Search

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

Regulation of important cellular functions via signaling pathways is a fundamental property of the cell. Intracellular Ca(2+) is probably a best known second messenger in cell biology. In mechanosensory cells of the inner ear, the hair cells, intracellular Ca(2+) participates in a variety of functions including mechano-electrical transduction, synaptic transmission, and efferent regulation of the outer hair cells, one of two types of hair cells in the mammalian cochlea. The outer hair cells are responsible for the amplification of sound-induced vibrations within the cochlea, which determines the sensitivity of mammalian hearing. Besides Ca(2+), another intracellular ion, Cl(-) may have very specific function in the same outer hair cells. Intracellular Cl(-) is required for the motor function of prestin, a unique plasma membrane molecular motor of these cells. The goal of this article is to review practical aspects of the techniques suitable for imaging of Ca(2+) and Cl(-) in live mammalian cochlear hair cells.

Abstract Despite all recent achievements in identification of the molecules that are essential for the structure and mechanosensory function of stereocilia bundles in the auditory hair cells of mammalian species, we still have only a rudimentary understanding of the mechanisms of stereocilia formation, maintenance, and repair. Important molecular differences distinguishing mammalian auditory hair cells from hair cells of other types and species have been recently revealed. In addition, we are beginning to solve the puzzle of the apparent life-long stability of the stereocilia bundles in these cells. New data link the stability of the cytoskeleton in the mammalian auditory stereocilia with the normal activity of mechanotransduction channels. These data suggest new ideas on how a terminally-differentiated non-regenerating hair cell in the mammalian cochlea may repair and tune its stereocilia bundle throughout the life span of the organism. Highlights • Differentially expressed proteins regulate the hair bundle staircase architecture. • The stability of the stereocilia actin core depends on intrastereocilia Ca2+ levels. • Changes in intrastereocilia Ca2+ levels may trigger signals for stereocilia repair. • Ca2+-sensitive proteins within stereocilia could be potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor (Fgf) signaling is involved in the exquisite cellular patterning of the developing cochlea, and is necessary for proper hearing function. our previous data indicate that Fgf signaling disrupts actin, which impacts the surface stiffness of sensory outer hair cells (ohCs) and non-sensory supporting pillar cells (PCs) in the organ of Corti. here, we used Atomic Force microscopy (AFm) to measure the impact of loss of function of Fgf-receptor 3, on cytoskeletal formation and cell surface mechanical properties. We find a 50% decrease in both ohC and PC surface stiffness, and a substantial disruption in microtubule formation in PCs. moreover, we find no change in ohC electromotility of Fgfr3- deficient mice. to further understand the regulation by Fgf-signaling on microtubule formation, we treated wild-type cochlear explants with Fgf-receptor agonist Fgf2, or antagonist Su5402, and find that both treatments lead to a significant reduction in β-tubulin isotypes I and ii. to identify downstream transcriptional targets of Fgf-signaling, we used QPCr arrays to probe 84 cytoskeletal regulators. of the 5 genes significantly upregulated following treatment, Clasp2, Mapre2 and Mark2 impact microtubule formation. We conclude that microtubule formation is a major downstream effector of Fgf-receptor 3, and suggest this pathway impacts the formation of fluid spaces in the organ of Corti. [ABSTRACT FROM AUTHOR]

In inner ear hair cells, activation of mechanotransduction channels is followed by extremely rapid deactivation that depends on the influx of Ca2+ through these channels. Although the molecular mechanisms of this "fast" adaptation are largely unknown, the predominant models assume Ca22+ sensitivity as an intrinsic property of yet unidentified mechanotransduction channels. Here, we examined mechanotransduction in the hair cells of young postnatal shaker 2 mice (Myo15sh2/sh2). These mice have no functional myosin-XVa, which is critical for normal growth of mechanosensory stereocilia of hair cells. Although stereocilia of both inner and outer hair cells of Myo15sh2/sh2 mice lack myosin-XVa and are abnormally short, these cells have dramatically different hair bundle morphology. Myo15sh2/sh2 outer hair cells retain a staircase arrangement of the abnormally short stereocilia and prominent tip links. Myo15sh2/sh2 inner hair cells do not have obliquely oriented tip links, and their mechanosensitivity is mediated exclusively by "top-to-top" links between equally short stereocilia. In both inner and outer hair cells of Myo15sh2/sh2 mice, we found mechanotransduction responses with a normal "wild-type" amplitude and speed of activation. Surprisingly, only outer hair cells exhibit fast adaptation and sensitivity to extracellular Ca22+. In Myo15sh2/sh2 inner hair cells, fast adaptation is disrupted and the transduction current is insensitive to extracellular Ca22+.We conclude that the Ca22+ sensitivity of the mechanotransduction channels and the fast adaptation require a structural environment that is dependent on myosin-XVa and is disrupted in Myo15sh2/sh2 inner hair cells, but not in Myo15sh2/sh2 outer hair cells. [ABSTRACT FROM AUTHOR]

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 two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin. [ABSTRACT FROM AUTHOR]