Cochlear outer hair cells in a dominant-negative connexin26 mutant mouse preserve non-linear capacitance in spite of impaired distortion product otoacoustic emission.

Mutations in the connexin26 gene (GJB2) are the most common genetic cause of congenital bilateral non-syndromic sensorineural hearing loss. Transgenic mice were established carrying human Cx26 with the R75W mutation that was identified in a deaf family with autosomal dominant negative inheritance [Kudo T et al. (2003) Hum Mol Genet 12:995-1004]. A dominant-negative Gjb2 R75W transgenic mouse model shows incomplete development of the cochlear supporting cells, resulting in profound deafness from birth [Inoshita A et al. (2008) Neuroscience 156:1039-1047]. The Cx26 defect in the Gjb2 R75W transgenic mouse is restricted to the supporting cells; it is unclear why the auditory response is severely disturbed in spite of the presence of outer hair cells (OHCs). The present study was designed to evaluate developmental changes in the in vivo and in vitro function of the OHC, and the fine structure of the OHC and adjacent supporting cells in the R75W transgenic mouse. No detectable distortion product otoacoustic emissions were observed at any frequencies in R75W transgenic mice throughout development. A characteristic phenotype observed in these mice was the absence of the tunnel of Corti, Nuel's space, and spaces surrounding the OHC; the OHC were compressed and squeezed by the surrounding supporting cells. On the other hand, the OHC developed normally. Structural features of the lateral wall, such as the membrane-bound subsurface cisterna beneath the plasma membrane, were intact. Prestin, the voltage-dependent motor protein, was observed by immunohistochemistry in the OHC basolateral membranes of both transgenic and non-transgenic mice. No significant differences in electromotility of isolated OHCs during development was observed between transgenic and control mice. The present study indicates that normal development of the supporting cells is indispensable for proper cellular function of the OHC.

Usefulness of heavily T(2) weighted magnetic resonance images for the differential diagnosis of parotid tumours.

OBJECTIVES: To evaluate the usefulness of heavily T(2) weighted (T2W) magnetic resonance (MR) images for the differential diagnosis of parotid tumours. METHODS: Conventional T2W and heavily T2W images obtained from 43 patients with parotid tumours were reviewed retrospectively. Tumours were classified pathologically into three types: pleomorphic adenoma (25 cases), Warthin's tumour (7 cases) and malignant tumour (11 cases). During interpretation of each MR image, special attention was paid to the homogeneity and signal intensity displayed by the solid portions of the tumours. RESULTS: Heavily T2W images could distinguish pleomorphic adenoma from Warthin's tumour and malignant tumours based on the homogeneity and signal intensity of the solid portions, whereas conventional T2W images could not. On conventional T2W images, neither homogeneity nor signal intensity differed significantly among these three tumour types. On heavily T2W images, malignant and Warthin's tumours appeared more homogeneous than pleomorphic adenoma (P<0.0061); signal intensity from pleomorphic adenoma was significantly different to signal intensity from malignant tumours (P<0.05) and Warthin's tumour (P<0.005). CONCLUSIONS: Heavily T2W MR images can be useful in distinguishing pleomorphic adenoma from Warthin's tumour and malignant tumours.

Distribution of protein motors along the lateral wall of the outer hair cell.

The outer hair cell (OHC) plays an important role in the normal functioning of the cochlea, and cochlear amplification is believed to be based on OHC electromotility. This electromotility putatively arises from a conformational change of molecules, i.e., 'protein motors', which would be distributed along the plasma membrane. Although it has been assumed that protein motors are distributed in a restricted area of the plasma membrane, details of such distribution remain unclarified. In this study, first, in order to understand the difference in the stiffness along the cell axis, the local deformation of the OHC in response to hypotonic stimulation is analyzed by measuring the displacement of microspheres attached randomly to the lateral wall of the cell. As a result, the stiffness is expected to be constant throughout the region except in the apical part of the cell, and the stiffness of the apical part is expected to be higher than that of the other regions. Then, the local elongation and contraction of the OHC in response to sinusoidal voltage stimulation are analyzed by measuring the displacement of the microspheres in the same way as in the case of the hypotonic stimulation. From the two measurements mentioned above, it is concluded that there are no motors in the apical and basal parts of the cell, and that the motors are equally distributed along the cell lateral wall in the middle part of the cell.

Furosemide alters nonlinear capacitance in isolated outer hair cells.

The outer hair cell (OHC) from the organ of Corti plays a crucial role in hearing through its unique voltage-dependent mechanical responses. Furosemide, one of the loop diuretics, disrupts normal cochlear function. Here we report on direct effects of furosemide on OHC motility-related, voltage-dependent capacitance using the whole-cell patch-clamp technique. Extracellularly applied furosemide reversibly shifted the voltage at peak capacitance (V(pkC(m))) to positive levels. The shift, whose maximum approached 90 mV, evidenced a Hill coefficient of 1.5 and K(1/2) of 10 mM. Changes in the magnitude of nonlinear capacitance were not fully reversible. While it is clear that the overwhelming effect of furosemide on hearing results via its effects on the endolymphatic potential, the present results indicate that furosemide directly alters OHC motility and may, in part, contribute to sensory dysfunction.

Measurement of stapedius contraction during vocalization effort in patients after laryngectomy or tracheostomy.

The contraction of the stapedius muscle during the effort of vocalization was examined by measurement of acoustic compliance in subjects who had undergone laryngectomy or tracheostomy. No significant level of persistent compliance change was recorded in any of the subjects, indicating the absence of effective contraction of the stapedius during the vocalization effort. In two subjects who use an electrolarynx, although no significant compliance change resulted from the simple vocalization effort without the electrolarynx (no actual vocalized sound), a remarkable level of persistent compliance change was observed during the vocalization using the electrolarynx. These results seem to indicate that a simple vocalization effort without actual voice cannot elicit effective contraction of the middle ear muscles, and that sound generation during vocalization is essential for effective contraction of the middle ear muscles during vocalization, at least in some human subjects.

Density of motility-related charge in the outer hair cell of the guinea pig is inversely related to best frequency.

Whole cell voltage clamp and freeze fracture were used to study the electrophysiological and ultrastructural correlates of the outer hair cell (OHC) lateral membrane molecular motors. We find that specific voltage-dependent capacitance, which derives from motility-related charge movement, increases as cell length decreases. This increasing non-linear charge density predicts a corresponding increase in sensor-motor density. However, while OHC lateral membrane particle density increases, a quantitative correspondence is absent. Thus, the presumed equivalence of particle and motor is questionable. The data more importantly indicate that whereas the voltage driving OHC motility, i.e. the receptor potential, may decrease with frequency due to the OHC's low-pass membrane filter, the electrical energy (Q x V) supplied to the lateral membrane will tend to remain stable. This conservation of energy delivery is likely crucial for the function of the cochlear amplifier at high frequencies.

Effects of membrane potential on the voltage dependence of motility-related charge in outer hair cells of the guinea-pig.

1. Isolated outer hair cells (OHCs) from the guinea-pig were whole-cell voltage clamped to study the influence of initial voltage on the voltage dependence of motility-related gating current or, equivalently, on the voltage dependence of membrane capacitance. 2. Prepulse delivery caused changes in the magnitude of motility-related gating currents, which are due predominantly to shifts in the voltage at peak capacitance (VpkCmm). Depolarization shifts VpkCm in the hyperpolarizing direction, and hyperpolarization does the opposite. The mean shift between -120 and +40 mV prepulse states with long-term holding potentials (> 2 min) at -80 mV was 14. 67 +/- 0.95 mV (n = 10; mean +/- s.e.m.). 3. The effect of initial membrane potential is sigmoidal, with a voltage dependence of 23 mV per e-fold change in VpkCm, and maximum slope within the physiological range of OHC resting potentials. This indicates that the cell is poised to respond maximally to changes in resting potential. 4. The kinetics of prepulse effects are slow compared with motility-related gating current kinetics. High-resolution measurement of membrane capacitance (Cm) using two voltage sinusoids indicates that shifts in VpkCm induce Cm changes with time courses fitted by two exponentials (tau0, 0.070 +/- 0.003 s; tau1, 1.28 +/- 0.07 s; A0, 1.54 +/- 0.13 pF; A1, 1.51 +/- 0.13 pF; means +/- s.e.m. ; n = 22; step from +50 to -80 mV). Recovery of prepulse effects exhibits a similar time course. 5. Prepulse effects are resistant to intracellular enzymatic digestion, to fast intracellular calcium buffers, and to intracellular pressure. Through modelling, we indicate how the effect may be explained by an intrinsic voltage-induced tension generated by the molecular motors residing in the lateral membrane.

Effects of potassium channel blockers on the acetylcholine-induced currents in dissociated outer hair cells of guinea pig cochlea.

Much physiological evidence is available to show that acetylcholine (ACh) hyperpolarizes the outer hair cells (OHCs) of guinea pig cochlea and induces Ca2+-activated K+ currents. In this study, using the nystatin perforated patch-clamp technique, we investigated the effects of various K+ channel blockers on the ACh-induced currents (I[ACh]) in dissociated OHCs of guinea pig cochlea. The I(ACh) were inhibited by apamin in a concentration-dependent manner. The half-maximal inhibitory concentration for apamin on the ACh-induced response was 1.59 x 10(-9) M. Charybdotoxin and iberiotoxin had no inhibitory effect on the I(ACh) The inhibitory potency of the various K+ channel blockers on the I(ACh) was as follows: apamin >> quinine approximately quinidine approximately d-tubocurarine > tetraethylammonium chloride > 4-aminopyridine approximately Ba2+ > Cs2+. It is proposed that the Ca2+-activated K+ channels of mammalian cochlear OHCs should be classified as small conductance Ca2+-activated K+ (SK) channels according to their pharmacological properties.

A case of leiomyosarcoma of the sphenoid sinus.

A 43-year-old man with a primary leiomyosarcoma of the left sphenoid sinus is presented. To our knowledge, this is an unusual case of leiomyosarcoma, which has never been reported in the literature. Accurate and safe diagnosis was obtained by an endonasal endoscopic approach with minimal tissue invasion.

Effects of salicylate and lanthanides on outer hair cell motility and associated gating charge.

Salicylate, one of the most widely used drugs, is known to induce reversible tinnitus and hearing loss. Salicylate interferes with outer hair cells (OHCs), which are believed to underlie normal auditory frequency selectivity and sensitivity. In the present experiments, the effects of salicylate and lanthanides on OHC motility and nonlinear capacitance were investigated by using isolated guinea-pig OHCs while attempting to avoid inadvertent intracellular pressure change, which itself can affect OHC motility and capacitance. Either extracellularly or intracellularly applied salicylate reduced nonlinear peak capacitance (Cmpk) and shifted the voltage at peak capacitance to depolarized levels. Concentration-response curves for reduction in Cmpk by salicylate and GdCl3 revealed a half-maximal concentration and Hill coefficient of 1.6 mM and 1.0, and 0.6 mM and 1.2, respectively. In comparable groups of OHCs, the normal Cmpk values of which were near 40 pF, average Cmpk decreased to 28 and 36 pF for intracellularly and extracellularly applied salicylate, respectively. Salicylate reduced, but did not completely block, the voltage-induced length change. Extracellularly, but not intracellularly, applied lanthanide blocked voltage-induced movement and capacitance almost completely. After intracellular trypsin treatment, salicylate reduced voltage-dependent capacitance reversibly, suggesting that salicylate directly acts on the sensor/motor and not via effects on intracellular structures, such as the subsurface cisternae. The results are consistent with the hypothesis that the dissociated, charged form of salicylate directly interacts with the sensor/motor on the inner aspect of the OHC plasma, whereas lanthanides interact on the outer aspect.

Suppression of a nonselective cation conductance by substance P in cochlear outer hair cells.

Substance P (SP) hyperpolarizes outer hair cells (OHCs) of guinea pig cochlea. The cellular mechanisms of the SP response were investigated with the whole cell patch-clamp technique. SP induced outward currents in a dose-dependent manner at a holding potential of -60 mV in a concentration range between 3 x 10(-6) and 10(-4) M. SP decreased slope conductance between -60 and +20 mV. Ion substitutions in the external medium revealed that SP suppresses nonselective cation conductance with high permeability for Ca2+. The relative ion permeability of the channel modulated by SP was as follows: Ca2+ > Li+ approximately Cs+ approximately Na+ > Tris+. The potency of the agonist action was as follows: SP >> neurokinin A > neurokinin B. Peptide antagonists induced currents similar to those of SP. CP-96345, a selective nonpeptide antagonist for the neurokinin type 1 receptor, did not inhibit the SP-induced current. Intracellular dialysis of guanosine 5'-O-(2-thiodiphosphate) and pertussis toxin (PTX) suggests that a PTX-insensitive G protein is involved in the SP response. Neither the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid nor staurosporine (10(-6) M) affects the SP response. Local application of SP by a puffer pipette indicates that the SP receptors are distributed along the side of the OHC. These results suggest the possibility that the action of SP on the OHCs may not be mediated by the tachykinin receptors but rather by a tachykinin receptor-independent pathway. It is proposed that SP suppresses the nonselective cation conductance in the lateral wall of OHCs via a PTX-insensitive G protein.

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".

Caffeine rapidly decreases potassium conductance of dissociated outer hair cells of guinea-pig cochlea.

The effects of caffeine on the outer hair cells (OHCs) freshly dissociated from guinea-pig cochlea were investigated with the whole-cell patch-clamp technique, in both the conventional and the nystatin perforated patch-clamp configurations under voltage-clamp condition. Application of caffeine (> 1 mM for 10-30 s) induced an inward current (Icaffeine) with decrease of conductance in a dose-dependent manner at a holding potential (VH) of -60 mV. The reversal potential of Icaffeine (Ecaffeine) was close to the K+ equilibrium potential. The Icaffeine was not affected by Ca(2+)-free external solution. The internal perfusion of the Ca2+ chelator BAPTA had no effect on Icaffeine. The Icaffeine was not modulated by the external application of H-8 or staurosporine and by the internal perfusion of GDP-beta S. The amplitude of Icaffeine was the largest at the basal region of OHCs when caffeine was locally applied by the 'puffer' method. These results suggest that caffeine induces a decrease in membrane potassium conductance of the OHCs mainly at the basal region without mediating the intracellular signaling pathway.

Ionic properties of IK,n in outer hair cells of guinea pig cochlea.

The ionic properties of voltage-dependent K+ current activated at the resting membrane potential (IK,n) of outer hair cells (OHC) isolated from the guinea pig cochlea were studied using a patch-clamp technique in a whole-cell recording mode. The reversal potential of IK,n indicated a high selectivity for K+, and the relative permeability ratios for various monovalent cations were K+:Rb+:NH4+ = 1:1.21:0.13. Decrease in extracellular Cl- inhibited the IK,n. IK,n was blocked by Cs+ and Ba2+, although the inhibitory manner of Cs+ and Ba2+ were voltage-dependent and voltage-independent, respectively. By the use of puff-application method, the local application of Ba2+ to basolateral surface of OHC shifted the holding current level in an inward direction, whereas the application to apex and hair showed little change. Indicating that the IK,n channels preferentially locate at the basolateral region of cell membrane.

Cisplatin blocks voltage-dependent calcium current in dissociated outer hair cells of guinea-pig cochlea.

The effect of the ototoxic molecule cisplatin (cis-DDP) on the voltage-dependent Ca2+ channel in dissociated outer hair cells (OHCs) of guinea-pig cochlea was investigated using a whole-cell patch-clamp technique. Cis-DDP had antagonistic effect on the Ca2+ channel and reversibly suppressed the Ca2+ current in a concentration-dependent manner. These results suggested that one of the ototoxic mechanisms of cis-DDP is involved in the inhibition of the Ca2+ channel in OHCs.

Voltage-dependent channels in dissociated outer hair cells of the guinea pig.

Voltage-dependent channels in outer hair cells (OHCs) dissociated from the guinea pig cochlea were investigated by the use of a whole-cell patch-clamp technique. Two types of K+ current were recorded from OHCs. One was a slowly inactivating K+ current that was activated at a potential more positive than -30 mV. Another is a K+ current that was already activated at resting membrane potential. After suppressing both K+ currents, depolarizing voltage steps elicited a slowly inactivating inward current that was dependent on external Ca2+ and was indicative of an L-type Ca2+ channel in OHCs. Aminoglycoside antibiotics known to be ototoxic selectively inhibited the Ca2+ current.

Cellular mechanism of acetylcholine-induced response in dissociated outer hair cells of guinea-pig cochlea.

1. The acetylcholine (ACh)-induced currents (IACh) in dissociated outer hair cells (OHCs) of guinea-pig cochlea were investigated using the whole-cell patch-clamp technique, in both conventional and nystatin perforated-patch configurations. 2. ACh and carbamylcholine (CCh) induced outward currents at a holding potential (VH) of -60 mV in the perforated-patch configuration. The IACh increased in a sigmoidal fashion over the concentration range between 3 x 10(-6) and 10(-3) M. The dissociation constant (KD) was 1.7 x 10(-5) M and the Hill coefficient (n) was 2.7. The KD and n for CCh were 8.7 x 10(-5) M and 2.2, respectively. Neither nicotine nor muscarine induced any detectable current up to a concentration of 10(-3) M. 3. Various muscarinic agonists such as oxotremorine-M, McN-A-343 and oxotremorine could also induce the outward currents, although these current amplitudes were about one-third that of ACh, indicating that they were partial agonists. 4. The muscarinic antagonists atropine, 4-DAMP, AF-DX 116 and pirenzepine inhibited the IACh in a concentration-dependent manner. The half-inhibitory concentrations (IC50) for atropine, 4-DAMP, AF-DX 116 and pirenzepine were 4.8 x 10(-6), 6.2 x 10(-6), 2.1 x 10(-5) and 2.9 x 10(-4) M, respectively. 5. When the extracellular Ca2+ concentration ([Ca2+])o) was reduced to lower than 1 mM, the amplitude of IACh, abruptly decreased. In a nominally Ca(2+)-free external solution ACh did not induce any current. The increase of [Ca2+]o beyond 1 mM did not change the IACh. 6. When OHCs were perfused intracellularly with a pipette solution containing 10 mM BAPTA in the conventional whole-cell mode, ACh could not induce outward K+ currents. The Ca2+ ionophore A23187 induced an outward current. These results indicate that intracellular Ca2+ is involved in the ACh response. 7. Calmodulin inhibitors such as chlorpromazine, W-7 and trifluoperazine inhibited the IACh in a concentration-dependent manner. 8. When OHCs were dialysed with either 100 microM GDP beta S or 1 micrograms/ml pertussis toxin (PTX) through the patch pipette at a VH of -60 mV, the IACh diminished within 10 min, whereas the IACh of the control remained steady for over 20 min, suggesting that a PTX-sensitive G-protein is involved in the ACh response.(ABSTRACT TRUNCATED AT 400 WORDS)