Review Resources
Salamanca Study Abroad Program:
Neurobiology of Hearing
Must-See Websites:
Journey into the world of Hearing
(http://www.cochlea.org/en/spe)
Auditory Animations, Univ. of Wisconsin
(http://www.neurophys.wisc.edu/animations/)
The cochlea, transduction, and auditory periphery
The Cochlea, Fabio Mammano
(http://147.162.36.50/cochlea/index.htm)
R. Keith Duncan
University of Michigan
rkduncan@umich.edu
HHMI lecture from Jim Hudspeth
(http://media.hhmi.org/hl/97Lect3.html)
Reviews:
Gillespie et al. (2009) Cell, 139:33-44
Nouvian et al. (2006) J Membrane Biol, 209:153-165
Fettiplace and Hackney (2006) Nat Rev Neurosci, 7:19-29
Outline
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The auditory periphery
Cochlear structure and function
Cochlear fluids and endocochlear
potentials
Hair cell transduction
Active cochlear mechanics
Prestin and outer hair cell motility
What does the ear have to do?
Sound transmission
Sound pressure transmits through
incompressible fluids
The identity of sounds is conveyed largely by their frequency content. The cochlea
analyzes sound for frequency content, mapping frequency and energy level to a specific
place and transmitting this along with timing to the CNS.
spectra of the
sounds coming
out of the mouth
spectrum of the sound
produced by the glottis
Overview of the cochlea
Helicotrema
1.
2.
3.
4.
5.
Cochlear Partition
Scala Vestibuli
Scala Tympani
Spiral Ganglion
Auditory Nerve Fibers
1
Sensory cells reside in the organ of Corti
along the reticular lamina
TM
1. Scala media
2. Scala
vestibuli
3. Scala
tympani
4. Reissner’s
membrane
5. Basilar
membrane
6. Tectorial
membrane
7. Stria
Vascularis
8. Auditory
Nerve
9. Bony Spiral
Lamina
HeC
PC
DC
BM
Reticular
Lamina
Cochlear Fluids and the Endocochlear
Potential
Perilymph
Driving
Force
For
Potassium
Organ of Corti structure hints at a
division of labor among sensory cells
TM: tectorial membrane
BM: basilar membrane
PC: Pillar cells
DC: Dieter’s cells
HeC: Hensen’s cells
How do we know that hair cells are the transducer elements for hearing?
(1) innervated, (2) loss associated with deafness
Inner hair cells are primarily responsible for transmitting sound stimuli
because these receive 95% of primary afferents and are more tightly
associated with profound deafness.
Passive cochlear mechanics
Sound transmission into the
inner ear sets the cochlear
partition into motion. (more
on this later)
In mM:
145 Na+
3 K+
130 Cl1 Ca2+
This stimulates the sensory
hair cells.
Endolymph
+80 mV
1: Inner hair cell
3,500 in human cochlea
Primarily afferently innerv.
2: Outer hair cells
12,000 in human cochlea
Primarily efferently innerv.
In mM:
5 Na+
160 K+
130 Cl~ 0.02 Ca2+
-60 mV
0 mV
General Hair Cell Structure
Hair Cell & Hair Bundle Examples
Hair Bundle:
• Staircase (pipe-organ) of
actin-filled microvilli called
stereocilia
• Bilateral symmetry
• Stereocilia tapered at base
• Tilted inward
• Stereocilia interconnected
• Kinocilium (true cilium) not
present in mature cochlea
of mammal
IHC
OHC
Turtle VHC
Bullfrog VHC
Turtle VHC
From Geisler text
Functional differences in
bundle shape?
2
Stereocilium Structure
Tilney et al.
(1983)
Stereocilia are interconnected
At least 3 types of linkages:
• Tip links are arranged along
staircase.
• Lateral-links (or top connectors) and
ankle links interconnect all neighbors
(rows and staircase).
• Ankle links appear transiently during
development
• Dense actin core
• ~3000 filaments per
• 18-30 form rootlet (pivot)
• Hexagonally packed
• Incorporation at tips
• Support myosin motility
• Mutations in actin
assembly can cause
thin/thick, tall/short, fused,
or cytocauds
• Links are differentially susceptible to
enzyme treatment and noise trauma.
• Links are important for cohesion and
angled geometry of stereocilia
arrangement. Breakage causes
splaying of the bundle.
The hair bundle
moves as a unit
Transduction: Methods
Semi-intact preparations are used for
whole-cell patch-clamp studies.
Hair bundles stimulated with stiff
probes or water-jets.
Chick Tall Hair Cell
Water-jet Stimulation
500 Hz
15º displacement
Stroboscopic Lamp
Transduction: Asymmetric, Saturating,
Highly Sensitive
Hudspeth and Corey, 1977
Bullfrog sacculus; Two-electrode recording (current-clamp)
• 10 Hz triangle wave (Why use a triangle wave?)
• +displ = toward tallest stereocilia
• Astonishing sensitivity: threshold for change in receptor potential is ~1 pm.
That corresponds to a 10 cm movement of the tip of the Eiffel Tower.
• Intensity of sound (displacement of bundle) coded by a change in receptor
potential…NOT in spike rate like for a neuron.
Voltage-clamp:
• Constrain voltage
• Measure current
Probe
• Used to determine voltage
dependence
Patch
Pipette
Current-clamp:
• Inject current
• Measure voltage
• Used to measure action potentials
and receptor potentials
Transduction: Directional
Shotwell et al.,
1981
3
Transduction: Fast Gating
Transduction channels are located at the
tip of the hair bundle
Bullfrog sacculus, Voltage-clamp
Activation latency: 25 ms
2 orders of magnitude smaller than
latency in photoreceptors. Excludes
involvement of 2nd messengers.
More like mechanotransduction
channels.
Relaxation slower than activation,
requires two exponential
components, and is sensitive to
Ca2+. There is an as yet unknown
viscous or damping element involved
in deactivation of the transduction
channel.
Specializations at Hair Bundle Tip
Gentamicin (aminoglycoside
antibiotic) used as open channel
blocker of transduction channel.
Jaramillo & Hudspeth, 1991
Tip-links are sensitive to Ca2+ chelators.
Tip-links regenerate!
Freeze
Etch
EM
50 nm
Surface
Rendering
Kachar et al., 2000
Transduction channels are specifically
associated with the shorter rows of stereocilia
The Gating-Spring Hypothesis
Beurg et al., 2009
Note: At this point, it is still unclear whether the tip-link can be the stretchy element..
Note: Transduction channel now known to be at the other end of the link.
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The Gating-Spring Model Predicts
Nonlinear Stiffness
Molecular composition of the
mechanotransduction apparatus
• Tip-link = Cadherin23 and
protocadherin15
• Both form dimers to give helical
structure
• Mutations in CDH23 and PCDH15
lead to deafness and tip-link loss
• Binding of may be Ca2+ dependent
(explains why BAPTA breaks links)
• Combined structure would be VERY
stiff
• What then is the gating-spring?
What then is the “gating spring?”
Two hypothetical
mechanisms have
been proposed.
What is the molecular nature of the
transduction channel?
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What is the elastic
element?
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Adaptation in transducer currents
TRP?
Unclear…best candidate was TRPA1; KO still transduces
New possibilities?
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P2X?
No…not right localization and not ATP gated
Classic mechanically gated channels.
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No…ENaC KOs still transduce
Blocked by tubocurarine, PPADS, Suramin.
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And how is it tied to
channel gating?
Relatively non-selective though prefers Ca2+ (200:1).
Passes some large inorganic molecules (FM1-43).
High single channel conductance (100 pS).
Not voltage-gated.
Blocked by amiloride and aminoglycosides.
ENaC?
Tmc1/2?
Promising…Double KO eliminates transduction. Expression matches
development and localization.
Much work remains.
Adaptation Models
Displacement Stimulus
 fast ~ 0.1-1 ms
(slow, ~ 20 ms)
(fast, <1ms)
Schwander et al., JCB, 2010
 slow ~ 10-100 ms
Fast adaptation: due to a Ca2+ dependent channel closure ( ~ 0.1 ms)
Slow adaptation: depends on mysoin, ATP, and Ca2+ ( ~ 10 ms)
Motor models of slow adaptation include myosin-dependent
climbing/slipping of the upper tip-link density. Tension established
by this process sets the resting open state of MET channels (Po ~ 0.1)
5
Hair Cell Receptor Potential Shaped by Transduction
Current and Basolateral Conductances
The receptor potential is governed by a
variety of ion channel conductances,
many of which are illustrated here.
Open Questions
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• Mechanoelectrical transduction
initiates a change in receptor potential.
• Basolateral condutances set the
resting potential, shape the receptor
potential, and govern synaptic
transmission.
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Lecture 2: Passive and active
cochlear mechanics
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A place code is a general principle of many sensory systems
(somatotopic, retinotopic, others?)
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Damage (hair cell loss) at particular locations along the cochlea
is correlated with hearing loss at a particular frequency.
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But how is this frequency “tuning” established?
What is the molecular composition of the
transduction channel?
Is the channel directly coupled to tip-links or is it
gated by membrane stretch? In both cases, what is
the equivalent of the gating-spring? Does membrane
composition alter adaptation, gating-spring stiffness?
Do tip-link proteins treadmill as connected side-links
or form at stereocilium tip?
What role does slow adaptation have for highfrequency stimuli?
“Traveling Wave Era”
Bekesy Cochlea Models
1 kHz
Georg von Békésy (1899-1972)
Awarded a Nobel prize in 1961 for
studies on cochlear mechanics.
See “Experiments in hearing”
compiled by Glen Weaver.
“Traveling Wave Era”
“In situ” Measurements
Tonotopic Distribution of Frequency
• Improve
visualization with
silver grains and
strobe illumination
The cochlea is a frequency analyzer, breaking complex waves
into frequency components.
Tonos = tone; Topia = place
Tonotopic = frequency place code
4 kHz
• Measure amp. of
motion for constant
input pressure at
various frequencies.
1 kHz
250 Hz
Passive mechanics largely
due to gradual changes in the
stiffness of the basilar
membrane.
Base: thick and narrow
Apex: thin and wide
6
A (Nonlinear) Amplifier in the Ear
Gain = Amplitude of basilar membrane
Amplitude of stapes motion
Nonlinear Cochlear Mechanics:
Compression
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Displacement (nm)
Use laser to measure membrane at one
tonotopic position.
If you double the input to the basilar membrane, the output less
than doubles…at some stimulus levels.
Compare with stapes motion to get gain.
Vary frequency of sound stimulus while
keeping input (stapes motion) constant.
Linear growth
1 dB/dB
Sound pressure level (dB)
Passive mechanics gives the “dead or
high level shape” (like with Békésy).
Something else happens at low sound
levels in an alive animal (like with
psychoacoustics).
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Although the cochlea is extremely sensitive, it can withstand
sound pressures that are 1,000,000 times greater than sounds
at the threshold for detection (120 dB).
It survives by compressing the output of the cochlea for a large
range of mid to high levels of input (~40 to 100 dB SPL)
Cochlear mechanics is highly nonlinear.
Linear vs Nonlinear Systems
Nonlinear Cochlear Mechanics:
Distortion Product Otoacoustic Emissions
Acoustic
Basilar membrane
Distortion is a key feature of
nonlinear systems. All combinations
are generated (n*f1 ± m*f2) but cubic
(2f1 – f2) is largest.
Cubic Distortion
Distortion products can also be
measured directly on the basilar
membrane! Therefore, it cannot be
some strange effect of the speakers,
microphones, or middle ear.
OHC motility is a major contributor to
the cochlear amplifer
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Loss of OHCs
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+50 mV
OHC Motility
-150 mV
gain looks like a passive
(dead) cochlea.
nonlinearities (DPOAEs) lost.
Bill Brownell causes a
paradigm shift in 1980’s when
he describes OHC motility
(Scanning Electron
Microscopy, 1984; Science,
1985)
Change in voltage across the
OHC membrane induces
change in OHC length.
Holley and Ashmore, 1988
• Constant volume (not
osmotic effect).
• Produces gating
current without ion
flux (not conventional
ion channel).
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OHC Motor Units
OHC Nonlinear Capacitance
Motor unit must be:
• in the membrane,
• tied to the cytoskeleton,
• capable of generating large forces,
• able to sense membrane voltage.
IHC
OHC1
OHC2
Freeze fracture electron microscopy
shows densely packed particles, up
to 3,000 per mm2….or about
300,000 to 6,000,000 particles per
cell.
OHC3
Block dominant ionic currents (K+, Ca2+).
Voltage-clamp.
Measure membrane capacitance (like in exocytosis). But now the change
in capacitance is because of charged particles in the membrane, not fusion.
(A) results from a mouse OHC.
(B) Cm-Vm curves in an OHC compared with IHC.
To sense membrane voltage, some part of the motor must be charged.
When 6 million charges move in concert, they produce a measurable effect.
We measure this as a nonlinear capacitance.
The Prevailing View of the Cochlear Amplifier:
Positive Feedback from OHC Motility
What is the molecular nature of the
OHC motor?
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Motility does not require ATP. Not a traditional motor.
Phase-locked responses over 70 kHz. No 2nd messengers.
Voltage-dependent and nonlinear.
Requires extracellular anions (Cl-).
Prestin, an anion sulfate transporter
Active Hair Bundle Motion:
Prestin is capable of motility and NLC
Another Candidate for Cochlear Amplifier
Motility in TSA201 cells
Resonant frequency,
transduction kinetics,
and hair bundle
stiffness changes with
hair cell position
along cochlea.
NLC in HEK293 cells
prestin
Mutant and Non-transfected controls
Crawford and Fettiplace, 1985
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Active hair bundle movement involves
calcium and “fast” adaptation
Attempts at synergy
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Stimulus
Added
Energy
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Prestin knockout and knock-in models have
established that this molecule is necessary
for amplification, but they cannot address
whether Prestin is sufficient.
Current models suggest that hair bundle
amplification acts as a pre-filter for somatic
motility, but this issue is hotly debated.
Hudspeth et al., 2000
Supplementary Information
Open questions
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Membrane capacitance in OHCs acts like a
low-pass filter with a cut-off around 4 kHz.
How can the cochlear amplifier work above
this frequency?
Prestin is found in lower vertebrates. Are
their hair cells motile? What role does it play
there?
External Ear Function
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Pinna, ear canal, and outer surface of tympanic
membrane
Charles Darwin (1907): the human pinna is
vestigial and of no functional significance.
Functions:
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Adornment
Protect eardrum from foreign objects
Cerumen (antimicrobial moisturizer)
Collect sound
Resonator (20 dB gain at 2.5 kHz)
Sound localization cue
Middle Ear Function
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Consists of inner surface of
tympanic membrane plus
ossicular chain (maleus,
incus, and stapes)
Conducts sound energy to
inner ear.
Eustachian tube equalizes
pressure in tympanic cavity
with atmospheric pressure
(in children it’s a path for
infection, leading to otitis
media)
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A model of K+ flux and electrogenics
in stria vascularis and endolymph
Middle Ear Function:
Impedance Mismatch
Ossicles arranged to pivot around a
fulcrum.
Cochlear fluids are denser than air.
The impedance mismatch causes a
loss of ~36 dB
In Humans…
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Lever Ratio (1.3:1)
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Area Ratio (20:1)
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Another 2:1 from geometry of
eardrum
Lever Ratio
malleus
incus
Area Ratio
Oval
Window
sound
waves
Area = A
Ear
Drum
Area = 20A
Total 52:1 gain or 34.3 dB
Electrocochleaography
Cl
Marginal Cell R.P. is about +90 mV!
The high K+ in intermediate cells and extremely low K+ in intrastrial
space gives rise to the +90 mV E.P.
Auditory Brainstem Response (ABR)
Cochlear microphonic
What are the single-channel properties of
the transduction channel?
OHC Motility and Gating Charge
• Pre-incubate with BAPTA.
Often, single channel events
could be seen.
• Open probability increased
when stimulated (positive
displacement).
• Single channel conductance is
about 100 pS.
• Knowing the maximum
conductance of an intact hair
cell gives about 1-2 channels
per stereocilium.
Crawford et al., 1991
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Gating charge as a nonlinear
capacitor?
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Is the capacitance of a cell dependent on membrane voltage?
Capacitance is defined as dQ/dV.
We typically ignore gating charges from the few thousand ion
channels in a membrane.
Therefore, membrane capacitance is generally given by:
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Extrinsic voltage sensor?
C = eA/4pd.
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I- = Br- > NO3- > Cl- > HCO3- > F-.
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But if there are 300,000 to 6,000,000 charges all moving in
concert, we must go back to C = dQ/dV.
We often describe this gating current as a nonlinear
capacitance.
Q(Vm ) 
Cm 
dQ
dVm
Cytoplasmic monovalent anions are required for NLC
Hypothesis: Voltage sensor and conformation change linked to
anion translocation across membrane.
The hypothesis (and model below) turned out to be false when
Bai et al. (2009) identified and mutated the Cl- binding site,
negating anion binding but preserving motility and nonlinear
capacitance.
Qmax
1  e (Vm V1 / 2 ) /  )
Plot the shape
of the derivative.
Intrinsic voltage sensor?
Shown:
Only 1 of 4
suggested
topologies
Bai et al. (2009) neutralized alone or in combination many charged residues
within putative transmembrane domains, causing changes in voltage response.
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