The author is grateful to the research team (Maria Fernanda Andrade, Allison Kim, Robin Heemels).

All study procedures were performed at the Spaulding Neuromodulation Center/Spaulding Cambridge Hospital. Ethical approval for this protocol was obtained from Mass General Brigham IRB (Number Protocol #:2022P003200). Informed consent was obtained from all subjects using the encrypted Research Electronic Data Capture (REDCap) platform (see Table of Materials). Trial registration number: NCT05801809.
1. Subject selection and screening
<ol>
	<li>Identify potential subjects by several sources. 
	NOTE: For the present study, the human subjects were identified from (1) flyers in public areas across the Boston-land region, (2) internet and newspaper advertisements, (3) advertisements posted on public transportation (The T), (4) Via the Rally platform by Mass General Brigham Research (see Table of Materials). Forty-four healthy subjects were selected for the present study.</li>
	<li>Contact eligible subjects or ask permission electronically to contact them to provide more information about the study.</li>
	<li>At the first point of contact (usually a phone call or Zoom Enterprise call), a study co-investigator administers an online pr-screening questionnaire. Once the online pre-screening process is completed, take the information gathered by the co-investigator to the PI of the study for further review to confirm eligibility. Then, store the data obtained from the pre-screening in an encrypted web-based platform (REDCap, see Table of Materials).
	<ol>
		<li>Include subjects older than 18 years and naive to the stimulation (taVNS).</li>
		<li>Exclude pregnant women, the presence of medical conditions, and presence of any contraindication to transauricular vagus nerve stimulation.</li>
	</ol>
	</li>
</ol>
2. Equipment details
<ol>
	<li>Use a Transcutaneous Auricular Vagus Nerve Stimulation (taVNS) device (Figure 1), which consists of an earset (Figure 2) with conductive ear tips placed on the auricular concha of the ears (Figure 3).</li>
	<li>Connect the electrodes to a stimulator, and during active stimulation, stimulate both the cymbal conchae of the auricular at 30 Hz, 200-250 &#181;s, for 60 min. 
	NOTE: For the commercial details of the device and the related accessories, please see Table of Materials.</li>
</ol>
3. taVNS procedure
NOTE: The protocol consists of two visits: Visit 1 (consent, screening, and collection of demographics information), and Visit 2 (assessments and intervention). The flow of the study can be found in Figure 4.
<ol>
	<li>On Visit 2, randomize the subjects to receive the intervention. 
	NOTE: The active group receives active taVNS, and the sham group receives sham taVNS.</li>
	<li>Blind the subjects, intervention team (co-investigators/ CO-Is who performed the taVNS intervention), and outcome assessors (CO-Is who performed the assessments or analyzed the data) during the trial. Ensure that one non-involved staff member will generate the allocation sequence, seal the envelopes, and randomly assign individuals to interventions using external and visual display identical devices that differ on whether they are active (active current) or not (sham) by another staff member who is not involved in data collection or analysis.</li>
	<li>Collect the data for this study from subjects using an electronic format capture system (REDCap). The following assessments performed are displayed in Table 1.</li>
	<li>When the subject arrives, provide information regarding the procedure. First, assess the pain levels and pain modulation, using heat stimuli on the right forearm for the pain threshold, and cold water for the Conditioned Pain Modulation (CPM), following the adapted protocol suggested by Granot17 and Nirl18.
	<ol>
		<li>First, determine the pain-60 test temperature (temperature that instigates pain experience at a magnitude of 60 on a 60-100 NPS) by applying a Peltier thermode (see Table of Materials) on the right forearm of subjects and deliver short heat stimuli (41-48 &#176;C), each temperature lasting for 7 s starting from the time the stimulus intensity reaches the destination temperature.</li>
		<li>Ask the subjects to rate the level of pain intensity using a numerical pain scale (NPS) ranging from 0 = &#39;&#39;no pain&#39;&#39; to 100 = &#39;&#39;the worst pain imaginable&#39;&#39;.</li>
		<li>Once the pain-60 temperature is determined, administer the test stimulus by applying the same for 30 s at that temperature, and ask the subjects to rate their levels of pain intensity 3 times: at 10 s, 20 s, and 30 s after the thermode reaches the pain-60 temperature (mean scores of the three pain rating will be calculated).</li>
		<li>5 min after delivering the test stimulus, immerse&#160;the left hand of the subject in a bath of water set at 10 &#176;C to 12 &#176;C for 30 s for the conditioned stimulus. Then, apply the same pain-60 temperature on the right forearm of the subject (left hand will still be immersed) for 30 s and again ask the subject to rate their levels of pain intensity 3 times after the thermode reaches the pain-60 temperature: at 10 s, 20 s and 30 s. 
		NOTE: CPM (Conditioned Pain Modulation) response will be calculated as the difference between the average of pain ratings from the test stimulus minus the average of pain ratings during the conditioned stimulus.</li>
	</ol>
	</li>
	<li>Ask subjects to place an HRV monitor (displayed in Figure 5 and Figure 6).</li>
	<li>Next, assess baseline HRV for 5 min (to analyze frequency HF, LF, LF/HF, and time domains metrics) recording with the monitor connected by Bluetooth to a tablet.</li>
	<li>Set up the EEG connected to a computer system, and start the assessments (resting and task-related), which&#160;lasts about 30 min.</li>
	<li>Next, set up the taVNS device.
	<ol>
		<li>Examine, clean with a 70% alcohol pad, and prepare the ear skin of the subject to place the electrodes.</li>
		<li>Next, apply the saline solution to the eatips, place the electrodes on the ear, and start the stimulation, which lasts 60 min.</li>
	</ol>
	</li>
	<li>When the taVNS reaches 30 min, record HRV and EEG again for 5 min only.</li>
	<li>After 60 min of stimulation, assess the subject for EEG, HRV, and pain, and repeat the pretrial procedures (as mentioned below):
	<ol>
		<li>Perform EEG and HRV assessment, which lasts about 30 min.</li>
		<li>Perform CPM assessment following step 3.4.</li>
	</ol>
	</li>
	<li>Perform assessments regarding side effects, fatigue and mood.</li>
	<li>Complete the session.</li>
</ol>
4. Follow up procedures
<ol>
	<li>After randomizing the subjects and completing the data collection, perform data analysis3.</li>
</ol>

Measurement of Neuromodulation by Transcutaneous Auricular Vagus Nerve  Stimulation

<ol>
	<li>Ben-Menachem, E., Revesz, D., Simon, B. J., Silberstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgically+implanted+and+non-invasive+vagus+nerve+stimulation:+a+review+of+efficacy,+safety+and+tolerability.">Surgically implanted and non-invasive vagus nerve stimulation: a review of efficacy, safety and tolerability.</a> Eur J Neurol. 22 (9), 1260-1268 (2015).</li><li>Johnson, R. L., Wilson, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+vagus+nerve+stimulation+as+a+therapeutic+intervention.">A review of vagus nerve stimulation as a therapeutic intervention.</a> J Inflamm Res. 11, 203-213 (2018).</li><li>Gianlorenco, A. C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroencephalographic+patterns+in+taVNS:+A+systematic+review.">Electroencephalographic patterns in taVNS: A systematic review.</a> Biomedicines. 10 (9), 2208 (2022).</li><li>Ruhnau, P., Zaehle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+Auricular+Vagus+Nerve+Stimulation+(taVNS)+and+Ear-EEG:+Potential+for+closed-loop+portable+non-invasive+brain+stimulation.">Transcranial Auricular Vagus Nerve Stimulation (taVNS) and Ear-EEG: Potential for closed-loop portable non-invasive brain stimulation.</a> Front Hum Neurosci. 15, 699473 (2021).</li><li>Coan, J. A., Allen, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frontal+EEG+asymmetry+as+a+moderator+and+mediator+of+emotion.">Frontal EEG asymmetry as a moderator and mediator of emotion.</a> Biol Psychol. 67 (1-2), 7-49 (2004).</li><li>Davidson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+asymmetry,+emotion,+and+affective+style.">Cerebral asymmetry, emotion, and affective style.</a> Brain Asymmetry. , 361-387 (1995).</li><li>de Aguiar Neto, F. S., Rosa, J. L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depression+biomarkers+using+non-invasive+EEG:+A+review.">Depression biomarkers using non-invasive EEG: A review.</a> Neurosci Biobehav Rev. 105, 83-93 (2019).</li><li>Rao, R. P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Brain-Computer Interfacing: An Introduction. , (2013).</li><li>Machetanz, K., Berelidze, L., Guggenberger, R., Gharabaghi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=brain-heart+interaction+during+Transcutaneous+Auricular+Vagus+Nerve+Stimulation.">brain-heart interaction during Transcutaneous Auricular Vagus Nerve Stimulation.</a> Front Neurosci. 15, 632697 (2021).</li><li>Spyer, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annual+review+prize+lecture.+Central+nervous+mechanisms+contributing+to+cardiovascular+control.">Annual review prize lecture. Central nervous mechanisms contributing to cardiovascular control.</a> J Physiol. 474 (1), 1-19 (1994).</li><li>Jarczok, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+associations+of+self-rated+health:+heart+rate+variability+is+more+strongly+associated+than+inflammatory+and+other+frequently+used+biomarkers+in+a+cross+sectional+occupational+sample.">Investigating the associations of self-rated health: heart rate variability is more strongly associated than inflammatory and other frequently used biomarkers in a cross sectional occupational sample.</a> PLoS One. 10 (2), 0117196 (2015).</li><li>Shaffer, F., Ginsberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+heart+rate+variability+metrics+and+norms.">An overview of heart rate variability metrics and norms.</a> Front Public Health. 5, 258 (2017).</li><li>Haensel, A., Mills, P. J., Nelesen, R. A., Ziegler, M. G., Dimsdale, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+heart+rate+variability+and+inflammatory+markers+in+cardiovascular+diseases.">The relationship between heart rate variability and inflammatory markers in cardiovascular diseases.</a> PNEC. 33 (10), 1305-1312 (2008).</li><li>Wolf, V., K&#252;hnel, A., Teckentrup, V., Koenig, J., Kroemer, N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+transcutaneous+auricular+vagus+nerve+stimulation+affect+vagally+mediated+heart+rate+variability?+A+living+and+interactive+Bayesian+meta-analysis.">Does transcutaneous auricular vagus nerve stimulation affect vagally mediated heart rate variability? A living and interactive Bayesian meta-analysis.</a> Psychophysiol. 58 (11), e13933 (2021).</li><li>Geng, D., Liu, X., Wang, Y., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+transcutaneous+auricular+vagus+nerve+stimulation+on+HRV+in+healthy+young+people.">The effect of transcutaneous auricular vagus nerve stimulation on HRV in healthy young people.</a> PLoS One. 17 (2), 0263833 (2022).</li><li>Garrido, M. V., Prada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KDEF-PT:+Valence,+emotional+intensity,+familiarity+and+attractiveness+ratings+of+angry,+neutral,+and+happy+faces.">KDEF-PT: Valence, emotional intensity, familiarity and attractiveness ratings of angry, neutral, and happy faces.</a> Front Psychol. 8, 2181 (2017).</li><li>Granot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+endogenous+analgesia+magnitude+in+a+diffuse+noxious+inhibitory+control+(DNIC)+paradigm:+do+conditioning+stimulus+painfulness,+gender+and+personality+variables+matter.">Determinants of endogenous analgesia magnitude in a diffuse noxious inhibitory control (DNIC) paradigm: do conditioning stimulus painfulness, gender and personality variables matter.</a> Pain. 136 (1-2), 142-149 (2008).</li><li>Nirl, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+psychophysical+study+of+endogenous+analgesia:+the+role+of+the+conditioning+pain+in+the+induction+and+magnitude+of+conditioned+pain+modulation.">A psychophysical study of endogenous analgesia: the role of the conditioning pain in the induction and magnitude of conditioned pain modulation.</a> EJP. 15 (5), 491-497 (2011).</li><li>Santomauro, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+prevalence+and+burden+of+depressive+and+anxiety+disorders+in+204+countries+and+territories+in+2020+due+to+the+COVID-19+pandemic.">Global prevalence and burden of depressive and anxiety disorders in 204 countries and territories in 2020 due to the COVID-19 pandemic.</a> Lancet. 398 (10312), 1700-1712 (2021).</li><li>Tan, C., Yan, Q., Ma, Y., Fang, J., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognizing+the+role+of+the+vagus+nerve+in+depression+from+microbiota-gut+brain+axis.">Recognizing the role of the vagus nerve in depression from microbiota-gut brain axis.</a> Front. Neurol. 13, 1015175 (2022).</li><li>Kim, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+transcutaneous+auricular+vagus+nerve+stimulation+(taVNS):+A+systematic+review+and+meta-analysis.">Safety of transcutaneous auricular vagus nerve stimulation (taVNS): A systematic review and meta-analysis.</a> Sci. Rep. 12 (1), 22055 (2022).</li><li>Martins, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+vagus+nerve+in+fibromyalgia+syndrome.">The role of the vagus nerve in fibromyalgia syndrome.</a> Neurosci. Biobehav. Rev. 131, 1136-1149 (2021).</li><li>Fr&#248;kjaer, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+vagal+tone+enhances+gastroduodenal+motility+and+reduces+somatic+pain+sensitivity.">Modulation of vagal tone enhances gastroduodenal motility and reduces somatic pain sensitivity.</a> J Neurogastroenterol Motil. 28 (4), 592-598 (2016).</li></ol>

<span style="font-size: 11pt; font-family: Arial, sans-serif; color: rgb(34, 34, 34); font-variant-numeric: normal; font-variant-east-asian: normal; font-variant-alternates: normal; font-variant-position: normal; vertical-align: baseline; white-space-collapse: preserve;">H.C. and J.S. are directly associated with Neurive Co, a company developing neuromodulation technologies, such as taVNS, to treat common brain diseases. F.F. is supported by NIH grants and also consulting from Neurive. Spaulding Rehabilitation Hospital has received a significant gift from Neurive, Co., Ltd., the sponsor of the study. The financial interest was reviewed and managed in accordance with the conflict of interest policies of Mass General Brigham, the owner of SRH.

Transauricular Vagus Nerve Stimulation (taVNS) is emerging as a promising therapeutic avenue for addressing several neuropsychiatric conditions. Mood disorders, such as depression and anxiety, pose a significant global health burden, especially after the COVID-19 pandemic19. Recent studies exploring taVNS have exhibited the potential to alleviate symptoms associated with these disorders. 
The vagus nerve plays a pivotal role in the brain-gut axis and the regulation of e...

Transauricular vagus nerve stimulation (taVNS) is a recent neuromodulation technique that does not require surgery and utilizes a non-invasive stimulation device placed at the concha or tragus of the ear. Consequently, it is more accessible and safer for patients1. In recent years, the taVNS field has rapidly expanded, primarily focusing on clinical trials demonstrating potential therapeutic advantages for various pathological conditions, including epilepsy, depression, tinnitus, Parkinson's disease, impaired glucose tolerance, schizophrenia, and atrial fibrillation2. There is much to discuss about taVNS and its effects on biological processes in the central and peripheral systems. Ideally, a biological marker might demonstrate that the auricular branch of the vagus was stimulated, affecting intracranial structures and allowing researchers to analyze how taVNS affects physiological function. Nevertheless, without a trustworthy biomarker, it is not easy to understand what the taVNS data mean and how to interpret them effectively.
Electroencephalography (EEG) is an encouraging imaging tool to provide biomarkers for taVNS. It is a non-invasive, reliable, inexpensive approach to measure and quantify cortical activity3,4. Following this process, our group performed a systematic review, demonstrating elementary details that taVNS could influence cortical activity, mainly increasing EEG power spectrum activity in lower frequencies (delta and theta). However, diverse results in higher frequencies (alpha) and changes in early ERP components related to inhibitory tasks were also detected. High heterogeneity between the studies was found; therefore, more homogeneous, more significant, and well-planned studies are essential to make more robust conclusions about the effects of taVNS on brain activity measured by EEG3. Assessing EEG during taVNS could advance future research on integrating the two techniques for a mobile, closed-loop, monitoring, and non-invasive stimulation tool to affect brain oscillatory activity4.
Alpha asymmetry, which assesses the relative alpha band activity between the brain hemispheres, particularly at frontal electrodes, is a frequently researched EEG biomarker. Previous literature has used this biomarker to analyze the approach-withdraw hypothesis5,6, which holds that the right frontal side of the brain is associated with withdrawal behaviors. In contrast, the left frontal side is associated with approach behaviors. Since alpha is associated with low brain activity, an increase in alpha on the left side of the brain suggests lower activity and may show a lack of approach behavior. This concept helps to explain some results in the alpha band at the left side hemisphere in depressed patients7. Additionally, EEG electrodes record the activity of neuronal populations, examining Functional Connectivity (FC) or changes in large-scale brain networks, such as the default mode network (DMN)7,8.
Based on that, quantitative electroencephalography can be employed to assess the effects of taVNS on brain activity; however, more studies are required to systematically demonstrate the specific metrics and effects that would highlight the non-invasive stimulation through the auricular branch of the vagus nerve.
Peripherally, the vagus nerve and sympathetic nervous system mediate the heart's contractile and electrical function9. This regulation promotes the heart's pacemaker ability and controls it through physiological manifestations of the body, known as sinus depolarizations. Heart rate variability (HRV) records the changes per beat of sinus depolarization, thus non-invasively describing vagal influences on the sinus node10. Given this function, HRV has been seen and studied as a prominent neurocardiac function biomarker associated with an individual's well-being and the likelihood of morbidity, mortality, and stress11,12.
In the context of taVNS, HRV has been recorded in many trials, and stimulation has been thought to modulate HRV9,11,12. Considering that decreased HRV has been related to the morbidity and mortality of different diseases through mechanisms such as over-activity of the sympathetic nervous system, inflammatory response, and oxidative stress, the vagal nerve modulation of taVNS is thought to directly impact HRV and its sinus regulation13,14. In fact, some trials have already indicated that taVNS can increase HRV in healthy subjects, thus supporting this hypothesis15,16. However, there is still a need to understand better whether different taVNS parameters can affect HRV differently.
Currently, no mechanistic studies have investigated the taVNS neural network and autonomic nervous system effects of this technique together. Therefore, this protocol aims to assess how taVNS can affect EEG metrics and HRV and evaluate its safety. Additionally, this also aims to identify predictors that can influence the response to taVNS. Understanding the variables associated with the response to taVNS can help design future clinical trials to maximize the effects of this intervention.

<table><tbody><tr><td>Articulated arm</td><td>Electrical Geodesics, Inc.</td><td>20090645</td><td></td></tr><tr><td>Baby shampoo</td><td>Dynarex</td><td>1396</td><td></td></tr><tr><td>Charge Cable</td><td>NEURIVE Co.</td><td>HV12303003</td><td></td></tr><tr><td>Computer</td><td>Apple</td><td>YM92704U4PC</td><td></td></tr><tr><td>Condutive eartip</td><td>NEURIVE Co.</td><td>HV12303003</td><td></td></tr><tr><td>Earset</td><td>NEURIVE Co.</td><td>HV12303003</td><td></td></tr><tr><td>EEG 64-channel cap&amp;nbsp;</td><td>Electrical Geodesics, Inc.</td><td>H11333</td><td></td></tr><tr><td>Heart rate sensor</td><td>Polar</td><td>M311370175396</td><td></td></tr><tr><td>Monitor</td><td>Dell</td><td>REVA01</td><td></td></tr><tr><td>Net Amps 300</td><td>Electrical Geodesics, Inc.</td><td>A09370244</td><td></td></tr><tr><td>Peltier thermode</td><td>Advanced Medical Systems, Ramat Yishai, Isreal</td><td></td><td></td></tr><tr><td>Potassium Chloride (dry)</td><td>Electrical Geodesics, Inc.</td><td>820127755</td><td></td></tr><tr><td>Rally</td><td>Mass General Brigham Research</td><td></td><td>online platform</td></tr><tr><td>Research Electronic Data Capture (REDCap)</td><td>Vanderbilt</td><td></td><td>web-based software platform</td></tr><tr><td>Thermosensory Stimulator</td><td>Medoc Ltd</td><td>1241</td><td></td></tr><tr><td>Transauricular vagus nerve stimulator</td><td>NEURIVE Co.</td><td>HV12303003</td><td></td></tr></tbody></table>

understanding the effects of non&#45;invasive transauricular vagus nerve stimulation on eeg and hrv

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no mechanistic studies have investigated this technique's neural network and autonomic nervous system effects. This study aims to describe how taVNS can affect EEG metrics, HRV, and pain levels. Healthy subjects were randomly allocated into two groups: the active taVNS group and the sham taVNS group. Electroencephalography (EEG) and Heart Rate Variability (HRV) were recorded at baseline, 30 min, and after 60 min of 30 Hz, 200-250 &#181;s taVNS, or sham stimulation, and the differences between the metrics were calculated. Regarding vagal projections, some studies have demonstrated the role of the vagus nerve in modulating brain activity, the autonomic system, and pain pathways. However, more data is still needed to understand the mechanisms of taVNS on these systems. In this context, this study presents methods to provide data for a deeper discussion about the physiological impacts of this technique, which can help future therapeutic investigations in various conditions.

We performed a preliminary descriptive analysis of the first randomized subject without unblinding the study. For this reason, which arms this subject was allocated to is unknown. The first subject is a 69-year-old woman, non-Hispanic, Caucasian, with a college degree, who did not report any adverse event during or after the stimulation session. The clinical data are displayed in Table 2.
Besides, a topographic distribution of scalp plots was created in resting-state EEG for t...

Read this Scientific Article Protocol about Author Spotlight: Exploring the Effects of Transauricular Vagus Nerve Stimulation at JoVE.com

Author Spotlight: Exploring the Effects of Transauricular Vagus Nerve Stimulation

This protocol provides information on how to apply transcutaneous auricular vagus nerve stimulation (taVNS) in a clinical trial, including potential biomarkers such as EEG metrics and heart rate variability (HRV) to measure the effect of this treatment on the autonomic nervous system.

Understanding the Effects of Non&#45;Invasive Transauricular Vagus Nerve Stimulation on EEG and HRV

Several studies have demonstrated promising results of transcutaneous auricular vagus nerve stimulation (taVNS) in treating various disorders; however, no ...

understanding-effects-non-invasive-transauricular-vagus-nerve

Research

JoVE Journal

Neuroscience

2.0K Views.  Spaulding Rehabilitation Hospital and Massachusetts General Hospital, Harvard Medical School. Transauriculaire nervus vagus stimulatie, kortweg taVNS, is een veilige, niet-invasieve stimulatietechniek die zich richt op de auriculaire tak van de nervus vagus. Door de elektroden aan de achterkant van het oor te plaatsen als alternatief voor invasieve stimulatie, kan taVNS verschillende hersenfuncties beïnvloeden door de vagale afferente vezels te stimuleren. Verschillende onderzoeken hebben veelbelovende resultaten van taVNS aangetoond.Er is echter een gebrek aan mechanistische ...

Video: Auteur in de schijnwerpers: onderzoek naar de effecten van stimulatie van de nervus vagus transauriculaire vagus

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Behavior

Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation (tACS)

Oscillatory brain activities are considered to reflect the basis of rhythmic changes in transmission efficacy across brain networks and are assumed to integrate cognitive neural processes. Transcranial alternating current stimulation (tACS) holds the promise to elucidate the causal link between specific frequencies of oscillatory brain activity and cognitive processes. Simultaneous electroencephalography (EEG) recording during tACS would offer an opportunity to directly explore immediate neurophysiological effects of tACS. However, it is not trivial to measure EEG signals during tACS, as tACS creates a huge artifact in EEG data. Here we explain how to set up concurrent tACS-EEG experiments. Two necessary considerations for successful EEG recording while applying tACS are highlighted. First, bridging of the tACS and EEG electrodes via leaking EEG gel immediately saturates the EEG amplifier. To avoid bridging via gel, the viscosity of the EEG gel is the most important parameter. The EEG gel must be viscous to avoid bridging, but at the same time sufficiently fluid to create contact between the tACS electrode and the scalp. Second, due to the large amplitude of the tACS artifact, it is important to consider using an EEG system with a high resolution analog-to-digital (A/D) converter. In particular, the magnitude of the tACS artifact can exceed 100 mV at the vicinity of a stimulation electrode when 1 mA tACS is applied. The resolution of the A/D converter is of importance to measure good quality EEG data from the vicinity of the stimulation site. By following these guidelines for the procedures and technical considerations, successful concurrent EEG recording during tACS will be realized.

Concurrent Electroencephalography Recording During Transcranial Alternating Current Stimulation tACS

In this article we explain how to set up a concurrent transcranial alternating current stimulation and EEG experiment.

Oscillatory brain activities are considered to reflect the basis of rhythmic changes in transmission efficacy across brain networks and are assumed to ...

Simultaneous Transcranial Alternating Current Stimulation and Functional Magnetic Resonance Imaging

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs frequency-specific stimulation of the human brain through current applied to the scalp with surface electrodes. Most current knowledge of the technique is based on behavioral studies; thus, combining the method with brain imaging holds potential to better understand the mechanisms of tACS. Because of electrical and susceptibility artifacts, combining tACS with brain imaging can be challenging, however, one brain imaging technique that is well suited to be applied simultaneously with tACS is functional magnetic resonance imaging (fMRI). In our lab, we have successfully combined tACS with simultaneous fMRI measurements to show that tACS effects are state, current, and frequency dependent, and that modulation of brain activity is not limited to the area directly below the electrodes. This article describes a safe and reliable setup for applying tACS simultaneously with visual task fMRI studies, which can lend to understanding oscillatory brain function as well as the effects of tACS on the brain.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations, though its effects are not completely understood. This article describes a safe and reliable setup for applying tACS simultaneously with functional magnetic resonance imaging, which can increase understanding oscillatory brain function and effects of tACS.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

Calculating Heart Rate Variability from ECG Data from Youth with Cerebral Palsy During Active Video Game Sessions

The aim of this study was to generate a method for calculating heart rate variability (HRV) from electrocardiogram (ECG) waveforms. The waveforms were recorded by a HR monitor that participants (youth with cerebral palsy (CP)) wore during active video game (AVG) sessions. The AVG sessions were designed to promote physical activity and fitness (aerobic performance) in participants. The goal was to evaluate the feasibility of AVGs as a physical therapy (PT) intervention strategy. The maximum HR (mHR) was determined for each participant and the Target Heart Rate Zone (THRZ) was calculated for each of three exercise phases in the 20 min AVG session: (warm-up at 40-60% mHR, conditioning at 60-80% mHR, and cool down at 40-60% mHR). Each participant played three 20 min games during the AVG session. All games were played while sitting on a bench because many youth with CP cannot stand for extended periods of time. Each game condition differed with participants using hand icons only, hand and feet icons together or feet icons only to collect objects. The objective of the game (called KOLLECT) is to collect objects to gain points and avoid hazards to not lose points. Hazards were used in the warm-up and cool down phases only to promote slower, controlled movement to maintain HR in the target heart rate zone (THRZ). There were no hazards in the conditioning phase to promote higher levels and more intense physical activity. Analytic methods were used to generate HRV (selected time-domain and frequency-domain measures) from ECG data to examine aerobic workload. Recent applications of HRV indicate that short-term measurements (5 min bouts) are appropriate and that HRV biofeedback may help improve symptoms and the quality of life in a variety of health conditions. Although HR is a well-accepted clinical measure to examine aerobic performance and intensity in PT interventions, HRV may provide information of the autonomic system functions, recovery and adaptation during AVG sessions.

This protocol describes a method for calculating Heart Rate Variability (HRV) from electrocardiogram (ECG) waveforms. Waveforms from continuous heart rate (HR) recordings during active video game (AVG) sessions were used to measure the aerobic performance of youth with cerebral palsy (CP).

The aim of this study was to generate a method for calculating heart rate variability (HRV) from electrocardiogram (ECG) waveforms. The waveforms were ...

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus nerve stimulation (taVNS). Unlike cervically-implanted VNS, taVNS is an inexpensive and non-surgical method used to modulate the vagus system. taVNS is appealing as it allows for rapid translation of basic VNS research and serves as a safe, inexpensive, and portable neurostimulation system for the future treatment of central and peripheral disease. The background and rationale for taVNS is described, along with electrical and parametric considerations, proper ear targeting and attachment of stimulation electrodes, individual dosing via determination of perception threshold (PT), and safe administration of taVNS.

Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation taVNS: Technique, Targeting, and Considerations

A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described.

Non-invasive vagus nerve stimulation (VNS) may be administered via a novel, emerging neuromodulatory technique known as transcutaneous auricular vagus ...

Vagus Nerve Stimulation As an Adjunctive Neurostimulation Tool in Treatment-resistant Depression

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression (TRD). VNS exhibits antidepressive and stabilizing effects. This method is particularly useful as a long-term treatment, in which up to two-thirds of patients respond. The vagus nerve stimulator is positioned on the left vagus nerve during a surgical procedure and is activated telemetrically by a wand connected to a handheld computerized device. The treating physician can perform various adjustments of the vagus nerve stimulator during in-office visits (e.g., by modifying stimulation intensity or stimulation frequency) to achieve maximum therapeutic effects with low side effects. Set-up of the device usually takes several months. Typical side effects include wound infection, temporary salivation, coughing, paralysis of the vocal cords, bradycardia, or even asystole. The patient can stop the VNS by placing a magnet over the generator. The current protocol describes delivery of the specific stimulation tool and methods for adjusting the tuning parameters to achieve the best remission rates in patients with TRD.

Vagus nerve stimulation (VNS) has been shown to be effective as an adjunct treatment for treatment-resistant depression (TRD). VNS leads to antidepressive and antisuicidal effects and quality of life improvements. This protocol offers a step-by-step guide to managing and adjusting a vagus nerve stimulator for efficient treatment of TRD.

Vagus nerve stimulation (VNS) is an approved neurostimulation therapy. The purpose of the method is to treat patients with therapy-resistant depression ...

Medicine

Preparation of Peripheral Nerve Stimulation Electrodes for Chronic Implantation in Rats

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. Several recent studies have demonstrated the effectiveness of chronic VNS in enhancing central nervous system plasticity to improve motor rehabilitation, extinction learning, and sensory discrimination. Construction of chronically implantable devices for use in such studies is challenging due to rats&#8217; small size, and typical protocols require extensive training of personnel and time-consuming microfabrication methods. Alternatively, commercially available implantable cuff electrodes can be purchased at a significantly higher cost. In this protocol, we present a simple, low-cost method for construction of small, chronically implantable peripheral nerve cuff electrodes for use in rats. We validate the short and long-term reliability of our cuff electrodes by demonstrating that VNS in ketamine/xylazine anesthetized rats produces decreases in breathing rate consistent with activation of the Hering-Breuer reflex, both at the time of implantation and up to 10 weeks after device implantation. We further demonstrate the suitability of the cuff electrodes for use in chronic stimulation studies by pairing VNS with skilled lever press performance to induce motor cortical map plasticity.

Existing approaches for constructing chronically implantable peripheral nerve cuff electrodes for use in small rodents often require specialized equipment and/or highly trained personnel. In this protocol we demonstrate a simple, low-cost approach for fabricating chronically implantable cuff electrodes, and demonstrate their effectiveness for vagus nerve stimulation (VNS) in rats.

Peripheral nerve cuff electrodes have long been used in the neurosciences and related fields for stimulation of, for example, vagus or sciatic nerves. ...

Abdominal VNS for Treating Inflammatory Conditions in Rat Models

Implantation Surgery for Abdominal Vagus Nerve Stimulation and Recording Studies in Awake Rats

Abdominal vagus nerve stimulation (VNS) can be applied to the subdiaphragmatic branch of the vagus nerve of rats. Due to its anatomical location, it does not have any respiratory and cardiac off-target effects commonly associated with cervical VNS. The lack of respiratory and cardiac off-target effects means that the intensity of stimulation does not need to be lowered to reduce side effects commonly experienced during cervical VNS. Few recent studies demonstrate the anti-inflammatory effects of abdominal VNS in rat models of inflammatory bowel disease, rheumatoid arthritis, and glycemia reduction in a rat model of type 2 diabetes. Rat is a great model to explore the potential of this technology because of the well-established anatomy of the vagus nerve, the large size of the nerve that allows easy handling, and the availability of many disease models. Here, we describe the methods for cleaning and sterilizing the abdominal VNS electrode array and surgical protocol in rats. We also describe the technology required for confirmation of suprathreshold stimulation by recording evoked compound action potentials. Abdominal VNS has the potential to offer selective, effective treatment for a variety of conditions, including inflammatory diseases, and the application is expected to expand similarly to cervical VNS.

Author Spotlight: Exploring Abdominal VNS for Inflammatory Conditions

The present protocol describes the surgical technique for implanting an electrode array onto the abdominal vagus nerve in rats, along with methods for chronic electrophysiology testing and stimulation using the implanted device.

Abdominal vagus nerve stimulation (VNS) can be applied to the subdiaphragmatic branch of the vagus nerve of rats. Due to its anatomical location, it does ...

Unveiling Brain-Heart Interplay with EEG and Cardio Signals

BrainBeats as an Open-Source EEGLAB Plugin to Jointly Analyze EEG and Cardiovascular Signals

The interplay between the brain and the cardiovascular systems is garnering increased attention for its potential to advance our understanding of human physiology and improve health outcomes. However, the multimodal analysis of these signals is challenging due to the lack of guidelines, standardized signal processing and statistical tools, graphical user interfaces (GUIs), and automation for processing large datasets or increasing reproducibility. A further void exists in standardized EEG and heart-rate variability (HRV) feature extraction methods, undermining clinical diagnostics or the robustness of machine learning (ML) models. In response to these limitations, we introduce the BrainBeats toolbox. Implemented as an open-source EEGLAB plugin, BrainBeats integrates three main protocols: 1) Heartbeat-evoked potentials (HEP) and oscillations (HEO) for assessing time-locked brain-heart interplay at the millisecond accuracy; 2) EEG and HRV feature extraction for examining associations/differences between various brain and heart metrics or for building robust feature-based ML models; 3) Automated extraction of heart artifacts from EEG signals to remove any potential cardiovascular contamination while conducting EEG analysis. We provide a step-by-step tutorial for applying these three methods to an open-source dataset containing simultaneous 64-channel EEG, ECG, and PPG signals. Users can easily fine-tune parameters to tailor their unique research needs using the graphical user interface (GUI) or the command line. BrainBeats should make brain-heart interplay research more accessible and reproducible.

Author Spotlight: Advancing the Study of Brain-Heart Interplay with a Comprehensive EEGLAB Plugin for Multimodal Signal Analysis

The BrainBeats toolbox is an open-source EEGLAB plugin designed to jointly analyze EEG and cardiovascular (ECG/PPG) signals. It includes heartbeat-evoked potentials (HEP) assessment, feature-based analysis, and heart artifact extraction from EEG signals. The protocol will aid in studying brain-heart interplay through two lenses (HEP and features), enhancing reproducibility and accessibility.

The interplay between the brain and the cardiovascular systems is garnering increased attention for its potential to advance our understanding of human ...

Understanding the Effects of Non-Invasive Transauricular Vagus Nerve Stimulation on EEG and HRV

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