Active Hair Bundle Movements and the Cochlear Amplifier

Transcription

1 Active Hair Bundle Movements and the Cochlear Amplifier Anthony Ricci* Abstract The active process is a term used to describe amplification and filtering processes that are essential for obtaining the exquisite sensitivity of hearing organs. Understanding the components of the active process is important both for our understanding of the normal physiology of hearing and because perturbations of the cochlear amplifier may lead to such maladies as threshold shifts (both temporary and permanent), tinnitus, sensorineural hearing loss and presbicusis. To date the cochlear amplifier has largely been attributed to outer hair cell electromotility; however, recent evidence suggests, that active properties of the hair bundle may also be important. Most likely both somatic motility and active hair bundle movements contribute to establishing the cochlear active process. This paper reviews recent evidence regarding known active processes in the hair bundle gating compliance, and fast and slow adaptation. Key Words: Active process, adaptation, gating compliance, hair bundle, met channels Abbreviations: met = mechano-electric transduction, OHC = outer hair cell, IHC =inner hair cell Sumario: El proceso activo es un término utilizado para describir los procesos de amplificación y filtrado que son esenciales para lograr la exquisita sensibilidad de los órganos auditivos. Es importante entender los componentes de este proceso activo, tanto para comprender la fisiología normal de la audición, como las perturbaciones del amplificador coclear, que pueden llevar a trastornos tales como los cambios de umbrales (temporales y permanentes), el acúfeno, los trastornos auditivos sensorineurales y la presbiacusia. En la actualidad, el efecto amplificador coclear ha sido atribuido a la electromotilidad de las células ciliadas externas, aunque la evidencia reciente sugiere que las propiedades activas del haz ciliar también pueden ser importantes. Posiblemente, tanto la motilidad somática como los movimientos activos del haz ciliar contribuyen a establecer este proceso activo coclear. Este artículo revisa la evidencia reciente con relación a los procesos activos conocidos de este haz ciliar, incluyendo la compliancia de paso y las adaptaciones rápidas y lentas. Palabras Clave: proceso activo, haz ciliar, canales MET, adaptación, compliancia de paso, MET = transducción mecano-eléctrica; OHC = células ciliadas externas; IHC = células ciliadas internas. *Neuroscience Center and Kresge Hearing Labs, Louisiana State Health Sciences Center Reprint requests: Anthony Ricci, Neuroscience Center and Kresge Hearing Labs, Louisiana State Health Sciences Center, 2020 Gravier St. Suite D, New Orleans, LA 70112; Ph: ; Fax: ; aricci@lsuhsc.edu 325

2 Journal of the American Academy of Audiology/Volume 14, Number 6, 2003 HEARING Airborne sound is the propogation of mechanical pressure vibrations. The middle ear converts these airborne vibrations into pressure waves in fluid. The cochlea is specialized to create a pressure differences between compartments that results in a vibration of the basilar membrane. The organ of Corti, which houses the sensory apparatus, rides on top of the basilar membrane, thus sensing the vibrations imparted by sound (Fig. 1). Measurements from primary afferent neurons demonstrate that the cochlea can be described as a series of highly tuned sensors. Each sensory cell responds with high selectivity and sensitivity to a particular frequency of sound, its characteristic frequency. The organ of Corti is organized tonotopically with a high frequency basal region and a low frequency apical region. A variety of mechanical and electrical mechanisms are involved in establishing the tonotopic organization of the cochlea. These mechanisms can vary between species, but there are some fundamental similarities used by hair cells to overcome difficulties that are common to each. One common problem faced by all hair cells is that the energy associated with sound at threshold is small. In order to obtain such low threshold levels (high sensitivity), the signal must be first selected for frequency and then amplified. The purpose of this manuscript is first to explain the role of cochlear amplification to auditory detection and second to describe mechanisms thought to contribute to the cochlear amplifier. The primary focus will be on processes associated with hair cell sensory hair bundles. Sensitivity of Hearing Requires Amplification and Tuning In order to appreciate the extraordinary performance of auditory sensory hair cells, one must first recognize the thermodynamic barriers confronting these cells. Every biological sensor has a baseline energy (thermal noise) associated with it. The energy associated with the sensory stimulus, whether it is mechanical, chemical, or light adds to the existing thermal energy associated with the sensor. Detection is dependent on the relative difference between the baseline intrinsic energy (noise) and the energy associated with the incoming sound (signal). The challenge to the auditory system is that the energy associated with a sound at threshold is much smaller than that associated with the thermal energy of the sensory hair bundle (Denk et al., 1989) implying that these low energies cannot be detected. In part, this limitation is overcome by utilizing the sinusoidal nature of sound to signal average multiple cycles. Paradoxically, displacement of the basilar membrane at auditory threshold are subnanometer, values that are smaller than thermally driven hair bundle movements, predicted to be ~2nm (Sellick et al., 1982; Crawford and Fettiplace, 1985). To understand how it is possible to discriminate movements <1nm on top of a 2nm noise floor, one must realize that the power associated with thermal noise movement is uniform across frequencies. Much smaller movements are observed at any particular frequency. A detection system that has a narrowed bandwidth would have smaller thermal energies to combat. In contrast to the noise constraints imposed by a passive system, an active resonant system in the hair bundle that is tuned over a narrow frequency range can have the required detection limit (Bialek, 1987). The ability of hair bundles to detect signals with energies comparable to thermal noise energy and to use the energy associated with Brownian motion has been demonstrated, attesting to presence of active amplification and tuning mechanisms in the sensory hair bundle (Denk and Webb, 1992; Jaramillo and Wiesenfeld, 1998) and to the remarkable specialization of the sensory hair cell. Additional Evidence for an Active Process The mechanical passive properties of the cochlea produce a traveling wave along the basilar membrane that is broadly tuned and linear: the traveling wave is relatively insensitive to signal intensity. Measurements from the living, active cochlea are quite different, however; basilar membrane motion is highly nonlinear and steeply sensitive to stimulus intensity, suggesting a role for active amplification. Tuning curves measured at the basilar membrane, inner hair cell, and primary afferent neuron are significantly sharper than predicted by passive cochlear mechanics, again suggesting a role for active tuning (Narayan et al., 1998; Robles and Ruggero, 2001). Sounds generated by the ear, called 326

3 Active Hair Bundle Movements and the Cochlear Amplifier/Ricci otoacoustic emissions, are also thought to be a manifestation of the active process, demonstrating that the cochlea can generate basilar membrane motion (Kemp, 1978; Kemp, 1998). This finding supports the argument that the amplifier has a mechanical component. Together these data argue that an amplification and tuning process must exist in the cochlea. Significance of Amplification Arguments regarding cochlear amplification and filtering may appear to be focused on details of how the system operates and not of practical importance to those working at the clinical end of the field. This is not the case, however, as the loss of the cochlear amplifier, whether it be through noise induced lesions or ototoxic agents such as aminoglycoside antibiotics or cancer treating agents like cisplatin leads to significant irrecoverable hearing loss (Ajodhia and Dix, 1976; Lerner and Matz, 1980; Schaefer et al., 1985; Schweitzer, 1993; Seligmann et al., 1996). Age-related hearing loss is also often linked to perturbations of OHC function, a direct consequence of which is the loss of the cochlear amplifier. In general, cochlear deafness is due to either a failure of hair cell vibration detection (i.e., inner hair cell transmission) or by a deficit in the vibration caused by a failure at some level in the active process. Figure 1. Schematic of the organ of Corti illustrating the organization of the sensory cells, supporting cells, and important structural membranes. Sound pressure differences between scala vestibuli and scala tympani causes vibration of the basilar membrane. Movement of the basilar membrane results in a shearing of the OHC stereocilia that are imbedded in the tectorial membrane. The active process sets the operating range of the ear so perturbations that either increase or decrease amplification can lead to significant auditory deficits. Understanding the details of the mechanisms associated with the active process will provide the tools from which to devise both preventative and therapeutic treatment regimes. Mechanisms Underlying the Active Outer Hair Cell Motility The active process is the term used when referring to mechanisms involved in active cochlear amplification and tuning (see Robles and Rugger [2001] for review). A variety of experimental evidence suggests that OHCs are an essential component of the active process. Lesions of the OHC layer result in an elevation of threshold and a loss of otoacoustic emissions (Davis, 1983). The discovery that OHC membranes are specialized to change shape, that is, extend and contract with voltage, focused the scientific community on understanding and characterizing this process as the site for cochlear amplification (Brownell, 1984; Ashmore, 1987; Holley and Ashmore, 1988; Dallos and Corey, 1991; Santos-Sacchi, 1991). Agents such as salicylate that alter otoacoustic emissions target OHCs lateral wall motility (Tunstall et al., 1995; Kakehata and Santos- Sacchi, 1996; Hallworth, 1997; Lue and Brownell, 1999). Movement of the OHC can provide the mechanical energy required for amplification (Ashmore, 1987; Dallos et al., 1997). The movements are in the same direction as movement of the basilar membrane and so may sum with stimuli to increase the gain (see Fig. 1). OHC motility is thought to be part of a positive feedback loop that enhances basilar membrane movement in response to low intensity sound (Robles and Ruggero, 2001). OHC movement may directly alter basilar membrane motion on a cycle by cycle basis or may indirectly alter motion by changing the stiffness of the basilar membrane. The recent identification of a unique protein called prestin that may be the voltage sensor causing membranes to change shape has brought the characterization of OHC motility to the molecular level (Zheng et al., 2000; Santos Sacchi et al., 2001). 327

4 Journal of the American Academy of Audiology/Volume 14, Number 6, 2003 Additional Sources of Amplification Is OHC motility the sole source of the active process? That is, does OHC motility have all the required features to account for the known properties of the cochlear amplifier? To date neither electrical nor mechanical tuning of OHCs has been described suggesting that tuning, a critical component of cochlear amplification, is absent. What provides the sharpness of tuning of the basilar membrane? In addition, OHC motility is driven by voltage bringing up two limiting factors. One is the membrane time constant that will filter high frequency responses. The other is simply the theoretical arguments presented earlier requiring amplification at the site of mechanical transduction to limit amplification of thermal energy and to raise the mechanical stimulus above the noise floor. OHC motility cannot amplify a signal it cannot sense or discriminate. If there is no filtering at the sensory hair bundle, OHCs will amplify currents generated by the Brownian motion associated with the hair bundle. Experiments have demonstrated that tuning curves measured from primary afferent nerve recordings are comparable in sharpness to basilar membrane motion, suggesting no additional tuning mechanisms are required or specifically suggesting electrical tuning mechanisms of OHCs are not required (Narayan et al., 1998). What is the origin of the basilar membrane tuning that is reflected in the primary afferent neuron? It is likely that OHC motility provides the mechanical positive feedback but that this feedback is tuned by some other component, namely the sensory hair bundle. Experimental paradigms that targeted OHCs for the site of the active process could not target motility separate from mechano-electric transduction and so cannot test for multiple mechanisms within the OHCs. There is no doubt that OHC motility plays a pivotal role in establishing the cochlear amplifier, but it is unlikely that motility is the sole mechanism responsible for this process. Otoacoustic emissions, a signature of the active process, have been recorded from a variety of species that do not have OHCs, including birds and lizards (Manley et al., 1987; Manley et al., 1996; Taschenberger and Manley, 1997; Stewart and Hudspeth, 2000), suggesting that an additional source Figure 2. (A) Diagram of stereocilia showing actin core, tip-links, and side links. Deflection of the stereocilia toward the tall edge exerts force on the tip-link, that then exerts force either directly or indirectly onto the mechano-electric transduction (met) channels. (B) DIC image of the top of the turtle auditory papilla focused at the apical surface looking down onto the hair bundle. Individual stereocilia can be seen. (C) Mechano-electric-transducer current measured from a turtle hair cell recorded from the intact papilla preparation. The hair cell was voltage-clamped at 80mV. Deflection of the hair bundle toward its tall edge opens channels resulting in inward current. Larger deflections produce larger currents. The rapid decay of the current is fast adaptation and is thought to underlie a mechanical tuning mechanism in the sensory hair bundle. Larger deflections elicit multiple components of adaptation. 328

5 Active Hair Bundle Movements and the Cochlear Amplifier/Ricci of amplification exists. The affects of salicylate on OAEs has been postulated to be on outer hair cell motility (Brownell, 1990; Tunstall et al., 1995; Kakehata and Santos-Sacchi, 1996; Lue and Brownell, 1999), yet OAEs from gecko show similar pharmacological effects (Stewart and Hudspeth, 2000), implying a common mechanism of amplification in a system void of OHCs. Auditory thresholds in species without OHCs are equivalent or better than those of cochleated animals, indicating that amplification and tuning are comparable between species with and without outer hair cells (Taschenberger and Manley, 1998; Koppl and Yates, 1999). All of the above data suggest that additional sources of amplification and tuning are necessary. The data also suggests that the additional source is ubiquitous across species and hair cell types. The one feature common to all hair cells is the sensory hair bundle, the site of mechano-electric transduction. Hair Bundle Tuning Mechanisms Hair Bundle Structure and Mechanoelectric Transduction Hair bundles come in different shapes and sizes but share some common features. The hair bundle consists of a series of stereocilia that increase in height toward a tall edge. The stereocilia are rigid structures, bending at their base creating a shearing force between stereocilia at the tops (Crawford and Fettiplace, 1985). Deflection of the hair bundle toward its tall edge increases shearing and results in mechano-electric-transducer (met) channels opening, while deflection away from the tall edge reduces shearing closing channels (Fig. 2). The stereocilia are connected to each other by a matrix of extracellular proteins that run the length of the cilia (Furness and Hackney, 1985). One in particular, called the tip-link (Pickles et al., 1984; Osborne et al., 1988; Pickles et al., 1989), is thought to translate the movement associated with deflection of the hair bundle into a force applied to met channels (see Fig. 2). The tiplink is thought to provide directional sensitivity to the hair bundle in that they are present only on one side of the stereocilia so that movement of the bundle side to side does not affect the tip-link, only movement along the long axis of the bundle will impart force onto these links (Shotwell et al., 1981; Pickles et al., 1984; Pickles et al., 1989). The tallest row of stereocilia of OHC and hair cells of other species are embedded in the tectorial membrane (Kimura, 1966; Lim, 1972, 1986). The tectorial membrane is relatively rigid. Movement of the basilar membrane results in a lateral force being applied to the hair bundle relative to its apical surface. The lateral force results in a shearing of the hair bundle in the appropriate direction to either increase or decrease force on the tip-links. Inner hair cells are thought not to be embedded in the tectorial membrane, being more sensitive to fluid flow stimuli (velocity) generated in the space between the tectorial membrane and apical surface of the cells lining the basilar membrane. In this scheme active movements of the OHC could be imagined to act like a bellows increasing fluid flow across the inner hair cell hair bundles. Evidence That the Hair Bundle Can Provide Cochlear Amplification To be a candidate for cochlear amplifier, a process must provide tuning or frequency selectivity, amplification or gain, and there must be a mechanical correlate or force-generating component. Evidence suggests that the hair bundle meets these criteria. Data that hair bundles are tuned comes from several sources. Turtle auditory papilla hair bundles oscillate at the characteristic frequency of the hair cell (Crawford and Fettiplace, 1985). Frog saccule hair bundles also show oscillations (Benser et al., 1996). Hair cell receptor potentials reflect the oscillation suggesting the met current is also oscillating (Crawford and Fettiplace, 1985; Benser et al., 1996). Direct measurements of met currents demonstrate that they can oscillate near the characteristic frequency of the hair cell (Ricci et al., 1998) (Fig. 3). The time course of fast adaptation, the process that drives hair bundle tuning, varies tonotopically (Fig. 3) (Ricci and Fettiplace, 1997; Fettiplace et al., 2001). Both theoretical arguments and direct measurements suggest the calcium gradient across the stereocilia provides the energy required for this process (Choe et al., 1998; Ricci et al., 1998; Wu et al., 1999). That the hair bundle can provide amplification has also been demonstrated (Jaramillo et al., 1993; Markin and Hudspeth, 1995b; Martin et al., 2000). 329

6 Journal of the American Academy of Audiology/Volume 14, Number 6, 2003 Figure 3. Data supporting the hypothesis that hair bundles and fast adaptation provide a mechanical tuning mechanism. (A) Example of a met current obtained in a low calcium environment (50mM). The oscillation in the current is a resonance corresponding to the characteristic frequency of the cell and suggestive of a mechanical filter and amplifier in the bundle. (B) Plot of the time constant of adaptation against papilla location demonstrating a tonotopic distribution. Fast adaptation is thought to underlie the mechanical filter of the hair bundle. The solid line represents the tonotopic organization of the papilla derived from primary afferent nerve recordings (Crawford and Fettiplace, 1980). (C) Measurements of transducer current (middle) and hair bundle movement (lower) in response to a stimulus (top) with a flexible fiber. This data demonstrates a mechanical correlate to fast adaptation. (D) Depolarizations induce movement of the hair bundle by decreasing in intraciliary calcium levels. That this movement generates force can be seen by the ability of the hair bundle to move a flexible fiber. These types of experiments have been used to quantitatively assess force generated by hair bundles. In fact, the hair bundle uses the energy associated with noise or Brownian motion to drive amplification (Denk and Webb, 1992; Jaramillo and Wiesenfeld, 1998). Hair bundles can also generate force (Howard and Hudspeth, 1988; Hudspeth and Gillespie, 1994; Hudspeth, 1997; Ricci et al., 2002). Forces associated with channel gating have been measured (Howard and Hudspeth, 1988; Jaramillo and Hudspeth, 1993; Ricci et al., 2000; Ricci et al., 2002). Mechanical correlates to fast adaptation have been identified (Benser et al., 1996; Ricci et al., 2000; Ricci et al., 2002) (Fig. 3). Together strong evidence is available to support the hypothesis that the sensory hair bundle contains elements that underlie in part the cochlear amplifier. Recent evidence from lizard, using electrically driven otoacoustic emissions, has targeted the site of cochlear amplification to the sensory hair bundle (Manley et al., 2001). Active Properties of the Sensory Hair Bundle What Are the Underlying Mechanisms Involved in Hair Bundle Tuning and Amplification? Several active properties have been described in sensory hair bundles, most linked to the mechanically gated ion channels. First, a change in hair bundle stiffness has been reported to be directly linked to the state of the met channel (Howard and Hudspeth, 1988; Markin and Hudspeth, 1995a; Martin et al., 2000; Ricci et al., 2002). Second, two forms of adaptation have been described that serve to set the resting hair bundle tension and may underlie a mechanical tuning mechanism (Wu et al., 1999; Hudspeth et al., 2000). Third, a role for myosin VIIA in setting the resting hair bundle position has been characterized (Kros et al., 2002). This result is unusual in 330

7 Active Hair Bundle Movements and the Cochlear Amplifier/Ricci that myosin VIIA has been found at various points along the stereocilia in mammalian OHCs (Kros et al., 2002), locations away from the site of mechanically gated channels. What follows is a description of each of these processes and a discussion of how they may be involved in cochlear amplification. Mechano-electric Transduction The coupling of a mechanical stimulus to the electrical response must be direct because it is fast (Corey and Hudspeth, 1979; Crawford et al., 1989). Channel activation occurs with little or no delay, suggesting the hair bundle is poised at a position where channels can be opened or closed with movement either toward or away from its tall edge (Corey and Hudspeth, 1979; Crawford et al., 1989). In fact, at a hair bundle s resting position a portion of the met current is activated; treatments that disrupt the hair bundle result in a hair bundle movement toward the kinocilium (Assad et al., 1991), suggesting the hair bundle is under a standing tension. Resting tension is important because it implies the rate-limiting step in channel activation will be the activation kinetics of the channel. As long as there is a resting tension in the bundle there should be no delay other than the conformational change required to open the channel (Corey and Hudspeth, 1983; Crawford et al., 1989). A variety of processes are involved in establishing the resting tension in the hair bundle and in controlling the resting open probability of the met channel. Gating Spring Theory The gating spring theory posits that an elastic element tethered to the met channel exerts a force that opens the channel (Howard and Hudspeth, 1988; Markin and Hudspeth, 1995a) (see Fig. 4). The activation gate is hypothesized to be in series with an elastic element such that opening the channel results in an increase in hair bundle compliance (compliance is the inverse of stiffness) (Howard and Hudspeth, 1988; Markin and Hudspeth, 1995a; van Netten and Kros, 2000; Ricci et al., 2002). A prerequisite to the change in hair bundle compliance is that the channel compliance is a significant portion of the hair bundle s compliance (van Netten and Kros, 2000). Estimates suggest that met channels contribute between 30-80% of the hair bundle s compliance (Howard and Hudspeth, 1988; Jaramillo and Hudspeth, 1993; Markin and Hudspeth, 1995a; van Netten and Kros, 2000; Ricci et al., 2002). The gating force represents the decrease in gating spring force that occurs with channel opening. Experiments have been performed using flexible fibers that allow a force to be exerted onto the hair bundle and for the hair bundle to respond to this force Howard and Ashmore, 1986; Howard and Hudspeth, 1987; Howard and Hudspetht, 1988; Ricci et al., 2000; Ricci et al., 2002). Initial work done by Howard and Hudspeth (1988) demonstrated an increase in hair bundle compliance over stimulus ranges predicted to be where the met channels were activating. Using a displacementclamp system demonstrated that the stiffness minimum shifted under conditions that would be expected to shift the met channel activation curve (Martin et al., 2000). More recent work directly demonstrates the relationship between met channel activation and the measured stiffness minimum (Ricci et al., 2002). Estimates of single channel gating force can also be obtained from force-displacement plots. Good agreement between measurements of single channel force from cells in different tissue preparations have been observed with values ranging between pN/channel (Howard and Hudspeth, 1988; Markin and Hudspeth, 1995a; van Netten and Kros, 2000; Ricci et al., 2002). The amount of force a channel can generate is important because this force will be used by the bundle for amplification. The gating spring theory suggests that the met channels can generate force. The ability of gating compliance to create a region of negative stiffness has been argued to underlie the oscillatory nature of the hair bundle (Martin et al., 2000). The magnitude of force or the depth of the well of negative stiffness will be directly determined by the number of met channels present; therefore, accurate measurements of the number of met channels present per hair bundle is important for determining how much force a bundle can generate. Channel force can be used by the hair bundle for amplification. Adaptation, Two Types By definition, adaptation is a decrease in response during a constant stimulus (Figs. 2, 5). Adaptation has been observed in hair 331

8 Journal of the American Academy of Audiology/Volume 14, Number 6, 2003 cell met currents (Eatock et al., 1987; Crawford et al., 1989). Classically, adaptation is thought to extend the dynamic range and prevent saturation of the met channels. Adaptation is a shift in the setpoint of the met channels operating range. An example of a hair cell s response to a protocol used to elicit an adaptive response is given in Figure 5. The activation curve shifts to the right during a constant stimulus. In general, adaptation is both stimulus and calcium dependent (Eatock et al., 1987; Crawford et al., 1991; Ricci and Fettiplace, 1998). Two forms of adaptation have been described, fast adaptation and motor or slow adaptation. Fast adaptation is hypothesized to be a calcium driven process directly coupled to the met channel (Crawford et al., 1989; Crawford et al., 1991; Wu et al., 1999; Ricci et al., 2000). Slow adaptation is hypothesized to be a calcium-dependent process that uses a molecular motor, most likely myosin 1b, to move the met channel along the stereocilia (Eatock et al., 1987; Assad et al., 1989; Assad and Corey, 1992; Gillespie and Corey, 1997; Holt et al., 2002). The movement results in a change in force detected by the channel. The two forms of adaptation vary in several respects. First, fast adaptation has time constants that vary between 0.1 and about 5ms while slow adaptation has time constants in the 10s to 100s of milliseconds (Eatock et al., 1987; Assad et al., 1989; Crawford et al., 1989; Assad and Corey, 1992; Ricci and Fettiplace, 1997). Third, fast adaptation operates around the most sensitive portion of the hair cell s activation while the slower form of adaptation requires larger displacements of the hair bundle (Wu et al., 1999; Ricci et al., 2002). As defined by its kinetics, fast adaptation has been identified in both auditory and vestibular hair cells but is the predominant form of adaptation in auditory cells (Crawford et al., 1989; Kros et al., 1992; Kros et al., 1995; Wu et al., 1999; Holt et al., 2002; Ricci et al., 2002). Slow adaptation has been identified in all but mammalian cochlear hair cells and is the predominant form of adaptation found in vestibular hair cells (Eatock et al., 1987; Howard and Hudspeth, 1987; Geleoc et al., 1997; Holt et al., 2002). Fast Adaptation, a Tuning Mechanism Fast adaptation is a process hypothesized to be directly coupled to the met channels (Crawford et al., 1989). Calcium entering the channel is thought to bind either to the channel or to a protein in direct contact with the channel resulting in a conformational change closing the channel (Crawford et al., 1989; Ricci et al., 2000) (see schematic of Fig. 4). Fast adaptation may be a force generating Figure 4. Cartoon schematizing present hypothesis regarding mechanisms involved in the gating spring hypothesis of channel activation as well as fast and slow adaptation. The gating spring theory is represented as the activation gate being in series with an elastic element that applies force to the channel. Fast adaptation is depicted by the calcium binding site located on the channel. Calcium binding results in channel closure. Motor adaptation is depicted by the myosin attached to the channel and the actin core. This depiction is different than other cartoons of the motor in not requiring movement of the channel, rather using reorganization of the attachment to the cytoskeleton. 332

9 Active Hair Bundle Movements and the Cochlear Amplifier/Ricci process that can move the hair bundle (Ricci et al., 2000; Ricci et al., 2002). A rapid rebound in hair bundle movement has been identified in both turtle auditory (Ricci et al., 2000) (Fig. 3) and frog vestibular hair cells (Howard and Hudspeth, 1987; Benser et al., 1996). Fast adaptation predominates around the hair bundle s resting position and during small hair bundle deflections. Since bundle compliance is an important factor in determining the resonant frequency of the hair bundle, a mechanism for generating active bundle movements that does not require a change in hair bundle stiffness may be essential. Decreases in hair bundle stiffness would be predicted to broaden the frequency of the hair bundle oscillation. Hair bundle oscillations observed in turtle auditory hair cells were the first clue that a mechanical tuning mechanism might exist in the sensory hair bundle (Crawford and Fettiplace, 1985). That fast adaptation might underlie a mechanical tuning mechanism was first suggested based on a tonotopic distribution in the adaptation rate (Ricci and Fettiplace, 1997). Later it was observed that oscillations in the met current at frequencies comparable to the cell s characteristic frequency could be generated by bathing the hair bundle in a low micromolar concentrations of calcium (Ricci et al., 1998) (Fig. 3). Further evidence in support of a mechanical tuning mechanism was the identification of a mechanical correlate of fast adaptation that also varied tonotopically (Ricci et al., 2000) (Fig. 3). Low frequency spontaneous and evoked oscillations have also been observed in saccular hair cell bundles (Benser et al., 1996). Modeling the hair bundle oscillations from turtle has demonstrated that the kinetics of fast adaptation determine the resonant frequency of the oscillations (Wu et al., 1999). Slow (Motor) Adaptation Slow adaptation results in a decrease in met current amplitude on a time scale of tens to hundreds of milliseconds (Eatock et al., 1986). It is proposed to be an increase in hair bundle compliance as flexible fiber experiments demonstrate a slow movement of the hair bundle toward the kinocilia on a time course similar to that of the current decline (Howard and Hudspeth, 1988); however, much as fast adaptation may be due to a shift in the compliance curve and not a change in hair bundle stiffness, so too could an argument be made that slow adaptation does not directly result in a compliance change. The prevailing theory regarding slow adaptation is that it is a myosin-based process (Assad and Corey, 1992) that links the met channel to the actin cytoskeleton via a myosin isozyme (see schematic of Fig. 4). Myosin drags the channel up and down the actin as a function of intraciliary calcium concentration and force applied to the bundle (Hudspeth and Gillespie, 1994; Gillespie and Corey, 1997). Simplistically, calcium enters the stereocilia through the met channels triggering myosin to release from the actin, sliding down the actin core reducing tension on the met channels. The reduced tension results in channel closure. As calcium is reduced in the stereocilia, the myosin climb back up the actin restoring tension in the met channel. A variety of evidence both direct and indirect support this model (see the recent work of Holt et al., 2002 for a review). The role slow adaptation plays in mechanical tuning and amplification is not clear. Certainly the presence of myosin would be an excellent source of mechanical force, and theoretical arguments demonstrate the ability of myosin to generate the requisite force to drive hair bundle oscillations (Hudspeth and Gillespie, 1994; Manley and Gallo, 1997). Whether the kinetics of the process will be adequate for the high frequencies needed in the cochlea remains to be determined. No evidence for tonotopic variations in slow adaptation has been identified. Also, the presence of myosin 1β has not been demonstrated in mammalian cochlea. Additional Complexities Traditionally, adaptation is thought to set the resting position of the hair bundle (Eatock et al., 1987; Crawford et al., 1989). The position of the met activation curve will be essential in any tuning or amplification process. Recent evidence has suggested that a myosin VIIa is also important in setting the resting hair bundle tension Myosin VIIa has been identified in OHC hair bundles (Richardson et al., 1999). Mice mutants lacking myosin VIIA are deaf (Hasson et al., 1995; Friedman et al., 1999; Redowicz, 1999). Mutations of the myosin VIIa gene result in deafness 333

10 Journal of the American Academy of Audiology/Volume 14, Number 6, 2003 (Hasson et al., 1995; el-amraoui et al., 1996; Weil et al., 1996; Mburu et al., 1997). Myosin VIIa is located further down the stereocilia than the met channels (Hasson et al., 1995; Kros et al., 2002). The myosin VIIa may be associated with crosslinks between stereocilia, connecting stereocilia at various points along their length (Kros et al., 2002, see Fig. 2). The met activation curve for OHCs is shifted dramatically to the right so that larger than physiological stimuli are needed to activate the channels (Richardson et al., 1999). A model incorporating both forms of myosin has been suggested where the myosin VIIa, located away from the met channels serve to regulate resting hair bundle tension (Kros et al., 2002). Yet myosin VIIa plays a pivotal role is setting resting tension. These results are surprising and suggest that the original concept of the resting open probability of the met channels being affected only by proteins directly affiliated with the channel may be an oversimplification. In addition the description of a slow movement associated with the sensory hair bundle called a sag supports the argument that further complexities exist in the hair bundle (Ricci et al., 2002). Depolarizations of the hair bundle produced a movement toward the kinocilium that was due to fast adaptation. Longer depolarizations often resulted in the hair bundle moving back away from the kinocilium (Ricci et al., 2002). This was not accompanied by any change in the met current. Whether this movement is a manifestation of motor adaptation is not known. One difference is the time course of this process is about an order of magnitude slower than that reported for slow adaptation in turtle hair cells (Wu et al., 1999; Ricci et al., 2002). Perhaps the sag represents a separate form of adaptation, one that may act at sites further from the met channels, possibly the myosin VIIa. Additional myosins, whose functions have yet to be resolved, have also been identified in the cuticular plate region, some at the stereociliary insertional points and others forming a periculticular ring (Gillespie et al., 1996; Hasson et al., 1997). Perhaps this slower component is a reflection of a mechanism that allows for the hair bundle to rock about the top of the cell. Rocking would alter force translated to the met channels but have no significant effect on hair bundle stiffness. Of course this is all speculation but represents a new area for future work. Hair bundles have at least three active force generating systems, gating compliance, fast and slow adaptation and possibly a fourth slower type of adaptation. Most likely these mechanisms work in conjunction with each other to establish mechanical tuning. Each has advantages for tuning. Fast adaptation can be extremely fast and so may be responsible for setting the frequency of tuning, yet its operating range is small and the amount of force generated is limited by the number of channels present. Slow adaptation is limited in kinetics but can generate significant force and has an extended operating range. Perhaps slow adaptation serves to maintain the met channels at the steepest portion of their activation curve, thereby allowing fast adaptation to drive mechanical oscillations. Gating compliance may also serve as part of Figure 5. Stimulus protocol shown above the current records (A). Typical protocol used to investigate adaptation. An activation curve is generated about the hair bundle s resting position and also around a position that elicits an adaptive response. Peak current vs. displacement plots (B) demonstrate that adaptation results in a shift in the steady-state plot that serves to extend the dynamic range and limit saturation. 334

11 Active Hair Bundle Movements and the Cochlear Amplifier/Ricci the active process coupled with fast adaptation to generate mechanical oscillations. The speculated slower adaptation may be the only true adaptation, coming into play when large stimuli saturate other components. Tilting of the cuticular plate would be expected to rotate the hair bundle in the direction of the stimulus, thus reducing the shearing force on the hair bundle and the force on the met channel. Mammalian Systems Gating compliance as well as fast and slow adaptation have been identified in mammalian vestibular hair cells (Geleoc et al., 1997; Holt et al., 1997; Eatock, 2000). Gating compliance and fast adaptation have been identified in outer hair cells (OHC) (Russell and Richardson, 1987). Slow adaptation has yet to be observed, and myosin 1B has not been seen on OHC stereocilia questioning whether slow adaptation processes are relevant in this system. It is clear that there are active processes in auditory hair cell bundles and that the machinery for hair bundle tuning is present in OHCs as is electromotility. It seems likely that both would be used in a concerted manner to generate the active process and the amazing sensitivity of the mammalian cochlea in that both have distinct advantages. Lateral wall motility can generate much greater forces than can the hair bundle (Fettiplace et al., 2001). However, hair bundles may provide the tuning and initial amplification required for enhancing the signal to noise ratio at the hair bundle, and OHC motility may provide the additional force needed to tune the basilar membrane and thus the stimulus to the inner hair cells. OHC electromotility must be driven electrically and must at some level be tuned. If hair bundle amplification tunes the bundle, the electrically tuned signal generated by the bundle will drive OHC motility and thus provide requisite tuning to the basilar membrane. If the met channels are not tuned and the signal at the bundle not amplified, then the electrical signal driving OHC motility will also not be tuned. On the other hand, OHC motility may serve a similar role as slow adaptation in vestibular organs, that being to maintain the hair bundle at its optimal, linear position for met channel activation. More careful in-vivo and in-vitro experiments are required to really determine how these processes might interact. Most of the work discussed represents data from hair cells of vestibular or auditory papilla systems and not true cochlea. The work on cochlea systems is technically difficult due to higher temperatures, bone rather than cartilage and a good deal more connective tissue in adult animals. Because of these difficulties, the data pertaining to the properties of mammalian hair bundles is limited. Yet the existing body of data is suggestive of a conserved system so that extrapolation from these more robust turtle and frog preparations is reasonable. However, the need for adult mammalian preparations where molecular and genetic techniques can be applied is growing. Undoubtedly, over the next several years these preparations will pave the way into new and exciting areas of hair cell tuning and amplification. Future Directions Several critical questions need to be explored in order to better understand mechanisms involved in generating the active process. Molecular identification and characterization of the mechanically gated channel is critical for better, more quantitative assessments of its function. Careful pharmacological characterization of the native met channel is also needed in order to help in the molecular identification of the channel and possibly to give insight into therapeutics. Determining the number of met channels present per hair cell and per stereocilia is also critical in that the force generated by the gating properties is directly proportional to the number of available channels. In addition, hair bundle stiffness is most likely grossly underestimated due to the lack of a full complement of channels during measurements. As more information is obtained regarding the multiple processes responsible for cochlear amplification, the specific roles of each should also become more apparent. With this information should come the ability to design better treatments, whether they be pharmacological, in hearing aids, or even cochlear implants that better reproduce the native signal. Acknowledgment. This work was funded by NIDCD R01 DC03896 and the Tinnitus Foundation. Most of the presented data is from work done in collaboration with Robert Fettiplace and Andrew Crawford. Thanks to Michael Schnee, Christopher LeBlanc, and Hamilton Farris for advice and editing. 335

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