Detecting Central Auditory Processing Disorders in Awake Mice.

Mice are increasingly used as models of human-acquired neurological or neurodevelopmental conditions, such as autism, schizophrenia, and Alzheimer's disease. All these conditions involve central auditory processing disorders, which have been little investigated despite their potential for providing interesting insights into the mechanisms behind such disorders. Alterations of the auditory steady-state response to 40 Hz click trains are associated with an imbalance between neuronal excitation and inhibition, a mechanism thought to be common to many neurological disorders. Here, we demonstrate the value of presenting click trains at various rates to mice with chronically implanted pins above the inferior colliculus and the auditory cortex for obtaining easy, reliable, and long-lasting access to subcortical and cortical complex auditory processing in awake mice. Using this protocol on a mutant mouse model of autism with a defect of the Shank3 gene, we show that the neural response is impaired at high click rates (above 60 Hz) and that this impairment is visible subcortically-two results that cannot be obtained with classical protocols for cortical EEG recordings in response to stimulation at 40 Hz. These results demonstrate the value and necessity of a more complete investigation of central auditory processing disorders in mouse models of neurological or neurodevelopmental disorders.

Characterizing subcutaneous cortical auditory evoked potentials in mice.

Auditory Brainstem Responses (ABRs) are a reliably robust measure of auditory thresholds in the mammalian hearing system and can be used to determine deficits in the auditory periphery. However, because these measures are limited to the lower stages of the auditory pathway, they are insensitive to changes or deficits that occur in the thalamic and cortical regions. Cortical Auditory Evoked Potentials (CAEPs), as longer latency responses, capture information from these regions. However they are less frequently used as a diagnostic tool, particularly in rodent models, due to their inherent variability and subsequent difficult interpretation. The purpose of this study was to develop a consistent measure of subcutaneous CAEPs to auditory stimuli in mice and to determine their origin. To this end, we investigated the effect on the CAEPs recorded in response to different stimuli (noise, click, and tone (16 kHz) bursts), stimulus presentation rates (2/s, 6/s, 10/s) and electrode placements. Recordings were examined for robust CAEP components to determine the optimal experimental paradigm. We argue that CAEPs can measure robust and replicable cortical responses. Furthermore, by deactivating the auditory cortex with lidocaine we demonstrated that the contralateral cortex is the main contributor to the CAEP. Thus CAEP measurements could prove to be of value diagnostically in future for deficits in higher auditory areas.

Spontaneous Mouse Behavior in Presence of Dissonance and Acoustic Roughness.

According to a novel hypothesis (Arnal et al., 2015, Current Biology 25:2051-2056), auditory roughness, or temporal envelope modulations between 30 and 150 Hz, are present in both natural and artificial human alarm signals, which boosts the detection of these alarms in various tasks. These results also shed new light on the unpleasantness of dissonant sounds to humans, which builds upon the high level of roughness present in such sounds. However, it is not clear whether this hypothesis also applies to other species, such as rodents. In particular, whether consonant/dissonant chords, and particularly whether auditory roughness, can trigger unpleasant sensations in mice remains unknown. Using an autonomous behavioral system, which allows the monitoring of mouse behavior over a period of weeks, we observed that C57Bl6J mice did not show any preference for consonant chords. In addition, we found that mice showed a preference for rough sounds over sounds having amplitude modulations in their temporal envelope outside the "rough" range. These results suggest that some emotional features carried by the acoustic temporal envelope are likely to be species-specific.

Ultrarare heterozygous pathogenic variants of genes causing dominant forms of early-onset deafness underlie severe presbycusis.

Presbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls (P = 0.001); half were previously unknown (AF < 0.000002). MYO6, MYO7A, PTPRQ, and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1N321I/+ mice, carrying the TMC1:p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy.

Otogelin, otogelin-like, and stereocilin form links connecting outer hair cell stereocilia to each other and the tectorial membrane.

The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.

CIB2, defective in isolated deafness, is key for auditory hair cell mechanotransduction and survival.

Defects of CIB2, calcium- and integrin-binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type-IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2-/- mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2-/- mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair-cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2-/- mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.

Local gene therapy durably restores vestibular function in a mouse model of Usher syndrome type 1G.

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.

Auditory cortex interneuron development requires cadherins operating hair-cell mechanoelectrical transduction.

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23-/- and Cdhr15-/- mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an "early adhesion code" targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients.

Class III myosins shape the auditory hair bundles by limiting microvilli and stereocilia growth.

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.

The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells.

Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/-) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects.