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Review Article
Cochlear synaptopathy in acquired sensorineural hearing loss:
Manifestations and mechanisms
M. Charles Liberman
a
,
b
, Sharon G. Kujawa
a
,
b
,
*
a
Department of Otology and Laryngology, Harvard Medical School, Boston MA, USA
b
Eaton-Peabody Laboratories, Massachusetts Eye &Ear Infirmary, Boston MA, USA
article info
Article history:
Received 13 June 2016
Received in revised form
19 December 2016
Accepted 5 January 2017
Available online 10 January 2017
Keywords:
Auditory nerve
Cochlear synaptopathy
Cochlear neuropathy
Hidden hearing loss
Noise-induced hearing loss
abstract
Common causes of hearing loss in humans - exposure to loud noise or ototoxic drugs and aging - often
damage sensory hair cells, reflected as elevated thresholds on the clinical audiogram. Recent studies in
animal models suggest, however, that well before this overt hearing loss can be seen, a more insidious,
but likely more common, process is taking place that permanently interrupts synaptic communication
between sensory inner hair cells and subsets of cochlear nerve fibers. The silencing of affected neurons
alters auditory information processing, whether accompanied by threshold elevations or not, and is a
likely contributor to a variety of perceptual abnormalities, including speech-in-noise difficulties, tinnitus
and hyperacusis. Work described here will review structural and functional manifestations of this
cochlear synaptopathy and will consider possible mechanisms underlying its appearance and progres-
sion in ears with and without traditional ‘hearing loss’arising from several common causes in humans.
©2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents
1. Overt vs. ‘hidden’hearing loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 138
2. Cochlear synaptopathy and neurodegeneration in noise-exposed and aging mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 139
3. Glutamate excitotoxicity as an instigating factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 140
4. Functional effects of synaptopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 141
5. Cochlear neurodegeneration and SR types: special vulnerability of low-SR neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 141
5.1. Single unit evidence for low-SR vulnerability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................142
5.2. Morphology of synaptic vulnerability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..........................................142
6. Cochlear synaptopathy and relevance to human SNHL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 143
6.1. Synaptopathy in human temporal bones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................................143
6.2. Synaptopathy, low-SR neuropathy and human auditory function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........................143
6.3. Monitoring for synaptic injury and treatment efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........................144
7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 145
Acknowledgments ............................................................ . . ..................................................145
References ................................................................. .... . ..................................................145
1. Overt vs. ‘hidden’hearing loss
A longstanding view of acquired sensorineural hearing loss
(SNHL) has been that cochlear hair cells are among the most
vulnerable elements in the cochlea and that, in the vast majority of
cases, cochlear nerve fibers degenerate if, and only long after, the
loss of their peripheral hair cell targets. This view arose, funda-
mentally, because of the temporal offset between post-insult
degeneration of hair cells and loss of the spiral ganglion cell
(SGC) bodies of the primary auditory neurons with which they
*Corresponding author. Eaton-Peabody Laboratories, Massachusetts Eye and Ear
Infirmary, 243 Charles St., Boston, MA 02114-3096, USA.
E-mail address: Sharon_Kujawa@meei.harvard.edu (S.G. Kujawa).
Contents lists available at ScienceDirect
Hearing Research
journal homepage: www.elsevier.com/locate/heares
http://dx.doi.org/10.1016/j.heares.2017.01.003
0378-5955/©2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Hearing Research 349 (2017) 138e147
communicate. In animal models exposed to noise or ototoxic drugs,
hair cell loss can be widespread within hours (Bohne and Harding
2000; Lawner et al., 1997; Suzuki et al., 2008; Wang et al., 2002;
Webster and Webster, 1978), whereas the loss of SGCs is typically
not detectable for weeks to months after insult and can progress for
years (Johnsson, 1974; Miller et al., 1997; Sugawara et al., 2005;
Webster and Webster, 1978).
Threshold elevations accompany hair cell damage and loss; for
human assessments, the behavioral pure tone audiogram is a key
metric of this overt hearing loss, providing documentation of the
magnitude of the audibility loss, its pattern as a function of fre-
quency, and to some extent underlying site(s) of dysfunction (e.g.
middle ear, inner ear). It has long been known, however, that
audiometric thresholds do not always reflect reported or demon-
strated auditory perceptual difficulties and that thresholds and
otopathology are not always well aligned (Bharadwaj et al., 2015;
Felder and Schrott-Fischer, 1995; Gordon-Salant, 2005; Grose and
Mamo, 2010; Halpin et al., 1994; Moore, 2004; Lobarinas et al.,
2013; Ruggles et al., 2011; Schuknecht and Gacek, 1993).
Recent work in animal models has shed new light on this
disconnect. It is now clear, at least in the noise-exposed and aging
ear, 1) that cochlear neurons are a primary target, 2) that their
peripheral synaptic connections are the most vulnerable elements
and 3) that cochlear nerve synapses can be destroyed even when
hair cells survive. Although threshold shift is a sensitive metric of
underlying hair cell damage, it is relatively insensitive to this
diffuse loss of inner hair cell (IHC) synapses or of the cochlear nerve
fibers they drive; indeed, behavioral detection thresholds for tones
are little changed until neural loss exceeds about 80e90%
(Schuknecht and Woellner, 1955). Thus, cochlear synaptopathy can
be widespread in ears with intact hair cell populations and normal
audiograms, where it has been called “hidden”hearing loss
(Schaette and McAlpine, 2011).
This basic result has been observed in multiple mammalian
species, including compelling preliminary observations in human
temporal bones (Viana et al., 2015) and in noise-damage created by
both continuous (Rybalko et al., 2015; Singer et al., 2013; Wang and
Ren, 2012) and impulsive/blast exposures (Cho et al., 2013) and in
ears with, and without permanent threshold shifts (Kujawa et al.,
2011). Beyond noise and aging, gentamicin-treated mice (Ruan
et al., 2014) and temporal bones of humans who received amino-
glycosides in life (Hinojosa and Lerner, 1987; Sone et al., 1998) can
display diffuse cochlear neuropathy for treatments not sufficient to
cause hair cell loss. To date, findings have been most thoroughly
described in mouse models of noise and aging, as discussed in the
following sections.
2. Cochlear synaptopathy and neurodegeneration in noise-
exposed and aging mice
In recent years, results of a study aiming to investigate whether
noise can have delayed or progressive consequences in humans
(Gates et al., 2000) motivated a series of experiments in an inbred
strain of good-hearing, normally aging mice (CBA/CaJ), where
intended exposures could be rigidly specified, unintended expo-
sures avoided, and a variety of other potentially cofounding vari-
ables controlled in genetically ~ identical individuals. Mice were
exposed at various ages and were held with age-matched controls
for varying post-exposure times. Contrary to existing dogma, re-
sults demonstrated that noise can cause ongoing changes in
cochlear structure and function long after it has ceased. An unan-
ticipated finding of these initial studies was a dramatic loss of
cochlear neurons as young-exposed animals aged after a noise
exposure that produced moderate, permanent threshold shift
(PTS), but no hair cell loss (Kujawa and Liberman, 2006).
To explore this finding of noise-induced primary neuropathy
further, and to uncomplicate interpretation, the observations were
repeated for an exposure that produced only temporary threshold
shift (TTS) in fully adult animals (Kujawa and Liberman, 2009). In
this work, mice from the same inbred strain were exposed to a band
of noise placed in the region of best threshold sensitivity. The noise
was titrated in level and duration to produce a large, acute
threshold shift (30e40 dB at 24 h), but one that recovered by 2
weeks, without hair cell loss. Immunostained cochlear whole
mounts and plastic-embedded sections (Fig. 1AeD), imaged by
confocal and conventional light microscopy, were assessed to
quantify hair cells, cochlear neurons, and synaptic structures
providing the communication conduits. Hair cell-based distortion
product otoacoustic emissions (DPOAEs) and neural-based auditory
brainstem responses (ABRs) or compound action potentials (CAPs)
of the auditory nerve were used to assess the peripheral conse-
quences of the noise on function (Fig. 3A and B).
Presaging the ganglion cell losses, results of these studies
revealed an acute loss of synapses between IHCs and the peripheral
terminals of the spiral ganglion neurons that contact them (Kujawa
and Liberman, 2009). Although thresholds recovered, by design,
and no hair cells were lost, IHC synaptic losses were greater than
40% in basal cochlear regions, when assessed 24 h post noise, and
were stable 2 and 8 weeks later. Losses were proportional in
magnitude and cochlear location to the SGC loss observed in the
previous series, suggesting that this interruption of IHC-to-neural
communication set the stage for the neurodegeneration.
Subsequent studies showed that cochlear synaptopathy also
precedes hair cell loss and threshold shift in the aging mouse ear
(Sergeyenko et al., 2013). In the same normally aging inbred strain,
IHC synapse counts decline steadily throughout life, with losses
reaching ~50% in oldest ears and beginning well before significant
loss of threshold sensitivity or outer hair cells (OHCs) (compare
Fig. 1E and F). SGC losses follow, ultimately reaching about 40%
although IHC losses are only ~5% in oldest ears. SGC losses also are
closely parallel to those reported in an age-graded series of human
temporal bones with preserved hair cells (Makary et al., 2011; see
Fig. 6). Thus, the neural loss in these aging ears, as in the TTS ears, is
primary rather than a secondary consequence of the loss of their
IHC targets. Moreover, when animals received a single, TTS- and
synaptopathy-producing exposure as young adults, ongoing syn-
aptic and neural losses were larger than those that otherwise
Abbreviations
ABR Auditory Brainstem Response
AMPA
a
-Amino-3-hydroxy-5-methyl-4-
isoxazolepropionic Acid
ANF Auditory Nerve Fiber
CAP Compound Action Potential
CtBP2 C-terminal Binding Protein 2
DPOAE Distortion Product Otoacoustic Emission
GLAST Glutamate Aspartate Transporter
GluR Glutamate Receptor
IHC Inner Hair Cell
OHC Outer Hair Cell
PTS Permanent Threshold Shift
SGC Spiral Ganglion Cell
SNHL Sensorineural Hearing Loss
SPL Sound Pressure Level
SR Spontaneous Rate
TTS Temporary Threshold Shift
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147 139
occurred in aging ears (Fernandez et al., 2015).
3. Glutamate excitotoxicity as an instigating factor
The IHC - cochlear nerve synapse is the primary conduit
through which information about the acoustic environment is
transmitted to the auditory nervous system. In the normal ear, 95%
of cochlear nerve fibers make synaptic connection only with IHCs
(Spoendlin, 1972). Each cochlear nerve fiber has a cell body in the
spiral ganglion, a peripheral axon in the osseous spiral lamina
(OSL) and an unmyelinated terminal dendrite in the organ of Corti,
with a terminal bouton that forms a synapse with the IHC. The
synapse is comprised of a presynaptic ribbon surrounded by a halo
of neurotransmitter-containing vesicles within the IHC (Nouvian
et al., 2006) and a postsynaptic active zone on the cochlear
nerve terminal, with glutamate (AMPA-type) receptors for the
released neurotransmitter (Puel, 1995; Glowatzki and Fuchs,
2002). Collectively, these synapses convey information about
stimulus intensity and temporal properties over a wide dynamic
range (Moser et al., 2006). As summarized in a recent review
(Reijntjes and Pyott, 2016), the mechanisms supporting the di-
versity and breadth of afferent firing are likely resident within this
complex, determining the intrinsic excitability of the neural ele-
ments, and the modulation of this excitability by chemical
transmitters.
The time course of the initial events after exposure suggested a
role for an excitotoxic process. Work by Puel and colleagues has
shown that local application of glutamate receptor (GluR) agonists
can produce dose-dependent swelling of cochlear nerve terminals
contacting IHCs, as shown in Fig. 2. The dendritic swelling is
observed under IHCs, but not OHCs, and is prevented by prior
intracochlear perfusion of glutamate antagonists (see Ruel et al.,
2007 for review).
There is similar longstanding evidence that cochlear neurons are
directly targeted by noise, through excess sound-induced release of
the endogenous neurotransmitter. Morphological studies have
documented similar swelling of type I cochlear nerve terminals in
the region of their synaptic contact with IHCs (Spoendlin, 1971;
Robertson, 1983; Puel et al., 1998). Such terminal swelling can be
seen for exposures that produce PTS or TTS, including the exposure
producing the neuropathy described here (8e16 kHz at 100 dB SPL
for 2 h; Kujawa and Liberman, 2009). As for the glutamate agonist-
induced excitotoxicity, the ultrastructural pathology in cochlear
nerve terminals immediately after noise exposure is dramatic.
Protection against the noise-induced swelling is provided by
cochlear perfusion of the AMPA/kainate antagonist, kynurenate and
by Riluzole, which may protect by inhibiting glutamate release
(Ruel et al., 2005).
One working hypothesis (Kujawa and Liberman, 2009) is that
this excitotoxicity is a primary initial event in the degenerative
cascade observed after noise: 1) in the hours and days immediately
post exposure, some unmyelinated terminal dendrites of SGCs
degenerate back to the habenula as a direct effect of glutamate
excitotoxicity, associated dendritic swelling and possible terminal
rupture; 2) the loss of these peripheral terminals interrupts the
Fig. 1. Noise-induced and age-related loss of synapses and SGNs. Evaluating synaptopathy by triple-staining cochlear whole mounts for a pre-synaptic marker (CtBP2-red),a post-
synaptic marker (GluA2-green) and a hair cell marker (Myosin VIIa-blue). Confocal z-stacks in the IHC area from a control (A) and a noise-exposed mouse (B), 2 wks post exposure.
Light micrographs of osmium-stained plastic sections from noise-exposed ears, 2 wks (C) or 2 yrs (D) post exposure. Exposure in Band Dwas 8e16 kHz, 2 h, 100 dB SPL, delivered at
16 wk to CBA/CaJ mice. (E) In aging ears from the same inbred strain, synaptic counts at IHCs decrease steadily from 4 to 144 wks and parallel ganglion cell loss follows whereas, (F)
threshold loss begins comparatively later and accelerates beyond 80 wks, mirrored by accelerating loss of OHCs. IHC loss is trivial at any age. Red symbols flag 80 wk data points for
all measurements. After Kujawa and Liberman 2006, 2009; Sergeyenko et al. (2013).
Fig. 2. Excitotoxic swelling in the cochlea. Infusion of AMPA (200
m
M) in the cochlea
triggers massive swelling of afferent endings (*) underneath the inner hair cell (IHC).
Scale bar ¼1
m
M. From Ruel et al. (2007), with permission.
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147140
neurotrophin signaling required for normal development and
maintenance of the cochlear innervation (Fritzsch et al., 2004;
Ramekers et al., 2012; Stankovic et al., 2004) by removing the
intimate association between cochlear supporting cells (or hair
cells) and the neuronal Trk receptors for the neurotrophins; and 3)
this interruption of neurotrophin signaling compromises the long-
term viability of those neurons, essentially sealing their fate at an
early stage of the process (though the subsequent intracellular
balancing act between cell death and cell survival pathways may
take months to resolve).
A key test of the hypothesized role of neurotrophins in the
neurodegeneration that follows synaptic and terminal loss in these
ears is provided by rescue experiments demonstrating synapto-
genesis and recovery of function in a noise model (Wan et al., 2014;
Suzuki et al., 2016). Given that loss of SGCs and their central pro-
jections is very slow after such insults, and IHC targets often sur-
vive, results suggest the exciting possibility of hair cell eneuron
reconnection over a long therapeutic window in human
application.
Although it is easy to imagine excess glutamate release resulting
from prolonged, high-level acoustic stimulation, the glutamate
excitotoxicity hypothesis must be reconciled with recent studies
suggesting that IHC synaptopathy is also a primary effect of ami-
noglycoside antibiotics. As we have reported for noise exposure,
others have shown that when aminoglycoside doses are titrated to
levels below those causing hair cell loss, there can nevertheless be
significant loss of synaptic terminals on IHCs (Ruan et al., 2014) and
basal turn IHC synapses and SGCs (Oishi et al., 2015). Classic studies
of aminoglycoside ototoxicity focused on the hair cells as primary
targets and considered neural losses to be a secondary consequence
of hair cell loss (McFadden et al., 2004; Takeno et al., 1998; Bae
et al., 2008; Dodson and Mohuiddin, 200 0). However,
aminoglycoside-induced excitotoxic swelling of nerve terminals
also has been reported in both cochlear and vestibular end organs
(Basile et al., 1996; Duan et al., 2000; Sedo-Cabezon et al., 2014;
Smith, 1999), suggesting direct, excitotoxic effects of these drugs
on neural elements.
Recent studies also suggest that IHC synaptopathy may result
from impulse noise exposure (Cho et al., 2013). Again, although it is
easy to imagine high-level impulsive stimuli damaging by direct
mechanical effects, it is not obvious why a stimulus lasting only
microseconds should lead to over-release of neurotransmitter.
Clearly, more research is necessary to understand whether all these
elicitors of synaptopathy act via the same mechanism.
4. Functional effects of synaptopathy
The diffuse synaptic and neural loss observed in both noise-
exposed and aging ears does not elevate thresholds. However, if
DPOAE responses return to normal (after TTS-producing noise;
Kujawa and Liberman, 2009,Fig. 3A) or have not yet deteriorated
(in aging; Sergeyenko et al., 2013), the suprathreshold amplitude of
ABR wave 1 (Fig. 3B) can be highly predictive of the degree of
cochlear synaptopathy (Fig. 3C), as affected neurons are silenced
with the loss of their synaptic connection to the IHC. Consistent
with the innervation schema of a single auditory neuron commu-
nicating with a single IHC via a single synapse (Stamataki et al.,
2006), and the basic idea that each fiber contributes a tiny cur-
rent to ensemble far-field potentials (Antoli-Candela and Kiang,
1978; Buchwald and Huang, 1975), the fractional decrease in ABR
wave 1 amplitude scales linearly with the fractional loss of synaptic
connections in aging mice (Sergeyenko et al., 2013,Fig. 3C). And,
demonstrating the specificity as well as the sensitivity of the wave 1
assay, such permanent neural response amplitude declines are not
seen after noise exposures that fail to produce synapse loss
(Fernandez et al., 2015). The robustness of the correlation in inbred
mice, reviewed here, is likely enhanced by low inter-subject vari-
ability due to genetic homogeneity, as well as strict experimental
control of intended and untended exposures. These variables will
introduce challenges to the study of primary neurodegeneration in
the human. Moreover, this correspondence is only straightforward
if uncomplicated by hair cell damage, since disruption of mecha-
noelectric transduction also will reduce the ABR amplitudes.
5. Cochlear neurodegeneration and SR types: special
vulnerability of low-SR neurons
In all studies completed thus far, neural loss has been subtotal,
raising the possibility that cochlear insults are targeting a sub-
population of cochlear neurons. Auditory nerve fibers (ANFs) con-
tacting IHCs differ in spontaneous rates (SR) of firing (low, medium,
high), and their sound-driven firing rates vary over different ranges
to support a large dynamic range of neural response (Liberman,
1978). Threshold sensitivity of ANFs is inversely correlated with
SR; high-SR fibers have low thresholds, but saturate at levels where
high threshold, low-SR fibers continue to code level with increasing
firing rate (Winter et al.,1990). In addition to their higher pure-tone
thresholds, low-SR ANFs tend to have larger dynamic ranges
(Schalk and Sachs, 1980) and reduced susceptibility to excitatory
masking by continuous noise stimuli (Costalupes et al., 1984). Thus,
Fig. 3. Response amplitudes and synapse counts. Permanent reductions in ABR, but not DPOAE amplitudes in ears with recovered thresholds after noise. Shown are DPOAE (A)
and ABR wave 1 (B) response growth functions in the region of maximum acute TTS 1 d and 2 wk after exposure (as in Fig. 1) to 16 wk CBA/CaJ mice; unexposed controls shown for
comparison. Neural response amplitude declines are proportional to synaptic and neural losses in aging CBA/CaJ, where synapses are plotted vs mean wave 1 amplitudes (at 80 dB
SPL in 4e128 wk animals (C). Panels A,B from Fernandez et al., 2015; Panel C from Sergeyenko et al., 2013.
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147 141
although low-SR fibers are not needed for threshold detection, they
are likely important for hearing in noise and for fine temporal
precision at suprathreshold levels.
Two findings in work presented thus far suggested that the
primary neural degeneration that inevitably follows noise-induced
and age-related synapse loss might be biased toward the low-SR
subgroup, which comprises roughly 40% of the ANF population
(Liberman, 1978; Tsuji and Liberman, 1997). First, maximum
neuronal loss is roughly 40e50% for a broad range of noise ex-
posures (Kujawa et al., 2011) and in the unexposed, aged ear
before hair cell loss is significant (Sergeyenko et al., 2013). Second,
a selective loss of high-threshold fibers would provide a natural
explanation for the full recovery of thresholds in ears with
persistent suprathreshold neural amplitude declines after TTS.
Subsequent studies have probed these relationships, as described
below.
5.1. Single unit evidence for low-SR vulnerability
Neurophysiological studies suggest that neurons from the
different SR classes are not equally represented in the noise-
induced neuropathy (Furman et al., 2013). In these studies, re-
cordings were obtained from single ANFs in guinea pigs after a
noise exposure known to produce temporary threshold shifts with
acute loss of synapses, as in the mouse model (Kujawa and
Liberman, 2009). The proportion of fibers with low SR was signif-
icantly smaller in exposed than in control ears, particularly in
cochlear frequency regions relevant to the exposure (Fig. 4A). Sur-
viving high-SR fibers showed normal response properties,
including normal thresholds and tuning (Fig. 4B), supporting the
notion that OHCs were functionally normal and that low-SR neu-
rons with high thresholds were selectively eliminated. Studies in
gerbil provide two additional observations of the particular
vulnerability of low SR neurons; to aging (Schmiedt et al., 1996) and
to ouabain-induced neuropathy (Bourien et al., 2014). In the latter,
the dose-response relation revealed first effects on low-SR neurons
followed by medium- and then high-SR with increasing drug dose.
The apparent vulnerability of low-SR neurons remains unex-
plained. Low- and high-SR neurons and their synapses distribute
differently at IHCs; we speculate that different distributions of
glutamate receptor subtypes may contribute to differences in the
excitotoxic response to noise. Additionally, low-SR fibers are poor
in mitochondria, which are important in buffering intracellular
Ca2þ; this characteristic might also increase their vulnerability to
damage.
5.2. Morphology of synaptic vulnerability
Morphologic support for the preferential loss of low-SR neurons
comes from studies in which 1) SR-related spatial distributions of
ANFs at IHCs (Liberman, 1980, 1982), 2) presynaptic ribbons and
postsynaptic glutamate receptor patches (Yin et al., 2014) and 3)
post-noise reorganization of synaptic locations (Liberman et al.,
2015) all suggest preferential vulnerability of low-SR neurons and
their synapses after noise. Low- and high-SR fibers differ in syn-
aptic position on the IHC and in the size of synaptic ribbons and
associated AMPA-receptor patches (Liberman et al., 2011; Merchan-
Perez and Liberman, 1996); low-threshold, high-SR fibers tend to
synapse on the pillar side of the IHC, whereas the high-threshold,
low-SR fibers tend to synapse on the modiolar side (Liberman,
1982). This physiological gradient also appears in confocal images
from immunostained cochlear whole mounts as complementary
gradients in ribbon and GluR-patch size on the pillar vs. modiolar
sides of the IHC; large ribbons and small receptor patches tend to be
localized to the IHC's modiolar side compared to small ribbons and
large receptor patches on the pillar side (Yin et al., 2014). These
gradients appear to be part of the morphological substrate for the
low-SR/high-SR gradient in cochlear nerve response (Liberman,
1978).
In normal ears, the density of synapses tends to be greater on
the modiolar side of the IHC (Fig. 5A). After noise, loss of syn-
apses also appears greater on the modiolar side (Liberman et al.,
2015), consistent with physiological reports of selective loss of
low-SR fibers in this noise damage model (Furman et al., 2013).
However, synaptic positions along the IHC's basolateral mem-
brane appear to transiently redistribute along the habenular-
cuticular and modiolar-pillar axes after noise, particularly
within the region of greatest noise-induced synaptopathy,
recovering by 1 wk post exposure. Thus, interpreting synaptic
position after noise is complicated by dynamic changes that
occur in the acute post-exposure time frame. Spatial segregation
of high- and low-SR fibers in the OSL as shown in Fig. 5BandC
may be useful in assessing which fiber type has degenerated after
cochlear insult.
Other dynamic, post-noise changes to synaptic structure have
been observed. In the normal cochlea, confocal images document
Fig. 4. Low-SR neuron loss after noise. Single unit recordings were made in guinea pigs 10 days after a TTS-producing noise exposure that resulted in permanent ABR amplitude
declines and synapse loss but no hair cell loss. Spontaneous rate distributions suggest selective loss of low-SR fibers in the high-frequency region of maximum noise-induced injury
(A). In the same animals, thresholds and tuning of surviving nerve fibers, matched for CF, were not altered in noise exposed ears compared to controls (B). The single-fiber database
included 367 fibers from 14 control animals, and 382 fibers from 9 exposed animals. After Furman et al., 2013.
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147142
a one-to-one association between pre-synaptic ribbons and post-
synaptic glutamate receptor patches (Kujawa and Liberman,
2009), consistent with ultrastructural analyses (Liberman, 1980;
Stamataki et al., 20 06). After noise, there is a transient increase
in the number of ‘orphan’ribbons, restricted to basal cochlear
regions within the noise-damage focus (Fernandez et al., 2015;
Liberman et al., 2015). This change in the number of GluA2
puncta could reflect a transient internalization of surface gluta-
mate receptors, as documented previously in response to gluta-
mate agonists in vitro or noise in vivo (Chen et al., 2007). This
reversible down regulation of surface AMPA receptors may serve
a protective function (Chen et al., 2007, 2009) by modulating
synaptic strength.
Despite progress in describing morphological differences be-
tween low- and high-SR fibers and their contacts with the IHCs,
mechanisms underlying the apparent vulnerability of low-SR
neurons remain poorly understood. Neurotransmitter released
from the IHC must be maintained at levels low enough to ensure
high signal-to-noise ratio and to prevent excitotoxic damage to
afferent neurons. Rapid clearance of synaptic glutamate is
accomplished by the uptake system of glutamate transporters
(Bridges and Esslinger, 2005; Danbolt, 2001; Hakuba et al., 2000;
Seal and Amara, 1999) and immunostaining for glutamate
transporters is less intense on the low-SR side of the IHC (Furness
and Lawton, 2003). Low-SR fibers also have fewer mitochondria
which, in the central nervous system, are well documented to be
of fundamental importance to Ca
þþ
buffering mechanisms and
thus to the control of excitotoxicity (Szydlowska and Tymianski,
2010).
6. Cochlear synaptopathy and relevance to human SNHL
6.1. Synaptopathy in human temporal bones
Against this backdrop of animal studies, our working hypothesis
is that partial de-afferentation of IHCs is widespread in human ears
across a range of acquired SNHL etiologies, with or without overt
hearing loss. Using immunostaining for pre- and post-synaptic el-
ements as performed in the animal models, temporal bones from
individuals 55e89 years of age with no explicit otopathology
revealed dramatic cochlear synaptopathy, with afferent innervation
density ranging from 15 synapses per IHC in a 55 yr old to only 2.5
synapses per IHC in an 89 yr old, despite no significant loss of IHCs
or OHCs (Fig. 6B). As in normal-aging mice (Sergeyenko et al., 2013),
SGC counts decrease throughout the lifespan and throughout the
cochlea (Viana et al., 2015;Fig. 4B. In mice, the SGC counts un-
derestimate the degree of IHC de-afferentation, because the SGCs
survive for months after the loss of their peripheral synapses with
IHCs. Similarly, observations in human temporal bones suggest that
the loss of IHC synapses in normal-aging humans also can be
significantly greater than the loss of SGCs (Fig. 6A). These data
suggest that cochlear synaptopathy may be a major cause of func-
tional impairment in age-related hearing loss in humans.
6.2. Synaptopathy, low-SR neuropathy and human auditory
function
In summary, synapses are lost first as noise dose increases, and
synapses are lost first as age progresses. This may be a general
Fig. 5. Gradients in synaptic and afferent fiber morphology. IHC synapses in confocal z-stacks, acquired in the x-y plane (A) and re-projected into the y-z (B) plane. APre- and
post-synaptic elements in the IHC area are counted in cochlear whole mounts quadruple-immunostained for CtBP2 (red), GluA2 (green), NaK ATPase (blue), and myosin VIIa
(white). BSize gradients in pre- and post-synaptic elements are quantified according to location along habenul ar-cuticular and modiolar-pillar axes (Liberman et al., 2015). (C)
Tracing of peripheral axons from a cross section through the osseous spiral lamina (OSL; D) in a normal cat shows the SR-based gradient from thin (low-SR) to thick (high-SR) fibers
(Kawase and Liberman, 1992).
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147 143
finding in other forms of acquired SNHL common in humans, as
well. Affected neurons are silenced by the loss of this synaptic
connection, even if it takes months to years for the loss to be re-
flected in SGC loss. Audiometric thresholds are unaffected by
diffuse synaptopathy; however, such dramatic disconnection of
hair cells and ANFs must have significant perceptual consequences.
Normal response properties of low-SR neurons, in quiet and in
noise, have led to speculation regarding functional consequences of
their targeted loss. Low-SR neuropathy may be a major contributor
to a classic impairment in SNHL, speech-in-noise difficulty (see
Kujawa and Liberman, 2015; Plack et al., 2014 for discussion). This
notion is not new; low-SR neuropathy has been suggested to
contribute to well-documented performance declines with age that
include decreased speech understanding in noise and reduced
ability to utilize stimulus timing and amplitude modulation cues
(Schmiedt et al., 1996). It also may be important in limiting psy-
chophysical performance in “normal hearing”human listeners;
that is, those with good threshold sensitivity, and it may help ac-
count for performance differences in individuals with similar,
elevated audiometric thresholds. In support, deficits in binaural
temporal processing, seen as a decrease in the detectability of
interaural phase differences in amplitude modulated tones, are
highly correlated with changes in ABR responses consistent with
the selective loss of low-SR fibers (Bharadwaj et al., 2014, 2015).
Cochlear synaptopathy also may be a key elicitor of what are
commonly the most troubling sensory anomalies associated with
SNHL, tinnitus and hyperacusis. This may be the result of a
compensatory plasticity, wherein the synaptic gain in auditory
central circuits is increased when neural signals from the periphery
are attenuated (Bauer et al., 2007; Gu et al., 2010; Hickox and
Liberman, 2014; Kaltenbach and Afman, 2000; Knipper et al.,
2013; Roberts et al., 2010; Schaette and McAlpine, 2011). Results
support the long-standing hypothesis that reduced afferent
outflow from a damaged cochlea and the associated diminished
input to higher auditory centers drives increases in central gain that
may, in turn, underlie tinnitus.
Work in this area is in its infancy, and ultimately will be crucial
to the translation of these findings to humans, where the histopa-
thology will not be available in life. The TTS animal model of pri-
mary neuropathy has provided a powerful platform to characterize
synaptopathic/neurodegenerative consequences of noise exposure
and to begin to test hypotheses about the special role(s) of low-SR
fibers in auditory processing without the confounding variables of
hair cell damage and threshold shift. The recording of thresholds
and suprathreshold amplitudes of OHC-based DPOAEs and neural-
based ABRs in the same ears provides a valuable window into the
underlying histopathology in ears with normal thresholds; ABR
wave 1 amplitudes recorded in such ears scale closely with the
underlying synaptopathy. However, acquired SNHL in humans will
encompass a range of threshold losses and underlying damage that
may include mixed loss of hair cells and synaptic contacts on sur-
viving IHCs. Metrics robust to such mixed involvements and
accompanying threshold elevations will be required. Experiments
underway have undertaken assessment of synaptic and functional
losses for a range of TTS- and PTS-producing exposures, with and
without hair cell loss.
6.3. Monitoring for synaptic injury and treatment efficacy
Pure tone threshold audiometry serves as the standard metric
for assaying the effects of noise, ototoxic drugs and other agents on
hearing in clinical and occupational settings. Threshold measure-
ment protocols have undergone extensive vetting and standardi-
zation. Such measurements also form the basis for population
sensitivity norms to which individual sensitivity is compared, and
threshold-based estimates of noise risk have informed recom-
mended and enforced occupational exposure standards (e.g.
NIOSH, 1998;OSHA, 1983).
Against this backdrop, the standard of care in clinical and
occupational monitoring for hearing damage in noise- and ototoxic
drug-induced injury is assessment of exposure-related changes in
threshold sensitivity (OSHA, 1983; ASHA, 1994; AAA, 20 09;ACOEM
et al., 2012). Such a strategy, particularly if it includes extended
high-frequency threshold and OAE monitoring, should be valuable
as an early warning of hair cell injury and loss as well as impending
performance declines due to reduced audibility. If synapse loss in
humans precedes threshold elevation and OHC loss after noise or
ototoxic drugs, as it does in all animal models evaluated thus far,
clinical decision making and occupational health monitoring pro-
tocols would require revision to identify earliest injury, with the
goal of preserving hearing function. Similarly, should treatments
aimed at preserving, protecting or regenerating cochlear synaptic
connections materialize, assays of function with sensitivity to the
functional integrity of the synaptic machinery will be required.
Fig. 6. Cochlear de-afferentation in human temporal bones. Cochlear de-afferentation is seen in human temporal bones as a function of age (A) and cochlear location (B) in cases
with no hair cell loss and no explicit otologic disease. The small pink symbols (A) are estimated total SGC counts from archival sections (Makary et al., 2011); the five large symbols
(A) are the estimated total peripheral axon counts from the same five cases shown in B. Counts of cochlear nerve terminals per IHC (B) in 5 normal aging temporal bones
(55e89 yrs) with no history of otologic disease (Viana et al., 2015). Blue symbols are counts of synapses per IHC from an electron microscopic study of a normal middle-aged human
(Nadol, 1988).
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147144
Preliminary studies in humans have suggested several non-invasive
assays that may provide important clues to underlying synaptop-
athy (Liberman et al., 2016; Mehraei et al., 2016), as has been shown
directly in the animal models of noise and aging reviewed here.
7. Summary
New insights from animal studies of noise-induced and age-
related hearing loss suggest that the most vulnerable elements in
the inner ear are the synaptic connections between hair cells and
sensory neurons. Subtotal cochlear synaptopathy, and the primary
neural degeneration that follows, does not elevate thresholds. Thus,
it can be widespread in ears with intact hair cell populations and
normal audiograms. It also occurs in ears with sensory cell injury
and loss, resulting in a mixed sensory-neural pathology. We hy-
pothesize that de-afferentation of surviving IHCs may be a major
contributor to auditory dysfunction in numerous etiologies of ac-
quired SNHL. Thus, the result has profound human health ramifi-
cations. These discoveries are recent, and much remains to be
clarified. In our laboratories, the synaptopathy has been largely
permanent, indeed progressive, in multiple species. There are re-
ports, however, that spontaneous re-innervation can be seen (Puel
et al., 1995; Pujol and Puel, 1999; Sun et al., 2001), or that some of
the immediate synapse loss may be reversible (Liu et al., 2012; Shi
et al., 2013, 2015; 2016; Song et al., 2016). The source(s) of these
discrepancies remain to be identified. Work is ongoing to study the
phenomenon, to probe its mechanisms, and to assess the efficacy of
a possible therapeutic intervention, using cochlear insults that are
highly relevant to the human condition.
Acknowledgments
Research reported here was supported by grants from the DOD
(W81XWH-15-1-0103) and the NIH (R01 DC0188, R01 DC08577
and P30 DC 05209).
References
ACOEM Task Force on Occupational Hearing Loss, Kirchner, D.B., Evenson, E.,
Dobie, R.A., Rabinowitz, P., Crawford, J., Kopke, R., Hudson, T.W., 2012. Occu-
pational noise-induced hearing loss: ACOEM Task Force on Occupational
Hearing Loss. J. Occup. Environ. Med. 54, 106e108 .
American Academy of Audiology Position Statement and Clinical Practice Guide-
lines: Ototoxicity Monitoring, October 2009. Available at: http://www.
audiology.org/publications-resources/document-library/ototoxicity-
monitoring.
American Speech-Language-Hearing Association, 1994. Audiologic Management of
Individuals Receiving Cochleotoxic Drug Therapy [Guidelines]. Available at:
www.asha.org/policy.
Antoli-Candela Jr., F., Kiang, N.Y.S., 1978. Unit activity underlying the N1 potential.
In: Naunton, R.F., Fernandez, C. (Eds.), Evoked Electrical Activity in the Auditory
Nervous System. Academic Press, New York, pp. 165e191.
Bae, W.Y., Kim, L.S., Hur, D.Y., Jeong, S.W., Kim, J.R., 2008. Secondary apoptosis of
spiral ganglion cells induced by aminoglycoside: Fas-Fas ligand signaling
pathway. Laryngoscope 118 (9), 1659e1668.
Basile, A.S., Huang, J.M., Xie, C., Webster, D., Berlin, C., Skolnick, P., 1996. N-methyl-
D-aspartate antagonists limit aminoglycoside antibiotic-induced hearing loss.
Nat. Med. 2 (12), 1338e1343.
Bauer, C.A., Brozoski, T.J., Myers, K., 2007. Primary afferent dendrite degeneration as
a cause of tinnitus. J. Neurosci. Res. 85 (7), 1489e149 8.
Bharadwaj, H.M., Verhulst, S., Shaheen, L., Liberman, M.C., Shinn-Cunningham, B.G.,
2014. Cochlear neuropathy and the coding of supra-threshold sound. Front.
Syst. Neurosci. 8, 26. http://dx.doi.org/10.3389/fnsys.2014.00026. Feb 21.
Bharadwaj, H.M., Masud, S., Meharie, G., Verhulst, S., Shinn-Cunningham, B.G., 2015.
Individual differences reveal correlates of hidden hearing deficits. J. Neurosci.
35 (5), 2161e2172.
Bohne, B.A., Harding, G.W., 2000. Degeneration in the cochlea af ter noise damage:
primary versus secondary events. Am. J. Otol. 21 (4), 505e509.
Bourien, J., Tang, Y., Batrel, C., Huet, A., Lenoir, M., Ladrech, S., Desmadryl, G.,
Nouvian, R., Puel, J.L., Wang, J., 2014 Sep 1. Contribution of auditory nerve fibers
to compound action potential of the auditory nerve. J. Neurophysiol. 112 (5),
1025e1039. http://dx.doi.org/10.1152/jn.00738.2013.
Bridges, R.J., Esslinger, C.S., 2005. The excitatory amino acid transporters:
pharmacological insights on substrate and inhibitor specificity of the EAAT
subtypes. Pharmacol. Ther. 107 (3), 271e285 (Review).
Buchwald, J.S., Huang, C., 1975. Far-field acoustic response: origins in the cat. Sci-
ence 189, 382e384.
Chen, Z., Kujawa, S.G., Sewell, W.F., 2007. Auditory sensitivity regulation via rapid
changes in expression of surface AMPA receptors. Nat. Neurosci. 10 (10),
1238e1240.
Chen, Z., Peppi, M., Kujawa, S.G., Sewell, W.F., 2009. Regulated expression of surface
AMPA receptors reduces excitotoxicity in auditory neurons. J. Neurophysiol.
102, 1152e115 9 .
Cho, S.I., Gao, S.S., Xia, A., Wang, R., Salles, F.T., Raphael, P.D., Abaya, H., Wachtel, J.,
Baek, J., Jacobs, D., Rasband, M.N., Oghalai, J.S., 2013. Mechanisms of hearing
loss after blast injury to the ear. PLoS One 8 (7), e67618.
Costalupes, J.A., Young, E.D., Gibson, D.J., 1984. Effects of continuous noise back-
grounds on rate response of auditory nerve fibers in cat. J. Neurophysiol. 51,
1326e1344.
Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65 (1), 1e105 (Review).
Dodson, H.C., Mohuiddin, A., 2000. Response of spiral ganglion neurones to
cochlear hair cell destruction in the Guinea pig. N. J. Neurocytol. 29 (7),
525e537.
Duan, M., Agerman, K., Ernfors, P., Canlon, B., 2000. Complementary roles of neu-
rotrophin 3 and N-methyl-D-aspartate antagonist in the protection of noise and
aminoglycoside-induced ototoxicity. Proc. Natl. Acad. Sci. U. S. A. 97 (13),
7597e7602.
Felder, E., Schrott-Fischer, A., 1995. Quantitative evaluation of myelinated nerve
fibres and hair cells in cochleae of humans with age-related high-tone hearing
loss. Hear Res. 91 (1e2), 19e32.
Fernandez, K.A., Jeffers, P.W., Lall, K., Liberman, M.C., Kujawa, S.G., 2015. Aging after
noise exposure: acceleration of cochlear synaptopathy in “recovered”ears.
J. Neurosci. 35 (19), 7509e7520.
Fritzsch, B., Tessarollo, L., Coppola, E., Reichardt, L.F., 2004. Neurotrophins in the ear:
their roles in sensory neuron survival and fiber guidance. Prog. Brain Res. 146,
265e278.
Furman, A.C., Kujawa, S.G., Liberman, M.C., 2013. Noise-induced cochlear neurop-
athy is selective for fibers with low spontaneous rates. J. Neurophysiol. 110 (3),
577e586.
Furness, D.N., Lawton, D.M., 2003. Comparative distribution of glutamate trans-
porters and receptors in relation to afferent innervation density in the
mammalian cochlea. J. Neurosci. 23, 11296e11304.
Gates, G.A., Schmid, P., Kujawa, S.G., Nam, B., D'Agostino, R., 2000. Longitudinal
threshold changes in older men with audiometric notches. Hear Res. 141 (1e2),
220e228.
Glowatzki, E., Fuchs, P.A., 2002. Transmitter release at the hair cell ribbon synapse.
Nat. Neurosci. 5 (2), 147e154.
Gordon-Salant, S., 2005. Hearing loss and aging: new research findings and clinical
implications. J. Rehabil. Res. Dev. 42, 9e24.
Grose, J.H., Mamo, S.K., 2010. Processing of temporal fine structure as a function of
age. Ear Hear 31, 755e760.
Gu, J.W., Halpin, C.F., Nam, E.C., Levine, R.A., Melcher, J.R., 2010. Tinnitus, diminished
sound-level tolerance, and elevated auditory activity in humans with clinically
normal hearing sensitivity. J. Neurophysiol. 104 (6), 3361e3370.
Hakuba, N., Koga, K., Gyo, K., Usami, S.I., Tanaka, K., 2000. Exacerbation of noise-
induced hearing loss in mice lacking the glutamate transporter GLAST.
J. Neurosci. 20 (23), 8750e8753.
Halpin, C., Thornton, A., Hasso, M., 1994. Low-frequency sensorineural loss: clinical
evaluation and implications for hearing aid fitting. Ear Hear. 15, 71e81.
Hickox, A.E., Liberman, M.C., 2014. Is noise-induced cochlear neuropathy key to the
generation of hyperacusis or tinnitus? J. Neurophysiol. 111 (3), 552e564.
Hinojosa, R., Lerner, S.A., 1987. Cochlear neural degeneration without hair cell loss
in two patients with aminoglycoside ototoxicity. J. Infect. Dis. 156 (3), 449e455.
Johnsson, L.G., 1974. Sequence of degeneration of Corti's organ and its first-order
neurons. Ann. Otol. Rhinol. Laryngol. 83 (3), 294e303.
Kaltenbach, J.A., Afman, C.E., 2000. Hyperactivity in the dorsal cochlear nucleus
after intense sound exposure and its resemblance to tone-evoked activity: a
physiological model for tinnitus. Hear. Res. 140 (1e2), 165e172.
Kawase, T., Liberman, M.C., 1992. Spatial organization of the auditory nerve ac-
cording to spontaneous discharge rate. J. Comp. Neurol. 319 (2), 312e318.
Knipper, M., Van Dijk, P., Nunes, I., Rüttiger, L., Zimmermann, U., 2013. Advances in
the neurobiology of hearing disorders: recent developments regarding the basis
of tinnitus and hyperacusis. Prog. Neurobiol. 111, 17e33.
Kujawa, S.G., Liberman, M.C., 2006. Acceleration of age-related hearing loss by early
noise exposure: evidence of a misspent youth. J. Neurosci. 26 (7), 2115e2123.
Kujawa, S.G., Liberman, M.C., 2009. Adding insult to injury: cochlear nerve
degeneration after “temporary”noise-induced hearing loss. J. Neurosci. 29 (45),
14077e14085.
Kujawa, S.G., Liberman, M.C., 2015. Synaptopathy in the noise-exposed and aging
cochlea: primary neural degeneration in acquired sensorineural hearing loss.
Hear Res. 330 (Pt B), 191e199.
Kujawa, S.G., Micucci, S., Liberman, M.C., 2011. Noise-induced primary neural
degeneration: effects of spectrum, duration, intensity and survival. In:
Midwinter Meeting of the Association for Research in Otolaryngology, p. 56.
Lawner, B.E., Harding, G.W., Bohne, B.A., 1997. Time course of nerve-fiber regener-
ation in the noise-damaged mammalian cochlea. Int. J. Dev. Neurosci. 15 (4e5),
601e617.
Liberman, M.C., 1978. Auditory-nerve response from cats raised in a low-noise
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147 145
chamber. J. Acoust. Soc. Am. 63 (2), 442e455.
Liberman, M.C., 1980. Morphological differences among radial afferent fibers in the
cat cochlea: an electron microscopic study of serial sections. Hear Res. 3 (1),
45e63.
Liberman, M.C., 1982. Single-neuron labeling in the cat auditory nerve. Science 216
(4551), 1239e1241.
Liberman, L.D., Wang, H., Liberman, M.C., 2011. Opposing gradients of ribbon size
and AMPA receptor expression underlie sensitivity differences among cochlear-
nerve/hair cell synapses. J. Neurosci. 31 (3), 801e808.
Liberman, L.D., Suzuki, J., Liberman, M.C., 2015. Dynamics of cochlear synaptopathy
after acoustic overexposure. J. Assoc. Res. Otolaryngol. 16 (2), 205e219. Erratum
in: J Assoc Res Otolaryngol. 2015 16(2): 221. Suzuki, Jun [added].
Liberman, M.C., Epstein, M.J., Cleveland, S.S., Wang, H., Maison, S.F., 2016. Toward a
differential diagnosis of hidden hearing loss in humans. PLoS One 11 (9),
e0162726. http://dx.doi.org/10.1371/journal.pone.0162726. Sept 12.
Liu, L., Wang, H., Shi, L., Almuklass, A., He, T., Aiken, S., et al., 2012. Silent damage of
noise on cochlear afferent innervation in Guinea pigs and the impact on tem-
poral processing. PLoS One 7, e49550. http://dx.doi.org/10.1371/
journal.pone.0049550.
Lobarinas, E., Salvi, R., Ding, D., 2013. Insensitivity of the audiogram to carboplatin
induced inner hair cell loss in chinchillas. Hear Res. 302, 113e120.
Makary, C.A., Shin, J., Kujawa, S.G., Liberman, M.C., Merchant, S.N., 2011. Age-related
primary cochlear neuronal degeneration in human temporal bones. J. Assoc.
Res. Otolaryngol. 12 (6), 711e717.
McFadden, S.L., Ding, D., Jiang, H., Salvi, R.J., 2004. Time course of efferent fiber and
spiral ganglion cell degeneration following complete hair cell loss in the
chinchilla. Brain Res. 997 (1), 40e51.
Mehraei, G., Hickox, A.E., Bharadwaj, H.M., Goldberg, H., Verhulst,S., Liberman, M.C.,
Shinn-Cunningham, B.G., 2016. Auditory brainstem response latency in noise as
a marker of cochlear synaptopathy. J. Neurosci. 36 (13), 3755e3764.
Merchan-Perez, A., Liberman, M.C., 1996. Ultrastructural differences among afferent
synapses on cochlear hair cells: correlations with spontaneous discharge rate.
J. Comp. Neurol. 371 (2), 208e221.
Miller, J.M., Chi, D.H., O'Keeffe, L.J., Kruszka, P., Raphael, Y., Altschuler, R.A., 1997.
Neurotrophins can enhance spiral ganglion cell survival after inner hair cell
loss. Int. J. Dev. Neurosci. 15 (4e5), 631e643.
Moore, B.C.J., 2004. Dead regions in the cochlea: conceptual foundations, diagnosis,
and clinical applications. Ear Hear 25 (2), 98e116 .
Moser, T., Neef, A., Khimich, D., 2006. Mechanisms underlying the temporal preci-
sion of sound encoding at the inner hair cell ribbon synapse. J. Physiol. 576.1,
55e62.
Nadol Jr., J.B., 1988. Application of electron microscopy to human otopathology.
Ultrastructural findings in neural presbycusis, Meniere's disease and Usher's
syndrome. Acta Otolaryngol. 105 (5e6), 411e419.
National Institute for Occupational Safety and Health, 1998. Criteria for a Recom-
mended Standard: Occupational Noise Exposure, revised criteria 1998. U.S.
Department of Health and Human Services, Centers for Disease Control and
Prevention, National Institute for Occupational Safety and Health Publication
No. 98e126. NIOSH, Cincinnati.
Nouvian, R., Beutner, D., Parsons, T.D., Moser, T., 2006. Structure and function of the
hair cell ribbon synapse. J. Membr. Biol. 209, 153e165.
Oishi, N., Duscha, S., Boukari, H., Meyer, M., Xie, J., Wei, G., Schrepfer, T.,
Roschitzki, B., Boettger, E.C., Schacht, J., 2015. XBP1 mitigates aminoglycoside-
induced endoplasmic reticulum stress and neuronal cell death. Cell Death
Dis. 6, e1763. http://dx.doi.org/10.1038/cddis.2015.108.
OSHA, 1983. Occupational Noise Exposure; Hearing Conservation Amendment, vol.
46, pp. 9738e9785. Final Rule. 29 C.F.R. 1910.95. Federal Register 48.
Plack, C.J., Barker, D., Prendergast, G., 2014. Perceptual consequences of “hidden”
hearing loss. Trends hear. Sep. 9, 18. http://dx.doi.org/10.1177/
2331216514550621 pii: 2331216514550621 (Review).
Puel, J.L., 1995. Chemical synaptic transmission in the cochlea. Prog. Neurobiol. 47,
449e476.
Puel, J.-L., Safieddine, S., D'Aldin, C., Eybalin, M., Pujol, R., 1995. Synaptic regener-
ation and functional recovery after excitotoxic injury in the Guinea pig cochlea.
C. R. Acad. Sci. Paris Ser. III 318, 67e75.
Puel, J.-L., Ruel, J., d'Aldin, C.G., Pujol, R., 1998. Excitotoxicity and repair of cochlear
synapses after noise-trauma induced hearing loss. NeuroReport 9, 2109e2114.
Pujol, R., Puel, J.-L., 1999. Excitotoxicity, synaptic repair, and functional recovery in
the mammalian cochlea: a review of recent findings. Ann. N. Y. Acad. Sci. 884,
249e254.
Ramekers, D., Versnel, H., Grolman, W., Klis, S.F., 2012. Neurotrophins and their role
in the cochlea. Hear Res. 288, 19e33.
Reijntjes, D.O.J., Pyott, S.J., 2016. The afferent signaling complex: regulation of type I
spiral ganglion neuron responses in the auditory periphery. Hear Res. 336,
1e16.
Roberts, L.E., Eggermont, J.J., Caspary, D.M., Shore, S.E., Melcher, J.R., Kaltenbach, J.A.,
2010. Ringing ears: the neuroscience of tinnitus. J. Neurosci. 30 (45),
149 72e14979.
Robertson, D., 1983. Functional significance of dendritic swelling after loud sounds
in the Guinea pig cochlea. Hear Res. 9, 263e278.
Ruan, Q., Ao, H., He, J., Chen, Z., Yu, Z., Zhang, R., Wang, J., Yin, S., 2014. Topographic
and quantitative evaluation of gentamicin-induced damage to peripheral
innervation of mouse cochleae. Neurotoxicology 40, 86e96.
Ruel, J., Wang, J., Pujol, R., Hameg, A., Dib, M., Puel, J.L., 2005. Neuroprotective effect
of riluzole in acute noise-induced hearing loss. Neuroreport 16 (10),1087e1090.
Ruel, J., Wang, J., Rebillard, G., Eybalin, M., Lloyd, R., Pujol, R., Puel, J.L., 2007.
Physiology, pharmacology and plasticity at the inner hair cell synaptic complex.
Hear Res. 227 (1e2), 19e27.
Ruggles, D., Bharadwaj, H., Shinn-Cunningham, B.G., 2011. Normal hearing is not
enough to guarantee robust encoding of suprathreshold features important in
everyday communication. Proc. Natl. Acad. Sci. U. S. A. 108 (37), 15516e
15521.
Rybalko, N., Chumak, T., Bure
s, Z., Popel
a
r, J.,
Suta, D., Syka, J., 2015. Development of
the acoustic startle response in rats and its change after early acoustic trauma.
Behav. Brain Res. 286, 212e221.
Schaette, R., McAlpine, D., 2011. Tinnitus with a normal audiogram: physiological
evidence for hidden hearing loss and computational model. J. Neurosci. 31 (38),
13452e13457.
Schalk, T.B., Sachs, M.B., 1980. Nonlinearities in auditory-nerve fiber responses to
bandlimited noise. J. Acoust. Soc. Am. 67 (3), 903e913.
Schmiedt, R.A., Mills, J.H., Boettcher, F.A., 1996. Age-related loss of activity of
auditory-nerve fibers. J. Neurophysiol. 76 (4), 2799e2803.
Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Ann. Otol.
Rhinol. Laryngol. 102, 1e16.
Schuknecht, H.F., Woellner, R.C., 1955. An experimental and clinical study of deaf-
ness from lesions of the cochlear nerve. J. Laryngol. Otol. 69 (2), 75e97.
Seal, R.P., Amara, S.G., 1999. Excitatory amino acid transporters: a family in flux.
Ann. Rev. Pharmacol. Toxicol. 39, 431e456.
Sedo-Cabezon, L., Boadas-Vaello, P., Soler-Martin, C., Llorens, J., 2014. Vestibular
damage in chronic ototoxicity: a mini-review. NeuroToxicology 43, 21e27.
Sergeyenko, Y., Lall, K., Liberman, M.C., Kujawa, S.G., 2013. Age-related cochlear
synaptopathy: an early-onset contributor to auditory functional decline.
J. Neurosci. 33 (34), 13686e13694.
Shi, L., Liu, L., He, T., Guo, X., Yu, Z., Yin, S., Wang, J., 2013. Ribbon synapse plasticity
in the cochleae of Guinea pigs after noise-induced silent damage. PLoS One 8,
e81566. http://dx.doi.org/10.1371/journal.pone.0081566.
Shi, L., Liu, K., Wang, H., Zhang, Y., Hong, Z., Wang, M., Wang, X., Jiang, X., Yang, S.,
2015. Noise induced reversible changes of cochlear ribbon synapses contribute
to temporary hearing loss in mice. Acta Otolaryngol. 135, 1093e1102 .
Shi, L., Chang, Y., Li, X., Aiken, S.J., Liu, L., Wang, J., 2016. Coding deficits in noise-
induced hidden hearing loss may stem from incomplete repair of ribbon syn-
apses in the cochlea. Front. Neurosci. 25. http://dx.doi.org/10.3389/
fnins.2016.00231.
Singer, W., Zuccotti, A., Jaumann, M., Lee, S.C., Panford-Walsh, R., Xiong, H.,
Zimmermann, U., Franz, C., Geisler, H.S., K€
opschall, I., Rohbock, K., Varakina, K.,
Verpoorten, S., Reinbothe, T., Schimmang, T., Rüttiger, L., Knipper, M., 2013.
Noise-induced inner hair cell ribbon loss disturbs central arc mobilization: a
novel molecular paradigm for understanding tinnitus. Mol. Neurobiol. 47 (1),
261e279.
Smith, P.F., 1999. Are vestibular hair cells excited to death by aminoglycoside an-
tibiotics? J. Vestib. Res. 10, 1e5.
Sone, M., Schachern, P.A., Paparella, M.M., 1998. Loss of spiral ganglion cells as a
primary manifestation of aminoglycoside ototoxicity. Hear Res. 115, 217e223.
Song, Q., Shen, P., Li, X., Shi, L., Liu, L., Wang, J., et al., 2016. Coding deficits in hidden
hearing loss induced by noise: the nature and impacts. Front. Neurosci. 10, 231.
http://dx.doi.org/10.3389/fnins.2016.00231.
Spoendlin, H., 1971. Primary structural changes in the organ of Corti after acoustic
overstimulation. Acta Otolaryngol. 71, 166e176 .
Spoendlin, H., 1972. Innervation densities of the cochlea. Acta Otolaryngol. 73,
235e248.
Stamataki, S., Francis, H.W., Lehar, M., May, B.J., Ryugo, D.K., 2006. Synaptic alter-
ations at inner hair cells precede spiral ganglion cell loss in aging C57BL/6J
mice. Hear Res. 221, 104e118 .
Stankovic, K., Rio, C., Xia, A., Sugawara, M., Adams, J.C., Liberman, M.C., Corfas, G.,
2004. Survival of adult spiral ganglion neurons requires erbB receptor signaling
in the inner ear. J. Neurosci. 24, 8651e8661.
Sugawara, M., Corfas, G., Liberman, M.C., 2005. Influence of supporting cells on
neuronal degeneration after hair cell loss. J. Assoc. Res. Otolaryngol. 6 (2),
136e147.
Sun, H., Hashino, E., Ding, D.L., Salvi, R.J., 2001. Reversible and irreversible damage
to cochlear afferent neurons by kainic acid excitotoxicity. J. Comp. Neurol. 430
(2), 172e181.
Suzuki, M., Ushio, M., Yamasoba, T., 2008. Time course of apoptotic cell death in
Guinea pig cochlea following intratympanic gentamicin application. Acta Oto-
laryngol. 128 (7), 724e731.
Suzuki, J., Corfas, G., Liberman, M.C., 2016. Round-window delivery of neurotrophin
3 regenerates cochlear synapses after acoustic overexposure. Sci. Rep. 6, 24907.
http://dx.doi.org/10.1038/srep24907. Apr 25.
Szydlowska, K., Tymianski, M., 2010. Calcium, ischemia and excitotoxicity. Cell.
Calcium 47, 122e129.
Takeno, S., Wake, M., Mount, R.J., Harrison, R.V., 1998. Degeneration of spiral gan-
glion cells in the chinchilla after inner hair cell loss induced by carboplatin.
Audiol. Neurootol. 3 (5), 281e290.
Tsuji, J., Liberman, M.C., 1997. Intracellular labeling of auditory nerve fibers in
Guinea pig: central and peripheral projections. J. Comp. Neurol. 381, 188e202.
Viana, L.M., O'Malley, J.T., Burgess, B.J., Jones, D.D., Oliveira, C.A., Santos, F.,
Merchant, S.N., Liberman, M.C., 2015. Cochlear neuropathy in human presby-
cusis: confocal analysis of hidden hearing loss in post-mortem tissue. Hear Res.
327, 78e88.
Wan, G., G
omez-Casati, M.E., Gigliello, A.R., Liberman, M.C., Corfas, G., 2014. Neu-
rotrophin-3 regulates ribbon synapse density in the cochlea and induces
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147146
synapse regeneration after acoustic trauma. Elife 3.
Wang, Y., Ren, C., 2012. Effects of repeated “benign”noise exposures in young CBA
mice: shedding light on age-related hearing loss. J. Assoc. Res. Otolaryngol. 13
(4), 505e515.
Wang, Y., Hirose, K., Liberman, M.C., 2002. Dynamics of noise-induced cellular
injury and repair in the mouse cochlea. J. Assoc. Res. Otolaryngol. 3 (3),
248e268.
Webster, D.B., Webster, M., 1978. Cochlear nerve projections following organ of corti
destruction. Otolaryngol 86 (2), 342e353.
Winter, I.M., Robertson, D., Yates, G.K., 1990. Diversity of characteristic frequency
rate-intensity functions in Guinea pig auditory nerve fibres. Hear Res. 45,
191e202.
Yin, Y., Liberman, L.D., Maison, S.F., Liberman, M.C., 2014. Olivocochlear innervation
maintains the normal modiolar-pillar and habenular-cuticular gradients in
cochlear synaptic morphology. J. Assoc. Res. Otolaryngol. 15 (4), 571e583.
M.C. Liberman, S.G. Kujawa / Hearing Research 349 (2017) 138e147 147
