Participants

All experimental procedures were conducted on human volunteers in accordance with protocols guidelines approved by an Institutional Review Board (Solutions IRB, Little Rock, AR). All subjects provided written informed consent prior to experimentation. We conducted three independent experiments in this study using different groups of volunteer subjects. All experiments were designed using a between subjects approach. All subjects were blinded to the study conditions. For all experimental conditions, exclusion criteria were as follows: neurological or psychiatric disorder, cranial or facial metal plate or screw implants, severe face or head trauma, recent concussion or brain injury, recent hospitalized for surgery/illness, high blood pressure, heart disease, diabetes, acute eczema on the scalp and uncorrectable vision or hearing.

Experiment 1 (Fig. 2c) was designed to study the influence of TEN on a component of the sympathetic skin response by implementing functional infrared thermography to assay changes in emotional thermoregulation expressed in several facial regions as described below. The sample pool was consisted of 19 healthy right-handed subjects (7 male, 12 female) between the ages of 18 to 27 (mean age = 22.16 ± 2.09 years). Subjects were randomly assigned to receive sham (n = 10) or TEN (n = 9). The ethnicity of the subjects was as follows: 42% of participants were Asian, 37% were Caucasian, 10% were African-American and 5% were Hispanic and 5% of mixed ethnic background. The educational background of subjects was as follows: 47% had completed some college, 32% had a Bachelor’s degree and 21% had completed some post-graduate work or had a post-graduate degree.

Experiment 2 (Fig. 2d) was designed to test the impact of TEN on affective mood as reported by the Profile of Mood States (POMS) survey36. The sample pool consisted of 45 healthy subjects (16 male, 29 female) that ranged in age from 18 to 43 (mean age = 22.46 ± 4.23 years). Subjects were randomly assigned to receive sham (n = 20) or TEN (n = 25). The ethnicity of subjects was as follows: 47% were Caucasian, 31% were Asian, 16% were African-American, 4% were Hispanic and 2% were of a mixed ethnic background. The educational background of subjects was as follows: 11% completed high school, 44% completed some college, 29% had a Bachelor’s degree and 16% completed some post-graduate work or had a post-graduate degree.

Experiment 3 (Fig. 2e) was designed to study the influence of TEN on psychophysiological arousal and the mobilization salivary biochemicals in response to acute stress induced by a classical fear conditioning paradigm and a series of time pressured cognitive tasks. The sample pool consisted of 20 male subjects (sham n = 10; TEN n = 10) to avoid the introduction of confounds related to hormonal variance across menstrual cycles on stress biochemical profiles. The subjects were between the ages 19 to 27 (mean age = 22.3 ± 2.2 years). The ethnicity of subjects was as follows: 50% of the subjects were Asian, 35% were Caucasian, 10% were African American and 5% were Hispanic. The educational background of subjects was as follows: 5% completed highs school, 40% of participants had completed some college, 40% had a Bachelor’s degree and 15% had completed some post-graduate work.

Transdermal electrical neuromodulation

Prior to this study, we spent two years developing and investigating a variety of electrical neuromodulation waveforms and approaches. The transdermal electrical neuromodulation (TEN) waveform developed for use in this study was a pulsed (7–11 kHz; 50% duty cycle) biphasic current producing average amplitude of 5–7 mA for 15 min (Experiments 1 and 2; Fig. 2c,d) or 14 min (Experiment 3; Fig. 2e). The TEN waveform pulse frequency linearly ramped from 7–11 kHz during the first 30 sec of the protocol and ramped down linearly from 11–7 kHz during the last 30 sec of the stimulus. The sham waveform was an active stimulation control as described above, but pulsed at a frequency of 1–2 kHz (50% duty cycle; < 4 mA average current amplitude) for 15 min (Experiments 1 and 2; Fig. 2c,d) or 14 min (Experiment 3; Fig. 2e) to mimic skin sensations similar to those experienced throughout the real TEN stimulation protocol (Table 2). Subjects were only able to control the stimulus intensity within the ranges indicated, but not the frequency of stimuli. Subjects were not able to distinguish any differences between the sensations elicited by the real TEN or sham waveforms. During both the real TEN and sham stimulus protocols, subjects were instructed to adjust the current output of a medical-grade wearable TEN device (Thync, Inc., Los Gatos, CA) using an iPod touch connected to the device over a Bluetooth low energy network such that it was comfortable. TEN and sham waveforms were delivered to the right temple (10/20 site F8) and base of the neck (5 cm below the inion) using custom-designed electrodes comprising a hydrogel material and a conductive Ag/AgCl film secured to the wearable TEN device. The anterior electrode positioned over F8 was a 4.9 cm2 oval having a major axis of 2.75 cm and a minor axis of 2.25 cm while the posterior electrode was a 12.5 cm2 rectangle with a length of 5 cm and a height of 2.5 cm. The average current density was <2 mA/cm2 at all times to keep in accordance with general safety practices to prevent any damage to the skin. Subjects were assigned to experimental conditions using a randomization method or a counterbalancing approach. Subjects were always kept blind to all experimental conditions.

Functional infrared thermography

In Experiment 1 (Fig. 2c), after providing brief demographic information, subjects were seated in front of a calibrated infrared thermal imaging camera (Fig. 2a; FLIR T450sc, FLIR Systems Inc., Nashua, NH) positioned 1.5 meters from subjects face in a thermally stable testing room (Fig. 2b) maintained at 24 °C. Time-lapsed (30 Hz) infrared (λ = 7.5 to 13 μm) images were acquired during the 5 min baseline period, during the 15 min sham or TEN treatment period and for a recovery period up to 10 min following the termination of treatment (Supplementary Videos 1 and 2). Images were then stored for later offline image analysis. Regions of interest were positioned and tracked on the forehead, cheeks, nose and chin region of the face (Fig. S1). From radiographic datasets the average baseline temperature was calculated for each subject and facial location across the 5 min baseline period. Average temperatures were calculated for each subject from each facial region during the 2, 5, 10 and 20 min period following the onset of sham or TEN stimulation (Fig. 3a). These average temperatures were analyzed and expressed as a percent change from baseline (Fig. 3b) for each subject to account for any variation in baseline temperatures across subjects.

Profile of Mood States Survey

In Experiment 2 (Fig. 2d), after providing brief demographic information, subjects were seated in a testing room (Fig. 2b). Subjects had a 5 min resting period prior to receiving a 15 min sham or TEN treatment. Immediately following stimulation, participants completed the POMS survey36, which is a 65-item scale designed to assess transient affective states and comprises six subscales: Anger-Hostility (11 items), Depression-Dejection (14 items), Fatigue-Inertia (8 items), Vigor-Activity (6 items), Tension-Anxiety (3 items) and Confusion-Bewilderment (3 items). Items were scored on a 5-point scale, ranging from 0 = not at all to 4 = extremely and were indexed to how participants felt in that moment. Reliabilities of the subscales ranged from 0.50 to 0.91.

Acute stress induction

In Experiment 3 (Fig. 2e) we implemented an acute stress induction paradigm to study the effects of TEN on physiological and biochemical stress responses. Subjects received either TEN or sham treatment in a between subjects design (N = 10 subjects per group). All participants were tested between the hours of 13:00 and 16:00 to limit variability introduced by circadian fluctuations in salivary analytes (see Salivary collection and stress biomarker assays below). Following informed consent, subjects were allowed to acclimate for 20 min before providing a baseline saliva sample. After providing the initial saliva sample, subjects were connected to a wearable TEN device as described above, a peripheral nerve stimulator (MiniStim MS-IVA, Life-Tech, Inc., Dallas, TX) was positioned over the median nerve of the right wrist and a Shimmer3 (Shimmer, Dublin, Ireland) optical heart rate (HR) monitor and galvanic skin conductance (GSC) sensor was placed on the index, middle and ring fingers of the opposite hand. The timing, presentation of stimuli and acquisition of HR and GSC data was accomplished using Attention Tool (iMotions, Inc., Cambridge, MA).

The stress trial (Fig. 2e) commenced 30 sec prior to the onset of TEN or sham treatment. On the beginning of the stress trial there was a 6 min pre-trial period to give the TEN or sham treatment time to begin exerting an effect before the induction of acute stress began. The stress trial comprised a 6 min classical fear-conditioning paradigm immediately followed by a 6 min time-constrained cognitive battery. Immediately following the time pressured cognitive tests the stress trial concluded with a 3 min neutral video of a nature scene. Both the classical fear-conditioning paradigm and the time-constrained series of cognitive tests are known to induce acute stress and increase sympathetic activity69,70,71,72. Before the beginning of the stress trial, participants were instructed that when the computer monitor they were seated in front of began to flash still images (instead of the baseline period video) that they would be given an electrical shock every time an image of lightning appeared. The transition from baseline videos to still images during the fear-conditioning portion of the stress trail induced an anticipatory increase in acute stress as reflected by instantaneous changes in GSC (Fig. 6b) in every subject. During the fear-conditioning component of the stress trial participants were randomly presented with 40 still images of nature scenes for 6 seconds each: 10 images of lightning paired with electrical shock (0.5 sec, 4–6.5 mA) and 30 neutral nature scenes.

The second component of the stress trial included a time-constrained series of three cognitive tests (2 min each) including a Flanker test, n-back working-memory test and a Stroop task. The Flanker task is a selective attention task in which participants indicate the direction of a target stimulus that is flanked by stimuli that are oriented in the same direction (congruent), in the opposite direction (incongruent), or a neutral direction as the response target. The n-back, which assesses working memory, has participants view a sequence of stimuli and indicate when the current stimulus matches the stimuli from n steps earlier in the sequence. In this case, subjects were instructed to get to 2 back as quickly and accurately as possible. The Stroop task tests sematic memory by having participants indicate the color of the ink in which a color word is written as fast as possible. Trials can be congruent, the text color and the word refer to the same color, or incongruent, the ink color and the word refer to different colors, which can lead to frustration and itself induce acute stress71,72. Reaction times and accuracy were measured for all tests and analyzed off-line in subsequent analyses. Following the stress trial, there was a 30 min. recovery period during which subjects reported the presence, duration (in minutes) and severity (0 to 8 scale) of skin redness or irritation, headache, dizziness, nausea and vision or hearing changes and two additional saliva samples were collected as described below.

Heart rate variability and galvanic skin conductance metrics

We acquired cardiac activity and electrodermal activity during the stress trial using a Shimmer3 optical heart rate monitor integrated with a GSC sensor. Raw electrocardiogram data were collected using Attention Tool before being processed offline using Kubios HRV (University of Eastern Finland, Kuopio, Finland). From these data we quantified the average HR, R-R interval, standard deviation of the normal-to-normal heartbeat (SDNN), power in the low-frequency (0.04–0.15 Hz) and high-frequency (0.15–0.4 Hz) bands of the HRV spectra and the LF/HF ratio in response to TEN and sham treatments during the stress trial. All GSC data were acquired using Attention Tool and stored offline for analysis using Igor Pro (Wavemetrics, Inc., Portland, OR). We quantified the peak-to-peak changes for GSC on raw data, where: ΔGSC = (GSC peak − GSC base )/GSC base occurring at the onset of the fear-conditioning component of the stress trial (ΔGSC fear ; Fig. 6b) and the average peak-to-peak change in response to the delivery of the 10 unconditioned stimuli or electrical shocks (ΔGSC shock ; Fig. 6b) for each subject.

Salivary collection and stress biomarker assays

Prior to arrival, participants were instructed to not brush their teeth within 45 minutes, eat within one hour, consume caffeine or alcohol within 12 hours or have dental work performed within 24 hours of their scheduled appointment. After providing informed consent, participants rinsed their mouths in preparation for contributing a saliva assay and were then seated in a quiet room during which they self-reported basic demographic information. After 20 minutes, participants provided a baseline saliva sample via the passive drool method. As per manufacturer’s instructions (SalivaBio, Inc., State College, PA), saliva is pooled at the front of the mouth and eased through a tube, centered on the lips, directly into a cryovial and immediately stored at −20 °C. The same collection procedure was used to collect additional saliva samples 10 min and 30 min following the end of the stress trial. Saliva samples were coded and sent to Salimetrics, LLC (State College, PA) where ELISA methods were employed to assess α-amylase (Salimetrics 1–1902) and cortisol levels (Salimetrics 3002) in a blinded manner using a subject coding procedure.

The protein α-amylase is widely recognized as a biochemical marker of sympathetic nervous system activity and sympathoadrenal medullary (SAM) axis activation. More specifically, salivary levels of α-amylase directly correlate with plasma norepinephrine (NE) concentrations following the induction of acute stress including when electrical shock is used as a stressor39,40,41,42,43,44,45. Cortisol is a prototypical stress hormone reflective of hypothalamus pituitary adrenal (HPA) axis activation, which has slower onset and longer lasting effects compared to SAM axis activation.

Statistical analyses

All statistical analyses were conducted using one-way ANOVA’s, repeated measures ANOVA’s, or independent t-tests with IBM SPSS Statistics Software (IBM Corporation, Armonk, NY) as indicated. Experimenters analyzing data were blinded to treatment groups using a subject coding procedure. Prior to analysis all group data were confirmed to be normally distributed using a test for normality with the Shapiro-Wilk procedure (P > 0.05) and visual inspection of Q-Q plots in SPSS. The variance on all between group data was examined using Levene’s test for homoscedasticity with no significant differences at P > 0.05. Analyses of thermographic data from different facial regions were conducted using a repeated measures ANOVA with time as the within subjects factor and treatment as the between subjects factor. The POMS data and cognitive data were analyzed using a series of one-way ANOVA’s with SPSS. The HR, HRV and GSC data were analyzed using independent t-tests. Thresholds for statistical significance were set at P < 0.05. All data reported and shown are mean ± SD unless otherwise indicated.