Hair cells in mouse cochlear cultures are selectively labeled by brief exposure to FM1-43, a styryl dye used to study endocytosis and exocytosis. Real-time confocal microscopy indicates that dye entry is rapid and via the apical surface. Cooling to 4 degrees C and high extracellular calcium both reduce dye loading. Pretreatment with EGTA, a condition that breaks tip links and prevents mechanotransducer channel gating, abolishes subsequent dye loading in the presence of calcium. Dye loading recovers after calcium chelation with a time course similar to that described for tip-link regeneration. Myo7a mutant hair cells, which can transduce but have all mechanotransducer channels normally closed at rest, do not label with FM1-43 unless the bundles are stimulated by large excitatory stimuli. Extracellular perfusion of FM1-43 reversibly blocks mechanotransduction with half-blocking concentrations in the low micromolar range. The block is reduced by high extracellular calcium and is voltage dependent, decreasing at extreme positive and negative potentials, indicating that FM1-43 behaves as a permeant blocker of the mechanotransducer channel. The time course for the relief of block after voltage steps to extreme potentials further suggests that FM1-43 competes with other cations for binding sites within the pore of the channel. FM1-43 does not block the transducer channel from the intracellular side at concentrations that would cause complete block when applied extracellularly. Calcium chelation and FM1-43 both reduce the ototoxic effects of the aminoglycoside antibiotic neomycin sulfate, suggesting that FM1-43 and aminoglycosides enter hair cells via the same pathway.

The most serious side-effect of the widely used aminoglycoside antibiotics is irreversible intracellular damage to the auditory and vestibular hair cells of the inner ear. The mechanism of entry into the hair cells has not been unequivocally resolved. Here we report that extracellular dihydrostreptomycin not only blocks the mechano-electrical transducer channels of mouse outer hair cells at negative membrane potentials, as previously shown, but also enters the cells through these channels, which are located in the cells' mechanosensory hair bundles. The voltage-dependent blocking kinetics indicate an open-channel block mechanism, which can be well described by a two barrier-one binding site model, quantifying the antibiotic's block of the channel as well as its permeation in terms of the associated rate constants. The results identify the open transducer channels as the main route for aminoglycoside entry. Intracellularly applied dihydrostreptomycin also blocks the transducer channels, but at positive membrane potentials. However, the potency of the block was two orders of magnitude lower than that due to extracellular dihydrostreptomycin. Extracellular Ca 2+ increases the free energy of the barrier nearest the extracellular side and of the binding site for dihydrostreptomycin. This reduces both the entry of dihydrostreptomycin into the channel and the channel's affinity for the drug. In vivo, where the extracellular Ca 2+ concentration in the endolymph surrounding the hair bundles is < 100 µM, we predict that some 9000 dihydrostreptomycin molecules per second enter each hair cell at therapeutic drug concentrations.

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.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of a particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells 1, is responsible for a substantial proportion of patients with hearing impairment 2. While the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss since poor innervation limits the prospective performance of an implant 3. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here, we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory evoked response (ABR) thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.