Selective Inner Hair Cell Loss in a Neonate Harbor Seal (Phoca vitulina)

Congenital hearing loss is recognized in humans and other terrestrial species. However, there is a lack of information on its prevalence or pathophysiology in pinnipeds. It is important to have baseline knowledge on marine mammal malformations in the inner ear, to differentiate between congenital and acquired abnormalities, which may be caused by infectious pathogens, age, or anthropogenic interactions, such as noise exposure. Ultrastructural evaluation of the cochlea of a neonate harbor seal (Phoca vitulina) by scanning electron microscopy revealed bilateral loss of inner hair cells with intact outer hair cells. The selective inner hair cell loss was more severe in the basal turn, where high-frequency sounds are encoded. The loss of inner hair cells started around 40% away from the apex or tip of the spiral, reaching a maximum loss of 84.6% of hair cells at 80–85% of the length from the apex. Potential etiologies and consequences are discussed. This is believed to be the first case report of selective inner hair cell loss in a marine mammal neonate, likely congenital.

Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

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.

Endolymphatic Hydrops is a Marker of Synaptopathy Following Traumatic Noise Exposure

After acoustic trauma, there can be loss of synaptic connections between inner hair cells and auditory neurons in the cochlea, which may lead to hearing abnormalities including speech-in-noise difficulties, tinnitus, and hyperacusis. We have previously studied mice with blast-induced cochlear synaptopathy and found that they also developed a build-up of endolymph, termed endolymphatic hydrops. In this study, we used optical coherence tomography to measure endolymph volume in live CBA/CaJ mice exposed to various noise intensities. We quantified the number of synaptic ribbons and postsynaptic densities under the inner hair cells 1 week after noise exposure to determine if they correlated with acute changes in endolymph volume measured in the hours after the noise exposure. After 2 h of noise at an intensity of 95 dB SPL or below, both endolymph volume and synaptic counts remained normal. After exposure to 2 h of 100 dB SPL noise, mice developed endolymphatic hydrops and had reduced synaptic counts in the basal and middle regions of the cochlea. Furthermore, round-window application of hypertonic saline reduced the degree of endolymphatic hydrops that developed after 100 dB SPL noise exposure and partially prevented the reduction in synaptic counts in the cochlear base. Taken together, these results indicate that endolymphatic hydrops correlates with noise-induced cochlear synaptopathy, suggesting that these two pathologic findings have a common mechanistic basis.

Noise Exposure Potentiates Exocytosis From Cochlear Inner Hair Cells

Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15–16 were exposed to 1–12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔCm showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3KO mice showed a noise-induced reduction in ΔCm and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.

C1ql1 is expressed in adult outer hair cells of the cochlea in a tonotopic gradient

Hearing depends on the transduction of sounds into neural signals by the inner hair cells of the cochlea. Cochleae also have outer hair cells with unique electromotile properties that increase auditory sensitivity, but they are particularly susceptible to damage by intense noise exposure, ototoxic drugs, and aging. Although the outer hair cells have synapses on afferent neurons that project to the brain, the function of this neuronal circuit is unclear. Here, we created a novel mouse allele that inserts a fluorescent reporter at the C1ql1 locus which revealed gene expression in the outer hair cells and allowed creation of outer hair cell-specific C1ql1 knockout mice. We found that C1ql1 expression in outer hair cells corresponds to areas with the most sensitive frequencies of the mouse audiogram, and that it has an unexpected adolescence-onset developmental timing. No expression was observed in the inner hair cells. Since C1QL1 in the brain is made by neurons, transported anterogradely in axons, and functions in the synaptic cleft, C1QL1 may serve a similar function at the outer hair cell afferent synapse. Histological analyses revealed that C1ql1 conditional knockout cochleae may have reduced outer hair cell afferent synapse maintenance. However, auditory behavioral and physiological assays did not reveal a compelling phenotype. Nonetheless, this study identifies a potentially useful gene expressed in the cochlea and opens the door for future studies aimed at elucidating the function of C1QL1 and the function of the outer hair cell and its afferent neurons.

Inner hair cell stereocilia are embedded in the tectorial membrane

Mammalian hearing depends on sound-evoked displacements of the stereocilia of inner hair cells (IHCs), which cause the endogenous mechanoelectrical transducer channels to conduct inward currents of cations including Ca2+. Due to their presumed lack of contacts with the overlaying tectorial membrane (TM), the putative stimulation mechanism for these stereocilia is by means of the viscous drag of the surrounding endolymph. However, despite numerous efforts to characterize the TM by electron microscopy and other techniques, the exact IHC stereocilia-TM relationship remains elusive. Here we show that Ca2+-rich filamentous structures, that we call Ca2+ ducts, connect the TM to the IHC stereocilia to enable mechanical stimulation by the TM while also ensuring the stereocilia access to TM Ca2+. Our results call for a reassessment of the stimulation mechanism for the IHC stereocilia and the TM role in hearing.