Auditory Mechanics
著者 和田 仁
Auditory Mechanics
東北大学 大学院工学研究科
バイオロボティクス専攻
Master’s dissertation:
外力を受ける円板の非線形強制振動
Doctoral dissertation:
棒，ばね－質量系の振動挙動に及ぼす支持
部の影響に関する研究
Human auditory system
Eustachian
tube
Secretory otitis media
成人と幼児の耳管 auditory tube, eustachian tube, tuba auditiva
成人の耳管 (右) にくらべて幼児 (左) では短く， ひろく， かつ水平に
位置している．
Five senses
http://www.info.gov.hk/dh/diseases/CD/ScarletF everc.htm http://www.yunphoto.net/jp/photobase/yp2363.html http://www.answers.com/topic/nose http://konicaminolta.jp/entertainment/femmage/ 200305/special/sp01_02.html http://www.gadling.com/Hearing
Sight
Taste
Touch
Five senses
Smell
OUTLINE
1. Mechanism of our auditory system
2. Research
2.1 Sweep frequency impedance meter (SFI)
2.2 Cochlear amplification
2.3 Outer hair cell
2.4 Prestin
Human auditory system
Eustachian
tube
Computer-aided reconstruction of
auditory system
A temporal bone is extracted from a fresh cadaver, sliced at intervals of 0.2 mm, and traced by a digitizer. Data are put into a computer. From this picture, the relationship of size and location among the various components can be clearly understood.
Measurement
system
Time-averaged speckle pattern interferometry. A beam emitted from the laser generator is divided into the object and reference beams. These two beams are combined with each other, and an interference speckle pattern is generated. This image is accumulated by use of a CCD camera.
Displacement distribution of the left TM vibrations. Frequency f = 1.0 kHz and sound pressure level P = 85 dB SPL. (a) Perspective plots. (b) Contour maps corresponding to (a). The contours represent lines of the constant vibration amplitude. Intervals are 3 nm. The positive and negative digits mean the upward and downward maximum values of the displacements at the specific points, respectively. The displacement amplitude of tympanic membrane vibrations is of nanometer order of two digits.
(nm) (a) f = 1.0 kHz, P = 85 dB SPL (b) Posterior Anterior Inferior Manubrium
Middle ear model
1, TM (pars tensa)
2, TM (pars flaccida)
3, malleus
4, incus
5, stapes
6, anterior malleal ligament
7, posterior incudal ligament
8, tensor tympani tendon
9, stapedial tendon
10, annular ligament
DC, damping constant
of the cochlea
KL, linear springs
KT, torsional springs
232 flat triangular plate elements and 82 hexahedral elements are applied to the tympanic membrane and the ossicles, respectively.
500 Hz
Malleus and incus rotate around the axis between the anterior malleal ligament and the posterior incudal ligament, and the umbo and stapes have a piston-like movement.
Dynamic behavior of the middle ear
Arrows indicate the directions of the movements. The ratio of the area of the TM to that of the stapes is 17:1, and the ratio of the arm of the malleus to that of the incus is 1.3:1.0. The main role of the middle ear is to match the low impedance of the air in the external auditory canal to the high impedance of the cochlear fluids. In other words, the middle ear is an impedance transformer.
Cochlea and its cross section
The cochlea has three fluid-filled compartments, which are divided by Reissner’s membrane and the basilar membrane.
Cochlear straight model
2 kHz
6 kHz
Vibrations of the stapes generate movement of the cochlear fluids that interacts with the basilar membrane. This interaction produces progressive traveling waves on the basilar membrane, which are similar to waves beating upon a seashore. Traveling waves on the basilar membrane have a peak near the apex when low frequency sound enters the cochlea, while high frequency sound develops the traveling waves on the basilar membrane, which have a peak near the base.
Cochlea and its cross section
The cochlea has three fluid-filled compartments, which are divided by Reissner’s membrane and the basilar membrane.
Structure of the organ of Corti
Hairlike structures, i.e., stereocilia, extend from the top of IHCs and OHCs. The organ of Corti is covered by the tectorial membrane and given rigidity by the pillar cells.
f = 16 kHz
Displacement × 500 for clarity
Vibration mode of the organ of Corti
The organ of Corti undergoes a rocking motion. The shear motion between the tectorial membrane and the reticular lamina induces the flow of fluid, which leads to the deflection of the free-standing IHC stereocilia.
IHC
Impulses
SV
ST
Structure of the stereocilia
Tip link
IHC
Stereocilia
OHC
Side link
100 nm
OHC stereocilia
10 µm
IHC stereocilia
Hackney and Furness, 1995 B. Kachar et al., 2000
Mechanoelectrical transduction process
Auditory nerve fiber
Stereocilia
Inner hair cell (IHC) Tip link Ion channel Fluid flow Lateral link Depolarization Action potential (Kachar et al., 2000) Tip link Stereocilia + +
The tip link is under tension and the mechanoelectrical transduction channel is thought to open. Due to this, an influx of ions into the IHC is generated, which in turn depolarizes the membrane potentials of the IHC and then produces action potentials in the auditory nerve fibers.
Lateral link
Stereocilia
Inner hair cell (IHC)
Auditory nerve
Mechanoelectrical transduction process
Ion channel
Response in the auditory nerve fiber. The upper waveform shows and
example of spontaneous activity. When the stimulus tone is delivered to
the external auditory meatus, a spike rate rises with an increase in
the stimulus level.
Auditory cortex Cerebrum
Medial geniculate body
Inferior colliculus Lateral laminiscus Nucleus of superior olive Cochlea Cochlear nucleus Cerebellum
A u d i t o r y p a t h w a y s o f t h e
brainstem. In the ascending
auditory pathways, neurons are
specialized and some of the
information is unified, while
other information is suppressed.
聴覚路と上位中枢
蝸牛核，上オリーブ核，下
丘，内側膝状体へと進むにつ
れて，情報の統合や抑制が行
われ，神経は特殊化していく．
聴覚野 内側膝状体 下丘 外側毛帯 上オリーブ 核 蝸牛核 蝸牛 大脳 小脳Normal subject (2D)
Normal
FREQUENCY(kHz) SPL(dB) 0.0 0.4 0.8 1.2 1.6 2.0 –12 –8 –4 0 0 –200 200 press.(daPa) ∆S P LSeparation
Separation
(2D)
FREQUENCY(kHz) SPL(dB) 0.0 0.4 0.8 1.2 1.6 2.0 0 6 12 –21 –100 –200 press.(daPa)Separation
Improvement of SFI meter
10 mm
Previous probe New probe
Probe set
Pressure sensor Syringe pump Stepping motor Power Probe 4 m m 5 m m
Subjects
10
Number: 24 normal hearing neonates
(14 males and 10 females)
Age: 1 day to 4 days
Weight: 2518- 4380 g
Neonates
5
10
5
20
SPL
10
2
P
20
SPL
10
2
P
10
−
−
×
=
×
=
log
where P is pressure (Pa).
Definition of sound pressure level
S
ound pr
es
sur
e
le
ve
l
(dB
S
P
L
)
Frequency (Hz)
Maximum audible curve
Minimum audible curve
Audible field
Microbeads
Laser
Basilar membrane
Diagram of BM vibration measurement
To confirm the cochlear amplification, an attempt is made to directly measure the basilar membrane vibrations in both living and dead guinea pigs by a Laser Doppler Velocimeter. A hole with a diameter of 0.5 mm is opened at the bony wall of the cochlea, and glass microbeads with a diameter of 20 µm are placed on the basilar membrane in order to increase reflections of the laser beam.
Frequency (kHz)
ALIVE
DEAD
8
6
0
10
12
100
200
300
V
elo
city
(
µm)
BM velocity response
Frequency (kHz)
S
en
si
ti
v
it
y
(
d
Br
e
1
(µ
m
/s
)/
2
0
µP
a)
Sensitivity of BM velocity responses to harmonic stimulation. Vertical
unit is velocity per unit pressure. The parameter is sound pressure level
(dB SPL). Curves of the post mortem condition were obtained one hour
after the respirator was stopped.
alive dead 20 30 40 60 40 50 60
OHC
external solution
Ag-AgCl electrode
pipette
high speed video system
Whole-cell voltage clamp technique
Instead of bending stereocilia, intracellular potentials are charged by the whole-cell voltage clamped technique.
Input-output function
The input-output function of the OHC is not expressed by a straight line but a curved one, i.e., the function is non-linear.
Protein motor
Pillars
Plasma membrane
Spectrin filaments
Actin filaments
Cortical lattice Subsurface cisternae
Lateral wall
The OHC lateral wall has a unique trilaminate structure: the outermost plasma membrane, the cortical lattice, and the innermost subsurface cisternae. The motor protein is thought to be embedded in the plasma membrane. The source of the somatic length change of the OHCs is considered to be the conformational changes of these motor proteins. In 2000, the motor protein was identified in the gerbil cochlea and termed “Prestin.”
time Intr ac ellu la r pote nti al
Dynamic behavior of the OC
f = 16 kHz
50 dB SPL
Active -135 deg. Passive Displacement × 10,000 Displacement × 100The magnified deflection of the organ of Corti leads to increase in the movement of the fluids in the space near the stereocilia of the IHCs and in the deflection of IHC stereocilia. Owing to this mechanism, our auditory system is characterized by high sensitivity, sharp tuning, and compressive nonlinearity.
Peter Dallos and myself in his retirement symposium
in Evanston in 2010
Protein motor
Pillars
Plasma membrane
Spectrin filaments
Actin filaments
Cortical lattice Subsurface cisternae
Lateral wall
The OHC lateral wall has a unique trilaminate structure: the outermost plasma membrane, the cortical lattice, and the innermost subsurface cisternae. The motor protein is thought to be embedded in the plasma membrane. The source of the somatic length change of the OHCs is considered to be the conformational changes of these motor proteins. In 2000, the motor protein was identified in the gerbil cochlea and termed “Prestin.”
58
Motor protein prestin was identified in 2000.
Zheng, J. et al., 2000. Prestin is the motor protein of cochlear outer hair cells. Nature 405, 149-155.
Prestin-encoding cDNA correspond to a polypeptide of 744
amino-acid residues with a molecular weight of about 80 kDa.
Prestin includes 12 transmembrane domains.
Membrane topology of prestin
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Prestin gene
Mammalian expression vector
CHO cell
CHO-Pres
Prestin
Prestin cDNA is transfected into Chinese hamster ovary (CHO) cells using
plasmid vectors. The transfected cells then express prestin. We generated stably
prestin-expressing cell lines using CHO cells.
Iida, K., Wada, H. et al., 2005. Construction of an expression
system for the motor protein prestin in Chinese hamster ovary cells.
Hear. Res. 205, 262-270.
Ideal Sample
Actin
filament
Other membrane
protein (not
prestin)
CHO cell
Prestin
Substrate
Plasma membrane
Tip
Prestin
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(Milhiet et al., 2006)
Reconstitution
Reconstitution method of prestin into an artificial lipid bilayer. Prestin molecules were purified from the
prestin-expressing CHO
cells. Lipid bilayer treated with detergent was prepared on mica. Prestin was reconstituted into the lipid bilayer and observed by AFM.
62
Three dimensional AFM image at high magnification
High-magnification AFM image of cytoplasmic side of prestin reconstituted into the artificial lipid bilayer. Many ring-like structure shown by white arrowhead, which is considered to show prestin molecules, can be detected.
63
Structure of prestin
A: Alanine
G: Glycine
T: Threonine
S: Serine
R: Arginine
H: Histidine
Prestin is known to be one of 11 members of the anion transporter family SLC26. Six proteins of this family have the same sequence.
S i x m u t a n t s w e r e e n g i n e e r e d t o b e e x p r e s s e d i n
HEK 293 cells.
G127A is a prestin mutant where glycine (G) at
position 127 was replaced by alanine (A). Normal prestin is called wild-type (WT) prestin.
G127A T128A
S129A
R130A
H131A S129T
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Patch-clamp recording
Mutations in the GTSRH sequence resulted in a decrease
of nonlinear capacitance.
65
M122I M225Q T428L
Prestin
specific amino acids
M122I is a prestin mutant where methionine (M) at position
122 was replaced by isoleucine (I).
M
Valine
Leucine
Methionine
Threonine
Isoleucine
Glutamine
T
I
Q
:
:
:
:
V
L
:
:
66
Nonlinear capacitance of the prestin mutants
Methionine at position 122 (M122) and threonine at
position 428 (T428) may not be essential for prestin.
67
Voltage-dependent motility expressed in TSA201 cells
Video image of a TSA201 cell partially drawn into a microchamber. Motility was measured at the farthest excluded membrane segment.
Zheng, J. et al., 2000. Prestin is the motor protein of cochlear outer hair cells. Nature 405, 149-155. Transfected cells Stimulus waveform Control cell 150 nm 440 m V 100 ms
68
Motility measurement of prestin-expressing cells
Methionine at position 225 (M225) is somehow involved in
the control of nonlinear capacitance and motility.
Compact state
Extended state
OHC
Lipid bilayer
Motor
(Iwasa, 1994)Two state model
Two states of the membrane motor unit.
The motor unit has two states with different membrane areas. The compact state has less membrane area than the extended state. A transition between these states is accompanied by a transfer of an electrical charge q across the membrane.
q q
72
Solute carrier 26 family -prestin-
Scala media
Outer hair cells
744 amino acids, 81.4 kDa
Inner hair cells
SLC26A1 Sat-1 SLC26A2 DTDST SLC26A7 SUT2 SLC26A8 Tal1 SLC26A3 DRA SLC26A4 Pendrin SLC26A9 None SLC26A5 Prestin SLC26A6 CFEX
73
Pendrin
I- Reissner’s membrane Scala media Spiral prominence epithelial cells Outer sulcus epithelial cellsOuter hair cells
Root cells
780 amino acids, 85.7 kDa Inner hair cells
74
Hereditary hearing loss
Heredeitary hearing loss is occurred in one out of
every 1,000 people.
Pendred syndrome
Sensorineural hearing loss
Goiter
Non-syndromic hearing loss with
an enlarged vestibular aqueduct
Pendrin
-derived hearing loss (~10%)
75
Pendrin mutants
10 pendrin mutants are reported in Japanese patients.
P123S, M147V, K369E, A372V, N392Y, C565Y,
S657N, S666F, T721M, H723R.
P123S M147V K369E A372V C565Y N392Y T721M H723R S666F S657NIntracellular localization of pendrin and its mutants in HEK293 cells
N392Y C565Y S657N S666F T721M H723R TRITC DIC Empty (pcDNA3.1) Merge Pendrin (Wild-type) M147V K369E A372V P123S TRITC DIC MergeIntracellular localization of pendrin was analyzed by immunofluorescent staining. This figure shows that wild –type pendrin and two pendrin mutants K369E and C565Y were localized in the plasma membrane, whereas the other 8 pendrin mutants were retained in the cytoplasm.
Effect of salicylate on the intracellular localization of mutant pendrin
TRITC DIC Merge 10 mM Salicylate Vehicle Control P123S TRITC DIC Merge M147V N392Y A372V S657N S666F T721M H723RIntracellular localization of pendrin was analyzed by immunofluorescent staining. This figure reveals that after incubation with salicylate, fluorescence intensity of TRITC of plasma membrane is stronger than that of the cytoplasm in the cells expressing P123S, M147V, S657N and H723R, but not in those expressing A372V, N392Y, S666F and T721M.
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Anion exchanger activity of pendrin mutants
** ** ** ** ## ## ## ## ## ## ## ## 0 1000 2000 3000 4000 5000 6000 Re m a ini ng Ra di oa c ti v it y i n Ce ll s (cp m/ mg ) Emp ty - + - + - + - + - + - + W ild -t yp e P 12 3 S M 14 7 V S 65 7 N H 72 3 R - - K 36 9 E C 56 5 Y Salicylate (10 mM)
Effects of salicylate on the iodide efflux from HEK293 cells. The remaining radioactivity in cells was defined by dividing intracellular iodide content in the lysates (cpm/ml) by protein content in the lysates (mg/ml). The remaining radioactivity in the cells transfected with P123S, M147V, S657N and H723R was significantly decreased by salicylate. ** P < 0.01 vs. corresponding control (the cells lacking salicylate), ## P < 0.01 vs. empty without salicylate.
Summary of pendrin mutants and in vivo effects of salicylate
OH OH O Wild-type Pendrin P123S (P) M147V (N) S657N (N) H723R (P/N) K369E (N) C565Y (N) Plasma Membrane Cytoplasm Salicylate Pendrin Mutants A372V (P/N) N392Y (P) S666F (N) T721M (N)Iodide Efflux Activity
+ - + + Misfolding Refolding
Salicylate induced the transport of 4 pendrin mutants, i.e., P123S, M147V, S657N and H723R, to the plasma membrane and the recovery of their anion exchanger activity, while 4 other pendrin mutants, i.e., A372V, N392Y, S666F and T721M, were retained in the cytoplasm.
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