Testing the Noradrenergic Hypothesis of Transcutaneous Vagus Nerve Stimulation
Phase: NA
Conditions: no Condition, Electroencephalography, Pupil Diameter
Sponsor: Arkansas Tech University

Study Protocol and Statistical Analysis Plan NCT03854539 August 27, 2020 
 
Protocol. Participants. Healthy male and female adult participants (ages 18+) will be recruited 
from the Arkansas Tech University psychology student test pool and may receive course credit 
for participating. Participants fill out a questionnaire screening for mental disorders and use of 
substances which interfere with accurate assessment of salivary alpha amylase. The 
questionnaire also includes items on age, ethnicity and gender. In both experiments participants 
will be randomly assigned to groups with stimulation order determined using a Latin 
square design. Protection of human subjects. The IRB protocol is under review. Women and 
minorities are included and their data will be analyzed in accordance with the described data 
analysis plan. Stimulation procedures. We will apply sham stimulation and tVNS using identical 
stimulation parameters with the exception of electrode placement. Electrode Placement. 
Participants in tVNS condition groups will receive stimulation via electrodes attached on the 
anterior and posterior ear at the concha, an area richly innervated with vagal fibers [10]. Sham-
stimulated participants will be stimulated with identical stimulus parameters as the 
tVNS group with the electrodes placed on the helix region. The helix region of the ear is thought 
to be free of vagal or other cranial nerve fibers. Non-stimulation participants will receive no 
stimulation. In the nonstimulation condition electrodes will be applied randomly to one of the 
above locations without electrical stimulation. The purpose of this group is to control for the 
possibility that mild stimulation will increase arousal through non-vagal pathways. Stimulation 
parameters. In the first study, to establish a stimulation intensity effect curve, participants will 
receive 0.0, 0.25, 0.5, or 1.0mA stimulation either to the concha or helix region (sham 
stimulation). The stimulus will consist of trains of 30Hz, with 0.5mS pulse width. Trains will 
alternate 30 seconds on and 30 seconds off for five minutes. In a second study to determine the 
relative effects of intermittent vs constant stimulation, participants will receive helix (sham) or 
concha (tVNS) stimulation at the 0.5mA level. Intermittent stimulation will consist of trains of 
30Hz, with 0.5mS pulse width. Trains will alternate 30 seconds on and 30 seconds off for ten 
minutes. Constant stimulation will be identical with the exception that instead of alternating on-
off periods, the stimulation will be a single five minute on block (holds total number of 
impulses equal). Salivary Alpha Amylase. Saliva samples will be collected using the passive 
drool method, considered to be the superior method for collection of alpha amylase samples 
[11]. Participants allow drool to pool inside the oral cavity and use a straw to provide 2mL of 
saliva into a cryogenic sample container. Following collection samples are placed on ice and 
then frozen until assay. Salivary alpha amylase assay. Samples are assayed using kinetic 
reaction assay, without modifications to the manufacturer’s recommended protocol (Salimetrics, 
State College, PA) will be used. Details on this assay can be found in Gordis et al [11]. For sAA, 
sample test volumes are 10 μl and the lower limit of sensitivity is 0.4 U/mL. Samples are tested 
in duplicate with the average of the duplicates used for analysis. Duplicates exceeding 10% 
variance will be re-assayed. As a secondary measure, heart rate will be monitored by ECG and 
continuously recorded (3 lead) throughout the experiment using an iWorx TA device. Recoding 
electrodes are placed on the right and left clavicles andcostal margin. Heart rate will be used to 
confirm sympathetic induction and effects of heart rate variability may 
later be investigated as a primary measure in future studies. 
 
Data analysis Within-subject percent change from immediately preceding baseline scores will 
be calculated for each level of stimulation ([baseline/stimulation] *100). Statistical analysis of 
percent changes in salivary alpha amylase concentrations by stimulation group will be 
performed with one-way repeated measures ANOVA with Bonferroni correction for multiple 
comparisons of each stimulation level to the sham stimulation level. Statistical 
analysis to determine changes in baseline concentration of salivary alpha amylase within each 
intensity will be conducted using repeated measures ANOVA with Bonferroni correction for 

multiple <comparisons. A significance level of p <0.05 will be used for all statistical tests 
conducted. Within-subject percent change from immediately preceding baseline scores will be 
calculated foreach level of stimulation ([baseline/stimulation] *100). Statistical analysis of 
percent changes in salivary alpha amylase concentrations by stimulation group will be 
performed with one-way repeated measures ANOVA with Bonferroni correction for multiple 
comparisons of each stimulation level to the sham stimulation level. Statistical 
analysis to determine changes in baseline concentration of salivary alpha amylase within each 
intensity will be conducted using repeated measures ANOVA with Bonferroni correction for 
multiple <comparisons. Asignificance level of p <0.05 will be used for all statistical tests 
conducted.