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The Journal of Neuroscience, December 15, 2002, 22(24):10838-10846
Efferent Protection from Acoustic Injury Is Mediated via  9
Nicotinic Acetylcholine Receptors on Outer Hair Cells
Stéphane F.
Maison1,
Anne E.
Luebke2,
M. Charles
Liberman1, and
Jian
Zuo3
1 Department of Otology and Laryngology, Harvard
Medical School and Eaton-Peabody Laboratory, Massachusetts Eye and Ear
Infirmary, Boston, Massachusetts 02114, 2 Department of
Otolaryngology and Neuroscience Program, University of Miami School of
Medicine, Miami, Florida 33136, and 3 Department of
Developmental Neurobiology, St. Jude Children's Research Hospital,
Memphis, Tennessee 38105
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ABSTRACT |
Exposure to intense sound can damage the mechanosensors of the
inner ear and their afferent innervation. These neurosensory elements
are innervated by a sound-activated feedback pathway, the olivocochlear
efferent system. One major component of this system is cholinergic, and
known cholinergic effects are mediated by the  9/10 nicotinic
acetylcholine receptor (nAChR) complex. Here, we show that
overexpression of 9 nAChR in the outer hair cells of bacterial
artificial chromosome transgenic mice significantly reduces acoustic
injury from exposures causing either temporary or permanent damage,
without changing pre-exposure cochlear sensitivity to low- or
moderate-level sound. These data demonstrate that efferent protection
is mediated via the 9 nAChR in the outer hair cells and provide
direct evidence for a protective role, in vivo, of a
member of the nAChR family.
Key words:
olivocochlear; nicotinic; cholinergic; noise-induced hearing loss; transgenic mouse; cochlea
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INTRODUCTION |
The 9 nicotinic acetylcholine
receptor (nAChR) is a subunit of a receptor family with 17 known
members. As a group, they are widely and differentially expressed in
the CNS and PNS (Jones et al., 1999 ). In vitro, these
subunits form pentameric complexes surrounding a nonspecific cation
channel (Cordero-Erausquin et al., 2000). The functional roles of these
subunits range from mediating fast synaptic transmission in the
autonomic nervous system to transducing neuroprotective and
antinociceptive effects in vivo and in vitro
(Jones et al., 1999).
Expression of the 9 nAChR is restricted to the anterior pituitary,
the nasal epithelium, and the inner hair cells (IHCs) and outer hair
cells (OHCs), the mechanosensors of the inner ear (Elgoyhen et al.,
1994; Zuo et al., 1999). In the cochlea, the 9 subunit may act in
concert with 10, the newest member of the nAChR family (Elgoyhen et
al., 2001). However, the 9 subunit, per se, is essential for the
best-studied cholinergic effect on cochlear function: the suppression
of cochlear responses normally induced by activation of the cholinergic
efferent system known as the olivocochlear (OC) pathway (Vetter et al.,
1999).
In hair cells, 9/10 complexes are functionally coupled to
KCa channels; thus, ligand-gated
Ca2+ entry through the nAChR (Fuchs and
Murrow, 1992) is coupled to K+ efflux and
intracellular hyperpolarization (Housley and Ashmore, 1991). This
hyperpolarization of OHCs, in turn, affects their electromotile
responses (Dallos, 1992), decreasing cochlear mechanical responses to
low-level sounds (Dolan and Nuttall, 1988; Murugasu and Russell, 1996)
and elevating auditory thresholds (Wiederhold and Kiang, 1969).
The functional significance of this efferent feedback system remains
controversial; however, a role in protection from acoustic injury has
been proposed. In anesthetized animals, electrical stimulation of the
efferent pathway reduces temporary hearing loss from simultaneous
acoustic overexposure (Rajan, 1988; Reiter and Liberman, 1995); chronic
surgical de-efferentation renders awake animals more vulnerable to
permanent hearing loss from high-level noise (Kujawa and Liberman,
1997). Although an efferent role in protection is well established, the
complexity of the OC system has hampered understanding of the
mechanisms underlying this effect. At the neuroanatomical level, the
efferent pathway consists of two subsystems (see Fig. 1): a medial
component projecting primarily to OHCs and a lateral component
primarily innervating afferent terminals on IHCs (Warr et al., 1986).
At the cytochemical level, there is evidence for both GABAergic and
cholinergic transmission in both medial and lateral systems (Eybalin,
1993) and evidence for dopaminergic and peptidergic transmission in the
latter (Eybalin, 1993). To directly assess the role of the 9 nAChR
in efferent-mediated protection from acoustic injury, we studied a
transgenic (Tg) mouse line in which 9 is overexpressed. This report
documents (1) the overexpression at the protein level, (2) the
localization to OHCs via a green fluorescent protein (GFP)
reporter, (3) the lack of effect of overexpression on baseline cochlear
sensitivity, (4) the enhancement of electrically evoked OC effects on
the cochlea, and (5) the resultant enhancement of resistance to both
temporary and permanent acoustic injury.
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MATERIALS AND METHODS |
Experimental procedures. FVB/NJ mice were obtained
from The Jackson Laboratory (Bar Harbor, ME). Transgenic lines
were created (see below), maintained as heterozygotes in the original
FVB/NJ strain background, and genotyped in the Zuo laboratory in
Memphis [an extensive description of the methods is described by Zuo
et al. (1999)]. Transgenic mice (both homozygous and heterozygous) and
their wild-type littermates were shipped to Miami and Boston, where
investigators were blinded to the genotype until all data acquisition
was complete. For all physiological experiments, including baseline
testing auditory sensitivity and magnitude of OC suppression, as well
as in acoustic overexposure experiments, mice ranged in age from 10 to
12 weeks. Mice used for Western blot ranged in age from 4 to 12 weeks.
Transgenic construct and genotyping. Two homologous
fragments that contain the coding portion and the 3' untranslated
region of exon 5 were used for modification of the transgene. An
internal ribosome entry site/GFP cassette (1.3 kb) was inserted
after the stop codon (see Fig. 2A) (Zuo et al.,
1999). A second transgenic line was created with an identical construct
except that exon 4 (E4) of the 9 nAChR gene was deleted, as
in the 9 knock-out strategy (Vetter et al., 1999). This was achieved
with two homologous fragments flanking the E4. The previously modified
bacterial artificial chromosome (BAC), mK9, was further modified for
such a deletion. The resulting BAC DNA, mK9E4, was used for transgenic
injection as described previously. Mice were genotyped using both PCR
and interphase fluorescence in situ hybridization (FISH)
assays (see Fig. 2B) (Zuo et al., 1999). For the
interphase FISH analysis, the ends (~0.5-1 cm) of tails were removed
from mice that were between the ages of 1 week and 9 months. The tail
fragments were immersed in complete tissue culture medium (Roswell Park
Memorial Institute 1640) at 4°C for up to 3 d. The tail
fragments were minced and then enzymatically disaggregated by
collagenase (400 U/ml in complete culture medium) overnight at room
temperature on a rocker. The liberated cells were then centrifuged and
washed once with PBS. The cells were centrifuged, and the cell
pellets were resuspended in a 0.075 M KCl
solution for 5 min and centrifuged again. The cell pellets were
resuspended in Carnoy's fixative (methanol/glacial acetic acid ratio
of 3). The fixative was changed once, and air-dried slides were
prepared on wet microscope slides. The BAC DNA used as a probe was
labeled with digoxigenin-deoxyUTP by nick translation. The labeled
probe was combined with sheared mouse DNA (catalog number 18440-016;
Invitrogen, Gaithersburg, MD) and hybridized to the fixed cells
in a solution containing 50% formamide, 10% dextran sulfate, and 2×
SSC (0.03 M trisodium citrate, pH 7.0, and 0.3 M NaCl). A specific probe signal was detected by
incubating the hybridized slides in fluorescein-labeled antidigoxigenin
antibodies followed by counterstaining with
4',6-diamidino-2-phenylindole. Our rapid interphase FISH
genotyping results on >163 mice were consistent with previous
metaphase FISH and PCR results (Zuo et al., 1999). To further determine
the copy number of the BAC transgene, we performed quantitative FISH
analysis on 40 interphase tail cells of one heterozygote (Poon et al.,
1999). These experiments were performed using a CytoVision Image
analysis system (Applied Image, Inc., Rochester, NY). All images were
captured in one session to eliminate fluctuations in fluorescence
attributable to fading of the dyes. The signal area was measured in
pixels, and the signal intensity was measured in arbitrary units. The
signal intensity was then multiplied by the signal area to yield a
total fluorescence emission per signal. From a total of 40 interphase
cells, the mean fluorescence emission for the transgene was divided by
the mean fluorescence emission from the endogenous locus to estimate the copy number of the transgene relative to the endogenous locus. Because the endogenous signal represents a single copy of the BAC, the
ratio of the endogenous signal and the transgenic signal would indicate
the copy number of the BAC transgene. We obtained the ratio of 3.6 ± 1.4 (mean ± SD) (n = 40). Thus, the copy
number of the BAC transgene is ~4. Similar measurements on the BAC
transgenic line mK9E4 showed that the copy number of the BAC transgene
is ~1 (data not shown).
Western blot analysis. Mouse cochleas were homogenized in
0.5 ml of radioimmunoprecipitation assay buffer (0.15 M NaCl, 0.05 M Tris, 0.1% NP-40, 0.05%
deoxycholate, and 0.01% SDS). Equal volumes of each cochlea (40 µl; ~40 µg of tissue) and 30 µg of control tissues (i.e.,
brain, skeletal muscle, pituitary, and eye) were separated by SDS-PAGE
and electroblotted to Immobilon P membranes (Millipore, Bedford, MA),
and Western blotting was performed as described in the fast-blot
protocol for Immobilon P membranes using either an antibody generated
against 9 nAChR (MU43f) or an antimyosin VI antibody. The resulting
autoradiographs were scanned, and band densities were determined using
BioMax ID image analysis software (Eastman Kodak, Rochester, NY). To ensure that this semiquantitative analysis remained in the dynamic range of the measurement system, band densities were analyzed for a
10-fold dilution series performed on cochlear samples from both
wild-type and transgenic ears.
Coimmunoprecipitation from mouse cochleas. Mouse cochleas
were frozen and placed in PTN50 buffer (50 mM
sodium phosphate, pH 7.4, 1% Triton X-100, and 50 mM NaCl) containing protease inhibitors and
homogenized at high speed in a Brinkmann Polytron (Brinkmann Instruments, Westbury, NY) for 30 sec. The homogenate was
freeze-thawed, and the lysate was centrifuged at 12,000 × g in a microfuge for 5 min to pellet cellular debris. 9
nAChRs were immunoprecipitated from the lysate overnight at 4°C using
the antibody MU43f (9 rabbit polyclonal); 10 nAChRs were
immunoprecipitated from the lysate using the antibody MU81
(10 chicken polyclonal). The immune complexes were captured
using immobilized protein A-Sepharose beads (Sigma, St. Louis, MO) or
anti-chicken IgY-conjugated Sepharose beads (Promega, Madison, WI); the
immunoprecipitated material was extracted by boiling in SDS Laemmli
sample buffer. The samples were resolved by 4-15% SDS-PAGE and
analyzed by Western blot analysis using antibody MU81 (anti-10
antibody) or MU43 (anti-9 antibody).
Auditory brainstem responses. Auditory brainstem responses
(ABRs) were measured in each animal both before and after the acoustic overexposure. For the measurement, mice were anesthetized with xylazine
(20 mg/kg, i.p.) and ketamine (100 mg/kg, i.p.). Needle electrodes were
inserted at vertex and pinna, with a ground near the tail. ABR
potentials were evoked with 5 msec tone pips (0.5 msec rise-fall with
a cos2 onset envelope, delivered at
35/sec). The response was amplified (10,000×), filtered (0.1-3
kHz), and averaged with an analog-to-digital board in a
LabVIEW-driven data-acquisition system (National Instruments, Austin,
TX). The sound level was raised in 5 dB steps from 10 dB below
threshold up to 80 dB sound pressure level (SPL). At each sound level,
1024 responses were averaged (with stimulus polarity alternated), using
an "artifact reject," whereby response waveforms were discarded
when the peak-to-peak amplitude was >15 µV. On visual inspection of
stacked waveforms, the threshold was defined as the lowest SPL level at
which any wave could be detected, usually corresponding to the step
just below that at which the peak-to-peak response amplitude rose
significantly above the noise floor (~0.25 µV).
Cochlear immunostaining and quantification of immunopositive
terminals. After intracardial perfusion with 10% formalin,
cochleas were decalcified in EDTA and half-turns were dissected and
immunostained as whole mounts. Tissue was incubated in primary antisera
overnight [rabbit anti-vesicular acetylcholine transporter (VAT);
Sigma], followed by a biotinylated secondary antibody,
avidin-biotin-HRP complex (ABC kit; Vector Laboratories, Burlingame,
CA) and DAB/H2O2; the
tissue was then embedded in plastic and mounted on glass slides. For
each cochlea, each dissected piece was measured by computerized planimetry and the cochlear location was converted to frequency (Ehret, 1983). To quantify immunopositive terminals, outlines were
traced via a drawing tube using high-numerical-aperture objectives (2000× total magnification). When tracing, the fine focus was continually adjusted to optimize the imaging of each terminal cluster.
The traces were digitized, and the areas were computed with NIH Image
software. In the OHC area, all immunopositive terminals were traced;
the values from each row were averaged within bins corresponding to 100 µm of cochlear length.
Distortion product otoacoustic emissions-based assay of
OC-mediated suppression. Animals were anesthetized as for ABR
testing, and posterior craniotomy and partial cerebellar aspiration
were performed to expose the floor of the IVth ventricle. To stimulate the OC, shocks (monophasic pulses of 150 µsec duration presented at
200 per sec) were applied through fine silver wires (0.4 mm spacing)
placed along the midline, spanning the OC decussation. The shock
threshold for facial twitches was determined, muscle paralysis was
induced with -D-tubocurarine (1.25 mg/kg,
i.p.), and the animal was connected to a respirator via a tracheal
cannula. Shock levels were raised to 6 dB above the twitch threshold.
The distortion product otoacoustic emission (DPOAE) at
2f1  f2
[f1 and f2 are the
values of the two primary frequencies presented to the ear
(f2 > f1)] was recorded with a
custom acoustic assembly consisting of two 0.25 inch condenser
microphones to generate primary tones
(f1 and
f2 with
f2/f1 = 1.2 and an f2 level 10 dB less than
the f1 level) and a Knowles miniature
microphone (EK3103; Knowles Electronics, Franklin Park, IL) to
record sound pressure in the ear canal. Stimuli were generated
digitally (AO-6; National Instruments, Austin, TX). The sound pressure
in the ear canal was amplified and digitally sampled at 20 µsec
(A-2000; National Instruments). Fast Fourier transforms were
computed and averaged over five consecutive waveform traces, and
2f1-f2
DPOAE amplitude and the surrounding noise floor were extracted, a
procedure requiring ~6 sec of data acquisition and processing time.
DPOAE thresholds were defined by the visual inspection of amplitude
versus level functions as the lowest
f2 level above which the DPOAE
amplitude was always greater than the surrounding noise floor (which
varied from 20 to 0 dB SPL, depending on the frequency; threshold
amplitudes were typically 3-4 dB above the noise). During the OC
suppression assay, the f2 level was
set to produce a DPOAE ~10-15 dB greater than the noise floor. To
measure OC effects, repeated measures of baseline DPOAE amplitude were
first obtained (n = 12), followed by a series of 12 interleaved periods during which DPOAE amplitudes were measured with
(n = 6) and without (n = 6)
simultaneous shocks to the OC. The magnitude of the OC effect was
defined as the mean decrease in DPOAE amplitude (in decibels) for all
six trials that included shocks compared with the mean baseline amplitude.
Acoustic injury. Animals were exposed, awake, and
unrestrained; they were kept within cages suspended inside a small
reverberant sound-exposure box (Liberman and Gao, 1995). The exposure
stimulus was generated by a custom-made white-noise source, filtered
with a 60 dB/octave slope, amplified (Crown power amp; Crown Audio, Elkhart, IN), and delivered (JBL compression driver; JBL Scientific, San Luis Obispo, CA) through an exponential horn fitted securely to a
hole in the top of a reverberant box. Sound exposure levels were
measured at four positions within each cage using a 0.25 inch Bruel and
Kjaer (Copenhagen, Denmark) condenser microphone; the sound
pressure was found to vary by <0.5 dB across these measurement positions. The sound pressure was calibrated daily by positioning the
microphone at the approximate position of the animal's head.
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RESULTS |
Overexpression of 9 nAChR in transgenic animals
In the present report, we studied a
transgenic mouse line in which 9 is overexpressed via insertion of a
modified BAC containing the mouse 9 nAChR gene and its promoter,
along with a reporter gene, GFP (Fig.
2A). Mice were
genotyped using both PCR and interphase FISH assays (Fig.
2B) (Zuo et al., 1999).
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Figure 1.
Cross section of the sensory epithelium of the
inner ear (organ of Corti) showing one row of IHCs, three rows of OHCs,
a single auditory nerve afferent contacting an IHC, a representative
efferent fiber from the medial OC (MOC) system,
contacting all three rows of OHCs, and an efferent fiber from the
lateral OC (LOC) system contacting the peripheral
terminal of an auditory nerve fiber. Arrows indicate the
direction of information propagation along the neurons.
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Figure 2.
Construction and characterization of transgenic
mice overexpressing 9 nAChR. A, The schematic
illustrates the site-directed mutagenesis of a BAC containing the mouse
9 nAChR gene. IRES, Internal ribosome entry
site; UTR, untranslated region.
B, FISH genotyping using mouse 13K9 BAC DNA and
interphase chromosomes of a cell from the tail of a 1-week-old
wild-type, adult heterozygote (Tg+/), or adult homozygote (Tg+/+)
mouse. The endogenous locus is indicated by the white
arrows, and the transgenic locus is indicated by the
red arrows. C,
Fluorescence microscopy shows that expression of GFP reporter in
transgenic animals is restricted to cochlear OHCs, with a stronger
signal in homozygotes (Tg+/+) than in heterozygotes (Tg+/). The
images represent identical exposures of surface preparations of the
organ of Corti from the apical half of the cochlea in adult mice from
each of three genotypes. D, E, Western blots show 9
nAChR protein expression in transgenic mice restricted to the pituitary
and cochlea; in the cochlea, the expression is stronger in transgenic
animals than in wild-type littermates. F, Quantitative
analysis of Western blots. Expression levels were quantified by
measuring band density (60 kDa band) and normalizing to myosin VI (148 kDa band). Error bars indicate means ± SEM; n,
number of animals in each sample; asterisks indicate
statistical significance (p < 0.001;
Student's t test).
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Transgenic animals showed normal growth and body weights, and their
open-field behavior was indistinguishable from wild-type littermates.
GFP expression in this transgenic line has been shown to recapitulate
the endogenous expression of 9 nAChR during neonatal development, as
determined by in situ hybridization (Zuo et al., 1999).
The pattern of transgene expression in the adult inner ear was
investigated via endogenous fluorescence of the GFP reporter (Fig.
2C) and anti-GFP immunostaining (data not shown). Both
methods suggested that 9 overexpression was driven only in OHCs.
There was no clear radial gradient among the three OHC rows; however, GFP signals decreased in intensity from apex to base.
Western blot analysis of cochlear homogenates (Fig.
2D-F) suggested that levels of 9 protein,
normalized to hair-cell number, were ~1.6 times higher in transgenic
mice than in wild-type littermates: expressed in arbitrary units of
optical density, values of 1.33 ± 0.07 in wild types
(n = 13) versus 1.98 ± 0.21 for heterozygotes (n = 6) versus 2.12 ± 0.27 for homozygotes
(n = 6) were obtained (Fig. 2D).
Differences between wild types and either transgenic genotype were
statistically significant (p < 0.001;
Student's t test); however, the small mean difference
between the homozygous and heterozygous animals was not.
Coimmunoprecipitation studies (Fig. 3)
suggest that overexpression of 9 protein led to the formation of
more 9/10 complexes in cochlear tissues. When cochlear lysates
were precipitated with a rabbit anti-9 antibody and then probed with
chick anti-10 (Fig. 3C), the band density indicating
10 levels on the resultant gels was higher in transgenic cochleas
than in wild types. In the example shown, the densities of the
wild-type, heterozygote, and homozygote transgenic mice were 38.9, 61.9, and 76.8 arbitrary units, respectively. Similarly, when lysates
were precipitated with anti-10 and probed with anti-9 (Fig.
3B), the band densities were higher in transgenic mice than
in wild types. Similar results were obtained with two independent runs
from different sets of cochleas for each of the two complementary
coimmunoprecipitation tests. Control lanes show a lack of staining in
antibody-only controls; the two tests were run identically, except that
the controls were run without the cochlear lysates, to
demonstrate the lack of cross-reactivity between the two primary
antibodies themselves.
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Figure 3.
Coimmunoprecipitation tests show that there is an
increase in 9/10 protein complexes in the transgenic cochleas.
A, Fragments of the peptide alignments for 9 and
10 proteins from humans, rats, and guinea pigs illustrate the
peptide regions used to create the anti-9 and anti-10 antibodies.
Dark gray regions denote absolute identity with 9
from guinea pigs; light gray denotes conserved protein
identity. gp, Guinea pig. B, C, Western
blots for two complementary coimmunoprecipitation tests: precipitate
with 10 and probe with 9 (A) and
precipitate with 9 and probe with 10. Each data set is derived
from cochlear lysates pooled from eight cochleas from four animals of
each genotype. WT, Wild type; +/+, homozygotes; +/,
heterozygotes; C, controls; ipt,
immunoprecipitation.
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Cochlear function before acoustic injury
Assessment of baseline cochlear function, conducted in
double-blind manner, revealed no significant differences between
transgenic mice and wild-type littermates. Cochlear function was
assessed by measurement of both ABRs and DPOAEs. The ABR measurement
represents synchronous neural activity generated at several levels of
the ascending auditory pathways, including the auditory nerve. The DPOAEs are sounds created within the cochlea, amplified by the action
of OHCs and propagated through the middle ear back to the ear canal,
where they can be measured with a microphone (Kemp, 1986). Although the
ear creates DPOAEs at a number of frequencies, the largest is that at
the frequency
2f1-f2.
For ABRs and DPOAEs, data were gathered in such a way as to allow both
a measure of the threshold of response (Fig.
4A,B) (i.e., the lowest
stimulus level required to produce a criterion response chosen to be
just above the measurement noise floor) and the growth of response
magnitude with sound pressure level (Fig. 4C,D). For growth
functions, data are shown at only one test frequency; however, the
results were similar for all test frequencies measured (all frequencies
tested can be read from Fig. 4A,C). Thus, the
normality of all of these response measures in the transgenic animals
indicates that cochlear mechanics, transduction, and synaptic
transmission at low and moderate sound levels are not significantly
altered by the 9 overexpression.
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Figure 4.
Characterization of baseline cochlear function in
transgenic mice versus littermate wild types before acoustic
overexposure. Group means ± SEM are plotted; the numbers of
animals in each group are indicated in the key.
A, C, Pre-exposure thresholds for ABRs and ABR
amplitude-versus-level functions are unaffected by the transgene. ABR
thresholds are determined by visual inspection of waveforms obtained at
5 dB increments in SPL. ABR amplitude-versus-level functions are shown
only for the test frequency at 22.62 kHz. B, D, DPOAE
thresholds and amplitude-versus-level functions are also unaffected by
the transgene. DPOAE thresholds are determined by visual inspection of
waveforms obtained at 5 dB increments in SPL. Amplitude-versus-level
functions are shown only for f2 = 14.14 kHz. WT, Wild type.
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OC morphology and function before acoustic injury
A second set of pre-exposure baseline experiments demonstrated
that classic efferent suppressive effects were significantly enhanced
in the transgenic animals (Fig. 5). To
evaluate the effects of 9 overexpression on these efferent effects,
DPOAEs were measured while electrically stimulating the efferents at
the floor of the IVth ventricle, where they run close to the surface of
the brainstem (Warr et al., 1986). Given the close relationship between
OHC function and distortion product amplitude, the degree of DPOAE suppression can be a sensitive measure of these cholinergic actions on
OHCs. As illustrated in Figure 5B, our assay of classic
efferent cochlear suppression involved repeated measures of DPOAE
magnitude without (Fig. 5, open triangles) versus with (Fig.
5, closed triangles) simultaneous electrical activation of
the efferent bundle with 200 shock trains per second. The efferent
effect was then defined as the difference (in decibels) between the
average DPOAE amplitude for the 12 trials before the first shock trains
and the average DPOAE amplitude for the six trials during shocks to the
efferent bundle. Note that for the individual data run shown in Figure 5B, the efferent effect magnitude tends to decrease with
repeated stimulation. Similar adaptation of OC-mediated cochlear
suppression has been reported previously (Wiederhold and Kiang,
1969).
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Figure 5.
Efferent-mediated suppression of cochlear
DPOAEs is enhanced in transgenic animals. A, Data show
mean suppression ± SEM of the DPOAE at
2f1-f2
attributable to efferent electrical stimulation. The numbers of animals
tested in each group are indicated in the key.
Techniques for deriving efferent effect magnitude are illustrated in
B. Dashed line indicates no efferent effect.
B, Measurement of efferent effect magnitude in one
animal at f2 = 22.62 kHz. For all
measurements, the f2 level was set to
produce a DPOAE ~10-15 dB above the noise floor. Repeated
measurements of baseline DPOAE amplitude were first obtained
(n = 12), followed by a series of 12 interleaved periods in which DPOAE amplitudes were measured with
(n = 6) and without (n = 6)
simultaneous shocks to the OC. The black arrow indicates
the magnitude of the efferent effect, defined as the mean decrease in
DPOAE amplitude (in decibels) for all six with-shock trials
(bottom dashed line) compared with the mean baseline
amplitude (top dashed line). The noise floor indicates the
average spectral level for the six frequency points surrounding
2f1-f2.
WT, Wild type.
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The data in Figure 5A summarize the mean effects seen in a
number of wild-type versus transgenic animals. These mean data show
that 9 overexpression increased the magnitude of efferent-mediated cochlear suppression for test frequencies between 16 and 32 kHz (two-way ANOVA; F(1,13) = 8.961;
p = 0.01), with smaller effects seen above and below
that region.
To determine whether these enhanced peripheral effects in the
transgenic line arise from fundamental alterations in efferent innervation patterns, the size and number of cholinergic OC terminals on OHCs were quantified in one homozygous transgenic animal (Fig. 6). To visualize these terminals,
cochleas were immunostained for VAT, and the silhouette areas of
immunopositive terminals were measured throughout the cochlear spiral.
As shown in Figure 6, this estimate of total terminal volume for the
cholinergic innervation of OHCs normally peaks in the middle of the
cochlear spiral, at cochlear regions tuned near 12 kHz. Results from
the transgenic cochlea were not significantly different.
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Figure 6.
Density of cholinergic OC terminals on OHCs as a
function of cochlear location for transgenic versus normal mice. Data
show the total silhouette area of immunostained terminals (anti-VAT)
from all three rows. Transgenic data are from a single homozygous
animal. Control data were obtained from four CBA/CaJ mice. Error bars
indicate SEM. Innervation densities are expressed as total silhouette
area per OHC. CF, Cochlear frequency.
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Protection from acoustic injury
To evaluate the effects of 9 overexpression on the
vulnerability to both reversible and irreversible forms of acoustic
injury, groups of transgenic and wild-type animals were exposed for 2 hr to a noise band (8-16 kHz) at either 92 or 110 dB SPL and allowed to recover for 12 hr or 1 wk before
assessing the degree of temporary (Fig. 7) or permanent noise-induced
hearing loss (Fig. 8), respectively, via
measurement of thresholds for auditory evoked potentials. All exposures
and subsequent physiological analyses were performed by the
Boston-based team, who were kept unaware of the genotyping data.
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Figure 7.
9 nAChR overexpression reduces temporary
noise-induced damage. A, Protective effects of the
transgene for an exposure designed to cause TTS: 2 hr of exposure to an
8-16 kHz noise band at 92 dB SPL. When measured 12 hr after exposure,
the mean TTS was significantly larger in wild types than in
transgenics. WT, Wild type. B, C,
ABR amplitude-versus-level functions are shown for two test
frequencies. Data are expressed as group means ± SEM. Gray
band indicates passband of the noise used for acoustic
overstimulation. Dashed line indicates no threshold shift.
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Figure 8.
9 nAChR overexpression reduces permanent
noise-induced damage. A, Protective effects of the
transgene for an exposure designed to cause PTS: 2 hr of exposure to an
8-16 kHz noise band at 110 dB SPL. When measured 1 wk after exposure,
the mean PTS was significantly larger in wild-type than in
transgenic animals. Data are expressed as group means ± SEM.
WT, Wild type. B, C, ABR
amplitude-versus-level functions are shown for two test
frequencies. Data are expressed as group means ± SEM. Gray
band indicates passband of the noise used for acoustic
overstimulation. Dashed line indicates no threshold shift.
|
|
When measured 12 hr after exposure at 92 dB, temporary threshold shifts
(TTSs) were maximal near 16 kHz for all genotypes (Fig. 7A);
1 wk later, mean thresholds had returned to normal (data not shown),
demonstrating the reversibility of the damage from the 92 dB exposure.
The mean TTS was significantly smaller in transgenic than in wild-type
animals over the entire range of tested frequencies (two-way ANOVA;
F(1,12) = 10.03; p = 0.008). Pairwise comparisons between either heterozygote or homozygote groups and wild types also showed statistically significant differences (two-way ANOVA; F(1,10) = 7.293, p = 0.022, and F(1,6) = 21.251, p = 0.004, respectively). Although the trend
of the means suggests that homozygotes were slightly more resistant
than heterozygotes, the differences were not significant. When viewed
as amplitude-versus-level functions, ABR data show consistently higher
suprathreshold response amplitudes for transgenic versus wild-type
animals. Three octave-spaced test frequencies are shown (Fig.
7B,C); however, the results were similar for the other test
frequencies measured (8, 16, and 32 kHz).
The 110 dB exposure produced significantly more acute threshold shift,
and it resolved, among wild-type controls, to a permanent threshold
shift (PTS) of 15-25 dB across all frequencies tested, when measured 1 week after exposure (Fig. 8A). Among the animals exposed at 110 dB, there was also a clear reduction in PTS
vulnerability among the transgenic animals (two-way ANOVA;
F(1,12) = 5.968; p = 0.032). By chance, the littermates used in this arm of the study
contained no animals homozygous for the transgene. Measurements of the
growth of response magnitude with SPL also revealed consistent and
dramatic differences between genotypes, with higher response amplitudes
at all levels for transgenic mice. Three octave-spaced test frequencies
are illustrated in Figure 8B,C; similar results were
seen at three other test frequencies (8, 16, and 32 kHz)
Two previous experimental series also showed significantly enhanced
resistance to acoustic injury in transgenic animals (data not shown).
One series (with five wild-type, three Tg+/, and six Tg+/+ animals)
produced a slightly more severe TTS than that shown in Figure
7A (noise band at 94 instead of 92 dB). A second series
(with 12 wild-type, 11 Tg+/, and 7 Tg+/+ animals) produced a smaller
PTS than that shown in Figure 8A (noise band at 104 instead of 110 dB). In the second series, PTS differences between wild
type and each transgenic genotype were statistically significant in the
range 4-22.62 kHz by ANOVA (F(1,21) = 5.587, p = 0.028 for wild type vs Tg+/ and
F(1,17) = 4.795, p = 0.043 for wild type vs Tg+/+), whereas differences between heterozygous
and homozygous animals were not
(F(1,16) = 0.120; p > 0.05).
A final series of control experiments was performed to exclude a
possible contribution to the observed resistance of other factors, such
as the expression of GFP or other genes in the BAC construct. To this
end, a control BAC construct was designed differing only by deletion of
exon 4 of the 9 nAChR gene (Fig. 2A). This new
transgenic line expressed GFP without overexpressing the 9 protein
(as quantified by Western blot), and after exposure to the 110 dB noise
band showed a PTS pattern that was statistically indistinguishable from
that seen in wild-type animals (data not shown).
| |
DISCUSSION |
These results represent a key step in dissecting the contributions
of different transmitter/receptor complexes to the observed effects of
the structurally and cytochemically diverse OC efferent system. Data
from these mutant mice provide direct evidence that efferent-mediated
protection from acoustic injury is mediated via the 9 nAChR
complexes on OHCs. Furthermore, the fact that the magnitude of the
heightened resistance associated with 9 overexpression is comparable
with the magnitude of the heightened PTS vulnerability seen after
surgical de-efferentation (Kujawa and Liberman, 1997) and with the
increased TTS resistance associated with massive electrical stimulation
of the efferent bundle (Rajan, 1988) is consistent with the idea that
9 participates directly in all of the efferent-mediated protective
effects observed to date. For example, in the pioneering study by Rajan
(1988) of electrically evoked OC activity and TTS in guinea pigs, the
largest protection reduced peak TTS by only 16 dB (from ~22 dB in
control ears to ~6 dB in ears with 400/sec shocks to the OC bundle
during acoustic overexposure). In comparison, the transgenic
overexpression of 9 in the present study reduced TTS by as much as
22 dB (compare 22 kHz values for wild-type vs transgenic animals in
Fig. 7A).
The additional observation that 9 overexpression was restricted to
the OHCs also strongly implicates these cells, and thus the medial OC
system, in the phenomenon of efferent-mediated protection. Although
in situ hybridization studies have shown a clear 9 signal in the IHCs of adult rats (Simmons and Morley, 1998; Luo et al., 1998),
the native expression pattern in adult mice has never been investigated; the lack of 9 expression in our transgenic mice is
consistent with the transition of efferent innervation from IHCs to
OHCs during postnatal development. Thus, the present data show that
there is no reason to invoke any noncholinergic effects of the medial
OC system on OHCs, nor any action of the lateral OC system in the IHC
area to explain OC-mediated protection from acoustic injury.
Work on the 9 knock-out mouse suggests that the 9 nAChR is also
essential for the generation of classic efferent-mediated cochlear
suppression of responses to low and moderate sound levels (Vetter et
al., 1999). The present results are consistent in that overexpression
of 9 led to enhancements of this type of efferent-mediated suppression (Fig. 5). Thus, both of the best-characterized efferent effects appear interrelated via a common dependence on the 9 subunit. The observed frequency dependence of OC suppressive effects (Fig. 5A) is consistent with the longitudinal distribution
of OC cholinergic terminals in the mouse (Fig. 6): both appear to peak
at approximately the 12 kHz region. Our measures of OC suppression are
limited to frequencies of >11.3 kHz, because DPOAE amplitudes in mice
are very small for f2 at  8 kHz (S. F. Maison and M. C. Liberman, unpublished observations). Thus,
the correlation between the frequency dependence of the peripheral
effects of OC and the cochlear distribution of OC terminals is not as
compelling as in cats or guinea pigs (Liberman et al., 1990).
The apparent frequency dependence of the enhancements of OC suppression
in the transgenic mice (i.e., largest at 22.6 kHz) (Fig. 5A)
must be viewed cautiously. In particular, the effects of 9
overexpression may be underestimated at 11.3 kHz, at which OC effects
peak, because of saturation in the assay. To maximize OC effects, we
minimized the sound pressure of the primaries, because OC effects are
maximal near threshold (Wiederhold, 1970). Thus, the primary levels
were set to produce a DPOAE only 10 dB above the noise floor, and, when
the OC effects were large, DPOAEs were often driven into the noise
(e.g., first point with OC shocks) (Fig. 5B), thus
saturating the assay and reducing measured enhancements.
Enhanced OC effects in the transgenic animals are not attributable to
fundamental changes in the efferent innervation pattern associated with
9 overexpression (Fig. 6). Thus, they are presumed to arise from
changes in the number, nature, or distribution of 9 receptor
complexes in the postsynaptic membrane. Evidence suggests that 9 in
OHCs in vivo is complexed with 10 nAChR subunits: in vitro coexpression of 10 with 9 produces
ligand-gated currents and desensitization behavior more like native
receptors; 9 homomers show smaller currents but less desensitization
(Elgoyhen et al., 2001). Our coimmunoprecipitation data (Fig. 3) show
that the 9 upregulation driven by the transgene is coupled with an
increased formation of 9/10 complexes in the cochlea. Thus, the
enhancement of classic OC suppression, observed in transgenic animals
(Fig. 5), is well explained by an increased level of functional
9/10 complexes. Whereas 9 mRNA in our transgenic cochleas was
increased by more than fivefold with regard to wild-type animals (Zuo
et al., 1999), 9 protein was increased only 1.5-1.7 times (Fig. 2F). Although Western blot estimates of expression
changes are semiquantitative at best, increases in protein expression
comparable with the mRNA enhancement may be limited by space
constraints at the postsynaptic membrane. Transgenic overexpression
studies of rhodopsin in retinal photoreceptor cells have also noted
modest (i.e., 20-25%) changes in protein levels despite clear-cut
changes in the phenotype (Tan et al., 2001).
The idea that 9 overexpression could have dramatic effects on the
response of the ear to high-level sounds (Figs. 7, 8), with no
changes in threshold sensitivity or responses to moderate-level stimuli
(Fig. 4), fits well with the known characteristics of this sound-evoked
feedback pathway. The efferent neurons that project to these
cholinergic synapses on OHCs have low levels of spontaneous activity
and begin to respond to sound at levels 20-30 dB above afferent fiber
thresholds (Liberman, 1988). As the sound level increases, efferent
discharge rates increase monotonically; thus the magnitude of their
peripheral effects should also increase with increasing sound level.
There are two fundamentally different ways in which 9 activation
could reduce acoustic injury: (1) directly, via protective effects on
the OHCs themselves, because these cells are both necessary for the
exquisite threshold sensitivity and are among the elements most
vulnerable to acoustic overexposure, or (2) indirectly, by reducing the
OHC contribution to amplification of mechanical vibrations of the
cochlear sensory epithelium.
The case for mechanical effects (hypothesis 2) lacks key empirical
data. Although OC activation can reduce cochlear vibrations to
low-level sounds (Murugasu and Russell, 1996), evidence concerning the
magnitude of these efferent effects for high levels of acoustic stimulation is sparse and contradictory (Guinan and Stankovic, 1996).
Clarifying these mechanical effects is key to understanding the
mechanisms. In all previously studied mammalian models of acoustic
injury (including FVB/N mice), the magnitude of noise-induced PTS grows
by 5-10 dB for every 1 dB increase in exposure level once a damaging
exposure level is reached (Yoshida et al., 2000; see below). Thus,
efferent protective effects as large as 30 dB can arise from a
suppression of cochlear vibration equivalent to only a 3 dB decrease in
the input stimulus level.
The alternative hypothesis (i.e., that 9 activation leads
directly to hair cell protection) is interesting in light of the hypothesized role of the 7 subunit in mediating a variety of neuroprotective effects in vitro (Cordero-Erausquin et al.,
2000). Previous studies of OC effects in vivo have suggested
that 9 activation leads to both a rapid ( = 100 msec)
calcium-activated K+ efflux as well as a
slow ( = 10 sec) wave of calcium-induced calcium release in
hair cells (Sridhar et al., 1997). Additional circumstantial evidence
has linked the magnitude of this slow effect with the magnitude of
OC-mediated protection (Reiter and Liberman, 1995).
According to the present conclusions, the 9 knock-out mouse should
be exceptionally vulnerable to acoustic injury. A previous attempt to
address this question (Yoshida et al., 1999) failed to show significant
effects of the knock-out on acoustic vulnerability. However, the study
was inconclusive about the role of 9, because it was also shown that
the knock-out background strain (129/SvEv) is uniquely resistant to
acoustic injury (Yoshida et al., 2000). In 129/SvEv mice, the growth
rate of PTS with exposure level is only 1 dB/dB, rather than the 5-10
dB/dB discussed above. Thus, if efferent-mediated protective effects
arise via sound-evoked feedback inhibition of cochlear mechanical
vibration equivalent to only 1-2 dB of stimulus reduction, the effects
on PTS will not appear significant in the uniquely resistant 129/SvEv
ear, in which that small effective attenuation is not amplified into a
PTS reduction 5-10 times larger.
Conclusions
Our investigations provide direct evidence that efferent-mediated
protection from acoustic injury is mediated via the 9 nAChR complexes on OHCs. The present results focus future investigation of
efferent-mediated protection on clarification of the downstream effects
of ligand binding to the 9 receptors. In a broader context, the
results also constitute the first direct demonstration of in
vivo modulation of a protective effect of nAChRs by transgenic modulation of the expression level of a particular receptor subunit. The fact that 9 receptor expression is limited to very few sites in
the nervous system and the fact that the transgenic overexpression in
the ear can be clearly limited to one cell class provide a unique
opportunity to isolate and characterize the role of one member of the
nAChR receptor subunit family in the generation of a protective effect
in vivo.
| |
FOOTNOTES |
Received Aug. 22, 2002; revised Sept. 24, 2002; accepted Sept. 27, 2002.
This work was supported by National Institutes of Health (NIH) Grants
R01 DC-0188, R01 DC-03086, R01 EY12950, R21 DC 05168, and R03 DC-04761,
NIH Cancer Center Grant CA-21765, Chiles Endowment Biomedical Research
Program of the Florida Department of Health Grant BM028, March of Dimes
Birth Defects Foundation Grant 5-FY98-0725, the American Lebanese
Syrian Associated Charities, and a Long-Term Fellowship of the Human
Frontier Science Program Organization (S.F.M.). We thank J. He, A. Lowrey, J. Treadaway, M. Valentine, and V. Valentine for assistance;
Dr. T. Hasson (University of California, San Diego, CA) for providing
the Myosin VI antibody; and Dr. J. J. Guinan Jr. for comments on
this manuscript.
Correspondence should be addressed to Dr. M. Charles Liberman,
Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114-3096. E-mail:
mcl{at}epl.meei.harvard.edu.
| |
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ABSTRACT
INTRODUCTION
