]>HEARES3195S0378-5955(99)00009-X10.1016/S0378-5955(99)00009-XElsevier Science B.V.Fig. 1Velocity responses of the BM for an applied current to the cochlea (average of 256 stimuli). Positive velocity values represent motion toward scala tympani. The rectangular pulse of current (400 μA, 1.5 ms duration) was passed between electrodes located in the scala tympani and the scala vestibuli of the first turn. (GP033, bead location on BM is at the zona arcuata.) The duration of the current is given by the bar graphic in the center of the figure. A: The polarity of the voltage applied to the electrodes was scala vestibuli positive. B: The polarity of the voltage was scala vestibuli negative.Fig. 2Displacement responses (positive values are toward the scala vestibuli) of the basilar membrane for current applied to the cochlea as described in the caption of Fig. 1. Data were collected from a bead located at the basilar membrane radial location of the outer pillar cell/first row of OHCs. Positive voltage applied to the scala vestibuli resulted in the response given by the dotted line. Negative voltage to the scala vestibuli gave a response given by the solid line. These responses are the average from 2048 stimuli. The unit of BM displacement is nanometer (nm). (GP033, bead location on BM is at the zona arcuata.)Fig. 3Input/output functions for BM displacement (peak value) as a function of the current delivered to the cochlea. Positive voltage to the scala vestibuli and negative voltage to the scala tympani resulted in motion toward the scala vestibuli for beads located on the pectinate area of the BM (outer pillar cell area (OPC), outer hair cell one area (OHC1), outer hair cell three area (OHC3)), while the bead on the arcuate area at the margin of the spiral osseous lamina (on the zona arcuata (ZA)) moved toward scala tympani. The functions are linear for the range of current tested. Current level is presented in μA.Fig. 4Basilar membrane (BM) velocity responses from four locations (spiral osseous lamina edge (SOL), outer hair cell one (OHC1), outer hair cell two (OHC2), Claudius cells) across the width of the BM. The stimulus was a 500 μA rectangular pulse (1.5 ms duration) applied to the basal turn starting at 1.0 ms (see bar) with scala vestibuli having a positive voltage and scala tympani a negative voltage. For the topmost trace and the second trace, arrows mark the occurrences of onset and offset pulses. These pulses correspond to BM displacement steps (see Fig. 2) at the initiation and termination of the current. The relative size of the onset and offset velocity pulses indicate the relative magnitude of BM displacement at the various radial locations which, in this animal (GP 036), are not of typical relative magnitude as shown in Fig. 5. Note the phase reversal of the velocity pulse between the SOL location on the zona arcuata and the other locations, which are all on the zona pectinata of the BM. Ringing transient responses in this less sensitive cochlea are smaller and less delayed than the more sensitive example of Fig. 1.Fig. 5The composite data from the animals in this study as given in Table 1. When a current of 400 μA was applied to the cochlea, displacement and peak velocity of the ringing responses both reached a maximum at the same radial location; near the outer pillar cell or the first row of OHCs. Filled circles and open squares are the mean and bars are ±1 S.D. from the mean. The numbers over each bar are the numbers of measurement animals in the mean calculation.Table 1The direction of BM displacement with current stimulationAnimal #dB loss at 18 kHznear SOLOPCOHC1OHC2OHC3Claudius cellsGP00820↓↓GP01023↓↓GP01525↓↓↓GP016>20↑↓GP01718↓GP02014↑↓↓↓GP0217↑↓GP02525↓↓↓GP026>40↓↓GP02828↑↓GP03246↓↑↑GP0339↑↓↓GP034>30↑↑↑GP03510↓GP036>30↑↓↓↓GP03711↑↓↓↓GP0398↑↓GP0468↑↓GP04712↑↓Direction trend⇑⇓⇓⇓⇓⇓⇑Various current levels ‘500 μA, scala tympani positive, scala vestibuli negative. ↑=displacement toward scala media; ↓=displacement toward scala tympani. SOL, spiral osseous lamina; OPC, outer pillar cell; OHC, outer hair cell.The radial pattern of basilar membrane motion evoked by electric stimulation of the cochleaAlfred L.Nuttallac*nuttall@ohsu.eduMengheGuobTianyingRenaaOregon Hearing Research Center, NRC04, Department of Otolaryngology/Head and Neck Surgery, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098, USAbDepartment of Otolaryngology, Zhujiang Hospital, The First Military Medical University, Guangzhou 510282,ChinacKresge Hearing Research Institute, The University of Michigan, 1301 East Ann Street, Ann Arbor, MI 48109-0506, USA*Corresponding authorAbstractElectric 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 μA 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 μA (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.KeywordsGuinea pigOtoacoustic emissionLaser Doppler velocimetryOuter hair cellElectromotility1IntroductionThe electromotility of outer hair cells (OHCs) has been characterized in a variety of in vitro studies beginning with the seminal paper by Brownell et al. (1985). It has been shown that the voltage sensitivity of somatic length change is about 2 nm/mV at a membrane resting potential of −70 mV (Santos-Sacchi, 1989). A measurable force from OHCs can be inferred when there is a dimension change of the organ of Corti (e.g., Zenner et al., 1992). It has also been determined that when operating under load, OHC elongation is considerably smaller than when a hair cell is unloaded (Hallworth, 1995).Little is known about the effects of any force or displacement that might result from OHC membrane polarization changes when OHCs are operating in their natural environment within the organ of Corti in vivo. According to current hypotheses the mechanical role of OHCs is that they could act as acoustic force amplifiers in a cycle-by-cycle positive or negative feedback amplifier fashion (Dallos, 1992; Mountain and Hubbard, 1989) or as active mechanical stiffness regulators (Dallos and He, 1997).The result of OHC force or displacement in the organ of Corti is not only dependent on their inherent mechanical properties but also on the structural elements of the organ that permit the delivery of force, or the coupling of stiffness, into the motion of the basilar membrane (BM) and ultimately the displacement of inner hair cell stereocilia. The OHCs are arranged in complex association with supporting cells, particularly Deiters’ cells and pillar cells. Models of cochlear mechanics that attempt to provide a deeper understanding of normal sensitivity and frequency tuning require incorporation of organ of Corti micromechanical features (e.g., Geisler, 1986; Steele and Taber, 1981). This is especially true for finite element modeling approaches where mechanical properties are needed for the various nodes in the model (Kolston and Ashmore, 1996; Zhang et al., 1997).In this study we examined the displacement and velocity of the BM that is produced by putative forces produced by OHCs when electrically stimulated. The measurements were made in the intact and living guinea pig organ of Corti. We observed that rectangular displacements of at least 1–2 nm could be elicited. The radial pattern of BM motion was complex (bi-directional) for a given polarity of electric polarization across the organ. The data are consistent with the hypothesis that the morphologically thin arcuate zone region of the basilar membrane is very compliant by comparison with the pectinate zone.2MethodsHealthy young adult pigmented guinea pigs weighing 250–400 g were used in this study. Animals were from the Murphy Breeding Laboratory or the Charles River Company and were housed in American Association for Accreditation of Laboratory Animal Care approved facilities. Experimental protocols were approved by the Committee on Use and Care of Animals at the University of Michigan and at the Oregon Health Sciences University. The animals were preanesthetized with an initial injection of pentobarbital (15 mg/kg i.p.) followed 15 min later by an injection of ketamine/xylazine (ketamine 40 mg/kg; xylazine 10 mg/kg i.m.). Supplemental doses of ketamine and xylazine were given on a schedule or as needed, judging by a slight withdrawal in response to toe pinch. Pentobarbital supplements (7 mg/kg i.p.) were given every 2 h.The animal’s rectal temperature was maintained at 38±18°C with a servo-regulated heating blanket. The cochlear temperature was additionally controlled by supplemental heat to the head from a lamp and a heated head holder. The electrocardiogram and heart rate were continuously monitored as an additional measure of anesthesia level and animal general condition. All presented data were collected from animals with a normal electrocardiogram.The guinea pig’s head was firmly fixed in a heated headholder that was mounted on a custom-made manipulator to allow positioning of the cochlea in the visual field of the BM velocity-measuring microscope. A tracheotomy was performed and a ventilation tube inserted into the trachea to insure free breathing. A ventral and postauricular surgical dissection exposed the auditory bulla on the left side and the external ear was removed (exposing the lateral 2/3 of the external auditory canal) to facilitate placement of the acoustic speculum. The bulla was widely opened to expose the cochlea and the middle ear muscle tendons were sectioned. An Ag-AgCl ball electrode with about 200–300 μm diameter was placed in the round window niche for recording of sound-evoked cochlear potentials. An Ag-AgCl ground electrode was inserted into neck soft tissue medial to the exposed bulla. The round window signal, relative to the neck ground electrode, was amplified 10 times by a P16 Grass amplifier and 100 times by a custom-designed AC amplifier. The amplified CAP signal from the round window was displayed on an oscilloscope and the N1 detection of 10 μV was used as a threshold criterion.An opening approximately 300 μm in diameter was made on the lateral wall of the scala tympani of the basal cochlear turn for measurements of the BM velocity. The surgical opening into the cochlea to observe BM motion was made by thinning the bone over the scala tympani of the basal turn with a knife and picking out the bone pieces with a small hook.Two electrodes were made of Teflon-insulated 75 μm diameter platinum-iridium wire for the cochlear electrical stimulation. One stimulation electrode was inserted inside the scala vestibuli through a 100 μm diameter hole made on the lateral wall of the scala vestibuli of the basal cochlear turn. The electrode was fixed and the hole was sealed with tissue cement. Another platinum-iridium electrode was positioned in the scala tympani through the opening on the lateral wall of the scala tympani. To evoke basilar membrane motion, a 1.5 ms electrical pulse was generated by a D/A converter and was delivered as an electrical stimulus to the cochlea by an optical-isolated constant current stimulator. The current level of the electrical stimulus was controlled by a programmable attenuator.After the electrodes were fixed in position and the animal’s head was maneuvered into position presenting the BM approximately in the horizontal plane, gold-coated glass beads (<20 μm diameter) were placed on the BM to serve as reflective objects for the laser beam of the laser Doppler velocimeter (Polytec OFV-1101).The radial positions of these beads across the width of the basilar membrane were random. Their radial locations were noted in relation to visible morphological features of the organ of Corti such as the OHCs, the spiral capillary of the BM and the osseous spiral lamina. Generally, it was possible to achieve more than two radial bead locations in a given experiment (see Table 1). The laser beam of the laser Doppler velocimeter was coupled into a compound microscope and the focused laser beam was directed at a selected glass microbead on the BM. Velocity signals from the instrument were analyzed and measured with the aid of a lock-in amplifier (Stanford Instruments SR530) and a spectrum analyzer (Stanford Instruments SR760). Experiments were accomplished on the top of a vibration isolation table and inside a double-walled soundproof booth. Details of the surgical method and the measurement of BM velocity can be found in Brown et al. (1983) and Nuttall et al. (1991).For the measurement of electrically evoked BM vibration, the velocity or displacement signal of the laser Doppler velocimeter was digitized by an A/D converter at the rate of 125 000 samples per second with a 4 ms time window synchronized with electrical stimuli without time delay. The laser Doppler velocimeter signal was averaged by custom software. A velocity or displacement waveform of the BM vibration could be obtained from the instrument. The directions and amplitudes of the BM step responses and of transient ringing responses were determined. The relationship between the vibration and the radial positions of the beads across the BM was observed.3ResultsThe results presented in this report are derived from 19 guinea pig experiments. Individual examples of typical responses are shown in the figures for the purpose of qualitatively conveying the nature of BM displacement and velocity. The purpose of this report is to describe the pattern of BM displacement that results from a mechanical force acting within the organ of Corti when the cochlea is stimulated electrically. Since electrically evoked BM responses are greatly reduced in cochleas of guinea pigs treated with ototoxic drugs to destroy OHCs, the mechanical force is likely due to the electromotile activity of these cells (Nuttall and Ren, 1995).The application of electric current pulses to the cochlea evokes BM motion. Fig. 1 illustrates the general form of this motion. The velocity responses of the BM are shown in response to 400 μA positive (Fig. 1A) and negative (Fig. 1B) current pulses of 1.5 ms duration. Initially, an onset transient occurs (a velocity spike), the direction of which is dependent on the polarity of the electric current. These velocity transients correspond to a displacement step of BM motion. An example of displacement steps is given in Fig. 2 (obtained from the same animal). The displacement waveforms are considerably more noisy than the velocity waveform, reflecting the animal motion artifact introduced by cardiovascular, respiratory, and skeletal muscle activity and the poorer signal-to-noise ratio of the interferometer for displacement measurements. In these experiments, using bipolar electrodes between the scala tympani and scala vestibuli, a positive voltage applied to the scala vestibuli relative to a negative voltage in scala tympani evoked motion of the BM near outer hair cells toward the scala media.Note in Fig. 1 that the velocity ‘spikes’ which occur at the beginning of each current pulse in Fig. 1A,B are approximately equal in size (height). This indicates that the displacement motion of the BM for the two polarities of stimulation was the same and therefore that the length of the putative OHC elongation or contraction was symmetrical. Fig. 2 illustrates the pattern of the actual displacement measured with amplitude-sensitive interferometry. The time-dependent shape of the displacement pattern in Fig. 2 was found to be variable between animals. In cochleas with poor sensitivity, the displacement pattern tended to be rectangular in shape. Sensitive cochleas tended to have displacement responses that had the appearance of being high pass filtered. We define the sensitive cochlea as the condition where the sound level required for a criterion response of the cochlear action potential (CAP) evoked by acoustic tone bursts (i.e., the 10 μV N1 threshold at 18 kHz, the best frequency of the BM measurement location) is within about 10 dB of normal. Most of the cochleas studied (Table 1) have greater loss than 10 dB but their sensitivity is generally better than would be the result of a complete loss of the cochlear amplifier (>40 dB loss). The time-dependent changes of the displacement were not systematically studied in this investigation.Following the onset of the displacement (or the onset velocity spike) a ringing response of the BM is observed. A second ringing response occurs following the offset of current. The polarity of the current (i.e., scala vestibuli positive related to scala tympani) alters the size of the ringing response. Positive current enhances the BM ringing motion while negative current decreases the response. The ringing responses are the local resonant vibration of the BM at the tonotopic best frequency (BF), which in this case is about 18 kHz. We have shown that these transient, ringing responses are equivalent to those that result from acoustic transient stimuli (clicks) and that they are the result of intracochlearly generated acoustic energy from OHCs stimulated by the current (Nuttall and Ren, 1995). That current modulates the transient ringing response has previously been reported by us (Nuttall and Dolan, 1993; Nuttall et al., 1995). The cause of the amplitude modulation of the ringing is not known but is possibly related to the influence of the current on OHC membrane polarization and changes in the gain of the cochlear amplifier.The symmetric displacements of the BM are linear functions of current in the range used in this study (up to 500 μA). Fig. 3 shows input/output curves for BM displacement for levels of current from 100 to 500 μA. The functions are approximately straight. The three lines plotted, however, illustrate that the amount of displacement is a function of the radial position across the BM. The highest sloped line is for a location at the junction of the zona arcuata and the zona pectinata areas of the BM and at the location of the OHCs.The radial pattern of velocity across the BM is illustrated by the data plotted in Fig. 4 for fortuitous distribution of glass measurement beads on the surface of the BM. The figure shows that relative displacement (as measured by the size of the initial velocity spike) and the ringing transient response are functions of the radial distance across the width of the BM. Fig. 5 graphs the mean (±1 S.D.) displacement and ringing response peak velocity (for positive current) responses of a number of animals in this study. Note that the maximum displacement and velocity motion of the BM is at a location close to the junction of zona arcuata and pectinata or near the first row of OHCs. The large variance of the data at the pillar cell, OHC1 and OHC2 locations, could be due to position assessment inaccuracies. Close to the margin of the spiral osseous lamina, the displacement reversed in direction. Therefore, there must be a transition point of no motion that is located somewhere between the outer pillar cell and the spiral osseous lamina. We were not able to define this point with any greater precision. Only in one animal were three beads fortuitously next to one another across the width of the zona arcuata. The center bead did have a ‘neutral’ motion while the other two had opposite phase. Since the three beads were touching, or nearly so, it is possible that they influenced the motion of one another.Fig. 5 also depicts that the radial position of maximum velocity of the ringing response corresponds to the position displacement response maximum. The junction between the anatomically different regions of the BM (zona arcuata and zona pectinata) is the location of large amplitude motion. Our results on the position of peak displacement appear similar to data reported by Nilsen and Russell (1998).Since only a few reflective beads are placed on the BM in a given animal, it is difficult to obtain information on all locations across the BM. It is therefore useful to examine the group data of all the measured guinea pigs in this report. The direction of BM motion is summarized in Table 1, which shows that the area near the osseous spiral lamina is opposite (in general) from that which occurs near OHCs when the cochlea is electrically stimulated by a rectangular pulse of current. The place of transition between the opposing motion patterns is near the junction of the zona arcuata and the zona pectinata.4DiscussionIn this study we have made an initial exploration of the micromechanics of the organ of Corti, in situ. The means to explore the mechanics is through the application of electric current to the cochlea. In an ear not stimulated by sound, electric current evokes motion of the BM. A putative elongation and force produced by the OHCs from the electrically induced polarization changes of their membranes is most likely responsible for moving cellular elements of the organ of Corti. A motion of the BM so induced is a demonstration that OHCs have the strength to displace the BM (acting against a stiffness in this case), and the pattern of the motion would reveal some properties about the transfer of the OHC-produced force within the structure of the organ of Corti.Xue et al. (1995) observed displacements between 5 and 10 nm for a current of 50 μA delivered into the scala media. This is a larger displacement level than we achieved and is possibly due to a more effective OHC membrane polarization by the scala media delivered current. Xue et al. (1995), using estimates of the electrical resistances of the scala media and OHCs, calculated that 50 μA would produce a membrane potential shift of 40 mV which would be depolarizing for positive voltage in the scala media. When current is applied across the cochlear duct (the method of this paper), electroanatomical considerations dictate that perhaps only 10% of the applied current would pass into scala media (Honrubia and Ward, 1968). The effective current would therefore be similar to that used by Xue et al. (1995).We observed that the BM displacement responses were linear over ±500 μA1 (Fig. 3). This linearity also suggests that the membrane potential shifts induced were small because the OHC length-to-membrane potential relationship is nonlinear (Evans et al., 1991; Santos-Sacchi, 1993). It is consistent that Hallworth (1995) has shown that mechanically loaded or unloaded isolated OHCs give linear displacements for estimated membrane potentials of up to 15 mV2. Moreover, we have observed linear electrically evoked otoacoustic emissions, a finding that implies linear tissue vibration (Ren and Nuttall, 1995; Ren et al., 1996; Ren, 1996).1The limiting factors of current delivery in our experiments are both biological and electrochemical. With the typical size platinum wire we use (3T), noticeable gassing occurs at the wire surface between 800 and 1200 μA. From the biological point of view, we have empirically determined in our experiments that damage to the cochlear amplifier causing reduced sensitivity occurs above 500 μA. The pathophysiology of the damage is not known at this time.2The 15 mV estimate was obtained from voltage divider model of the microchamber stimulated OHC in the Hallworth (1995) paper using the parameters: 4 μm radius and 30 μm length.The measured BM displacement is considerably less than that expected from studies of isolated OHCs and the estimated membrane potential shift induced in this study. The slope gains of isolated OHCs are between about 1 and 4 nm/mV and strongly dependent on resting membrane potential (Evans et al., 1991; Santos-Sacchi, 1989). Mammano and Ashmore (1993) have determined that current induced in situ displacements of OHCs of the cochlear apical turn cause up to a five times larger antiphasic movement of the reticula lamina than the BM. Since this antiphasic motion of the reticular lamina and the BM will be a function of the compliance of both structures, it may be possible that their displacement relationship is changed considerably in the base of the cochlea where the BM is as much as 100 times stiffer than in the apex (von Békésy, 1960). Nevertheless, the clear displacement of the BM indicates that OHCs can produce a physiologically relevant force (causing displacements of the same order as evoked by moderate-level sounds). Xue et al. (1995), based on BM point stiffness measurements from Olson and Mountain (1991), calculated the single OHC slope gain for force to be about 2 pN/mV, which is comparable to that obtained in other studies (Hallworth, 1995; Russell and Schauz, 1995).In the present study, we observed that the pattern of motion across the width of the organ of Corti is biphasic. These results extend our earlier report (Nuttall et al., 1995, 1997) and confirm the similar finding of Xue et al. (1993). The concept of a second mode of vibration across the width of the BM can be found in the speculations by von Békésy (1960) based on anatomical considerations. The OHCs are located in association with the thicker pectinate zone of the BM.The point stiffness of the pectinate and arcuate zones of the BM have been measured by Olson and Mountain (1994). They found a highly asymmetric stiffness distribution about the position of the outer pillar cell. The outer pillar cell location was about five times stiffer than the BM in the arcuate zone. They attribute this stiffness difference to cellular elements of the organ of Corti rather than the inherent properties of the two zones of the BM. The outer pillar cell may be responsible for the peak in stiffness at its radial location across the width of the BM. An antiphasic motion vibration mode of the BM could be achieved by the deflection and rotation of this cell. Antiphasic motion also could result from fluid pressure acting within an isovolumetric organ of Corti. Conservation of volume could require the relatively compliant arcuate zone of the BM to move in the opposite direction relative to the pectinate zone when the outer hair cells distend the reticular lamina and the BM in opposite directions.The stimulus protocol of this study (pulses) is sufficiently different from that of Xue et al. (1993, 1995) (sinusoids) that direct comparison of the results is difficult. Xue et al. (1993; their Fig. 4B) show the antiphasic motion of the BM in the gerbil as a vibration mode is present at high frequencies, and we also observe the phase of the two zones to be different in our stimulation protocol. Moreover, Xue et al. (1995) found a significant phase lead of the pectinate zone relative to the current at low frequencies. This was attributed either to velocity tracking of the length changes of OHCs or a phase shift of the OHC membrane potential. In the current study, the displacement responses of the BM were in phase with the current when the cochlea was insensitive but in cochleas with preserved sensitivity (i.e., a functional cochlear amplifier) the responses were sometimes velocity-like. The study of velocity-like responses is an area of future work.Although this was not specifically investigated in the current study, we have previously reported that both acoustic and electrical stimulation of the organ of Corti with sinusoids resulted in equivalent phase vs. frequency functions. In contrast Xue et al. (1995) found that electrical stimulation caused much smaller total phase shifts. The difference could be due to electrode configurations. However, our interpretation of the mode of stimulation of the local activity of the BM for high frequencies is that the current causes an intracochlear differential acoustic pressure by OHC-caused shape change of the organ of Corti that is equivalent to an external stimulus (Nuttall and Ren, 1995). In the current study we observed a related phenomenon where the ringing responses following onset or offset of a given polarity of current stimulation always had the same phase in the two zones even though the rectangular BM displacement due to the current was antiphasic. There was no phase shift of the ringing responses as a function of radial distance across the BM. We attribute this result to the forced oscillation of the two zones experiencing the same initial pressure.The direction of motion of the BM from a current applied across the cochlea with positive voltage applied to the scala vestibuli relative to the scala tympani is pectinate zone deflection toward the scala media. This direction is consistent with Xue et al. (1995) for positive voltage in the scala media. Previously we reported that this polarity moved the BM toward the scala tympani but that was for a reflective bead on the BM at the zona arcuata. 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