Onset Of Hearing Research Articles

Phosphoinositide Modulation of Heteromeric Kv1 Channels Adjusts Output of Spiral Ganglion Neurons from Hearing Mice.

Rapid spike adaptation is an important feature of auditory neurons that preserves temporal precision. In spiral ganglion neurons, the primary afferents in the cochlea, adaptation is regulated by heteromeric ion channels composed of Kv1.1 and Kv1.2 subunits. These subunits colocalize to common functional microdomains, such as juxtaparanodes and the somatic membrane. Activity of the heteromeric channels is controlled by cellular availability of PIP2, a membrane phospholipid. This mechanism provides an intrinsic regulation of output from the auditory nerve, which could be targeted for therapeutic adjustment of hearing sensitivity.

Re-Emergent Inhibition of Cochlear Inner Hair Cells in a Mouse Model of Hearing Loss.

Synaptic changes in the auditory periphery are increasingly recognized as important factors in hearing loss. To date, anatomical work has described the loss of afferent contacts from cochlear hair cells. However, relatively little is known about the efferent innervation of the cochlea during hearing loss. We performed intracellular recordings from mouse inner hair cells across the lifespan and show that efferent innervation of inner hair cells arises in parallel with the loss of afferent contacts and elevated hearing threshold during aging. These efferent neurons inhibit inner hair cells, raising the possibility that they play a role in the progression of age-related hearing loss.

Adenomatous Polyposis Coli Protein Deletion in Efferent Olivocochlear Neurons Perturbs Afferent Synaptic Maturation and Reduces the Dynamic Range of Hearing.

This study identifies novel molecules and cellular interactions that are essential for the proper maturation of afferent ribbon synapses in sensory cells of the inner ear, and for normal hearing.

The Structural Development of the Mouse Dorsal Cochlear Nucleus.

The dorsal cochlear nucleus (DCN) is a major subdivision of the mammalian cochlear nucleus (CN) that is thought to be involved in sound localization in the vertical plane and in feature extraction of sound stimuli. The main principal cell type (pyramidal cells) integrates auditory and non-auditory inputs, which are considered to be important in performing sound localization tasks. This study aimed to investigate the histological development of the CD-1 mouse DCN, focussing on the postnatal period spanning the onset of hearing (P12). Fluorescent Nissl staining revealed that the three layers of the DCN were identifiable as early as P6 with subsequent expansion of all layers with age. Significant increases in the size of pyramidal and cartwheel cells were observed between birth and P12. Immunohistochemistry showed substantial changes in synaptic distribution during the first two postnatal weeks with subsequent maturation of the presumed mossy fibre terminals. In addition, GFAP immunolabelling identified several glial cell types in the DCN including the observation of putative tanycytes for the first time. Each glial cell type had specific spatial and temporal patterns of maturation with apparent rapid development during the first two postnatal weeks but little change thereafter. The rapid maturation of the structural organization and DCN components prior to the onset of hearing possibly reflects an influence from spontaneous activity originating in the cochlea/auditory nerve. Further refinement of these connections and development of the non-auditory connections may result from the arrival of acoustic input and experience dependent mechanisms.

Cellular glutathione content in the organ of Corti and its role during ototoxicity.

Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation.

Specific synaptopathies diversify brain responses and hearing disorders: you lose the gain from early life.

Before hearing onset, inner hair cell (IHC) maturation proceeds under the influence of spontaneous Ca2+ action potentials (APs). The temporal signature of the IHC Ca2+ AP is modified through an efferent cholinergic feedback from the medial olivocochlear bundle (MOC) and drives the IHC pre- and post-synapse phenotype towards low spontaneous (spike) rate (SR), high-threshold characteristics. With sensory experience, the IHC pre- and post-synapse phenotype matures towards the instruction of low-SR, high-threshold and of high-SR, low-threshold auditory fiber characteristics. Corticosteroid feedback together with local brain-derived nerve growth factor (BDNF) and catecholaminergic neurotransmitters (dopamine) might be essential for this developmental step. In this review, we address the question of whether the control of low-SR and high-SR fiber characteristics is linked to various degrees of vulnerability of auditory fibers in the mature system. In particular, we examine several IHC synaptopathies in the context of various hearing disorders and exemplified shortfalls before and after hearing onset.

Mechanisms of Mammalian Middle Ear Ossicle Transition from the Reptilian Jaw Joint

During synapsid evolution, postdentary elements in the reptilian jaw transitioned into the middle ear of early mammals. Separation from the dentary allowed unconstrained evolution of the middle ear ossicles, resulting in increased hearing sensitivity and amplified frequency range. Given the importance of such an innovation, it is essential to understand the evolutionary pathway that led to the current phenotype, as this has implications for understanding the development of all mammals. Using an extant model system, Monodelphis domestica, we investigate the developmental basis of the definitive mammalian middle ear. We utilize micro‐CT imaging to characterize the morphological events underpinning the transition, cryosectioning and immunohistochemistry to identify cellular processes behind the morphological events, and laser capture microscopy followed by RNA Sequencing to identify changes in gene expression driving the cellular processes. Among our findings are: decreasing size and rearward movement of the middle ear ossicles appear to be false illusions created by the continued growth and expansion of the surrounding skull elements; separation of Meckel's cartilage from the malleus, occurring at postnatal day 20, is facilitated by apoptosis and is a prerequisite for the onset of hearing; separation and functional changes are due to alterations in timing and expression levels of key genes.Supported by NSF Graduate Research Fellowship and Doctoral Dissertation Improvement Grant.

Calcium-Induced Calcium Release during Action Potential Firing in Developing Inner Hair Cells

In the mature auditory system, inner hair cells (IHCs) convert sound-induced vibrations into electrical signals that are relayed to the central nervous system via auditory afferents. Before the cochlea can respond to normal sound levels, developing IHCs fire calcium-based action potentials that disappear close to the onset of hearing. Action potential firing triggers transmitter release from the immature IHC that in turn generates experience-independent firing in auditory neurons. These early signaling events are thought to be essential for the organization and development of the auditory system and hair cells. A critical component of the action potential is the rise in intracellular calcium that activates both small conductance potassium channels essential during membrane repolarization, and triggers transmitter release from the cell. Whether this calcium signal is generated by calcium influx or requires calcium-induced calcium release (CICR) is not yet known. IHCs can generate CICR, but to date its physiological role has remained unclear. Here, we used high and low concentrations of ryanodine to block or enhance CICR to determine whether calcium release from intracellular stores affected action potential waveform, interspike interval, or changes in membrane capacitance during development of mouse IHCs. Blocking CICR resulted in mixed action potential waveforms with both brief and prolonged oscillations in membrane potential and intracellular calcium. This mixed behavior is captured well by our mathematical model of IHC electrical activity. We perform two-parameter bifurcation analysis of the model that predicts the dependence of IHCs firing patterns on the level of activation of two parameters, the SK2 channels activation and CICR rate. Our data show that CICR forms an important component of the calcium signal that shapes action potentials and regulates firing patterns, but is not involved directly in triggering exocytosis. These data provide important insights into the calcium signaling mechanisms involved in early developmental processes.

The timecourse of apoptotic cell death during postnatal remodeling of the mouse cochlea and its premature onset by triiodothyronine (T3)

Apoptosis underlies various forms of tissue remodeling during development. Prior to the onset of hearing, thyroid hormone (T3) promotes cochlear remodeling, which involves regression of the greater epithelial ridge (GER), a transient structure of columnar cells adjacent to the mechanosensory hair cells. We investigated the timecourse of apoptosis in the GER and the influence of ectopic T3 on apoptosis. In saline-treated mice, activated caspase 3-positive cells were detected in the GER between postnatal days 7 and 13 and appeared progressively along the cochlear duct from base to apex over developmental time. T3 given on P0 and P1 advanced the overall program of apoptosis and remodeling by ~4 days. Thyroid hormone receptor β was required for these actions, suggesting a receptor-mediated process of initiation of apoptosis. Finally, T3 given only at P0 or P1 resulted in deafness in adult mice, thus revealing a transient period of susceptibility to long-term damage in the neonatal auditory system.

Activation of BK and SK channels by efferent synapses on outer hair cells in high-frequency regions of the rodent cochlea.

Cholinergic neurons of the brainstem olivary complex project to and inhibit outer hair cells (OHCs), refining acoustic sensitivity of the mammalian cochlea. In all vertebrate hair cells studied to date, cholinergic inhibition results from the combined action of ionotropic acetylcholine receptors and associated calcium-activated potassium channels. Although inhibition was thought to involve exclusively small conductance (SK potassium channels), recent findings have shown that BK channels also contribute to inhibition in basal, high-frequency OHCs after the onset of hearing. Here we show that the waveform of randomly timed IPSCs (evoked by high extracellular potassium) in high-frequency OHCs is altered by blockade of either SK or BK channels, with BK channels supporting faster synaptic waveforms and SK channels supporting slower synaptic waveforms. Consistent with these findings, IPSCs recorded from high-frequency OHCs that express BK channels are briefer than IPSCs recorded from low-frequency (apical) OHCs that do not express BK channels and from immature high-frequency OHCs before the developmental onset of BK channel expression. Likewise, OHCs of BKα(-/-) mice lacking the pore-forming α-subunit of BK channels have longer IPSCs than do the OHCs of BKα(+/+) littermates. Furthermore, serial reconstruction of electron micrographs showed that postsynaptic cisterns of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs, and immunofluorescent quantification showed that efferent presynaptic terminals of BKα(-/-) OHCs were smaller than those of BKα(+/+) OHCs. Together, these findings indicate that BK channels contribute to postsynaptic function, and influence the structural maturation of efferent-OHC synapses.

Cross-modal and multisensory training may distinctively shape restored senses.

Cross-modal and multisensory training may distinctively shape restored senses.

Loss of auditory activity modifies the location of potassium channel KCNQ5 in auditory brainstem neurons.

KCNQ5/Kv7.5, a low-threshold noninactivating voltage-gated potassium channel, is preferentially targeted to excitatory endings of auditory neurons in the adult rat brainstem. Endbulds of Held from auditory nerve axons on the bushy cells of the ventral cochlear nucleus (VCN) and calyces of Held around the principal neurons in the medial nucleus of the trapezoid body (MNTB) are rich in KCNQ5 immunoreactivity. We have previously shown that this synaptic distribution occurs at about the time of hearing onset. The current study tests whether this localization in excitatory endings depends on the peripheral activity carried by the auditory nerve. Auditory nerve activity was abolished by cochlear removal or intracochlear injection of tetrodotoxin (TTX). Presence of KCNQ5 was analyzed by immunocytochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. After cochlear removal, KCNQ5 immunoreactivity was virtually undetectable at its usual location in endbulbs and calyces of Held in the anteroventral CN and in the MNTB, respectively, although it was found in cell bodies in the VCN. The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies. Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection. These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem. This may contribute to adaptations in the excitability and neurotransmitter release properties of these presynaptic endings under altered input conditions. © 2014 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Development of intrinsic connectivity in the central nucleus of the mouse inferior colliculus.

The inferior colliculus (IC) in the mammalian midbrain is the major subcortical auditory integration center receiving ascending inputs from almost all auditory brainstem nuclei as well as descending inputs from the thalamus and cortex. In addition to these extrinsic inputs, the IC also contains a dense network of local, intracollicular connections, which are thought to provide gain control and contribute to the selectivity for complex acoustic features. However, in contrast to the organization of extrinsic IC afferents, the development and functional organization of intrinsic connections in the IC has remained poorly understood. Here we used laser-scanning photostimulation with caged glutamate to characterize the spatial distribution and strength of local synaptic connections in the central nucleus of the inferior colliculus of newborn mice until after hearing onset (P2-P22). We demonstrate the presence of an extensive excitatory and inhibitory intracollicular network already at P2. Excitatory and inhibitory synaptic maps to individual IC neurons formed continuous maps that largely overlapped with each other and that were aligned with the presumed isofrequency axis of the central nucleus of the IC. Although this characteristic organization was present throughout the first three postnatal weeks, the size of input maps was developmentally regulated as input maps underwent an expansion during the first week that was followed by a dramatic refinement after hearing onset. These changes occurred in parallel for excitatory and inhibitory input maps. However, the functional elimination of intrinsic connections was greater for excitatory than for inhibitory connections, resulting in a predominance of intrinsic inhibition after hearing onset.

Spontaneous activity in the developing auditory system.

Spontaneous electrical activity is a common feature of sensory systems during early development. This sensory-independent neuronal activity has been implicated in promoting their survival and maturation, as well as growth and refinement of their projections to yield circuits that can rapidly extract information about the external world. Periodic bursts of action potentials occur in auditory neurons of mammals before hearing onset. This activity is induced by inner hair cells (IHCs) within the developing cochlea, which establish functional connections with spiral ganglion neurons (SGNs) several weeks before they are capable of detecting external sounds. During this pre-hearing period, IHCs fire periodic bursts of Ca(2+) action potentials that excite SGNs, triggering brief but intense periods of activity that pass through auditory centers of the brain. Although spontaneous activity requires input from IHCs, there is ongoing debate about whether IHCs are intrinsically active and their firing periodically interrupted by external inhibitory input (IHC-inhibition model), or are intrinsically silent and their firing periodically promoted by an external excitatory stimulus (IHC-excitation model). There is accumulating evidence that inner supporting cells in Kölliker's organ spontaneously release ATP during this time, which can induce bursts of Ca(2+) spikes in IHCs that recapitulate many features of auditory neuron activity observed in vivo. Nevertheless, the role of supporting cells in this process remains to be established in vivo. A greater understanding of the molecular mechanisms responsible for generating IHC activity in the developing cochlea will help reveal how these events contribute to the maturation of nascent auditory circuits.

Synaptic transmission between end bulbs of Held and bushy cells in the cochlear nucleus of mice with a mutation in Otoferlin.

Mice that carry a mutation in a calcium binding domain of Otoferlin, the putative calcium sensor at hair cell synapses, have normal distortion product otoacoustic emissions (DPOAEs), but auditory brain stem responses (ABRs) are absent. In mutant mice mechanotransduction is normal but transmission of acoustic information to the auditory pathway is blocked even before the onset of hearing. The cross-sectional area of the auditory nerve of mutant mice is reduced by 54%, and the volume of ventral cochlear nuclei is reduced by 46% relative to hearing control mice. While the tonotopic organization was not detectably changed in mutant mice, the axons to end bulbs of Held and the end bulbs themselves were smaller. In mutant mice bushy cells in the anteroventral cochlear nucleus (aVCN) have the electrophysiological hallmarks of control cells. Spontaneous miniature excitatory postsynaptic currents (EPSCs) occur with similar frequencies and have similar shapes in deaf as in hearing animals, but they are 24% larger in deaf mice. Bushy cells in deaf mutant mice are contacted by ∼2.6 auditory nerve fibers compared with ∼2.0 in hearing control mice. Furthermore, each fiber delivers more synaptic current, on average 4.8 nA compared with 3.4 nA, in deaf versus hearing control mice. The quantal content of evoked EPSCs is not different between mutant and control mice; the increase in synaptic current delivered in mutant mice is accounted for by the increased response to the size of the quanta. Although responses to shocks presented at long intervals are larger in mutant mice, they depress more rapidly than in hearing control mice.

Postnatal expression of neurotrophic factors accessible to spiral ganglion neurons in the auditory system of adult hearing and deafened rats.

Spiral ganglion neurons (SGNs) receive input from cochlear hair cells and project from the cochlea to the cochlear nucleus. After destruction of hair cells with aminoglycoside antibiotics or noise, SGNs gradually die. It has been assumed that SGN death is attributable to loss of neurotrophic factors (NTFs) derived from hair cells or supporting cells in the organ of Corti (OC). We used quantitative PCR (qPCR) to assay NTF expression-neurotrophin-3 (NT-3), BDNF, GDNF, neurturin, artemin, and CNTF-in the OC and cochlear nucleus at various ages from postnatal day 0 (P0) to P90 in control hearing and neonatally deafened rats. NT-3, neurturin, and CNTF were most abundant in the postnatal hearing OC; CNTF and neurturin most abundant in the cochlear nucleus. In the OC, NT-3 and CNTF showed a postnatal increase in expression approximately concomitant with hearing onset. In rats deafened by daily kanamycin injections (from P8 to P16), surviving inner hair cells were evident at P16 but absent by P19, with most postsynaptic boutons lost before P16. NT-3 and CNTF, which normally increase postnatally, had significantly reduced expression in the OC of deafened rats, although CNTF was expressed throughout the time that SGNs were dying. In contrast, neurturin expression was constant, unaffected by deafening or by age. CNTF and neurturin expression in the cochlear nucleus was unaffected by deafening or age. Thus, NTFs other than NT-3 are available to SGNs even as they are dying after deafening, apparently conflicting with the hypothesis that SGN death is attributable to lack of NTFs.

Timed conditional null of connexin26 in mice reveals temporary requirements of connexin26 in key cochlear developmental events before the onset of hearing

Mutations in the Gjb2 gene, which encodes a gap junction protein connexin26 (Cx26), are the most prevalent form of hereditary deafness in humans and represent about half of non-syndromic congenital deafness cases in many ethnic populations. Cochlear potassium (K+) recycling in mature cochlea is required for normal hearing. It is thought that gap junctions are essential for K+ recycling and that Gjb2 mutations cause Gjb2-associated deafness by disrupting K+ recycling in mature cochlea. Here we present evidence showing that Gjb2 is required for normal development of the neonatal organ of Corti prior to the onset of the hearing in mice. In the conditional Gjb2 null (cCx26 null) mice, ribbon synapses in inner hair cells remained poorly developed, the afferent type I fibers failed to finish the refinement process to form convergent innervation to individual inner hair cells. The spontaneous depolarizing activities in the supporting cells, which normally diminish in the wild type cochleae after postnatal day 8 (P8), remained strong in the cochlea after P8 in the mutant mice. Furthermore, the deafness phenotype was readily generated only if the Cx26 expression in the organ of Corti was significantly reduced before P6. Similar amount of Cx26 reduction in more mature cochleae had a much weaker effect in damaging the hearing sensitivity. Our findings indicated that Cx26 plays essential roles in the maturation process of the organ of Corti prior to the establishment of high K+ in the endolymph and the onset of hearing. These results suggest that successful treatment of Cx26 deafness requires early intervention before the cochlea fully matures.

Distribution of glial cells in the auditory brainstem: Normal development and effects of unilateral lesion

Auditory brainstem networks facilitate sound source localization through binaural integration. A key component of this circuitry is the projection from the ventral cochlear nucleus (VCN) to the medial nucleus of the trapezoid body (MNTB), a relay nucleus that provides inhibition to the superior olivary complex. This strictly contralateral projection terminates in the large calyx of Held synapse. The formation of this pathway requires spatiotemporal coordination of cues that promote cell maturation, axon growth, and synaptogenesis. Here we have examined the emergence of distinct classes of glial cells, which are known to function in development and in response to injury. Immunofluorescence for several astrocyte markers revealed unique expression patterns. Aldehyde dehydrogenase 1 family member L1 (ALDH1L1) was expressed earliest in both nuclei, followed by S100ß, during the first postnatal week. Glial fibrillary acidic protein (GFAP) expression was seen in the second postnatal week. GFAP-positive cell bodies remained outside the boundaries of VCN and MNTB, with a limited number of labeled fibers penetrating into the margins of the nuclei. Oligodendrocyte transcription factor 2 (OLIG2) expression revealed the presence of oligodendrocytes in VCN and MNTB from birth until after hearing onset. In addition, ionized calcium binding adaptor molecule 1 (IBA1)-positive microglia were observed after the first postnatal week. Following hearing onset, all glial populations were found in MNTB. We then determined the distribution of glial cells following early (P2) unilateral cochlear removal, which results in formation of ectopic projections from the intact VCN to ipsilateral MNTB. We found that following perturbation, astrocytic markers showed expression near the ectopic ipsilateral calyx. Taken together, the developmental expression patterns are consistent with a role for glial cells in the maturation of the calyx of Held and suggest that these cells may have a similar role in maturation of lesion-induced connections.

Developmental profiles of the intrinsic properties and synaptic function of auditory neurons in preterm and term baboon neonates.

The human fetus starts to hear and undergoes major developmental changes in the auditory system during the third trimester of pregnancy. Although there are significant data regarding development of the auditory system in rodents, changes in intrinsic properties and synaptic function of auditory neurons in developing primate brain at hearing onset are poorly understood. We performed whole-cell patch-clamp recordings of principal neurons in the medial nucleus of trapezoid body (MNTB) in preterm and term baboon brainstem slices to study the structural and functional maturation of auditory synapses. Each MNTB principal neuron received an excitatory input from a single calyx of Held terminal, and this one-to-one pattern of innervation was already formed in preterm baboons delivered at 67% of normal gestation. There was no difference in frequency or amplitude of spontaneous excitatory postsynaptic synaptic currents between preterm and term MNTB neurons. In contrast, the frequency of spontaneous GABA(A)/glycine receptor-mediated inhibitory postsynaptic synaptic currents, which were prevalent in preterm MNTB neurons, was significantly reduced in term MNTB neurons. Preterm MNTB neurons had a higher input resistance than term neurons and fired in bursts, whereas term MNTB neurons fired a single action potential in response to suprathreshold current injection. The maturation of intrinsic properties and dominance of excitatory inputs in the primate MNTB allow it to take on its mature role as a fast and reliable relay synapse.

Multisensory training improves auditory spatial processing following bilateral cochlear implantation.

Cochlear implants (CIs) partially restore hearing to the deaf by directly stimulating the inner ear. In individuals fitted with CIs, lack of auditory experience due to loss of hearing before language acquisition can adversely impact outcomes. For example, adults with early-onset hearing loss generally do not integrate inputs from both ears effectively when fitted with bilateral CIs (BiCIs). Here, we used an animal model to investigate the effects of long-term deafness on auditory localization with BiCIs and approaches for promoting the use of binaural spatial cues. Ferrets were deafened either at the age of hearing onset or as adults. All animals were implanted in adulthood, either unilaterally or bilaterally, and were subsequently assessed for their ability to localize sound in the horizontal plane. The unilaterally implanted animals were unable to perform this task, regardless of the duration of deafness. Among animals with BiCIs, early-onset hearing loss was associated with poor auditory localization performance, compared with late-onset hearing loss. However, performance in the early-deafened group with BiCIs improved significantly after multisensory training with interleaved auditory and visual stimuli. We demonstrate a possible neural substrate for this by showing a training-induced improvement in the responsiveness of auditory cortical neurons and in their sensitivity to interaural level differences, the principal localization cue available to BiCI users. Importantly, our behavioral and physiological evidence demonstrates a facilitative role for vision in restoring auditory spatial processing following potential cross-modal reorganization. These findings support investigation of a similar training paradigm in human CI users.