Non-Invasive Vagus Nerve Stimulation Enhances Cooperative Behavior In Humans

Carina R. Oehrn (  carina.oehrn@staff.uni-marburg.de ) Philipps-University Marburg Lena Molitor Philipps-University Marburg Kristina Krause Philipps-University Marburg Hauke Niehaus Philipps-University Marburg Laura Schmidt Philipps-University Marburg Lukas Hakel Philipps-University Marburg Lars Timmermann Philipps-University Marburg Katja Menzler Philipps-University Marburg Susanne Knake Philipps-University Marburg Immo Weber Philipps-University Marburg


Introduction
The vagus nerve is a central part of the gut-brain axis and bi-directionally links the vegetative and the central nervous system [1]. A range of cognitive and emotional processes can in uence vegetative processes via the vagus nerve, i.e. by changes in heart rate or respiration [2]. The impact of vagus nerve activity on cognitive and emotional processes, i.e., the other direction of information ow, is still largely unknown. Electrical stimulation of the vagus nerve (vagus nerve stimulation, VNS) can be used to study causal effects of vagus nerve activity on functions of the central nervous system. As it constitutes a common treatment for medication-resistant epilepsy, it can be safely applied in humans [3].
Evidence from rodents [4,5] and humans [6-8] provide converging evidence that invasive and noninvasive VNS is associated with enhanced reward processing, reinforcement learning and recognition memory. Animal studies indicate that these alterations in behavior are associated with enhanced dopamine release in the midbrain [4,5,9]. Based on current evidence, these effects are function-speci c, as studies failed to observe effects on working memory [10], implicit learning [11] and con ict processing [12].
The polyvagal theory represents a bio-behavioral model that relates vagus nerve activity to social interaction [13]. Based on phylogenetic reasoning and anatomical ndings of vagus nerve connectivity, it implicates the efferent part of the vagus nerve in the expression of social behaviors, e.g., through its projections to laryngeal, pharyngeal and facial muscles essential for verbal and non-verbal communication. However, the role of afferent projections of the vagus nerve to the brain for social cognition is still unclear.
Studies that indirectly assessed vagus nerve activity by means of heart-rate variability (HRV) in humans report that high HRV, and thus parasympathetic tonus, at baseline is predictive for enhanced social engagement and cooperative behavior [14][15][16][17]. One meta-analysis of 16 studies demonstrated that compassion, an important emotional aspect of social interaction [18], positively correlates with HRV [19].
However, the causal relationship between vagus nerve activity and social behavior and the direction of interaction between vagus nerve and the central nervous system during these processes are still unknown.
Several studies show that social interaction recruits similar neural networks as reward processing (for a review see 20). Rewarding social stimuli, e.g. positive facial expressions, own social reputation and positive social feedback, are associated with activity in dopaminergic basal-ganglia-thalamo-cortical circuits similar to non-social rewards [21][22][23][24][25]. In particular, cooperation with other humans is perceived as rewarding and recruits brain regions involved in reward processing, such as the medial orbitofrontal cortex [26,27], caudate nucleus[28] and anterior cingulate cortex [27].
Based on human and animal studies demonstrating effects of the VNS on dopaminergic brain circuits and reward processing, one might hypothesize that the stimulation of afferent bers of the vagus nerve enhances rewards derived from social interactions. On a behavioral level, this might be re ected by enhanced cooperative behavior with other humans.
Recent studies indicate that speci c personality traits, in particular neuroticism and extraversion, are associated with social behavior [29]. While personality traits are undoubtedly in uenced by multiple neurotransmitter systems, speci cally extraversion and neuroticism have been linked to dopaminedependent reward-processing [30,31]. Thereby, a highly reactive dopaminergic system, e.g. as measured by dopamine-relevant genes, structural volume of dopamine-rich brain regions or dopamine receptor availability, has been associated with high extraversion, whereas the opposite has been suggested for neuroticism [32][33][34]. If afferent VNS effects are mediated via the dopaminergic system, one could thus hypothesize individual stimulation effects interact with these personality traits.
Decision making processes can be disentangled into several sub-processes based on choices and reaction time. Drift-diffusion modelling (DDM) constitutes one of the most common methods for the assessment of value-based choices [35]. DDM dissects the decision process into several sub-processes including a starting bias towards response options, the rate of accumulation of information (i.e., the drift rate), the amount of information needed for a decision (i.e., boundary separation) and non-decision operations re ecting perceptual and motor computations. While the drift-diffusion model is commonly used to make inferences on classic perceptual decision making tasks like the anker [36] or color discrimination task 37,38. Only few studies have analyzed sub-components of social decision making in humans [39][40][41][42]. Previous studies show an association between shifts in starting bias and reward value expectation [38,43]. These studies report associations between pro-social social behavior and changes in starting bias and drift rate, both relatively early parts of the decision process [39,42]. Thus, one could hypothesize that VNS effects on cooperative behavior occur at these early stages of the decision process.
Here, we assess the causal relationship between vagus nerve activity and social interaction. To this end, we applied transcutaneous VNS (tVNS) to 19 long-standing seizure-free epilepsy patients, who had never received invasive or non-invasive VNS treatment. We applied auricular VNS in order to stimulate afferent parts of the vagus nerve, while patients performed a computerized version of the well-established prisoner's dilemma task [44]. Based on trial-by-trial choices of participants, we assessed effects of stimulation on cooperative behavior. Further, we assessed the impact of subject characteristics, e.g., sex and age, on stimulation effects. We also included the big ve personality traits into our analysis, i.e., neuroticism, extraversion, openness, agreeableness, and conscientiousness. To understand the impact of tVNS on social decision making in more detail, we used behavioral modelling.

Participants
In order to estimate effect size of tVNS on social behavior, we rst conducted a pilot study with three patients. We subsequently estimated sample size by means of a power analysis implemented in Matlab (function sampsizepwr of the Statistics and Machine Learning Toolbox), which indicated a minimum of 18 participants required to reveal an effect of stimulation on cooperations (α = 0.05) with 95% power. As our study design entailed the completion of the paradigm on two separate study days with an interval of 14 days between measurements, we expected a 20% drop out rate. We therefore recruited 23 VNS-naïve patients with temporal lobe epilepsy throughout the course of one year. We excluded patients with neurological or psychiatric comorbidities by means of the medical history, the Beck Depression Inventory (BDI-II 45) and the Quality of Life in Epilepsy questionnaire (QoLiE 46). To minimize the impact of epilepsy on the results, we exclusively included participants with a minimum seizure-free period of one year and without hippocampal sclerosis. Epileptic medication was stable across testing sessions. Four participants withdrew from the study between testing days, resulting in a nal number of 19 participants with complete data sets (13 females; mean age ± SD: 45 ± 12 years). Participants completed a neuropsychological test battery including measures of executive functioning, memory, recall and implicit memory. Further, we obtained self-ratings on the NEO Personality Inventory by Costa und McCrae (NEO-PI) assessing the big ve personality traits [47] and the positive and negative affect schedule (PANAS 48, Supplemental Table S1). All participants signed written informed consent. The study protocol was approved by the ethics committee of the Faculty of Medicine at the University of Marburg and conducted in accordance with the latest version of the Declaration of Helsinki.

Sham and vagus nerve stimulation
We applied sham and transcutaneous vagus nerve stimulation on two separate days with an interval of 14 days between measurements. To avoid order effects, we applied stimulation in a double-blind, pseudorandomized manner, i.e., nine patients received tVNS during the rst, ten during the second testing session. In both conditions, we stimulated participants for two hours before and during the behavioral experiment and followed the same experimental protocol including the identical stimulation frequency (25 Hz), duty cycle (50%), pulse width (30 s) and amplitude (below individual pain threshold: (mean±SD amplitude: tVNS: 1.17±0.51mA, sham: 1.17±0.46mA).
In the active tVNS condition, we applied stimulation to the left cymba conchae to stimulate the auricular branch of the vagus nerve according to the guidelines of the manufacturer (see Fig. 1Ai). This area of the external ear is innervated exclusively by the sensory branch of the vagus nerve, while other parts receive afferent innervation shared with other nerves [49,50]. Current evidence including anatomical and neuroimaging studies, as well as investigations of autonomic parameters in response to auricular tVNS suggests that the cymba conchae constitutes a suitable location for vagal modulation [51]. During sham stimulation, we attached the probe at the center of the left lobule (see Fig. 1Aii). An independent clinician, who was not involved in the acquisition and analysis of data, attached the device on each testing day and subsequently covered the ear using a headband. The location of stimulation was therefore neither visible to the experimenters, nor the participants, who were unaware of the current stimulation condition. Participants were told that the purpose of the study was to test different stimulation settings of tVNS [52].

Experimental paradigm
Subjects performed a computerized version of the iterated prisoner's dilemma paradigm (Fig. 1B, 53) presented on a 24-inch monitor using the PsychToolbox-3 [54] implemented in Matlab (R2016b, Mathworks) in an acoustically shielded chamber. Participants indicated their responses by button press on a standard keyboard. All participants received written and oral instructions and performed several practice trials, in which different stimuli were used than in the subsequent experiment. The experimenter ensured that each participant understood the task before commencing the main part of the experiment.
During the task, subjects played against eight different opponents and could earn points by either cooperating or deceiving their counterpart. The number of awarded points depended on the choice of the player and opponent (Fig. 1C). We instructed subjects to score as many points as possible. To disentangle effects of TVNS on reward-seeking from speci c effects on social behavior, we ensured that deception yielded the greatest payoff using predetermined playing strategies of the computerized opponents (maximal possible points per testing day across all opponents: 2175, points achieved when always deceiving: 1725, points achieved when always cooperating: 1500).Subjects completed a total of 120 trials. In 60 trials, subjects were led to believe that they played live against four human opponents (15 trials each). To this end, we implemented a cover story in which the subject played live against university students including delayed logins of one opponent implemented in the Matlab code. In the other half of the trials, subjects knew that they played against four computers (15 trials each). In all trials, subjects played against computers with one of four predetermined game strategies. We assigned the same game strategies to "humans" and computer opponents: cooperative style (70% cooperation), deceitful style (70% deception), tit for tat (replicating the response of the player) and random (drawn from a uniform distribution). Against three of the four opponents, deception represented the most rewarding game strategy, i.e., resulted in the greatest number of points (Supplemental Figure 1). All trials were presented in a randomized order (across opponents and strategies). To make opponents more relatable to the subjects, each opponent (humans and computers) was introduced in the beginning of the experiment. To this end, we presented images of four humans and computers pseudo-randomly chosen from a pool of 20 human photos of volunteering colleagues and laboratory members (10 women, 10 men) and 10 computer pictures. These images were accompanied by a randomly selected name (from a list of the most common names given between 1985 and 1995), birthplace (selected from a list of medium-sized cities in Germany) and age (between 20 and 30 years) to match the cover story. After introduction of the opponents, we asked subjects to rate the likability of each opponent on a visual rating scale using the Self-Assessment Manikin (SAM, 55, Fig. 1D).
During the experiment, subjects randomly played against each opponent for 15 trials. In each trial, the rst screen consisted of a presentation of the opponent. Thereafter, the subject and opponent were successively asked to decide whether they choose to cooperate or deceive each other. The subjects and opponent's decision were requested in a pseudo-randomized order and the result of both decisions was presented after the second decision had been made. We presented the result alongside the number of achieved points for the last and for all trials. Simulated response times of opponents were randomly drawn from an interval of one to three seconds to match the cover story of live opponents. After completion of all trials, the total number of points gained against each opponent was presented. After both testing days, the experimenters assessed whether subjects believed that they played live against human opponents and whether they had noticed differences between stimulation conditions.

Statistical analysis
We performed all calculations with Matlab 2016b (Mathworks), the DMAT toolbox [56], SPSS (IBM SPSS Statistics, V26) and self-written code. The alpha level for all tests was set to 0.05.

Effects of stimulation on cooperation
We discarded trials with invalid responses, i.e., when subjects pressed an unde ned key or exceeded the time limit (tVNS: 0-1 trials per subject, sham: 0-6 trials per subject, 16 trials across conditions and subjects). Thereafter, we investigated effects of stimulation (tVNS vs. sham) and opponent (human vs. computer) on the number of cooperations (in percent) and reaction times by means of a repeated measures ANOVA. We further assessed the effect of stimulation on the likeability of opponents, which might indirectly affect cooperation, and on gaming performance measured as total achieved points.

Prediction of cooperation
When we found effects in the repeated measures ANOVA, we assessed the impact of several parameters on trial-by-trial task performance by means of a mixed effects logistic regression model. We thereby incorporated several parameters into the model that could impact cooperative behavior either independently or in interaction with the stimulation effect. To this end, we de ned the factor "subject" as random effect and assessed xed effects of opponent characteristics (likability, gaming strategy of opponent, last decision of opponent), subject characteristics (sex, age, NEO-PI scores, believed that they played live against real opponents) and the factor time on the subject's decision to cooperate or betray.
We further incorporated interaction effects between stimulation and all xed parameters. To avoid multicollinearity, we rst performed a feature selection assuring that predictors were not highly correlated (Pearson correlations, R 2 >0.7).

Behavioral modelling
We dissected the decision process into several cognitive components using DDM. To this end, we included the behavioral parameters "reaction time" and "choice" (cooperation/deception) of all trials and tted seven nested models for both stimulation conditions separately. For each model, we allowed one model parameter to vary freely for all conditions and compared it to a model with completely xed parameters using a chi-square difference test at an alpha-level of 0.05 [56,57]. The estimated parameters include starting point, drift-rate, non-decision time and boundary separation. Starting point was normalized by the individual boundary separation to improve comparability between individuals and conditions resulting in the starting bias with a range of zero to one with 0.5 indicating no initial preference for either choice. For starting bias and drift-rate, we additionally performed a one-sample t-test against 0.5 and against zero, respectively. We corrected for multiple comparisons by means of Bonferroni correction.

Results
Our post-experimental questionnaire revealed that none of the patients noticed a difference in stimulation or behavioral performance between the two sessions. Further, patients did not perceive gastrointestinal, cardiac, or other sensations during either stimulation condition. 12 out of 19 patients believed that they were playing against live opponents.

Effects of stimulation on cooperations
The repeated measures ANOVA revealed that subjects cooperated more frequently during tVNS compared to sham stimulation ( Fig. 2A Figure 2A, main effect opponent: F 1,18 =54.72, p<0.001) and scored less points against human opponents compared to computers (Supplemental Figure 2B, main effect opponent: F 1,18 =5.59, p=0.03).  Figure 3A, R=-0.48, p=0.038), but no correlation with extraversion (Supplemental Figure 3B, R=0.23, p=0.35). Please refer to Supplemental Table S2 for a complete overview of the results.

Effects of tVNS on mood and other bodily sensations
A paired two sample t-tests revealed no effect of tVNS on acute affect as measured by positive or

Discussion
We assessed the causal relationship between vagus nerve activity and social interaction in humans by means of tVNS. Recent evidence from rodents [4,5] and humans [6,7] indicate that tVNS is associated with enhanced reward-seeking and reinforcement learning. To disentangle general effects on reward processing from speci c effects on social behavior, we used a paradigm in which deception yielded the greatest payoff. A repeated measures ANOVA and a logistic mixed effects regression model provide converging evidence that tVNS enhanced cooperative behavior compared to sham simulation independent of obtained rewards, i.e., points attained in the game. This indicates that tVNS has a speci c effect on social interaction that can be dissociated from general effects on reward-processing. Our logistic mixed effects regression model further revealed that participants cooperated less frequently when opponents had deceived them in the preceding trial and more frequently with opponents they liked, according to the pre-game likability assessment. This emphasizes the impact of social factors on behavior in this computerized task, i.e., that participants did not solely rely on a rational game strategy.
Our control analyses demonstrated that tVNS effects were not indirectly mediated by effects on mood and other bodily sensations.
We further investigated the impact of subject characteristics on cooperative behavior and the stimulation effect and found that women cooperated more frequently than men. Previous studies found mixed effects of an association between sex, gender norms and cooperation [58]. However, a discussion hereof would exceed the scope of this paper. Further, we found interactions between the effect of tVNS on cooperation and speci c personality traits. Our analyses revealed that the stimulation effect decreased as a function of participants' neuroticism, a personality trait associated with a less functional dopamine system in the brain [32,33]. Due to our sample size, this result is preliminary and should be interpreted with caution. However, based on recent evidence from rodents suggesting that VNS boots activity in dopaminergic brain regions [4,5], one could speculate that VNS exerts a particularly strong effect on social behavior in humans with a less active dopaminergic system. Further, our mixed effects model revealed a positive association between extraversion and the stimulation effect, which, however, was not signi cant in a post hoc correlation analysis.
To disentangle which sub-components of social decision-making were affected by tVNS, we calculated behavioral models based on choices and reaction times. Behavioral modelling suggests that tVNS in uences starting bias, and therefore early stages of the decision process. Human studies demonstrated that the parameter starting point re ects a bias in reward value expectation [38,43] as well as prior reward probability [43] independent of the cognitive processing of sensory evidence [59]. These ndings indicate that tVNS mediates cooperative behavior by biasing participants' expectation toward cooperative behavior even before further information about the current opponent is accumulated. Further, we found effects of tVNS on drift rates in line with a previous study showing that a lower absolute drift-rate is associated with pro-social, i.e. altruistic, decision making [42]. Drift rate is thought to re ect the quality of information extracted from the presented stimulus [38,60]. This indicates that after presentation of the opponent, the extraction and accumulation of information leading to cooperative behavior is enhanced. We did nd a statistical difference in non-decision time between stimulation conditions, the effect size was close to zero (d=0.051) and thus its in uence on cooperative behavior probably negligible.
One limitation of our study is that we assessed effects in epilepsy patients, as the device had no CE certi cate for the use in healthy participants. To reduce possible effects of the disease, we only included patients, who had been seizure-free for at least a year and did not exhibit macroscopically visible lesion in the hippocampus. However, we cannot rule out that microlesions and local changes in neurotransmitter systems impacted the results. Thus, future studies should replicate this nding in healthy controls. Further, it is of interest to assess neural networks involved in the enhancement of cooperative behavior by VNS. Based on human and animal studies demonstrating effects of VNS on dopaminergic brain circuits (for a review see 20) and studies showing that social interaction recruits similar networks as reward processing, one could hypothesize the effect of tVNS on social behavior is mediated by dopaminergic neural networks. However, the effect of tVNS, as well as social interaction itself, is likely to be a more complex process involving alterations in other neuropeptides, such as oxytocin and noradrenaline (for a review see 61). The investigation of neural networks associated with VNS effects on social interaction will be subject to future studies.

Conclusion
Taken together, our results indicate that enhanced vagus nerve activity plays a causal role for mediating social interaction and biases participants towards cooperative behavior. This effect is more pronounced in participants with higher scores in neuroticism. Behavioral modelling revealed that the effect of VNS on stimulation occurs at early stages of decision-making, even before stimulus processing. Thus, our results indicate that alterations in vagal tone are not merely an adaptive process in response to social situations, but can also, in return, in uence social behavior. The interaction between vagal activity and social behavior is therefore bidirectional. On the one hand, brain to vagus nerve information ow can elicit changes in autonomic activity, such as respiration and heart rate [2]. On the other hand, vagus stimulation can in uence cognitive processes and mediate behavior in social contexts. Thus, one could speculate that effects of VNS on cooperative behavior dates to an early survival mechanism. Vagal activity could therefore be crucial for survival by allowing for social bonding, when ght and ight (i.e., the sympathetic vegetative response) is not the most adaptive response.

Declarations Data Availability Statement
The datasets generated and analyzed during the current study and the analysis code are available from the corresponding author upon reasonable request.  Repeated measures ANOVA demonstrated a main effect "opponent" on reaction times (F 1,18 =4.76, p=0.043). Bar graphs illustrate mean±SEM parameters across participants.

Figure 3
Effect of stimulation on DDM parameters. A chi-square difference test comparing the goodness of t of the two competing models used in DDM analysis demonstrated stimulation effects on A) starting bias (p=0.01) and B) drift-rate (p<0.001). Stimulation increased the starting bias towards cooperation and shifted drift-rate towards zero. Bar graphs illustrate mean±SEM parameters across participants.

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