This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and

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1 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier s archiving and manuscript policies are encouraged to visit:

2 PAIN Ò 154 (2013) The acquisition and generalization of cued and contextual pain-related fear: An experimental study using a voluntary movement paradigm Ann Meulders a,, Johan W.S. Vlaeyen a,b,c a Research Group on Health Psychology, University of Leuven, Leuven, Belgium b Center of Excellence Generalization Research in Ill Health and Psychopathology, University of Leuven, Leuven, Belgium c Department of Clinical Psychological Science, Maastricht University, The Netherlands Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article article info abstract Article history: Received 21 April 2012 Received in revised form 3 September 2012 Accepted 30 October 2012 Keywords: Contextual pain-related fear Cued pain-related fear Fear conditioning Fear generalization Unpredictability Voluntary movement paradigm Recent evidence indicates that pain-related fear can be acquired through associative learning. In the clinic, however, spreading of fear and avoidance is observed beyond movements/activities that were associated with pain during the original pain episode. One mechanism accounting for this spreading of fear is stimulus generalization. In a voluntary movement-conditioning paradigm, healthy participants received predictable pain (ie, one movement predicts pain, another does not) in one context, and unpredictable pain in another context. The former procedure is known to induce cued pain-related fear to the painful movement, whereas the latter procedure generates contextual pain-related fear. In both experimental pain contexts, we subsequently tested fear generalization to novel movements (having either proprioceptive features in common with the original painful movement or nonpainful movement). Results indicated that in the predictable pain context, pain-related fear spreads selectively to novel movements proprioceptively related to the original painful movement, and not to those resembling the original nonpainful movement. In the unpredictable context, nondifferential fear generalization was observed, suggesting persistent contextual pain-related fear and poor safety learning. These data illustrate that spreading of pain-related fear is fostered by previously acquired movement-pain contingencies. Based on recent advances in anxiety research, we proposed an innovative approach conceptualizing predictable pain as a laboratory model for fear of movement in regional musculoskeletal pain, and unpredictable pain generating contextual pain-related fear as a prototype of widespread musculoskeletal pain. Consequently, fear generalization might play an important role in spreading of pain-related fear and avoidance behavior in regional and widespread musculoskeletal pain. Ó 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. 1. Introduction Evidence from cross-sectional studies with chronic pain sufferers [20] and prospective studies in acute pain [10,16,40] corroborates the fear-avoidance model s assumption that pain-related fear is a key factor in the origins of chronic musculoskeletal pain [46,47]. A recent conditioning study using joystick movements as conditioned stimuli (CSs) and a painful electrocutaneous stimulus as unconditioned stimulus (US) demonstrated the role of associative learning in the development of pain-related fear in healthy individuals [27]. That is, due to propositional knowledge relating pain to an initially neutral movement [31,34], this movement starts to signal Corresponding author. Address: Department of Psychology, University of Leuven, Tiensestraat 102, Box 3726, Leuven 3000, Belgium. Tel.: ; fax: address: ann.meulders@ppw.kuleuven.be (A. Meulders). danger/harm and comes to elicit autonomic fear responses and to spur avoidance behavior. In chronic pain patients, fear and avoidance are often not restricted to movements/activities that were associated with pain during the initial pain episode [21]. An intriguing yet empirically under-investigated question entails how spreading of pain-related fear and avoidance in chronic pain occurs. From an associative learning perspective, conditioned fear responses might extend to a range of novel stimuli resembling the original fear-eliciting CS. In essence, stimulus generalization is highly adaptive, because the ability to detect similarities between unique but related stimuli may contribute to avoiding harm in a dynamic environment [13,15,17]. Yet, together with reducing the risk of missing positive threat alarms, generalization bears an increased risk to respond to false threat alarms, which might be the case in persistent fear and avoidance behavior in chronic pain. It is recognized that in other psychological disorders (eg, posttraumatic stress disorder, panic /$36.00 Ó 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

3 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) disorder), overgeneralization of fearful responding is critically involved [7,8,24,25,38]. The identification of the dimensional and componential structures of pain-related fear challenges the view that it is a unitary concept [42]. Based on contextual fear-conditioning literature [11,12,29,43], 2 types of pain-related fear can be distinguished, depending on the temporal (un)predictability of the US: cued and contextual pain-related fear. Cued pain-related fear is typically induced by predictable pain (ie, pairings of movement and pain-us), whereas chronic contextual pain-related fear is induced by unpredictable pain (ie, pain-us explicitly unpaired with movements) [27,28]. Consistent with these observations, we argued that predictable pain may be viewed as a laboratory model for cued pain-related fear (sometimes termed kinesiophobia [19]) in regional musculoskeletal pain (eg, back pain) and unpredictable pain may be a prototype for widespread musculoskeletal pain (eg, fibromyalgia) [9,14,35]. To date, generalization of fear acquisition has never been systematically investigated in the chronic pain domain. Using the Voluntary Joystick Movement Paradigm [27,28,30], we tested fear generalization to novel diagonal movements (generalization stimuli; GSs) in both a predictable and an unpredictable pain context. We expected (a) generalization to novel movements proprioceptively related to the original painful movement (GS+) but not to those proprioceptively resembling the nonpainful movement (GS ) in the predictable context, and (b) that contextual pain-related fear fosters nonspecific fear generalization to novel movements in the unpredictable context (GS+ = GS ). Because category membership of novel stimuli is more elusive when the context acts as a predictor for the pain-us, fear might generalize to a broader range of stimuli than in the predictable context. 2. Methods and materials 2.1. Participants In total, 40 healthy individuals (28 females; M age = 21 years, SD age = 2.81, range = years) participated in this study and were compensated as follows: (A) psychology students of the University of Leuven received course credits (n = 7), and (B) volunteers (mostly nonpsychology university students and highly educated working people) were paid 10 (n = 33). Participants completed a general health checklist to make sure they did not suffer from respiratory or cardiovascular diseases, neurological diseases (eg, epilepsy), psychiatric disorders, or any other minor or major illness, and they did not have a chronic pain condition nor were pregnant. Additional exclusion criteria were hearing problems and pain at the dominant hand or wrist. The experimental protocol was approved by the Ethical Committee of the Department of Psychology of the University of Leuven. All participants signed the informed consent form, which explicitly stated that they were allowed to decline participation at any time during the experiment Stimulus material and measures The experiment was run on a Windows XP (Microsoft Corporation, Redmond, WA, USA) computer (Dell Optiplex 755; Dell Inc., Round Rock, TX, USA) with 2 GB RAM and an Intel Core2 Duo processor (Intel, Santa Clara, CA, USA) at 2.33 GHz and an ATI Radeon 2400 graphics card (Advanced Micro Devices, Sunnyvale, CA, USA) with 256 MB of video RAM, using Affect 4.0 [39]. We used 4 proprioceptive stimuli (ie, moving a Logitech [Newark, CA, USA] Attack 3 joystick in the horizontal plane [left/right] and in the vertical plane [upward/downward]) as CSs. The GSs were diagonal movements, (ie, left-top, right-top, left-bottom, right-bottom). Proprioception is restrictively defined as the perception of posture and movement, also referred to as postural somesthesis. Therefore, it can be argued that the most relevant features that the GSs share with the original CSs are proprioceptive in nature. However, in the current study, visuospatial features (ie, location/direction of movement in a 3- dimensional space) of course coincide with proprioception and may be involved as well. The pain-us was an electrocutaneous stimulus (2-ms duration), which was delivered by a commercial constant current stimulator (DS7A, Digitimer, Welwyn Garden City, England) through surface Sensor Medics (Homestead, FL, USA) electrodes (8 mm) filled with K-Y gel (Johnson & Johnson, New Brunswick, NJ, USA) that were attached to the wrist of the dominant hand. During the calibration procedure, participants received a series of electrocutaneous stimuli of increasing intensity and were asked to indicate how intense/painful each stimulus was on a scale from 1 to 10 where 1 means: you feel something but this is not painful, it is merely a sensation ; 2 means: this sensation starts to be painful, but it is still a very moderate pain ; up to 10, which means: this is the worst pain you can imagine. A subjective stimulus intensity of 8, which refers to a stimulus that is significantly painful and demanding some effort to tolerate was targeted (mean subjective stimulus intensity was 8.31, SD = 0.82, range 5-10). The mean stimulus intensity was 27.8 ma (SD = 13.65, range 8-64 ma). Conditioned pain-related fear was measured through subjective self-reports as well as a more objective psychophysiological correlate of fear learning, that is, the eyeblink startle response. The startle reflex is a cross-species, full-body reflex involved in defensive response mobilization. It is a very short latency reflex triggered by startle-evoking stimuli (eg, acoustic startle probe) that is mediated by a simple brainstem and spinal cord pathway, both directly and indirectly connected to the amygdala [4]. In anxiety research, electromyographic (EMG) activity of the orbicularis oculi triggered by an acoustic startle probe is often used as an approximation of the eyeblink component of the startle response. Startle modulation refers to the increase or potentiation of the startle reflex during fear states elicited by the anticipation of an aversive stimulus (eg, an electrocutaneous stimulus). In the present setup, the startle probe was a 100-dBA burst of white noise with instantaneous rise time presented binaurally for 50 ms through headphones. Eyeblink startle responses elicited by startle probes delivered during the CS/GS movements served as an index of cued pain-related fear. Eyeblink startle responses elicited by startle probes during the intertrial interval (ITI) served as an index of contextual pain-related fear Procedure The experiment was conducted during an 80-minute session and consisted of a preparation phase, a practice phase, a habituation phase, an acquisition phase, a transfer-of-acquisition phase, and a generalization phase. In a within-subjects design (Table 1), participants received both predictable pain stimuli in one context and unpredictable pain stimuli in another context. Half of the participants moved the joystick horizontally (left/right) in the predictable context, and vertically (upward/downward) in the unpredictable context. The other half of the participants had the reverse combination: they moved the joystick vertically in the predictable context, and horizontally in the unpredictable context. In the predictable context, one movement (CS p +) was consistently followed by the pain-us, and the other movement (CS p ) was never followed by the pain-us. Note that the direction of joystick movement that served as the CS p + and the CS p in the predictable context was counterbalanced across participants. In the unpredictable context, however, the pain-us was never delivered contingent on either of the joystick movements (CS u1 and CS u2 ), but was presented during the context (ITI). During acquisition training, participants voluntarily initiated their movements, so they freely

4 274 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) Table 1 Experimental design summary. Context Practice Habituation Acquisition Transfer of acquisition Generalization Predictable 4 CS p + only 6 probes 12 CS p + 4 CS p + 2 GS p + u1 4 CS p 12 CS p 4 CS p 2 GS p u1 2 GS p + u2 2 GS p u2 Unpredictable 4 CS u1 6 probes 12 CS u1 4 CS u1 2 GS p + u1 4 CS u2 12 CS u2 4 CS u2 2 GS p u1 12 US (during ITI) 4 US (during ITI) 2 GS p + u2 2 GS p u2 CS, conditioned stimuli; GS, generalization stimuli; US, unconditioned stimulus. Note: Conditions were run within subjects. CS p+ and CS p, respectively, refer to the movement that is followed by the pain-us and the movement that is never followed by the pain-us in the predictable context, whereas CS u1 and CS u2 both refer to movements that are never followed by the pain-us in the unpredictable context. GS movements are novel diagonal movements that have either one feature in common with the original CS p + [GS p + u1 and GS p + u2 ] or the original CS p [GS p u1 and GS p u2 ]. GSs are never reinforced (ie, generalization test under extinction). The suffix only is used to indicate nonreinforcement of the CS p + movement (ie, during the practice phase). chose in which direction they were going to move on a certain trial. During the transfer-of-acquisition phase, however, they could no longer choose the order of the movements themselves, but the movement direction was signaled. During the generalization phase, the same signaling procedure was used to test conditioned responding to novel diagonal movements (GSs) under extinction Preparation phase Participants were informed (orally and in writing) that painful electrocutaneous stimuli (pain-us) and loud noises (acoustic startle probes) would be administered during the experiment. After providing informed consent, participants went to the experimental room. The electrodes for eyeblink startle responses were attached and the intensity level of the pain-us was selected following the calibration procedure (see Section 2.2. Stimulus material and measures) Practice phase Before initiating the practice phase, participants received detailed instructions about the experimental task. In each block, participants had to move the joystick 8 times as quickly and accurately as possible when prompted by a starting signal + (fixation cross presented in the middle of the computer screen), in whatever order they freely chose. The position of counter bars on the computer screen indicated in which movement plane (horizontal vs. vertical) they had to move. The counter bars, each divided into 4 equal segments, always appeared on 2 sides of the computer screen (left/ right or top/bottom) (Fig. 1). In a horizontal block, these bars were displayed on the left and right side of the computer screen, whereas in the vertical block, these bars appeared at the top and the bottom of the computer screen. Successful movements always resulted in changing the color of the segments of the corresponding counter bar. That way, participants could instantly evaluate how many movements in each direction remained to be carried out. In total, 2 blocks of 8 trials were run: the first block comprised 4 trials in each of both directions in the horizontal movement plane (4 left/ 4 right), and the second block comprised 4 trials in each of both directions in the vertical movement plane (4 top/4 bottom), or vice versa. During the practice phase, no acoustic startle probes or pain- USs were presented, and online verbal feedback about the task performance was provided by the experimenter Startle habituation phase A habituation phase was included to prevent possible confounds in the data because the responses to the first startle probes usually are relatively large. The habituation phase included 12 trials, each lasting for 24 seconds (with a variable ITI of, on average, 5 seconds [±2 seconds]). During each trial, one startle probe is presented (100-dBA burst of white noise), either between the 1 st and the 2 nd second (4 trials), at the 10 th second (4 trials), or between the 15 th and 17 th second (4 trials) of the trial. The timing of probes during trials was randomized across participants. During this phase, the participants wore headphones, and only dimmed light was available. Note that no pain-uss were delivered during this phase Acquisition phase This phase was largely identical to the practice phase, with the exception that (1) pain-uss and startle probes were presented, (2) 8 blocks (4 predictable and 4 unpredictable) of 8 trials were run instead of 2 blocks, and (3) instructions now emphasized to pay close attention to the starting signal + and to respond as fast and accurately as possible upon its presentation. Although a CS movement was of variable length depending on the participants movement speed, a trial typically included an ITI consisting of a pre-cs interval of 3.5 seconds and a post-cs interval of 8 seconds. The pain-us was presented within each CS p + trial in the predictable context (100% contingency) and in half of the trials during ITI in the unpredictable context. In the predictable context, the US occurred immediately after the CS p + movement. In the unpredictable context, the pain-us occurred 1-3 seconds before the presentation of the starting signal +, or seconds after the CS movement. In each block with 8 CS movements, 4 of the startle probes were presented during the CS movements (2 during CS p +orcs u1, and 2 during CS p or CS u2 ), and 4 during the ITI (2 probe positions before the CS and 2 probe positions after the CS). To avoid startle facilitation during ITI being confounded by direct responses to presentations of the pain-us in the unpredictable context, startle probes were presented before the CS when the pain-us was presented after the CS, and after the CS when the pain-us was presented before the CS. Note that we did not inform the participants about the contingencies between the joystick movements (CSs) and the pain-us. After each conditioning block, the participants rated the cued painrelated fear elicited by each of the CS movements Transfer of acquisition Trials during the transfer of acquisition phase were basically built up the same way as during the acquisition phase. Yet, CS movements were no longer voluntarily initiated but signaled. That is, 50 ms after trial onset, a red asterisk ( ) appeared for 500 ms at one of the CS movement directions (left/right in a horizontal block; top/bottom in a vertical block), indicating in which direction participants had to move. Before actually performing the signaled movement, participants rated their US-expectancy and cued pain-related fear. After completing the ratings, they waited for the + starting signal to start moving into the signaled direction. After successfully performing the signaled CS movement, a post- CS ITI of 8 seconds followed (cfr. timing acquisition). During the transfer phase, 2 blocks (one predictable and one unpredictable)

5 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) Fig. 1. Schematic overview of the experimental task in the (A) predictable context and (B) unpredictable context during the acquisition, the transfer of acquisition and the generalization phases. Note: The white + serves as the starting signal to initiate the voluntary movements during the acquisition phase. During the transfer of acquisition and the generalization phases, the order of the movements is no longer chosen freely, a red indicates which CS/GS (diagonal) movement ought to be performed. Bluecolored segments of the counter bars represent the number of performed movements and white-colored segments of the counter bars indicate the movements that still ought to be performed. CSp+ and CSp, respectively, refer to the movement that is followed by the pain-us (ie, left) and the movement that is never followed by the pain-us (ie, right) in the predictable condition, whereas CSu1 and CSu2 both refer to movements that are never followed by the pain-us in the unpredictable condition (ie, upwards and downwards). GS movements are novel diagonal movements that have either one feature in common with the original CSp+ [GSp+u1 and GSp+u2 ] or the original CSp [GSp u1 and GSp u2]. CS, conditioned stimuli; GS, generalization stimuli. of 8 trials were run. Startle probes were delivered following the same timing as during acquisition Generalization phase The procedure of the generalization phase was mainly the same as the transfer of acquisition phase. The difference was that participants now had to perform 4 novel diagonal movements (GSs), which each have a feature in common with either the original CS p + or the original CS p, namely left-top, right-top, left-bottom, or right-bottom. Again, 50 ms after trial onset, a red asterisk ( ) appeared for 500 ms in one of the corners of the screen to signal which movement had to be performed in randomized order. After successfully performing a movement, a post-gs ITI of 8 seconds followed (cfr. timing acquisition). On each trial, a startle probe was delivered during the GS movement and no pain-uss were delivered. The 4 GS movements were performed 2 times in each experimental context (predictable vs. unpredictable); the order was randomized across participants Manipulation checks and outcome variables Manipulation checks Retrospective US-expectancy. As a manipulation check, we assessed retrospective US-expectancy after the entire experiment. Participants indicated for both CS movements in each context how much they expected the painful stimulus to occur on an 11-point Likert scale (range 0-10) with labels not at all to very much Online US-expectancy during transfer of acquisition. During the transfer-of-acquisition phase, participants indicated before each movement to what extent they expected the painful stimulus to occur when performing the signaled movements (CSs) on an 11-point Likert scale (range 0-10) with labels not at all to very much. That way, we could assess whether the contingencies learned during the acquisition phase using the voluntary movement set-up transfer to the signaled movement set-up SAM ratings of unhappiness, arousal, and control. After the experiment, the participants also filled out 3 Self-Assessment Manikin scales (SAM) [3], measuring (un)happiness, arousal, and the control they experienced when performing the respective CS movements. These self-assessment scales each consisted of 5 different pictographs and were accompanied by the following questions: How (un)happy did you feel when performing the left/right/ upward/downward movement?, How excited/calm where you when performing the left/right/upward/downward movement?, and To what extent did you feel in control of the situation when performing

6 276 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) the left/right/upward/downward movement? Responses were scored from 1 to Self-reported cued pain-related fear After each block, participants answered the following question: How afraid were you to perform the left/right/upward/downward movement? on an 11-point Likert scale ranging from 0 to 10 with anchors not fearful at all to the worst fear you can imagine. Note that only questions that applied to the previous block were asked (ie, after horizontal block ratings for the left/right movements and after vertical block ratings to the upwards/downwards movements). During the transfer of acquisition and the generalization phases, participants rated before each movement how afraid they were to actually perform the signaled movements (CSs/GSs) Online US-expectancy during generalization During the generalization phase, participants rated before each movement to what extent they expected the painful stimulus to occur when performing the signaled movements (GSs) on an 11- point Likert scale (range 0-10) with labels not at all to very much Eyeblink startle modulation Orbicularis oculi EMG activity was recorded with 3 Ag/AgCl Sensor Medics electrodes (4 mm) filled with electrolyte gel. After cleaning the skin with exfoliating peeling cream to reduce interelectrode resistance, electrodes were placed on the left side of the face according to the site specifications proposed by Blumenthal et al. [2]. The raw signal was amplified by a Coulbourn isolated bioamplifier (Coulbourn Instruments, Whitehall, PA, USA) with bandpass filter (LabLinc v75 04). The recording bandwidth of the EMG signal was between 90 Hz and 1 khz (±3 db). The signal was rectified online and smoothed by a Coulbourn multifunction integrator (LabLinc v76 23 A) with a time constant of 20 ms. The EMG signal was digitized at 1000 Hz from 200 ms before the onset of the auditory startle probe until 1000 ms after probe onset Response latency We defined movement-onset latency (time to start to move the joystick) as the time from the moment the starting signal ( + = fixation cross) is presented until participants leave the start region, which is defined as an invisible and relatively small circle around the fixation cross presented in the middle of the computer screen. Coordinates (in pixels) for this start region are calculated for a 17- inch computer screen: X = 512, Y = 384, and the radius of this circle, r = Preference for predictable pain The experiment ended with a behavioral task. As a cover story, we told participants that something went wrong in the data acquisition of one of the blocks during the experiment and to make sure enough data were collected for the statistical analysis, they were requested to perform one extra block. We told participants that they could choose between an extra horizontal or vertical block (forced choice). Next, we encoded their choice in terms of preference for performing an extra block in the predictable or unpredictable context Experimental setting Participants were seated in an armchair (0.6-m screen distance) in a sound-attenuated and dimmed experimental room, adjacent to the experimenter s room. Further verbal communication was possible through an intercom system; the experimenter observed the participants and their physiological responses online by means of a closed-circuit TV installation and computer monitors Response definition Startle modulation Using psychophysiological analysis (PSPHA) [5], a modular script-based program, we calculated the peak amplitudes defined as the maximum of the response curve within ms after the startle probe onset. All startle waveforms were visually inspected off line, and technical abnormalities and artifacts were eliminated using the PSPHA software. Every peak amplitude was scored by subtracting its baseline score (averaged EMG level between 1 and 20 ms after the probe onset). The raw scores were transformed to z-scores to account for interindividual differences in physiological reactivity. In order to optimize the visualization of the startle data and avoid negative values on the Y-axis, T-scores a linear transformation of the z-scores were used in the figures. Averages were calculated for responding during CS/GS movements and ITI separately for the predictable and the unpredictable contexts Reaction time measures For the statistical analysis of the response latencies, data from the practice phase were omitted. To eliminate outliers, trials with reaction times above 3000 ms were discarded, as well as trials with reaction times deviating more than 2.5 SDs from the truncated mean of its corresponding cell in the experimental design. From the remaining data, the mean reaction times for each participant were calculated for each CS movement in each experimental context per block, and averaged over the 4 movements in that block. For the generalization phase, means were calculated for both GS movements having a feature in common with the CS p +orcs p for each experimental context separately. 3. Results We carried out a series of repeated-measures analyses of variance (ANOVAs) to examine the effects of unpredictable versus predictable pain-uss on the respective dependent measures. Because we had clear a priori hypotheses, we further analyzed the data using planned comparisons. Following Kirk [18], mean square error terms and degrees of freedom appropriate for the specific contrasts were used. Greenhouse-Geisser corrections are reported when appropriate. Uncorrected degrees of freedom and corrected P-values are reported together with e and the effect size indication g 2 P. Statistical analyses for all dependent measures were run with Statistica 10 software (Tulsa, OK, USA) Manipulation checks Retrospective US-expectancy Mean retrospective US-expectancy ratings (Table 2) were analyzed as a manipulation check in a 2 2 (Context [Predictable/ Unpredictable] Stimulus Type [CS+/CS ]) repeated-measures ANOVA (Note that throughout this paper, the notations CS+ and CS used in the descriptions of the statistical analyses and the figures, respectively, refer to the CS p + and the CS p in the predictable context, and to the unreinforced CSs [ie, CS u1 and CS u2 in the unpredictable context]. With respect to the GSs in the figures and analyses, including the generalization data, for the sake of simplicity, GS p + u1 and GS p + u2 and GS p u1 and GS p u2 are referred to, respectively, as GS+/GS ). There was a significant main effect of Stimulus Type, F(1,39) = 22.58, P < , g 2 P ¼ :37, and a significant Context Stimulus Type interaction, F(1,39) = 12.59, P < 0.01, g 2 P ¼ :24. Planned comparisons confirmed that US-expectancy ratings were higher for the CS+ movement than for the CS movement, F(1,39) = 30.07, P < , g 2 P ¼ :44, in the

7 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) Table 2 Manipulation check measures: SAM unhappiness, arousal, and control ratings, and retrospective US-expectancy ratings for the CS p + and CS p in the predictable condition, and the CS u1 and CS u2 in the unpredictable condition. n = 40 Predictable condition Unpredictable condition CS p + CS p CS u1 CS u2 SAM unhappiness 3.90 (0.13) 2.50 (0.16) 3.38 (0.13) 3.43 (0.14) SAM arousal 3.38 (0.17) 2.83 (0.16) * 3.30 (0.13) 3.30 (0.13) SAM control 3.00 (0.18) 2.76 (0.20) 3.38 (0.16) 3.43 (0.18) Retrospective US-expectancy 6.00 (0.68) 1.58 (0.98) 3.95 (0.50) 3.80 (0.44) SAM, Self-Assessment Manikin scales; CS, conditioned stimuli; US, unconditioned stimulus. * P < ** P < ; for the CS p +/CS p differences and the CS u1 /CS u2 differences in the predictable and the unpredictable condition, respectively. predictable context. In the unpredictable context, US-expectancies for both CS movements did not differ, F < Online US-expectancy during transfer of acquisition To check whether participants transferred their knowledge about the stimulus contingencies acquired in the voluntary to the signaled movement set-up, we collected US-expectancy ratings before each movement during the transfer-of-acquisition phase. We analyzed the mean online US-expectancy ratings in a 2 2 (Context [Predictable/Unpredictable] Stimulus Type [CS+/CS ]) repeated-measures ANOVA. This analysis yielded significant main effects for Context, F(1,39) = 4.35, P < 0.05, g 2 P ¼ :10, and Stimulus Type, F(1,39) = , P < , g 2 P ¼ :77, as well as a significant Context Stimulus Type interaction, F(1,39) = , P < , g 2 P ¼ :78. Planned comparisons showed that higher US-expectancies were given for the CS+ movement than for the CS movement, F(1,39) = , P < , g 2 P ¼ :86, in the predictable context (Fig. 2). In the unpredictable context, we observed no differences in US-expectancy for both CS movements, F < SAM ratings of unhappiness, arousal, and control We performed 3 separate 2 2 (Context [Predictable/Unpredictable] Stimulus Type [CS+/CS ]) repeated-measures ANOVAs on the mean SAM ratings of unhappiness, arousal, and control (Table 2). The first analysis revealed significant main effects for Context, F(1,39) = 5.24, P < 0.05, g 2 P ¼ :12, and Stimulus Type, F(1,39) = 18.10, P < 0.001, g 2 P ¼ :32, as well as a significant Fig. 2. Mean online US-expectancy (+SEs) during the CS/GS movements, respectively, during transfer of acquisition and generalization in the predictable and the unpredictable context. Note: P < 0.05; P < 0.01; P < 0.001; P < CS, conditioned stimuli; GS, generalization stimuli. Context Stimulus Type interaction, F(1, 39) = 27.82, P < , g 2 P ¼ :42. Planned comparisons demonstrated that participants felt more unhappy in the predictable context when they were performing the CS+ movement compared with performing the CS movement, F(1,39) = 35.72, P < , g 2 P ¼ :44. In the unpredictable context, no such differences in unhappiness between both movements were reported, F < 1. In the second analysis examining the mean SAM arousal ratings, no significant Context Stimulus Type interaction emerged, F(1,39) = 2.95, P = 0.09, g 2 P ¼ :07. Yet, planned comparisons demonstrated that participants were more aroused when performing the CS+ movement than when performing the CS movement in the predictable context, F(1,39) = 4.37, P < 0.05, g 2 P ¼ :10, whereas in the unpredictable context, both CS movements were rated as equally arousing, F < 1. The last analysis only yielded a significant main effect of Context, F(1, 39) = 7.62, P < 0.01, g 2 P ¼ :16, indicating that participants felt less in control in the unpredictable context than in the predictable context Outcome variables Self-reported cued pain-related fear We conducted a (Context [Predictable/Unpredictable] Stimulus Type [CS+/CS ] Block [ACQ1-3, ACQ4 ]) repeated-measures ANOVA on the mean cued pain-related fear ratings for the CS movements in both contexts (predictable and unpredictable) separately for the 3 acquisition blocks and the transfer of acquisition block (Fig. 3). There was a significant main effect of Stimulus Type, F(1,39) = 92.23, P < , g 2 P ¼ :70, and a significant Context Block interaction, F(3,117) = 10.17, P < , e =.98, g 2 P ¼ :21, suggesting that fearful responding to the movements developed differently in predictable and unpredictable contexts. Of crucial importance, there was a significant Context Stimulus Type interaction, F(1, 39) = , P < , g 2 P ¼ :76, but this interaction was not modulated by Block, F < 1. Furthermore, planned comparisons revealed that in the predictable context, participants reported more cued pain-related fear in response to the CS+ movement than in response to the CS movement, F(1,39) = , P < , g 2 P ¼ :79. As expected, no such differences in pain-related fear were observed for both unreinforced CS movements in the unpredictable condition, F < 1. To examine generalization of cued pain-related fear to the novel diagonal GS movements, we ran a 2 2 (Context [Predictable/ Unpredictable] Stimulus Type [GS+/GS ]) repeated-measures ANOVA (Fig. 4). This analysis showed a significant main effect of Stimulus Type, F(1,39) = 7.20, P < 0.05, g 2 P ¼ :16, and a significant Context Stimulus Type interaction, F(1,39) = 5.27, P < 0.05, g 2 P ¼ :12. In line with our expectations, planned comparisons showed that in the predictable context, GS+ movements (diagonal movements proprioceptively related to the original CS+) elicited higher fear-of-pain reports during the generalization test than

8 278 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) Fig. 3. Mean self-reported cued pain-related fear (+SEs) in response to the CS movements in the predictable and the unpredictable context assessed after each block during acquisition (ACQ1-3) and before each trial during the transfer of acquisition block (ACQ4 ). Note: P < 0.05; P < 0.01; P < 0.001; P < CS, conditioned stimuli. Fig. 4. Mean self-reported cued pain-related fear (+SEs) in response to the diagonal GS movements in the predictable and the unpredictable context assessed before each trial during the generalization phase. Note: P < 0.05; P < 0.01; P < 0.001; P < GS, generalization stimuli. the GS movements (diagonal movements proprioceptively related to the original CS ), F(1,39) = 8.80, P < 0.01, g 2 P ¼ :18. In the unpredictable context, the same diagonal GS+ and GS movements did not elicit different fear-of-pain ratings, F < 1. comparing autonomic fear responding elicited during the CSs and the ITI in both contexts revealed significant main effects for Context, F(1,39) = 45.04, P < , g 2 P ¼ :54, for Block, F(3,117) = 8.58, P < , e =.77, g 2 P ¼ :18, as well as for Stimulus Type, F(2,78) = 7.39, P < 0.01, e =.97, g 2 P ¼ :16. Importantly, the Stimulus Type Condition interaction was significant, F(2,78) = 19.16, P < , e =.97, g 2 P ¼ :33, confirming that the startle responses elicited during the ITI and the respective CSs differed significantly in both the predictable and the unpredictable context. Planned comparisons further demonstrated that mean startle amplitudes during the CS+ movement were higher than during the CS movement, F(1,39) = 13.42, P < 0.001, g 2 P ¼ :26. In the unpredictable context, no such difference was observed, F < 1. Interestingly, startle reflexes during the ITIs in the unpredictable context were higher than in the predictable context, F(1,39) = 99.42, P < , g 2 P ¼ :72, suggesting that more chronic contextual pain-related fear emerged in the unpredictable context. To examine generalization of autonomic fearful responding to the novel diagonal GS movements, we carried out a 2 2 (Context [Predictable/Unpredictable] Stimulus Type [GS+/GS] repeatedmeasures ANOVA) (Fig. 6). The pattern of differential startle Online US-expectancy during generalization We analyzed the mean online US-expectancy ratings during the generalization test in a 2 2 (Context [Predictable/Unpredictable] Stimulus Type [GS+/GS ]) repeated-measures ANOVA. This analysis revealed a significant main effect for Stimulus Type, F(1,39) = 8.04, P < 0.01, g 2 P ¼ :17, as well as a significant Context Stimulus Type interaction, F(1,39) = 11.46, P < 0.01, g 2 P ¼ :23. Planned comparisons showed that US-expectancies for the GS+ movements were elevated compared with those for the GS movements, F(1,39) = 11.19, P < 0.01, g 2 P ¼ :22 (Fig. 2). In the unpredictable context, the same GS movements did not generate such differences in US-expectancy ratings, F < Eyeblink startle modulation We analyzed the mean startle responses during acquisition and transfer of acquisition with a (Context [Predictable/ Unpredictable] Stimulus Type [CS+/CS /ITI] Block [ACQ1-3, ACQ4 ]) repeated-measures ANOVA (Fig. 5). The analysis Fig. 5. Mean eyeblink startle amplitudes (+SEs) during the CS movements and the ITI in the predictable and the unpredictable contexts during the acquisition (ACQ1-3) and transfer of acquisition (ACQ4 ) separately. Note that for graphic purposes, T- scores were used. CS, conditioned stimuli; ITI, intertrial interval.

9 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) painful stimulus. The response latencies did not significantly differ between both CS movements in the unpredictable context, F(1,39) = 3.74, P = 0.06, g 2 P ¼ :09. To examine possible generalization effects, we ran a 2 2 repeated-measures ANOVA (Context [Predictable/Unpredictable] Stimulus Type [GS+/GS ]). This analysis only revealed a significant main effect for Context, F(1,39) = 6.64, P < 0.05, g 2 P ¼ :15, suggesting that participants in the unpredictable condition were more hesitant to initiate the same GS movements than in the predictable context. Planned comparisons showed that during the generalization test, no differences were found between GS+ and GS movements, either in the predictable context or in the unpredictable context, both Fs < 1. Fig. 6. Mean eyeblink startle amplitudes (+SEs) during the diagonal GS movements in the predictable and the unpredictable contexts during the generalization phase. Note that for graphic purposes, T-scores were used. CS, conditioned stimuli; ITI, intertrial interval; GS, generalization stimuli. responding was similar to that of the verbal fear ratings, but the analysis failed to show a significant Context Stimulus Type interaction, F(1,39) = 1.66, P = Because we had clear a priori hypotheses, planned comparisons were further calculated. Startle amplitudes elicited during the GS+ movements were more elevated compared with those during the GS movements in the predictable context, but the difference barely failed to reach statistical significance, F(1,39) = 4.05, P = 0.05, g 2 P ¼ :09. No different startle amplitudes were elicited by the same GS movements in the unpredictable context, F < Response latency We performed a (Context [Predictable/Unpredictable] Stimulus Type [CS+/CS ] Block [ACQ1-3, ACQ4 ]) repeated-measures ANOVA on the mean response latencies for the different CS movements (Fig. 7). This analysis showed a main effect for Stimulus Type, F(1,39) = 9.37, P < 0.01, g 2 P ¼ :19, and Block, F(3,117) = 2.84, P < 0.05, e =.64, g 2 P ¼ :07. The Context Stimulus Type interaction was not significant, F(1,39) = 1.86, P = As expected, participants reaction times were slower during the CS+ movement than during the CS movement in the predictable context, F(1,39) = 6.96, P < 0.05, g 2 P ¼ :15, suggesting that participants were more hesitant initiating the movement associated with the Forced choice preferences We told participants after the experiment that they would have to perform one extra block, but that they could choose whether they wanted to perform a horizontal or a vertical block; 82.5% (33/40) of participants preferred to perform an extra block in the predictable context, whereas only 17.5% (7/40) of participants chose to do an extra block in the unpredictable context. These results seem to suggest that, although in both contexts an equal number of pain stimuli with identical intensity were delivered, the majority of the participants had a preference for the signaled pain context Correlations between cued pain-related fear at the end of acquisition and during generalization We calculated Pearson correlation coefficients between the level of cued pain-related fear in response to the CS movements at the end of acquisition and the level of cued pain-related fear elicited by the novel diagonal GS movements during generalization (Table 3). We hypothesized that better discriminative fear learning during acquisition might be related to less fear generalization (ie, negative correlation), because if the specific features of the CS+ are better encoded, differences between the GSs would be magnified. This analysis however, revealed a significant positive correlation between the CS+/CS difference scores at the end of acquisition and the GS+/GS difference scores during generalization, Pearson s r = 0.38, P < This correlation can be interpreted as follows: more differential fear learning during acquisition is related to better discrimination during fear generalization. Furthermore, the analysis showed a positive correlation between cued pain-related fear ratings in response to the CS+ movement at the Fig. 7. Mean response latencies (+SEs) in ms during CS/GS movements averaged over blocks during the acquisition and the generalization phase, respectively. Note: P < 0.05; P < 0.01; P < 0.001; P < CS, conditioned stimuli; GS, generalization stimuli.

10 280 A. Meulders, J.W.S. Vlaeyen / PAIN Ò 154 (2013) Table 3 Pearson correlation coefficients for stimuli of interest in the predictable and unpredictable context. r value P value Predictable pain context pain-related fear ratings r(cs p +, GS+) 0.66 <0.001 r(cs p,gs ) 0.66 <0.001 r(d[cs p +, CS p ], D[GS+, GS ]) 0.38 < 0.05 Predictable pain context startle eyeblink measures r(cs p +, GS+) r(cs p,gs ) r(d[cs p +, CS p ], D[GS+, GS ]) Unpredictable pain context pain-related fear ratings r(m[cs u1,cs u2 ], M[GS+, GS ]) 0.78 <0.001 Unpredictable pain context startle eyeblink measures r(m[cs u1,cs u2 ), M[GS+, GS ]) 0.36 <0.05 CS, conditioned stimuli; GS, generalization stimuli. Note: Pearson correlation coefficients between pain-related fear ratings for the stimuli of interest at the end of acquisition and generalization, with D(x, y) representing the difference score between x and y, and M(x, y) representing the mean of x and y. end of acquisition and cued pain-related fear ratings in response to the GS+ movements during generalization, Pearson s r = 0.66, P < There was also a significant positive correlation between cued pain-related fear ratings in response to the CS movement at the end of acquisition and cued pain-related fear ratings in response to the GS movements during generalization, Pearson s r = 0.66, P < With respect to pain-related fear ratings in the unpredictable context, we calculated the Pearson correlation coefficients between the mean pain-related fear elicited by both unpredictable CS movements at the end of acquisition and the mean pain-related fear elicited by the novel GS movements during generalization. We observed a significant positive correlation, Pearson s r = 0.78, P < 0.001, indicating that participants who did not trust the technically safe movements during acquisition also are more afraid of new movements during generalization. In the psychophysiological fear index, most of the reported correlations failed to reach significance, however, the latter finding was corroborated in startle eyeblink measures with a significant positive correlation, Pearson s r = 0.36, P < Discussion The present study examined spreading of fear towards novel movements sharing proprioceptive features with the original (non)painful movements, in both a predictable and an unpredictable pain context. As proprioceptive fear conditioning is particularly relevant for patients with pain in the musculoskeletal system, we employed a voluntary movement paradigm using joystick movements as CSs, and a painful electrocutaneous stimulus as US. In a between-subjects design, healthy participants received predictable pain in one context, and unpredictable pain in another context. In the predictable context, one movement was followed by the pain-us (CS+), and the other was not (CS ). In the unpredictable context, both movements were never reinforced, but pain- USs were presented unsignaled. Subsequently, we tested fear generalization to novel diagonal movements (GSs) in both pain contexts. First, we successfully induced cued pain-related fear to the painful movement, but not to the nonpainful movement in the predictable context. This effect was apparent in elevated fear ratings, higher eyeblink startle amplitudes, and higher US-expectancies for the CS+ movement compared with the CS movement. We also successfully established contextual pain-related fear as indexed by elevated ITI startle reflexes in the unpredictable context. As expected, there were no differences in the fear ratings, US-expectancy measures, and startle responses elicited during each unpredictable movement. Intriguingly, these unpredictable movements generally yielded higher fear responses than the functionally equivalent CS in the predictable context, suggesting a deficiency in safety learning or spillover effects from the threatening/unsafe context (see also [27]). Furthermore, we observed longer movement-onset latencies for the painful movement than for the nonpainful movement in the predictable context, which might be indicative of freezing-like guarding [26,36] or avoidance behavior (for a similar argument, see [27,28]). No such difference between both unreinforced movements was present in the unpredictable context. Second and more importantly, we demonstrated differential fear generalization in response to the novel diagonal movements in the predictable context. That is, the GS+ movements generated higher fear ratings and higher US-expectancy ratings than the GS movements. The same data pattern emerged in the eyeblink startle measures, however, the statistical test just failed to reach significance (P = 0.05). Taken together, however, these data strongly support the notion that cued pain-related fear can spread following a generalization gradient (ie, more fear generalization to novel movements with greater proprioceptive similarity to the initial fearevoking movement than to novel movements sharing proprioceptive features with the original safe movement). In the unpredictable context, however, the same GS movements did not reveal such a differential fear generalization effect. Fear ratings, USexpectancy ratings, and startle eyeblink measures did not differ between the GS+ and GS movements in the unpredictable context. These findings demonstrate robust contextual modulation: the same movements acquired a different meaning in each experimental context and thus elicit dissimilar conditioned fear responses. Importantly, these effects can only be attributed to previously acquired CS-US contingencies. More specifically, in the predictable context, movements that share proprioceptive features, but have never been paired with the painful stimuli themselves, come to elicit fear. In the unpredictable context, the lack of safety identification and contextual pain-related fear spreads to a broader range of novel GS movements. Fear generalization in the daily life context of pain might work as follows: if lifting an object is followed by pain, one might learn that leaning forward is dangerous. As a result, other harmless movements (eg, lifting a baby) that share this leaning-forward aspect may be avoided as well, disregarding other important aspects (eg, weight of the object). This example illustrates how spreading of fear of movement might occur in regional musculoskeletal pain (eg, low back pain). In contrast, spreading of contextual pain-related fear is less stimulus-specific, and therefore corresponds more to the clinical picture of widespread musculoskeletal pain. In the absence of clear predictors of increasing pain episodes, one might become more generally afraid of pain increases, inducing a more general and sustained form of distress. Consequently, one might start avoiding a broader range of activities in a broader range of situations that share features of the initial pain context (eg, household-related or work-related tasks). Third, with regard to the movement-onset latencies, the differential generalization effect did not materialize in the predictable context. Yet, participants initiated the same GS movements slower when carried out in the unpredictable/threatening context than when carried out in the predictable/safe context. Possibly, the contextual pain-related fear generated by the unpredictable context has detrimental effects on performance, that is, participants may be reluctant to initiate all kinds of movements because in that context, painful events can occur at any time (ie, human freeze-like behavior [26,36]). Moreover, the test of fear generalization under extinction might impact performance: in the predictable context, previously learned CS-US knowledge is more straightforwardly