Noninvasive Vagus Nerve Stimulation (VNS) for Neuromotor Adaptations
Phase: NA
Conditions: Healthy Young Adults
Sponsor: Georgia Institute of Technology

Study Protocol and 
Statistical Analysis Plan (SAP) 
 
 
 
Official Title:   
 
Noninvasive Vagus Nerve Stimulation (VNS) for Neuromotor Adaptations 
 
Principal Investigator: 
Minoru Shinohara, PhD 
Associate Professor 
School of Biological Sciences 
Georgia Institute of Technology 
 
Author: 
 
 
Minoru Shinohara, PhD 
 
NCT Number:  
 
NCT03628976 
 
Document date:  
 
04/16/2021 
 
IRB approval date:   
04/16/2021 
 
 
 
 
 

Study Protocol 
 
Noninvasive Vagus Nerve Stimulation (VNS) for Neuromotor Adaptations 
 
Aims 
Motor function is compromised with advanced age, and motor impairment is involved in various neuromotor 
injuries and disorders including stroke, spinal cord injury, and amputation. Development of effective 
interventions for facilitating neuromotor adaptation for motor skills and strength is essential for accelerating or 
augmenting rehabilitation outcomes in the voluntary control of impaired limbs or attached assistive devices. 
Our ultimate goal is to find non-pharmacological, noninvasive, and affordable neuromodulating interventions 
for enhancing the rehabilitation outcome. Our idea stems from animal studies in which invasive (implanted) 
afferent vagus nerve stimulation (VNS, i.e. stimulation of cranial nerve X) paired with motor training enhanced 
neuromotor plasticity and motor recovery, most likely through increased releases of central neuromodulators 
from the brainstem (e.g. norepinephrine). In a rat model of stroke, implanted VNS paired with rehabilitative 
training facilitated and enhanced motor recovery and retention in motor function substantially (full recovery 
with implanted VNS vs. half recovery without implanted VNS). Not only in the injured model, but also in non-
injured rats, implanted VNS paired with motor training facilitated neural plasticity in the motor cortex. 
Applicability of these facilitating effects of VNS in injured/non-injured rats to human neuromotor adaptation is 
unknown. Additionally, implanted VNS in humans would involve potential risks, higher costs, and 
inconvenience for rehabilitation. Hence, it is important to investigate the efficacy of adding noninvasive VNS to 
motor training for neuromotor adaptation in motor skills and strength in humans. As a noninvasive form of VNS 
for humans, transcutaneous VNS (tVNS) at the outer ear has been developed for studying its potential effect 
on several neurological conditions including epilepsy, depression, tinnitus, and pain, but not on motor 
impairment. tVNS can be applied by electrically stimulating the afferent auricular branch of the vagus nerve 
located medial of the tragus at the entry of the ear canal. In healthy humans, tVNS activates the brainstem 
including locus coeruleus, where norepinephrine (i.e. neuromodulator) is synthesized. It is unknown whether 
the noninvasive VNS (i.e. tVNS) actually leads to the facilitation of neuromotor adaptation in humans when 
combined with motor training in healthy or clinical populations. With potential applicability of this novel 
intervention to various clinical populations (e.g. stroke survivors, spinal cord injury, amputees, and older 
adults) in future vision, it is essential to start with the basic understanding about the effect of paired tVNS on 
the training-induced neuromotor adaptations in non-disabled humans. We have preliminary observations that 
would suggest the facilitating effect of tVNS on neuromotor adaptations in non-disabled humans. The 
overarching hypothesis is that an addition of paired tVNS to motor training increases central noradrenaline and 
facilitates training-induced neuromotor adaptations in human adults. In line with the concept of an R03 
mechanism, the scope of this proposal is to produce pilot data demonstrating the proof of concept that would 
allow us to further investigate this promising approach in humans. To demonstrate the proof of concept of the 
overarching hypothesis, the specific aim of the study is to examine the effects of paired tVNS during motor 
training on central norepinephrine and neuromotor adaptations in humans. 
 
Project design 
 
Population. Non-disabled young adults (18-39 years old, men and women) 
 
Intervention. Subjects will be randomly assigned to the tVNS or Sham group.  There will be 5 experimental 
days, and the tasks on each day are summarized as below. 
 
Day 1 (Baseline): Visuomotor Test  TMS (preparation & Test)  Visuomotor Training  TMS (Test) 
Day 2: Visuomotor Test  Visuomotor Training 
Day 3: Visuomotor Test  Visuomotor Training and Alpha-Amylase Activity Measurement  
Day 4: Visuomotor Test  Visuomotor Training 
Day 5 (Post): Visuomotor Test  TMS (preparation & Test)  Visuomotor Training  TMS (Test) 
 
Visuomotor tasks with the index finger will be performed for the test and training.  The first day is primarily the 
test day for assessing the baseline skill and neural excitability before training.  Subjects will perform training 
after the initial tests on the first day and subsequent days.  The last day is the final test day to assess the 
effects of training with tVNS or Sham on neuromotor adaptations.  On the first and last days, the TMS test will 
be performed at the beginning and after the visuomotor tasks in both groups.  In between these days, paired 
tVNS and Sham will be applied during training execution in the tVNS and Sham groups, respectively.  The 

changes in visuomotor skill and corticospinal excitability will be compared between tVNS and Sham groups.  
On Day 3, salivary alpha-amylase activity will be determined as a biomarker of central norepinephrine. 
 
Protocol. All subjects performed visuomotor task as training and test.  TMS test and central noradrenaline test 
were also performed.  
 
Visuomotor tasks.  Visuomotor tasks will be performed to induce and examine the adaptations in the 
visuomotor skill and neural excitability due to training.  Visuomotor tasks include 1) visuomotor test on the first 
and last days and 2) visuomotor training on all days.  The setup is the same across tasks.  Subjects will 
receive tVNS or Sham during the training but not during the tests.  A sensitive force transducer will be 
attached to the lateral aspect of the index finger and the little finger.  The metacarpophalangeal joint will be in 
the neutral position.  Subjects will adjust the abduction force of the index finger and the little finger to track 
the corresponding target force trajectory as accurately as possible with visual feedback.  The target force 
trajectory for the test and training will be the same complex trajectory composed of multiple sinusoidal waves 
of various frequencies (<5 Hz).  Every trial will use the same waves.  The target force level will range from 0-
10% of their maximal voluntary contraction force.  All subjects will perform 50 trials (10 trials × 5 sets) with the 
same target trajectory across days.  During training, subjects in the tVNS and Sham groups will receive paired 
tVNS and Sham during the execution phase of these training trials, respectively.  All subjects will perform the 
visuomotor test without tVNS or Sham. 
 
Paired tVNS and Sham during training.  In the tVNS group, tVNS will be applied electrically to the afferent 
auricular branch of the vagus nerve located medial of the tragus at the entry of the left ear canal.  One small 
surface electrode will be attached to the inner surface and another to the outer surface of the tragus.  In the 
Sham group, the same stimulation will be applied to the ear lobe, where the vagus nerve is absent.  The 
electrodes will be connected to an isolated electrical stimulator.  In both groups, electrodes will be attached to 
the tragus and the earlobe.  Subjects will receive stimulation in one of the electrode locations.  Stimulation will 
be delivered only during the execution of the motor task. 
 
TMS test.  TMS test will be performed immediately before and after the visuomotor training on the first and last 
days to examine corticospinal excitability.  Subjects will not receive tVNS or Sham during the TMS test.  Basic 
procedures with TMS will follow those in our previous studies.  The head of the subjects will be secured with a 
vacuum cushion molded for their neck and head.  Surface EMG electrodes will be attached over the first 
dorsal interosseous muscle in the hand in a belly-tendon configuration.  Baseline EMG and motor evoked 
potential (MEP) will be obtained from surface EMG using a high-gain EMG preamplifier.  A figure-of-eight TMS 
coil will be placed over the primary motor cortex.  A TMS coil navigation system and a coil holder will be used 
to maintain the coil position in 3-dimensional space relative to the head.  After identifying the hot spot for 
evoking MEP in the first dorsal interosseous muscle, the resting motor threshold will be determined.  To 
examine corticospinal excitability in the resting muscle, TMS will be applied at 8 TMS intensities ranging from 
90%-160% of the resting motor threshold in random order.  At each TMS intensity,13 TMS will be applied with 
5-7 s randomized intervals, and the first response in each intensity will be discarded.  Subjects will maintain 
relaxed muscles during the TMS test, and the relaxation will be ensured by monitoring and quantifying their 
background EMG.  To obtain the reference value for normalizing MEP amplitude across days, compound 
muscle action potentials (M-waves) of the first dorsal interosseous muscle will be obtained just before the 
TMS test.  Stimulation electrodes will be attached to the skin overlying the ulnar nerve of the forearm just 
proximal to the wrist.  Using a constant-current isolated electrical stimulator, single supramaximal electrical 
stimuli will be applied to the nerve to obtain the maximal peak-to-peak amplitude of the M-wave (Mmax). 
 
Central noradrenaline. Central noradrenaline will be assessed indirectly with the salivary measure of alpha-
amylase activity as a biomarker.  Saliva will be sampled via salivette strips before and after the training on 
Day 3. Three saliva samples will be analyzed for each set and averaged. 
 

Statistical Analysis Plan 
 
Noninvasive Vagus Nerve Stimulation (VNS) for Neuromotor Adaptations 
 
Aim 
The aim of the study is to examine the effects of paired tVNS during motor training on central norepinephrine 
and neuromotor adaptations in humans. 
 
Sample size 
24 participants 
 
Outcomes and analyses (all primary outcomes) 
For assessing the visuomotor skill, the mean squared error of the produced force trajectory in reference to the 
target will be determined.  For assessing corticospinal excitability, peak-to-peak amplitude of MEP will be 
averaged across responses.  MEP amplitude will be normalized to the corresponding Mmax. As the dependent 
variable for visuomotor skill, mean squared error, normalized to the baseline value, will be compared for group 
and day using a two-way ANOVA with repeated measures.  As the dependent variable for corticospinal 
excitability, MEP amplitude will be compared for group, time, and day using a three-way ANOVA with repeated 
measures.  As the dependent variable for central norepinephrine activity, alpha-amylase activity will be 
compared for group and time using a two-way ANOVA with repeated measures.  An alpha value of 0.05 will 
be used for statistical significance.  Post-hoc comparison will be performed when appropriate.