an extremely rare histopathological pattern, in the preterm vs. the full-term babies (27% vs. 3%, respectively). The findings suggest that a common cause of non-genetic auditory neuropathy is selective loss of inner hair cells rather than primary damage to the cochlear nerve.
auditory neuropathy; deafness; cochlea; histopathology
Recent work in mouse showed that acoustic overexposure can produce a rapid and irreversible loss of cochlear nerve peripheral terminals on inner hair cells (IHCs) and a slow degeneration of spiral ganglion cells, despite full recovery of cochlear thresholds and no loss of inner or outer hair cells (Kujawa and Liberman, J Neurosci 29:14077–14085, 2009). This contrasts with earlier ultrastructural work in guinea pig suggesting that acute noise-induced neural degeneration is followed by full regeneration of cochlear nerve terminals in the IHC area (Puel et al., Neuroreport 9:2109–2114, 1998; Pujol and Puel, Ann N Y Acad Sci 884:249–254, 1999). Here, we show that the same patterns of primary neural degeneration reported for mouse are also seen in the noise-exposed guinea pig, when IHC synapses and cochlear nerve terminals are counted 1 week post-exposure in confocal images from immunostained whole mounts and that the same slow degeneration of spiral ganglion cells occurs despite no loss of IHCs and apparent recovery of cochlear thresholds. The data cast doubt on prior claims that there is significant neural regeneration and synaptogenesis in the adult cochlea and suggest that denervation of the inner hair cell is an important sequela of “reversible” noise-induced hearing loss, which likely applies to the human ear as well.
acoustic injury; excitotoxicity; inner ear
Pharmacological studies suggest that dopamine release from lateral olivocochlear efferent neurons suppresses spontaneous and sound-evoked activity in cochlear nerve fibers and helps control noise-induced excitotoxicity; however, the literature on cochlear expression and localization of dopamine receptors is contradictory. To better characterize cochlear dopaminergic signaling, we studied receptor localization using immunohistochemistry or RT-PCR and assessed histopathology, cochlear responses and olivocochlear function in mice with targeted deletion of each of the five receptor subtypes. In normal ears, D1, D2 and D5 receptors were detected in microdissected immature (P10–P13) spiral ganglion cells and outer hair cells but not inner hair cells. D4 was detected in spiral ganglion cells only. In whole cochlea samples from adults, transcripts for D1, D2, D4 and D5 were present, whereas D3 mRNA was never detected. D1 and D2 immunolabeling was localized to cochlear nerve fibers, near the first nodes of Ranvier (D2) and in the inner spiral bundle region (D1 and D2) where presynaptic olivocochlear terminals are found. No other receptor labeling was consistent. Cochlear function was normal in D3, D4 and D5 knockouts. D1 and D2 knockouts showed slight, but significant enhancement and suppression, respectively, of cochlear responses, both in the neural output (ABR wave 1) and in outer-hair cell function (DPOAEs). Vulnerability to acoustic injury was significantly increased in D2, D4 and D5 lines: D1 could not be tested, and no differences were seen in D3 mutants, consistent with a lack of receptor expression. The increased vulnerability in D2 knockouts was seen in DPOAEs, suggesting a role for dopamine in the OHC area. In D4 and D5 knockouts, the increased noise vulnerability was seen only in ABRs, consistent with a role for dopaminergic signaling in minimizing neural damage.
dopamine; cochlea; hair cell; olivocochlear; acoustic injury
Acetylcholine is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. To study the role of cochlear muscarinic receptors, we studied receptor localization with immunohistochemistry and reverse transcription-PCR and measured olivocochlear function, cochlear responses, and histopathology in mice with targeted deletion of each of the five receptor subtypes. M2, M4, and M5 were detected in microdissected immature (postnatal days 10–13) inner hair cells and spiral ganglion cells but not outer hair cells. In the adult (6 weeks), the same transcripts were found in microdissected organ of Corti and spiral ganglion samples. M2 protein was found, by immunohistochemistry, in olivocochlear fibers in both outer and inner hair cell areas. M3 mRNA was amplified only from whole cochleas, and M1 message was never seen in wild-type ears. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were unaffected by loss of Gq-coupled receptors (M1, M3, or M5), as were shock-evoked olivocochlear effects and vulnerability to acoustic injury. In contrast, loss of Gi-coupled receptors (M2 and/or M4) decreased neural responses without affecting DPOAEs (at low frequencies). This phenotype and the expression pattern are consistent with excitatory muscarinic signaling in cochlear sensory neurons. At high frequencies, both ABRs and DPOAEs were attenuated by loss of M2 and/or M4, and the vulnerability to acoustic injury was dramatically decreased. This aspect of the phenotype and the expression pattern are consistent with a presynaptic role for muscarinic autoreceptors in decreasing ACh release from olivocochlear terminals during high-level acoustic stimulation and suggest that muscarinic antagonists could enhance the resistance of the inner ear to noise-induced hearing loss.
Cochlear sensory cells and neurons receive efferent feedback from the olivocochlear (OC) system. The myelinated medial component of the OC system, and its effects on outer hair cells (OHCs), has been implicated in protection from acoustic injury. The unmyelinated lateral (L)OC fibers target ipsilateral cochlear nerve dendrites, and pharmacological studies suggest the LOC's dopaminergic component may protect these dendrites from excitotoxic effects of acoustic overexposure. Here, we explore LOC function in vivo via selective stereotaxic destruction of LOC cell bodies in mouse. Lesion success in removing the LOC, and sparing the MOC, was assessed by histological analysis of brainstem sections and cochlear whole-mounts. Auditory brainstem responses (ABR), a neural-based metric, and distortion product otoacoustic emissions (DPOAEs), an OHC-based metric, were measured in control and surgical mice. In cases where the LOC was at least partially destroyed, there were increases in suprathreshold neural responses that were frequency- and level-independent, and not attributable to OHC-based effects. These interaural response asymmetries were not found in controls, or in cases where the lesion missed the LOC. In LOC-lesion cases, after exposure to a traumatic stimulus, temporary threshold shifts were greater in the ipsilateral ear, but only when measured in the neural response; OHC-based measurements were always bilaterally symmetric, suggesting OHC vulnerability was unaffected. Interaural asymmetries in threshold shift were not found in either unlesioned controls or in cases which missed the LOC. These findings suggest that the LOC modulates cochlear nerve excitability and protects the cochlea from neural damage in acute acoustic injury.
Cochlea; Excitotoxicity; Noise-induced hearing loss; Feedback control
Immunostaining mouse cochleas for tyrosine hydroxylase (TH) and dopamine β-hydroxylase suggests that there is a rich adrenergic innervation throughout the auditory nerve trunk and a small dopaminergic innervation of the sensory cell areas. Surgical cuts in the brainstem confirm these dopaminergic fibers as part of the olivocochlear efferent bundle. Within the sensory epithelium, TH-positive terminals are seen only in the inner hair cell area, where they intermingle with other olivocochlear terminals expressing cholinergic markers (vesicular acetylcholine transporter; VAT). Double immunostaining suggests little colocalization of TH and VAT; quantification of terminal volumes suggests that TH-positive fibers constitute only 10–20% of the efferent innervation of the inner hair cell area. Immunostaining of mouse brainstem revealed a small population of TH-positive cells in and around the lateral superior olive. Consistent with cochlear projections, double staining for the cholinergic marker acetylcholinesterase suggested that TH-positive somata are not cholinergic and vice versa. All observations are consistent with the view that a small dopaminergic subgroup of lateral olivocochlear neurons 1) projects to the inner hair cell area, 2) is distinct from the larger cholinergic group projecting there, and 3) may correspond to lateral olivocochlear “shell” neurons described by others (Warr et al.  Hear. Res 108:89–111).
tyrosine hydroxylase; colocalization; hearing; feedback control; olivocochlear
Efferent auditory pathways have been implicated in sound localization and its plasticity. We examined the role of the olivocochlear system (OC) in horizontal sound localization by the ferret and in localization learning following unilateral earplugging. Under anesthesia, adult ferrets underwent olivocochlear bundle section at the floor of the fourth ventricle, either at the midline or laterally (left). Lesioned and control animals were trained to localize 1 s and 40ms amplitude-roved broadband noise stimuli from one of 12 loudspeakers. Neither type of lesion affected normal localization accuracy. All ferrets then received a left earplug and were tested and trained over 10 d. The plug profoundly disrupted localization. Ferrets in the control and lateral lesion groups improved significantly during subsequent training on the 1 s stimulus. No improvement (learning) occurred in the midline lesion group. Markedly poorer performance and failure to learn was observed with the 40 ms stimulus in all groups. Plug removal resulted in a rapid resumption of normal localization in all animals. Insertion of a subsequent plug in the right ear produced similar results to left earplugging. Learning in the lateral lesion group was independent of the side of the lesion relative to the earplug. Lesions in all reported cases were verified histologically. The results suggest the OC system is not needed for accurate localization, but that it is involved in relearning localization during unilateral conductive hearing loss.
Two mouse models, the CochG88E/G88E or “knock-in” and the Coch−/− or “knock-out” (Coch null), have been developed to study the human late-onset, progressive, sensorineural hearing loss and vestibular dysfunction known as DFNA9. This disorder results from missense and in-frame deletion mutations in COCH (coagulation factor C homology), encoding cochlin, the most abundantly detected protein in the inner ear. We have performed hearing and vestibular analyses by auditory brainstem response (ABR) and vestibular-evoked potential (VsEP) testing of the Coch−/− and CochG88E/G88E mouse models. Both Coch−/− and CochG88E/G88E mice show substantially elevated ABRs at 21 months of age, but only at the highest frequency tested for the former and all frequencies for the latter. At 21 months, 9 of 11 Coch−/− mice and 4 of 8 CochG88E/G88E mice have absent ABRs. Interestingly Coch−/+ mice do not show hearing deficits, in contrast to CochG88E/+, which demonstrate elevated ABR thresholds similar to homozyotes. These results corroborate the DFNA9 autosomal dominant mode of inheritance, in addition to the observation that haploinsufficiency of Coch does not result in impaired hearing. Vestibular evoked potential (VsEP) thresholds were analyzed using a two factor ANOVA (Age X Genotype). Elevated VsEP thresholds are detected in Coch−/− mice at 13 and 21 months, the two ages tested, and as early as seven months in the CochG88E/G88E mice. These results indicate that in both mouse models, vestibular function is compromised before cochlear function. Analysis and comparison of hearing and vestibular function in these two DFNA9 mouse models, where deficits occur at such an advanced age, provide insight into the pathology of DFNA9 and age-related hearing loss and vestibular dysfunction as well as an opportunity to investigate potential interventional therapies.
Coch; cochlin; DFNA9 mouse models; ABR; VsEP
The vasculature and neurons of the inner ear receive adrenergic innervation from the cervical sympathetic chain, and adrenergic receptors may be expressed by cells of the organ of Corti and stria vascularis, despite a lack of direct sympathetic innervation. To assess the functional role of adrenergic signaling in the auditory periphery, we studied mice with targeted deletion of the gene for dopamine β-hydroxylase (DBH), which catalyzes the conversion of dopamine to noradrenaline; thus, these mutant mice have no measurable adrenaline or noradrenaline. Dbh−/− mice were more susceptible to spontaneous middle-ear infection than their control littermates, consistent with a role for sympathetics in systemic and/or local immune response. At 6–8 weeks of age, cochlear thresholds and suprathreshold responses assessed by auditory brainstem responses and distortion product otoacoustic emissions, as well as light-microscopic morphology, were indistinguishable from controls, if ears with conductive hearing loss were eliminated. Dbh−/− mice were no more susceptible to acoustic injury than controls, despite prior reports that sympathectomy reduces noise damage. Dbh−/− mice showed enhancement of shock-evoked olivocochlear suppression of cochlear responses, which may arise from the loss of adrenergic inputs to olivocochlear neurons in the brainstem. However, adrenergic modulation of olivocochlear efferents does not mediate the protective effect of contralateral cochlear destruction on ipsilateral response to acoustic overexposure.
dopamine β-hydroxylase; sympathetic nervous system; otitis media; olivocochlear
Mutations in whirlin cause either Usher syndrome type II (USH2), a deafness-blindness disorder, or nonsyndromic deafness. The molecular basis for the variable disease expression is unknown. We show here that only the whirlin long isoform, distinct from a short isoform by virtue of having two N-terminal PDZ domains, is expressed in the retina. Both long and short isoforms are expressed in the inner ear. The N-terminal PDZ domains of the long whirlin isoform mediates the formation of a multi-protein complex that includes usherin and VLGR1, both of which are also implicated in USH2. We localized this USH2 protein complex to the periciliary membrane complex (PMC) in mouse photoreceptors that appears analogous to the frog periciliary ridge complex. The latter is proposed to play a role in photoreceptor protein trafficking through the connecting cilium. Mice carrying a targeted disruption near the N-terminus of whirlin manifest retinal and inner ear defects, reproducing the clinical features of human USH2 disease. This is in contrast to mice with mutations affecting the C-terminal portion of whirlin in which the phenotype is restricted to the inner ear. In mice lacking any one of the USH2 proteins, the normal localization of all USH2 proteins is disrupted, and there is evidence of protein destabilization. Taken together, our findings provide new insights into the pathogenic mechanism of Usher syndrome. First, the three USH2 proteins exist as an obligatory functional complex in vivo, and loss of one USH2 protein is functionally close to loss of all three. Second, defects in the three USH2 proteins share a common pathogenic process, i.e., disruption of the PMC. Third, whirlin mutations that ablate the N-terminal PDZ domains lead to Usher syndrome, but non-syndromic hearing loss will result if they are spared.
Usher syndrome is a devastating genetic disorder affecting both vision and hearing. It is classified into three clinical types. Among them, type II (USH2) is the predominant form accounting for about 70% of all Usher syndrome cases. Three genes, USH2A, USH2C, and USH2D, underlie the development of USH2; and they encode usherin, Very Large G protein-coupled Receptor-1 (VLGR1), and whirlin, respectively. In this study, we show that the long whirlin isoform organizes the formation of a multi-protein complex in vivo that includes usherin and VLGR1. Targeted disruption of whirlin long isoform abolishes the normal cellular localization of the two partner USH2 proteins in the retina and in the inner ear and causes visual and hearing defects. We present the first definitive evidence that the USH2 proteins mark the boundary of the periciliary membrane complex, which was first described in frog photoreceptors and is thought to play a role in regulating intracellular protein transport. We propose that defects in all USH2 proteins share a common pathogenic pathway by disrupting the periciliary membrane complex in photoreceptors.
Overexposure to intense sound can cause temporary or permanent hearing loss. Post-exposure recovery of threshold sensitivity has been assumed to indicate reversal of damage to delicate mechano-sensory and neural structures of the inner ear and no persistent or delayed consequences for auditory function. Here we show, using cochlear functional assays and confocal imaging of the inner ear in mouse, that acoustic overexposures causing moderate, but completely reversible, threshold elevation leave cochlear sensory cells intact, but cause acute loss of afferent nerve terminals and delayed degeneration of the cochlear nerve. Results suggest that noise-induced damage to the ear has progressive consequences that are considerably more widespread than are revealed by conventional threshold testing. This primary neurodegeneration should add to difficulties hearing in noisy environments, and could contribute to tinnitus, hyperacusis and other perceptual anomalies commonly associated with inner ear damage.
noise-induced hearing loss; primary neural degeneration; excitotoxicity; synaptic ribbon; spiral ganglion cell; noise damage risk
Despite pharmacological and immunohistochemical evidence for GABA as a neurotransmitter in the olivocochlear efferent bundle, a clear functional role of GABA in the inner ear has not emerged. To explore the role of metabotropic GABAB receptors, we characterized the cochlear phenotype of mice with targeted deletion of the GABAB1 subunit and determined its tissue localization using a mouse line expressing a GFP-tagged GABAB1 subunit under the endogenous promoter. Immunostaining revealed GABAB1 expression in both type I and type II ganglion cells and in their synaptic terminals under inner and outer hair cells, respectively. No GABAB1 expression was observed in hair cells. Mean cochlear thresholds, measured via both auditory brainstem responses and distortion product otoacoustic emissions (DPOAEs), were elevated by ∼10 dB in GABAB1-deficient mice, consistent with outer hair cell dysfunction. Olivocochlear efferent function, assessed via DPOAE suppression during efferent electrical stimulation, was unaffected by GABAB1 deletion. GABAB1-deficient mice showed increased resistance to permanent acoustic injury, with mean threshold shifts ∼25 dB smaller than wild-types after exposure to 8–16-kHz noise at 100 dB for 2 h. In contrast, there was no vulnerability difference to temporary acoustic injury following exposure to the same noise at 94 dB for 15 min. Our results suggest that GABAergic signaling in type II afferent neurons may be required for normal outer hair cell amplifier function at low sound levels and may also modulate outer hair cell responses to high-level sound.
inner ear; feedback; efferent
Cochlear hair cells use SK2 currents to shape responses to cholinergic efferent feedback from the brain. Using SK2-/- mice, we demonstrate that, in addition to their previously defined role in modulating hair cell membrane potentials, SK2 channels are necessary for long-term survival of olivocochlear fibers and synapses. Loss of the SK2 gene also results in loss of electrically driven olivocochlear effects in vivo, and down regulation of ryanodine receptors involved in calcium-induced calcium release, the main inducer of nAChR evoked SK2 activity. Generation of double-null mice lacking both the α10 nAChR gene, loss of which results in hypertrophied olivocochlear terminals, and the SK2 gene, recapitulates the SK2-/- synaptic phenotype and gene expression, and also leads to down regulation of α9 nAChR gene expression. The data suggest a hierarchy of activity necessary to maintain early olivocochlear synapses at their targets, with SK2 serving an epistatic, upstream, role to the nAChRs.
cochlea; small conductance potassium channels; nicotinic receptors; synaptic degeneration; synaptogenesis
Cochlear outer hair cells (OHCs) serve both as sensory receptors and biological motors. Their sensory function is poorly understood because their afferent innervation, the type-II spiral ganglion cell, has small unmyelinated axons and constitutes only 5% of the cochlear nerve. Reciprocal synapses between OHCs and their type-II terminals, consisting of paired afferent and efferent specialization, have been described in the primate cochlea. Here, we use serial and semi-serial-section transmission electron microscopy to quantify the nature and number of synaptic interactions in the OHC area of adult cats. Reciprocal synapses were found in all OHC rows and all cochlear frequency regions. They were more common among third-row OHCs and in the apical half of the cochlea, where 86% of synapses were reciprocal. The relative frequency of reciprocal synapses was unchanged following surgical transection of the olivocochlear bundle in one cat, confirming that reciprocal synapses were not formed by efferent fibers. In the normal ear, axo-dendritic synapses between olivocochlear terminals and type-II terminals and/or dendrites were as common as synapses between olivocochlear terminals and OHCs, especially in the first row, where, on average, almost 30 such synapses were seen in the region under a single OHC. The results suggest that a complex local neuronal circuitry in the OHC area, formed by the dendrites of type-II neurons and modulated by the olivocochlear system, may be a fundamental property of the mammalian cochlea, rather than a curiosity of the primate ear. This network may mediate local feedback control of, and bidirectional communication among, OHCs throughout the cochlear spiral.
inner ear; auditory nerve; spiral ganglion
Mutations in COCH (coagulation factor C homology) are etiologic for the late-onset, progressive, sensorineural hearing loss and vestibular dysfunction known as DFNA9. We introduced the G88E mutation by gene targeting into the mouse and have created a CochG88E/G88E mouse model for the study of DFNA9 pathogenesis and cochlin function. Vestibular-evoked potential (VsEP) thresholds of CochG88E/G88E mice were elevated at all ages tested compared with wild-type littermates. At the oldest ages, two out of eight CochG88E/G88E mice had no measurable VsEP. Auditory brainstem response (ABR) thresholds of CochG88E/G88E mice were substantially elevated at 21 months but not at younger ages tested. At 21 months, four of eight CochG88E/G88E mice had absent ABRs at all frequencies tested and two of three CochG88E/+ mice had absent ABRs at three of four frequencies tested. Distortion product otoacoustic emission amplitudes of CochG88E/G88E mice were substantially lower than Coch+/+ mice and absent in the same CochG88E/G88E mice with absent ABRs. These results suggest that vestibular function is affected beginning as early as 11 months when cochlear function appears to be normal, and dysfunction increases with age. Hearing loss declines substantially at 21 months of age and progresses to profound hearing loss at some to all frequencies tested. This is the only mouse model developed to date where hearing loss begins at such an advanced age, providing an opportunity to study both progressive age-related hearing loss and possible interventional therapies.
Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.
mimecan/osteoglycin; proteoglycan; hearing loss
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9′T line of knockin mice with a threonine for leucine change (L9′T) at position 9′ of the second transmembrane domain of the α9 nicotinic cholinergic subunit, rendering α9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9′T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9′T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the α9α10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9L9′T/L9′T mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter α9α10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.
Nicotinic cholinergic receptors are essential to higher order brain function. Structurally, these operate through a myriad of ligand-gated pentameric arrangements of different homologous subunits. Here, we report progress in understanding the structural properties of a neuronal nicotinic receptor at the synapse. Receptors assembled from two nicotinic cholinergic subunits (α9 and α10) serve exclusively at the synapse between central nervous system descending fibers and cochlear hair cells. This enabled us to show direct causality between a point mutation of the α9 subunit, and predicted alterations in the synaptic strength in sensory hair cells of the cochlea of α9 point mutant mice. Furthermore, this single mutation results in profound enhancement of central nervous system feedback to the cochlea. And finally, as a consequence, mutant mice possessing this altered receptor have substantially improved resistance to traumatic sound. Thus, central neuronal feedback on cochlear hair cells provides an opportunity to define one specific role that nicotinic receptors can play in the nervous system, enabling study from biophysical to behavioral levels and promoting a target for the prevention of noise-induced hearing loss.
A point mutation in the cochlear hair cell nicotinic cholinergic receptor leads to strengthened central nervous system feedback to the cochlea and enhances protection from noise-induced hearing loss.