Developmental changes in electrophysiological membrane properties of mouse cochlear inner hair cells (IHCs) were studied from just after terminal differentiation up to functional maturity. As early as embryonic day 14.5 (E14.5) newly differentiated IHCs express a very small outward K+ current that is largely insensitive to 4-aminopyridine (4-AP). One day later the inward rectifier, IK1, is first observed. These immature cells initially exhibit only slow graded voltage responses under current clamp. From E17.5 spontaneous action potentials occur. During the first week of postnatal development, the outward K+ current steadily increases in size and a progressively larger fraction of the current is sensitive to 4-AP. During the second postnatal week, the activation of the 4-AP-sensitive current, by now contributing about half of the outward K+ current, shifts in the hyperpolarizing direction. Together with an increase in size of IK1, this hyperpolarizes the cell, thus inhibiting the spontaneous spike activity, although spikes could still be evoked upon depolarizing current injection. Starting at about the onset of hearing (postnatal day 12, P12) immature IHCs make the final steps towards fully functional sensory receptors with fast graded voltage responses. This is achieved mainly by the expression of the large-conductance Ca2+-activated K+ current IK,f, but also of a current indistinguishable from the negatively activating IK,n previously described in mature outer hair cells (OHCs). The 4-AP-sensitive current continues to increase after the onset of hearing to form the major part of the mature delayed rectifier, IK,s. By P20 IHCs appear mature in terms of their complement of K+ conductances.

Auditory outer hair cells can elongate and shorten at acoustic frequencies in response to changes ofplasma membrane potential. We show that this fast bidirectional contractile activity consists of an electromechanical transduction process that occurs at the lateral plasma membrane and can be activated and analyzed independently in small membrane patches inside a patch electrode. Bidirectional forces are generated by increases and decreases in membrane area in response to hyperpolarization and depolarization, respectively. We suggest that the force generation mechanism is driven by voltage-dependent conformational changes within a dense array of large transmembrane proteins associated with the site of electromechanical transduction.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

BackgroundInsulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness.Methodology/Principal FindingsA combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1−/− null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1−/− and Igf1+/+ transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1+/+ mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27Kip1.Conclusions/SignificanceWe have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I.