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

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

Sensory cells reside in the organ of Corti

along the reticular lamina

TM

1. Scala media
2. Scala
vestibuli
3. Scala
tympani
4. Reissners
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: Dieters cells
HeC: Hensens 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?

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
15 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
potentialNOT 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

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.

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?

What is the elastic

element?

Adaptation in transducer currents

TRP?

Unclearbest candidate was TRPA1; KO still transduces

New possibilities?

P2X?

Nonot right localization and not ATP gated

Classic mechanically gated channels.

NoENaC KOs still transduce

Blocked by tubocurarine, PPADS, Suramin.

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?

PromisingDouble 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)

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

Mechanoelectrical transduction
initiates a change in receptor potential.
Basolateral condutances set the
resting potential, shape the receptor
potential, and govern synaptic
transmission.

Lecture 2: Passive and active

cochlear mechanics

A place code is a general principle of many sensory systems

(somatotopic, retinotopic, others?)

Damage (hair cell loss) at particular locations along the cochlea

is correlated with hearing loss at a particular frequency.

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 Bksy (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

A (Nonlinear) Amplifier in the Ear

Gain = Amplitude of basilar membrane
Amplitude of stapes motion

Nonlinear Cochlear Mechanics:

Compression

Displacement (nm)

Use laser to measure membrane at one

tonotopic position.

If you double the input to the basilar membrane, the output less
than doublesat 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 Bksy).
Something else happens at low sound
levels in an alive animal (like with
psychoacoustics).

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

Loss of OHCs

+50 mV

OHC Motility

-150 mV

gain looks like a passive

(dead) cochlea.
nonlinearities (DPOAEs) lost.

Bill Brownell causes a

paradigm shift in 1980s 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).

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?

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

Active hair bundle movement involves

calcium and fast adaptation

Attempts at synergy

Stimulus
Added
Energy

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

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

Pinna, ear canal, and outer surface of tympanic

membrane
Charles Darwin (1907): the human pinna is
vestigial and of no functional significance.
Functions:

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

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 its a path for
infection, leading to otitis
media)

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

Lever Ratio (1.3:1)

Area Ratio (20:1)

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

10

Gating charge as a nonlinear

capacitor?

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:

Extrinsic voltage sensor?

C = eA/4pd.

I- = Br- > NO3- > Cl- > HCO3- > F-.

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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