Mia Thomaidou

How negative experiences influence the brain in pain: Neuroimaging & biobehavioral insights Mia A. Thomaidou

How negative experiences influence the brain in pain: Neuroimaging and biobehavioral insights Mia Thomaidou PhD dissertation

Author Mia Thomaidou Printing Ridderprint | www.ridderprint.nl Cover Design Mia Thomaidou, based on visualizations of our pain fMRI data ISBN 978-94-6458-249-9 The research presented in this thesis was carried out at the Faculty of Social and Behavioral Sciences, Health, Medical and Neuropsychology Unit, Leiden University, the Netherlands. Research was supported by an NWO Vici grant, granted to A.W.M. Evers, as well as the Institute of Psychology, Leiden University. The authors report no conflicts of interest. Copyright © Mia A. Thomaidou, 2022. All rights reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without written permission from the author.

3 How negative experiences influence the brain in pain: Neuroimaging and biobehavioral insights Dissertation ter verkrijging van de graad van doctor aan de Universiteit Leiden, op gezag van rector magnificus prof.dr.ir. H. Bijl, volgens besluit van het college voor promoties te verdedigen op woensdag 7 september 2022 klokke 16:15 uur door Athina Mia Thomaidou geboren te Athene

4 Promotor prof.dr. A.W.M. Evers Co-promotor dr. D.S. Veldhuijzen Promotion committee prof.dr. Paul F. Wouters (Decaan/voorzitter) prof.dr. Sander Nieuwenhuis prof.dr. Monique Steegers (Amsterdam UMC) prof.dr. Johan Vlaeyen (KU Leuven) prof.dr. Jos Brosschot

5 Dedication For Oma Mia. Through the darkness of the deepest pain you shine a light so bright for us to find the way.

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7 Table of contents Chapter 1. ........................................................................................ 9 General introduction...................................................................................... 9 Chapter 2. ...................................................................................... 29 Learned nocebo effects on cutaneous sensations: Meta-analysis of experimental behavioral findings. .............................................................. 29 Chapter 3. ...................................................................................... 63 How negative experience influences the brain: A comprehensive neurobiology review..................................................................................... 63 Chapter 4. ....................................................................................109 Learning mechanisms in nocebo hyperalgesia: The role of conditioning and extinction processes. .......................................................................... 109 Chapter 5. ....................................................................................147 An experimental investigation into the mediating role of pain-related fear in nocebo hyperalgesia....................................................................... 147 Chapter 6. ....................................................................................189 Temporal structure of brain oscillations predicts learned nocebo responses to pain. ....................................................................................... 189 Chapter 7. ....................................................................................219 A pharmacological fMRI investigation of brain plasticity mechanisms in nocebo hyperalgesia............................................................................... 219 Chapter 8. ....................................................................................253 Summary and general discussion ............................................................. 253 Curriculum Vitae........................................................................287 Brief CV....................................................................................................... 288 Articles published in peer-reviewed journals ......................................... 289 Articles in preparation ............................................................................... 290 Acknowledgments .....................................................................291 Dutch Summary .........................................................................293

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Chapter 1. General introduction

Learning decisively shapes the way individuals experience the world around them and how they respond to various external stimuli, including pain 1–3. Increased pain sensitivity can result from negative experiences creating negative expectations about an environmental stimulus, such as a treatment, a phenomenon termed nocebo hyperalgesia 1–3. Nocebo has been described as the negative counterpart to placebo. Nearly a century into the proliferation of placebo-controlled studies 4–6, research has shown that learned expectations regarding inert treatments may not only have positive placebo effects, but may also mimic negative treatment outcomes, such as medication side-effects 7–9. Nocebo responses may thus produce deleterious effects on a variety of symptoms, as a result of learning mechanisms that are not yet fully understood. For example, it remains unclear how negative expectations on a cognitive level influence pain processing in the brain, or what the involvement of relevant emotions may be. Due to a known involvement of learning in the experience of pain 1,10–12, it is important to study and better understand the biobehavioral mechanisms that underly nocebo hyperalgesia. In this general introduction, first, nocebo hyperalgesia will be framed as a multifaceted phenomenon that can be part of the intricate mechanisms of pain processing. Cognitive-emotional pain processing is described in the context of learned nocebo responses and as complimentary to sensory-discriminatory nociceptive processing. The state of the art in experimental nocebo research and the relevance of experimental learning mechanisms are then outlined. Experimental models that are typically implemented to investigate the cognitive and emotional aspects of nocebo hyperalgesia are described. Cognitive processes such as learning, as well as emotional processes such as fear, are then presented as major putative underlying factors in nocebo hyperalgesia, as indicated by experimental findings. Biobehavioral underpinnings of nocebo effects are then discussed in relation to current neurobiological literature; gaps in knowledge are highlighted. Finally, an outline of the current dissertation is presented.

Chapter 1 – General introduction 11 Nocebo hyperalgesia and its involvement in pain Nocebo hyperalgesia seems to be an intricate component of pain processing. Due to the multifaceted, subjective, and often unpredictable nature of pain, recovery from pain and chronic conditions are especially difficult to manage 13. Pain has been described to arise in response to a nociceptive signal from the body, but this is not always a direct path. Rather, the experience of pain is heavily influenced by an array of processes within the nervous system 14. These intricate processes do not merely rely on information regarding the nature of the nociceptive stimulus, but may include pain processing based on past experiences or cognitive-emotional factors such as fear 13. We start by outlining the basic mechanisms of pain processing, in order to examine how these may be influenced by cognitive and emotional elements under nocebo hyperalgesic conditions. Due to the multifactorial nature of pain, processing of nociceptive input involves a large, distributed neural network that is not yet fully understood 15. Pain pathways have been extensively investigated via neuroimaging methods such as functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) and are thought to encompass numerous brain regions and processes. One neural system that has been implicated in pain processing relays sensory-discriminatory functions that are mainly involving nociceptive stimulus information. A second, more distributed system, is thought to be involved in cognitiveevaluative processes 16,17 and these could relate to learning and behavioral underlying factors. It should be noted that these networks and systems are not clearly defined, and that the literature contains inconsistencies regarding the exact brain areas that may be included in these systems 17. Nevertheless, sensory-discriminatory processes and cognitive-evaluative processes have been extensively researched and are often found to play important roles in pain perception 16–18 and in nocebo hyperalgesia 19–21.

12 Further insights come from biobehavioral studies illustrating that the experience of pain arises from a combination of bottom-up processes (for example, the type and intensity of nociceptive stimulation) 22–27 and top-down processing (for example nocebo-related processes such as learning or emotional modulation of incoming pain signals) 23,26,28–31. As one potential product of this interplay between sensory perception, cognition, and emotion, nocebo hyperalgesia is a complex phenomenon, and sophisticated experimental methods are required to understand its influence on pain. Experimental learning mechanisms Research typically induces nocebo effects by use of experimental learning models. Experimental models refer to simulations of conditions or responses to treatment that resemble clinical conditions and are induced in healthy individuals, artificially, in a laboratory. In nocebo research, learning manipulations consistently induce nocebo hyperalgesic responses. Typically, conditions in which a sham treatment is associated with pain aggravation are created by use of well-established learning techniques such as classical conditioning or providing negative verbal or written information. Experimentally induced nocebo hyperalgesia in healthy subjects enables researchers to examine these effects, in order to disentangle the mechanisms by which learning can affect pain sensitivity. In the most robust experimental models of nocebo hyperalgesia, classical conditioning forms and reinforces pain expectations through associative learning 11,32–34. In conditioning models of nocebo hyperalgesia, an association between a high-intensity pain stimulus (unconditioned stimulus, UCS) and a nocebo (inert treatment) conditioned stimulus (CS) is formed by repeatedly pairing the two stimuli. After repeated trials, an association between the nocebo

Chapter 1 – General introduction 13 stimulus and the worsening of pain is formed and the nocebo stimulus can evoke changes in perceived pain (conditioned response, CR), similar to the previous pain stimulus (Figure 1) 35,36. The powerful associative learning mechanisms employed by classical conditioning serve to recreate learned hyperalgesic responses to a specific stimulus. Classical conditioning thus attempts to recreate a putative clinical context in which not only physical injury but also memories of previous experiences and expectations about the future could have a strong impact on pain symptoms. Verbally delivered negative information can also alter pain expectations through instructional learning. Negative suggestions typically involve explaining the pain-enhancing effect of a (sham) treatment. Suggestions are used to induce nocebo hyperalgesia by themselves or to enhance the effectiveness of nocebo conditioning 9,37,38. The combination of conditioning and verbal suggestions is found to create the strongest experimental model of nocebo hyperalgesia 7,39. This points towards a complex interplay of diverse learning processes that may underlie nocebo effects. Experimental models are thus suited for examining pain aggravation under nocebo hyperalgesic conditions, but they can also shed light on pain chronification due to nocebo. Experimental attenuation models of conditioned effects have shown that learning may be involved in the persistence of pain over time. Paradigms that employ extinction or counterconditioning methods may provide valuable insights into the factors that contribute to nocebo responses 40–43. In a typical extinction paradigm, associations between the UCS and CS are discontinued, and learned effects would be expected to become extinct over multiple trials that are no longer negatively reinforced 7,39. This type of behavioral paradigm represents that, even classical conditioning has been discontinued, increased pain sensitivity can persist in response to a learned nocebo stimulus, over a prolonged period of extinction 42,44. The

14 clinical implications of negative pain effects that are resistant to extinction or other forms of nocebo attenuation remain unclear 1. Cognitive-emotional factors Experimental nocebo models that include conditioning and negative suggestions also enable the study of diverse cognitive and emotional factors that may be involved in nocebo hyperalgesia. Cognitive manipulations, such as varying the type of learning, can shed a light on the intricate processes that give rise to the experience of pain. Factors that contribute to the formation or the persistence of nocebo effects can be studied using manipulations within experimental models. For instance, studies have shown that consistent and repetitive learning methods, such as classical conditioning with continuous reinforcement of an association, induces the strongest nocebo responses 42. However, interrupted or inconsistent learning is still able to induce nocebo hyperalgesia to some extent 42,45, which may be important from a clinical perspective, where pain and learning may be less consistent than in experimental settings. Partially reinforced learning has been shown to slow down the extinction or minimization of learned effects 45, including nocebo effects 46. Learning as an underlying cognitive factor contributing to the formation of nocebo effects may also be investigated at a neurobiological level. Brain plasticity has long been shown to be non-unitary 47–50, with multiple mechanisms and processes being involved in different types of memorization, learning, as well as recall 48,51,52. Given this multifaceted nature of learning, precise manipulations, targeting associative aspects of learning, hold the potential of enriching our understanding of hyperalgesic pain responses. For example, structures such as the NMethyl-D-aspartate (NMDA) receptor and neurochemicals such as glutamate have been consistently implicated in a broad array of brain

Chapter 1 – General introduction 15 plasticity processes, including learning by association 53–54. Pharmacological manipulation of these receptors has been proven fruitful for enhancing learning during exposure therapy 53–55 and may also prove useful in the examination of the precise neurocognitive mechanisms, such as specific receptors and localized learning processes, that may facilitate nocebo hyperalgesia. Emotional underlying factors, such as fear of pain symptoms, may additionally tie into the processing of pain based on prior experiences and expectations. Through the common theme of learning, nocebo hyperalgesia bares some similarities to the formation of phobias 56–58. Nocebo and phobic responses may both be characterized by a key involvement of cognitive-affective components such as aversive learning and fear of a stimulus 12,53,59. Indeed, neural correlates of nocebo hyperalgesia show some involvement of fear processing. This is especially evident by the consistent involvement of the amygdala in both nocebo responses 19,20,60 and fear responses 61–64. Similarly to nocebo conditioning, pain-related fear can be acquired through associative learning 65–68. Pain-related fear may thus be relevant to nocebo effects because it may arise in experimental models as a result of experienced pain or as a result of threatening information regarding upcoming pain. Fear experienced during nocebo conditioning may thus potentially augment the acquisition of negative expectations, but experimental studies on this are lacking so far. This makes fear an especially important factor to study in relation to nocebo hyperalgesia.

16 Figure 1. A representation of typical experimental procedures for delivering classical conditioning and negative suggestions. At baseline before any intervention, an inert treatment has no effect. In the case of a conditioning paradigm this becomes a conditioning stimulus, CS, but in verbal suggestion paradigms no conditioning takes place and suggestions can be delivered once, verbally or in writing. A nocebo treatment is here represented as an inert pill. During a learning paradigm, the negative association between the nocebo treatment and pain aggravation (unconditioned stimulus, UCS) is experienced by the subject (unconditioned response, UCR). If learning a negative association comes in form of a verbal suggestion, continuous learning is not required, and one suggestion can in principle suffice. Thereafter, a nocebo effect on pain (conditioned response, CR in the case of classical conditioning) is formed, as a result of learned negative expectations regarding the nocebo treatment. Biobehavioral underpinnings In recent years, fundamental research has focused on unravelling how nocebo responses are formed and how they may integrate in pain processing. In order to reach a better understanding of the neurocognitive components of nocebo effects, it is imperative to build on previous nocebo research, by utilizing consistent experimental models. In this way, results are comparable, and the reliability of findings can be tested over multiple studies. Theories and findings from the nocebo literature should also be connected to what is currently known about overlapping cognitive and emotional processes. Comprehensive reviews of the current state of research into the neural correlates of

Chapter 1 – General introduction 17 nocebo hyperalgesia can thus be very valuable, next to neurobiological studies that build upon this accumulation of knowledge. Research to date has highlighted some key areas that may be involved in nocebo hyperalgesia. Pain-specific processing has been implicated in the presentation of nocebo hyperalgesic responses. For instance, the dorsal horn of the spinal cord, the secondary somatosensory cortex, and the dorsolateral prefrontal cortex (dlPFC) all seem to be activated during nocebo responses 11,15. This may indicate that pain reports under nocebo hyperalgesic conditions closely resemble typical pain processing. Concurrently, nocebo responses have also been shown to involve brain areas such as the anterior cingulate cortex (ACC), amygdala, and hippocampus 11,69, which supports an involvement of cognitiveemotional factors in nocebo effects, which are also involved in pain processing and integration. Nocebo hyperalgesia also seems to involve chemical systems, such as prostaglandins, cortisol, and dopamine 69,70, while electrophysiological correlates point towards an involvement of alpha and gamma brain rhythms 71–73. It is thus evident, albeit not surprising, that nocebo hyperalgesia largely overlaps with pain processing. The involvement of a wide network of cognitive-emotional processing is also supported by neurophysiological findings. However, replicating findings from one nocebo study to the next seems challenging due to the implementation of diverse experimental models and distinct learning mechanisms between different studies. Employing diverse methods helps clarify specific aspects of nocebo hyperalgesia but also leads to inconsistencies and gaps in the literature, rendering nocebo hyperalgesia a phenomenon that is still poorly understood on a neurocognitive level. With learning and the formation of expectations being at the heart of the most robust nocebo theories 1,11,72,74,75 it is important to observe and interpret similarities between these cognitive processes and nocebo effects. Limbic system structures, and especially the amygdala, are

18 known to play a crucial role in learning and memory formation 76–79. More specifically, the amygdala and hippocampus, structures sometimes implicated in nocebo effects 20,21, play essential roles in the formation of new memories based on past experiences 80–82. At the same time, the consistent involvement of the ACC in nocebo effects may be drawing together prior memories, expectations, and information processing. The ACC is an area that is largely interconnected to the limbic system and may play a key role in cognitive control and conflict monitoring 83,84. These neuroscientific similarities may indicate that nocebo hyperalgesia involves a complex cognitive network that is responsible both for memory formation and for the recall and integration of learned expectations and incoming sensory information. Brain plasticity, learning, and cognitive-emotional factors, seem to form an interconnected, cortical-subcortical network that may be involved in nocebo hyperalgesia and in pain processing 21,60,62,85 (Figure 2). Yet, inconsistencies and a lack of replicability between nocebo studies do not permit for firm conclusions to be drawn. Cognitive and emotional components, such as learning and fear, seem have a critical role in the formation of nocebo hyperalgesia. Nevertheless, there is uncertainty regarding the specific learning mechanisms that may be involved, how they affect brain plasticity, and how learning networks may integrate with pain processing. This is evident in the large disparity in neurophysiological findings of fMRI and EEG studies. Inconsistencies in the literature also lead to uncertainty regarding the impact of fear, as compared to related cognitive-emotional responses such as anxiety. It is important for systematic experimental research to actively manipulate states of brain plasticity, learning, memory, and other cognitive processes. While a comprehensive overview of the state-ofthe-art in nocebo research is valuable, pharmacological manipulations and other methods that directly manipulate cognitive states may be central components in the strive to unravel the specific neurocognitive mechanisms of nocebo hyperalgesia.

Chapter 1 – General introduction 19 Figure 2. The working theoretical model of this dissertation. In the process of pain perception, incoming information to the Peripheral Nervous System (PNS) regarding a pain stimulus (bottom-up information), in combination with negative experiences, are influenced by nocebo-related cognitive-emotional factors (top-down information). Based on these factors of interest, integrative processing of pain takes place in the Central Nervous System (CNS), with the potential of giving rise to negative pain outcomes related to learned nocebo responses. The current dissertation In this dissertation, we address biobehavioral aspects of nocebo hyperalgesia, using neuroimaging as well as behavioral science methods. First, a systematic review and meta-analysis provides a novel examination of nocebo effect sizes and relevant factors in experimental studies. Thereafter, a literature review aims to comprehensively summarize what is currently known about the neurobiological correlates of nocebo hyperalgesia. Subsequently, a series of experimental studies attempt to directly manipulate and study cognitive and emotional factors involved in nocebo, by use of innovative methods. Fear and pharmacological manipulations of specific learning mechanisms are utilized for the first time in these studies, while original 3DLQ VWLPXODWLRQ LQFRPLQJ WR 316 1HJDWLYH H[SHULHQFHV DVVRFLDWHG WR SDLQ )HDU ([SHFWDWLRQV %HOLHIV 3DLQ FRJQLWLYH SURFHVVLQJ LQWHJUDWLRQ LQ &16 3DLQ RXWFRPH H[SHULHQFHG SDLQ ,QFRPLQJ LQIRUPDWLRQ &RJQLWLYH HPRWLRQDO FRPSRQHQWV %LREHKDYLRUDO LQWHJUDWLRQ 3DLQ SHUFHSWLRQ &KDSWHUV &KDSWHUV &KDSWHUV &KDSWHUV /HDUQLQJ

20 electrophysiological methods reveal biomarkers of nocebo effects. The aim of this dissertation was to further the knowledge on the neurochemical, electrophysiological, and cognitive-emotional processes that underlie nocebo hyperalgesia, with a specific focus on cognition, emotion, and brain plasticity. In Chapter 2 we explore the state of the art in behavioral nocebo research with a systematic review and meta-analysis of nocebo literature on somatosensory sensations, including pain. We aim to address the efficacy of different experimental learning methods for the induction of nocebo effects. We systematically summarize results from dozens of studies that investigated these effects, and we discuss the implications of their findings. This meta-analysis showed that across sensations, the magnitude of nocebo responses is affected by the type of learning, with classical conditioning being more potent than verbal suggestions alone. We discuss the lack of explanatory or moderation factors found in the literature. Our analysis served to illuminate the extent to which learning processes induce nocebo effects on different sensations and what the practical and theoretical implications of a lack of moderating factors identified may be for research and clinical practice. In Chapter 3, we review the neurobiological literature on nocebo hyperalgesia. This comprehensive review article summarizes neurobiological findings from studies that utilized (f)MRI, EEG, magnetoencephalography (MEG), as well as pharmacological and biochemical measures. The review provides a comprehensive overview and serves to highlight consistent neural correlates of nocebo hyperalgesia across a variety of different experimental nocebo models. In this way, this overview aims to provide an up-to-date picture of the biobehavioral correlates of nocebo effects. We outline the evidence from this field and give an overview of similarities and differences between nocebo research and learning/memory research.

Chapter 1 – General introduction 21 Building on previous nocebo studies as described in the first chapters of this dissertation, Chapter 4 presents a randomized controlled trial investigating distinct learning schedules. This experiment aimed to examine the role of continuous versus partial learning and the consequences of such learning schedules for the persistence of nocebo hyperalgesia. For this purpose, both the induction and the attenuation of nocebo hyperalgesia are experimentally manipulated via distinct learning methods. Healthy participants were randomized to receive conditioning on nocebo effects with continuous reinforcement, partial reinforcement, or sham conditioning. In attenuation, counterconditioning (i.e., positive conditioning of the nocebo conditioned stimulus) was compared to extinction for the attenuation of nocebo hyperalgesia. This study provided important insights into the effect that different learning schedules may have on the acquisition and attenuation of nocebo responses. In Chapter 5 an experimental study of cognitive-emotional processes is presented. Here, we aimed to investigate how fear can augment nocebo responses and how this may affect the persistence of these responses over time. We experimentally manipulated fear of pain during the induction of nocebo responses. We used two distinct fear inductions. One fear induction method was to manipulate pain levels with the aim of inducing fear of the high pain stimulations. The other fear induction included a threat manipulation that convinced participants that their skin is critically sensitive to pain. This study provided insights into the role of specific types of fear in the acquisition and extinction of nocebo hyperalgesia, which may be of important relevance given the relationship between fear and pain in clinical practice. Chapter 6 presents an electrophysiological investigation into nocebo hyperalgesia. We aimed to explore alterations in EEG biomarkers during the anticipation, acquisition, and evocation of nocebo hyperalgesia. This thorough investigation served to unravel multiple electrophysiological

22 aspects of nocebo effects, thereby shedding light on novel aspects of brain processing under nocebo hyperalgesic conditions. We induced nocebo hyperalgesia by use of conditioning and negative suggestions and recorded EEGs before, during, and after nocebo acquisition and evocation. This EEG study enriched our understanding of the role that learning and nociceptive processing play in nocebo hyperalgesia. In Chapter 7 we present a pharmacological fMRI study that investigated neurochemical correlates of learning in nocebo hyperalgesia. In this randomized clinical trial, we aimed to pharmacologically manipulate NMDA receptors, known for mediating certain types of learning, such as associative learning involved in classical conditioning. fMRI methods allowed for the exploration of brain activations during nocebo acquisition and extinction. We used classical conditioning and negative suggestions to induce nocebo hyperalgesia in a group receiving a low dose of D-cycloserine (a known partial NMDA receptor agonist) and in a group receiving placebo. These manipulations and imaging methods served to explore how NMDA-dependent learning influences the formation of nocebo effects and several potentially relevant brain processes were identified. Chapter 8 is a general discussion relating to this dissertation. In this chapter the results of the conducted studies are summarized and connected to the aims of this PhD project. We then further discuss these aims in light of theoretical and practical implications.

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Chapter 2. Learned nocebo effects on cutaneous sensations: Meta-analysis of experimental behavioral findings. Submitted for publication as Thomaidou, MA, Blythe JS, Peerdeman KJ, van Laarhoven AIM, Van Schothorst MME, Veldhuijzen DS, Evers AWM. Learned nocebo effects on cutaneous sensations: A systematic review and meta-analysis of experimental behavioral findings.

30 Abstract In past decades, the field of nocebo research has focused on studying how sensory perception can be shaped by learning. Behavioral conditioning processes as well as mere verbal suggestions of a negative treatment outcome may aggravate pain and itch perception. Gaining a comprehensive view of the magnitude of nocebo effects and the factors that contribute to their formation will help steer nocebo research towards fruitful directions for better understanding complex sensory phenomena. We conducted a systematic review and meta-analysis of a total of 37 distinct experimental nocebo studies on healthy participants, with four separate meta-analyses for nocebo effects on pain or itch, induced with classical conditioning and verbal suggestion, or verbal suggestion alone. We conducted subgroup analyses and meta-regression on factors such as the type and intensity of sensory stimuli, and the length of learning paradigms. This meta-analysis showed that on average, effect sizes of nocebo effects were moderate to large (Hedges g between 0.26-0.71 for the four primary outcomes). The combination of conditioning and verbal suggestions yielded stronger nocebo responses on pain in particular. Subgroup analyses, including factors such as the type of sensory stimulation, did not explain the moderate heterogeneity in nocebo magnitudes between different studies. Risk of bias was generally low and was not related to nocebo magnitudes either. We discuss these results in relation to the role of conditioning as well as aversive learning, and we recommend more consistency in designing and reporting nocebo experiments.

Chapter 2 – Meta-analysis 31 Introduction Negative expectations regarding the effects of a treatment can result in the aggravation of cutaneous sensations such as pain and itch 1–3. Such learned responses can be induced experimentally, allowing for the study of processes by which nocebo effects lead to symptom amplification 4– 10. In experimental studies, nocebo responses are defined as a significant increase in a sensation after a nocebo treatment, relative to no-treatment or a control treatment. Negative expectations leading to such responses are typically induced through classical conditioning, verbal suggestions, or their combination 4,5,10–13. Classical conditioning induces nocebo effects by building implicit associations between an (inert) treatment and the worsening of sensations such as pain or itch 14–16. Verbal suggestions explicitly provide negative information regarding the pain- or itchincreasing effects of a treatment 7. Because nocebo studies employ diverse methods, to better understand their potential impact on nocebo outcomes these methodological features warrant a systematic investigation. Learning has consistently been shown to underlie induced nocebo effects 5–7,9,17, and verbal suggestions seem to induce stronger nocebo responses when combined with conditioning 18. The positive counterpart to nocebo, placebo effects, also appear to be stronger when induced through a combination of conditioning with verbal suggestions, compared to conditioning alone, both on pain 19 and itch (Bartels et al., 2014; Blythe et al., 2019). One meta-analysis included results from ten nocebo experiments published up to 2013 and reports that the overall magnitude of the nocebo effect was moderate to large and effects were generally larger when verbal suggestions were used in combination with conditioning 18. That early meta-analysis had a limited sample of studies

32 available, and an up-to-date review is needed to examine how different types of learning may induce nocebo effects of different magnitudes. At the same time, other variables, such as the type of sensation (i.e., pain or itch), stimulus modality (e.g., thermal, electrical), the intensity of pain or itch stimulations, and the length of learning in different behavioral paradigms, also require a systematic examination across studies. For example, in experimental nocebo research, some nocebo conditioning paradigms include as few as four associative learning trials (Blythe et al., 2021), while others employ much longer paradigms 6,8,21. A diverse set of cutaneous sensory induction methods are also used, such as thermal 17, electrical 6,20, or laser pain stimulations 22. Such methodological choices, often meant to target specific underlying processes in nocebo experiments, can potentially influence nocebo responding and thus merit further investigation. Given the recent growth of nocebo research, we conducted a systematic review and meta-analysis of experimental nocebo studies in healthy participants to provide novel insights into distinct contributions of methodological factors in the induction of nocebo responses. We focused on cutaneous sensations, aiming to examine nocebo responses induced with comparable sensory inductions externally on the skin. First, we examined nocebo magnitudes between pain and itch and based on the learning method used. Then, we conducted subgroup analyses and meta-regression to assess how the type and intensity of stimulations, the length of learning, the timing of measurement of nocebo magnitudes, and risk of bias in studies may impact nocebo magnitudes.

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