Transauricular vagus nerve stimulation at auricular

Accepted Manuscript

Transauricular vagus nerve stimulation at auricular acupoints Kindey (CO10), Yidan(CO11), Liver (CO12) and Shenmen (TF4) can induce auditory and limbic corticesactivation measured by fMRI

Liyan Peng, Ketao Mu, Aiguo Liu, liangqiang Zhou, Yueyue Gao, Imrit TejvanshShenoy, Zhigang Mei, Qingguo Chen

PII: S0378-5955(17)30364-7

DOI: 10.1016/j.heares.2017.12.003

Reference: HEARES 7461

To appear in: Hearing Research

Received Date: 27 July 2017

Revised Date: 3 December 2017

Accepted Date: 5 December 2017

Please cite this article as: Peng, L., Mu, K., Liu, A., Zhou, l., Gao, Y., Shenoy, I.T., Mei, Z., Chen, Q.,Transauricular vagus nerve stimulation at auricular acupoints Kindey (CO10), Yidan (CO11), Liver(CO12) and Shenmen (TF4) can induce auditory and limbic cortices activation measured by fMRI,Hearing Research (2018), doi: 10.1016/j.heares.2017.12.003.

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https://doi.org/10.1016/j.heares.2017.12.003

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Transauricular vagus nerve stimulation at auricular acupoints Kindey (CO10), Yidan

(CO11), Liver (CO12) and Shenmen (TF4) can induce auditory and limbic cortices

activation measured by fMRI

Liyan Peng1, Ketao Mu2, Aiguo Liu1, liangqiang Zhou1, Yueyue Gao1, Imrit Tejvansh Shenoy1, ZhigangMei3, Qingguo Chen1

1Department of Otorhinolaryngology, Tongji hospital, Tongji medical college, Huazhong University of Science and technology, Wuhan, China; 2Department of Radiology, Tongji hospital, Tongji medical college, Huazhong University of Science and technology, Wuhan, China.

3Yichang Hospital of Traditional Chinese Medicine, Clinical Medical College of Traditional Chinese Medicine, China Three Gorges University, Yichang, Hubei, China Liyan Peng and Ketao Mu contribute equally to this paper. Qingguo Chen and Zhigang Mei are the Corresponding authors who contribute equally to this paper.

Address correspondence to Qingguo Chen, Department of Otorhinolaryngology,Tongji medical college, Huazhong University of Science and technology, 430030, Jie Fang Avenue 1095, Wuhan, China. Email:[email protected] Second correspondence to Zhigang Mei, Yichang Hospital of Traditional Chinese Medicine, Clinical Medical College of Traditional Chinese Medicine, China Three Gorges University, Yichang, Hubei, China. Email: [email protected]

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Transauricular vagus nerve stimulation at auricular acupoints Kindey (CO10), Yidan

(CO11), Liver (CO12) and Shenmen (TF4) can induce auditory and limbic cortices

activation measured by fMRI

Abstract

The purpose of this study was to explore the central mechanism of transauricular

vagus nerve stimulation (taVNS) to human by fMRI and to find a suitable taVNS site

for potential tinnitus treatment. 24 healthy subjects aged between 28 and 38 years

were enrolled in the experiment. 8 subjects were stimulated in the auricular acupoints

Kindey (CO10), Yidan (CO11), Liver (CO12) and Shenmen (TF4) in the left ear, 8

subjects were stimulated at the anterior wall of the auditory canal and left lower limb

as an anterior stimulation group; 8 persons who were arranged in a sham group

received taVNS at the left ear lobe and tail of the helix. Functional magnetic

resonance imaging (fMRI) data from the cortices was collected and an Alphasim

analysis was performed. We found that taVNS at auricular acupoints CO10-12, TF4

can instantly and effectively generate blood oxygenation level dependent (BOLD)

signal changes in the prefrontal, auditory and limbic cortices of healthy subjects by

fMRI. When comparing the acupoints group and the sham group in the left brain, the

signals from the prefrontal cortex, the auditory ascending pathway including superior

temporal gyrus, middle temporal gyrus, thalamus and limbic system regions such as

putamen, caudate, posterior cingulate cortex, amygdala and parahippocampal gyrus

were increased under our stimulation. The difference of the BOLD signal in the left

brain between acupoints group and anterior group was in the superior temporal gyrus.

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We could also find signal differences in several regions of right brain among the

groups. In conclusion, taVNS at acupoints CO10-12, TF4 could activate the prefrontal,

auditory and limbic cortices of healthy brain and this scheme could be a promising

tool for tinnitus treatment.

Key words

Transauricular vagus nerve stimulation; Acupoints; fMRI; BOLD

1. Introduction

Vagus nerve stimulation (VNS) in the modern literature refers to a technique where a

surgeon or researcher wraps a unidirectional wire around the vagus nerve in the neck

(George et al., 2010). With the success of several clinical trials, VNS was approved

for the treatment of refractory epilepsy and depression in 1997 (Yuan and Silberstein,

2016). To date, a wide variety of disorders were under consideration of VNS

treatment including anxiety, pain and Alzheimer’s disease. Owing to the similarities

between the pathogenesis of pain and tinnitus, scientists have recently tried to treat

tinnitus with VNS. De Ridder reported a case and demonstrated that vagus nerve

stimulation paired with tones could be an effective therapy for tinnitus (De ridder et

al., 2015).

The rationale for using the VNS therapy is based on the findings that VNS triggers the

release of several neuromodulators that are thought to enhance plastic changes in the

cerebral cortex; when paired with motor or sensory stimuli, it promotes a substantial

reorganization of cortical maps (Seol et al., 2007; Engineer et al., 2011; Hays et al.,

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2013; Yakuina et al., 2017). On the other hand, the neuromodulator pathways

involved in VNS therapy could be that stimulation of the vagus nerve activates the

nucleus tractus solitarius, which in turn activates locus coeruleus (norepinephrine) and

nucleus basalis (acetylcholine) (Lulic et al., 2009; Engineer et al., 2013).

As an easy, non-invasive and low cost technique for probing neurocircuitry and

treating illness, transauricular vagus nerve stimulation (taVNS) seems to be suitable in

dealing with the diseases mentioned above. The anatomical studies demonstrated that

the ear is the only place on the surface of the human body where there is afferent

vagus nerve distribution (Peuker et al., 2012). Thus, direct stimulation of the afferent

nerve fibers on the ear should produce an effect similar to classic VNS but without the

burden of surgical intervention (George et al., 2010; Vanneste et al., 2012; Yuan and

Silberstein, 2016).

Cymba concha is the approved taVNS stimulation position for the epilepsy,

depression and pain management in Europe (Yuan and Silberstein, 2016). Auricular

acupoints Kidney (CO10), Yidan (CO11), Liver (CO12) in traditional Chinese

medicine, primary-like or core areas of cymba chonca and triangular fossa, are usually

selected to treat mental illnesses or psychiatric disorders, such as insomnia, anxiety or

tinnitus for thousands of years. The Shenmen point (TF4) has the effect of

tranquilizing the mind (Li et al., 2015). Therefore we would like to know what kind of

effects that taVNS at CO10-12 and TF4 could induce in the brain and whether such a

method approved by traditional Chinese medicine could be used to treat tinnitus.

In this experiment we tested and compared the brain response induced by taVNS

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between traditional Chinese acupoints CO10-12, TF4 and anterior wall of the outer

auditory canal to enhance our understanding of specific auricular sites for taVNS in

potential tinnitus treatment.

2. Methods

2.1. Subjects

Totally twenty-four healthy subjects aged 28–38 years took part in this study. The

participants had a mean age of 33 ± 2.7 (mean ± SD) years and included 10 males and

14 females. All subjects were screened for their general physical condition in advance

to ensure primary health. Present or past neurological disorders, psychiatric disorders

or concurrent therapy treatment were ruled out by history talking and self evaluation.

Ethics committee approval was obtained and granted from Huazhong University of

Science and Technology (HUST). The subjects gave their written informed consents

and were instructed that they could withdraw from the experiment at any time. Eight

subjects were arranged in the acupoints stimulation group where the stimulation site

was in the area of the acupuncture points CO10-12 and TF4 (Alimi et al., 2017) (Fig.

1); eight subjects were included in the anterior stimulation group and they were

stimulated at the anterior wall of the auditory canal and left lower limb (left middle

shank). Eight subjects took part in the sham group; the stimulation electrodes were

attached to the center of the left ear lobe and tail of the helix separately.

Before performing the experiment, we performed a training session for all the subjects.

The stimulation procedure and the protocols of the MRI measurements were

explained to the participants. All subjects reported that they were used to the stimulus

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amplitude after a few seconds and then the current was increased stepwise up to a

level where a constant sensation was reached. The stimulus intensity was adjusted to a

level between detection threshold (first perceptible or tingling sensation) and pain

threshold (first pricking or unpleasant sensation). The stimulation algorithm (see

section below) was performed as in the later fMRI session. For this procedure, the

subjects laid horizontally on an examination couch. Blood pressure and heart rate

were recorded continuously before and after the fMRI scan.

2.2. TaVNS stimulation procedure

Two electrodes were applied in the following taVNS procedure simultaneously. The

stimulation electrodes which were MRI compatible consisted of a silver plate (5 mm

in diameter) and an elongated cylindrical silver stimulation electrode (8 mm in length，

3mm in diameter). In the acupoints group, the silver plate was placed in the left ear

triangular fossa; the cylindrical electrode was placed in the left cymba concha. In the

anterior stimulation group, the plate electrode was placed in the left external acoustic

meatus on the inner side of the tragus; the cylindrical electrode was placed on the left

lower limb (left middle shank). In the sham group, the stimulation electrodes were

attached to the center of the left ear lobe and tail of the helix. All electrodes were

connected to the stimulation device by copper cables. The stimulus was a

monophasic-modified rectangle impulse with a pulse width of 250 µs. Typically, the

electrical current amplitude threshold was around 5 mA on the acupuncture points

(varied individually between 4 and 8 mA). Stimulation frequency was kept at 20 Hz,

which was known to activate nerve fibers. The individual thresholds of perception and

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pain were determined for each subject. Electrical stimulus was applied from a widely

used transcutaneous electrical nerve stimulation device (TENS200, HUATUO GmbH,

Hangzhou, China).

2.3. Experimental protocol

Functional MRI sessions were performed using the following protocol: the

experiment lasted for 420 seconds and was started with a baseline lasting for 60

seconds. This was followed by a first stimulation period of 30 seconds and a

break/baseline of 60 seconds. Four alternating stimulation and baseline sequences

were performed in total.

2.4. Functional magnetic resonance imaging of the brain

Functional MRI was performed with a three dimensional 3.0-Tesla MRI scanner (GE

Medical Systems, Milwaukee, WI) at the institute of Radiology Department, Tongji

hospital, Wuhan, China. The head of the subject was fixed in a head coil by rubber

pads and both ears were plugged and eyes were closed. Three dimensional (3D) Fast

Imaging Employing FSPGR was performed with the following parameters: repetition

time (TR)=6.6ms; echo time (TE)=2.8ms; flip angle=60; contiguous 1.0mm slice

thickness; FOV=16cm/image; matrix=256*256; number of excitation (NEX)=1.

Possible head movements of the subjects were corrected using the motion correction

function of the SYNGO scanner software (Siemens Medical Solutions).

2.5. Functional MRI Data processing

Standard professional data post-processing software, Data Processing Assistant for

Resting-state fMRI (DPARSF 3.2 advanced edition,

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http://restfmri.net/forum/DPARSF), was used for data analysis. DPARSF is a plug-in

software based on statistical parametric mapping (SPM 8,

http://www.fil.ion.ucl.ac.uk/spm), which runs on a matrix laboratory platform

(MATLAB R2012a). Preprocessing included data format conversion, slice timing,

realignment, normalization, smoothing with a 6mm full width at half-maximum

gaussian kernel, and filtering (0.06–0.11Hz). Then dcm2nii package of MRIcroN

software was used to transfer each 4D.nii file to 150 paired .img and .hdr files which

could be distinguished by SPM 8. After specifying 1st-level analysis, .mat file were

generated, followed by the step of “estimate” and “results”. Finally, .con files were

extracted and reorganized into group A, group B and group C. ANOVA was used to

analyze inter-group differences among group A, group B and group C, and the

significantly different brain area was picked up to be set as a mask. Two-sample t-test

was further used to analyze the inter-group differences between group A & group B,

group C & group B and group A & group C, based on the mask extracted in the

ANOVA. Set cluster size > 54 voxel, volume > 1458 mm3, rmm=4 (surface connected)

and correct thresholds by AlphaSim, when viewing the result files.

3. Results

3.1. Psychophysics

The psychophysiological parameters (heart rate and blood pressure) were evaluated

during training session. After the stimulation, all subjects reported a relaxed yet

focused condition. Heart rate, systolic blood pressure and diastolic blood pressure

were measured immediately before and after taVNS. A one-sample

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Kolmogorov-Smirnov test was used to compare the distributions of our data to the

normal distribution. Paired Student T-test was used to compare the results before and

after the stimulation. The mean values and standard deviations of heart rate, systolic

blood pressure and diastolic blood pressure before the stimulation were 72 ± 6

beats/min, 105 ± 7 mmHg and 68 ± 5 mmHg, respectively. After the stimulation, the

mean values and standard deviations of heart rate, systolic blood pressure and

diastolic blood pressure were 70 ± 5 beats/min, 102 ± 5 mmHg and 67 ± 3 mmHg,

respectively. When comparing the data before and after the stimulation, the P values

of the heart rate (p=0.836), systolic blood pressure (p=0.696) and diastolic blood

pressure (p=0.410) showed no statistical differences (p > 0.05).

3.2. Cortical activation

The results were summarized in table 1 and table 2. In general, stimulation of the

acupoints CO10-12 and TF4 in the left ear led to activations of certain brain regions,

especially of left temporal and limbic areas. Almost the same results were found in the

anterior stimulation group. The only difference between these two groups was the

superior temporal gyrus of the left cerebrum. This region was not activated in anterior

stimulation group.

3.2.1. Acupoints vs. sham stimulation (Fig. 2 - 4)

There were significant activations in the left superior temporal gyrus, left and right

middle temporal gyrus, left and right thalamus in our experiment. The left and right

amygdala, left caudate, left posterior cingulum cortex, left parahippocampal gyrus and

left putamen, which were thought to be part of the limbic system, were also activated.

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We also found the left and right prefrontal cortex activated. Notably, there was

significant activation in Brodmann area 41 (MNI coordinates: -45, -46, 14) (Fig. 10),

which was the primary auditory cortex in the auditory pathway.

3.2.2. Anterior vs. sham stimulation (Fig. 5 - 7)

We could find significant activations in anterior stimulation compared with sham

stimulation group in the ascending auditory pathway regions such as the left middle

medial temporal gyrus, the left and right thalamus. The putamen and amygdala on

both sides, caudate, posterior cingulum cortex and parahippocampal gyrus in the left

side were also activated. The left side of the prefrontal cortex was activated.

3.2.3. Acupoints vs. anterior stimulation (Fig. 8 and 9)

The only different brain region between these two groups was the superior temporal

cortex. In the anterior stimulation group, this area was not significantly activated

compared to the acupoints group.

4. Discussion

The most significant finding of this study was that taVNS at auricular acupoints

Kidney (CO10), Yidan (CO11), Liver (CO12) and TF4 can instantly and effectively

generate BOLD signal changes in the auditory, limbic and prefrontal cortices of

healthy subjects as shown by fMRI. In our experiment the signals from the left

temporal gyrus including primary auditory cortex and the limbic system region such

as left putamen, left caudate and left posterior cingulate cortex, amygdala, left

parahippocampal gyrus were obviously increased following stimulation.

Firstly, left ear taVNS induced activations of the cortices in the ascending auditory

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transduction pathway. The signals from the left superior temporal gyrus, left middle

temporal gyrus and left thalamus increased robustly (Fig. 2 and 5). In the right brain,

the activated areas were the middle temporal gyrus (Fig. 3). Primary and auditory

association cortex (Brodmann Area 21, 22, 41, 42) (Fig. 4 and 10), or near medial

Heschl’s gyrus, were significantly activated in our experiments. Primary auditory

cortex region, which increased stimulus-evoked BOLD responses were demonstrated

in tinnitus patients in many human neuroimaging investigations (Gu et al., 2010;

Leaver et al., 2011, 2012), was thought to be an important area where the neural

synchrony and neural plasticity occurred to generate tinnitus (Wienbruch et al., 2006;

Weisz et al., 2007). The neural plasticity was evidenced by excitation and inhibition in

the receptive field of primary auditory cortical neurons after limited receptor organ

damage, which causes cortical map modifications that may result in tinnitus (Rajan et

al., 1998; Roberts et al., 2008). The secondary auditory cortex was involved in the

auditory non-classical pathway, which was the anatomical basis of the cross modal

interaction that provides the input of the somatosensory system to the auditory

pathway (Dehmel et al., 2008). These meant that taVNS was an effective input that

directly regulates the central auditory pathway. Therefore, we inferred that taVNS

could be a useful method to arouse the neural changes in auditory cortex of tinnitus

patients.

At the same time, left cingulate gyrus, left putamen, left caudate, left

parahippocampal gyrus, left and right amygdala were activated (Fig. 2 and 5). These

regions are known to be of importance in the pathophysiology of depressive states

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where limbic activity might underlie abnormal emotional processing (Drevets et al.,

2008; Cole et al., 2011). Many models of tinnitus pathophysiology proposed that

negative or emotional reactions to tinnitus may be involved in the progress of tinnitus.

It was considered that dysregulation of the auditory system by specific structures of

the limbic system converted subjective tinnitus to chronic form (Rauschecker et al.,

2010; De Ridder et al., 2011; Leaver et al., 2012). Typically, transient tinnitus can be

assessed by limbic frontostriatal networks as an unwanted stimulus and thus

suppressed. In patients with chronic tinnitus, this regulatory mechanism does not

function properly: the initial tinnitus signal could enter limbic networks via

projections from the auditory thalamus (MGN, medial geniculate nucleus) and/or

auditory cortex to the amygdala and nucleus accumbens. Similarly, limbic structures

could suppress auditory activity via projections between ventromedial prefrontal

cortex and MGN (Leaver et al. 2016). Although the specific methods and mechanisms

for the associated brain junctions need to be further defined, it was certain that the

auditory center of the tinnitus patient interacts with the limbic system. Combined with

our finding that taVNS could activate limbic system of normal subjects, we deduce

that taVNS could make changes to the limbic network of tinnitus patients.

It is interesting to note that our experiments also found activation of the prefrontal

cortex, which plays an important role in the cognitive and attention process (Fig. 4

and 7). Cognitive and attention processes beyond stress and affection are clearly

relevant to tinnitus. Patients must deal with the tinnitus percept as it interferes with

incoming auditory stimulus in their daily lives. Tinnitus signal may be under

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attentional control and some therapies such as cognitive behavior therapy train patient

to lessen the impact of tinnitus by attentional and cognitive strategies (Jastreboff,

2007; Roberts et al., 2013). Neuroimaging findings in regions outside the ascending

auditory pathway may support these processes. Sdywell et al had reported correlations

between patient-reported tinnitus loudness and increases in stimulus-evoked fMRI

signal in lateral prefrontal cortex (Seydell-Greenwald et al., 2012).

Auricular acupoints mentioned above are distributed in the regions supplied by

auricular branch of vagus nerve and trigeminal nerve. They have been used to treat

tinnitus for thousands of years in China (Chen, 2010). Putting new wine in old bottles,

we scientifically deduced that taVNS at CO10-12, TF4 could be a prospective

treatment for tinnitus patients through these ways: 1. TaVNS can activate primary and

secondary auditory cortex where the neural synchrony and plasticity occurred to

produce tinnitus; 2. Limbic network can be influenced by taVNS, a place which

causes subjective tinnitus to become chronic; 3. Prefrontal cortex activation was

changed under taVNS treatment, here cognitive and attentional processes are clearly

relevant to tinnitus process. 4. There are currently several clinical studies on taVNS

for the treatment of tinnitus and their results are promising.

Some results of our experiments differed from previous similar experiments. Kraus

and Stefan had reported that the signal from the limbic system was deactivated, but

our experiment showed that the limbic system was activated (Kraus et al., 2007, 2013;

Dietrich et al., 2008). In these observations, brain regions like left amygdala, middle

temporal gyrus were deactivated. But our findings showed the opposite. Yakunina et

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al summed up all the BOLD fMRI studies on vagus nerve stimulation (VNS) since

September 2016, with a total of seven VNS and four taVNS literatures included

(Yakunina et al., 2017). The results showed that the activation or deactivation status of

each brain region were not exactly the same in these studies. For example, about the

amygdala, kraus et al found it was deactivated after taVNS, but Frangos study

reported that it was activated (Frangos et al., 2015); secondly, about the cingulate

gyrus posterior, kraus reported deactivation and Dietrich and Frangos found it was

activated (Dietrich et al., 2008; Frangos et al., 2015); lastly, about the middle temporal

gyrus, most of the taVNS experiments did not report this area; Kraus reported and

found it was deactivated, but Bohning and Nahas found this region was activated after

VNS (Bohning et al., 2001; Nahas et al., 2007).

The possible reasons of the discrepancies among these results of the taVNS fMRI

studies, besides the different stimulus schemes, stimulation instruments and small

samples in the experiments, could be that the stimulus in these experiments induced

only transient responses which may be different from the long term stable changes of

the brain. For example, Henry et al published papers on acute and prolonged effects of

brain blood flow alterations induced by therapeutic vagus nerve stimulation in partial

epilepsy in 1998 and 2004 respectively (Henry et al., 1998, 2004). The results showed

that during immediate-effect studies, VNS decreased bilateral hippocampal, amygdala,

and cingulate cerebral blood flow and increased bilateral insular cerebral blood flow;

but no significant cerebral blood flow changes were observed in these regions during

prolonged studies. Therefore, our opinions on these discrepancies were that this

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deactivation or activation status was only a transient unstable brain reaction under

taVNS; the final effect of taVNS on brain determined by the BOLD should be based

on the fMRI results after long period treatment of taVNS.

There are several limitations to this pilot study that should be noted. Firstly, the

samples used in the current study were not large and our observations solely reflected

the acute changes in brain induced by taVNS. Parallel studies of VNS were inclined

to involve in the long-term effects, which will be addressed in our future taVNS

studies. Another limitation was that acupoint group and anterior stimulation group

were conducted in two different series, so inter-individual anatomical diversity has to

be taken into consideration when estimating the difference. Third, we didn’t show

brainstem activations (for example, locus coeruleus and solitary tract) that are

considered as mandatory for further subcortical and cortical activities. Last, it remains

unclear whether vagal afferences were the decisive cause of the displayed fMRI

hyperactivations by taVNS. Alternatively, nerves other than the vagus nerve, such as

the great auricular nerve or the auriculotemporal nerve, might also be responsible for

the observed effects, because the outer ear has several innervations. Therefore, further

studies are necessary to confirm taVNS induced activations of brain stem nuclei like

the tractus nucleus solitaries.

5. Conclusions

TaVNS at acupoints CO10-12, TF4 could activate primary and secondary auditory

cortex, limbic cortices and prefrontal cortices of healthy brain and this scheme could

be a promising tool for tinnitus treatment.

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Disclosure

The authors report no conflicts of interest. The authors alone are responsible for the

content and writing of the paper.

Funding information

This study was supported by grants from the National Natural Science Foundation of

China (project number: 81700909).

Acknowledgment

We are grateful to doctor Hui Dai for image processing and data statistics.

References

Alimi, D., Chelly, JE., 2017. New Universal Nomenclature in Auriculotherapy. J

Altern Complement Med. DOI:10.1089/acm.2016.0351

Bohning, D.E., Lomarev, M.P., Denslow, S., Nahas, Z., Shastri, A., George, M.S.,

2001. Feasibility of vagus nerve stimulation-synchronized blood oxygenation

level-dependent functional MRI. Invest Radiol. 36, 470–479.

Cheng, X.N., 2010. Chinese acupuncture and moxibustion. Foreign Language Press,

Beijing.

Cole, J., Costafreda, S.G., McGuffin, P., Fu, C.H., 2011. Hippocampal atrophy in first

episode depression: a meta-analysis of magnetic resonance imaging studies. J. Affect.

Disord. 134, 483-487.

De Ridder, D., Elgoyhen, A.B., Romo, R., Langguth, B., 2011. Phantom percepts:

tinnitus and pain as persisting aversive memory networks. Proc. Natl. Acad. Sci. USA.

108, 8075-8080.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

De Ridder, D., Kilgard, M., Engineer, N., 2015. Placebo-controlled vagus nerve

stimulation paired with tones in a patient with refractory tinnitus: a case report. Otol

Neurotol. 36, 575-580.

Dehmel, S., Cui, Y.L., Shore, S.E., 2008. Cross-modal interactions of auditory and

somatic inputs in the brainstem and midbrain and their imbalance in tinnitus and

deafness. Am. J. Audiol. 17, S193-209.

Dietrich, S., Smith, J., Scherzinger, C., Hofmann-Preiss, K., Freitag, T., Eisenkolb, A.,

Ringler, R., 2008. A novel transcutaneous vagus nerve stimulation leads to brainstem

and cerebral activations measured by functional MRI. Biomed Tech (Berl). 53, 104–

111.

Drevets, W.C., Price, J.L., Furey, M.L., 2008. Brain structural and functional

abnormalities in mood disorders: implications for neurocircuitry models of depression.

Brain. Struct. Funct. 213, 93-118.

Engineer, N.D., Møller, A.R., 2013. Directing neural plasticity to understand and treat

tinnitus. Hear Res. 295, 58-66.

Engineer, N.D., Riley, J.R., Seale, J.D., Vrana, W.A., Shetake, J.A., Sudanagunta, S.P.,

Borland, M.S., Kilgard, M.P., 2011. Reversing pathological neural activity using

targeted plasticity. Nature. 470, 101-104.

Frangos, E., Ellrich, J., Komisaruk, B.R., 2015. Non-invasive access to the vagus

nerve central projections via electrical stimulation of the external ear: fMRI evidence

in humans. Brain Stimul. 8, 624–636.

George, M.S., Aston-Jones, G., 2010. Noninvasive techniques for probing

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

neurocircuitry and treating illness: vagus nerve stimulation (VNS), transcranial

magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS).

Neuropsychopharmacology. 35, 301-316.

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, 3361-3370.

Hays, S.A., Rennaker, R.L., Kilgard, M.P., 2013. Targeting plasticity with vagus nerve

stimulation to treat neurological disease. Prog Brain Res. 207, 275–299.

Henry, T.R., Bakay, R.A.,Pennell, P.B., Epstein, C.M., Votaw, J.R., 2004. Brain

blood-flow alterations induced by therapeutic vagus nerve stimulation in partial

epilepsy: II. prolonged effects at high and low levels of stimulation. Epilepsia. 45,

1064-1070.

Henry, T.R., Bakay, R.A., Votaw, J.R., Pennell, P.B., Epstein, C.M., Faber, T.L.,

Grafton, S.T., Hoffman, J.M., 1998. Brain blood flow alterations induced by

therapeutic vagus nerve stimulation in partial epilepsy: I. Acute effects at high and

low levels of stimulation. Epilepsia. 39, 983-990.

Jastreboff, P.J., 2007. Tinnitus retraining therapy. Prog. Brain Res. 166, 415-423.

Kraus, T., Hosl, K., Kiess, O., Schanze, A., Kornhuber, J., Forster, C., 2007. BOLD

fMRI deactivation of limbic and temporal brain structures and mood enhancing effect

by transcutaneous vagus nerve stimulation. J Neural Transm (Vienna). 114, 1485–

1493.

Kraus, T., Kiess, O., Hosl, K., Terekhin, P., Kornhuber, J., Forster, C., 2013. CNS

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BOLD fMRI effects of sham-controlled transcutaneous electrical nerve stimulation in

the left outer auditory canal-a pilot study. Brain Stimul. 6, 798–804.

Leaver, A.M., Renier, L., Chevillet, M.A., Morgan, S., Kim, H.J., Rauschecker, J.P.,

2011. Dysregulation of limbic and auditory networks in tinnitus. Neuron. 69, 33-43.

Leaver, A.M., Seydell-Greenwald, A., Rauschecker, J.P., 2016. Auditory-limbic

interactions in chronic tinnitus: Challenges for neuroimaging research. Hear Res. 334,

49-57.

Leaver, A.M., Seydell-Greenwald, A., Turesky, T.K., Morgan, S., Kim, H.J.,

Rauschecker, J.P., 2012. Cortico-limbic morphology separates tinnitus from tinnitus

distress. Front. Syst. Neurosci. 6:21.

Li, T.T., Wang, Z.J., Yang, S.B, Zhu, J.H., Zhang, S.Z., Cai, S.J., Ma, W.H., Zhang,

D.Q., Mei, Z.G., 2015. Transcutaneous electrical stimulation at auricular acupoints

innervated by auricular branch of vagus nerve pairing tone for tinnitus: study protocol

for a randomized controlled clinical trail. Trails. 16:101.

Lulic, D., Ahmadian, A., Baaj, A.A., Benbadis, S.R., Vale, F.L.,2009. Vagus nerve

stimulation. Neurosurg Focus. 27:E5.

Nahas, Z., Teneback, C., Chae, J.H., Mu, Q., Molnar, C., Kozel, F.A., Walker, J.,

Anderson, B., Koola, J., Kose, S., Lomarev, M., Bohning, D.E., George, M.S., 2007.

Serial vagus nerve stimulation functional MRI in treatment-resistant depression.

Neuropsychopharmacology. 32, 1649–1660.

Peuker, E.T., Filler, T.J., 2002. The nerve supply of the human auricle. Clin. Anat. 15,

35-37.

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Rajan, R., Irvine, D.R., 1998. Neuronal responses across cortical field A1 in plasticity

induced by peripheral auditory organ damage. Audiol. Neurootol. 3,123-144.

Rauschecker, J.P., Leaver, A.M., Mühlau, M., 2010. Tuning out the noise:

limbic-auditory interactions in tinnitus. Neuron. 66, 819-826.

Roberts, L.E., Husain, F.T., Eggermont, J.J., 2013. Role of attention in the generation

and modulation of tinnitus. Neurosci. Biobehav. Rev. 37, 1754-1773.

Seol, G.H., Ziburkus, J., Huang, S., Song, L., Kim, I.T., Takamiya, K., Huganir, R.L.,

Lee, H.K., Kirkwood, A., 2007. Neuromodulators control the polarity of

spiketiming-dependent synaptic plasticity. Neuron. 55, 919-929.

Seydell-Greenwald, A., Leaver, A.M., Turesky, T.K., Ward, L.M., Bosnyak, D.J., 2012.

Functional MRI evidence for a role of ventral prefrontal cortex in tinnitus. Brain Res.

1485,22-39.

Vanneste, S., De Ridder, D., 2012. Noninvasive and invasive neuromodulation for the

treatment of tinnitus: an overview. Neuromodulation. 15, 350-360.

Weisz, N., Müller, S., Schlee, W., Dohrmann, K., Hartmann, T., Elbert, T., 2007. The

neural code of auditory phantom perception. J. Neurosci. 27, 1479-1484.

Wienbruch, C., Pau,l I., Weisz, N., Elbert, T., Roberts, L.E., 2006. Frequency

organization of the 40-Hz auditory steady-state response in normal hearing and in

tinnitus. Neuroimage. 33, 180-194.

Yakunina, N., Kim, S.S., 2017. Optimization of Transcutaneous Vagus Nerve

Stimulation Using Functional MRI. Neuromodulation. 20, 290-300.

Yuan, H., Silberstein, S.D., 2016. Vagus Nerve and Vagus Nerve Stimulation, a

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Comprehensive Review: Part II. Headache. 56, 259-266.

Legend

Fig. 1.

Top left: This illustration of the right anterior ear was labeled to indicate the

anatomical terminology used for different regions of the auricle. Top right: This

drawing of the external ear showed the broad projection of three cranial nerves to

three different territories of auricle. Below left: The ear acupuncture nomenclature

system developed in China after the WHO international conference in 1990 and the

whole concha region of the ear is represented by the letters “CO”. Below right:

Sketch showing the positions of the specific auricular acupoints CO10-12, TF4 and

the innervation of the human auricle, including auricular vagus nerve and trigeminal

nerve (blue grid), great auricular nerve (green grid) and auriculotemporal nerve (red

grid). The first three pictures came from Auriculotherapy manual 4rd written by Terry

Oleson.

Fig. 2. Activation map which showed the contrasts of acupoints vs. sham stimulation

group of the left brain. Brain regions of red color meant the differentially activated

areas between two groups. MNI coordinate of the activated brain slices were recorded

below the MRI pictures respectively and marked by red crosshairs in the pictures.

Fig. 3. Activation map which showed the contrasts of acupoints vs. sham stimulation

group of the right brain. Brain regions of red color meant the differentially activated

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Fig. 4. The results of figure 2 were comprehensively analyzed in axial, coronal and

sagittal slices, the numbers denote the respective region in the Brodmann Area (BA).

Middle temporal gyrus (BA21) corresponded to green color and superior temporal

gyrus (BA22) meant pink, primary and auditory association cortex (BA41, 42) was

brown and red. Cingulate cortex (BA23) was green. Prefrontal gyrus (BA9)

corresponded to yellow. Parahippocampal gyrus (BA34) was showed in purple.

Fig. 5. Activation map which showed the contrasts of anterior vs. sham stimulation

group of the left brain. Brain regions of red color meant the differentially activated



Fig. 6. Activation map which showed the contrasts of anterior vs. sham stimulation

group of the right brain. Brain regions of red color meant the differentially activated




sagittal slices, the numbers denote the respective region. Prefrontal gyrus (BA46)

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corresponded to green and precuneus (AAL67) corresponded to crimson in

Anatomical Automatic Labeling (AAL) space.

Fig. 8. Activation map which showed the contrasts of anterior vs. acupoints

stimulation group. MNI coordinate of the differentially activated brain slices were

recorded below the MRI pictures and marked by red crosshairs in the pictures.


sagittal slices, the numbers denote the respective region in the Brodmann Area (BA).

Middle temporal gyrus (BA21) corresponded to green color.

Fig. 10. MNI cordinate of Brodmann Area 41 (primary and auditory association

cortex) in axial, coronal and sagittal slices.

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Table 1 Changes in BOLD signal of left brain by acupoints vs. anterior vs. sham stimulation

Acupoints VS Sham Anterior VS Sham Acupoints VS Anterior

Brain region Voxel Tvalue MNI coordinate

Voxel Tvalue MNI coordinate


X Y Z X Y Z X Y Z

superior Temporal gyrus 165 2.849 -59 -47 15 - - - - - 94 3.1301 -45 -48 15 middle temporal gyrus 137 4.369 -46 -47 15 24 2.39 -42 -68 21 - - - - - thalamus 93 4.385 -7 -26 7 51 2.67 -7 -17 -1 - - - - - putamen 89 2.1443 -22 20 6 89 2.75 -14 10 -9 - - - - - caudate 65 2.1783 -19 20 6 62 3.46 -17 22 8 - - - - - posterior cingulum cortex 143 3.4609 -9 -46 35 50 4.33 -10 -49 33 - - - - - amygdala 2 3.5988 -25 1 -12 5 2.75 -20 0 -11 - - - - - parahippocampal gyrus 13 2.119 -23 -3 -7 6 2.35 -16 4 -18 - - - - - prefrontal cortex 169 3.630 -14 52 35 4 4.09 -14 64 20 - - - - -

Note: Results of taVNS in the left ear. Voxel number, t-values and p-values (p < 0.05) were obtained from the statistical parametric mapping of the Alphasim analysis.

Positive t-values represented an increase of the BOLD signal during stimulation in the respective brain region. The MNI coordinates reflected the center of gravity of

the cluster as found by the AlphaSim analysis. The minimum cluster size was 1458mm3 during the fMRI data analysis.

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Table 2 Changes in BOLD signal of right brain by acupoints vs. anterior vs. sham stimulation

Acupoints VS Sham Anterior VS Sham Acupoints VS Anterior

Brain region Voxel Tvalue MNI coordinate



X Y Z X Y Z X Y Z

middle temporal gyrus 90 3.559 53 -66 15 - - - - - - - - - - thalamus 48 3.35 7 -26 5 24 2.82 7 -11 -1 - - - - - amygdala 4 3.075 25 4 -12 2 3.6 18 0 -12 - - - - - prefrontal cortex 87 3.23 14 58 35 - - - - - - - - - -

Note: Results of taVNS in the left ear. Voxel number, t-values and p-values (p < 0.05) were obtained from the statistical parametric mapping of the alphasim analysis.

Positive t-values represented an increase of the BOLD signal during stimulation in the respective brain region. The MNI coordinates reflected the center of gravity of

the cluster as found by the AlphaSim analysis. The minimum cluster size was 1458mm3 during the fMRI data analysis.

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HIGHLIGHTS TaVNS at acupoints CO10-12, TF4 can arouse BOLD signals by fMRI in temporal gyrus and limbic cortices TaVNS at acupoint CO10-12, TF4 can induce signals from the prefrontal cortex TaVNS at acupoint CO10-12, TF4 could be a potential treatment for tinnitus.

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Transauricular vagus nerve stimulation at auricular