Local mechanical properties of guinea pig outer hair cells measured by atomic force microscopy

In this study, mechanical properties of guinea pig outer hair cells (OHCs) were measured by atomic force microscopy (AFM). First, in order to confirm the availability of AFM for measurement of the mechanical properties of the OHC, Young's moduli of the OHCs measured in this study were converted into stiffnesses using a one-dimensional model of the cell and then compared with the values reported in the literature. Next, the difference in local mechanical properties of the OHC along the cell axis was measured. Finally, the relationship between Young's modulus in the middle region of the OHC and the cell length was evaluated. The results were as follows. (1) AFM is an adequate tool for the measurement of mechanical properties of the OHC. (2) Mechanical properties in the apical region of the OHC are a maximum of three times larger than those in the basal and middle regions of the cell. (3) Young's modulus in the middle region of a long OHC obtained from the apical turn of the cochlea and that of a short OHC obtained from the basal turn or the second turn are 2.0+/-0.81 kPa (n=10) and 3.7+/-0.96 kPa (n=10), respectively. In addition, it was found that Young's modulus decreases with an increase in the cell length.     

Acetylcholine-induced calcium oscillation in isolated outer hair cells in guinea pig

Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2 stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2 ]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2 ]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2 ]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2 ]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.     

Relationship between the local stiffness of the outer hair cell along the cell axis and its ultrastructure observed by atomic force microscopy

As electromotility may arise from a conformational change of the molecules' 'protein motors', which might be distributed along the outer hair cell (OHC) lateral wall, the force generated by the OHC electromotility would be related not only to the conformational change of the protein motors but also to the mechanical properties of the lateral wall. Therefore, a detailed understanding of the mechanical properties of the OHC lateral wall is important. In our previous reports, to understand the difference in the stiffness along the cell axis, the local deformation of the OHC in response to hypotonic stimulation was analyzed by measuring the displacement of microspheres attached randomly to the cell lateral wall, and the distribution of Young's modulus along the cell axis was obtained using the contact mode of an atomic force microscope (AFM). These investigations revealed that the stiffness of the cell in the apical region was greater than that in other regions where the stiffness is constant. In this study, the ultrastructure of the OHC lateral wall was investigated with the oscillation imaging mode of the AFM (Tapping Mode), and the relationship between the stiffness along the cell axis and the ultrastructure that was observed by the AFM imaging was analyzed. From the analysis, it was concluded that the circumferential filaments observed in the tapping mode AFM are actins which are part of the cortical lattice, and that the difference between the intervals of the circumferential filaments in the apical region and those in other regions is one factor that causes the high stiffness in the apical region.     

Mechanical properties of sensory and supporting cells in the organ of Corti of the guinea pig cochlea--study by atomic force microscopy

Mammalian hearing is refined by amplification of the motion of the cochlear partition. To understand the cochlear amplification, mechanical models of the cochlea have been used. When the dynamic behavior of the cochlea is analyzed by a model, elastic properties of the cells in the organ of Corti must be determined in advance. Recently, elastic properties of outer hair cells (OHCs) and pillar cells have been elucidated. However, those of other cells have not yet been clarified. Therefore, in this study, using an atomic force microscope (AFM), elastic properties of Hensen's cells, Deiters' cells and inner hair cells (IHCs) in the apical turn and those in the basal and second turns were estimated. As a result, slopes indicative of cell elastic properties were (8.9 +/- 5.8) x 10(3) m(-1) for Hensen's cells (n = 30), (5.5 +/- 5.3) x 10(3) m(-1) for Deiters' cells (n = 20) and (3.8 +/- 2.6) x 10(3) m(-1) for IHCs (n = 20), and Young's modulus were 0.69 +/- 0.45 kPa for Hensen's cells and 0.29 +/- 0.20 kPa for IHCs. There was no significant difference between elastic properties of each type of cell in the apical turn and those in the basal and second turns. However, it was found that there is a significant difference between Young's moduli of cells estimated in this study and those of the OHCs and pillar cells reported previously.     

Thyroid hormone is not necessary for the development of outer hair cell electromotility

The outer hair cell (OHC), one of two receptor cell types in the organ of Corti, plays a critical role in mammalian hearing. OHCs enhance basilar membrane motion through a local mechanical feedback process within the cochlea, termed the 'cochlear amplifier'. It is generally believed that the basis of cochlear amplification is a voltage-dependent electromotile response of OHCs. Measurements of electromotility in developing animals indicate that the onset of motility normally occurs around 7 days after birth in altricial rodents such as gerbils and rats. Thyroid hormone (TH) plays a crucial role in the development of the auditory system. Deficiency of the hormone between the late embryonic stage and the second postnatal week can cause severe hearing loss. Several studies suggest that TH deficiency might also affect the development of the cochlear amplifier. The goal of this study was therefore to examine whether TH was necessary for the development of OHC motility. The organ of Corti of gerbils was dissected out at birth and grown in culture with defined concentration of triiodothyronine (T3), the active ligand for the TH receptor. Motility was measured from OHCs isolated from 7-, 11- and 14-day-old cultures. Motility did indeed develop in OHCs deprived of normal concentration of T3. This suggests that the defective auditory function seen in TH-deficient animals is most likely due to morphological and physiological changes in the cochlea, rather than the motor function of the OHCs.     

Differences in the distribution of F-actin in outer hair cells along the organ of Corti

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.     

Prestin forms tetramer with each subunit being mechanically independent

Prestin is the motor protein of cochlear outer hair cells (OHCs). It is able to perform rapid and recipro-cal electromechanieal conversion that underlies OHC electromotility. Due to the inadequate size of a single prestin molecule to form the ～12 nm intramembraneous protein particles (IMPs) in the OHC lateral membrane (LM), the possibility of prestin oligomerization has been proposed. It has been suggested that prestin molecules form high-order oligomers, most likely as the tetramer, in heterologous systems. In OHCs, however, the oligomeric structure of prestin remains unelear. Here we calculated the prestin-related charge density in both gerbil and guinea pig OHCs through measuring their nonlinear capacitance (NLC) and LM surface area, showing that the average charge den-sity (22, 608 μm-2 in gerbils; 19, 460 μm-2 in guinea pigs) is statistically 4 times the average density of IMPs (5,686 μm-2 in gerbils; 5, 000 μm-2 in guinea pigs). This suggests that each IMP contains four prestin molecules based upon the notion that each prestin transfers a single elementary charge, implying that prestin forms tetramers in OHCs. To determine whether the prestin tetramer functions as a mechanical unit, we subsequently compared the slope factors (or) of electromotility and NLC simultaneously measured from the same OHC, showing that the α val-ues of the two are statistically the same. This suggests that each prestin molecule in the tetramer is mechanically independent and equally contributes to OHC electromotility.     

A subpopulation of outer hair cells possessing GABA receptors with tonotopic organization

The olivocochlear innervation has been postulated to regulate active mechanical processes in the mammalian cochlea. Histochemical studies led to the suggestion that a subpopulation of these efferent nerves, which predominantly terminate on outer hair cells (OHCs), are gamma-aminobutyric acid (GABA)-ergic. By means of two monoclonal antibodies, we were able to visualize GABAA-receptor immunoreactivity at the basal pole of isolated sensory cells. Both subunits of the GABAA receptor, the alpha- and beta-subunit, are known to form the transmembranous GABA/benzodiazepine-receptor complex and were present on OHCs. In addition, these inhibitory receptors were more numerous in the apical turns of the cochlea, indicating another criterion for distinguishing the apical from basal turns of the cochlea. These results support the concept that a subpopulation of axosomatic synapses at the basal pole of OHCs liberate the inhibitory neurotransmitter GABA into the synaptic cleft. Binding of the transmitter to these newly observed subsynaptic receptors is possibly followed by a change in OHC motility and a subsequent modulation of the movement of the basilar membrane.     

The radial pattern of basilar membrane motion evoked by electric stimulation of the cochlea.

Electric current applied to the cochlea can evoke in situ electromotile responses of the organ of Corti. These nonsound-generated responses can give insight into the mechanics of the organ as the putative forces produced by outer hair cells (OHC) must couple to the modes of vibration of the basilar membrane (BM). In this study, platinum-iridium wire electrodes were positioned into the scala vestibuli and scala tympani of the first cochlear turn in the guinea pig. Current (1.5 ms rectangular-shaped pulses) was applied to these electrodes at levels to 500 microA peak. A laser Doppler velocimeter was used to record the velocity or displacement of the basilar membrane at the tonotopic 18 kHz place via an opening into the scala tympani of the first cochlear turn. Beads were positioned across the width of the BM so that the velocity or displacement of the BM could be studied in the radial direction. It was found that the current pulses evoked linear displacements of up to 2 nm for current levels of 500 microA (higher levels were damaging to the organ of Corti). The pattern of motion across the width of the BM was such that maximum displacement and velocity was located near the first row of OHCs and the position of the outer pillar cell footplate. The BM motion was biphasic in that the zona arcuata moved in the opposite direction to that of the zona pectinata. The results of this study demonstrate that the level of force produced by OHCs is effective in moving the BM and that the distribution of force within the organ of Corti leads to a multimodal motion pattern of the BM for this experimentally artificial means of evoking OHC motion.     

Dimensions of the cochlear stereocilia in man and the guinea pig

The tuning properties of the basilar membrane and the presence of acoustic emissions from the cochlea suggest that an energy consuming, mechanically active cochlear amplifier exists. Some models of this amplifier demand a mechanical resonator within the cochlea. The lengths of the stereocilia of the inner and outer hair cells in man and the guinea pig have been measured from scanning electron micrographs using a stereometric technique. In both species there is a linear increase in the length of the longest inner hair cell stereocilia with distance along the cochlea. There are, however, marked differences between the dimensions of the outer hair cell stereocilia in the two species. In man, there is an increase in length which is is a hyperbolic function of distance along the cochlear duct. The picture is more complicated in the guinea pig. This could account for some of the differences in auditory physiology between the two groups. The mechanical resonance properties of the human OHC stereocilia have been assessed, and, with certain assumptions, these properties are such that resonance of the stereocilia of the OHCs could form part of the cochlea amplifier, at least in man.     

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

The outer hair cell (OHC) lateral plasma membrane houses the transmembrane protein prestin, a necessary component of the yet unknown molecular mechanism(s) underlying electromotility and the exquisite sensitivity and frequency selectivity of mammalian hearing. The importance of the plasma membrane environment in modulating OHC electromotility has been substantiated by recent studies demonstrating that membrane cholesterol alters prestin activity in a manner consistent with cholesterol-induced changes in auditory function. Cholesterol is known to affect membrane material properties, and measurements of lipid lateral mobility provide a method to asses these changes in living OHCs. Using fluorescence recovery after photobleaching (FRAP), we characterized regional differences in the lateral diffusion of the lipid analog di-8-ANEPPS in OHCs and investigated whether lipid mobility, which reflects membrane fluidity, is sensitive to membrane cholesterol. FRAP experiments revealed quantitative differences in lipid lateral mobility among the apical, lateral, and basal regions of the OHC and demonstrated that diffusion in individual regions is uniquely sensitive to cholesterol manipulations. Interestingly, in the lateral region, both cholesterol depletion and loading significantly reduced the effective diffusion coefficient from control values. Thus, the fluidity of the OHC lateral plasma membrane is regulated by cholesterol levels in a non-monotonic manner, suggesting that the overall material properties of the lateral plasma membrane are optimally tuned for OHC function in the native state. These results support the idea that the cholesterol-dependent regulation of prestin function and electromotility correlates with changes in the properties of the lipid environment that surrounds and supports prestin.     

Effects of intense noise exposure on the outer hair cell plasma membrane fluidity

Outer hair cells (OHCs) play an important role in cochlear amplification via their length changes (electromotility). A noise-induced cochlear amplification loss leading to a permanent threshold shift (PTS) was observed without a significant hair cell loss in rats [Chen, G.D., Liu, Y., 2005. Mechanisms of noise-induced hearing loss potentiation by hypoxia. Hear. Res. 200, 1-9.]. Since motor proteins are inserted in the OHC lateral membrane, any change in the OHC plasma membrane may result in a loss of OHC electromotility, leading to a loss of cochlear amplification. In this study, the lateral diffusion in the OHC plasma membrane was determined in vitro in guinea pigs by fluorescent recovery after photobleaching (FRAP) after an in vivo noise exposure. The lateral diffusion in the OHC plasma membrane demonstrated a length-dependence, which increased as OHC length increased. A reduction in the lateral diffusion was observed in those OHCs with lengths of 50-70 microm after exposure to an 8-kHz octave band noise at 110 dB SPL for 3h. This membrane fluidity change was associated with the selective PTS at frequencies around 8 kHz. The reduction of the lateral diffusion in the OHC lateral wall indicated that noise could impair the micromechanics of the OHC lateral wall and might consequently impair OHC electromotility to induce threshold shift.     

Quantitative Relations between Outer Hair Cell Electromotility and Nonlinear Capacitance

The electrically evoked somatic motility of outer hair cells (OHC), briefly termed OHC electromotility, plays a crucial role in cochlear amplification that underlies the remarkably high sensitivity and frequency selectivity of the mammalian hearing. Accompanying OHC electromotility is a voltage-dependent gating charge movement within the cell lateral membrane, manifested as a measurable nonlinear capacitance (NLC) in OHCs. The electromotility and NLC of OHCs are highly correlated by sharing a common molecular substrate, the motor protein prestin. In this study, we systematically characterized the quantitative relationship between OHC electromotility and NLC in their voltage dependences for the purpose of further understanding the electromechanical transduction in OHCs. The results demonstrated that the two possess differing voltage dependences with the V1/2 of electromotility consistently being ～20 mV depolarized in comparison with that of NLC although their slope factors α are statistically identical. Further investigations showed that the initial state of OHCs influences the voltage dependence of electromotility but not that of NLC, indicating that some biophysical factors other than the motor protein per se are involved in the process of OHC length changes. We proposed that the cytoskeletal spectrin-actin framework underneath the OHC plasma membrane and the cell’s turgor are the two most probable factors that cause the voltage-dependence discrepancy between OHC electromotility and NLC.     

Biophysical Mechanisms Underlying Outer Hair Cell Loss Associated with a Shortened Tectorial Membrane

The tectorial membrane (TM) connects to the stereociliary bundles of outer hair cells (OHCs). Humans with an autosomal dominant C1509G mutation in alpha-tectorin, a protein constituent of the TM, are born with a partial hearing loss that worsens over time. The Tecta ^C1509/+ transgenic mouse with the same point mutation has partial hearing loss secondary to a shortened TM that only contacts the first row of OHCs. As well, Tecta ^C1509G/+ mice have increased expression of the OHC electromotility protein, prestin. We sought to determine whether these changes impact OHC survival. Distortion product otoacoustic emission thresholds in a quiet environment did not change to 6 months of age. However, noise exposure produced acute threshold shifts that fully recovered in Tecta ^+/+ mice but only partially recovered in Tecta ^C1509G/+ mice. While Tecta ^+/+ mice lost OHCs primarily at the base and within all three rows, Tecta ^C1509G/+ mice lost most of their OHCs in a more apical region of the cochlea and nearly completely within the first row. In order to estimate the impact of a shorter TM on the forces faced by the stereocilia within the first OHC row, both the wild type and the heterozygous conditions were simulated in a computational model. These analyses predicted that the shear force on the stereocilia is ~50% higher in the heterozygous condition. We then measured electrically induced movements of the reticular lamina in situ and found that while they decreased to the noise floor in prestin null mice, they were increased by 4.58 dB in Tecta ^C1509G/+ mice compared to Tecta ^+/+ mice. The increased movements were associated with a fourfold increase in OHC death as measured by vital dye staining. Together, these findings indicate that uncoupling the TM from some OHCs leads to partial hearing loss and places the remaining coupled OHCs at higher risk. Both the mechanics of the malformed TM and the increased prestin-related movements of the organ of Corti contribute to this higher risk profile.     

Slow motility, electromotility and lateral wall stiffness in the isolated outer hair cells

Slow motile length changes of isolated, apical turn outer hair cells (OHCs) (n=36) were induced by perfusion of saline (flow rate: 0.6 microl/min) as a mechanical challenge or by perfusion of 12.5 mM KCl solution for 90 s as a chemical and mechanical challenge with and without ocadaic acid (OA), a serine/threonine protein phosphatase inhibitor. Electromotility was evoked by square pulses from +/-35 mV to +/-240 mV during the slow shortening and recovery period (n=36). Stiffness of the lateral wall was measured by the micropipette aspiration technique (n=20). Saline perfusion caused a reversible shortening of 774+/-87 nm (n=9) as well as K+ of 1465+/-159 nm (n=9). Slow shortening increased lateral wall stiffness (1.25+/-0.02 to 1.52+/-0.03 nN/microm) (n=5-5). Simultaneously, electromotility magnitude decreased (n=9). Ocadaic acid blocked slow shortening, increased lateral wall stiffness, and decreased the magnitude of electromotility. Mechanical or mechanical+chemical stimulation of ocadaic acid treated OHCs do not further change stiffness or electromotility. Isolated OHCs respond with slow shortening and consutive cell stiffness increase to mechanical insult. This phenomenon seems operating with calcium-, and phosphorylation-dependent modifications of the cytoskeletal proteins. The subsequent electromotility gain decrease suggests a slow OHC shortening driven regulation of the cochlear amplifier with simultaneous safety control of the auditory periphery against overstimulation.     

Measurement of Ca2+ Flow in Cochlear Cells Using Non-Invasive Micro-Test Technique

Objective To test Calcium ion(Ca2+)flow at the head and end of outer hair cells(OHCs)in resting state and in response to Nimodipine treatment.Methods Non-invasive micro-test techniques were used to study Ca2+ in isolated OHCs in adult guinea pigs.Results Four types of Ca2+ transport were identified in OHCs on basilar membrane tissue fragments:influx at the head of with efflux at the bottom(type 1):efflux at the head of OHCs with influx at the bottom(type 2);influx at the both head and bottom(type 3);and efflux at the both head and bottom(type 4).However,only type 1 and type 3 of Ca2+ ion transport were detected in the cochlea.We propose that Ca2+ ion transport exists in adult guinea pig cochlear OHCs in resting state and is variable.Ca 2+flow in OHC can be inhibited by Nimodipine in resting state.     

Electromotile performance of isolated outer hair cells during slow motile shortening

CONCLUSION: The electromotile performance of isolated OHCs does not seem to be dependent on slow motile shortening alone; other mechanisms, such as phosphorylation, are also involved. OBJECTIVE: To elucidate the relationship between the magnitude of electromotile displacements and magnitude of slow motile shortening of outer hair cells (OHCs) induced by mechanical or chemical stimulation over a reversible range. MATERIAL AND METHODS: Isolated guinea pig OHCs were mechanically (0.6 microl/min perfusion of saline; n =4) or chemically and mechanically (0.6 microl/min perfusion of 12.5 mM KCl; n =4) stimulated in a glass microchamber to evoke slow motile shortening. RESULTS: Combined mechanical and chemical stimulation evoked greater OHC shortening than mechanical stimulation alone. Both forms of stimulation resulted in reversible shortening. Electromotility was measured using low voltage (+/- 35 mV) and higher voltage (up to +/- 240 mV) electrical pulses mimicking the receptor potential at different stages of cell shortening. The magnitude of electromotility decreased simultaneously with slow motile shortenings of OHCs. Irrespective of the character of the stimulus (mechanical or mechanical + chemical), the decrease in the magnitude of electromotility was dependent on the degree of cell shortening. Ocadaic acid, a protein phosphatase inhibitor, blocked slow motility and decreased the magnitude of electromotility.     

High-Frequency Force Generation in the Constrained Cochlear Outer Hair Cell: A Model Study

Cochlear outer hair cell (OHC) electromotility is believed to be responsible for the sensitivity and frequency selectivity of the mammalian hearing process. Its contribution to hearing is better understood by examining the force generated by the OHC as a feedback to vibration of the basilar membrane (BM). In this study, we examine the effects of the constraints imposed on the OHC and of the surrounding fluids on the cell's high-frequency active force generated under in vitro and in vivo conditions. The OHC is modeled as a viscoelastic and piezoelectric cylindrical shell coupled with viscous intracellular and extracellular fluids, and the constraint is represented by a spring with adjustable stiffness. The solution is obtained in the form of a Fourier series. The model results are consistent with previously reported experiments under both low- and high-frequency conditions. We find that constrained OHCs achieve a much higher corner frequency than free OHCs, depending on the stiffness of the constraint. We analyze cases in which the stiffness of the constraint is similar to that of the BM, reticular lamina, and tectorial membrane, and find that the force per unit transmembrane potential generated by the OHC can be constant up to several tens of kHz. This model, describing the OHC as a local amplifier, can be incorporated into a global cochlear model that considers cochlear hydrodynamics and frequency modulation of the receptor potential, as well as the graded BM stiffness and OHC length.     

Chlorpromazine inhibits cochlear function in guinea pigs

Outer hair cell (OHC) electromotility provides mechanical positive feedback that functions as the cochlear amplifier. In isolated OHCs, chlorpromazine shifts the electromotility voltage-displacement transfer function in a depolarizing direction without affecting its magnitude. This study sought to measure the effects of chlorpromazine on cochlear function in vivo. Salicylate, a drug that greatly reduces the magnitude of electromotility, was used for comparison. Perilymphatic perfusion of the guinea pig cochlea with chlorpromazine or salicylate increased the compound action potential (CAP) threshold across the frequency spectrum (1-20 kHz). Both drugs also increased distortion product otoacoustic emission (DPOAE) thresholds in the higher frequencies (10-20 kHz). Complete reversibility of these effects occurred after washout. Both drugs demonstrated concentration-dependent reductions in cochlear function that followed sigmoidal curves with similar fits to previously reported results in isolated OHCs. The endolymphatic potential was not affected by either of these drugs. Thus, chlorpromazine inhibits cochlear function in a manner consistent with what would be expected from data in isolated OHCs. This suggests that shifting the electromotility transfer function correspondingly reduces the gain of the cochlear amplifier.     

豚鼠冲击波负压暴露后耳蜗毛细胞损害定量观察

目的 探讨冲击波负压（blast underpressure,BUP）暴露后豚鼠耳蜗毛细胞损害特点.方法将豚鼠暴露实验性BUP 14天后处死,硝酸银染色硬铺片法计数观察耳蜗基底膜毛细胞损伤情况.结果压力峰值介于-22.4kPa和-63.3kPa之间的实验性BUP暴露后,豚鼠耳蜗外毛细胞出现了明显的病理性改变,损伤的程度以第二转最重,第二排和第三排的病变比第一排更为严重.BUP强度越高,毛细胞损害越重.各实验组动物的外毛细胞总缺失率明显高于正常对照组（P＜0.01）;重复暴露3次的动物外毛细胞缺失率明显高于暴露1次的动物（P＜0.01）.结论BUP暴露可引起明显的豚鼠耳蜗外毛细胞缺失等损害,其损害程度与负压峰值及暴露次数密切相关;毛细胞损害越重,ABR阈移也就越明显.