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Inner ear hair cells detect sound-induced displacements and transduce these stimuli into electrical signals in a hair bundle that consists of stereocilia that are arranged in rows of increasing height. When stereocilia are deflected, they tug on tiny (~5 nm in diameter) extracellular tip links interconnecting stereocilia, which convey forces to the mechanosensitive transduction channels. Although mechanotransduction has been studied in live hair cells for decades, the functionally important ultrastructural details of the mechanotransduction machinery at the tips of stereocilia (such as tip link dynamics or transduction-dependent stereocilia remodeling) can still be studied only in dead cells with electron microscopy. Theoretically, scanning probe techniques, such as atomic force microscopy, have enough resolution to visualize the surface of stereocilia. However, independent of imaging mode, even the slightest contact of the atomic force microscopy probe with the stereocilia bundle usually damages the bundle. Here we present a detailed protocol for the hopping probe ion conductance microscopy (HPICM) imaging of live rodent auditory hair cells. This non-contact scanning probe technique allows time lapse imaging of the surface of live cells with a complex topography, like hair cells, with single nanometers resolution and without making physical contact with the sample. The HPICM uses an electrical current passing through the glass nanopipette to detect the cell surface in close vicinity to the pipette, while a 3D-positioning piezoelectric system scans the surface and generates its image. With HPICM, we were able to image stereocilia bundles and the links interconnecting stereocilia in live auditory hair cells for several hours without noticeable damage. We anticipate that the use of HPICM will allow direct exploration of ultrastructural changes in the stereocilia of live hair cells for better understanding of their function.

Mechanosensory outer hair cells play an essential role in the amplification of sound-induced vibrations within the mammalian cochlea due to their ability to contract or elongate following changes of the intracellular potential. This unique property of outer hair cells is known as electromotility. Selective efferent innervation of these cells within the organ of Corti suggests that regulation of outer hair cell electromotility may be the primary function of the efferent control in the cochlea. A number of studies demonstrate that outer hair cell electromotility is indeed modulated by the efferent neurotransmitter, acetylcholine. The effects of acetylcholine on outer hair cells include cell hyperpolarization and a decrease of the axial stiffness, both mediated by intracellular Ca2+. This article reviews these results and considers other potential mechanisms that may regulate electromotility, such as direct modification of the plasma membrane molecular motors, alteration of intracellular pressure, and modification of intracellular chloride concentration. [ABSTRACT FROM AUTHOR]

The mammalian inner ear is a sensory organ that has specialized hair cells that detect sound, as well as orientation and movement of the head. The 'hair' bundle on the apical surface of these cells is a mechanosensitive organelle that consists of precisely organized actin-filled projections known as stereocilia. Alterations in hair-bundle morphogenesis can result in hearing loss, balance defects or both. Positional cloning of genes that underlie hereditary hearing loss, coupled with the characterization of corresponding mouse models, is revealing how hair cells have adapted the molecular mechanisms of intracellular motility and intercellular adhesion for the morphogenesis of their apical surfaces. [ABSTRACT FROM AUTHOR]

Hair cells are specialized mechanoreceptors common to auditory and vestibular sensory organs of mammalian and non-mammalian species. Different hair cells are believed to share common features related to their mechanosensory function. It has been shown that hair cells possess various forms of motile properties that enhance their receptor function. Membrane-based electromotility is a form of hair cell motility observed in isolated outer hair cells (OHCs) of the cochlea. A novel membrane protein, prestin, recently cloned from gerbil and rat tissues, is presumably responsible for electromotility. We cloned prestin from mouse organ of Corti and confirmed strong homology of this protein among different rodent species. We explored whether or not prestin is present in hair cells of the vestibular system. Using reverse transcription-polymerase chain reaction, we demonstrated that prestin is expressed in mouse and rat auditory and vestibular organs, but not in chicken auditory periphery. In situ hybridization and immunolocalization studies confirmed the presence of prestin in OHCs as well as in vestibular hair cells (VHCs) of rodent saccule, utricle and crista ampullaris. However, in the VHCs, staining of varying intensity with anti-prestin antibodies was observed in the cytoplasm, but not in the lateral plasma membrane or in the stereociliary membrane. Whole-cell patch-clamp recordings showed that VHCs do not possess the voltage-dependent capacitance associated with membrane-based electromotility. We conclude that although prestin is expressed in VHCs, it is unlikely that it supports the form of somatic motility observed in OHCs. [Copyright &y& Elsevier]