The effects of substrate composition and topography on the characteristics and growth of cell cultures of cochlear fibrocytes.

Spiral ligament fibrocytes of the cochlea play homoeostatic roles in hearing and their degeneration contributes to hearing loss. Culturing fibrocytes in vitro provides a way to evaluate their functional characteristics and study possible therapies for hearing loss. We investigated whether in vivo characteristics of fibrocytes could be recapitulated in vitro by modifying the culture substrates and carried out proof of concept studies for potential transplantation of culture cells into the inner ear.

Ultrastructural localization of the likely mechanoelectrical transduction channel protein, transmembrane-like channel 1 (TMC1) during development of cochlear hair cells.

Transmembrane channel like protein 1 (TMC1) is likely to be a pore-forming subunit of the transduction channel of cochlear hair cells that is mechanically gated by tension on tip links in the stereocilia bundle. To localise TMC1 precisely, we labelled mice cochleae of different ages using custom-made polyclonal antibodies to TMC1 for light and transmission electron microscopy (TEM). Immunofluorescence revealed stereocilia labelling at P9 but not at P3 in apical hair cells.

Spatiotemporal changes in the distribution of LHFPL5 in mice cochlear hair bundles during development and in the absence of PCDH15.

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain.

The role of transmembrane channel-like proteins in the operation of hair cell mechanotransducer channels.

Sound stimuli elicit movement of the stereocilia that make up the hair bundle of cochlear hair cells, putting tension on the tip links connecting the stereocilia and thereby opening mechanotransducer (MT) channels. Tmc1 and Tmc2, two members of the transmembrane channel-like family, are necessary for mechanotransduction. To assess their precise role, we recorded MT currents elicited by hair bundle deflections in mice with null mutations of Tmc1, Tmc2, or both.

Electrical tuning and transduction in short hair cells of the chicken auditory papilla.

The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction, or electrical properties of SHCs. To address this problem, we made patch-clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C.

The development, distribution and density of the plasma membrane calcium ATPase 2 calcium pump in rat cochlear hair cells.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling.

Subcellular distribution and relative expression of fibrocyte markers in the CD/1 mouse cochlea assessed by semiquantitative immunogold electron microscopy.

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic.

Relative time course of degeneration of different cochlear structures in the CD/1 mouse model of accelerated aging.

Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy.

The ultrastructural distribution of prestin in outer hair cells: a post-embedding immunogold investigation of low-frequency and high-frequency regions of the rat cochlea.

Outer hair cells (OHCs) of the mammalian cochlea besides being sensory receptors also generate force to amplify sound-induced displacements of the basilar membrane thus enhancing auditory sensitivity and frequency selectivity. This force generation is attributable to the voltage-dependent contractility of the OHCs underpinned by the motile protein, prestin. Prestin is located in the basolateral wall of OHCs and is thought to alter its conformation in response to changes in membrane potential.

The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane.

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy.

The concentrations of calcium buffering proteins in mammalian cochlear hair cells.

Calcium buffers are important for shaping and localizing cytoplasmic Ca2+ transients in neurons. We measured the concentrations of the four main calcium-buffering proteins (calbindin-D28k, calretinin, parvalbumin-alpha, and parvalbumin-beta) in rat cochlear hair cells in which Ca2+ signaling is a central element of fast transduction and synaptic transmission. The proteins were quantified by calibrating immunogold tissue counts against gels containing known amounts of each protein, and the method was verified by application to Purkinje cells in which independent estimates exist for some of the protein concentrations.

The distribution of calcium buffering proteins in the turtle cochlea.

Hair cells of the inner ear contain high concentrations of calcium-binding proteins that limit calcium signals and prevent cross talk between different signaling pathways during auditory transduction. Using light microscope immunofluorescence and post-embedding immunogold labeling in the electron microscope, we characterized the distribution of three calcium-buffering proteins in the turtle cochlea. Both calbindin-D28k and parvalbumin-beta were confined to hair cells in which they showed a similar distribution, whereas calretinin was present mainly in hair-cell nuclei but also occurred in supporting cells and nerve fibers.