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Evidence for a modulating effect of transcutaneous auricular vagus nerve stimulation (taVNS) on salivary alpha-amylase as indirect noradrenergic marker: A pooled mega-analysis

Open AccessPublished:September 29, 2022DOI:https://doi.org/10.1016/j.brs.2022.09.009

      Highlights

      • Data pooling across 10 studies showed that taVNS leads to increased salivary alpha-amylase release compared to sham.
      • These findings substantiate the assumption that vagal activation via taVNS triggers noradrenaline release.
      • The diurnal trajectory of salivary alpha-amylase activity was replicated.

      Abstract

      Background

      Non-invasive transcutaneous auricular vagus nerve stimulation (taVNS) has received tremendous attention as a potential neuromodulator of cognitive and affective functions, which likely exerts its effects via activation of the locus coeruleus-noradrenaline (LC-NA) system. Reliable effects of taVNS on markers of LC-NA system activity, however, have not been demonstrated yet.

      Methods

      The aim of the present study was to overcome previous limitations by pooling raw data from a large sample of ten taVNS studies (371 healthy participants) that collected salivary alpha-amylase (sAA) as a potential marker of central NA release.

      Results

      While a meta-analytic approach using summary statistics did not yield any significant effects, linear mixed model analyses showed that afferent stimulation of the vagus nerve via taVNS increased sAA levels compared to sham stimulation (b = 0.16, SE = 0.05, p = 0.001). When considering potential confounders of sAA, we further replicated previous findings on the diurnal trajectory of sAA activity.

      Conclusion(s)

      Vagal activation via taVNS increases sAA release compared to sham stimulation, which likely substantiates the assumption that taVNS triggers NA release. Moreover, our results highlight the benefits of data pooling and data sharing in order to allow stronger conclusions in research.

      Keywords

      1. Introduction

      Transcutaneous auricular vagus nerve stimulation (taVNS) has drawn tremendous attention as a promising non-invasive brain stimulation tool for the treatment of clinical disorders [
      • Broncel A.
      • Bocian R.
      • Kłos-Wojtczak P.
      • Kulbat-Warycha K.
      • Konopacki J.
      Vagal nerve stimulation as a promising tool in the improvement of cognitive disorders.
      ], such as pharmacoresistant epilepsy [
      • Rong P.
      • Liu A.
      • Zhang J.
      • Wang Y.
      • He W.
      • Yang A.
      • Li L.
      • Ben H.
      • Li L.
      • Liu H.
      • et al.
      Transcutaneous vagus nerve stimulation for refractory epilepsy: a randomized controlled trial.
      ,
      • Bauer S.
      • Baier H.
      • Baumgartner C.
      • Bohlmann K.
      • Fauser S.
      • Graf W.
      • Hillenbrand B.
      • Hirsch M.
      • Last C.
      • Lerche H.
      • et al.
      Transcutaneous vagus nerve stimulation (tvns) for treatment of drug-resistant epilepsy: a randomized, double-blind clinical trial (cmpse02).
      ,
      • Aihua L.
      • Lu S.
      • Liping L.
      • Xiuru W.
      • Hua L.
      • Yuping W.
      A controlled trial of transcutaneous vagus nerve stimulation for the treatment of pharmacoresistant epilepsy.
      ], depression [
      • Fang J.
      • Rong P.
      • Hong Y.
      • Fan Y.
      • Liu J.
      • Wang H.
      • Zhang G.
      • Chen X.
      • Shi S.
      • Wang L.
      • et al.
      Transcutaneous vagus nerve stimulation modulates default mode network in major depressive disorder.
      ] and chronic pain [
      • Napadow V.
      • Edwards R.R.
      • Cahalan C.M.
      • Mensing G.
      • Greenbaum S.
      • Valovska A.
      • Li A.
      • Kim J.
      • Maeda Y.
      • Park K.
      • et al.
      Evoked pain analgesia in chronic pelvic pain patients using respiratory-gated auricular vagal afferent nerve stimulation.
      ]. Given its non-invasive nature, taVNS has also more recently been used in non-clinical settings to modulate various affective and cognitive processes, such as emotion recognition, fear extinction, cognitive control, and attention (cf. [
      • Farmer A.D.
      • Strzelczyk A.
      • Finisguerra A.
      • Gourine A.V.
      • Gharabaghi A.
      • Hasan A.
      • Burger A.M.
      • Jaramillo A.M.
      • Mertens A.
      • Majid A.
      • et al.
      International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020).
      ,
      • Weymar M.
      • Zaehle T.
      Editorial: New Frontiers in Noninvasive Brain Stimulation: Cognitive, Affective and Neurobiological Effects of Transcutaneous Vagus Nerve Stimulation.
      ]). The effects of taVNS have been suggested to be related to the modulation of distinct brainstem, subcortical and cortical regions, and their associated neurotransmitter systems (cf. [
      • Colzato L.
      • Beste C.
      A literature review on the neurophysiological underpinnings and cognitive effects of transcutaneous vagus nerve stimulation: challenges and future directions.
      ]). Indeed, previous animal studies have shown that vagal afferents modulate serotonergic [
      • Dorr A.E.
      • Debonnel G.
      Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission.
      ,
      • Manta S.
      • Dong J.
      • Debonnel G.
      • Blier P.
      Enhancement of the function of rat serotonin and norepinephrine neurons by sustained vagus nerve stimulation.
      ], dopaminergic [
      • Tellez L.A.
      • Medina S.
      • Han W.
      • Ferreira J.G.
      • Licona-Limón P.
      • Ren X.
      • Lam T.T.
      • Schwartz G.J.
      • De Araujo I.E.
      A gut lipid messenger links excess dietary fat to dopamine deficiency.
      ,
      • Han W.
      • Tellez L.A.
      • Perkins M.H.
      • Perez I.O.
      • Qu T.
      • Ferreira J.
      • Ferreira T.L.
      • Quinn D.
      • Liu Z.-W.
      • Gao X.-B.
      • et al.
      A neural circuit for gut-induced reward.
      ], cholinergic [
      • Hulsey D.R.
      • Hays S.A.
      • Khodaparast N.
      • Ruiz A.
      • Das P.
      • Rennaker II, R.L.
      • Kilgard M.P.
      Reorganization of motor cortex by vagus nerve stimulation requires cholinergic innervation.
      ] and noradrenergic [
      • Roosevelt R.W.
      • Smith D.C.
      • Clough R.W.
      • Jensen R.A.
      • Browning R.A.
      Increased extracellular concentrations of norepinephrine in cortex and hippocampus following vagus nerve stimulation in the rat.
      ,
      • Raedt R.
      • Clinckers R.
      • Mollet L.
      • Vonck K.
      • El Tahry R.
      • Wyckhuys T.
      • De Herdt V.
      • Carrette E.
      • Wadman W.
      • Michotte Y.
      • et al.
      Increased hippocampal noradrenaline is a biomarker for efficacy of vagus nerve stimulation in a limbic seizure model.
      ] signaling. The exact neural mechanisms possibly mediating the effects of taVNS are, however, not fully understood yet.
      One of the hypothesized working mechanisms by means of which taVNS may exert some of its effects is through the activation of the locus coeruleus-noradrenaline (LC-NA) system. Afferent fibers of the vagus nerve forward information of the adrenergic release from the adrenal gland to the brain [
      • McIntyre C.K.
      • McGaugh J.L.
      • Williams C.L.
      Interacting brain systems modulate memory consolidation.
      ], where they project to the nucleus tractus solitarii (NTS) [
      • Izquierdo J.
      • Insua J.
      • Biscardi A.
      • Izquierdo I.
      Some observations on the responses to the afferent stimulation of the vagus.
      ,
      • Wan S.
      • Browning K.N.
      • Coleman F.H.
      • Sutton G.
      • Zheng H.
      • Butler A.
      • Berthoud H.-R.
      • Travagli R.A.
      Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons.
      ]. The NTS sends excitatory projections to the nucleus paragigantocellularis (PGi; [
      • Reyes B.A.
      • Van Bockstaele E.J.
      Divergent projections of catecholaminergic neurons in the nucleus of the solitary tract to limbic forebrain and medullary autonomic brain regions.
      ]), which, in turn, is linked to the noradrenergic brainstem nucleus LC [
      • Aston-Jones G.
      • Ennis M.
      • Pieribone V.A.
      • Nickell W.T.
      • Shipley M.T.
      The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network.
      ,
      • Nieuwenhuis S.
      • De Geus E.J.
      • Aston-Jones G.
      The anatomical and functional relationship between the p3 and autonomic components of the orienting response.
      ]. The LC-NA system projects to several brain regions through an extended neuronal network including frontal and medio-temporal regions [
      • Schwarz L.A.
      • Luo L.
      Organization of the locus coeruleus-norepinephrine system.
      ] and modulates behavior by tonic and phasic firing [
      • Aston-Jones G.
      • Rajkowski J.
      • Kubiak P.
      • Alexinsky T.
      Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task.
      ], thus exerting influence on perception, attention, motivation and memory processes [
      • Sara S.J.
      • Bouret S.
      Orienting and reorienting: the locus coeruleus mediates cognition through arousal.
      ]. Impairments in the LC-NA system have further been associated with cognitive decline in aging and some degenerative disorders, such as Alzheimer's disease [
      • Mather M.
      • Harley C.W.
      The locus coeruleus: essential for maintaining cognitive function and the aging brain.
      ,
      • Dahl M.J.
      • Mather M.
      • Werkle-Bergner M.
      • Kennedy B.L.
      • Guzman S.
      • Hurth K.
      • Miller C.A.
      • Qiao Y.
      • Shi Y.
      • Chui H.C.
      • et al.
      Locus coeruleus integrity is related to tau burden and memory loss in autosomal-dominant alzheimer's disease.
      ].
      Evidence for such a modulatory vagal influence on the LC-NA system activity comes from different lines of research. Animal studies showed increased LC-firing rates after invasive vagal nerve stimulation [
      • Dorr A.E.
      • Debonnel G.
      Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission.
      ,
      • Roosevelt R.W.
      • Smith D.C.
      • Clough R.W.
      • Jensen R.A.
      • Browning R.A.
      Increased extracellular concentrations of norepinephrine in cortex and hippocampus following vagus nerve stimulation in the rat.
      ,
      • Raedt R.
      • Clinckers R.
      • Mollet L.
      • Vonck K.
      • El Tahry R.
      • Wyckhuys T.
      • De Herdt V.
      • Carrette E.
      • Wadman W.
      • Michotte Y.
      • et al.
      Increased hippocampal noradrenaline is a biomarker for efficacy of vagus nerve stimulation in a limbic seizure model.
      ,
      • Groves D.A.
      • Bowman E.M.
      • Brown V.J.
      Recordings from the rat locus coeruleus during acute vagal nerve stimulation in the anaesthetised rat.
      ,
      • Hulsey D.R.
      • Riley J.R.
      • Loerwald K.W.
      • Rennaker II, R.L.
      • Kilgard M.P.
      • Hays S.A.
      Parametric characterization of neural activity in the locus coeruleus in response to vagus nerve stimulation.
      ] and reduced firing after vagotomy [
      • Svensson T.
      • Thoren P.
      Brain noradrenergic neurons in the locus coeruleus: inhibition by blood volume load through vagal afferents.
      ]. Various processes mediated by the LC-NA system have further been shown to be improved by invasive vagal stimulation in animals, including extinction learning [
      • Alvarez-Dieppa A.C.
      • Griffin K.
      • Cavalier S.
      • McIntyre C.K.
      Vagus nerve stimulation enhances extinction of conditioned fear in rats and modulates arc protein, camkii, and glun2b-containing nmda receptors in the basolateral amygdala, Neural Plasticity 2016.
      ,
      • Noble L.J.
      • Chuah A.
      • Callahan K.K.
      • Souza R.R.
      • McIntyre C.K.
      Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.
      ], memory retention [
      • Clark K.
      • Krahl S.
      • Smith D.
      • Jensen R.
      Post-training unilateral vagal stimulation enhances retention performance in the rat.
      ] and inhibitory avoidance learning [
      • Clark K.
      • Smith D.
      • Hassert D.
      • Browning R.
      • Naritoku D.
      • Jensen R.
      Posttraining electrical stimulation of vagal afferents with concomitant vagal efferent inactivation enhances memory storage processes in the rat.
      ], as well as in humans (see for verbal recognition memory [
      • Clark K.B.
      • Naritoku D.K.
      • Smith D.C.
      • Browning R.A.
      • Jensen R.A.
      Enhanced recognition memory following vagus nerve stimulation in human subjects.
      ,
      • Ghacibeh G.A.
      • Shenker J.I.
      • Shenal B.
      • Uthman B.M.
      • Heilman K.M.
      The influence of vagus nerve stimulation on memory.
      ]; but see [
      • Mertens A.
      • Gadeyne S.
      • Lescrauwaet E.
      • Carrette E.
      • Meurs A.
      • De Herdt V.
      • Dewaele F.
      • Raedt R.
      • Miatton M.
      • Boon P.
      • et al.
      The potential of invasive and non-invasive vagus nerve stimulation to improve verbal memory performance in epilepsy patients.
      ]).
      Further evidence comes from studies relating vagal activity to pupil dilation [
      • Bianca R.
      • Komisaruk B.R.
      Pupil dilatation in response to vagal afferent electrical stimulation is mediated by inhibition of parasympathetic outflow in the rat.
      ,
      • Jodoin V.D.
      • Lespérance P.
      • Nguyen D.K.
      • Fournier-Gosselin M.-P.
      • Richer F.
      • et al.
      Effects of vagus nerve stimulation on pupillary function.
      ,
      • Mridha Z.
      • de Gee J.W.
      • Shi Y.
      • Alkashgari R.
      • Williams J.
      • Suminski A.
      • Ward M.P.
      • Zhang W.
      • McGinley M.J.
      Graded recruitment of pupil-linked neuromodulation by parametric stimulation of the vagus nerve.
      ,
      • Lai J.
      • David S.V.
      Short-term effects of vagus nerve stimulation on learning and evoked activity in auditory cortex.
      ] and to the attention-related P300 amplitude of the event-related potentials (ERPs) [
      • Neuhaus A.
      • Luborzewski A.
      • Rentzsch J.
      • Brakemeier E.
      • Opgen-Rhein C.
      • Gallinat J.
      • Bajbouj M.
      P300 is enhanced in responders to vagus nerve stimulation for treatment of major depressive disorder.
      ,
      • De Taeye L.
      • Vonck K.
      • van Bochove M.
      • Boon P.
      • Van Roost D.
      • Mollet L.
      • Meurs A.
      • De Herdt V.
      • Carrette E.
      • Dauwe I.
      • et al.
      The p3 event-related potential is a biomarker for the efficacy of vagus nerve stimulation in patients with epilepsy.
      ], both representing physiological markers of LC-NA system activity (see for pupil dilation [
      • Joshi S.
      • Li Y.
      • Kalwani R.M.
      • Gold J.I.
      Relationships between pupil diameter and neuronal activity in the locus coeruleus, colliculi, and cingulate cortex.
      ,
      • Reimer J.
      • McGinley M.J.
      • Liu Y.
      • Rodenkirch C.
      • Wang Q.
      • McCormick D.A.
      • Tolias A.S.
      Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex.
      ,
      • Liu Y.
      • Rodenkirch C.
      • Moskowitz N.
      • Schriver B.
      • Wang Q.
      Dynamic lateralization of pupil dilation evoked by locus coeruleus activation results from sympathetic, not parasympathetic, contributions.
      ,
      • Breton-Provencher V.
      • Sur M.
      Active control of arousal by a locus coeruleus gabaergic circuit.
      ]; see for P300 [
      • Murphy P.R.
      • Robertson I.H.
      • Balsters J.H.
      • O’connell R.G.
      Pupillometry and p3 index the locus coeruleus–noradrenergic arousal function in humans.
      ,
      • Vazey E.M.
      • Moorman D.E.
      • Aston-Jones G.
      Phasic locus coeruleus activity regulates cortical encoding of salience information.
      ]; see for review [
      • Nieuwenhuis S.
      • Aston-Jones G.
      • Cohen J.D.
      Decision making, the p3, and the locus coeruleus–norepinephrine system.
      ]). For instance, invasive vagal stimulation was found to increase resting pupil diameter in epileptic patients [
      • Jodoin V.D.
      • Lespérance P.
      • Nguyen D.K.
      • Fournier-Gosselin M.-P.
      • Richer F.
      • et al.
      Effects of vagus nerve stimulation on pupillary function.
      ], an effect also found in animal data [
      • Bianca R.
      • Komisaruk B.R.
      Pupil dilatation in response to vagal afferent electrical stimulation is mediated by inhibition of parasympathetic outflow in the rat.
      ,
      • Mridha Z.
      • de Gee J.W.
      • Shi Y.
      • Alkashgari R.
      • Williams J.
      • Suminski A.
      • Ward M.P.
      • Zhang W.
      • McGinley M.J.
      Graded recruitment of pupil-linked neuromodulation by parametric stimulation of the vagus nerve.
      ] (see for review [
      • Burger A.M.
      • D'Agostini M.
      • Verkuil B.
      • Van Diest I.
      Moving beyond belief: a narrative review of potential biomarkers for transcutaneous vagus nerve stimulation.
      ]). With regard to the P300 amplitude, De Taeye and colleagues [
      • De Taeye L.
      • Vonck K.
      • van Bochove M.
      • Boon P.
      • Van Roost D.
      • Mollet L.
      • Meurs A.
      • De Herdt V.
      • Carrette E.
      • Dauwe I.
      • et al.
      The p3 event-related potential is a biomarker for the efficacy of vagus nerve stimulation in patients with epilepsy.
      ] observed that epileptic patients who responded favorably to invasive vagal stimulation showed an increase in P300 amplitude during stimulation. This effect was also found in depressive patients in an earlier study by Neuhaus and colleagues [
      • Neuhaus A.
      • Luborzewski A.
      • Rentzsch J.
      • Brakemeier E.
      • Opgen-Rhein C.
      • Gallinat J.
      • Bajbouj M.
      P300 is enhanced in responders to vagus nerve stimulation for treatment of major depressive disorder.
      ].
      In light of the substantial evidence towards a modulatory role of invasive vagal stimulation on LC-NA system activity (mostly in animals and human clinical contexts), recent studies have investigated whether non-invasive taVNS shows a similar impact on the LC-NA activity in healthy humans. Initial brain imaging studies confirmed enhanced functional LC activation during taVNS compared to active sham stimulation in healthy participants [
      • Kraus T.
      • Kiess O.
      • Hösl K.
      • Terekhin P.
      • Kornhuber J.
      • Forster C.
      Cns bold fmri effects of sham-controlled transcutaneous electrical nerve stimulation in the left outer auditory canal–a pilot study.
      ,
      • Frangos E.
      • Ellrich J.
      • Komisaruk B.R.
      Non-invasive access to the vagus nerve central projections via electrical stimulation of the external ear: fmri evidence in humans.
      ,
      • Yakunina N.
      • Kim S.S.
      • Nam E.-C.
      Optimization of transcutaneous vagus nerve stimulation using functional mri.
      ,
      • Sclocco R.
      • Garcia R.G.
      • Kettner N.W.
      • Isenburg K.
      • Fisher H.P.
      • Hubbard C.S.
      • Ay I.
      • Polimeni J.R.
      • Goldstein J.
      • Makris N.
      • et al.
      The influence of respiration on brainstem and cardiovagal response to auricular vagus nerve stimulation: a multimodal ultrahigh-field (7t) fmri study.
      ,
      • Borgmann D.
      • Rigoux L.
      • Kuzmanovic B.
      • Thanarajah S.E.
      • Münte T.F.
      • Fenselau H.
      • Tittgemeyer M.
      Modulation of fmri brainstem responses by transcutaneous vagus nerve stimulation.
      ,
      • Teckentrup V.
      • Krylova M.
      • Jamalabadi H.
      • Neubert S.
      • Neuser M.P.
      • Hartig R.
      • Fallgatter A.J.
      • Walter M.
      • Kroemer N.B.
      Brain signaling dynamics after vagus nerve stimulation.
      ]. Other studies, but not all, showed a modulatory effect of taVNS on various cognitive and affective processes potentially associated with noradrenergic signaling, with respect to fear extinction (see for positive effects [
      • Burger A.M.
      • Verkuil B.
      • Van Diest I.
      • Van der Does W.
      • Thayer J.F.
      • Brosschot J.F.
      The effects of transcutaneous vagus nerve stimulation on conditioned fear extinction in humans.
      ,
      • Burger A.
      • Verkuil B.
      • Fenlon H.
      • Thijs L.
      • Cools L.
      • Miller H.
      • Vervliet B.
      • Van Diest I.
      Mixed evidence for the potential of non-invasive transcutaneous vagal nerve stimulation to improve the extinction and retention of fear.
      ,
      • Szeska C.
      • Richter J.
      • Wendt J.
      • Weymar M.
      • Hamm A.O.
      Promoting long-term inhibition of human fear responses by non-invasive transcutaneous vagus nerve stimulation during extinction training.
      ]; but see for no effects [
      • Genheimer H.
      • Andreatta M.
      • Asan E.
      • Pauli P.
      Reinstatement of contextual conditioned anxiety in virtual reality and the effects of transcutaneous vagus nerve stimulation in humans.
      ,
      • Burger A.M.
      • Van Diest I.
      • van der Does W.
      • Hysaj M.
      • Thayer J.F.
      • Brosschot J.F.
      • Verkuil B.
      Transcutaneous vagus nerve stimulation and extinction of prepared fear: a conceptual non-replication.
      ]), memory (see for positive effects [
      • Jacobs H.I.
      • Riphagen J.M.
      • Razat C.M.
      • Wiese S.
      • Sack A.T.
      Transcutaneous vagus nerve stimulation boosts associative memory in older individuals.
      ,
      • Giraudier M.
      • Ventura-Bort C.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ]; but see for no effects [
      • Mertens A.
      • Gadeyne S.
      • Lescrauwaet E.
      • Carrette E.
      • Meurs A.
      • De Herdt V.
      • Dewaele F.
      • Raedt R.
      • Miatton M.
      • Boon P.
      • et al.
      The potential of invasive and non-invasive vagus nerve stimulation to improve verbal memory performance in epilepsy patients.
      ,
      • Mertens A.
      • Naert L.
      • Miatton M.
      • Poppa T.
      • Carrette E.
      • Gadeyne S.
      • Raedt R.
      • Boon P.
      • Vonck K.
      Transcutaneous vagus nerve stimulation does not affect verbal memory performance in healthy volunteers.
      ]), cognitive control (see for positive effects [
      • Sellaro R.
      • van Leusden J.W.
      • Tona K.-D.
      • Verkuil B.
      • Nieuwenhuis S.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation enhances post-error slowing.
      ,
      • Sellaro R.
      • de Gelder B.
      • Finisguerra A.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation (tvns) enhances recognition of emotions in faces but not bodies.
      ,
      • Steenbergen L.
      • Sellaro R.
      • Stock A.-K.
      • Verkuil B.
      • Beste C.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation (tvns) enhances response selection during action cascading processes.
      ,
      • Fischer R.
      • Ventura-Bort C.
      • Hamm A.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) enhances conflict-triggered adjustment of cognitive control.
      ,
      • Keute M.
      • Boehrer L.
      • Ruhnau P.
      • Heinze H.-J.
      • Zaehle T.
      Transcutaneous vagus nerve stimulation (tvns) and the dynamics of visual bistable perception.
      ]; but see for no effects [
      • Tona K.-D.
      • Revers H.
      • Verkuil B.
      • Nieuwenhuis S.
      Noradrenergic regulation of cognitive flexibility: no effects of stress, transcutaneous vagus nerve stimulation, and atomoxetine on task-switching in humans.
      ]) and attention (see for positive effects [
      • Rufener K.S.
      • Geyer U.
      • Janitzky K.
      • Heinze H.-J.
      • Zaehle T.
      Modulating auditory selective attention by non-invasive brain stimulation: differential effects of transcutaneous vagal nerve stimulation and transcranial random noise stimulation.
      ,
      • Maraver M.J.
      • Steenbergen L.
      • Hossein R.
      • Actis-Grosso R.
      • Ricciardelli P.
      • Hommel B.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation modulates attentional resource deployment towards social cues.
      ]; but see for no effects [
      • Burger A.M.
      • Van der Does W.
      • Brosschot J.F.
      • Verkuil B.
      From ear to eye? no effect of transcutaneous vagus nerve stimulation on human pupil dilation: a report of three studies.
      ]).
      Despite the promising indications for taVNS-related behavioral improvements, there is current uncertainty regarding the relation between NA markers and taVNS-mediated vagal activation due to a number of non-replicable or merely subtle findings (cf. [
      • Farmer A.D.
      • Strzelczyk A.
      • Finisguerra A.
      • Gourine A.V.
      • Gharabaghi A.
      • Hasan A.
      • Burger A.M.
      • Jaramillo A.M.
      • Mertens A.
      • Majid A.
      • et al.
      International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020).
      ,
      • Burger A.M.
      • D'Agostini M.
      • Verkuil B.
      • Van Diest I.
      Moving beyond belief: a narrative review of potential biomarkers for transcutaneous vagus nerve stimulation.
      ]). The modulatory effects of taVNS on pupil dilation [
      • Sharon O.
      • Fahoum F.
      • Nir Y.
      Transcutaneous vagus nerve stimulation in humans induces pupil dilation and attenuates alpha oscillations.
      ,
      • Urbin M.A.
      • Lafe C.W.
      • Simpson T.W.
      • Wittenberg G.F.
      • Chandrasekaran B.
      • Weber D.J.
      Electrical stimulation of the external ear acutely activates noradrenergic mechanisms in humans.
      ,
      • Villani V.
      • Finotti G.
      • Di Lernia D.
      • Tsakiris M.
      • Azevedo R.T.
      Event-related transcutaneous vagus nerve stimulation modulates behaviour and pupillary responses during an auditory oddball task.
      ] have not consistently been replicated [
      • Keute M.
      • Boehrer L.
      • Ruhnau P.
      • Heinze H.-J.
      • Zaehle T.
      Transcutaneous vagus nerve stimulation (tvns) and the dynamics of visual bistable perception.
      ,
      • Burger A.M.
      • Van der Does W.
      • Brosschot J.F.
      • Verkuil B.
      From ear to eye? no effect of transcutaneous vagus nerve stimulation on human pupil dilation: a report of three studies.
      ,
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Franssen M.
      • Claes N.
      • Weymar M.
      • von Leupoldt A.
      • Van Diest I.
      Effects of transcutaneous auricular vagus nerve stimulation on reversal learning, tonic pupil size, salivary alpha-amylase, and cortisol.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Villca Ponce G.
      • Claes S.
      • von Leupoldt A.
      • Van Diest I.
      No evidence for a modulating effect of continuous transcutaneous auricular vagus nerve stimulation on markers of noradrenergic activity.
      ] and studies on the effects of taVNS on the P300 amplitude have also yielded mixed results. Whereas some studies found an increase of the P300 during taVNS compared to sham stimulation [
      • Rufener K.S.
      • Geyer U.
      • Janitzky K.
      • Heinze H.-J.
      • Zaehle T.
      Modulating auditory selective attention by non-invasive brain stimulation: differential effects of transcutaneous vagal nerve stimulation and transcranial random noise stimulation.
      ,
      • Lewine J.D.
      • Paulson K.
      • Bangera N.
      • Simon B.J.
      Exploration of the impact of brief noninvasive vagal nerve stimulation on eeg and event-related potentials.
      ,
      • Warren C.V.
      • Maraver M.J.
      • de Luca A.
      • Kopp B.
      The effect of transcutaneous auricular vagal nerve stimulation (tavns) on p3 event-related potentials during a bayesian oddball task.
      ], others found an increase only in response to specific stimuli [
      • Ventura-Bort C.
      • Wirkner J.
      • Genheimer H.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
      ], or found no differences between stimulation conditions [
      • Fischer R.
      • Ventura-Bort C.
      • Hamm A.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) enhances conflict-triggered adjustment of cognitive control.
      ,
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ,
      • Gadeyne S.
      • Mertens A.
      • Carrette E.
      • Van den Bossche F.
      • Boon P.
      • Raedt R.
      • Vonck K.
      Transcutaneous auricular vagus nerve stimulation cannot modulate the p3b event-related potential in healthy volunteers.
      ]. Other attempts of finding reliable physiological markers include for instance vagally-mediated heart rate variability, which, however, did not show to be affected by taVNS (see for review [
      • Wolf V.
      • Kühnel A.
      • Teckentrup V.
      • Koenig J.
      • Kroemer N.B.
      Does transcutaneous auricular vagus nerve stimulation affect vagally mediated heart rate variability? a living and interactive bayesian meta-analysis.
      ]).
      In recent years, salivary alpha-amylase (sAA) has emerged as promising indirect marker of LC-NA system activity based on pharmacological studies showing an involvement of noradrenergic activity in sAA secretion [
      • Ehlert U.
      • Erni K.
      • Hebisch G.
      • Nater U.
      Salivary α-amylase levels after yohimbine challenge in healthy men.
      ,
      • Kuebler U.
      • von Känel R.
      • Heimgartner N.
      • Zuccarella-Hackl C.
      • Stirnimann G.
      • Ehlert U.
      • Wirtz P.H.
      Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.
      ,
      • Warren C.M.
      • van den Brink R.L.
      • Nieuwenhuis S.
      • Bosch J.A.
      Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.
      ] (see for review [
      • Nater U.M.
      • Rohleder N.
      Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
      ]). Although some studies exploring taVNS effects on sAA level changes demonstrated increased sAA levels after taVNS compared to sham stimulation [
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Genheimer H.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
      ], supporting sAA as a potential marker of central NA-enhancement modulated by taVNS, others reported no such enhancement [
      • Giraudier M.
      • Ventura-Bort C.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Franssen M.
      • Claes N.
      • Weymar M.
      • von Leupoldt A.
      • Van Diest I.
      Effects of transcutaneous auricular vagus nerve stimulation on reversal learning, tonic pupil size, salivary alpha-amylase, and cortisol.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Villca Ponce G.
      • Claes S.
      • von Leupoldt A.
      • Van Diest I.
      No evidence for a modulating effect of continuous transcutaneous auricular vagus nerve stimulation on markers of noradrenergic activity.
      ,
      • Koenig J.
      • Parzer P.
      • Haigis N.
      • Liebemann J.
      • Jung T.
      • Resch F.
      • Kaess M.
      Effects of acute transcutaneous vagus nerve stimulation on emotion recognition in adolescent depression.
      ,

      A. Sommer, R. Fischer, U. Borges, S. Laborde, S. Achtzehn, R. Liepelt. Unpublished results (n.d.).

      ]. Ultimately, possible reasons for this lack of replicability regarding physiological markers of LC-NA system activity might be small sample sizes, the heterogeneity of stimulation procedures (e.g., stimulation parameters, stimulation duration [
      • Farmer A.D.
      • Strzelczyk A.
      • Finisguerra A.
      • Gourine A.V.
      • Gharabaghi A.
      • Hasan A.
      • Burger A.M.
      • Jaramillo A.M.
      • Mertens A.
      • Majid A.
      • et al.
      International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020).
      ,
      • Weymar M.
      • Zaehle T.
      Editorial: New Frontiers in Noninvasive Brain Stimulation: Cognitive, Affective and Neurobiological Effects of Transcutaneous Vagus Nerve Stimulation.
      ]) or methodological differences in data collection and/or preprocessing across studies (e.g., in saliva collection for sAA level changes [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ]).
      An opportunity to overcome these limitations and accelerate progress in validating potential relations between reliable NA markers and taVNS-mediated vagal activation is data pooling. By increasing overall sample size, the pooling of several independent studies improves statistical power and the overall generalizability of results (e.g., by distinguishing generalizable findings from false positives that emerge from smaller-samples studies; [
      • Boedhoe P.S.
      • Heymans M.W.
      • Schmaal L.
      • Abe Y.
      • Alonso P.
      • Ameis S.H.
      • Anticevic A.
      • Arnold P.D.
      • Batistuzzo M.C.
      • Benedetti F.
      • et al.
      An empirical comparison of meta-and mega-analysis with data from the enigma obsessive-compulsive disorder working group.
      ]). It further allows for consideration of within- and between-study variance to possibly explain some of the heterogeneity in the data (i.e., based on differences in study characteristics). Data pooling also enhances the ability to construct predictive models that are more widely applicable and better powered to identify relevant predictive factors [
      • Debray T.P.
      • Moons K.G.
      • Abo-Zaid G.M.A.
      • Koffijberg H.
      • Riley R.D.
      Individual participant data meta-analysis for a binary outcome: one-stage or two-stage?.
      ].
      Therefore, the aim of the present study was to overcome the existing limitations by pooling raw data from a large sample of studies that collected sAA levels in the context of taVNS research. Our focus on sAA was primarily due to its widespread use across taVNS laboratories, its inexpensive and non-invasive measurement and ultimately, its potential to become a clinically meaningful and reliable marker that might shed further light on the efficacy of taVNS. In order to explore whether taVNS enhances sAA levels as putative marker of NA activity in the pooled data, and to investigate if, and to what extent, different factors (e.g., stimulation parameters, stimulation duration) may modulate the assumed relation between taVNS and sAA level changes, we conducted linear mixed model analyses based on a hypothesis-driven approach as well as on an exploratory approach. Mixed models allow the specification of fixed and (crossed) random factors (e.g., participants and studies), they further allow the incorporation of continuous variables (i.e., yielding for instance fixed effects of linear and quadratic trends) and their interactions with categorical factors [
      • Kliegl R.
      • Wei P.
      • Dambacher M.
      • Yan M.
      • Zhou X.
      Experimental effects and individual differences in linear mixed models: estimating the relationship between spatial, object, and attraction effects in visual attention.
      ]. Mixed models are also optimal to deal with missing data. Thus, conducting mixed model analyses with a sample of pooled sAA data may provide valuable information on the relation between taVNS-mediated afferent vagal activation and sAA as an inexpensive and non-invasive index of central noradrenergic activity.

      2. Material and methods

      2.1 Sample

      Authors of previous and ongoing taVNS studies collecting sAA data were contacted and invited to participate in the project. We received data from twelve studies and included ten studies that applied taVNS as stimulation method [
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Genheimer H.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
      ] (Exp. 1b) [
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ] (Exp. 2) [
      • Giraudier M.
      • Ventura-Bort C.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Franssen M.
      • Claes N.
      • Weymar M.
      • von Leupoldt A.
      • Van Diest I.
      Effects of transcutaneous auricular vagus nerve stimulation on reversal learning, tonic pupil size, salivary alpha-amylase, and cortisol.
      ,
      • Koenig J.
      • Parzer P.
      • Haigis N.
      • Liebemann J.
      • Jung T.
      • Resch F.
      • Kaess M.
      Effects of acute transcutaneous vagus nerve stimulation on emotion recognition in adolescent depression.
      ], including three unpublished studies [

      A. Sommer, R. Fischer, U. Borges, S. Laborde, S. Achtzehn, R. Liepelt. Unpublished results (n.d.).

      ,

      M. Giraudier, C. Ventura-Bort, M. Weymar. Unpublished results (n.d).

      ,

      C. Ventura-Bort, M. Weymar. Unpublished results (n.d.).

      ] (see Table 1 for details about study characteristics). Two studies that applied auricular acupuncture were excluded from analyses [
      • Schultz G.
      • Altenstein C.
      • Klausenitz C.
      • Hesse T.
      • Hacker H.
      • Petersmann A.
      • Hannich H.
      • Hahnenkamp K.
      • Usichenko T.
      Auricular acupuncture vs. progressive muscle relaxation and no intervention for exam anxiety in medical students–a randomized controlled trial with non-randomized condition.
      ,
      • Usichenko T.
      • Wenzel A.
      • Klausenitz C.
      • Petersmann A.
      • Hesse T.
      • Neumann N.
      • Hahnenkamp K.
      Auricular stimulation vs. expressive writing for exam anxiety in medical students–a randomized crossover investigation.
      ].
      Table 1Overview study characteristics and stimulation parameters.
      StudyReferenceNTaskDesignStimulation deviceStimulation lengthDuty cycleStimulation intensity methodsAA collection method
      1Ventura-Bort et al. (2018)N = 20, 17f, Mage = 20.4visual oddballwithin-subjectNEMOS, tVNS Technologies GmbH35mincontinuousdetermined individuallyswab collection
      2Ventura-Bort et al. (2021)N = 37, 20f, Mage = 23passive viewingwithin-subjectNEMOS, tVNS Technologies GmbH7mincontinuousdetermined individuallyswab collection
      3Ventura-Bort et al. (in prog.)N = 31, 27f, Mage = 21.3passive viewingwithin-subjectNEMOS, tVNS Technologies GmbH14min30s on/30s offdetermined individuallyswab collection
      4Giraudier et al. (in prep.)N = 62, 50f, Mage = 23.8visual oddball, serial reaction timewithin-subjectNEMOS, tVNS Technologies GmbH80mincontinuous, 30s on/30s offdetermined individuallyspitting method
      5Giraudier et al. (2020)N = 61, 47f, Mage = 23.4lexical decisionbetween-subjectNEMOS, tVNS Technologies GmbH23min30s on/30s offdetermined individuallyswab collection
      6D'Agostini et al. (2021)N = 71, 55f, Mage = 23.3reversal learningbetween-subjectNEMOS, tVNS Technologies GmbH, DS5 DIGITIMER, Welwyn Garden City, UK40min30s on/30s offdetermined individuallyswab collection
      7Koenig et al. (2021)N = 30, 24f, 14–17 yearsmorphing faces, emotion recognition, emotional go/nogowithin-subjectVITOS, tVNS Technologies GmbH28min30s on/30s offfixed at 0.5 mAswab collection
      8Warren et al. (2019)N = 20, Mage = 23.6visual and auditory oddball, task switchingwithin-subjectNEMOS, tVNS Technologies GmbH80min30s on/30s offfixed at 0.5 mAspitting method
      9Warren et al. (2019)N = 17, 0f, Mage = 22.1task switchingwithin-subjectNEMOS, tVNS Technologies GmbH80min30s on/30s offfixed at 0.5 mAspitting method
      10Sommer et al. (in prep.)N = 27, 16f, Mage = 25.6number categorization based dual taskwithin-subjectNEMOS, tVNS Technologies GmbH61min30s on/30s offdetermined individuallyspitting method
      From all included studies, sAA levels were available for a total of 371 healthy participants. All participants provided informed written consent for the experimental protocol, which was approved in accordance with the declaration of Helsinki. Participant characteristics are shown in Table 1. Information on participant pre-selection and data collection for published studies are available in more detail in each individual publication. All data have been made publicly available on the Open Science Framework and can be accessed at https://osf.io/rdpcs.

      2.2 Transcutaneous auricular vagus nerve stimulation

      In all included studies, taVNS stimulation was conducted using two titan electrodes attached to a mount and wired either to a stimulation unit (NEMOS, VITOS; see Table 1 for details) or to a bipolar constant current stimulator (DS5 DIGITIMER; see Table 1 for details). In the active vagus stimulation condition, the stimulator electrodes were placed in the left cymba conchae, an area exclusively innervated by the auricular branch of the vagus nerve [
      • Ellrich J.
      Transcutaneous vagus nerve stimulation european neurological review.
      ,
      • Peuker E.T.
      • Filler T.J.
      The nerve supply of the human auricle.
      ]. For the sham stimulation condition, the electrodes were positioned in the center of the left ear lobe, an area known to be free of vagal innervation [
      • Ellrich J.
      Transcutaneous vagus nerve stimulation european neurological review.
      ,
      • Peuker E.T.
      • Filler T.J.
      The nerve supply of the human auricle.
      ]. All studies applied stimulation on a single day. In studies 1, 2, and 4, stimulation was administered continuously, whereas in studies 3 and 5–10, stimulation alternated between on and off phases every 30 s. Stimulation intensity was either adjusted individually for each participant above the detection threshold and below the pain threshold [
      • Ellrich J.
      Transcutaneous vagus nerve stimulation european neurological review.
      ] (studies 1–6 and 10) or was fixed at 0.5 mA for all participants (studies 7–9). Across all ten studies, stimulation intensities varied from 0.1 mA to 5 mA for the sham (earlobe) condition (Msham = 1.20, SDsham = 0.82) and from 0.25 mA to 4 mA for the vagus (cymba conchae) condition (Mvagus = 1.03, SDvagus = 0.66). All stimulation characteristics are shown in Table 1.

      2.3 Salivary alpha-amylase

      Alpha-amylase is a salivary enzyme involved in the digestion of starch in the oral cavity [
      • Baum B.J.
      Principles of saliva secretion.
      ]. It can be measured through saliva collection in an inexpensive and non-invasive fashion and, as such, has emerged as a proxy measure of sympathetic arousal, likely reflecting stress-related changes in the body [
      • Nater U.M.
      • Rohleder N.
      Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
      ,
      • Chatterton Jr., R.T.
      • Vogelsong K.M.
      • Lu Y.-c.
      • Ellman A.B.
      • Hudgens G.A.
      Salivary α-amylase as a measure of endogenous adrenergic activity.
      ,
      • Nater U.M.
      The role of salivary alpha-amylase in stress research.
      ,
      • Rohleder N.
      • Nater U.M.
      • Wolf J.M.
      • Ehlert U.
      • Kirschbaum C.
      Psychosocial stress-induced activation of salivary alpha-amylase: an indicator of sympathetic activity?.
      ,
      • Granger D.A.
      • Kivlighan K.T.
      • El-Sheikh M.
      • Gordis E.B.
      • Stroud L.R.
      Salivary α-amylase in biobehavioral research: recent developments and applications.
      ]. It is important to note that sAA levels measured during stress might be influenced by activity of the sympathetic or parasympathetic nervous system or some combination of both [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ,
      • Ali N.
      • Nater U.M.
      Salivary alpha-amylase as a biomarker of stress in behavioral medicine.
      ]. In recent years, however, sAA has been accepted as promising marker of sympathetic nervous system activity based on pharmacological studies showing an involvement of noradrenergic activity in sAA secretion [
      • Ehlert U.
      • Erni K.
      • Hebisch G.
      • Nater U.
      Salivary α-amylase levels after yohimbine challenge in healthy men.
      ,
      • Kuebler U.
      • von Känel R.
      • Heimgartner N.
      • Zuccarella-Hackl C.
      • Stirnimann G.
      • Ehlert U.
      • Wirtz P.H.
      Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.
      ,
      • Warren C.M.
      • van den Brink R.L.
      • Nieuwenhuis S.
      • Bosch J.A.
      Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.
      ]. For instance, Ehlert and colleagues [
      • Ehlert U.
      • Erni K.
      • Hebisch G.
      • Nater U.
      Salivary α-amylase levels after yohimbine challenge in healthy men.
      ] reported that administration of yohimbine (i.e., an alpha-adrenergic receptor antagonist) activated sAA via adrenergic mechanisms, thus pointing to sAA as marker of the central sympathetic system. More recently, Warren and colleagues [
      • Warren C.M.
      • van den Brink R.L.
      • Nieuwenhuis S.
      • Bosch J.A.
      Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.
      ] administered atomoxetine, a highly selective NA transporter blocker that increases central NA levels, and validated the initial findings by Ehlert and colleagues [
      • Ehlert U.
      • Erni K.
      • Hebisch G.
      • Nater U.
      Salivary α-amylase levels after yohimbine challenge in healthy men.
      ] (see also [
      • Kuebler U.
      • von Känel R.
      • Heimgartner N.
      • Zuccarella-Hackl C.
      • Stirnimann G.
      • Ehlert U.
      • Wirtz P.H.
      Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.
      ]; see for review [
      • Nater U.M.
      • Rohleder N.
      Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
      ]).
      To assess the effects of taVNS on sAA level changes in our pooled data, in all included studies, sAA levels (U/ml) were collected before (i.e., prior to the application of the taVNS device) and after (i.e., after finalizing the psychological task(s) and removing the taVNS device) stimulation. Four studies also collected sAA levels during stimulation (studies 4, 7–9). Saliva samples were either collected using cotton swabs (i.e., 66.31% of participants were instructed to gently chew the cotton swab in their mouth and then place it into a sample tube) or by spitting (i.e., 33.69% of participants were instructed to spit out saliva either through a plastic straw or directly without straw into a sample tube). Of note, sAA levels are sensitive to sampling techniques because different salivary glands contribute to different rates of saliva secretion, which influences the quantity of sAA secreted into oral fluids [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ,
      • Ali N.
      • Nater U.M.
      Salivary alpha-amylase as a biomarker of stress in behavioral medicine.
      ]. The swab collection method requires chewing (i.e., stimulated saliva secretion), which affects sAA levels independently of central noradrenergic involvement [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ]. Therefore, the spitting method is generally favored when collecting saliva samples. For details about sample storage and analysis see each individual publication.

      2.4 Statistics

      All statistical analyses were carried out in the R environment [
      R Core Team
      R: a language and environment for statistical computing.
      ]. Pre- and post-processing of data was conducted using tidyverse [
      • Wickham H.
      • Averick M.
      • Bryan J.
      • Chang W.
      • McGowan L.D.
      • François R.
      • Grolemund G.
      • Hayes A.
      • Henry L.
      • Hester J.
      • et al.
      Welcome to the tidyverse.
      ].

      2.4.1 Mixed model analysis

      To test the effects of taVNS on sAA level changes, we conducted a series of linear mixed models (LMMs) using lme4 [
      • Bates D.
      • Mächler M.
      • Bolker B.
      • Walker S.
      Fitting linear mixed-effects models using lme4.
      ]. A Box-Cox distributional analysis [
      • Box G.E.
      • Cox D.R.
      An analysis of transformations.
      ] indicated that a logarithmic transformation brought the typically skewed sAA data [
      • Nater U.M.
      • Rohleder N.
      Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
      ] in line with the assumption of normal distribution.
      As fixed effects, we specified sequential-difference contrasts (i.e., a priori defined comparisons between specific conditions and/or groups; cf. [
      • Schad D.J.
      • Vasishth S.
      • Hohenstein S.
      • Kliegl R.
      How to capitalize on a priori contrasts in linear (mixed) models: a tutorial.
      ]) for time (post vs. pre, post vs. mid), for stimulation (vagus vs. sham) and for the interaction between time and stimulation respectively. We also included the effect of stimulation length, the effect of duty cycle (continuous vs. 30s on/30s off), the effect of stimulation intensity method (fixed at 0.5 mA vs. determined individually), the effect of sAA collection method (swab collection vs. spitting method), the effect of stimulation intensity (group mean-centered) and their associated interactions (included interactions vary between models). The model predictors gender (male vs. female) and time of day (i.e., timeslots I-VI based on the time of the sAA measurement) were only included as fixed effects in a separate analysis due to a large amount of missing data (lost or not provided) for those predictors, reducing the total amount of observations drastically when including them (N = 1092).
      As random factors, we included participant (N = 371) and study (N = 10) with a total amount of 1556 observations. The selected random-effect structure included theoretically relevant variance components and correlation parameters and was supported by the data (cf. [
      • Bates D.
      • Kliegl R.
      • Vasishth S.
      • Baayen H.
      Parsimonious mixed models.
      ]). We included random intercepts for participant and study and allowed the effect of time (post vs. pre) and the effect of stimulation (vagus vs. sham) to vary across subjects (random slope), constraining random intercept and random slope to be independent. We further allowed the effect of time (post vs. pre) to vary between studies, constraining uncorrelated random intercept and random slope within studies. The random slope time (post vs. mid) did not significantly improve model fit and was excluded from all models. The random-effect structure was identical for all models.
      Parsimonious model selection followed the general recommendations by Bates et al. [
      • Bates D.
      • Kliegl R.
      • Vasishth S.
      • Baayen H.
      Parsimonious mixed models.
      ] and was performed without knowledge or consideration of fixed-effect estimates. In a maximal to minimal-that-converges modeling process, fitted models were processed with random-effects principal component analysis to obtain loadings of the variance-covariance matrix of the random effects (i.e., an iterative reduction of random-effects structure complexity was performed).
      For assessment of relative differences in goodness of fit, we used the log-likelihood and, for model comparisons, the χ2-distributed likelihood ratio and its associated p-value. P-values for fixed effects were calculated using Satterthwaite's approximations [
      • Satterthwaite F.E.
      An approximate distribution of estimates of variance components.
      ]. Final models were estimated with restricted maximum likelihood. Pairwise post hoc comparisons were computed using lsmeans [
      • Lenth R.V.
      Least-squares means: the r package lsmeans.
      ] with Tukey-adjusted p-values. The report of results followed the recommendations by Meteyard & Davies [
      • Meteyard L.
      • Davies R.A.
      Best practice guidance for linear mixed-effects models in psychological science.
      ].

      2.4.2 Meta-analysis

      In addition to LMMs, we performed a meta-analysis of the current studies. We therefore calculated Hedges'g [
      • Hedges L.V.
      • Olkin I.
      Nonparametric estimators of effect size in meta-analysis.
      ] as effect sizes based on standardized mean differences (SMDs) using metafor [
      • Viechtbauer W.
      Conducting meta-analyses in r with the metafor package.
      ] and meta [
      • Schwarzer G.
      • et al.
      meta: an r package for meta-analysis.
      ]. Effect sizes were calculated for the sAA increase under taVNS (Δpost-pre) compared to sham (Δpost-pre) on the log-sAA data. Cohen's d and Cohen's dz [
      • Cohen J.
      Statistical power analysis for the behavioral sciences.
      ] have been uploaded as additional effect size estimates on the Open Science Framework and can be accessed at https://osf.io/rdpcs. A statistical power-analysis for the meta-analysis followed the recommendations by Valentine and colleagues [
      • Valentine J.C.
      • Pigott T.D.
      • Rothstein H.R.
      How many studies do you need? a primer on statistical power for meta-analysis.
      ].

      2.4.3 Test-retest reliability

      Test-retest reliability of sAA levels (pre vagus vs. pre sham) was tested using an intra-class correlation (ICC) coefficient using psych [
      • Revelle W.
      An overview of the psych package.
      ] and included all data from studies employing a within-subject design (Nparticipants = 233, Nstudies = 8).

      2.4.4 Bayesian evidence synthesis

      A Bayesian approach (protocol by Scheibehenne et al. [
      • Scheibehenne B.
      • Jamil T.
      • Wagenmakers E.-J.
      Bayesian evidence synthesis can reconcile seemingly inconsistent results: the case of hotel towel reuse.
      ]) was also performed. Results of this analysis, however, did not reveal additional information and were therefore not included in this paper (results can be found on the Open Science Framework (https://osf.io/rdpcs) where the project was pre-registered on March 2, 2021).

      3. Results

      3.1 Mixed models

      3.1.1 Model selection

      Overall, we explored a variety of modeling approaches in order to identify the most appropriate and best-performing predictive models and consequently, specified three models of increasing complexity that were supported by the data. See Supplement A for details about the model selection approaches.

      3.1.2 The core model

      As fixed effects in M1, we included the sequential-difference contrasts for time, for stimulation and their associated interaction. The model output from M1 showed no main effect of time on sAA, b = 0.08, SE = 0.05, p = 0.150, and no main effect of stimulation, b = 0.06, SE = 0.03, p = 0.067. Interestingly, the interaction between time and stimulation was significant, b = 0.12, SE = 0.04, p = 0.005, showing increased sAA levels for vagus, b = 0.16, SE = 0.05, p = 0.048, as opposed to sham stimulation, b = 0.03, SE = 0.05, p = 0.966 (Mvaguspre=4.47 U/ml, Mvaguspost=4.63 U/ml, Mshampre=4.50 U/ml, Mshampost=4.52 U/ml). The model output from M1 is displayed in Table 2.
      Table 2The core model M1 with Nobservations = 1556, Nparticipants = 371, Nstudies = 10.
      Fixed Effects
      Est (U/ml)SE (U/ml)95% CItp
      Intercept4.540.124.26–4.8137.26<0.001
      Time (post - pre)0.080.05−0.03 − 0.191.580.150
      Stimulation (vagus - sham)0.060.03−0.00 − 0.111.840.067
      Time X Stimulation0.120.040.04–0.212.810.005
      Random Effects
      VarianceS.D.Correlation
      Participant (Intercept)0.520.72
      Study (Intercept)0.130.36
      Time ‖ Participant0.090.30
      Stimulation ‖ Participant0.100.31−0.39
      Time ‖ Study0.020.13
      Model Fit
      R2MarginalConditional
      0.0030.813

      3.1.3 The full model

      As fixed effects in M2, we specified a priori defined comparisons for time, for stimulation and for the interaction between time and stimulation. We also included the effect of stimulation length, the effect of duty cycle, the effect of stimulation intensity method, the effect of sAA collection method, the effect of stimulation intensity and the interaction between time, stimulation and duty cycle. Similarly to the output of M1, the output from M2 showed a significant interaction between time and stimulation, b = 0.16, SE = 0.05, p = 0.001, revealing increased sAA levels for vagus, b = 0.19, SE = 0.06, p = 0.017, as opposed to sham stimulation, b = 0.01, SE = 0.06, p = 0.994 (see Fig. 1A) (see also Fig. S1 in Supplement B), and a significant interaction between time, stimulation and duty cycle, b = 0.19, SE = 0.10, p = 0.050, showing a stronger sAA increase for vagus than for sham with continuous stimulation as opposed to interval stimulation (see Fig. 1B). No further significant effects were found (0.05 < ps < 1). The model output of M2 is displayed in Table 3.
      Fig. 1
      Fig. 1A: Interaction between time and stimulation, B: Interaction between time, stimulation and duty cycle, C: Effect of time of day for vagus compared to sham stimulation.
      Table 3The full model M2 with Nobservations = 1556, Nparticipants = 371, Nstudies = 10.
      Fixed Effects
      Est (U/ml)SE (U/ml)95% CItp
      Intercept5.040.493.82–6.2610.27<0.001
      Time (post - pre)0.080.05−0.03 − 0.191.590.148
      Stimulation (vagus - sham)0.060.03−0.00 − 0.121.910.057
      Time X Stimulation0.160.050.07–0.263.380.001
      Stimulation intensity method (fixed at 0.5 mA - determined individually)0.430.23−0.13 − 1.001.840.112
      sAA collection method (swab collection - spitting method)0.050.50−1.19 − 1.280.090.928
      Stimulation intensity−0.050.04−0.13 − 0.02−1.340.180
      Stimulation length−0.010.01−0.03 − 0.01−1.120.308
      Duty cycle (continuous - 30s on/30s off)−0.160.15−0.47 − 0.14−1.080.284
      Time X Stimulation X Duty cycle0.190.100.00–0.381.960.050
      Random Effects
      VarianceS.D.Correlation
      Participant (Intercept)0.530.73
      Study (Intercept)0.070.26
      Time ‖ Participant0.090.30
      Stimulation ‖ Participant0.090.31−0.38
      Time ‖ Study0.020.13
      Model Fit
      R2MarginalConditional
      0.1480.829

      3.1.4 The iterative model

      We specified a final model M3 based on an iterative modeling approach. As fixed effects in M3, we specified a priori defined comparisons for time, for stimulation and for the interaction between time and stimulation. We also included the effect of sAA collection method and the interaction between time, stimulation and duty cycle. The model showed no main effect of time on sAA, b = 0.08, SE = 0.05, p = 0.148, and no main effect of stimulation, b = 0.06, SE = 0.03, p = 0.051. As in M1 and M2, we found a significant interaction of time and stimulation, b = 0.16, SE = 0.05, p = 0.001, indicating increased sAA levels for vagus, b = 0.19, SE = 0.06, p = 0.017, compared to sham stimulation, b = 0.01, SE = 0.06, p = 0.994. No further significant effects were found (0.05 < ps < 0.09). The model output of M3 is displayed in Table 4.
      Table 4The iterative model M3 with Nobservations = 1556, Nparticipants = 371, Nstudies = 10.
      Fixed Effects
      Est (U/ml)SE (U/ml)95% CItp
      Intercept4.490.114.24–4.7441.20<0.001
      Time (post - pre)0.080.05−0.03 − 0.201.590.148
      Stimulation (vagus - sham)0.060.03−0.00 − 0.121.960.051
      Time X Stimulation0.160.050.07–0.263.380.001
      sAA collection method (swab collection - spitting method)0.420.22−0.08 − 0.921.940.087
      Time X Stimulation X Duty cycle0.190.10−0.00 − 0.371.950.051
      Random Effects
      VarianceS.D.Correlation
      Participant (Intercept)0.530.72
      Study (Intercept)0.090.31
      Time ‖ Participant0.090.30
      Stimulation ‖ Participant0.100.31−0.39
      Time ‖ Study0.020.13
      Model Fit
      R2MarginalConditional
      0.0520.815

      3.2 Model comparison

      3.2.1 Best-performing model

      The comparison between M1, M2 and M3 revealed significant evidence for a difference in goodness of fit, showing that the full model M2 is the best-performing model as opposed to M1, χ2(2) = 7.65, p = 0.022, and M3, χ2(4) = 9.82, p = 0.043.

      3.2.2 Effects in the random structure

      The random-effect structure was identical for all models and revealed a negative, medium high correlation between slope of stimulation and slope of time in all models (see Table 2, Table 3, Table 4), i.e., participants with higher difference between measurements (pre and post stimulation) over both conditions showed a larger stimulation main effect (higher sAA levels in taVNS session) over both time points.

      3.3 Additional model predictors

      3.3.1 Gender and time of day in the full model

      Adding gender and time of day to the best-performing model M2 (with a total amount of 1092 observations, 285 participants and 6 studies due to a large amount of missing data for those predictors) did significantly contribute to goodness of fit, χ2(6) = 15.10, p = 0.019. However, neither the associated interaction between time, stimulation and gender, χ2(1) = 0.80, p = 0.371, nor the interaction between time, stimulation and time of day, χ2(5) = 4.92, p = 0.426, significantly improved model fit. Similar to the output of M2, the interaction between time and stimulation was significant when adding gender and time of day as fixed effects, b = 0.18, SE = 0.06, p = 0.002, revealing increased sAA levels for vagus compared to sham stimulation. The model further showed a significant main effect of stimulation, b = 0.09, SE = 0.04, p = 0.029, which, however, seemed to be driven by the significant interaction between time and stimulation. Moreover, a significant difference for time of day, b = 0.32, SE = 0.12, p = 0.007, showing significantly lower sAA levels for time of day I (i.e., early morning) as compared to later during the day, was significant (MtimeofdayI = 3.98 U/ml, MtimeofdayII = 4.30 U/ml, MtimeofdayIII = 4.39 U/ml, MtimeofdayIV = 4.32 U/ml, MtimeofdayV = 4.50 U/ml, MtimeofdayVI = 4.33 U/ml) (see Fig. 1C). No further significant effects were found (0.10 < ps < 0.80).

      3.4 Meta-analysis

      There was strong evidence for the null hypothesis across studies, g = 0.13, 95%CI [ − 0.07, 0.34], t = 1.52, p = 0.164, suggesting no effect of vagal stimulation on the sAA increase. There was no evidence for homogeneity, τ = 0.265, 95%CI [0.17, 0.51], I2 = 92%, p < 0.01. This meta-analysis, however, was shown to be underpowered to detect potentially meaningful effects significantly different from zero, with a power of 0.21. The forest plot of this analysis is represented in Fig. 2.
      Fig. 2
      Fig. 2Forest plot of standardized mean difference for all included studies for the sAA increase under taVNS compared to sham stimulation. The diamond shape represents the average effect and its length symbolizes the confidence interval of the pooled results. The red line below the diamond represents the length of the associated prediction interval. Note: g, effect estimate; SE, standard error; SMD, standardized mean difference; CI, confidence interval. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

      4. Discussion

      Previous work has suggested a modulatory role of taVNS on cognitive and affective functions, which might be mediated by activation of the LC-NA system. Reliable effects of taVNS on markers of LC-NA system activity, however, have not been demonstrated yet (cf. [
      • Farmer A.D.
      • Strzelczyk A.
      • Finisguerra A.
      • Gourine A.V.
      • Gharabaghi A.
      • Hasan A.
      • Burger A.M.
      • Jaramillo A.M.
      • Mertens A.
      • Majid A.
      • et al.
      International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020).
      ]). The present project, therefore, aimed to shed light on this recent controversy by pooling raw data from a large sample of taVNS studies that collected sAA levels as potential marker of central NA release. We explored a variety of modeling approaches and observed that taVNS, compared to sham stimulation, increased sAA levels in all generated predictive models, suggesting a modulatory role of taVNS on sAA. When considering potential confounders of sAA, we further replicated previous findings on the diurnal trajectory of sAA activity with lower levels in the morning and an increase during the course of the day.
      The enhancing effect of taVNS (prior compared to post stimulation) on sAA was consistent across all generated predictive models, suggesting that it is a highly relevant predictor. The release of central NA has previously been associated with increased sAA secretion in pharmacological studies [
      • Ehlert U.
      • Erni K.
      • Hebisch G.
      • Nater U.
      Salivary α-amylase levels after yohimbine challenge in healthy men.
      ,
      • Kuebler U.
      • von Känel R.
      • Heimgartner N.
      • Zuccarella-Hackl C.
      • Stirnimann G.
      • Ehlert U.
      • Wirtz P.H.
      Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.
      ,
      • Warren C.M.
      • van den Brink R.L.
      • Nieuwenhuis S.
      • Bosch J.A.
      Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.
      ] (see for review [
      • Nater U.M.
      • Rohleder N.
      Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
      ]). Consequently, sAA has emerged as a promising marker of sympathetic nervous system activity, orchestrated by the LC-NA system [
      • Dunn A.J.
      • Swiergiel A.
      • Palamarchouk V.
      Brain circuits involved in corticotropin-releasing factor-norepinephrine interactions during stress.
      ]. The current findings thus suggest that taVNS, through activation of afferent fibers of the vagus nerve, leads to the activation of the LC-NA system.
      Single studies, however, produced mixed results. In one study, Ventura-Bort and colleagues [
      • Ventura-Bort C.
      • Wirkner J.
      • Genheimer H.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
      ] reported increased sAA levels after taVNS but not after sham stimulation based on post hoc analysis. Similarly, Warren and colleagues [
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ] replicated this finding and further found no effects of taVNS on salivary flow rate (i.e., amount of saliva per minute), ruling out parasympathetic influence on sAA release (cf. [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ]). Nevertheless, there has also been a growing body of null findings in taVNS studies, challenging the reliability of sAA as potential NA marker and further questioning taVNS efficacy. Most recently, D'Agostini and colleagues [
      • D'Agostini M.
      • Burger A.M.
      • Villca Ponce G.
      • Claes S.
      • von Leupoldt A.
      • Van Diest I.
      No evidence for a modulating effect of continuous transcutaneous auricular vagus nerve stimulation on markers of noradrenergic activity.
      ] reported no evidence for a modulating effect of taVNS on sAA in a sample of 66 healthy participants performing a novelty auditory oddball task. Similarly, five other studies used in the current data pooling have added to the inconsistent evidence for a modulating effect of taVNS on sAA in humans [
      • Giraudier M.
      • Ventura-Bort C.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ,
      • D'Agostini M.
      • Burger A.M.
      • Franssen M.
      • Claes N.
      • Weymar M.
      • von Leupoldt A.
      • Van Diest I.
      Effects of transcutaneous auricular vagus nerve stimulation on reversal learning, tonic pupil size, salivary alpha-amylase, and cortisol.
      ,
      • Koenig J.
      • Parzer P.
      • Haigis N.
      • Liebemann J.
      • Jung T.
      • Resch F.
      • Kaess M.
      Effects of acute transcutaneous vagus nerve stimulation on emotion recognition in adolescent depression.
      ,

      A. Sommer, R. Fischer, U. Borges, S. Laborde, S. Achtzehn, R. Liepelt. Unpublished results (n.d.).

      ]. The inconsistency and lack of replicability across taVNS studies may be due to several reasons. First, as shown in our meta-analysis, most included studies had relatively low sample sizes and the investigated effects were small (as indicated by the wide CI in Fig. 2). This can lead to an increase in both false-negative and false-positive findings. Second, our meta-analysis showed a large heterogeneity between studies, which most likely is related to differences in study characteristics, including experimental designs (e.g., experimental tasks), stimulation procedures (e.g., stimulation length, stimulation intensity, stimulation duty cycle), methodological differences in data collection (e.g., sAA collection method), preprocessing and/or statistical analysis. This was further validated by the fact that the meta-analysis was underpowered (i.e., lack of power in meta-analyses has been proposed to be potentially caused by high heterogeneity rather than by the number of studies [
      • Jackson D.
      • Turner R.
      Power analysis for random-effects meta-analysis.
      ]). It is worth mentioning that our dataset included taVNS studies that collected sAA, which predominantly reported no significant effects of taVNS on sAA. By increasing overall sample size, however, the pooling of these independent studies led to evidence for a modulating effect of taVNS on sAA, suggesting that taVNS increases central noradrenergic release. An important implication of the fact that we find this effect even though most of the included studies reported null findings is that the effects of taVNS on sAA are rather delicate. Interestingly, the overall high variance between participants in sAA levels might suggest that participants tend to react differently to taVNS. The assessment of the distributions of sAA increases and decreases for vagus and sham stimulation, however, did not enable us to conclusively clarify whether we are looking at a small but generalizable effect or if a small percentage of responders drives the observed effect (see Fig. S2 and Fig. S3 in Supplement B). As suggested by the meta-analysis and Fig. S3 in Supplement B, the variability across studies is large. Some studies show an (almost identical) overlap between vagus and sham stimulation conditions (e.g., [

      M. Giraudier, C. Ventura-Bort, M. Weymar. Unpublished results (n.d).

      ]), whereas others show higher values for one of the conditions (see for vagus condition for instance [
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ]; see for sham condition for instance [
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ], Exp. 2). Although not conclusive, we interpret these results as not pointing towards a few responders. When looking closely at single distributions of studies showing the observed effect of vagus stimulation on sAA levels, the effect seems to be due to a general, small effect (see Refs. [
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ,
      • Warren C.M.
      • Tona K.D.
      • Ouwerkerk L.
      • Van Paridon J.
      • Poletiek F.
      • van Steenbergen H.
      • Bosch J.A.
      • Nieuwenhuis S.
      The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
      ,
      • Ventura-Bort C.
      • Wirkner J.
      • Genheimer H.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
      ,

      C. Ventura-Bort, M. Weymar. Unpublished results (n.d.).

      ]), rather than being driven by a group of responders. Fig. S2 in Supplement B further highlights that the overall distribution is not characterized by individual outliers. This needs to be further investigated in future studies, which should determine statistically valuable sample sizes in order to confirm meaningful increases of sAA after taVNS compared to sham stimulation. Based on our analyses, however, it is not possible to determine such statistically valuable sample sizes for future studies due to the large heterogeneity of the data. Although a power analysis revealed that statistical power was sufficient for conducting linear mixed model analyses in the present dataset (see Supplement C), the corresponding estimation of sample sizes to reach an acceptable power only applies to similarly heterogeneous datasets and thus, cannot be transferred to single study designs. The fact that some studies could find significant effects of taVNS on sAA levels with rather small sample sizes, however, suggests that this is generally possible and might depend on specific and possibly yet unknown study characteristics (e.g., stimulation length, task).
      In order to identify the most appropriate predictive models, we explored a variety of modeling approaches and consequently, determined our full model as best-performing model out of the three developed models. In addition to the already discussed enhancing effect of taVNS on sAA, this model also showed a significant interaction between time, stimulation and duty cycle, possibly indicating continuous stimulation to be more efficient as opposed to interval stimulation (30s on/30s off). It has been suggested that interval stimulation might lead to unwanted rapid decline in NA activity, thus possibly reducing the modulating effect of taVNS on markers of noradrenergic activity [
      • D'Agostini M.
      • Burger A.M.
      • Villca Ponce G.
      • Claes S.
      • von Leupoldt A.
      • Van Diest I.
      No evidence for a modulating effect of continuous transcutaneous auricular vagus nerve stimulation on markers of noradrenergic activity.
      ]. Although this is in line with some animal research showing such decline in NA after invasive vagal stimulation was turned off [
      • Mridha Z.
      • de Gee J.W.
      • Shi Y.
      • Alkashgari R.
      • Williams J.
      • Suminski A.
      • Ward M.P.
      • Zhang W.
      • McGinley M.J.
      Graded recruitment of pupil-linked neuromodulation by parametric stimulation of the vagus nerve.
      ,
      • Follesa P.
      • Biggio F.
      • Gorini G.
      • Caria S.
      • Talani G.
      • Dazzi L.
      • Puligheddu M.
      • Marrosu F.
      • Biggio G.
      Vagus nerve stimulation increases norepinephrine concentration and the gene expression of bdnf and bfgf in the rat brain.
      ], other electrophysiological studies in rats have reported enhanced firing rates of LC neurons and NA release after invasive vagal stimulation delivered in 30s on/30s off cycles [
      • Dorr A.E.
      • Debonnel G.
      Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission.
      ,
      • Manta S.
      • Dong J.
      • Debonnel G.
      • Blier P.
      Enhancement of the function of rat serotonin and norepinephrine neurons by sustained vagus nerve stimulation.
      ,
      • Groves D.A.
      • Bowman E.M.
      • Brown V.J.
      Recordings from the rat locus coeruleus during acute vagal nerve stimulation in the anaesthetised rat.
      ,
      • Manta S.
      • El Mansari M.
      • Debonnel G.
      • Blier P.
      Electrophysiological and neurochemical effects of long-term vagus nerve stimulation on the rat monoaminergic systems.
      ]. In humans, the impact of different duty cycles on effects of taVNS is also not well understood yet. Recent studies showed for both, continuous and interval stimulation, an improvement in memory (see for continuous stimulation [
      • Ventura-Bort C.
      • Wirkner J.
      • Wendt J.
      • Hamm A.O.
      • Weymar M.
      Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
      ]; see for interval stimulation [
      • Giraudier M.
      • Ventura-Bort C.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
      ]) and cognitive control (see for continuous stimulation [
      • Fischer R.
      • Ventura-Bort C.
      • Hamm A.
      • Weymar M.
      Transcutaneous vagus nerve stimulation (tvns) enhances conflict-triggered adjustment of cognitive control.
      ]; see for interval stimulation [
      • Sellaro R.
      • van Leusden J.W.
      • Tona K.-D.
      • Verkuil B.
      • Nieuwenhuis S.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation enhances post-error slowing.
      ,
      • Sellaro R.
      • de Gelder B.
      • Finisguerra A.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation (tvns) enhances recognition of emotions in faces but not bodies.
      ,
      • Steenbergen L.
      • Sellaro R.
      • Stock A.-K.
      • Verkuil B.
      • Beste C.
      • Colzato L.S.
      Transcutaneous vagus nerve stimulation (tvns) enhances response selection during action cascading processes.
      ,
      • Keute M.
      • Boehrer L.
      • Ruhnau P.
      • Heinze H.-J.
      • Zaehle T.
      Transcutaneous vagus nerve stimulation (tvns) and the dynamics of visual bistable perception.
      ]). In general, however, the majority of taVNS studies delivered stimulation in 30s on/30s off cycles, mostly due to technical reasons (i.e., tVNS Technologies GmbH has embedded this on/off cycle in their commercial device). This imbalance across studies is also reflected in our dataset, with three studies applying continuous stimulation, and eight studies applying stimulation alternating between on and off phases every 30 s. It must be mentioned though that the triple interaction observed in the present data may also be partly driven by differences in experimental designs. Of note, all studies that applied continuous stimulation also used emotionally laden (arousing) material (IAPS images [
      • Lang P.J.
      International affective picture system (iaps): affective ratings of pictures and instruction manual.
      ]), which also modulates sAA levels (e.g., [
      • Segal S.K.
      • Cahill L.
      Endogenous noradrenergic activation and memory for emotional material in men and women.
      ]). Thus, sAA levels may increase particularly under tonic stimulation and in the context of emotional arousal. Considering the heterogeneity of our data and the explorative character of the full modeling approach, the observed advantage of continuous stimulation, however, should be interpreted with caution and requires future verification.
      When further considering potential confounders of sAA levels by adding time of day and gender to the best-performing model, we found decreased sAA levels in the morning as compared to later during the day. This finding is consistent with previous literature suggesting that saliva composition varies rhythmically over the day [
      • Rohleder N.
      • Nater U.M.
      • Wolf J.M.
      • Ehlert U.
      • Kirschbaum C.
      Psychosocial stress-induced activation of salivary alpha-amylase: an indicator of sympathetic activity?.
      ,
      • Dawes C.
      The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human submandibular saliva.
      ,
      • Nater U.M.
      • Rohleder N.
      • Schlotz W.
      • Ehlert U.
      • Kirschbaum C.
      Determinants of the diurnal course of salivary alpha-amylase.
      ]. Specifically, animal studies showed that sAA levels are low at the beginning of the day and increase at the end of the afternoon [
      • Dawes C.
      The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human submandibular saliva.
      ,
      • Bellavia S.
      • Sanz E.
      • Chiarenza A.
      • Sereno R.
      • Vermouth N.
      Circadian rhythm of alpha-amylase in rat parotid gland.
      ]. In humans, similar effects have been found [
      • Jenzano J.W.
      • Brown C.
      • Mauriello S.M.
      Temporal variations of glandular kallikrein, protein and amylase in mixed human saliva.
      ,
      • Artino M.
      • Dragomir M.
      • Ionescu S.
      • Bădiţa D.
      • Niţă V.
      • Chiţoi E.
      Diurnal behaviour of some salivary parameters in patients with diabetes mellitus (protein concentration, amylase activity, density)–note i.
      ,
      • Rantonen P.J.
      • Meurman J.H.
      Correlations between total protein, lysozyme, immunoglobulins, amylase, and albumin in stimulated whole saliva during daytime.
      ]. More recently, Nater and colleagues [
      • Nater U.M.
      • Rohleder N.
      • Schlotz W.
      • Ehlert U.
      • Kirschbaum C.
      Determinants of the diurnal course of salivary alpha-amylase.
      ] investigated the diurnal profile of sAA in a field study with hourly samplings from morning to evening and confirmed a decrease of sAA in the first hour after awakening, along with rising levels towards the afternoon and evening. The authors further examined potentially influencing factors of sAA and found that the diurnal profile of sAA was rather robust against influence factors such as gender. This is consistent with our results showing a similar diurnal course of sAA (i.e., decreased levels in the morning and rising levels throughout the day) and no evidence for an effect of gender. These findings invite to consider potential confounders for a reliable measurement of sAA. Even though time of day did not seem to directly influence stimulation, researchers should consider scheduling experimental sessions at the same time of day in within-subject designs and preferably avoid the measurement of sAA early in the morning (i.e., before 10am) to control for the effects of circadian influence (cf. [
      • Nater U.M.
      • Rohleder N.
      • Schlotz W.
      • Ehlert U.
      • Kirschbaum C.
      Determinants of the diurnal course of salivary alpha-amylase.
      ]). Researchers should also control for other potentially influencing factors of sAA (e.g., age) to further investigate which confounders are statistically associated with the outcome, and if so, these factors should be entered as covariates in statistical analyses [
      • Strahler J.
      • Skoluda N.
      • Kappert M.B.
      • Nater U.M.
      Simultaneous measurement of salivary cortisol and alpha-amylase: application and recommendations.
      ].
      When interpreting the results of the present study, some limitations should be taken into consideration. First, the validity of our findings is limited to the noradrenergic pathway as potential working mechanism of taVNS. Future research may consider alternative pathways targeted by taVNS, such as serotonergic, dopaminergic and cholinergic signaling, and their associated physiological markers (cf. [
      • Colzato L.
      • Beste C.
      A literature review on the neurophysiological underpinnings and cognitive effects of transcutaneous vagus nerve stimulation: challenges and future directions.
      ]). Ideally, this should include more stable markers with less potentially confounding factors than sAA. Although an acceptable test-retest reliability was found for sAA (ICC = 0.79, CI[0.75; 0.83], p < 0.001), other NA markers could be further explored such as the P300 ERP component (see for review [
      • Nieuwenhuis S.
      • Aston-Jones G.
      • Cohen J.D.
      Decision making, the p3, and the locus coeruleus–norepinephrine system.
      ]) or pupil dilation (see for review [
      • Burger A.M.
      • Van der Does W.
      • Brosschot J.F.
      • Verkuil B.
      From ear to eye? no effect of transcutaneous vagus nerve stimulation on human pupil dilation: a report of three studies.
      ]). Second, although sAA levels can be measured in a non-invasive and inexpensive fashion, some methodological concerns of sAA as index of central noradrenergic activity must be taken into account. Based on the ongoing debate whether sAA levels measured during stress reflect purely sympathetic or parasympathetic activity or some combination of both [
      • Bosch J.A.
      • Veerman E.C.
      • de Geus E.J.
      • Proctor G.B.
      α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
      ,
      • Ali N.
      • Nater U.M.
      Salivary alpha-amylase as a biomarker of stress in behavioral medicine.
      ], it has been recommended to collect salivary flow rate as measure of parasympathetic activity [
      • Strahler J.
      • Skoluda N.
      • Kappert M.B.
      • Nater U.M.
      Simultaneous measurement of salivary cortisol and alpha-amylase: application and recommendations.
      ]. In the present study, however, we did not investigate the contribution of salivary flow rate and thus, cannot exclude parasympathetic influence on sAA secretion, as this data was not available for the majority of included studies. Third, all included studies used tasks that might induce additional levels of stress (arousal), possibly interacting with the observed taVNS stimulation effects of sAA. Thus, it remains unclear whether the sole application of taVNS without such engaging task would also lead to similar increases. Future research should therefore investigate if and to what extent the effects of taVNS on sAA levels might be task-dependent. Although our work emphasizes the advantages of data pooling and data sharing (especially of raw data) to overcome limitations of single studies (i.e., small sample size), and to accelerate progress in validating potential relations between reliable NA markers and taVNS-mediated vagal activation, disadvantages and shortcomings of data pooling should also be taken into consideration. Mega-analyses require homogeneous datasets and the establishment of a common centralized database [
      • Boedhoe P.S.
      • Heymans M.W.
      • Schmaal L.
      • Abe Y.
      • Alonso P.
      • Ameis S.H.
      • Anticevic A.
      • Arnold P.D.
      • Batistuzzo M.C.
      • Benedetti F.
      • et al.
      An empirical comparison of meta-and mega-analysis with data from the enigma obsessive-compulsive disorder working group.
      ]. Methodological differences in study characteristics, stimulation protocols, data collection, preprocessing and/or statistical analysis across studies therefore reduce comparability. Indeed, our meta-analysis showed high heterogeneity in the data, which in turn might explain why we were not able to detect any other effects of stimulation parameters (e.g., stimulation length, stimulation intensity) on sAA levels. Therefore, it is important to emphasize the explorative character of the present approach and further research is certainly necessary.
      To summarize, the present findings lead us to conclude that vagal activation via taVNS increases sAA release compared to sham stimulation, which likely substantiates the assumption that taVNS triggers NA release. Future taVNS studies with appropriate sample sizes, collecting sAA levels, along with other potentially confounding factors of sAA, are essential to further validate our findings in other contexts. Given the rather small effect size and the heterogeneity of our data, there are still numerous open questions and concerns that need to be addressed. Importantly, the generalizability of the observed effect of taVNS on sAA release remains unclear. Future studies need to account for the possibility of inter-individual differences of participants (i.e., non-responders) and should further determine statistically valuable sample sizes in order to confirm meaningful increases of sAA after taVNS compared to sham stimulation. Accordingly, the question arises as to the practicality of sAA as an indirect marker of NA system activation in the context of taVNS research since not all included studies showed a significant effect of taVNS on sAA. This work particularly emphasizes the benefits of data pooling and data sharing in order to publish more meaningful and valuable data in research and to further address these open questions together. In this line, we urge researchers to join forces in the search for essential stimulation parameters and reliable markers that might shed further light on the efficacy of taVNS.

      CRediT authorship contribution statement

      Manon Giraudier: Conceptualization, Methodology, Formal analysis, Data curation, Writing – original draft, Writing – review & editing, Visualization. Carlos Ventura-Bort: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing. Andreas M. Burger: Data curation, Writing – review & editing. Nathalie Claes: Data curation, Writing – review & editing. Martina D'Agostini: Data curation, Writing – review & editing. Rico Fischer: Data curation, Writing – review & editing. Mathijs Franssen: Data curation, Writing – review & editing. Michael Kaess: Data curation, Writing – review & editing. Julian Koenig: Data curation, Writing – review & editing. Roman Liepelt: Data curation, Writing – review & editing. Sander Nieuwenhuis: Data curation, Writing – review & editing. Aldo Sommer: Data curation, Writing – review & editing. Taras Usichenko: Data curation, Writing – review & editing. Ilse Van Diest: Data curation, Writing – review & editing. Andreas von Leupoldt: Data curation, Writing – review & editing. Christopher M. Warren: Data curation, Writing – review & editing. Mathias Weymar: Conceptualization, Methodology, Data curation, Writing – original draft, Writing – review & editing, Supervision, Project administration, Funding acquisition.

      Declaration of competing interest

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Acknowledgements

      This project is part of the scientific research ”Network for Transcutaneous Vagus Nerve Stimulation Research”, which is funded by the Research Foundation Flanders, Belgium (FWO; W001520 N). IvD and AvL were supported by infrastructure grants from the FWO and the Research Fund KU Leuven, Belgium (AKUL/19/06; I011320 N) and by the ”Asthenes” long-term structural funding Methusalem grant (METH/15/011) from the Flemish Government, Belgium. For funding of the publication fees we acknowledge the support of the Deutsche Forschungsgemeinschaft (DFG; 491466077).

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

      References

        • Broncel A.
        • Bocian R.
        • Kłos-Wojtczak P.
        • Kulbat-Warycha K.
        • Konopacki J.
        Vagal nerve stimulation as a promising tool in the improvement of cognitive disorders.
        Brain Res Bull. 2020; 155: 37-47
        • Rong P.
        • Liu A.
        • Zhang J.
        • Wang Y.
        • He W.
        • Yang A.
        • Li L.
        • Ben H.
        • Li L.
        • Liu H.
        • et al.
        Transcutaneous vagus nerve stimulation for refractory epilepsy: a randomized controlled trial.
        Clinical Science, 2014
        • Bauer S.
        • Baier H.
        • Baumgartner C.
        • Bohlmann K.
        • Fauser S.
        • Graf W.
        • Hillenbrand B.
        • Hirsch M.
        • Last C.
        • Lerche H.
        • et al.
        Transcutaneous vagus nerve stimulation (tvns) for treatment of drug-resistant epilepsy: a randomized, double-blind clinical trial (cmpse02).
        Brain Stimul. 2016; 9: 356-363
        • Aihua L.
        • Lu S.
        • Liping L.
        • Xiuru W.
        • Hua L.
        • Yuping W.
        A controlled trial of transcutaneous vagus nerve stimulation for the treatment of pharmacoresistant epilepsy.
        Epilepsy Behav. 2014; 39: 105-110
        • Fang J.
        • Rong P.
        • Hong Y.
        • Fan Y.
        • Liu J.
        • Wang H.
        • Zhang G.
        • Chen X.
        • Shi S.
        • Wang L.
        • et al.
        Transcutaneous vagus nerve stimulation modulates default mode network in major depressive disorder.
        Biol Psychiatr. 2016; 79: 266-273
        • Napadow V.
        • Edwards R.R.
        • Cahalan C.M.
        • Mensing G.
        • Greenbaum S.
        • Valovska A.
        • Li A.
        • Kim J.
        • Maeda Y.
        • Park K.
        • et al.
        Evoked pain analgesia in chronic pelvic pain patients using respiratory-gated auricular vagal afferent nerve stimulation.
        Pain Med. 2012; 13: 777-789
        • Farmer A.D.
        • Strzelczyk A.
        • Finisguerra A.
        • Gourine A.V.
        • Gharabaghi A.
        • Hasan A.
        • Burger A.M.
        • Jaramillo A.M.
        • Mertens A.
        • Majid A.
        • et al.
        International consensus based review and recommendations for minimum reporting standards in research on transcutaneous vagus nerve stimulation (version 2020).
        Front Hum Neurosci. 2021; 14: 409
        • Weymar M.
        • Zaehle T.
        Editorial: New Frontiers in Noninvasive Brain Stimulation: Cognitive, Affective and Neurobiological Effects of Transcutaneous Vagus Nerve Stimulation.
        Front. Psychol. 2021; 12: 694723https://doi.org/10.3389/fpsyg.2021.694723
        • Colzato L.
        • Beste C.
        A literature review on the neurophysiological underpinnings and cognitive effects of transcutaneous vagus nerve stimulation: challenges and future directions.
        J Neurophysiol. 2020; 123: 1739-1755
        • Dorr A.E.
        • Debonnel G.
        Effect of vagus nerve stimulation on serotonergic and noradrenergic transmission.
        J Pharmacol Exp Therapeut. 2006; 318: 890-898
        • Manta S.
        • Dong J.
        • Debonnel G.
        • Blier P.
        Enhancement of the function of rat serotonin and norepinephrine neurons by sustained vagus nerve stimulation.
        J Psychiatr Neurosci. 2009; 34: 272-280
        • Tellez L.A.
        • Medina S.
        • Han W.
        • Ferreira J.G.
        • Licona-Limón P.
        • Ren X.
        • Lam T.T.
        • Schwartz G.J.
        • De Araujo I.E.
        A gut lipid messenger links excess dietary fat to dopamine deficiency.
        Science. 2013; 341: 800-802
        • Han W.
        • Tellez L.A.
        • Perkins M.H.
        • Perez I.O.
        • Qu T.
        • Ferreira J.
        • Ferreira T.L.
        • Quinn D.
        • Liu Z.-W.
        • Gao X.-B.
        • et al.
        A neural circuit for gut-induced reward.
        Cell. 2018; 175: 665-678
        • Hulsey D.R.
        • Hays S.A.
        • Khodaparast N.
        • Ruiz A.
        • Das P.
        • Rennaker II, R.L.
        • Kilgard M.P.
        Reorganization of motor cortex by vagus nerve stimulation requires cholinergic innervation.
        Brain Stimul. 2016; 9: 174-181
        • Roosevelt R.W.
        • Smith D.C.
        • Clough R.W.
        • Jensen R.A.
        • Browning R.A.
        Increased extracellular concentrations of norepinephrine in cortex and hippocampus following vagus nerve stimulation in the rat.
        Brain Res. 2006; 1119: 124-132
        • Raedt R.
        • Clinckers R.
        • Mollet L.
        • Vonck K.
        • El Tahry R.
        • Wyckhuys T.
        • De Herdt V.
        • Carrette E.
        • Wadman W.
        • Michotte Y.
        • et al.
        Increased hippocampal noradrenaline is a biomarker for efficacy of vagus nerve stimulation in a limbic seizure model.
        J Neurochem. 2011; 117: 461-469
        • McIntyre C.K.
        • McGaugh J.L.
        • Williams C.L.
        Interacting brain systems modulate memory consolidation.
        Neurosci Biobehav Rev. 2012; 36: 1750-1762
        • Izquierdo J.
        • Insua J.
        • Biscardi A.
        • Izquierdo I.
        Some observations on the responses to the afferent stimulation of the vagus.
        Pharmacology. 1959; 1: 325-332
        • Wan S.
        • Browning K.N.
        • Coleman F.H.
        • Sutton G.
        • Zheng H.
        • Butler A.
        • Berthoud H.-R.
        • Travagli R.A.
        Presynaptic melanocortin-4 receptors on vagal afferent fibers modulate the excitability of rat nucleus tractus solitarius neurons.
        J Neurosci. 2008; 28: 4957-4966
        • Reyes B.A.
        • Van Bockstaele E.J.
        Divergent projections of catecholaminergic neurons in the nucleus of the solitary tract to limbic forebrain and medullary autonomic brain regions.
        Brain Res. 2006; 1117: 69-79
        • Aston-Jones G.
        • Ennis M.
        • Pieribone V.A.
        • Nickell W.T.
        • Shipley M.T.
        The brain nucleus locus coeruleus: restricted afferent control of a broad efferent network.
        Science. 1986; 234: 734-737
        • Nieuwenhuis S.
        • De Geus E.J.
        • Aston-Jones G.
        The anatomical and functional relationship between the p3 and autonomic components of the orienting response.
        Psychophysiology. 2011; 48: 162-175
        • Schwarz L.A.
        • Luo L.
        Organization of the locus coeruleus-norepinephrine system.
        Curr Biol. 2015; 25 (R1051–R1056)
        • Aston-Jones G.
        • Rajkowski J.
        • Kubiak P.
        • Alexinsky T.
        Locus coeruleus neurons in monkey are selectively activated by attended cues in a vigilance task.
        J Neurosci. 1994; 14: 4467-4480
        • Sara S.J.
        • Bouret S.
        Orienting and reorienting: the locus coeruleus mediates cognition through arousal.
        Neuron. 2012; 76: 130-141
        • Mather M.
        • Harley C.W.
        The locus coeruleus: essential for maintaining cognitive function and the aging brain.
        Trends Cogn Sci. 2016; 20: 214-226
        • Dahl M.J.
        • Mather M.
        • Werkle-Bergner M.
        • Kennedy B.L.
        • Guzman S.
        • Hurth K.
        • Miller C.A.
        • Qiao Y.
        • Shi Y.
        • Chui H.C.
        • et al.
        Locus coeruleus integrity is related to tau burden and memory loss in autosomal-dominant alzheimer's disease.
        Neurobiol Aging. 2022; 112: 39-54
        • Groves D.A.
        • Bowman E.M.
        • Brown V.J.
        Recordings from the rat locus coeruleus during acute vagal nerve stimulation in the anaesthetised rat.
        Neurosci Lett. 2005; 379: 174-179
        • Hulsey D.R.
        • Riley J.R.
        • Loerwald K.W.
        • Rennaker II, R.L.
        • Kilgard M.P.
        • Hays S.A.
        Parametric characterization of neural activity in the locus coeruleus in response to vagus nerve stimulation.
        Exp Neurol. 2017; 289: 21-30
        • Svensson T.
        • Thoren P.
        Brain noradrenergic neurons in the locus coeruleus: inhibition by blood volume load through vagal afferents.
        Brain Res. 1979; 172: 174-178
        • Alvarez-Dieppa A.C.
        • Griffin K.
        • Cavalier S.
        • McIntyre C.K.
        Vagus nerve stimulation enhances extinction of conditioned fear in rats and modulates arc protein, camkii, and glun2b-containing nmda receptors in the basolateral amygdala, Neural Plasticity 2016.
        2016
        • Noble L.J.
        • Chuah A.
        • Callahan K.K.
        • Souza R.R.
        • McIntyre C.K.
        Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats.
        Learn Mem. 2019; 26: 245-251
        • Clark K.
        • Krahl S.
        • Smith D.
        • Jensen R.
        Post-training unilateral vagal stimulation enhances retention performance in the rat.
        Neurobiol Learn Mem. 1995; 63: 213-216
        • Clark K.
        • Smith D.
        • Hassert D.
        • Browning R.
        • Naritoku D.
        • Jensen R.
        Posttraining electrical stimulation of vagal afferents with concomitant vagal efferent inactivation enhances memory storage processes in the rat.
        Neurobiol Learn Mem. 1998; 70: 364-373
        • Clark K.B.
        • Naritoku D.K.
        • Smith D.C.
        • Browning R.A.
        • Jensen R.A.
        Enhanced recognition memory following vagus nerve stimulation in human subjects.
        Nat Neurosci. 1999; 2: 94-98
        • Ghacibeh G.A.
        • Shenker J.I.
        • Shenal B.
        • Uthman B.M.
        • Heilman K.M.
        The influence of vagus nerve stimulation on memory.
        Cognit Behav Neurol. 2006; 19: 119-122
        • Mertens A.
        • Gadeyne S.
        • Lescrauwaet E.
        • Carrette E.
        • Meurs A.
        • De Herdt V.
        • Dewaele F.
        • Raedt R.
        • Miatton M.
        • Boon P.
        • et al.
        The potential of invasive and non-invasive vagus nerve stimulation to improve verbal memory performance in epilepsy patients.
        Sci Rep. 2022; 12: 1-13
        • Bianca R.
        • Komisaruk B.R.
        Pupil dilatation in response to vagal afferent electrical stimulation is mediated by inhibition of parasympathetic outflow in the rat.
        Brain Res. 2007; 1177: 29-36
        • Jodoin V.D.
        • Lespérance P.
        • Nguyen D.K.
        • Fournier-Gosselin M.-P.
        • Richer F.
        • et al.
        Effects of vagus nerve stimulation on pupillary function.
        Int J Psychophysiol. 2015; 98: 455-459
        • Mridha Z.
        • de Gee J.W.
        • Shi Y.
        • Alkashgari R.
        • Williams J.
        • Suminski A.
        • Ward M.P.
        • Zhang W.
        • McGinley M.J.
        Graded recruitment of pupil-linked neuromodulation by parametric stimulation of the vagus nerve.
        Nat Commun. 2021; 12: 1-14
        • Lai J.
        • David S.V.
        Short-term effects of vagus nerve stimulation on learning and evoked activity in auditory cortex.
        Eneuro. 2021; 8
        • Neuhaus A.
        • Luborzewski A.
        • Rentzsch J.
        • Brakemeier E.
        • Opgen-Rhein C.
        • Gallinat J.
        • Bajbouj M.
        P300 is enhanced in responders to vagus nerve stimulation for treatment of major depressive disorder.
        J Affect Disord. 2007; 100: 123-128
        • De Taeye L.
        • Vonck K.
        • van Bochove M.
        • Boon P.
        • Van Roost D.
        • Mollet L.
        • Meurs A.
        • De Herdt V.
        • Carrette E.
        • Dauwe I.
        • et al.
        The p3 event-related potential is a biomarker for the efficacy of vagus nerve stimulation in patients with epilepsy.
        Neurotherapeutics. 2014; 11: 612-622
        • Joshi S.
        • Li Y.
        • Kalwani R.M.
        • Gold J.I.
        Relationships between pupil diameter and neuronal activity in the locus coeruleus, colliculi, and cingulate cortex.
        Neuron. 2016; 89: 221-234
        • Reimer J.
        • McGinley M.J.
        • Liu Y.
        • Rodenkirch C.
        • Wang Q.
        • McCormick D.A.
        • Tolias A.S.
        Pupil fluctuations track rapid changes in adrenergic and cholinergic activity in cortex.
        Nat Commun. 2016; 7: 1-7
        • Liu Y.
        • Rodenkirch C.
        • Moskowitz N.
        • Schriver B.
        • Wang Q.
        Dynamic lateralization of pupil dilation evoked by locus coeruleus activation results from sympathetic, not parasympathetic, contributions.
        Cell Rep. 2017; 20: 3099-3112
        • Breton-Provencher V.
        • Sur M.
        Active control of arousal by a locus coeruleus gabaergic circuit.
        Nat Neurosci. 2019; 22: 218-228
        • Murphy P.R.
        • Robertson I.H.
        • Balsters J.H.
        • O’connell R.G.
        Pupillometry and p3 index the locus coeruleus–noradrenergic arousal function in humans.
        Psychophysiology. 2011; 48: 1532-1543
        • Vazey E.M.
        • Moorman D.E.
        • Aston-Jones G.
        Phasic locus coeruleus activity regulates cortical encoding of salience information.
        Proc Natl Acad Sci. 2018; 115: E9439-E9448
        • Nieuwenhuis S.
        • Aston-Jones G.
        • Cohen J.D.
        Decision making, the p3, and the locus coeruleus–norepinephrine system.
        Psychol Bull. 2005; 131: 510
        • Burger A.M.
        • D'Agostini M.
        • Verkuil B.
        • Van Diest I.
        Moving beyond belief: a narrative review of potential biomarkers for transcutaneous vagus nerve stimulation.
        Psychophysiology. 2020; 57e13571
        • Kraus T.
        • Kiess O.
        • Hösl K.
        • Terekhin P.
        • Kornhuber J.
        • Forster C.
        Cns bold fmri effects of sham-controlled transcutaneous electrical nerve stimulation in the left outer auditory canal–a pilot study.
        Brain Stimul. 2013; 6: 798-804
        • Frangos E.
        • Ellrich J.
        • Komisaruk B.R.
        Non-invasive access to the vagus nerve central projections via electrical stimulation of the external ear: fmri evidence in humans.
        Brain Stimul. 2015; 8: 624-636
        • Yakunina N.
        • Kim S.S.
        • Nam E.-C.
        Optimization of transcutaneous vagus nerve stimulation using functional mri.
        Neuromodulation: technology at the neural interface. 2017; 20: 290-300
        • Sclocco R.
        • Garcia R.G.
        • Kettner N.W.
        • Isenburg K.
        • Fisher H.P.
        • Hubbard C.S.
        • Ay I.
        • Polimeni J.R.
        • Goldstein J.
        • Makris N.
        • et al.
        The influence of respiration on brainstem and cardiovagal response to auricular vagus nerve stimulation: a multimodal ultrahigh-field (7t) fmri study.
        Brain Stimul. 2019; 12: 911-921
        • Borgmann D.
        • Rigoux L.
        • Kuzmanovic B.
        • Thanarajah S.E.
        • Münte T.F.
        • Fenselau H.
        • Tittgemeyer M.
        Modulation of fmri brainstem responses by transcutaneous vagus nerve stimulation.
        Neuroimage. 2021; 244118566
        • Teckentrup V.
        • Krylova M.
        • Jamalabadi H.
        • Neubert S.
        • Neuser M.P.
        • Hartig R.
        • Fallgatter A.J.
        • Walter M.
        • Kroemer N.B.
        Brain signaling dynamics after vagus nerve stimulation.
        Neuroimage. 2021; 245118679
        • Burger A.M.
        • Verkuil B.
        • Van Diest I.
        • Van der Does W.
        • Thayer J.F.
        • Brosschot J.F.
        The effects of transcutaneous vagus nerve stimulation on conditioned fear extinction in humans.
        Neurobiol Learn Mem. 2016; 132: 49-56
        • Burger A.
        • Verkuil B.
        • Fenlon H.
        • Thijs L.
        • Cools L.
        • Miller H.
        • Vervliet B.
        • Van Diest I.
        Mixed evidence for the potential of non-invasive transcutaneous vagal nerve stimulation to improve the extinction and retention of fear.
        Behav Res Ther. 2017; 97: 64-74
        • Szeska C.
        • Richter J.
        • Wendt J.
        • Weymar M.
        • Hamm A.O.
        Promoting long-term inhibition of human fear responses by non-invasive transcutaneous vagus nerve stimulation during extinction training.
        Sci Rep. 2020; 10: 1-16
        • Genheimer H.
        • Andreatta M.
        • Asan E.
        • Pauli P.
        Reinstatement of contextual conditioned anxiety in virtual reality and the effects of transcutaneous vagus nerve stimulation in humans.
        Sci Rep. 2017; 7: 1-13
        • Burger A.M.
        • Van Diest I.
        • van der Does W.
        • Hysaj M.
        • Thayer J.F.
        • Brosschot J.F.
        • Verkuil B.
        Transcutaneous vagus nerve stimulation and extinction of prepared fear: a conceptual non-replication.
        Sci Rep. 2018; 8: 1-11
        • Jacobs H.I.
        • Riphagen J.M.
        • Razat C.M.
        • Wiese S.
        • Sack A.T.
        Transcutaneous vagus nerve stimulation boosts associative memory in older individuals.
        Neurobiol Aging. 2015; 36: 1860-1867
        • Giraudier M.
        • Ventura-Bort C.
        • Weymar M.
        Transcutaneous vagus nerve stimulation (tvns) improves high-confidence recognition memory but not emotional word processing.
        Front Psychol. 2020; 11: 1276
        • Ventura-Bort C.
        • Wirkner J.
        • Wendt J.
        • Hamm A.O.
        • Weymar M.
        Establishment of emotional memories is mediated by vagal nerve activation: evidence from noninvasive tavns.
        J Neurosci. 2021; 41: 7636-7648
        • Mertens A.
        • Naert L.
        • Miatton M.
        • Poppa T.
        • Carrette E.
        • Gadeyne S.
        • Raedt R.
        • Boon P.
        • Vonck K.
        Transcutaneous vagus nerve stimulation does not affect verbal memory performance in healthy volunteers.
        Front Psychol. 2020; 11: 551
        • Sellaro R.
        • van Leusden J.W.
        • Tona K.-D.
        • Verkuil B.
        • Nieuwenhuis S.
        • Colzato L.S.
        Transcutaneous vagus nerve stimulation enhances post-error slowing.
        J Cognit Neurosci. 2015; 27: 2126-2132
        • Sellaro R.
        • de Gelder B.
        • Finisguerra A.
        • Colzato L.S.
        Transcutaneous vagus nerve stimulation (tvns) enhances recognition of emotions in faces but not bodies.
        Cortex. 2018; 99: 213-223
        • Steenbergen L.
        • Sellaro R.
        • Stock A.-K.
        • Verkuil B.
        • Beste C.
        • Colzato L.S.
        Transcutaneous vagus nerve stimulation (tvns) enhances response selection during action cascading processes.
        Eur Neuropsychopharmacol. 2015; 25: 773-778
        • Fischer R.
        • Ventura-Bort C.
        • Hamm A.
        • Weymar M.
        Transcutaneous vagus nerve stimulation (tvns) enhances conflict-triggered adjustment of cognitive control.
        Cognit Affect Behav Neurosci. 2018; 18: 680-693
        • Keute M.
        • Boehrer L.
        • Ruhnau P.
        • Heinze H.-J.
        • Zaehle T.
        Transcutaneous vagus nerve stimulation (tvns) and the dynamics of visual bistable perception.
        Front Neurosci. 2019; 13: 227
        • Tona K.-D.
        • Revers H.
        • Verkuil B.
        • Nieuwenhuis S.
        Noradrenergic regulation of cognitive flexibility: no effects of stress, transcutaneous vagus nerve stimulation, and atomoxetine on task-switching in humans.
        J Cogn Neurosci. 2020; 32: 1881-1895
        • Rufener K.S.
        • Geyer U.
        • Janitzky K.
        • Heinze H.-J.
        • Zaehle T.
        Modulating auditory selective attention by non-invasive brain stimulation: differential effects of transcutaneous vagal nerve stimulation and transcranial random noise stimulation.
        Eur J Neurosci. 2018; 48: 2301-2309
        • Maraver M.J.
        • Steenbergen L.
        • Hossein R.
        • Actis-Grosso R.
        • Ricciardelli P.
        • Hommel B.
        • Colzato L.S.
        Transcutaneous vagus nerve stimulation modulates attentional resource deployment towards social cues.
        Neuropsychologia. 2020; 143107465
        • Burger A.M.
        • Van der Does W.
        • Brosschot J.F.
        • Verkuil B.
        From ear to eye? no effect of transcutaneous vagus nerve stimulation on human pupil dilation: a report of three studies.
        Biol Psychol. 2020; 152107863
        • Sharon O.
        • Fahoum F.
        • Nir Y.
        Transcutaneous vagus nerve stimulation in humans induces pupil dilation and attenuates alpha oscillations.
        J Neurosci. 2021; 41: 320-330
        • Urbin M.A.
        • Lafe C.W.
        • Simpson T.W.
        • Wittenberg G.F.
        • Chandrasekaran B.
        • Weber D.J.
        Electrical stimulation of the external ear acutely activates noradrenergic mechanisms in humans.
        Brain Stimul. 2021; 14: 990-1001
        • Villani V.
        • Finotti G.
        • Di Lernia D.
        • Tsakiris M.
        • Azevedo R.T.
        Event-related transcutaneous vagus nerve stimulation modulates behaviour and pupillary responses during an auditory oddball task.
        Psychoneuroendocrinology. 2022; 105719
        • Warren C.M.
        • Tona K.D.
        • Ouwerkerk L.
        • Van Paridon J.
        • Poletiek F.
        • van Steenbergen H.
        • Bosch J.A.
        • Nieuwenhuis S.
        The neuromodulatory and hormonal effects of transcutaneous vagus nerve stimulation as evidenced by salivary alpha amylase, salivary cortisol, pupil diameter, and the p3 event-related potential.
        Brain Stimul. 2019; 12: 635-642
        • D'Agostini M.
        • Burger A.M.
        • Franssen M.
        • Claes N.
        • Weymar M.
        • von Leupoldt A.
        • Van Diest I.
        Effects of transcutaneous auricular vagus nerve stimulation on reversal learning, tonic pupil size, salivary alpha-amylase, and cortisol.
        Psychophysiology. 2021; 58e13885
        • D'Agostini M.
        • Burger A.M.
        • Villca Ponce G.
        • Claes S.
        • von Leupoldt A.
        • Van Diest I.
        No evidence for a modulating effect of continuous transcutaneous auricular vagus nerve stimulation on markers of noradrenergic activity.
        Psychophysiology. 2022; e13984
        • Lewine J.D.
        • Paulson K.
        • Bangera N.
        • Simon B.J.
        Exploration of the impact of brief noninvasive vagal nerve stimulation on eeg and event-related potentials.
        Neuromodulation: Technology at the Neural Interface. 2019; 22: 564-572
        • Warren C.V.
        • Maraver M.J.
        • de Luca A.
        • Kopp B.
        The effect of transcutaneous auricular vagal nerve stimulation (tavns) on p3 event-related potentials during a bayesian oddball task.
        Brain Sci. 2020; 10: 404
        • Ventura-Bort C.
        • Wirkner J.
        • Genheimer H.
        • Wendt J.
        • Hamm A.O.
        • Weymar M.
        Effects of transcutaneous vagus nerve stimulation (tvns) on the p300 and alpha-amylase level: a pilot study.
        Front Hum Neurosci. 2018; 12: 202
        • Gadeyne S.
        • Mertens A.
        • Carrette E.
        • Van den Bossche F.
        • Boon P.
        • Raedt R.
        • Vonck K.
        Transcutaneous auricular vagus nerve stimulation cannot modulate the p3b event-related potential in healthy volunteers.
        Clin Neurophysiol. 2022; 135: 22-29
        • Wolf V.
        • Kühnel A.
        • Teckentrup V.
        • Koenig J.
        • Kroemer N.B.
        Does transcutaneous auricular vagus nerve stimulation affect vagally mediated heart rate variability? a living and interactive bayesian meta-analysis.
        Psychophysiology. 2021; 58e13933
        • Ehlert U.
        • Erni K.
        • Hebisch G.
        • Nater U.
        Salivary α-amylase levels after yohimbine challenge in healthy men.
        J Clin Endocrinol Metab. 2006; 91: 5130-5133
        • Kuebler U.
        • von Känel R.
        • Heimgartner N.
        • Zuccarella-Hackl C.
        • Stirnimann G.
        • Ehlert U.
        • Wirtz P.H.
        Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men.
        Psychoneuroendocrinology. 2014; 49: 290-298
        • Warren C.M.
        • van den Brink R.L.
        • Nieuwenhuis S.
        • Bosch J.A.
        Norepinephrine transporter blocker atomoxetine increases salivary alpha amylase.
        Psychoneuroendocrinology. 2017; 78: 233-236
        • Nater U.M.
        • Rohleder N.
        Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: current state of research.
        Psychoneuroendocrinology. 2009; 34: 486-496
        • Koenig J.
        • Parzer P.
        • Haigis N.
        • Liebemann J.
        • Jung T.
        • Resch F.
        • Kaess M.
        Effects of acute transcutaneous vagus nerve stimulation on emotion recognition in adolescent depression.
        Psychol Med. 2021; 51: 511-520
      1. A. Sommer, R. Fischer, U. Borges, S. Laborde, S. Achtzehn, R. Liepelt. Unpublished results (n.d.).

        • Bosch J.A.
        • Veerman E.C.
        • de Geus E.J.
        • Proctor G.B.
        α-amylase as a reliable and convenient measure of sympathetic activity: don't start salivating just yet.
        Psychoneuroendocrinology. 2011; 36: 449-453
        • Boedhoe P.S.
        • Heymans M.W.
        • Schmaal L.
        • Abe Y.
        • Alonso P.
        • Ameis S.H.
        • Anticevic A.
        • Arnold P.D.
        • Batistuzzo M.C.
        • Benedetti F.
        • et al.
        An empirical comparison of meta-and mega-analysis with data from the enigma obsessive-compulsive disorder working group.
        Front Neuroinf. 2019; 12: 102
        • Debray T.P.
        • Moons K.G.
        • Abo-Zaid G.M.A.
        • Koffijberg H.
        • Riley R.D.
        Individual participant data meta-analysis for a binary outcome: one-stage or two-stage?.
        PLoS One. 2013; 8e60650
        • Kliegl R.
        • Wei P.
        • Dambacher M.
        • Yan M.
        • Zhou X.
        Experimental effects and individual differences in linear mixed models: estimating the relationship between spatial, object, and attraction effects in visual attention.
        Front Psychol. 2011; 1: 238
      2. M. Giraudier, C. Ventura-Bort, M. Weymar. Unpublished results (n.d).

      3. C. Ventura-Bort, M. Weymar. Unpublished results (n.d.).

        • Schultz G.
        • Altenstein C.
        • Klausenitz C.
        • Hesse T.
        • Hacker H.
        • Petersmann A.
        • Hannich H.
        • Hahnenkamp K.
        • Usichenko T.
        Auricular acupuncture vs. progressive muscle relaxation and no intervention for exam anxiety in medical students–a randomized controlled trial with non-randomized condition.
        Brain Stimulat: basic, Translational, and Clinical Research in Neuromodulation. 2017; 10: 431
        • Usichenko T.
        • Wenzel A.
        • Klausenitz C.
        • Petersmann A.
        • Hesse T.
        • Neumann N.
        • Hahnenkamp K.
        Auricular stimulation vs. expressive writing for exam anxiety in medical students–a randomized crossover investigation.
        PLoS One. 2020; 15e0238307
        • Ellrich J.
        Transcutaneous vagus nerve stimulation european neurological review.
        6. 2011: 262-264
        • Peuker E.T.
        • Filler T.J.
        The nerve supply of the human auricle.
        Clin Anat. 2002; 15: 35-37
        • Baum B.J.
        Principles of saliva secretion.
        Ann N Y Acad Sci. 1993; 694: 17-23
        • Chatterton Jr., R.T.
        • Vogelsong K.M.
        • Lu Y.-c.
        • Ellman A.B.
        • Hudgens G.A.
        Salivary α-amylase as a measure of endogenous adrenergic activity.
        Clin Physiol. 1996; 16: 433-448
        • Nater U.M.
        The role of salivary alpha-amylase in stress research.
        Cuvillier Verlag, 2004
        • Rohleder N.
        • Nater U.M.
        • Wolf J.M.
        • Ehlert U.
        • Kirschbaum C.
        Psychosocial stress-induced activation of salivary alpha-amylase: an indicator of sympathetic activity?.
        Ann N Y Acad Sci. 2004; 1032: 258-263
        • Granger D.A.
        • Kivlighan K.T.
        • El-Sheikh M.
        • Gordis E.B.
        • Stroud L.R.
        Salivary α-amylase in biobehavioral research: recent developments and applications.
        Ann N Y Acad Sci. 2007; 1098: 122-144
        • Ali N.
        • Nater U.M.
        Salivary alpha-amylase as a biomarker of stress in behavioral medicine.
        Int J Behav Med. 2020; 27: 337-342
        • R Core Team
        R: a language and environment for statistical computing.
        R Foundation for Statistical Computing, Vienna, Austria2019
        • Wickham H.
        • Averick M.
        • Bryan J.
        • Chang W.
        • McGowan L.D.
        • François R.
        • Grolemund G.
        • Hayes A.
        • Henry L.
        • Hester J.
        • et al.
        Welcome to the tidyverse.
        J Open Source softw. 2019; 4: 1686
        • Bates D.
        • Mächler M.
        • Bolker B.
        • Walker S.
        Fitting linear mixed-effects models using lme4.
        2014: 5823 (arXiv preprint arXiv:1406)
        • Box G.E.
        • Cox D.R.
        An analysis of transformations.
        J Roy Stat Soc B. 1964; 26: 211-243
        • Schad D.J.
        • Vasishth S.
        • Hohenstein S.
        • Kliegl R.
        How to capitalize on a priori contrasts in linear (mixed) models: a tutorial.
        J Mem Lang. 2020; 110104038
        • Bates D.
        • Kliegl R.
        • Vasishth S.
        • Baayen H.
        Parsimonious mixed models.
        2015 (arXiv preprint arXiv:1506.04967)
        • Satterthwaite F.E.
        An approximate distribution of estimates of variance components.
        Biometr Bull. 1946; 2: 110-114
        • Lenth R.V.
        Least-squares means: the r package lsmeans.
        J Stat Software. 2016; 69: 1-33
        • Meteyard L.
        • Davies R.A.
        Best practice guidance for linear mixed-effects models in psychological science.
        J Mem Lang. 2020; 112104092
        • Hedges L.V.
        • Olkin I.
        Nonparametric estimators of effect size in meta-analysis.
        Psychol Bull. 1984; 96: 573
        • Viechtbauer W.
        Conducting meta-analyses in r with the metafor package.
        J Stat Software. 2010; 36: 1-48
        • Schwarzer G.
        • et al.
        meta: an r package for meta-analysis.
        R News. 2007; 7: 40-45
        • Cohen J.
        Statistical power analysis for the behavioral sciences.
        revised ed. Lawrence earlbaum associates, hillsdale, nj1988
        • Valentine J.C.
        • Pigott T.D.
        • Rothstein H.R.
        How many studies do you need? a primer on statistical power for meta-analysis.
        J Educ Behav Stat. 2010; 35: 215-247
        • Revelle W.
        An overview of the psych package.
        2011
        • Scheibehenne B.
        • Jamil T.
        • Wagenmakers E.-J.
        Bayesian evidence synthesis can reconcile seemingly inconsistent results: the case of hotel towel reuse.
        Psychol Sci. 2016; 27: 1043-1046
        • Dunn A.J.
        • Swiergiel A.
        • Palamarchouk V.
        Brain circuits involved in corticotropin-releasing factor-norepinephrine interactions during stress.
        Ann N Y Acad Sci. 2004; 1018: 25-34
        • Jackson D.
        • Turner R.
        Power analysis for random-effects meta-analysis.
        Res Synth Methods. 2017; 8: 290-302
        • Follesa P.
        • Biggio F.
        • Gorini G.
        • Caria S.
        • Talani G.
        • Dazzi L.
        • Puligheddu M.
        • Marrosu F.
        • Biggio G.
        Vagus nerve stimulation increases norepinephrine concentration and the gene expression of bdnf and bfgf in the rat brain.
        Brain Res. 2007; 1179: 28-34
        • Manta S.
        • El Mansari M.
        • Debonnel G.
        • Blier P.
        Electrophysiological and neurochemical effects of long-term vagus nerve stimulation on the rat monoaminergic systems.
        Int J Neuropsychopharmacol. 2013; 16: 459-470
        • Lang P.J.
        International affective picture system (iaps): affective ratings of pictures and instruction manual.
        2005 (Technical report)
        • Segal S.K.
        • Cahill L.
        Endogenous noradrenergic activation and memory for emotional material in men and women.
        Psychoneuroendocrinology. 2009; 34: 1263-1271
        • Dawes C.
        The effects of flow rate and duration of stimulation on the concentrations of protein and the main electrolytes in human submandibular saliva.
        Arch Oral Biol. 1974; 19: 887-895
        • Nater U.M.
        • Rohleder N.
        • Schlotz W.
        • Ehlert U.
        • Kirschbaum C.
        Determinants of the diurnal course of salivary alpha-amylase.