Download Active Hair Bundle Movements and the Cochlear Amplifier

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: 504-599-0848; Fax: 504-599-0891; E-mail: [email protected]
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Journal of the American Academy of Audiology/Volume 14, Number 6, 2003
HEARING
A
irborne 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
326
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
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.
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
Significance of Amplification
Outer Hair Cell Motility
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” 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 SantosSacchi, 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).
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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 sharp-
ness 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.
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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).
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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).
330
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
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 0.2-0.4pN/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
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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
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 com-
pliance 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
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
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
Journal of the American Academy of Audiology/Volume 14, Number 6, 2003
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