Ian J. Russell

Phase-locking in the cochlear nerve of the guinea-pig and its relation to the receptor potential of inner hair-cells.

The high-frequency limit of phase-locking has been measured in fibres of the auditory nerve in the guinea-pig. It is shown that phase-locking begins to decline at about 600 Hz and is no longer detectable above 3.5 kHz which is about 1 octave lower than in the cat, squirrel monkey and some birds. Direct measurements of the cochlear afferent fibre synaptic delay are consistent with indirect estimates from phase-locking, both giving values of 0.7-0.8 ms. Measurements of the receptor potentials of inner hair-cells in the guinea pig cochlea indicate that as the stimulus frequency is increased there is a progressive decrease in the a.c. component compared to the steady depolarization. The cause of this decline is the low-pass filtering of the a.c. component by the hair-cell membrane. The cut-off and slope of the decline in the a.c. component is consistent with the suggestion that this process is the limiting factor in cochlear nerve fibre phase-locking. The implications of these findings for interspecies variation in phase-locking cut-off, for cochlear mechanisms and for the encoding of complex sounds are discussed.

A Targeted Deletion in α-Tectorin Reveals that the Tectorial Membrane Is Required for the Gain and Timing of Cochlear Feedback

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.

The effect of efferent stimulation on basilar membrane displacement in the basal turn of the guinea pig cochlea

Tone-evoked basilar membrane (BM) displacements were measured with a laser diode interferometer from the basal turn of the guinea pig cochlea. The olivocochlear bundle (OCB) was electrically stimulated for 60--80 msec periods at rates of < 200 sec-1 via electrodes placed at the point at which the OCB crosses the floor of the fourth ventricle. For tones close to the best or characteristic frequency (CF), OCB stimulation tended to linearize the highly compressive displacement- level functions and to displace the steep, low-level region toward higher intensities along the intensity axis by < 27 dB sound pressure levels. This shift resulted in a desensitization of the tip of the BM displacement tuning curve that was associated sometimes with downward shifts in the tuning curve CF of < 500 Hz. OCB-induced suppression of the BM response was not associated with a consistent broadening of the tuning curve or with major changes in the phase of the BM response. At frequencies in the low-frequency tail of the tuning curve, OCB stimulation had no observable effect on the motion of the BM. The effect of OCB stimulation on the BM response was blocked by perfusing the scala tympani with 1 microM strychnine. Thus, the effect of OCB stimulation on the frequency tuning of the BM is very similar to the effect of OCB stimulation on the sensitivity and frequency tuning of afferent fibers and inner hair cells. The results indicate that the postsynaptic action of the OCB may cause a change in gain of the voltage-dependent outer hair cell motility without observable changes in the stiffness of the cochlear partition or the position of the BM.

The responses of inner and outer hair cells in the basal turn of the guinea-pig cochlea and in the mouse cochlea grown in vitro

Until recently the responses of the mechanosensitive hair cells of the cochlea have been inferred from their morphology, morphological relationships with other structures in the cochlea, and by indirect electrophysiological measurements. With the advent of techniques for making intracellular recordings from hair cells in the cochleas of anaesthetised mammals it has been possible to measure the responses of hair cells to acoustic stimulation and to assess their roles in sensory transduction in the cochlea. Intracellular recordings of the responses of inner and outer hair cells in the basal turn of the guinea-pig cochlea show that they differ considerably from each other. The receptor potentials of inner hair cells are larger, predominantly depolarizing to low frequency tones and at their best frequencies (16-20 kHz) they generate depolarizing dc receptor potentials. Outer hair cells generate predominantly hyperpolarizing potentials to low frequency tones. They do not produce significant voltage responses at high frequencies except at high intensities when they generate slowly rising depolarizing potentials which are associated with loss of cochlear sensitivity. At low frequencies the receptor potentials of the inner hair cells phase lead those of the outer hair cell. Measurements of their frequency selectivity show that inner and outer hair cells are both sharply tuned. It is proposed that the responses of inner and outer hair cells are consistent with sensory and motor roles respectively in mechanoelectric transduction and that the outer hair cells are the site of an active mechanical process responsible for the frequency selectivity and sensitivity of the cochlea. Intracellular recordings from hair cells in the mouse cochlea maintained in vivo have provided a direct measure of the mechanosensitivity of cochlear hair cells (approximately 30 mV per degree of displacement of their stereociliary bundles) and indirect evidence that the transfer characteristics of the outer hair cells in vivo may be due to their mechanoelectrical interaction with the tectorial membrane. This is because the transfer characteristics of the inner and outer hair cells are similar in vitro in the absence of a tectorial membrane. Considerable importance is attributed to the shape of the transfer characteristics of the inner and outer hair cells. Changes in these characteristics during anoxia and following exposure to intense tones are associated with depolarization of the outer hair cells and loss of cochlear sensitivity and frequency selectivity. Current-voltage studies of hair cells in vivo show the inner and outer hair cells to be electrically nonlinear.(ABSTRACT TRUNCATED AT 400 WORDS)

Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane

Frequency tuning in the cochlea is determined by the passive mechanical properties of the basilar membrane and active feedback from the outer hair cells, sensory-effector cells that detect and amplify sound-induced basilar membrane motions. The sensory hair bundles of the outer hair cells are imbedded in the tectorial membrane, a sheet of extracellular matrix that overlies the cochleas sensory epithelium. The tectorial membrane contains radially organized collagen fibrils that are imbedded in an unusual striated-sheet matrix formed by two glycoproteins, α-tectorin (Tecta) and β-tectorin (Tectb). In Tectb−/− mice the structure of the striated-sheet matrix is disrupted. Although these mice have a low-frequency hearing loss, basilar-membrane and neural tuning are both significantly enhanced in the high-frequency regions of the cochlea, with little loss in sensitivity. These findings can be attributed to a reduction in the acting mass of the tectorial membrane and reveal a new function for this structure in controlling interactions along the cochlea.

Polypeptide composition of the mammalian tectorial membrane

The effects of the enzymes collagenase, pepsin, chondroitinase ABC and keratanase on the polypeptide composition of the mammalian tectorial membrane have been analysed using one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After reduction at least ten polypeptides can be consistently and clearly recognized in SDS gels with molecular weights relative to globular protein standards of 245, 235, 190, 165, 155, 145, 100, 93, 60-73 and 35-49 kDa. With the exception of the 60-73 and 35-49 kDa bands all these polypeptides are sensitive to digestion with bacterial collagenase. The 235, 165, 155, 145 and 93 kDa bands also resist degradation by cold, acidic pepsin. Amino acid analysis of whole tectorial membranes demonstrates that glycine accounts for nearly 25% of the total amino acid content, that proline, hydroxyproline and hydroxylysine are present and that amine sugars can be detected in fairly high concentrations. Estimates based on hydroxyproline content suggest that collagens account for 25-50% of the total tectorial membrane protein. Immunoblotting techniques demonstrate the presence of polypeptides cross reacting with antisera to Type II collagen, Type IX collagen and Type V collagen. Results from immunohistochemical studies confirm that these polypeptides are present in the tectorial membrane and are not contaminants of the isolation procedure. Collagenase treatment of tectorial membranes reveals the presence of an additional non-collagenous polypeptide with an apparent molecular weight of 173 kDa on 7.5% polyacrylamide gels, and polydisperse high molecular weight material spreading over a broad range at the top of the gels. This high molecular weight material and the 173, 60-73 and 35-49 kDa non-collagenous polypeptides are pepsin sensitive and all bind wheat germ agglutinin (WGA) suggesting that they contain N-acetyl glucosamine. The 173 kDa band also binds soybean agglutinin (SBA) suggesting the presence of N-acetyl galactosamine. In the absence of reducing agent the 173 and 60-73 kDa bands are no longer observed and high molecular weight material forming a broad band at the top of the separating gel is seen. The electrophoretic behaviour of this non-collagenous, glycosylated, disulphide bonded, high molecular weight material is altered by treatment with keratanase but not by chondroitinase ABC. The results of this study indicate the tectorial membrane contains at least three different collagen types and, in addition to these collagenous proteins, several non-collagenous, glycosylated polypeptides that may account for as much as 50% of the total tectorial membrane protein.

The response of hair cells in the basal turn of the guinea-pig cochlea to tones.

1. Intracellular recordings were made from inner and outer hair cells in the basal turn of the guinea‐pig cochlea. The resting membrane potentials of the inner hair cells are more positive than ‐50 mV while those of outer hair cells are usually more negative than ‐70 mV. 2. At low frequencies the receptor potentials of inner hair cells are predominantly depolarizing while those from outer hair cells are hyperpolarizing at low and moderate sound pressure (e.g. less than 90 dB re 2 X 10(‐5) Pa at 600 Hz). The potentials then become predominantly depolarizing at high sound pressure. 3. The asymmetry of the inner and outer hair cell receptor potentials are manifested instantaneously except at high stimulus levels when the depolarizing responses of outer hair cells take several cycles to develop. 4. At the offset of intense tones outer hair cell membrane potentials remain depolarized by 1‐2 mV above their resting value and return to normal over a period depending on the level and duration of the tone. 5. In response to tones above about 2 kHz and at levels below about 90 dB the wave forms of outer hair cell receptor potentials are virtually symmetrical without measurable d.c. components. In response to tones close to their best frequencies (16‐21 kHz), inner hair cells in the basal turn generate large depolarizing (d.c.) receptor potentials while outer hair cells from this region of the cochlea do not generate significant voltage responses. 6. Frequency tuning curves were derived for inner and outer hair cells from the amplitude‐intensity relationships of their d.c. and phasic (a.c.) receptor potentials respectively. When the latter were compensated for the low‐pass characteristics of the recording system and the hair cell time constant, the frequency selectivity of inner and outer hair cells are similar. 7. The response properties of inner and outer hair cells in the basal turn of the guinea‐pig cochlea are discussed in relation to their proposed roles in mechano‐electric transduction.

Two-tone suppression in cochlear hair cells

Abstract The phenomenon of two-tone suppression that is known to occur at the level of the auditory nerve is shown to also occur in the receptor potential of single presumed inner hair cells in the first turn of the guinea pig cochlea.

A deafness mutation isolates a second role for the tectorial membrane in hearing

α-tectorin (encoded by Tecta) is a component of the tectorial membrane, an extracellular matrix of the cochlea. In humans, the Y1870C missense mutation in TECTA causes a 50- to 80-dB hearing loss. In transgenic mice with the Y1870C mutation in Tecta, the tectorial membranes matrix structure is disrupted, and its adhesion zone is reduced in thickness. These abnormalities do not seriously influence the tectorial membranes known role in ensuring that cochlear feedback is optimal, because the sensitivity and frequency tuning of the mechanical responses of the cochlea are little changed. However, neural thresholds are elevated, neural tuning is broadened, and a sharp decrease in sensitivity is seen at the tip of the neural tuning curve. Thus, using TectaY1870C/+ mice, we have genetically isolated a second major role for the tectorial membrane in hearing: it enables the motion of the basilar membrane to optimally drive the inner hair cells at their best frequency.

The morphology and physiology of hair cells in organotypic cultures of the mouse cochlea

Organotypic explant cultures were prepared from the cochleas of 1 to 3 day post-natal mice and maintained in vitro for up to 5 days. The hair cells retain morphological integrity for the duration of the culture period although they exhibit embryological features such as a kinocilium and additional microvilli on their apical surfaces. The resting membrane potentials of mouse inner hair cells (IHCs) in vitro are similar to those of guinea-pig IHCs in vivo but the membrane potentials of outer hair cells (OHCs) in the mouse cochlea in vitro are less polarized than the resting membrane potentials of OHCs in the basal turn of the guinea-pig cochlea in vivo. The voltage responses of IHCs and OHCs to sinusoidal displacements of their stereocilia are similar to each other in waveform and dynamic range, although the responses of IHCs are larger than those of OHCs. The relationship between transducer conductance and stereocilia displacement in IHCs and OHCs is non-linear and largely accounts for the depolarizing asymmetry of the voltage response. The receptor potentials of IHCs and OHCs reverse close to 0 mV indicating that the transducer conductance is non-selective for cations. The voltage responses of IHCs and OHCs to intracellular current injection rectify when the membrane potentials are more depolarized than about -30 mV. This rectification is most pronounced in OHCs. OHCs also exhibit a time-dependent, voltage-sensitive conductance although they do not behave as electrical resonators.