Shanthini Mahendrasingam

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-α, and parvalbumin-β) 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 results showed that, in animals with fully developed hearing, inner hair cells had \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\frac{1}{10}\) \end{document} of the proteinaceous calcium buffer of outer hair cells in which the cell body contained parvalbumin-β (oncomodulin) and calbindin-D28k at levels equivalent to 5 mm calcium-binding sites. Both proteins were partially excluded from the hair bundles, which may permit fast unbuffered Ca2+ regulation of the mechanotransducer channels. The sum of the calcium buffer concentrations decreased in inner hair cells and increased in outer hair cells as the cells developed their adult properties during cochlear maturation. The results suggest that Ca2+ has distinct roles in the two types of hair cell, reflecting their different functions in auditory transduction. Ca2+ is used in inner hair cells primarily for fast phase-locked synaptic transmission, whereas Ca2+ may be involved in regulating the motor capability underlying cochlear amplification of the outer hair cell. The high concentration of calcium buffer in outer hair cells, similar only to skeletal muscle, may protect against deleterious consequences of Ca2+ loading after acoustic overstimulation.

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. During the first postnatal week, we observed a normal MT current in hair cells lacking Tmc1 or Tmc2; however, in the absence of both isoforms, we recorded a large MT current that was phase-shifted 180°, being evoked by displacements of the hair bundle away from its tallest edge rather than toward it as in wild-type hair cells. The anomalous MT current in hair cells lacking Tmc1 and Tmc2 was blocked by FM1-43, dihydrostreptomycin, and extracellular Ca2+ at concentrations similar to those that blocked wild type. MT channels in the double knockouts carried Ca2+ with a lower permeability than wild-type or single mutants. The MT current in double knockouts persisted during exposure to submicromolar Ca2+, even though this treatment destroyed the tip links. We conclude that the Tmc isoforms do not themselves constitute the MT channel but are essential for targeting and interaction with the tip link. Changes in the MT conductance and Ca2+ permeability observed in the absence of Tmc1 mutants may stem from loss of interaction with protein partners in the transduction complex.

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. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, β-actin, γ-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and β- and γ-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.

Differential distribution of β- and γ-actin in guinea-pig cochlear sensory and supporting cells

Abstract Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing β- and γ-actin isoforms which have been described as having differing intracellular distributions in chick cochlear hair cells. Here, we have used post-embedding immunogold labelling for β- and γ-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of β-actin decrease and γ-actin increase in the order, outer pillar cells, inner pillar cells, Deiters’ cells and hair cells. There is also more β-actin and less γ-actin in outer pillar cells in higher than lower frequency regions. In hair cells, β-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially in the rootlets and towards the periphery of their shafts; labelling densities for γ-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest β-actin and γ-actin form homomeric filaments. These data confirm differential distribution of these actin isoforms in the mammalian cochlea and reveal systematic differences between sensory and supporting cells. Increased expression of β-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.

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 precise ultrastructural distribution of prestin was determined using post‐embedding immunogold labelling and the density of the labelling was compared in low‐frequency and high‐frequency regions of the cochlea. The labelling was confined to the basolateral plasma membrane in hearing rats but declined towards the base of the cells below the nucleus. In pre‐hearing animals, prestin labelling was lower in the membrane and also occurred in the cytoplasm, presumably reflecting its production during development. The densities of labelling in low‐frequency and high‐frequency regions of the cochlea were similar. Non‐linear capacitance, thought to reflect charge movements during conformational changes in prestin, was measured in OHCs in isolated cochlear coils of hearing animals. The OHC non‐linear capacitance in the same regions assayed in the immunolabelling was also similar in both the apex and base, with charge densities of 10 000/μm2 expressed relative to the lateral membrane area. The results suggest that prestin density, and by implication force production, is similar in low‐frequency and high‐frequency OHCs.

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 Ca2+ homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post‐embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post‐natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High‐resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low‐ and high‐frequency OHC bundles despite larger MT currents in high‐frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low‐ and high‐frequency regions from calibration of immunogold particle counts as 2200/μm2 from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca2+ load through MT channels may constitute a major cause of OHC vulnerability and high‐frequency hearing loss.

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. We found that SHCs are electrically tuned by a Ca(2+)-activated K(+) current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K(+) current, but electrical tuning was observed only at resting potentials positive to -45 mV, where the A current is inactivated. We predict that the resting potential in vivo is approximately -40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability of ∼0.26. The resting open probability stems from a low endolymphatic Ca(2+) concentration (0.24 mM) and a high intracellular mobile Ca(2+) buffer concentration, estimated from perforated-patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindin D-28K with calibrated postembedding immunogold labeling, demonstrating >1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca(2+) pump isoform PMCA2a.

Ultrastructural localization of cadherin in the adult guinea-pig organ of Corti.

The apices of the majority of cells of the organ of Corti are connected together by junctional complexes to form the reticular lamina, a barrier that prevents the mixing of endolymph and perilymph. These complexes include tight junctions, adherens junctions and desmosomes. Further information is required about the identity and distribution of the molecules involved in these connections if the function and organization of the reticular lamina are to be well understood. One major category of molecules occurring in adherens junctions and desmosomes, and involved in the maintenance of tissue integrity, is the cadherins. However, although cadherin has been identified in junctions between supporting cells in the adult mammalian organ of Corti at the light microscopic level, its ultrastructural distribution has not so far been described. A post-embedding immunogold labelling technique has therefore been used in conjunction with a monoclonal antibody to cadherin to investigate its ultrastructural distribution in the adult guinea-pig reticular lamina. Immunolabelling is observed in hair cell-supporting cell junctions and in supporting cell-supporting cell junctions. In addition, there is more labelling associated with inner hair cell-supporting cell junctions than with outer hair cell-supporting cell junctions. This may indicate that the junctions associated with the two types of hair cell have different functional properties.

QUANTITATIVE ANALYSIS OF THE EXPRESSION OF THE GLUTAMATE-ASPARTATE TRANSPORTER AND IDENTIFICATION OF FUNCTIONAL GLUTAMATE UPTAKE REVEAL A ROLE FOR COCHLEAR FIBROCYTES IN GLUTAMATE HOMEOSTASIS

There are several subtypes of fibrocyte in the spiral ligament and spiral limbus of the cochlea that may contribute to fluid homeostasis. Immunocytochemical data suggest that these fibrocytes possess the glutamate-aspartate transporter, GLAST, as do supporting cells around the hair cells. However, functional glutamate uptake has not been demonstrated in fibrocytes. We used confocal and post-embedding immunogold electron microscopy to confirm that GLAST is expressed in adult fibrocytes of CD-1 mice with a relative expression: spiral limbus fibrocytes>type II>V>IV>I spiral ligament fibrocytes. Because they were sparsely present in most samples, type III fibrocytes were assessed only in one sample where their GLAST levels were similar to type I. Type II, type V and spiral limbus fibrocytes have many fine cellular processes that increase their surface area, those of the latter two coming into direct contact with perilymph, and type V fibrocytes contain the most glutamate. These data imply that glutamate uptake occurs in the fibrocytes. We assessed uptake of D-aspartate (a glutamate analogue) together with GLAST expression immunocytochemically and electrophysiologically. D-aspartate accumulated into GLAST expressing fibrocytes in vitro and evoked currents blockable by the GLAST inhibitor D,L-threo-beta-benzyloxyaspartate (TBOA), similar to those of supporting cells around inner hair cells. Currents were strongest in spiral limbus fibrocytes, progressively lower in type V and type II fibrocytes, and were negligible in type I fibrocytes in accordance with the relative expression levels of GLAST. We conclude that in addition to their known homeostatic functions, fibrocytes, in particular spiral limbus, type II and type V fibrocytes play a role in glutamate homeostasis in the cochlea.

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.