Inner Hair Cell and Neuron Degeneration Contribute to Hearing Loss in a DFNA2-Like Mouse Model.

DFNA2 is a progressive deafness caused by mutations in the voltage-activated potassium channel KCNQ4. Hearing loss develops with age from a mild increase in the hearing threshold to profound deafness. Studies using transgenic mice for Kcnq4 expressed in a mixed background demonstrated the implication of outer hair cells at the initial phase. However, it could not explain the last phase mechanisms of the disease. Genetic backgrounds are known to influence disease expressivity. To unmask the cause of profound deafness phenotype, we backcrossed the Kcnq4 knock-out allele to the inbred strain C3H/HeJ and investigated inner and outer hair cell and spiral ganglion neuron degeneration across the lifespan. In addition to the already reported outer hair cell death, the C3H/HeJ strain also exhibited inner hair cell and spiral ganglion neuron death. We tracked the spatiotemporal survival of cochlear cells by plotting cytocochleograms and neuronal counts at different ages. Cell loss progressed from basal to apical turns with age. Interestingly, the time-course of cell degeneration was different for each cell-type. While for outer hair cells it was already present by week 3, inner hair cell and neuronal loss started 30 weeks later. We also established that outer hair cell loss kinetics slowed down from basal to apical regions correlating with KCNQ4 expression pattern determined in wild-type mice. Our findings indicate that KCNQ4 plays differential roles in each cochlear cell-type impacting in their survival ability. Inner hair cell and spiral ganglion neuron death generates severe hearing loss that could be associated with the last phase of DFNA2.

MicroRNA-200 family members are weakly expressed in the neurosensory epithelia of the developing zebrafish (Danio rerio) inner ear.

MicroRNA-200 family members are expressed in the developing mouse inner ear and in zebrafish (Danio rerio) olfactory epithelia, taste buds, and neuromasts, and have also been shown to be associated with differentiation of olfactory and taste buds. However, the role of the miR-200 family in the inner ear of zebrafish had not been studied. We investigated the expression and function of the miR-200 family in the zebrafish inner ear via in situ hybridization and loss-of-function methods. Expression of the miR-200 family was weak and dispersed throughout the developing zebrafish inner ear. After knockdown of miR-200 family members in the developing inner ear, no significant differences in development were observed compared to the controls. Otic vesicles, otoliths, and semicircular canals appeared normal. Compared with less differentiated olfactory filaments in olfactory epithelia, the development of hair cells and statoacoustic ganglion neurons were normal. The kinocilia and stereocilia of hair cells, the innervation of hair cells, and the formation of ribbon synapses were also unaffected. Overall, we conclude that the miR-200 family has a negligible role in the development of zebrafish inner ear; the functions of the miR- 200 family may be organ-specific.

Transmitter release from cochlear hair cells is phase locked to cyclic stimuli of different intensities and frequencies.

The auditory system processes time and intensity through separate brainstem pathways to derive spatial location as well as other salient features of sound. The independent coding of time and intensity begins in the cochlea, where afferent neurons can fire action potentials at constant phase throughout a wide range of stimulus intensities. We have investigated time and intensity coding by simultaneous presynaptic and postsynaptic recording at the hair cell-afferent synapse from rats. Trains of depolarizing steps to the hair cell were used to elicit postsynaptic currents that occurred at constant phase for a range of membrane potentials over which release probability varied significantly. To probe the underlying mechanisms, release was examined using single steps to various command voltages. As expected for vesicular release, first synaptic events occurred earlier as presynaptic calcium influx grew larger. However, synaptic depression produced smaller responses with longer first latencies. Thus, during repetitive hair cell stimulation, as the hair cell is more strongly depolarized, increased calcium channel gating hurries transmitter release, but the resulting vesicular depletion produces a compensatory slowing. Quantitative simulation of ribbon function shows that these two factors varied reciprocally with hair cell depolarization (stimulus intensity) to produce constant synaptic phase. Finally, we propose that the observed rapid vesicle replenishment would help maintain the vesicle pool, which in turn would equilibrate with the stimulus intensity (and therefore the number of open Ca(2+) channels), so that for trains of different levels the average phase will be conserved.

Constitutive expression of the alpha10 nicotinic acetylcholine receptor subunit fails to maintain cholinergic responses in inner hair cells after the onset of hearing.

Efferent inhibition of cochlear hair cells is mediated by alpha9alpha10 nicotinic cholinergic receptors (nAChRs) functionally coupled to calcium-activated, small conductance (SK2) potassium channels. Before the onset of hearing, efferent fibers transiently make functional cholinergic synapses with inner hair cells (IHCs). The retraction of these fibers after the onset of hearing correlates with the cessation of transcription of the Chrna10 (but not the Chrna9) gene in IHCs. To further analyze this developmental change, we generated a transgenic mice whose IHCs constitutively express alpha10 into adulthood by expressing the alpha10 cDNA under the control of the Pou4f3 gene promoter. In situ hybridization showed that the alpha10 mRNA is expressed in IHCs of 8-week-old transgenic mice, but not in wild-type mice. Moreover, this mRNA is translated into a functional protein, since IHCs from P8-P10 alpha10 transgenic mice backcrossed to a Chrna10(-/-) background (whose IHCs have no cholinergic function) displayed normal synaptic and acetylcholine (ACh)-evoked currents in patch-clamp recordings. Thus, the alpha10 transgene restored nAChR function. However, in the alpha10 transgenic mice, no synaptic or ACh-evoked currents were observed in P16-18 IHCs, indicating developmental down-regulation of functional nAChRs after the onset of hearing, as normally observed in wild-type mice. The lack of functional ACh currents correlated with the lack of SK2 currents. These results indicate that multiple features of the efferent postsynaptic complex to IHCs, in addition to the nAChR subunits, are down-regulated in synchrony after the onset of hearing, leading to lack of responses to ACh.

Developmental regulation of nicotinic synapses on cochlear inner hair cells.

In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both alpha9 and alpha10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that alpha9 mRNA is expressed in adult IHCs but that alpha10 mRNA disappears after the onset of hearing. In the present study, antibodies to alpha10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of alpha10, but not alpha9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.