Accepted Manuscript. Learned Fear of Gastrointestinal Sensations in Healthy Adults

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1 Acceted Manuscrit Learned Fear of Gastrointestinal Sensations in Healthy Adults Erik Ceunen, Jonas Zaman, Nathalie Weltens, Ekaterina Sarafanova, Vicky Arijs, Johan W.S. Vlaeyen, Lukas Van Oudenhove, Ilse Van Diest PII: S (16) DOI: /j.cgh Reference: YJCGH To aear in: Clinical Gastroenterology and Heatology Acceted Date: 18 Aril 016 Please cite this article as: Ceunen E, Zaman J, Weltens N, Sarafanova E, Arijs V, Vlaeyen JWS, Van Oudenhove L, Van Diest I, Learned Fear of Gastrointestinal Sensations in Healthy Adults, Clinical Gastroenterology and Heatology (016), doi: /j.cgh This is a PDF file of an unedited manuscrit that has been acceted for ublication. As a service to our customers we are roviding this early version of the manuscrit. The manuscrit will undergo coyediting, tyesetting, and review of the resulting roof before it is ublished in its final form. Please note that during the roduction rocess errors may be discovered which could affect the content, and all legal disclaimers that aly to the journal ertain. All studies ublished in Clinical Gastroenterology and Heatology are embargoed until 3PM ET of the day they are ublished as corrected roofs on-line. Studies cannot be ublicized as acceted manuscrits or uncorrected roofs.

2 Title: Learned Fear of Gastrointestinal Sensations in Healthy Adults Ceunen, Erik a+ Zaman, Jonas a+ Weltens, Nathalie b Sarafanova, Ekaterina b Arijs, Vicky a Vlaeyen, Johan W.S. a,c Van Oudenhove, Lukas b Van Diest, Ilse a * Institutional Affiliations: a. Health Psychology, KU Leuven b. Laboratory for Brain-Gut Axis Studies (LABGAS), Translational Research Center for Gastrointestinal Disorders (TARGID), Deartment of Clinical & Exerimental Medicine, KU Leuven c. Clinical Psychological Science, Maastricht University, Netherlands + Shared first authorshi * Corresonding author: Ilse Van Diest Health Psychology KU Leuven Tiensestraat 10, box Leuven (BELGIUM) T Ilse.VanDiest@kuleuven.be Grant Suort: 1

3 EC was suorted by the grant for "the Psychology of Pain and Disability Research Program" funded by the Fund for Scientific Research Flanders awarded to JWSV. LVO is assistant research rofessor of the KU Leuven Secial Research Fund. JZ was suorted by a KU Leuven grant (PF/10/005) to the Centre for Excellence of Generalization Research in Ill Health and Psychoathology awarded to IVD and JWSV, and by an FWO research grant (G ) awarded to IVD. Abbreviations: CS = Conditioned Stimulus EMG = Electromyogram FGID = Functional Gastrointestinal Disorder IBS = Irritable Bowel Syndrome ISI = Inter-Stimulus Interval ISI ost-cs = Inter-Stimulus Interval occurring after the CS ISI re-cs = Inter-Stimulus Interval occurring rior to onset of the CS. US = Unconditioned Stimulus Disclosures: The authors have nothing to disclose that may be relevant to the manuscrit. Author Contributions: Study concet and design: EC, JV, LVO, IVD; acquisition of data: EC, ES, NW; data rearation: VA, EC, IVD; statistical analysis: JZ; drafting of the manuscrit: EC, JZ, IVD; revision of the manuscrit and accetation of the final draft: EC, JZ, ES, VA, NW, JV, LVO, IVD; material suort: NW.

4 Abstract: Background & Aims: Gastrointestinal symtom-secific fear and anxiety are imortant determinants of gastrointestinal symtom ercetion. We studied learning of fear toward innocuous gastrointestinal sensations as a utative mechanism in the develoment of gastrointestinal symtom-secific fear and anxiety. Methods: Fifty-two healthy subjects (6 women) received tyes of esohageal balloon distention at a ercetible but non-ainful intensity (conditioned stimulus [CS], the innocuous sensation) and at a ainful intensity (unconditioned stimulus [US]). Subjects were randomly assigned to 1 of grous. During the learning hase, the innocuous CS receded the ainful US in the exerimental grou (n=6). In the control grou (n=6), on the contrary, the US never followed the CS directly. During a subsequent extinction hase, both grous received only CS distention the ainful US was no longer administered. Indexes of fear learning towards the innocuous CS distention included the skin conductance resonse, fear-otentiated startle (measured by the eye blink electromyogram), and self-reorted exectancy of the US. Results: During the learning hase, only the exerimental grou learned to fear the innocuous gastrointestinal CS, based on the increase in US exectancy (comared to the control grou, P=.04), increased skin conductance resonse (comared to the control grou, P=.03), and otentiated startle reflex (comared to the control grou, P=.001) in resonse to the CS. The differences between exerimental and control grous in US exectancy and skin conductance, but not fear-otentiated startle, disaeared during the extinction hase. Conclusions: Fear towards innocuous gastrointestinal sensations can be established through associative learning in healthy humans. This may be an imortant mechanism in the develoment of fear of gastrointestinal symtoms, imlicated in the athohysiology of functional gastrointestinal disorders. KEY WORDS: functional gastro-intestinal disorders; visceral ain; interocetive conditioning; gastrointestinal symtom-secific fear Introduction Visceral ain is one of the rimary causes for seeking medical attention and the most common form of ain resulting from disease 1. Gastrointestinal symtoms, including visceral ain, occur often also in the absence of any detectable hysiological abnormalities, as is the case in functional gastrointestinal disorders (FGID). Stress-related affective and cognitive sychobiological rocesses lay an imortant role in the athohysiology of FGID through the brain-gut axis, the bidirectional neurohumoral communication system between the central nervous system and the gastrointestinal tract,3. Gastrointestinal symtom-secific fear, the arehension of secific visceral sensations, is one of the most imortant cognitive-affective rocesses in this 3

5 context 4,5 as it is associated with symtom severity and quality of life in FGID, secifically in irritable bowel syndrome (IBS) 6. Furthermore, decreases in gastrointestinal symtom-secific fear mediates the effect of exosure-based cognitive behavioral theray on IBS symtoms 7,8. How gastrointestinal symtom-secific fear develos remains unclear, but it is often assumed that fear learning lays a key role. More secifically, originally benign visceral sensations may become associated with unleasant or ainful visceral sensations. For examle, benign, non-ainful eigastric sensations may recede an eisode of stomach ache. As a consequence of this temoral contingency, the individual eventually exeriences these benign sensations as unleasant and may come to fear them, whereas before the same sensations were exerienced as relatively neutral. This natural learning rocess is a case of Pavlovian aversive conditioning in which a relatively neutral stimulus becomes a conditioned stimulus (CS) redicting the inherently unleasant unconditioned stimulus (US). When both the benign CS and the ainful US are exerienced at the same anatomical location as in the examle above, this is referred to as homoreflexive conditioning. When either the CS or US, or both, are erceived as informative about the internal state of the body, i.e. interocetive, this is referred to as interocetive conditioning 9. Homoreflexive interocetive fear conditioning is an interesting candidate mechanism in the develoment and maintenance of gastrointestinal symtom-secific fear, but to the best of our knowledge, this has not been directly studied The urose of the current study was therefore to study fear learning towards innocuous visceral sensations as a otential mechanism in the develoment of gastrointestinal symtomsecific fear. In order to address this void in the current knowledge, we set u a study with a ainful esohageal stimulus as US, and a detectable, non-ainful esohageal stimulus as CS. We exected fear learning to the CS to occur when the CS immediately recedes the ainful US (exerimental grou), but not when the CS and US are searated by a relatively long time interval (control grou). Materials and Methods Subjects Fifty-two healthy articiants (6 women) were recruited via advertisements on social media. Interested individuals received an informed consent in line with the declaration of Helsinki rior to deciding whether or not to articiate (more details in Sulementary 4

6 Material). Particiants were randomly assigned to the exerimental or the control grou (see later). Both grous were matched for age and gender. Esohageal Stimulation Both the CS and US consisted of mechanical stimulation of the distal, autonomously innervated art of the esohagus 13. The CS and the US lasted 5 and seconds, resectively. The intensity of stimulation was individually determined for both CS and US using a variation of the ascending methods of limits, with the CS being ercetible but non-ainful stimulation of the esohagus, and the US being ainful but still bearable stimulation at the same anatomical site (more details in Sulementary Material). A ediatric catheter (used for gavage) with a diameter of 3mm (TR-008, Pennine ) was inserted via the nose into the distal esohagus, 35 cm from the nostril. A deflated custom made, silicon medical balloon (diameter: 5mm, length: 5mm, Medasil ) was firmly attached to the end of the catheter ositioned in the esohagus (more details in Sulementary Material). Subjective exectancy of US onset Throughout the study, articiants osed their dominant hand on a custom-built dial 14,15, continuously rating the extent to which they exected the US in the following seconds. The scale of the dial ranged from 0 to 100. A score in the middle (50) meant the articiant totally did not know whether or not to exect the US. The more certain they were that the US would not come, the more articiants turned the dial below 50 and towards zero. The more certain they were to exect the US, the more they turned the dial from 50 uwards to 100. More details are rovided in Sulementary Material. Psychohysiological measures Eyeblink startle EMG The startle eyeblink reflex is a brief increase in activation of the muscle surrounding the eye, which can be elicited using a sudden burst of sound. The magnitude of the elicited muscle activation can be used as a measure of activation subcortical fear circuits 16. In fear conditioning studies, increased startle magnitudes during the CS relative to magnitudes during the absence of the CS are thought to reflect motor rearation (an asect of fear) in resonse to the CS (more details in Sulementary Material). Galvanic Skin Resonse 5

7 The skin conductance resonse is a measure of changes in electrodermal activity. These changes occur in resonse to activation of sweat glands. Sweat gland activity increases as a function of increase in emotional (and/or symathetic) arousal, with more exciting stimuli increasing skin conductance resonses 17. This measure was included based on the reasoning that an increase in skin conductance resonse would occur when the CS gains emotional significance, because articiants have learned it will be followed shortly by the ainful US (more details in Sulementary Material). Study Design The exeriment consisted of three hases: (a) a baseline hase (4 trials), (b) a learning hase (16 trials), and (c) an extinction hase (16 trials). During the baseline and extinction hases, both grous were treated identically and received one innocuous CS distention in every trial, and no ainful US distentions. During the learning hase, both grous received one innocuous CS in every trial and in addition one ainful US in 75% of the trials (the 3 rd, 8 th, 11 th, and 15 th trial of the learning hase had no US). Such artial (75%) reinforcement of the CS with the US during the learning hase is known to strengthen conditioning 18. In addition, it may better reflect clinical reality comared to a 100% reinforcement scheme, as also in atients, not every innocuous abdominal sensation is always followed by a ainful sensation. For the exerimental grou, the CS was followed almost immediately (with a s delay) by the US. The control grou had an interval of 6 seconds between the CS and the US onset (see Figure 1). In essence, in the exerimental grou the CS announces the imminence of the ainful US, whereas in the control grou it announces an imminent 'safe' and ain-free eriod. Every trial lasted 48 seconds, irresective of hase. The innocuous CS distention was administered always from the 15 th u to the 0 th second after trial onset. Acoustic startle robes occurred at the 19th second (during the CS) and the 43rd second (during the ost-cs inter stimulus interval, ISI ostcs ) of each trial (see Figure 1). More details are rovided in Sulementary Material. --- Please insert fig. 1 around here ---- Resonse definition and statistical analysis 6

8 As US-exectancy was measured continuously, data were reduced by selecting 5 time oints of interest for each trial: the 7 th second (rior to the CS, in the middle of ISI re-cs a ), 0 th second (during the CS), 4 th second (end of US for the exerimental grou / beginning of ISI ost-cs for the control grou), 33 rd second (middle of ISI ost-cs b ) and the 45 th second (end of trial; i.e., right before US onset for the control grou). Galvanic skin resonses were calculated by subtracting the mean skin conductance level during baseline (s before the CS onset) from the maximum value in the window 0-7 s following CS onset (more details in Sulementary Material). Eyeblink startle (EMG) resonses were calculated by taking the difference between the eak value in the ms time window and the mean value from the 0-0 ms time window following robe onset (more details in Sulementary Material). The learning and extinction hases were subdivided into an early and late block comrising 8 trials each. Hyotheses were tested with lanned comarisons in reeated measure ANOVAs with Block (baseline, early learning, late learning, early extinction, and late extinction) as a within-subject factor and Grou (exerimental, control) as a betweensubject factor. For US-exectancy, an additional within-subject factor Time was included (7 th, 0 th, 4 th, 33 rd, 45 th second) with the 7 th second (i.e., trial onset, rior to the CS) as reference. For startle EMG, a within-subject factor Stimulus (CS, ISI ost-cs ) was included. Greenhouse- Geisser corrections were alied where aroriate. Uncorrected degrees of freedom and corrected s are reorted together with η (as a measure of effect size) and ε (as a measure of shericity). All contrasts were tested two-tailed. Alha was set at.05. All statistical analyses were erformed using SPSS 0. To test the main hyothesis that fear learning to the CS would occur and extinguish again in the exerimental relative to the control grou, we tested for each measure secific lanned contrasts. We exected no grou differences to occur during the baseline and the last extinction block. During the late learning block, we exected the exerimental grou to have higher skin conductance resonses (GSRs) to the CS comared to the control grou in the learning hase, because the CS announced US-imminence only in the exerimental grou. In a similar vein, we exected that articiants from the exerimental grou would increase their US-exectancy during the CS (second 0 relative to second 7) to a greater extent than the control grou in the late learning block. For the control grou, the US was imminent towards the end of the ISI ostcs during the learning hase; therefore, we exected that only articiants a ISI re-cs = Inter Stimulus Interval from trial onset till onset of the CS. b ISI ost-cs = Inter Stimulus Interval from CS offset till US onset in the control grou. 7

9 from the control grou would have higher US-exectancies at second 45 relative to second 7 during the late learning block. For startle EMG, we exected that startle otentiation during the CS (startle eyeblink resonse magnitude during the CS relative to during the ISI) would increase from baseline to late acquisition in the exerimental grou only. The result section reorts on the lanned contrasts. Findings on the omnibus tests in the reeated measure ANOVAs can be found in Sulementary Materials. Results US-exectancy Baseline hase As exected, grous did not differ in their increase in US-exectancy during the CS (second 0 relative to second 7) or near the end of a trial (second 45 relative to second 7), F(1,50) =.31, =.58 and F(1,50) =.05, =.83, resectively. Learning hase As exected, both grous differed in their change in US exectancy during the CS during the late learning hase, F(1,50) = 4.54, =.038, η =.08: the exerimental grou had a greater increase in US-exectancy during the CS (second 0 relative to second 7) comared the control grou (see Figure ). In addition, the increase in US-exectancy from rior to the CS (second 7) towards the end of the trial (second 45) was greater for the control than for the exerimental grou, F(1,50) = 6.48, =.014, η =.1 (see Figure ). Extinction hase During the late extinction block, grou differences in US-exectancy during the CS (second 0 relative to second 7) and near the end of a trial (second 45 relative to second 7) were no longer significant, F(1,50) =.54, =.47 and F(1,50) =.0, =.87, resectively. --- Please insert Fig. around here --- Galvanic Skin Resonse Baseline hase As exected, no grou differences in skin conductance resonses to the CS were observed during baseline F(1, 50) =.5, =.6 (see Figure 3). 8

10 Learning hase As exected, the CS elicited significantly stronger skin conductance resonses in the exerimental grou comared to the control grou during the late learning hase, F(1,50) = 5.7, =.01, η =.1 (see Figure 3). Extinction hase As exected, there were no grou differences during the late extinction hase, in skin conductance resonses to the CS, F(1,50) = 1.98, =.17 (see Figure 3). Startle eye blink EMG Baseline hase -- Please insert fig. 3 around here -- During the baseline hase, no grou differences were observed in startle amlitudes to the CS relative to startle amlitudes during the ISI ost-cs, F(1, 43) = 1.4, =.7 (see Figure 4A & 4B). Learning hase During the late learning block, a significant grou difference in the startle magnitude during the CS relative to the ISI ost-cs was found, F(1,43) = 13.37, =.001, η =.4, with higher CS amlitudes comared to ISI amlitudes in the aired grou and the oosite attern in the unaired grou (see Figure 4A & 4B). Extinction hase Oosite to our hyothesis, there still was a significant grou difference in startle amlitudes during the CS relative to the ISI ost-cs during late extinction F(1,43) = 5.8, =.00, η =.1, with higher CS amlitudes comared to ISI amlitudes in the aired grou and the oosite in the unaired grou. (see Figure 4A & 4B). Discussion --- Please insert Fig. 4 around here --- 9

11 The current study sought to investigate for the first time whether fear towards innocuous gastrointestinal sensations can develo by means of associative learning between consecutive gastrointestinal events in healthy humans. To this end, a novel homoreflexive interocetive conditioning aradigm was develoed with exerimentally induced visceral sensations at the level of the distal esohagus, as CS and US. The general aim of the current study was to assess whether associative fear learning towards innocuous gastrointestinal sensation can be established, as such learning rocesses are considered central in the generation and maintenance of gastrointestinal symtom-secific fear, a key factor in the athohysiology of FGID. Princial findings We hyothesized that fear learning to the innocuous non-ainful visceral CS would be established by means of associative learning between interocetive events, which should be reflected in increases in subjective anticiation of the US during the CS, increased skin conductance resonses to the CS and fear otentiated startle resonses in the resence of the CS relative to the absence of the CS. We also hyothesized that these learned fear resonses would disaear again in the extinction hase, when the innocuous CS was no longer followed by the US. Associative fear learning. Particiants assigned to the exerimental grou learned to fear the innocuous gastrointestinal sensation (CS) during the learning hase, because for them it signaled the imminence of a ainful gastrointestinal sensation (US). Such fear learning to the CS was absent in the control grou for whom the innocuous sensation (CS) signaled a relatively safe eriod without ain. Imortantly, fear learning to the innocuous sensation (CS) was established at different levels of learning and for all outcomes. The effects on USexectancy indicate that in the late block of the learning hase, articiants from the exerimental grou had to some extent acquired exlicit knowledge of the CS-US contingency. In addition, the relatively increased skin conductance resonses to the CS in the exerimental comared to the control grou rovides evidence that the innocuous CS had gained emotional significance for articiants from the exerimental grou. Finally, we found also fear learning effects in startle eyeblink EMG, which is generally acceted to be an index of covert, subcortical activation of fear circuits 16. Together, these results convincingly demonstrate that fear for innocuous gastrointestinal sensations can arise from temoral contingencies between gastrointestinal events, giving rise to associative learning. This in turn can be linked to earlier hyotheses on the generation of gastrointestinal symtom-secific 10

12 fear, which attribute an imortant role to associative learning rocesses by which initially relatively 'neutral' bodily sensations start rovoking fear through activation of fear circuits in the brain Extinction of learned fear. Our hyothesis that fear would extinguish in the exerimental grou when the innocuous CS was no longer followed by the ainful US was only artially confirmed. Towards the end of extinction, both grous did no longer differ in the skin conductance resonses to or US-exectancies during the CS. Yet, the exerimental grou still resonded with a fear-otentiated startle to the CS, relative to the control grou. This is very much in line with earlier findings using resiratory sensations as CS and US in an interocetive associative learning aradigm 14. Whereas fear-conditioned changes in skin conductance resonses and in US exectancy rimarily reflect exlicit knowledge of the CS US contingency, startle otentiation is thought to more directly reflect subcortical, amygdaladeendent emotional learning that can dissociate from the former measures 19,0. Our findings suggest that extinction of unconscious, emotional learning to visceral sensations is articularly slow and rather difficult to establish, and may therefore require a more in-deth and rolonged extinction training. Clinical Imlications The resent findings on how fear towards innocuous gastrointestinal sensations can come about through an associative learning rocess is relevant for any gastrointestinal disorder but for FGID in articular, as many of those atients are characterized by excessive distress and fear towards certain tyes of gastrointestinal sensations 4. Recently, we have found that associative learning leading to gastrointestinal sensations does not only cause emotional distress, but also alters ercetual thresholds for those gastrointestinal sensations 13. Thus, gastrointestinal symtom-secific fear and visceral hyersensitivity may be closely related, likely because associative learning between gastrointestinal events is a key common mechanism underlying both henomena 11. Our findings further suort the value of exosure-based cognitive-behavioral treatment as an imortant treatment otion for FGID, articularly in atients with high levels of gastrointestinal symtom-secific fear. Previous research found exosure-based treatment to be effective in symtomatic imrovement of IBS, with its effects being mediated by reduction in gastrointestinal symtom-secific fear 8,10. In line with this, the resent study confirms that extinction learning as a rocess is not limited to external feared objects (e.g., 11

13 siders), but also alies to visceral sensations and can therefore considered the major active ingredient of successful interocetive exosure theraies. Interestingly, our findings from the extinction hase suggest that innocuous visceral sensations can activate subcortical emotional resonses, desite one is aware that the sensation will not be followed by or develos into a ainful sensation. Such dissociation between fear indices during extinction may have clinical relevance. For examle, even though a atient may have understood from the treating MD and clearly accets that a certain tye of gastrointestinal sensations is not harmful and does not reflect disease activity, a atient may still show fear resonses towards these innocuous gastrointestinal sensations, causing feelings of distress and otentially lowering the threshold to erceive the sensations. Therefore, indeth and rolonged exosure theray may be required to extinguish learned fear resonses to gastrointestinal sensations. Conclusion We can conclude from our study that innocuous gastrointestinal sensations can come to elicit fear once they have been associated to a ainful sensation that shares ercetual similarities to the innocuous sensation and has an identical anatomical origin (i.e. in this case, the gastrointestinal tract). The resent study demonstrated that it is ossible to form an association between an originally benign visceral sensation and an unleasant visceral sensation merely through the basic rocess of associative learning. Thus, the resent study established that classical conditioning is a viable mechanism to create gastrointestinal symtom-secific fear, which may in turn trigger the develoment of FGID and maintain or exacerbate symtoms. Furthermore, our findings suggest that a rolonged exosure theray may be necessary for an in-deth extinction of gastrointestinal symtom-secific fear. Figure legends Figure 1. Schematic illustration of trial structure during the learning hase. The conditioned stimulus (CS) was delivered from 15 to 0 seconds after trial onset, and receded and followed by an inter-stimulus interval (ISI), resectively labeled ISI re-cs and ISI ost-cs. An unconditioned stimulus (US) was delivered from to 4 seconds after trial onset for the exerimental grou, and from 46 to 48 seconds for the control grou. The sound symbols reresent acoustic startle robes, which were invariably administered at 19s and 43s 1

14 after trial onset. The black squares on the timelines are the oints in time which were included in the analysis of the subjective US-anticiation. 13

15 Figure. Mean US-anticiation ratings at the 7 th, 0 th, 4 th, 33 rd and 45 th second for the exerimental and control grou during the baseline hase, early learning hase (Early learn), late learning hase (Late learn), early extinction hase (Early ext) and late extinction hase (Late ext). On a scale, a rating of 50 reflects the oint of uncertainty, 100 reflects 100% certainty that the US is imminent, while 0 100% certainty that the US is not-imminent The light grey bars reresent the resentation of the CS, the darker grey bars reresent the resentation of the US. 14

16 Figure 3. Mean log transformed skin Conductance resonses of the exerimental and ccontrol grou during the baseline hase, early and late learning hase, early and late extinction hase. The error bars reresent the standard error. 15

17 Figure 4. Mean startle amlitudes (T-scores) for the (A) exerimental grou and the (B) control grou. The error bars reresent the standard error. See Results section for statistical details. CS = conditioned stimulus; ISI = Inter-stimulus interval (after CS); learn = learning hase; ext = extinction hase. 16

18 References 1. Cervero F, Laird JM. Visceral ain. Lancet. 1999;353(9170): doi: /s (99) Levy RL, Olden KW, Naliboff BD, et al. Psychosocial Asects of the Functional Gastrointestinal Disorders. Gastroenterology. 006;130(5): doi: /j.gastro Van Oudenhove L, Aziz Q. The role of sychosocial factors and sychiatric disorders in functional dysesia. Nat Rev Gastroenterol Heatol. 013;10(3): doi: /nrgastro Labus JS, Bolus R, Chang L, et al. The Visceral Sensitivity Index: develoment and validation of a gastrointestinal symtom-secific anxiety scale. Aliment Pharmacol Ther. 004;0(1): doi: /j x. 5. Labus JS, Mayer E a, Chang L, et al. The central role of gastrointestinal-secific anxiety in irritable bowel syndrome: further validation of the visceral sensitivity index. Psychosom Med. 007;69(1): doi: /psy.0b013e3180ef4. 6. Jerndal P, Ringström G, Agerforz P, et al. Gastrointestinal-secific anxiety: An imortant factor for severity of GI symtoms and quality of life in IBS. Neurogastroenterol Motil. 010;(6): doi: /j x. 7. Wolitzky-Taylor K, Craske MG, Labus JS, et al. Visceral sensitivity as a mediator of outcome in the treatment of irritable bowel syndrome. Behav Res Ther. 01;50(10): doi: /j.brat Ljótsson B, Andersson E, Lindfors P, et al. Prediction of symtomatic imrovement after exosure-based treatment for irritable bowel syndrome. BMC Gastroenterol. 013;13:160. doi: / x Razran G. The observable unconscious and the inferably conscious in current Soviet sychohysiology: Interocetive conditioning, semantic conditioning, and the orienting reflex. Psychol Rev. 1961;68(): Craske MG, Wolitzky-Taylor KB, Labus J, et al. A cognitive-behavioral treatment for irritable bowel syndrome using interocetive exosure to visceral sensations. Behav Res Ther. 011;49(6-7): doi: /j.brat Zaman J, Vlaeyen JWS, Van Oudenhove L, et al. Associative fear learning and ercetual discrimination: A ercetual athway in the develoment of chronic ain. Neurosci Biobehav Rev. 015;51: doi: /j.neubiorev Mayer E a. The neurobiology of stress and gastrointestinal disease. Gut. 000;47(6): doi: /gut

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24 Sulementary Material Methods Subjects The informed consent outlined the exerimental rocedure including stimuli to be delivered, guaranteed anonymity, and stated that articiation was voluntary (with a reimbursement of 50 Euros), and mentioned that articiation could be halted at any moment if the articiant so desired, without loss of the romised reimbursement. If still interested, articiants were required to indicate whether or not they had a history or resence of: (a) sychiatric conditions; (b) abdominal or thoracic surgery (excet aendectomy and cholecystomy); (c) neurological, endocrine, or digestive disorders, and/or (d) other medical disorders. Moreover they also had to indicate whether at the time of the exeriment they (e) were regnant, (f) had ain symtoms, (g) used medication affecting the function of the digestive tract and/or the nervous system, (h) had a recent accident of which they weren t fully recovered yet, and/or (i) had a serious hearing imairment. Anyone affirming any or several of these, was deemed unfit for articiation and was kindly thanked for their interest in articiating. Aroval for conducting the exeriment was obtained from the Medical Ethical Committee of the University of Leuven (reference number ML8570). Esohageal Stimulation To revent the catheter from moving due to eristaltic contractions of the esohagus that occur in resonse to balloon inflation, tae was used to gently attach the extraneous art of the catheter to the cheeks. The remainder of the catheter was draed over the ear and attached to an air filled syringe that was used for inflating the esohageal balloon. For this threshold determination, the volume of the balloon increased with 1 ml relative to each revious inflation. Between each 1 ml inflation, the balloon was deflated. Immediately after each inflation, subjects indicated whether they felt something, and rated what they felt on a scale from 0 to 10, with zero being no sensation at all, 1 indicating ossibly a sensation (not being entirely certain), indicating a sensation definitely being resent but not yet ainful, 8 being a clearly ainful but still tolerable sensation, and 10 being the maximally tolerable intensity of ain. Particiants were warned that intensity 10 would never be used, and that it was always ossible to reduce the volume if the subjective intensity was too high. During threshold determination, u to and including intensity 3, the balloon

25 was inflated for 5 seconds, equaling the duration of the CS to be used in the exeriment. Beyond this oint on the scale, the balloon was inflated for seconds only, which was the duration of the US to be used in the exeriment. The entire threshold determination rocedure was reeated a second time to make sure the thresholds were accurate. In case the second threshold determination yielded different results than the first, the thresholds obtained during the second determination were used as the first may be more rone to novelty effects. Subjective exectancy of US onset The osition of the dial in the scale was digitally registered at 10Hz and transmitted via a data acquisition card to a comuter throughout the entire exeriment. The recorded digital values give an indication of the subjective estimation of each articiant on how likely they felt they were to receive the US in the following seconds. As such, this dial can be used to assess whether articiants learned to make correct redictions of US onset. Psychohysiological Measures All signals described below were recorded using Affect 4.0 software 1 and transmitted via a 16-Bit PCI-61 data acquisition card (National Instruments, Austin, Texas) to a comuter, and treated offline with Psychohysiological Analysis software (PSPHA). Eyeblink startle EMG The startle was elicited and measured as based on the guidelines of Blumenthal et al. 3. A 50ms burst of white noise with a volume of 10dB was used as an acoustic startle robe. The raw EMG signal was amlified by a LabLinc v75-04 Coulbourn Isolated Bioamlifier with Bandass filter; the recording bandwidth was between 13Hz and 1 khz. This signal was transmitted to a LabLinc v76-4 Coulbourn 4 Channel Integrator, which rectified and smoothed the signal online with a time constant of 0ms. The EMG signal was digitized at 1kHz starting 500ms rior to onset of the acoustic robe, until 1000ms after robe onset. Galvanic Skin Resonse After cleaning the hyothenar side of the non-dominant hand with alcohol, two standard Ag/AgCl electrodes (diameter 1cm) filled with water-soluble KY*gel were attached here, saced aroximately.5cm aart. The galvanic skin resonse measured via these electrodes was transmitted to the LabLinc v71-3 Coulbourn Isolated Skin Conductance Couler, which maintained a constant voltage of 0.5V over the electrodes; the analog signal was digitized at 10Hz.

26 Study design Following the determination of the individualized thresholds for CS and US, electrodes for measuring startle and GSR were attached. Subjects were verbally informed what these electrodes would be used for, including information about the occurrence of acoustic startle robes throughout the exeriment. Following electrode attachment, the intended use of the dial was exlained to articiants, and after they indicated they had no more questions, earhones were mounted on their head. Throughout the entire exeriment, an exerimenter remained in the lab with the articiant in order to be able to administer the CS and US when required. Inflation and deflation of the esohageal balloon occurred outside the field of vision of the articiant for both CS and US, by means of a manually oerated air filled syringe. The exerimenter administering the CS and US was cued to do so via a monitor, which was also laced outside the field of vision of the articiant. On this monitor, a countdown occurred 5 seconds rior to inflation while indicating whether a CS or US had to be administered, and a second countdown occurred starting from onset of inflation, showing the remaining time till deflation. As the startle magnitude in resonse to the startle robe tends to be exaggerated uon initial resentation, and becomes more stable after reeated stimulation. Particiants were first exosed to twelve startle robes, all administered with a fixed interval of ten seconds immediately rior to the onset of the actual exeriment. After habituating to the robes, the articiant started using the US exectancy dial and continued doing so until the end of the exeriment. The dial was fixed in lace within arm s length in front of the articiant. Resonse definition and statistical analysis Startle eye blink EMG EMG signals were visually insected off-line to detect artifacts (e.g., excessive noise from muscular activity rior to the startle robe). Artifacts were rejected from analysis and defined as missing. The average ercentage of rejected resonses er articiant was 8% (SD 6%). If resonses to the robe were not visible, resonses were classified as a non-resonse and set at zero. Five articiants were excluded from the startle analysis because they either had more than 33% rejected resonses, or had no visible resonse to the robe more than 66% of the time. All startle resonses were T-transformed within ersons to correct for interindividual variability that was unrelated to the exerimental conditions of interest 3. Galvanic Skin Resonse

27 After skin conductance resonses were averaged across trials, skin conductance data were LOG 10 (1 + skin conductance resonse)-transformed before being analyzed. Results: Omnibus Test of Reeated Measure ANOVAs US-exectancy There was a main effect of Block F(4,00) = 9.45, <.001, η =.16, ε =.86, a main effect of Time F(4,00) = 8.3, <.001, η =.14, ε =.7 but no main effect of Grou F(1,50) =.1, =.8) and a trend towards significance for the Block Grou interaction F(4,00) =.06, =.099, η =.04, ε =.86. Furthermore, there was a significant Time Grou interaction F(4,00) = 8.87, <.001, η =.15, ε =.7, and a significant Block Time Interaction F(16,800) = 3.11, =.001, η =.06, ε =.56. The Block Time Grou interaction reached significance F(16,800) =.41, =.011, η =.05, ε =.56. Galvanic Skin Resonse There was a main effect of Block F(4, 00) = 16.48, <.001, η =.5, ε =.495 as skin resonses habituated across blocks. Furthermore, there was a trend for stronger skin resonses in the exerimental grou across blocks comared to the control grou (Main effect of grou: F(1,50) = 3.05, =.087, η =.06. The interaction between Block and Grou did not reach significance F(4,00) = 1.49, =.3. Startle eye blink EMG There was a main effect of Block F(4,17) = 5.03, =.005, η =.11, ε =.63, but no Block Grou interaction F(4,17) = 1.87, =.15, ε =.63. There was a significant Stimulus Block F(4,17) = 3., =.014, η =.07 and a Stimulus Grou interaction F(1,43) = 7.93, =.007, η =.16. The main effect of Stimulus F(1,43) = 3.58, =.065, η =. 08 as well as the three-way interaction between Block Stimulus Grou failed to reach significance F(4,17) =.05, =.089, η =.05. References

28 1. Sruyt A, Clarysse J, Vansteenwegen D, et al. Affect 4.0: a free software ackage for imlementing sychological and sychohysiological exeriments. Ex Psychol. 010;57(1): doi:10.107/ /a De Clercq A, Verschuere B, de Vlieger P, et al. Psychohysiological analysis (PSPHA): a modular scrit-based rogram for analyzing sychohysiological data. Behav Res Methods. 006;38(3): Blumenthal TD, Cuthbert BN, Filion DL, et al. Committee reort: Guidelines for human startle eyeblink electromyograhic studies. Psychohysiology. 005;4(1):1-15. doi: /j x.