Vagus Nerve Stimulation: Integration of Behavior and Cardiac Modulation
Phase: NA
Conditions: Major Depressive Disorder
Sponsor: Massachusetts General Hospital

Study Protocol 
Study: Vagus Nerve Stimulation: Integration of Behavior and Cardiac Modulation 
NCT number: NCT04467164 
Document date: 09-05-2017 

 
 
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PARTNERS HUMAN RESEARCH COMMITTEE 
DETAILED PROTOCOL 
 
 
Principal Investigator: Jill Goldstein, PhD 
Title: Vagus Nerve Stimulation: Integration of Behavior and Cardiac 
Modulation 
 
September 5, 2017 
 
 
I. BACKGROUND AND SIGNIFICANCE 
 
Implantable VNS (iVNS) has been safely used to treat thousands of patients with depression. 
Eighty percent of the fibers in the vagus nerve are afferents that project to the periaqueductal grey (PAG), 
nucleus tractus solitarius, limbic system and thalamus. Secondarily, there are projections to the prefrontal 
cortex, inferior and superior parietal cortex and basal ganglia. Imaging studies in MDD patients have 
confirmed the broad reach of vagal afferents. In depressed patients, iVNS/fMRI interleaving activated 
bilateral orbitofrontal cortex, parieto-occiptal cortex, hypothalamus, left amygdala and globus pallidus, 
areas implicated in the pathophysiology of MDD.  
However, iVNS has side effects that limit its attractiveness as a treatment. The location of the 
stimulating electrode is within the vicinity of the recurrent laryngeal nerve, thus can cause hoarseness 
during stimulation, i.e. every 5 minutes. In addition, it is an invasive procedure with associated anesthesia 
and surgical risks. Transcutaneous VNS (tVNS) (placed in the ear) is an alternative that has been studied 
in healthy subjects. The auricular branch of the vagus nerve carries only vagal afferents unlike the vagal 
branch used in iVNS, which contains 20% efferents to the heart. Stimulating the auricular branch thus 
carries no risk of direct suppression of heart rate. tVNS is a non-invasive procedure that can be easily 
removed from the ear. Studies have shown that tVNS stimulates overlapping neural networks in healthy 
controls as iVNS in depressed patients, including hippocampus, amygdala, anterior cingulate, insula and 
thalamus. Dr. Vitaly Napadow, collaborator, has demonstrated that tVNS decreases anxiety symptoms in 
women with chronic pain, a symptom highly comorbid in depression. Therefore, tVNS may be a lower 
risk and effective means of treating patients with MDD. Recently, a pilot study of tVNS was performed in 
depressed patients and demonstrated significant decreases in depressive symptoms compared to sham.  
The circuits stimulated by VNS (i.e. mood circuitry) overlap with the circuits for autonomic 
nervous system (ANS) regulation. Heart rate variability (HRV) is an important measure of the health of 
ANS. HRV analysis indirectly estimates sympathetic and parasympathetic modulation of the heart. The 
peak in low-frequency spectral band (LF) is thought to be influenced by both parasympathetic and 
sympathetic activity. The peak in high-frequency band (HF) is influenced solely by parasympathetic 
(vagal) activity and the LF/HF ratio approximates the balance between sympathetic and parasympathetic 
modulation of the heart. Deficits in vagal tone have been found in patients with MDD as evidenced by 
decreased HF band. Vagal output from the hypothalamus projects to the heart and releases acetylcholine 
to slow heart rate. Vagal afferents provide feedback regulation of the heart through the nucleus tractus 
solitaries, which then projects to the heart. This feedback on heart rate can come from any of the vagal 
afferent branches. The auricular branch of the vagus nerve is an easily accessible vagus branch that can be 
directly stimulated. The dorsal medullary vagal system operates in tune with respiration, and therefore 
stimulation gated to exhalation/inhalation might have modulatory effects on vagal tone. The systematic 

 
 
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study of respiratory-gated tVNS in MDD patients may reveal whether in addition to regulating mood, it 
can regulate HRV.  
Although deficits in vagal tone have been found in MDD, VNS has not yet been used in MDD 
patients to improve vagal tone. There have been reports of bradycardia and even asystole during 
implantation of iVNS. This may be due to direct stimulation of the vagal efferents of the target branch of 
the vagus. Although cardio-depressant effects have not been seen post operatively during active iVNS 
stimulation, modulation of vagal tone after iVNS has been reported. Complex studies of heart rate and the 
influences of vagal tone have not been performed in MDD with tVNS. Acupressure to the auricular 
branch of the vagus nerve restored sleep in post menopausal women. More importantly, the effect was 
only found in women who also had an increase in HF-HRV. If chronic tVNS normalizes HRV in MDD, it 
has the potential to have a significant impact on public health considering the high comorbidity between 
MDD and cardiovascular disease. Deficits in vagal tone have been associated with the comorbidity of 
MDD and CVD. Modulation of HRV chronically in MDD may thereby decrease risk for cardiac 
morbidity and mortality.  
 
II. SPECIFIC AIMS 
 
Major Depressive Disorder (MDD) is a leading cause of morbidity and mortality worldwide. 
Abnormalities in vagal tone are more common in MDD, particularly loss of parasympathetic cardiac 
regulation, and increase the risk for cardiovascular disease (CVD). Vagal nerve stimulation (VNS) is an 
FDA approved treatment for MDD that modulates circuitry implicated in mood regulation. However it is 
unclear whether therapeutic benefit of VNS on mood, can also reduce cardiac morbidity and mortality. The 
circuits for autonomic cardiac regulation overlap with those for mood regulation, but modulation of ANS 
has not been directly associated with mood response. As a first step, we will identify whether VNS effects 
on mood circuitry in MDD are associated with modulation of vagal tone and mood.  
In the proposed study in MDD patients, we will 1) identify the neural circuitry associated with tVNS, 
2) identify its acute effects on heart rate variability, and 3) assess the impact of brain activity and ANS 
function on mood. We will control for factors known to impact vagal tone, in particular gonadal hormones 
in women. This is the first study to map the neurobiology of mood and vagal response to VNS using a less 
invasive form of VNS that may have a wider application.  
 
Specific Aim 1: To determine the acute effects tVNS on cerebral blood flow in MDD patients. 
Hypothesis 1: During real tVNS/fMRI interleaving, tVNS will deactivate amygdala, parahippocampal 
gyrus, hippocampus, posterior cingulate, and precuneus and will activate insula, thalamus and anterior 
cingulate.  
Hypothesis 2: Brain activity deficits in stress response circuitry in MDD will be attenuated following 20 
minutes of tVNS, in hypothalamus, amygdala, hippocampus, and anterior cingulate gyrus. 
Specific Aim 2: To determine the acute effects of tVNS on vagal tone and mood in MDD patients.  
Hypothesis 1: tVNS will increase high frequency R-R variability and decrease mood symptoms compared 
to baseline. 
Specific Aim 3: To compare the effects of exhalation-gated tVNS with inhalation-gated tVNS on cerebral 
blood flow, vagal tone and mood regulation in MDD patients. 
Hypothesis 1: Exhalation-gated tVNS will have a significant greater impact on regulation of cerebral blood 
flow, vagal tone and mood in MDD patients compared to inhalation-gated tVNS.  
 
 
 
III. SUBJECT SELECTION 
 

 
 
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About 25 female, right-handed subjects will take part in this research study at Brigham and 
Women’s Hospital (BWH). (Up to) the first 5 subjects enrolled in the study will not have major depressive 
disorder (MDD). These non-MDD (test) subjects will do some of the study procedures so that we can make 
any changes before we enroll subjects with major depression. Following finalization of study procedures, 
up to 20 female subjects with MDD will be enrolled. Subjects with major depression will participate in 2 
telephone screenings and/or a REDCap online questionnaire, an evaluation visit, two MRI testing sessions, 
and four follow-up evaluations. The non-MDD test subjects will only participate in the telephone screening 
and one MRI session. 
 
Inclusion Criteria/Exclusion Criteria: 
Inclusion Criteria  
Patients with MDD will be women between 25-40 years of age meeting DSM criteria of recurrent 
MDD as assessed by SCID.  In addition, up to five non-MDD subjects will be enrolled to test all procedures 
prior to enrollment of MDD subjects. MDD patients with a comorbidity of PTSD will be considered for 
clinical applicability by our senior clinicians on a case by case basis. 
 
Exclusion Criteria 
• History of Axis I psychiatric diagnosis other than MDD or anxiety disorder - e.g., substance use 
disorder, psychotic disorder, or bipolar disorder. 
• Current Suicidal Ideation with intent and/or plan or history of suicide attempt within the last year 
• Use of psychotropic medications within four weeks prior to study with the exception of SSRI and 
SNRI class of antidpressant medication only 
• Use of Tricyclic antidepressants (TCAs), Monoamine oxidase inhibitors (MAOIs), and Atypical 
agents 
• History of cardiovascular disease 
• History of neuroleptic use 
• Past history of substance abuse or dependence within the past 12 months (excludes nicotine) 
• Bleeding disorder or use of anticoagulants. 
• Pregnancy 
• Metallic implants or devices contraindicating magnetic resonance imaging. 
• Use of beta blockers 
 
Verbal Screening 
Women of childbearing age will be asked if they think they are pregnant and if they are trying to 
get pregnant. Upon self-report, if they are not sure whether they are pregnant, OR if they are trying to get 
pregnant, then they will not be allowed to participate in this study. Negative pregnancy test allows study 
entrance.  
 
Source of subjects and recruitment methods 
Healthy adult female volunteers with MDD (and up to five test subject volunteers without MDD) 
will be recruited by poster, e-mail and website advertisements (see attached flier), phone call (with prior 
consent to contact), as well as from the outpatient psychiatry clinic and the registry Research Study 
Volunteer Program (RSVP for Health). The research assistant will first perform a phone interview with the 
subjects where an initial screening will take place to distinguish potential subjects from those not meeting 
eligibility criteria (as detailed above). If a potential subject responds to the REDCap online survey and 
meets eligibility criteria, the research assistant will contact the subject for further scheduling and to answer 
any questions. Efforts will be made to attain a mix of study participants in terms of racial/ethnic 
representation as determined by NIH guidelines. Children will not be included because of differences in 

 
 
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brain development with age. It would also be difficult for them to lie still throughout the scanning period. 
Patients will be recruited with the help of outpatient psychiatrists and colleagues. 
 
 
IV. SUBJECT ENROLLMENT 
 
Method of enrollment 
When contacted by a potential volunteer, the research assistant will either first perform an initial 
screening interview via phone.  This will be done to distinguish potential subjects from those not meeting 
eligibility criteria. During this initial interview, we will explain the purpose, procedures, and potential 
discomforts/risks of the study. If a potential subject responds to the REDCap online questionnaire and meets 
eligibility criteria, the research assistant will contact the subject to schedule her for a second phone 
screening and to answer any questions. If the subject is eligible by the initial phone screening or REDCap 
online questionnaire, a clinician Interviewer will have a second phone screening interview. This second 
phone screening interview will help determine if the subject is eligible for an in person evaluation. On the 
day of the evaluation, MDD subjects will again be explained the purpose, procedures, and potential 
discomforts/risks of the study. They will again undergo a deeper screening procedure and will be asked 
about their medical and psychiatric history and fill out a handedness questionnaire to assess that they are 
right handed (see attached forms/questionnaires). The evaluation is expected to take about 90 minutes. Only 
English speaking subjects will be enrolled as we do not have a Spanish speaking clinician to perform 
assessments.  Non-MDD test subjects will only participate in the telephone screening and one MRI session, 
and thus will not participate in the fill screening appointment. 
 
Procedure for obtaining informed consent  
After the initial phone screenings detailed above the post doctoral fellow will explain the purpose, 
procedures, and potential discomforts/risks of the study and then obtain a signed informed consent from the 
prospective subject.  
Subjects will be paid $25 for the Clinical Evaluation, $150 for each MRI appointment in which they 
participate and $25 for a telephone follow-up evaluation. If scanning is stopped due to subject discomfort, 
technical problems, or if a subject is found ineligible prior to entering the scanner, they will still be 
compensated for their visit.  
 
Confidentiality 
To respect confidentiality and the HIPAA rules, the subject’s name is not stored with their data. All 
the participant’s identities and data will be held in confidential files. On entry to the study subjects will be 
assigned a study ID#, and all files will use this ID for future references. All information regarding 
experimental subjects will be kept in a locked file cabinet in the office of the principal investigator. All 
study staff are IRB certified and all information and records are in compliance with HIPAA rules. 
 
 
V. STUDY PROCEDURES 
 
Study visits and parameters to be measured 
Experimental Design Overview 
The fMRI scanning session will take approximately 90 minutes. There are 2 conditions, i.e. two 
scanning sessions (note: 5 non-MDD test subjects have one scanning session only). Both conditions will 
consist of one 30 minute tVNS session, one inhalation-gated the other exhalation-gated. During both 

 
 
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conditions, we will perform electrical stimulation to the ear, to stimulate the auricular branch of the vagus. 
Stimulation parameters will be identical.  
 
tVNS Procedures for all Scans 
Periods of tVNS (inhalation-gated vs. exhalation-gated) stimulation will alternate with periods of 
rest, during which time the electrodes will remain in 
place.  
tVNS will be performed at the left ear by either 
Dr. Vitaly Napadow (trained in tVNS) or the post-
doctoral fellow. Two reusable MRI compatible 
electrodes will be attached to the cymba and cavum 
concha (Figure 2) of the auricle. Current will be 
delivered with UROstim transcutaneous electrical 
stimulator 
(Schwa 
Medico, 
Germany). 
Current 
amplitude will be set between the sensitivity threshold 
and the pain threshold for each subject. The waveform 
will consist of monophasic rectangular pulses. The 
stimulation parameters will include 30 Hz stimuli with 300 μsec pulse width. The output current will be 
increased gradually to a maximum of 8 mA (in 0.25-mA increments) to allow accommodation to the 
stimulation until a comfortable tolerance level is reached. Stimulation will be on for 30 seconds and off for 
30 seconds during the fMRI scan runs. These stimulation parameters and procedures are similar to those 
used in iVNS for depression. Respiratory gating for stimulation will require real-time evaluation of the 
respiratory cycle. An adaptive threshold detection method will be employed to detect end-inspiration and 
end-expiration in real-time and provide a signal controlling the delivery of the stimuli during the exhalatory 
or inhalatory pahse of respiration. Correct respiratory-gated stimulation will be confirmed by the 
experimenter via running chart of the respiration signal and stimulus pulse.  
Since receptors respond directly to current load and normal tissue response alters electrical 
resistance, it is important to use a current-constant EA stimulation device. A voltage-constant device 
(common in clinical practice) produces variable current load, and leads to variations in stimulation intensity.  
 
Clinical Assessments 
Outcomes will include whether acute stimulation with tVNS reduces mood symptoms. Our group 
has found tVNS diminishes anxiety in an acute paradigm. Measures at intake will include: Structured 
Clinical Interview for DSM Disorders, Hamilton Rating Scale for Depression 17, and the Montgomery 
Asperger Depression Rating Scale. Before and after tVNS/fMRI, we will assess mood with the Beck 
Depression Inventory-II,  Hamilton Rating Scale for Depression 17,  Spielberger State-Trait Anxiety 
Interview, and a Visual Analog Scale. 
 
Week 2 
Week 1 
Session 2: 
1)ECG and mood & anxiety 
assessments pre-post 
2)fMRI during mild stress 
task with ECG – pre-post  
3)fMRI and tVNS with ECG  
Session 3: 
1)ECG and mood & anxiety 
assessments pre-post 
2)fMRI during mild stress task 
with ECG – pre-post  
3)fMRI and tVNS with ECG  
Session 1: 
Screening, Evaluation 
and Questionnaires  
Day 0 
Figure 2 – tVNS electrode placement 

 
 
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Blood Draw 
Our on-site Phlebotomist will draw one sample [(approximately 5 teaspoons (26 ml)] at the 
beginning and another blood sample [approximately 3 teaspoons (13 ml)] after each of the two imaging 
visits. Previous studies from our group have demonstrated gonadal hormonal regulation of mood and 
autonomic nervous system circuitry. In addition, a significant body of evidence has suggested an 
interrelationship between the activity of the autonomic nervous system, particularly the vagus nerve and 
the immune system. We will use estradiol, progesterone, cytokines (IL-1b, IL-6, IL-10, TNFα), and C-
reactive protein (CRP) levels as covariates in the analysis of tVNS effects in the activation of brain 
regions related with mood and autonomic modulation. The results of the blood tests are not clinically 
relevant to individual persons; therefore, we will not return results to the subject. However, when the 
study analyses are complete, we will make study results available to the subject, if desired (although 
individuals are not identified in these analyses). 
 
Saliva Collection 
We would like to collect a sample of saliva (15 ml saliva tube) to examine cortisol levels in order 
to evaluate the state of the hypothalamic-pituitary-adrenal (HPA)-axis in depressed subjects. Collection 
will take place the evening before the scan (11pm), prior to the scan, and 60 minutes following the picture 
task.  This variable will be considered in the analysis of the effects of our intervention in the modulation 
of the stress response circuitry and central autonomic network. The samples will have no personal 
identifying information and will be placed in a storage bank in secure buildings in the Boston medical 
area that have limited access. They will be used only for research that is carefully reviewed and approved 
both by this study and the Institutional Review Board. 
 
 
Scanning 
Subjects will be randomized as to the order of scan dates (inhalation-gated vs. exhalation-gated), 
which will be separated by at least one week. Scanning begins with the subject at full rest then they will 
undergo our mild visual stress paradigm for 18 minutes. Then the electrodes will be placed. The stimulation 
block will have a total duration of 30 minutes. The electrodes will be withdrawn at 20 minutes after and the 
mild visual stress task repeated. Patients will first undergo a mild stress task where they view pictures during 
fMRI scanning. Our fMRI stress paradigm has been described in detail previously  and conducted in healthy 
subjects, MDD, and psychotic cases. Briefly, subjects view 3 stimulus blocks (6 images per block) for 30 
seconds (1 image every 5 seconds) in the following order: fixation, neutral, and negative images (see 
definitions below). Subjects view this sequence 4 times during each of 3 runs for a total of 72 images (24 
images per condition per run), and press a button each time a new image appears in order to ensure attention 
to the images, without adding cognitive load. Images were drawn from the International Affective Picture 
System (IAPS) according to affective valence and arousal (negative = unpleasant + high arousal, neutral = 
neutral + low arousal). Fixation images are based on Fourier transformations of each neutral image to create 
an image with the same physical properties of the original but without recognizable content. The stress task 
will be repeated after the tVNS portion of imaging. When the fMRI session is complete, subjects will be 
shown two blocks of negative and neutral images sampled from the set shown during imaging and will be 
asked to provide subjective ratings of arousal using the Self-Assessment Manikin (SAM). 
 
Post-Scan Follow Ups 
       After each fMRI scan we will follow up with each subject to assess their mood. We will follow up one 
day and seven days after each scan. The follow-ups one day after the scan will consist in the administration 
of a Visual Analogue Scale for mood states through a REDcap online survey.  For the 7th day follow-ups 
we will administer the Hamilton Depression Scale, Beck Depression Inventory-II, Spielberger State-Trait 
Anxiety Interview, and the Visual Analog Scale. The first 7th day follow up will be conducted in person at 
Imaging Visit #2. The second 7th day follow up will be conducted by phone. These follow ups will evaluate 
acute changes in mood following tVNS administration during the imaging visits. 

 
 
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Acquisition Parameters for FMRI and Physiological Data Collection 
General MRI Scanning Procedure for Subjects: When the subject arrives for the MRI scan, she will be 
asked to fill out a brief questionnaire in order to confirm that they can safely enter the MRI scanner.  She 
will be asked to remove any metallic items from her body and may be given non-metallic shirts and pants 
to wear in the scanning room.  The participants will be instructed to lie on the MRI table.  Once the 
participant is positioned comfortably on the MRI table, the table will be moved into the MRI scanner tube.  
The participant will be able to communicate with the operator outside of the MRI room. The participant 
will be asked to lie still during imaging. To help immobilize the participant's head, foam pillows will be 
placed under and around the head. Subjects may be asked to use a “bite bar” which conforms to the teeth, 
or a plastic mask that conforms to the face, to help keep the head still. Since the MRI scanner makes loud 
knocking or beeping sounds during the scan, the participant will be asked to wear earplugs during imaging.  
If the participant becomes uncomfortable at any time, the scan will be stopped and the problem eliminated.  
If it is not possible to alleviate the problem the scan will stopped and the participant will be free to 
discontinue participation.   
Structural MRI: To provide anatomical localization of the functional signal, high-resolution (voxel size= 1 
mm3) structural MRI scans will be acquired using a T1-weighted MP-RAGE pulse sequence (TR= 2300 
ms, TE= 2.95 ms, acceleration= GRAPPA factor 2, flip angle= 9º, FOV= 256 x 256 mm2, 176 axial slices).  
BOLD imaging: Whole-brain fMRI data will be acquired with gradient-echo echo-planar imaging (EPI) 
using Simultaneous Multi-Slice (SMS) acquisition with multi-band factor 5 (TR= 1250ms, TE= 33 ms, flip 
angle= 65º, FOV= 200 x 200 mm2, 75 axial slices, voxel size= 2 mm3). 
Physiological Monitoring During fMRI Scanning: Peripheral physiological data will be acquired using a 
Powerlab system (ML880; ADInstruments Inc, Colorado Springs, CO) at a 1000 Hz sampling rate.  We 
will use a magnetic resonance-compatible pneumatic belt placed around the subject’s lower thorax to 
evaluate changes in respiratory volume. In addition, cardiac pulsatility data will be acquired using a 
piezoelectric pulse transducer (AD instruments Inc., Colorado Springs, CO) attached to the right index 
finger.  
 
Data Storage  
Any data obtained from subjects during this study will not leave our institute in any form that would 
identify individual subjects. All subject data will be identified by a subject ID number. The PI will keep a 
master list of subject names and corresponding subject ID numbers in a locked cabinet at the BWH. 
Electronic data will be housed in secure, password protected computer accounts.     
 
 
VI. BIOSTATISTICAL ANALYSIS 
 
Specific data variables being collected for the study 
For this study data will be collected from MRI in electronic form. Subjects will also provide data in 
the form of questionnaires related to mood and medical history. Copies of these subject 
forms/questionnaires are included with this application.  
   
Sample Size (Power Analysis) 
The present study will recruit a total of 20 subjects (women age 25-40 with recurrent MDD). This 
will allow for the possibility of unusable data due to subject motion during scanning, scanner artifact 
problems as well as problems with the collection of behavioral data and human error.  
Group size power statistics: In a previous study of manual acupuncture (N = 11), we were able to detect 
changes in activation of limbic and subcortical gray structures in the approximate range of -0.7% to -0.2%.  

 
 
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Also based on previous data (amygdala, manual acupuncture stimulation), we will assume a standard 
deviation of BOLD signal change of 2.5% for the purpose of power calculation and sample size 
determination. We do not consider temporal autocorrelation in the power calculations that follow, though 
we will take autocorrelation into account during the data analysis. 
 
Statistical Analysis and Methods 
 
Behavioral Outcomes analysis  
  
The Beck Depression Inventory-II and the Spielberger State-Trait Anxiety Inventory will be used 
to evaluate acute changes in mood and anxiety symptoms. Behavioral data will be analyzed using repeated 
measures ANOVA [mood and anxiety ratings by time (PRE-, POST-stimulation) and session (exhalation-
gated, inhalation-gated)], controlled for baseline.  
 
Magnetic resonance imaging data analysis 
  
Comparisons of interest in the fMRI analysis (BOLD signal changes during exposure to negative vs 
neutral images) will be tested using linear contrasts and results from the individual subject level will be 
submitted to a second-level factorial analysis with two factors: time of visual stress task (PRE- or POST-
stimulation) and stimulation session (exhalation-gated or inhalation-gated). Significant interactions 
between these factors will be submitted to post-hoc analyses to determine directionality of the effect. Due 
to our a priori hypothesis of specific effects of tVNS on stress response circuitry, the analysis will be 
restricted to specific regions of interest (ROIs) using a small volume correction approach. A voxel-wise 
height threshold of p<0.001 (uncorrected for multiple comparisons), and a cluster correction with FWE p-
value<0.05 will be applied. Mean beta weights within each significant cluster will be extracted for each 
participant using the REX toolbox and will be included in regression analyses in STATA (StataCorp, 
College Station TX) to investigate the link between variations in stress response circuitry activity and 
changes in depressive symptoms (BDI score difference: POST-PRE) after tVNS administration. Adjusted 
regression coefficients will be calculated after including baseline BDI scores and use of antidepressant 
medications in the models. 
  
Functional connectivity will be evaluated during tVNS stimulation runs by extracting fMRI time 
series from the selected ROIs and using them in a weighted GLM and semi-partial correlation analyses. 
Resultant whole-brain parameter estimates and their variance from each individual will be passed to group-
level analyses to evaluate connectivity differences between exhalatory- and inhalatory-gated sessions. For 
evaluation of functional connectivity during IAPS, the extracted fMRI time series from the selected ROIs 
and its interaction with the regressors for negative and neutral content will be used to evaluate connectivity 
differences between the visual stress tasks during PRE- and POST-stimulation, for exhalatory- and 
inhalatory-gated sessions. General linear models (GLM) will be conducted in STATA (StataCorp, College 
Station TX) to investigate the link between variations in functional connectivity and changes in depressive 
symptoms in response to tVNS administration Adjusted regression coefficients will be calculated after 
including baseline BDI scores and use of antidepressant medications in the models.  
 
Cardiac Autonomic Analysis 
  
A point-process algorithm will be used to separate the heart rate variability dynamics in the classic 
spectral component within the high-frequency (HF) and low frequency (LF) ranges. Differences in 
normalized HF [HFn= (HF/(LF + HF))] will be estimated during exposure to negative images in the fMRI 
stress task as a metric of cardiac vagal activity.  
 
 
VII. RISKS AND DISCOMFORTS 
 
Blood Draw 

 
 
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Subjects may feel slight discomfort when we insert the needle and draw blood through a vein. 
Very rarely, the vein may become sore and red. In addition, a temporary, harmless bruise may develop. 
Although rare, some people faint when blood is drawn. In rare cases, the subject can get an infection 
where the blood was drawn. The infection can usually be treated with antibiotics. 
 
Saliva Collection 
 
 
At this time, there are no known risks for the saliva collection. 
 
MRI Scanning 
There are no known or foreseeable hazards or risks associated with MRI imaging except for people 
with electrically, magnetically, or mechanically activated implants (such as cardiac pacemakers) or those 
who have metallic clips in their brain – all of whom would be excluded from this study.  The powerful 
magnetic field created by the MRI can disturb these metal objects. In addition, since the effect of MRI on 
the fetus is unknown, women suspect pregnancy will be excluded from the study.  The investigator will ask 
the participant if they are prone to claustrophobia, if so they will not be scanned. The participant will be 
asked to fill out a screening questionnaire prior to the MRI scan in order to rule out any condition that may 
preclude MRI scanning.  Conventional MRI systems have been approved by the FDA and will be operated 
within the operating parameters reviewed and accepted by the FDA. The loud gradient noise if the MRI 
could potentially cause hearing damage if no protection were to be used, therefore, all subjects are required 
to wear earplugs to ensure scanner noise is reduced to safe levels. During the scan, voice contact with 
participants will be maintained. Participants will be instructed to notify the examiner of any discomfort. If 
the discomfort cannot be alleviated, the scanning session may be terminated prematurely and inquiries will 
be made to assess if subjects are well and comfortable. 
MRI could potentially uncover an anatomic abnormality in the brain. If this occurs, the established 
protocol for the BWH Research Imaging Center will be followed.  If any anatomic abnormality is suspected 
in the anatomic MRI images, the films will be reviewed by the staff radiologist in the scanning facility. The 
radiologist will decide whether the suspected abnormality is clinically significant and will discuss the 
findings as well as further recommendations with the participant. 
 
tVNS Stimulation 
There is a small possibility of slight and temporary discomfort at the site where the electrodes are 
placed. Potential vaso-vagal reactions to stimulation resulting in dizziness or light-headedness will be 
promptly recognized and treated by a study physician with prompt removal of the electrodes and 
repositioning the subject into a supine position. In some subjects, the experience of electrical stimulation 
may cause anxiety. Subjects will be required to remain under supervision until all symptoms subside. A 
study physician will be available at all times to discuss the study with subjects should they become 
concerned. 
 
In the proposed study, only non-magnetic electrodes will be used. The electrical stimulation 
procedure is without significant safety concerns. The electrical stimulator, checked by Biomedical 
Engineering, has a current limiter preventing harmful stimulation levels. Further, during the electrical 
stimulation, we will be closely monitoring that the current received by the subject is well tolerated. 
Throughout the measurement sessions, participants will be repeatedly asked if they are well and 
comfortable. Should the subject feel any discomfort in the fMRI measurements, she will be advised to 
squeeze the pneumatic signaling device (always held in her/his left hand) and the measurement will be 
immediately interrupted.  
 
VIII. POTENTIAL BENEFITS 

 
 
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There are no expected benefits for subjects participating in these studies.  However, the findings of 
this investigation may help advance the field of developing novel therapeutics as well as our understanding 
of pathways that may mediate the body’s mood and cardiac mechanisms. Therefore, we believe that the 
slight risks associated with these studies are reasonable in relation to the potential benefits.  
 
 
IX. MONITORING AND QUALITY ASSURANCE 
 
The investigator in charge of the study will perform safety monitoring during the entire imaging 
session.  The PI and co-investigators will also review the data for consistency and accuracy as well as 
monitor that analysis is conducted properly.  Furthermore, the technical staff at the Longwood Imaging 
Center will continually monitor the nature and quality of the data collected using the 3T MRI scanner. The 
participants data will be stored in a computer network in a secure, password protected, account. The 
participant will be identified by a unique participant number that is linked only to their name by a master 
data sheet that is kept in a secure locked filing cabinet.  
 
In the case of a serious adverse event (i.e. death, life threatening experience) the incident will be 
reported immediately (within 24 hours of event) by telephone, fax or e-mail followed by a full written report 
using the PHRC Adverse Event Form within 10 working days/14 calendar days. In the case of a mild (i.e. 
minor irritant type) or moderate (i.e. discomfort requiring treatment) adverse event that is unexpected but 
may be related to the experiment, the incident will be reported in writing using the PHRC Adverse Event 
Form within 20 working days/30 calendar days. In the case of a mild or moderate adverse event that is 
unexpected, but not related to the experiment, the PI will summarize the event in the progress report at 
continuing review. In the case of a mild or moderate adverse event that is expected the event will be 
summarized by the PI in the progress report at continuing review.