Auditory-somatosensory bimodal stimulation desynchronizes brain circuitry to reduce tinnitus in Guinea pigs and humans

Abstract

The dorsal cochlear nucleus is the first site of multisensory convergence in mammalian auditory pathways. Principal output neurons, the fusiform cells, integrate auditory nerve inputs from the cochlea with somatosensory inputs from the head and neck. In previous work, we developed a guinea pig model of tinnitus induced by noise exposure and showed that the fusiform cells in these animals exhibited increased spontaneous activity and cross-unit synchrony, which are physiological correlates of tinnitus. We delivered repeated bimodal auditory-somatosensory stimulation to the dorsal cochlear nucleus of guinea pigs with tinnitus, choosing a stimulus interval known to induce long-term depression (LTD). Twenty minutes per day of LTD-inducing bimodal (but not unimodal) stimulation reduced physiological and behavioral evidence of tinnitus in the guinea pigs after 25 days. Next, we applied the same bimodal treatment to 20 human subjects with tinnitus using a double-blinded, sham-controlled, crossover study. Twenty-eight days of LTD-inducing bimodal stimulation reduced tinnitus loudness and intrusiveness. Unimodal auditory stimulation did not deliver either benefit. Bimodal auditory-somatosensory stimulation that induces LTD in the dorsal cochlear nucleus may hold promise for suppressing chronic tinnitus, which reduces quality of life for millions of tinnitus sufferers worldwide.

Full text

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TINNITUS
Auditory-somatosensory bimodal stimulation
desynchronizes brain circuitry to reduce tinnitus in
guinea pigs and humans
Kendra L. Marks,1* David T. Martel,1,2* Calvin Wu,1* Gregory J. Basura,1 Larry E. Roberts,3
Kara C. Schvartz-Leyzac,1 Susan E. Shore1,2,4†
The dorsal cochlear nucleus is the first site of multisensory convergence in mammalian auditory pathways. Principal
output neurons, the fusiform cells, integrate auditory nerve inputs from the cochlea with somatosensory inputs
from the head and neck. In previous work, we developed a guinea pig model of tinnitus induced by noise exposure and
showed that the fusiform cells in these animals exhibited increased spontaneous activity and cross-unit synchrony,
which are physiological correlates of tinnitus. We delivered repeated bimodal auditory-somatosensory stimulation to
the dorsal cochlear nucleus of guinea pigs with tinnitus, choosing a stimulus interval known to induce long-term
depression (LTD). Twenty minutes per day of LTD-inducing bimodal (but not unimodal) stimulation reduced phys-
iological and behavioral evidence of tinnitus in the guinea pigs after 25 days. Next, we applied the same bimodal
treatment to 20 human subjects with tinnitus using a double-blinded, sham-controlled, crossover study. Twenty-
eight days of LTD-inducing bimodal stimulation reduced tinnitus loudness and intrusiveness. Unimodal auditory
stimulation did not deliver either benefit. Bimodal auditory-somatosensory stimulation that induces LTD in the
dorsal cochlear nucleus may hold promise for suppressing chronic tinnitus, which reduces quality of life for millions of
tinnitus sufferers worldwide.
INTRODUCTION
Tinnitus, the phantom perception of sound in the absence of external
stimuli, is a disorder that affects 15% of the population in the United
States (1) and is the most prevalent service-connected disability for
military personnel (2). Whereas some individuals are minimally
disturbed by their tinnitus, about 10% are bothered by it, and about
2 million individuals are debilitated (1). Negative impacts of tinnitus
include sleep disturbance, poor concentration, distress, depression,
and anxiety (1, 3). Current tinnitus therapies are more successful at
managing a patient’s reaction to their percept rather than addressing
the tinnitus, and no one therapy is effective for all patients. Even
when improving quality of life, none of the available tinnitus therapies
treat the underlying pathology, and few have reported reductions in
tinnitus loudness (4). A treatment that targets the underlying tinnitus
mechanisms would greatly improve clinical outcomes for patients.
Whereas tinnitus is commonly associated with acoustic over-
exposure, many patients with tinnitus have clinically normal audio-
metric thresholds (5, 6), and about 12% report a triggering event
such as a tooth abscess or head and neck injury precipitating their
tinnitus (7), indicating that events in addition to acoustic trauma
can modify neural activity in auditory pathways. Indeed, 60 to 80%
of tinnitus sufferers display a somatosensory component to their
tinnitus, evident in their ability to modulate their tinnitus pitch or
loudness by moving or applying pressure to their head or neck (8).
Tinnitus is thought to arise from dysregulated neural synchrony
across neural ensembles along the auditory pathway (9), beginning
in the dorsal cochlear nucleus (DCN) (10). The DCN is the first
central site for multisensory integration, receiving input from the
auditory nerve, auditory midbrain, auditory cortex, trigeminal and
cervical ganglia, spinal trigeminal nucleus, and dorsal column nuclei
(11–13). After noise exposure sufficient to temporarily elevate hearing
thresholds, spontaneous activity and cross-neural synchrony of DCN
output neurons, the fusiform cells, are increased in animals showing
behavioral evidence of tinnitus. Animals without behavioral evidence
of tinnitus do not show these neural correlates (14). Further, the tinnitus-
related neural changes can occur even in the absence of permanent
shifts in behavioral audiometric thresholds or electrophysiological
measures of peripheral hearing status (14, 15).
The DCN produces hypersynchronous output through its unique,
cerebellar-like circuit (fig. S1). In this circuit, auditory nerve fibers from
the cochlea form synapses with the fusiform cell basal dendrites, whereas
the nonauditory (for example, somatosensory) inputs are relayed by
granule cell axons that form synapses with the fusiform cell apical den-
drites (16). The apical dendritic synapses display spike timing–dependent
plasticity in which repeated elicitation of presynaptic excitatory post-
synaptic potentials (EPSPs) followed by post synaptic spikes produce
long-term potentiation (LTP), whereas postsynaptic spikes followed
by presynaptic EPSPs produce long-term depression (LTD) in vitro
(17). In vivo, auditory (sound) stimulation can be used to evoke post-
synaptic spikes, and somatosensory stimulation can be used to evoke
presynaptic activity in fusiform cells, such that paired auditory-
somatosensory stimulation produces long-term changes in fusiform
cell firing rates. In vivo, the resulting long-term effects are termed
“stimulus timing–dependent plasticity” (STDP). Whether LTP or LTD
occurs depends on the precise order and timing between the bimodal
stimuli (15). These “learning rules” are altered after noise exposure so
that animals with tinnitus show a broader range of stimulus intervals
that evoke LTP, have broader range OF intervals tha t evoke LTD (18).
Theoretical models of feedforward networks predict that LTP-driven
synaptic strengthening will increase circuit connectivity and result
in hypersynchrony (19). Hypersynchrony can also be driven by inhibitor y
1Kresge Hearing Research Institute, Department of Otolaryngology, University of Michigan,
Ann Arbor, MI 48109, USA. 2Department of Biomedical Engineering, University of Michigan,
Ann Arbor, MI 48109, USA. 3Department of Psychology, Neuroscience and Behavior
McMaster University, Hamilton, Ontario, Canada. 4Department of Molecular and Integrative
Physiology, University of Michigan, Ann Arbor, MI 48109, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: sushore@umich.edu
Copyright © 2018
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim
to original U.S.
Government Works
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network components (20), such as the cartwheel cells in the DCN
(fig. S1), which are also subjected to spike timing–dependent syn-
aptic modulation (17). Thus, increased LTP in the fusiform cell cir-
cuit could contribute to the hypersynchrony and increased sponta-
neous activity that are considered neural correlates of tinnitus (14).
Here, using a guinea pig model, we determined whether enhanced
LTP and reduced LTD in the fusiform cell circuit initiated hyper-
synchrony, resulting in behavioral evidence of tinnitus. We show,
in vivo, that auditory-somatosensory stimulation strengthened or
weakened neural synchrony between fusiform cells, depending on
the bimodal stimulus order and timing. Furthermore, in animals
with tinnitus, enhanced LTP correlated with increased synchrony and
spontaneous activity in fusiform cells. To counteract tinnitus, we
stimulated guinea pigs with repeated auditory-somatosensory bimodal
stimulation for 20 min/day for 25 days, choosing a bimodal interval
shown to produce LTD in the fusiform cell circuit. This noninvasive
approach resulted in decreased synchrony and spontaneous activity
in fusiform cells and reduced behavioral evidence of tinnitus. Fur-
thermore, neither unimodal sound nor unimodal somatosensory
stimulation reliably decreased behavioral or physiological evidence
of tinnitus in these animals. These findings demonstrated that fusi-
form cell spike timing–dependent plasticity may play a fundamental
role in regulating neural synchrony and perception and that LTD
could be harnessed to reverse pathological hypersynchrony to reduce
tinnitus.
Then, using stimulus protocols determined by the preclinical ani-
mal experiments, we conducted a similar study in 20 human partici-
pants with somatic tinnitus using a double-blinded, sham- controlled,
crossover design. We reasoned that, because the human cochlear nu-
cleus contains the cellular elements present in the DCN of rodents
(21), similar learning rules should be present in humans and guinea
pigs. We demonstrated that bimodal auditory-somatosensory stim-
ulation, but not unimodal auditory stimulation, effectively reduced
tinnitus loudness and intrusiveness cumulatively over the 4 weeks of
treatment.
RESULTS
STDP regulates synchrony among DCN fusiform cells in
guinea pigs
To test the role of STDP in regulating synchronous firing among
fusiform cells in the DCN, we recorded spontaneous spiking activity
from single fusiform cells in anesthetized normal-hearing guinea
pigs before and 15 min after bimodal stimulation (Fig.1A). Bimodal
stimulation consisted of sounds (tone bursts near the unit best fre-
quency) and transcutaneous electrical stimulation of the neck, pre-
sented within a ±20-ms interstimulus window (Fig.1A). Six bimodal
intervals were studied (sound preceding electrical stimulus by 5, 10,
or 20 ms or electrical stimulus preceding sound by 5, 10, or 20 ms)
in a separate series in a randomized order; physiological measurements
preceded and followed each series (see table S1 for STDP learning
rule types across unit/unit pairs). To quantify synchronous firing, we
measured peak cross-correlation coefficients between spontaneous
spike trains from fusiform cell pairs (Fig.1B). In one representative
unit pair, the peak cross-correlation coefficient decreased (Fig.1C,
top) after auditory-preceding-somatosensory stimulation (−10-ms
interval) but increased after somatosensory-preceding- auditory stim-
ulation (10-ms interval; Fig.1C, bottom). This unit pair exhibited a
Hebbian-like learning rule (Fig.1D) in which presynaptic, subthresh-
old activation of the parallel fibers by somatosensory stimulation fol-
lowed by postsynaptic activation of the basal dendrites by auditory
stimulation (sound) strengthened neural synchrony. In other unit pairs
(for example, Fig.1, E and F), the learning rule was anti–Hebbian-like,
where the same bimodal interstimulus interval as Fig.1C produced neu-
ral synchrony changes in the opposite direction. Other unit pairs exhib-
ited LTP-only learning rules (where all bimodal inter vals strengthened
synchrony) or LTD-only learning rules (where all bimodal intervals
weakened synchrony).
STDP regulates tinnitus-related increases in synchrony and
spontaneous activity
Increased synchrony, bursting, and spontaneous activity are established
neural correlates of tinnitus (14). To determine whether dysregulated
–10 –5 05
10
0
0.02
0.04
–10 –5 05
10
0
0.02
0.04
Time lag (ms)
x-corr coefficient
x-corr coefficient
–10 ms
Aud Som
–10 ms
10 ms
–10 –5 0510
0
0.01
0.02
C
Interval (ms)
–20 020
% ∆ (Peak x-corr
)
% ∆ (Peak x-corr
)
–50
0
50
100
Interval (ms)
–20
02
0
–20
0
20
40
Time lag (ms)
10 ms
–10 –5 0510
0
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F
D
Pre-BIS
Post-BIS
FC2
FC1 ISIH SA
Time lag (ms)
x-corr
–1
01
00
0
0.04
B
E
A
FC2
FC1
SA SABIS
150 s 60 s 15 min 150 s
Fig. 1. STDP regulates synchrony in fusiform cells of the guinea pig DCN.
(A) Spontaneous activity (SA) was recorded across the fusiform cell (FC) population
in 25 guinea pigs for 150 s, followed by 60 s (5 Hz) of bimodal stimulation (BIS) with
bimodal intervals (BI) from −20 to +20 ms. SA was recorded again 15 min after BIS
for 150 s. (B) Synchrony was assessed by cross-correlations (x-corr) of spikes in FC
pairs (FC1 and FC2). SA of FCs shows Poisson distributions in interspike interval
histograms (ISIHs). Synchronous unit pairs are defined by threshold cross-correlation
coefficients (x-corr coef) of 4 SD (dashed line). (C) In one representative FC unit pair,
a BI of −10 ms (auditory preceding somatosensory stimulus by 10 ms; pink) re-
duced the peak x-corr coef (top), whereas a BI of 10 ms (somatosensory preceding
auditory stimulus by 10 ms; blue) increased the peak x-corr coef 15 min after BIS
(bottom). (D) Changes in peak x-corr coef for the FC unit pair in (C) are plotted as a
function of BI (learning rule). (E) In a different FC unit pair, a BI of −10 ms increased
peak x-corr coef (top), whereas a BI of 10 ms decreased peak x-corr coef 15 min af-
ter BIS (bottom). (F) For the FC unit pair in (E), changes in x-corr coef after BIS were
opposite to that for the FC unit pair in (D).
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STDP contributes to tinnitus-related hypersynchrony, we induced
tinnitus in guinea pigs using noise exposure and assessed tinnitus
using gap-prepulse inhibition of the acoustic startle (GPIAS) response.
GPIAS measures the acoustic startle response in the presence of a
background narrow-band noise. When a gap is inserted into the
background noise before the startle stimulus, the startle response is
reduced in normal animals. However, in animals with tinnitus, the
tinnitus obscures the gap, and there is no decrement in the startle
response. By plotting the amplitude of the gap trials versus the no-
gap trials, an estimate of the animals’ tinnitus is obtained (see fig. S4)
(15, 22, 23). Noise exposure produced only temporary hearing threshold
elevations, which recovered after a few days (fig. S3), but resulted in
chronic tinnitus in 16 of 22 (72.7%) guinea pigs after 8 weeks (fig.
S4). Noise-exposed animals with tinnitus [ex posed with tinnitus
(ET)] exhibited significant increases in synchrony [one-way analysis
of variance (ANOVA), F(2) = 14.9, P = 2.3 × 10−6, post hoc P < 0.05;
Fig.2A] and spontaneous activity [F(2) = 17.5, P = 3.0 × 10−7, post
hoc P < 0.05; Fig.2B) across the fusiform cell population compared
to normal-hearing animals and the 27.3% of noise- exposed animals
that did not develop tinnitus [exposed no tinnitus (ENT)]. STDP for
synchrony was assessed and compared across the tinnitus, no-tinnitus,
and normal- hearing groups. The tinnitus group exhibited a greater
proportion of unit pairs with LTP-only learning rules, whereas the
normal- hearing and the non-tinnitus groups exhibited greater pro-
portions of anti-Hebbian–like and LTD-only learning rules (Fig.2C
and table S1) [2(3) = 15.8, P = 0.0013]. STDP for spontaneous activ-
ity of single units followed a similar trend (Fig.2D) [2(3) = 23.4,
P = 3.3 × 10−5]. To further quantify the learning rule distribution
shift from LTD toward LTP, we compared the LTD-LTP index
(Fig.2E, inset), which sums all positive/LTP integration phases and
all negative/LTD phases across all unit pairs or single units (see
Fig.2E for synchrony and Fig.2F for spontaneous activity). The tin-
nitus group showed more LTPs across all learning- rule types,
whereas the no-tinnitus group showed more LTD compared to the
normal-hearing group [F(2) = 10.33, P = 5.3 × 10−5 for synchrony;
F(2) = 91.7, P = 1.6 × 10−37 for spontaneous activity]. These findings
indicated that the tinnitus-driven circuit had a high probability for
LTP and strengthened neural synchrony.
Bimodal (but not unimodal) stimulation induces LTD to
reduce fusiform cell synchrony and spontaneous activity
Given that increased synchrony and spontaneous activity correlated
with an expansion of the LTP phase of the STDP learning rule, we
hypothesized that inducing LTD would reduce synchrony and spon-
taneous activity. First, we determined the bimodal interval that pro-
duced the strongest LTD in the animals with tinnitus by quantifying
LTD probability (overall proportion of units showing LTD at a given
bimodal interval). We found that more units responded with decreased
synchrony and spontaneous activity after bimodal stimulation in-
tervals of −5 and −10 ms. Whereas ±20-ms intervals showed slight
deviation from 0.5, they were not different from chance (Fig.3A).
Suppression of synchrony and spontaneous activity after −5-ms bi-
modal stimulation was significantly greater (due to less variance) than
unimodal auditory or unimodal somatosensory stimulation, neither
of which produced long-term effects [one-way ANOVA, F(2) = 11.3,
P = 1.1 × 10−6 for synchrony; F(2) = 142, P = 5.4 × 10−66 for sponta-
neous activity; Fig.3, B and C].
We next asked whether reducing syn-
chrony in the fusiform cell circuit would
affect the animal’s tinnitus behavior. We
hypothesized that repeated bimodal stim-
ulation with an LTD-inducing interval
(−5 ms) would reduce synchrony and
spontaneous activity as well as behavioral
evidence of tinnitus. Unimodal auditory
stimulation, on the other hand, should
not induce LTD because auditory synapses
on the basal dendrites are not plastic;
somatosensory input alone has been shown
to induce LTP (15, 18). To test this hypo th-
esis, we treated guinea pigs with tinnitus
with 20-min daily sessions of bimodal
stimulation consisting of an 8-kHz tone
burst (the frequency at which tinnitus was
most prevalent; see fig. S4) paired with
transcutaneous stimulation at the −5-ms
interval for 25 days (ET-treat group).
Three control groups were used, all ex-
pressing tinnitus after noise exposure. A
sham group received a sedative but no
bimodal or unimodal stimulation (ET-
sham); an auditory-only group received
the same 8-kHz tone but did not receive
transcutaneous somatosensory stimula-
tion (ET-audio); and a somatosensory- only
group received only electrical stimulation
(ET-som). After the 25-day treatment
LTD
LTD
LTP
LTP
Heb
Heb
aHeb
aHeb
N
N
ENT
ENT
ET
ET
A
B
C
D
E
F
0.50
0.5
0
0.5
0
0.50
Proportion
Proportion
∆ x-corr
–3.6
5.9
∆
t
N
ENT
ET
–5
0
5
LTD-LTP index (SA)
N
ENT
ET
–10
–5
0
5
LTD-LTP Index (x-corr)
N
ENT
ET
0
0.05
0.1
0.15
0.2
Weighted x-corr
**
N
ENT
ET
0
1
2
3
SA (1/s)
*
**
*
*
*
*
*
Fig. 2. STDP shifts toward LTP in guinea pigs with tinnitus. (A) Increased mean cross-correlation coefficient
(x-corr; weighted by the proportion of synchronous unit pairs) and (B) increased mean spontaneous firing rate (SA)
compared to the normal-hearing (N) and exposed-but-no-tinnitus (ENT) groups of animals. One-way analysis of vari-
ance (ANOVA), *P < 0.05; data are mean ± SEM. Spontaneous firing rates for the N, ENT, and ET (exposed tinnitus)
groups were 116, 93, and 167 unit pairs for x-corr and 106, 387, and 478 units, respectively. (C and D) A shift in the
proportion of learning rules toward Hebbian-like (Heb; x axis) and long-term potentiation (LTP) (y axis) in the ET group
for (C) synchrony and (D) SA. aHeb, anti-Hebbian. (E and F) Long-term depression (LTD)–LTP index: total magnitude of
LTP, that is, green area under the curve relative to total magnitude of LTD, that is, blue area above the curve of learn-
ing rules (E, inset), is increased in the ET group for (E) synchrony and (F) SA.
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period, we quantified tinnitus behavior in the four groups using the
tinnitus index (TI), which compared gap startle responses normalized
to the preexposure baseline before and after noise exposure (fig. S4).
Representative findings presented in Fig.4A (one animal per group)
show increased normalized startle responses after noise exposure,
indicating tinnitus, which was reduced after treatment only in the
animal receiving bimodal stimulation (ET-treat). Group analysis pre-
sented in Fig.4B showed that, compared to the pretreatment TI,
animals receiving bimodal stimulation (ET-treat) exhibited a signif-
icant reduction in the TI at the treated frequency of 8 kHz and not
at other tinnitus frequencies, whereas the sham group (ET-sham) and
the auditory-only group (ET-audio) showed no changes [two-way
ANOVA, F(2,1) = 3.70, P = 0.0069 for frequency × group). The
somatosensory-only group (ET-som) showed a small decrease in TI
at one frequency and a large increase in TI in another frequency but
no significant group mean change from control (post hoc P > 0.05).
Tinnitus reduction correlated with lower neural synchrony [Pearson’s
linear correlation: r(287) = 0.15, P = 0.010; correction of dependency
using linear mixed-effect model: P = 0.031; Fig.4C] and lower spon-
taneous activity [r(1125) = 0.20, P = 6.8 × 10−12; correction of depen-
dency using linear mixed-effect model: P = 0.0011; Fig.4D]. Together,
these results demonstrate that targeted LTD induction in guinea pigs
reduced tinnitus produced by dysregulated STDP, increased neuro-
nal synchrony, and spontaneous activity.
Bimodal (but not unimodal) auditory-somatosensory
stimulation reduces tinnitus loudness in humans
The positive animal study outcomes prompted the investigation of
bimodal treatment for humans suffering from tinnitus. A double-
blinded, sham-controlled, crossover study was used to evaluate the
effectiveness of bimodal auditory-somatosensory stimulation as a
tinnitus treatment. All subjects and investigators were blinded as to
whether subjects received an active (bimodal) or sham (unimodal-
auditory) treatment for the duration of the study. Upon enrollment,
participants were first assigned to either a sham group (n = 10,
group 1) or an active bimodal treatment group (n = 10, group 2;
Fig.5). Assignment was by a random number list that was precom-
puted before the start of the study. Take-home devices were pro-
grammed to deliver the bimodal or unimodal treatment protocols
by control software and data were encrypted to ensure blinding. The
sound stimuli were delivered through calibrated insert earphones,
and the electrical (somatosensory) stimuli were administered using
Ag-AgCl cups placed on the skin of the cervical spine or the cheek.
Participants used the devices for 30 min once a day for two 4-week
sessions with a 4-week washout period after each session. After the
washout period, subjects “crossed over” to receive the other treat-
ment for the second 4-week period so that all subjects received both
active and sham treatments. Participants returned to the laboratory
weekly for monitoring and tinnitus assessment: loudness was as-
sessed by matching tinnitus loudness to an external sound using
TinnTester software, and intrusiveness was assessed using the Tin-
nitus Functional Index (TFI; see Material and Methods).
The auditory stimulus (the same for bimodal and sham) was de-
rived from each individual’s tinnitus spectrum and audiogram (fig. S5,
see Materials and Methods). Devices provided either bimodal (auditory-
electric) stimulation (bimodal active treatment) or unimodal (auditory
alone) stimulation (sham treatment) for 30 min a day for 28 days. The
bimodal interval was the same as that shown to be effective in the
guinea pigs (−5 ms). Somatosensory stimulation alone was not pro-
vided because the animal study (Figs.3B and 4, A and B) indicated
that it could exacerbate the tinnitus.
The active bimodal treatment produced a significant (P < 0.05)
cumulative decrease in tinnitus loudness assessed by TinnTester
loudness matching each week of the active treatment (Fig.6A). The
greatest mean change in loudness occurred after the fourth and final
week of treatment. In contrast, loudness was stable (unchanged) during
sham treatment for both groups. There was no significant difference
between groups 1 and 2 (P = 0.88), demonstrating that treatment
order had no effect. Pooled groups showed a mean decrease of 8.035 ±
1.33 dB from a baseline of 54.42 ± 13.3 dB in loudness matches
during the 4 weeks of active treatment [two-way ANOVA, F(3,1) 7.768,
post hoc P = 5.5 × 10−5], significantly larger than the changes seen
in the other conditions (sham, active washout, and sham washout)
where changes from baseline were not significant (Fig.6B). Tinnitus
–5 ms
Uni. som
Uni. aud
LTPLTD
A
B
C
–20 –10 –5 51020
Interval (ms)
0
0.5
1
Probability of suppression
–100 –50 050
100
%∆ SA
0
10
20
30
# Unit
–100 –50 050 100
%∆ x-corr coef
0
20
40
# Unit pair
–5 ms
Uni. som
Uni. aud
x-corr
SA
Fig. 3. Targeted bimodal stimulation suppresses synchrony and spontaneous
activity in fusiform cells of guinea pigs. (A) Probability of synchrony (x-corr) or
SA suppression as a function of bimodal interval. Probability is computed by pro-
portion of unit pairs (total n = 159) or units (n = 251) showing decreased x-corr or
SA at a given bimodal interval. A probability of 0.5 indicates an equal number of units
showing increased or decreased x-corr or SA. The highest probability of suppression
occurs for the −10- and −5-ms intervals [error bar, confidence interval (CI) for binomial
proportion]. The −5-ms interval was chosen for the treatment. (B) The distributions
of suppression versus enhancement of synchrony are compared for the −5-ms bimodal
interval, unimodal somatosensory (uni. som), or unimodal auditory stimulation (uni.
aud). The bimodal stimulus clearly suppressed synchrony, whereas the unimodal
stimuli showed little deviation from zero. (C) Similar to synchrony, the bimodal
stimulus suppressed SA, whereas the unimodal stimulus showed little deviation
from zero (bar = 2% bin; shaded curve is fitted by Spline Interpolant).
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reduction reached an average of 12.2 dB in the fourth week of active
treatment. Of the 20 participants tested, 2 reported complete elimi-
nation of their tinnitus toward the end of the active treatment period.
Bimodal (but not unimodal) stimulation improves TFI scores
Mean overall TFI scores decreased from baseline of 29.2 ± 2.6 to
22.9 ± 1.8 units during the active treatment but remained un-
changed during the sham treatment (Fig. 6C). Improvements in
TFI scores were sustained beyond the active treatment and into the
washout period, unlike the changes in loudness matching. Because
treatment order also had no significant effect on TFI scores [general
linear mixed models (GLMM); P = 0.819], both groups were pooled
for statistical analysis. The mean TFI scores across the different
study periods (Fig.6D) were significantly improved (that is, reduced
relative to baseline) for both active and active washout periods (but
not sham periods) [7.33 ± 0.956 TFI units; two-way ANOVA, F(3,1) =
7.712, P = 6.14 × 10−5), indicating a di-
minished impact on daily life with mean
reductions of 7.51 and 6.71 points, re-
spectively. Eleven participants noted sub-
jective changes in volume, pitch, or
quality that resulted in their tinnitus
becoming less “harsh” or “piercing” and
more “mellow.” Even participants who
did not experience a complete elimina-
tion of their tinnitus reported anecdotally
that their tinnitus was noticeably less ob-
trusive and easier to ignore.
Ten of the 20 subjects had a clinically
significant reduction of at least 13 points
in their TFI scores during active treat-
ment, which is considered clinically mean-
i ngful for this questionnaire (24). There
were no demographic differences across
subjects showing significant TFI changes
compared to stable subjects (table S2).
Four participants had clinically significant
drops during the sham treatment, but
two of these also showed significant de-
creases in TFI during the active treatment.
Further, both participants reported that
their tinnitus improved more during the
active treatment. The two participants
who stated that the sham treatment was
more effective also had the shortest tin-
nitus duration (less than 1 year).
Reductions in loudness relative to
baseline correlated significantly with re-
ductions in overall TFI scores (linear mixed-
effects model: β = 0.169 ± 0.058, T =
2.94, P = 0.0035; fig. S7). Furthermore,
changes in loudness correlated with changes
in TFI subscores: sense of control, intrusive,
cognitive, and sleep (table S3).
DISCUSSION
Increases in synchrony, spontaneous
activity and bursting (14), and altered
STDP (15) are established neural cor-
relates of tinnitus. In animal models of tinnitus, increased synchrony
has been identified in the DCN (14), inferior colliculus (25), and
auditory cortex (26). These studies suggest that the tinnitus percept
emerges from increased spontaneous synchrony among neurons in
cortical and subcortical regions that contribute to perceptual binding
(the process of merging individual pieces of sensory information
into coherent representations) (27). Here, we first examined the re-
lationship between synchrony and STDP in normal-hearing guinea
pigs, which exhibited Hebbian and anti-Hebbian learning rules as
well as rules giving LTP or LTD. We then induced tinnitus in animals
using noise exposure that produced only temporary threshold shifts
and observed tinnitus-related increases in neural activity reflecting
an overall dominance of LTP. Subsequently, we applied the optimal
bimodal interval to induce LTD in sessions of 20-min duration for
25 days, which reversed hypersynchrony and behavioral evidence of
Pre-exp
Post-exp
Treat
0.2
0.4
0.6
0.8
1
0
Norm. startle
Tinnitus index (TI)
ET-sham
ET-audio
ET-treat
ET-som
AC
BD
1
2
∆ TI
∆ TI
∆ TI
∆ TI
ET-sham CI
ET-treat
ET-audio
ET-som
ET
-sham
ET-treat
ET-audi
o
Normal
Off-freq
On-freq
Off-freq
On-freq
ET-som
∆ TI
–1.5 –1 –0.5 00.5 11.5
TI
0
2
4
6
8
10
SA (1/s)
–1.5 –1 –0.5 00.5 11.5
∆ TI
0
0.02
0.04
0.06
0.08
0.1
0.12
Weighted x-corr
O-freq
On-freq
–
1
–
0.5
0
0.5
1
1.5
Fig. 4. LTD-induction reduces synchrony and spontaneous activity and reduces tinnitus in guinea pigs. (A) Four
representative animals (one from each group) showed increased normalized startles (norm. startle) after noise exposure
[from pre-exposure (pre-exp) to post-exposure (post-exp)] indicating tinnitus (left ordinate), quantified as the TI
(right ordinate). After LTD induction by application of a bimodal auditory-somatosensory stimulus to the fusiform
cells (ET-treat), there was a reduction in TI in the treated animal (ET-treat). Sham-treated (ET-sham; sedative only),
auditory stimulus–only (ET-audio), and somatosensory stimulus–only (ET-som) animals showed either no reduction
in TI or worsened TI. (B) Mean TI was significantly reduced in the ET-treat group at the treated frequency (on-freq; 8 kHz)
but not at the untreated frequencies (off-freq; 12 and 16 kHz). TI was not significantly reduced in the ET-sham, ET-audio,
or ET-som groups. Pink horizontal bar indicates the 95% CI for the ET-sham group. (C) The weighted mean cross-
correlation coefficient (x-corr) for FCs (at best frequencies within the TI bandwidth) is plotted as a function of TI (116,
36, 35, and 106 unit pairs for the ET-sham, ET-audio, ET-som, and ET-treat groups, respectively). Gray area indicates
the range of x-corr for nonexposed animals. Reduction in synchrony significantly correlated with TI reduction. (D) SA
plotted as a function of TI (446, 204, 202, and 696 units). Reduction in SA significantly correlated with TI reduction.
Data are mean ± SEM.
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Marks et al., Sci. Transl. Med. 10, eaal3175 (2018) 3 January 2018
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
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tinnitus at frequencies corresponding to the treatment frequency.
None of the control stimuli (sedative alone, unimodal somatosensory,
or unimodal auditory stimulation) had any effect on tinnitus behaviors
or tinnitus correlates. On the basis of the outcome of the animal
study, we used the same bimodal stimulus protocol to treat tinnitus
in humans.
STDP is essential for shaping sensory perception through input-
dependent learning. In the visual cortex, STDP modulates tuning of
visual neurons to orientation and motion (28). Similar STDP pro-
cesses shape map plasticity in the somatosensory and auditory cor-
tices for frequency selectivity, pitch encoding, and discrimination
(29–31). These context-dependent changes in sensory processing
can alter connectivity and synchrony of neural ensembles (32, 33).
In the fusiform cell circuit, multimodal inputs induce context-
dependent changes through STDP (34). Auditory-somatosensory
integration in DCN constitutes an adaptive filtering process through
which perception of behaviorally relevant sounds is amplified and
internally generated sounds are attenuated (17, 35, 36). Fusiform
cell synchrony regulation by STDP likely contributes to this perceptual
task, whereas dysregulated multimodal STDP gives rise to phantom
perception, as we show here.
Synaptic plasticity has been suggested as a foundation for network-
level homeostatic adaptation (37). In the fusiform cell circuit, glutamatergic
inputs to the granule cell–parallel fiber circuit are up-regulated after hear-
ing loss (38–40), resulting in increases in LTP (41). This homeo static
mechanism in response to altered input is not exclusive to the auditory
pathway (42). After light deprivation, visual-cortical neurons exhibit ex-
pansion in STDP due to increased N-methyl-d-aspartate (NMDA) re-
ceptor activation (43). Blocking NMDA receptors in the fusiform cell
circuit reduces neural synchrony (44). Muscarinic acetylcholine receptors,
whose expression is up-regulated after noise ex posure (45), also contrib-
ute to STdP (46, 47).
STDP can affect intrinsic membrane excitability by altering ion
channel conductance (48, 49). Maladaptive changes to fusiform cell
plasticity that decrease inhibition through reduced hyperpolarizing
currents could also contribute to increased synchrony and sponta-
neous activity. Reduced potassium channel activation and reduced
glycine and GABA (-aminobutyric acid) receptor activation of fusi-
form cells have been demonstrated in tinnitus models (50, 51). A
major source of GABA input and glycinergic input to fusiform cells
arises in cartwheel cells (fig. S1). These DCN interneurons, which
receive parallel-fiber synapses that exhibit STDP (17), provide recur-
rent inhibitory synapses onto fusiform
cells. Cartwheel cells, therefore, may play
an essential role in generating fusiform
cell synchrony (19, 20). Another poten-
tial player, the Golgi cell in the marginal
region of the cochlear nucleus, provides
feedback modulation of granule cell out-
put, which may entrain parallel fibers into
synchronized firing (52–54). These net-
work components are likely to work to-
gether to increase synchrony in fusiform
cells, thus potentially playing important
roles in tinnitus.
Because the human cochlear nucleus
contains all of the cellular elements pre-
sent in the DCN of rodents (21), we rea-
soned that the same bimodal protocol
might suppress tinnitus in humans. In both
the animal and the human studies, bi-
modal but not unimodal auditory stim-
ulation effectively suppressed tinnitus.
The documented failure of unimodal au-
ditory stimulation to produce long-term
changes in fusiform cell firing rates pre-
dicted that unimodal auditory stimulation
would be inefficient at reducing tinnitus
(15, 18, 34, 55). The significant reduc-
tion in tinnitus in animals and tinnitus
loudness and distress in humans suggests
Group 1 (n = 10)
Group 2 (n = 10)
Treatment
Baseline
Washout
period Treatment
ShamSham
Randomize
4 weeks 4 weeks 4 week 4 weeks
Final
washout
Fig. 5. Outline of crossover design for the human study. Subjects were randomly
assigned to group 1, in which the bimodal treatment was presented first, or group
2, in which the sham treatment was presented first. After 4 weeks of 30 min/day of
the treatment, there was a 4-week washout period. Thereafter, subjects crossed
over to receive the treatment that they had not yet received for 4 more weeks. This
was followed by a second, 4-week washout period. Loudness and Tinnitus Func-
tional Index (TFI) assessments were done weekly in the clinic.
Group 1
Group 2
Active
Active washout
Sham
Sham washout
–5 05101520
–20
–10
0
10
Change in loudness (dB SPL)
–5 05101520
Time (weeks)
–20
–10
0
10
Change in TFI
Active Active
washout
Sham Sham
washout
–10
–8
–6
–4
–2
0
Change in loudness (dB SPL)
Active Active
washout
Sham Sham
washout
–10
–5
0
5
Change in TFI
AB
CD
Fig. 6. Bimodal treatment results in reduced tinnitus loudness and reduced TFI scores in human patients.
(A) Mea n loudness by group. Group 1 (n = 10) received the active treatment first; group 2 (n = 10) received the sham
treatment first. Loudness was assessed using the interactive software TinnTester, in which subjects match their tinnitus
loudness and spectrum to an externally presented sound (see Materials and Methods). (B) Mean changes (normalized
to baseline) in loudness matching for each condition. (C) Mean TFI changes (relative to baseline) for groups 1 and 2.
(D) Mean changes (relative to baseline) in TFI scores. Error bars are SEM. dB SPL, decibels sound pressure level.
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that the bimodal treatment was successful at inducing frequency-
specific LTD, reversing the pathological neural activity responsible
for the generation of tinnitus.
Unimodal auditory treatment, in addition to being ineffective at
reducing tinnitus during the sham treatment phase, tended to cause
an increase in tinnitus loudness and TFI scores at the end of the sham
treatment, possibly due to the increased attention paid to the tinnitus
during the evaluation periods. Unimodal somatosensory stimulation,
on the other hand, shown to cause LTP and not LTD in animal studies
(15, 18, 34), predicted that unimodal somatosensory stimulation
could exacerbate tinnitus. Unimodal somatosensory stimulation did
exacerbate the tinnitus in some animals, preventing us from testing
the electrical-only stimulation condition in humans.
Bimodal auditory-somatosensory stimulation in humans had no
side effects, whereas invasive techniques such as deep brain stimula-
tion and vagal nerve stimulation can have severe side effects. Our
LTD induction approach is noninvasive, easy to implement, and
presents minimal risk. Although reduced tinnitus loudness did not
carry over into the washout period, this benefit was persistent enough
to accumulate over several days of treatment. Improved adjustment
to tinnitus, as reflected in the TFI scores, persisted during the washout
period, for up to 3 weeks. Furthermore, reductions in tinnitus loudness
correlated with TFI subscores regarding sense of control, intrusiveness,
cognition, and sleep, suggesting that tinnitus loudness reduction during
bimodal treatment conferred psychological benefits that outlasted
the treatment.
Other approaches to treat tinnitus, such as the coordinated reset
sound therapy or paired sound–vagal nerve stimulation also target
putative aberrant neural activity but have not yet yielded positive
results in the clinic. Paired vagal nerve stimulation, although showing
promising results in an animal model, requires invasive surgery with
accompanying risks and side effects, rendering it only suitable for
the most debilitated patients. Sound therapies do not consistently
reduce tinnitus loudness (56), perhaps because unimodal auditory
stimulation has no effect on modulating long-term plasticity in the
DCN (Fig.3, B and C) (15, 18).
There are some limitations to our study. Our study only tested
one subgroup of tinnitus patients, those with somatic tinnitus; thus,
it is unknown whether these results would translate to other sub-
groups. In addition, ethical considerations prevented us from testing
some protocol conditions in the human patients, such as the somato-
sensory stimulation–alone condition, which was observed to exac-
erbate tinnitus in the guinea pig study. Nevertheless, the neural de-
synchronization strategy presented here offers a new and accessible
treatment possibility for tinnitus sufferers.
MATERIALS AND METHODS
Study design
All animal procedures were performed per protocol established by the
National Institutes of Health Publication No. 80-23 and approved
by the University of Michigan’s University Committee on Use and
Care of Animals. First, noise-over exposure was used to induce tinnitus
in guinea pigs (see fig. S2). Evidence of tinnitus was provided by a
behavioral test (GPIAS) and confirmed with physiological signatures
of increased spontaneous rates of firing and synchrony in DCN fusi-
form cells. Twelve guinea pigs were used for physiological assessment
after noise exposure, and 13 were used for physiological assessment
after treatment. To the latter group (all expressing tinnitus), we applied
noninvasive, 20 min/day auditory-somatosensory stimulation (with
three different controls) for 25 days and assessed behavioral and neuro-
physiological correlates of tinnitus. Second (Fig.5), a double-blinded,
sham-controlled, crossover study was performed to evaluate the ef-
fectiveness of the auditory-somatosensory stimulation in humans
with tinnitus. The study was performed in accordance with the Uni-
versity of Michigan Institutional Review Board. Participants were
randomly assigned to either sham (n = 10) or active treatment first
(n = 10) groups. Participants were trained to use a small, customized
take- home device that provided the active and sham treatments.
Weekly tinnitus spectra estimation and self-reported questionnaires
were obtained on site. All 20 participants who completed the study
were included in the analysis.
Tinnitus assessment in guinea pigs
Tinnitus was assessed using GPIAS (fig. S4A) (14, 15, 41, 57). A
normalized startle ratio (NSR) was computed as the ratio of the
mean startle amplitude for the gap/prepulse trials and the mean of
the startle-only trials (fig. S4B). An animal was defined as having
tinnitus in a frequency band if the postexposure mean NSR value
for gap inhibition was significantly greater than the baseline value.
Neural recordings to evaluate spontaneous activity and synchrony
were performed after the completion of tinnitus assessments.
Human tinnitus assessment
A computerized procedure (TinnTester) (58) was used for weekly
loudness matching in the laboratory throughout the trial. The TFI
questionnaire was used to assess the impact of a subject’s tinnitus
on their quality of life (24).
Auditory-somatosensory treatment in guinea pigs
and humans
The somatosensory stimulation was provided by transcutaneous
active electrodes positioned on the skin overlying either the trigeminal
ganglion or the cervical spinal cord in the region of C2 (with the ground
electrode adjacent). In humans, electrode location depended on which
maneuvers induced the strongest change in tinnitus. In guinea pigs,
C2 was used throughout. Auditory stimulation was personalized
according to each subject’s tinnitus spectrum. In guinea pigs, 8 kHz
(most prevalent tinnitus frequency) was used. For the active treatment,
the auditory stimulus preceded the somatosensory stimulus by 5 ms.
Statistics
Two-tailed t test, 2 contingency tests, Pearson’s linear correlation,
one-way and two-way ANOVAs were used to determine statistical
differences ( = 0.05). Post hoc analyses for ANOVA were per-
formed using the Tukey-Kramer test where indicated. For statistical
significance evaluation of guinea pig’s tinnitus behavioral versus
neurophysiological results, patients’ loudness versus TFI, and loudness
matching measures, GLMM or linear mixed-effect models were used.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/10/422/eaal3175/DC1
Materials and Methods
Fig. S1. Development of STDP in the fusiform cell circuit.
Fig. S2. The experimental timeline for the animal study.
Fig. S3. Noise exposure produces only temporary threshold and suprathreshold shifts.
Fig. S4. GPIAS behavioral assessment of tinnitus in guinea pigs.
Fig. S5. Human treatment groups had similar hearing thresholds.
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Fig. S6. Reduction in tinnitus loudness in humans correlates with reductions in TFI.
Fig. S7. Tinnitus modulation maneuver checklist.
Table S1. Distribution of STDP learning rule type across unit/unit pairs in guinea pigs.
Table S2. Subject demographics.
Table S3. Correlations between changes in loudness and changes in TFI subscore.
References (59–70)
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Acknowledgments: We thank C. Ellinger, D. Valliencourt, and D. Thompson for technical
assistance, Consulting for Statistics, Computing and Analytics Research (CSCAR) for statistical
consultation, and S. Bledsoe, M. Roberts, A. Heeringa, and S. Koehler for the helpful comments
on a previous version of this manuscript. Funding: This study was supported by NIH grants
R01-DC004825 (to S.E.S.), T32-DC00011 (to D.T.M. and C.W.), and the Wallace H. Coulter
Translational Research Partnership (to S.E.S. and G.J.B.). Participation of L.E.R. was assisted by
funding from the Natural Sciences and Engineering Council of Canada. Author contributions:
S.E.S. conceived and designed the animal and human studies. S.E.S., C.W., and D.T.M. designed
the animal study. S.E.S., K.L.M., K.C.S.-L., and L.E.R. designed the human study. G.J.B. provided
subject medical clearance for study participation. K.L.M. collected the human data. D.T.M. and
C.W. collected the animal data. K.L.M., D.T.M., and S.E.S. analyzed the human data. C.W., D.T.M.,
and S.E.S. analyzed the animal data. K.L.M., D.T.M., C.W., L.E.R., K.C.S.-L., and S.E.S. wrote the
manuscript. Competing interests: S.E.S. and D.T.M. are inventors on U.S. patent no.
3A9242067 “Personalized auditory-somatosensory stimulation to treat tinnitus.” All other
authors declare that they have no competing interests.
Submitted 1 November 2016
Resubmitted 16 March 2017
Accepted 7 September 2017
Published 3 January 2018
10.1126/scitranslmed.aal3175
Citation: K. L. Marks, D. T. Martel, C. Wu, G. J. Basura, L. E. Roberts, K. C. Schvartz-Leyzac, S. E. Shore,
Auditory-somatosensory bimodal stimulation desynchronizes brain circuitry to reduce tinnitus in
guinea pigs and humans. Sci. Transl. Med. 10, eaal3175 (2018).
by guest on January 8, 2018http://stm.sciencemag.org/Downloaded from

tinnitus in guinea pigs and humans
Auditory-somatosensory bimodal stimulation desynchronizes brain circuitry to reduce
E. Shore
Kendra L. Marks, David T. Martel, Calvin Wu, Gregory J. Basura, Larry E. Roberts, Kara C. Schvartz-Leyzac and Susan
DOI: 10.1126/scitranslmed.aal3175
, eaal3175.10Sci Transl Med
promise for suppressing chronic tinnitus in patients.
tinnitus in the animals or the humans. Bimodal auditory-somatosensory stimulation that induces LTD may hold
subjects in a double-blinded, sham-controlled, crossover clinical study. Unimodal stimulation did not reduce
behavioral evidence of tinnitus in the animals. The same bimodal protocol reduced tinnitus loudness in human
minutes per day of bimodal stimulation to induce LTD in the cochlear nucleus reduced physiological and
stimulation designed to induce long-term depression (LTD) in the cochlear nucleus of these animals. Twenty
delivered precisely timed bimodal auditory-somatosensoryet al.tinnitus induced by noise trauma, Marks
Tinnitus reduces quality of life for millions of tinnitus sufferers worldwide. Using a guinea pig model of
The sound of silence
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REFERENCES http://stm.sciencemag.org/content/10/422/eaal3175#BIBL
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