The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Here, we report that Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and that lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, there are three, resulting in the formation of an ectopic tunnel of Corti. We demonstrate that these effects are due to a postnatal cell fate transformation of a Deiters' cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. Our results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing.

Cochlear gene transfer studies in animal models have utilized mainly two delivery methods: direct injection through the round window membrane (RWM) or intracochlear infusion through a cochleostomy. However, the surgical trauma, inflammation, and hearing loss associated with these methods lead us to investigate a less invasive delivery method. Herein, we studied the feasibility of a vector transgene-soaked gelatin sponge, Gelfoam, for transgene delivery into the mouse cochlea through an intact RWM. The Gelfoam absorbed with liposomes and adenovirus, but not with adeno-associated virus (AAV), was successful in mediating transgene expression across an intact RWM in a variety of cochlear tissues. The Gelfoam technique proved to be an easy, atraumatic, and effective, but vector-dependent, method of delivering transgenes through an intact RWM. Compared with the more invasive gene delivery methods, this technique represents a safer and a more clinically viable route of cochlear gene delivery in humans.

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.

Studies of the F344 rat have shown a variety of age-related auditory anatomy and physiology changes. The current study was undertaken to clarify the ARHL in the F344 rat, by examining the auditory pathway of the F344/NHsd substrain that is distributed by Harlan Laboratories for research in the United States. The F344/NHsd rat begins to lose its hearing at about 12 months, and by 24 months, there are 50-60 dB auditory brainstem response threshold shifts at 20 and 40 kHz and 20 dB losses at 5-10 kHz. Distortion product otoacoustic emissions (DPOAE) amplitudes at 1.8 to 12 kHz stimuli were depressed in the older (18-24 months) rats. Amplitude input-output functions of the compound action potential (CAP) were also depressed across frequency. The endocochlear potential (EP) was 90-100 mV in the 3 month old rats. All but one of the 24 month old rats' EPs were in the +75-85 mV range. Tympanometry revealed no differences in middle ear function between the young and older rats. Collectively, these findings suggest damage to the outer hair cells, but anatomical examination of the outer hair cells revealed a relative lack of cell loss compared to the magnitude of the hearing and DPOAE loss.