Åke 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.

Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells

Several structural and contractile proteins have been searched for with immunohistochemical methods using antibodies directed against these proteins. Three types of preparations from the guinea pig have been used: isolated stereocilia from the utricle, organ of Corti fragments obtained by cellular dissociation and 0.2-1 micrometer sections obtained by cryoultramicrotomy. The main finding is that different sets of proteins compose the cytoskeleton in supporting cells and the mechanoreceptor structures of the sensory cells. Thus, actin was found in association with fimbrin in the mechanoreceptive region of hair cells, whereas supporting cells, although rich in actin, did not reveal fimbrin. Instead tubulin was seen together with actin in supporting cells which also exhibited prekeratin. Fimbrin appears to function as a protein capable of making bundles and networks from actin filaments. Its exclusive presence in the mechanosensitive region of the sensory cells is possibly related to the function of these cells as mechanoreceptors.

Protection against noise trauma by pre-exposure to a low level acoustic stimulus

Guinea pigs were pre-exposed to a low level acoustic stimulus prior to exposure to a stimulus known to yield a permanent threshold shift. This pre-treatment resulted in: 1) approximately a 20 dB reduction in the threshold shift relative to animals not pre-exposed, and 2) complete recovery from the threshold shift after 2 months.

Stiffness of sensory-cell hair bundles in the isolated guinea pig cochlea

Stiffness of hair bundles on cochlear hair cells was measured in turns 2, 3 and 4 of isolated preparations of the guinea-pig organ of Corti maintained in tissue culture medium. Defined as the force required to produce a linear 1.0 micron deflection of the hair-bundle tip, stiffness is greater for deflection in the excitatory than in the inhibitory direction. The excitatory-to-inhibitory ratio for inner hair cells (IHC) is significantly lower than the ratio for outer hair cells (OHC). Hair-bundle stiffness decreases radially from the first to third rows of OHC. Over the measurement range of 9.0-18.0 mm from the stapes hair-bundle stiffness decreases much more for OHC (88-97%) than for IHC (50%). Although an increase in hair-bundle length with distance from the stapes accounts for some of the observed stiffness decrease, the major decrease is due to an increase in compliance of the sensory-hair attachment to the hair-cell surface.

Receptor potentials from hair cells of the lateral line.

Intracellular recordings from hair cells in the tail lateral line of mudpuppy Necturus maculosus show receptor potentials less than 800 microvolts, peak to peak, from stimuli that are considered large compared to natural stimuli. The hair cells are in neuromasts that are sensitive at the time of recording and are identified by both in vivo and in vitro examination of intracellular staining.

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.

Effects of caffeine and tetracaine on outer hair cell shortening suggest intracellular calcium involvement

Outer hair cell (OHC) shortening has previously been induced in vitro by the application of solutions containing high potassium (a depolarizing agent), acetylcholine (a suggested efferent transmitter) and cationized ferritin (a positively charged macromolecule), as well as by electrical current. The application of caffeine, which causes contractures in skeletal and smooth muscle by releasing calcium from intracellular stores to activate actin and myosin interaction, also causes shortening of OHCs. Tetracaine, which interferes with calcium movement in muscle and non-muscle cells, blocks potassium-induced and caffeine-induced shortening of OHCs, but does not block electrically-induced shortening. Sodium dantrolene which is an inhibitor of intracellular calcium release in skeletal muscle does not block potassium-induced OHC shortening. Immunocytochemical studies using antibodies to muscle-like contractile and regulatory proteins on unfixed, freeze-dried OHCs demonstrate the co-localization of calmodulin with actin throughout the OHC cytoplasm. These results support the ideas that in OHCs, intracellular calcium release is involved in the activation of shortening and that an actin-mediated cell shape change may be regulated by calmodulin in a manner similar to that which occurs in contraction of smooth muscle.

A temporal bone preparation for the study of cochlear micromechanics at the cellular level

An in vitro preparation of the guinea pig temporal bone was developed for studying the micromechanical behaviour of the cochlea. The preparation consists of the cochlea opened at the apex, allowing observation of cellular structures within the cochlear partition with an optical sectioning microscope and measurements of cellular vibration with laser interferometry. The middle ear ossicles and the tympanic membrane are left intact as well as the bony part of the external auditory canal, which is used for delivering a sound stimulus to the cochlea.

The lorenzinian ampullae of Polyodon spathula.

SummaryLight and electron microscopic observations on the ampullary organs of Polyodon spathula (Chondrostei, Osteichthyes) reveal a sensory epithelium similar to that found in the Lorenzinian ampulla, an electroreceptor found in marine Elasmobranchs.The sensory cells have a very small luminal part provided with a cilium. They are innervated by many nerve endings. Each nerve fibre apparently makes synaptic contact with several sensory cells. The synaptic structure in the sensory cell is composed of a flat sheet, the outermost part of which is surrounded by 3 or 4 annuli of densely staining material. The sheet extends into a protrusion of the sensory cell, and there is a corresponding invagination in the nerve terminal.The conclusion that these organs are electroreceptors, is supported by the finding that the fish responds to the introduction of an iron tube in the aquarium, whereas a wooden rod introduced in the same way causes no response.