Britta Flock

Mechanisms of movement in outer hair cells and a possible structural basis

SummaryIsolated outer hair cells were found to slowly shorten when subjected to a solution that would induce contraction in a muscle fibre. Two possible mechanisms underlying this behaviour emerge from ultrastructural and immunocytochemical investigations. Antibody labelling at the electron microscopic level demonstrates that actin is present not only in the stereocilia and in the cuticular plate but also along the wall of outer hair cells, between the plasma membrane and the subsurface fenestrated cisternae. The latter are interconnected by regularly spaced pillars, resembling those seen between the T-tubules and sarcoplasmic reticulum in muscle fibres. Contraction also results from the application of positively charged macromolecules to the bathing solution. This implies sensitivity of the membrane-associated complex (the cortex system) to an electrical current. A second contractile system may reside in the cytoplasm, where calmodulin is present in contracted hair cells. This protein is a calcium-binding control protein for contraction-like events in smooth muscle and non-muscle cells. The unique presence of the cortex system in outer hair cells, and its absence in inner hair cells, indicates a functional significance that relates to a motor function of outer hair cells in hearing.

Studies on the Sensory Hairs of Receptor Cells in the Inner Ear

The crista ampullaris of the semicircular canal in the frog can be isolated and mounted in a chamber so that the sensory hairs can be observed under high magnification in interference-contrast. The cupula is removed and the sensory hairs can be manipulated and their mechanical properties investigated by a microprobe held in a micromanipulator. The hairs appear quite stiff and pivot around their base. When subjected to force they break as if they are brittle. All the cilia within a bundle move together as if joined to one another. Labelling for electron-microscopy with polycationic ferritin reveals that the membrane surrounding the cilia has a surface coat of negatively charged molecules. When the organ is incubated with polycationic ferritin before fixation the sensory hairs agglutinate. Fusion of the membrane surrounding individual sensory hairs also occurs.

Three sets of actin filaments in sensory cells of the inner ear. Identification and functional orientation determined by gel electrophoresis, immunofluorescence and electron microscopy

SummaryReceptor cells in the ear are mechanically excited through displacement of sensory hairs, stereocilia, in relation to a sub-surface platform, the cuticular plate, into which rootlets of the stereocilia insert.The presence of actin in inner ear sensory organs and receptor cells was established by gel electrophoresis, by labelling with antibodies against actin, and by electron microscopy after decoration with subfragment-1 of Myosin. The latter method was used to determine the functional orientation of actin filaments found to be present in the mechanosensitive region of the receptor cells. Actin filaments were demonstrated in the stereocilia and their rootlets, in the cuticular plate and in relation to the zonula adherens surrounding the top of the cell. Filaments which run parallel to the cell surface were found in the cuticular plate and zonula adherens. Some filaments associated with the zonula adherens had a functional orientation opposite to that of more centrally located filaments in the cuticular plate. A structural complex consisting of a solid filament surrounded by actin filaments in hexagonal packing was found in the periphery of the cuticular plate. The possibility is suggested that the central filament is myosin.

Sound induced displacement response of the guinea pig hearing organ and its relation to the cochlear potentials

The sound induced motion of the cells within the fourth turn of the guinea pig organ of Corti was studied in an in vitro preparation (Ulfendahl et al. 1989). The cells were visualised by relief microscopy, achieved by an oblique illumination technique. The motion of the sensory cells was observed during the recording of the extracellular receptor potentials; the cochlear microphonics (CM) and the summating potential (SP). Our results show that the temporal bone preparation sustains an endocochlear potential and maintains the receptor potentials for 3-4 h. During the tone stimulus the outer hair cells were seen to elongate and the surface of the organ of Corti was displaced in the direction of scala vestibuli. The displacement response showed two frequency maxima, one at 150 and one at 300 Hz. The mechanical tuning of the sensory organ coincided with the tuning of the receptor potentials. Both the mechanical and the electrical responses at the 300 Hz peak were vulnerable to the administration of methylene blue suggesting cyclic GMP dependence, whereas the 150 Hz peak was unaffected. We conclude that the outer hair cells provide active tuning in the organ of Corti.

Ultrastructure of the Amphibian Papilla in the Bullfrog

The morphology of the amphibian papilla in the bullfrog has been studied with light and electron microscopy. The hair cells in the amphibian papilla are equipped with sensory hairs, one of which is a kinocilium. The orientation of the kinocilium varies in a complicated way along the sensory epithelium. At their basal ends, the hair cells are contacted by two types of nerve endings, morphologically identified as afferent‐ and efferent‐type terminals. Efferent‐type endings are more abundant in the broader anterior portion of the sensory epithelium than in the posterior portion. In the middle portion, tight junctions have been found between axons within the epithelium. In some cases, one of these axons could be traced to an afferent‐nerve ending. Tight junctions between neurons have been shown in a number of other systems to represent electrical synapses that transmit impulses with short delay.

Hydrops in the cochlea can be induced by sound as well as by static pressure

The Reissners membrane (RM) was visualised by confocal microscopy in the isolated temporal bone of the guinea pig. The function of the organ was followed by measuring its physiological response. Static pressure applied in the basal coil caused a distention of the RM in the apical coil into the scala vestibuli. The sensitivity to a test tone was reduced. When the pressure was relieved, the RM returned to its original position and the response recovered. If the increased pressure was maintained, the RM would bulge further. The RM could then be reversibly stretched and return gradually, with a delay, to its original position. Alternatively, it could be over-stretched and return with an over-shoot past its original position toward the organ of Corti. In response to repetitive tone pulses of above 80 dB, hydrops of the RM also developed. This was accompanied by a reduced sensitivity. A slow recovery to the original position, or over-shoot, and return of responsiveness could be seen. Above 106 dB sustained loss was generally seen. It is concluded that the RM can accommodate increased scala media pressure by distention. This will relieve the organ of Corti from part of the pressure and may protect the organ from trauma.

Methods for integrating fluorimetry in the study of hearing organ structure and function.

The measurement of function in the intact organ of Corti has up to now been achieved by three methods: electrophysiology, mechanical measurement and biochemical analysis. The two former methods have supplied information at the level of single identified cells. We have used a fourth method, optical fluorimetry, to measure hair cell function at the cellular level in the intact organ of Corti. Here we describe the methods involved in fluorescence labelling and video-enhanced microscopy in combination with electrophysiological recording of cochlear microphonic (CM) and summating potentials (SP). The guinea pig temporal bone containing an intact ear drum, ossicular chain and cochlea can be maintained in the isolated state by perfusion of the scala tympani with oxygenated tissue culture medium. Substances added to the perfusate readily diffuse through the basilar membrane into the organ of Corti. In this way cells in the organ can be stained by a number of fluorescent probes which label different structures and functions. Here we have used two dyes which label mitochondria and fluoresce with an intensity proportional to metabolic activity. By simultaneous measurement of CM and SP the functional state of the organ can be monitored.

Micro-Lesions in Reissner’s Membrane Evoked by Acute Hydrops

In some pathological conditions excessive amounts of endolymph can accumulate and cause swelling, hydrops, of the membranous labyrinth. Reissner’s membrane in the cochlea will distend and may even rupture. We have studied the effects of acute hydrops, followed for up to 5–6 h, in a preparation that allows continuous monitoring of structural alterations in individual cells in Reissner’s membrane. This is accomplished by using laser confocal microscopy on the membrane visualized by labeling its cells with fluorescent dyes. In specimens subjected to hydrops it was observed that discrete structural defects developed in Reissner’s membrane. These were seen as lesions in single cells or in groups of cells in the epithelial layer. It is suggested that through these micro-lesions the electro-chemical environment of the organ of Corti can be altered causing hearing loss and tinnitus during hydrops.

Ultrastructural Changes in the Outer Hair Cells of the Guinea Pig Cochlea after Exposure to Quinine

The outer hair cells have been shown to have motile properties which are likely to participate in the cochlear performance. Quinine is known to induce hearing loss as well as contraction of skeletal muscles. Isolated outer hair cells and isolated cochleae from guinea pigs have been exposed to quinine, which was also injected into living guinea pigs. When a physiological response was registered, the cells and cochleae were fixed and examined by transmission electron microscopy. In the isolated cells the formation of a central microtubule core occurred and in the cochleae a swelling of the subsurface cisternae in the outer hair cells was observed. The results are discussed in the context of a proposed effect of quinine on the contractile processes of the outer hair cells.

LASER SCANNING CONFOCAL MICROSCOPY OF THE HEARING ORGAN : FLUOROCHROME-DEPENDENT CELLULAR DAMAGE IS SEEN AFTER OVEREXPOSURE

In order to combine laser confocal microscopy with physiological measurements, a number of conditions have to be met: the dye must not be toxic to the cells the laser light itself must not damage the cells; and the excitation of the fluorochrome during imaging must not generate products with toxic effects. We have investigated these conditions the hearing organ of the guinea pig. Two dyes were used, namely, calcein-AM, which is metabolized in vital cells to a fluorescent product in the cytoplasm, and a lipophilic membrane dye. The effect of the dyes on cell function was tested in the intact hearing organ, maintained in the isolated temporal bone, by measuring the electrophysiological potentials generated by the sensory cells in response to tone pulses. The loading of the cells with the dyes had no adverse effects. The effect of the laser beam was explored on isolated coils from the cochlea. In two preparations, the specimens viewed in the confocal system were fixed and processed for electron microscopy. Identified cells were followed before, during, and after laser exposure and could ultimately be examined at the ultrastructural level. Exposure to the laser beam did not cause damage in unstained cells, even at high intensities. In stained tissue, confocal microscopy could safely be performed at normal beam intensity without causing ultrastructural changes. At high intensities, about 100 times normal for 60 times as long, irradiation damage was seen that was selective in that the cells stained with the different dyes exhibited damage at the different sites corresponding to the subcellular location of the dyes. Cells stained with calcein showed lysis of mitochondria and loss of cytoplasmic matrix, whereas cells stained with the styryl membrane dye showed swelling of subsurface cisternae, contortion of the cell wall, and shrinkage. The styryl dyes, in particular, which selectively stain the sensory and neuronal cells in the organ of Corti, could be exploited for phototoxic use.