Is conditioned pain modulation (CPM) affected by negative emotional state?

Conditioned pain modulation (CPM) is an experimental paradigm, which describes the inhibition of responses to a noxious or strong‐innocuous stimulus, the test stimulus (TS), by the additional application of a second noxious or strong‐innocuous stimulus, the conditioning stimulus (CS). As inadequate CPM efficiency has been assumed to be predisposing for clinical pain, the search for moderating factors explaining inter‐individual variations in CPM is ongoing. Psychological factors have received credits in this context. However, research concerning associations between CPM and trait factors relating to negative emotions has yielded disappointing results. Yet, the influence of anxious or fearful states on CPM has not attracted much interest despite ample evidence that negative affective states enhance pain. Our study aimed at investigating the effect of fear induction by symbolic threat on CPM.

Pain sensation can be inhibited by a second noxious or strong-innocuous stimulus applied at a remote site.This phenomenon was termed 'conditioned pain modulation' (CPM) (Yarnitsky et al., 2015) and is seen as psychophysical equivalent to the physiological mechanism 'diffuse noxious inhibitory controls' (DNIC) (Le Bars, 2002).In recent years, CPM has been assumed as risk factor for clinical pain, with inadequate CPM inhibition being observed in several chronic pain conditions (Lewis et al., 2012;Staud, 2012;Yarnitsky, 2015;Yarnitsky et al., 2014).
Although CPM is likely mediated by a circuit in the spinal cord reaching the brainstem (Bingel & Tracey, 2008;Youssef et al., 2016a), it is also open to higher brain regions as shown in imaging studies (Bogdanov et al., 2015;Moont et al., 2011;Piché et al., 2009;Youssef et al., 2016b).Further evidence for such influences is given by the association with higher order perceptual and cognitive processes (Bjørkedal & Flaten, 2012;Cormier et al., 2013;France et al., 2016;Goffaux et al., 2007;Larivière et al., 2007;Nir et al., 2011Nir et al., , 2012)).CPM may also be related to general (e.g., depression) or pain-specific affectivity (e.g., pain catastrophizing).Although such associations have been made plausible by previous findings (Bair et al., 2003;Burke et al., 2015;Quartana et al., 2009), strict evidence obtained just in CPM paradigms is largely missing as underscored by the results of a meta-analysis presenting only few and weak correlations (Nahman-Averbuch et al., 2016).We also found no relationship between CPM and trait fear (threat-potentiated startle) as well as pain-specific trait anxiety (questionnaires) (Horn-Hofmann et al., 2016).
Despite this negative evidence concerning a relationship between trait fear and anxiety on the one hand and CPM on the other hand, it may well be that CPM is altered by negative emotional states.The finding that pain perception is temporarily enhanced by negative emotional states has often been replicated (Bushnell et al., 2013;Wiech & Tracey, 2009).However, the effect of fear/anxiety states on CPM has yet been scarcely investigated.The effects of a threat manipulation on CPM have been investigated only once by Bernaba et al. (2014), who instructed participants that cold water used as conditioning stimulus (CS) might have harmful effects like frostbite and gangrene, without success because CPM could not be affected.A limitation of this threat induction might be additional information guaranteeing the subjects' safety for ethical reasons.
Our study aimed at testing the effects of affective picture viewing, which has proven efficacy for pain modulation (e.g., Meagher et al., 2001;Rhudy et al., 2005Rhudy et al., , 2013)), on CPM.Burn injuries were shown as aversive pictures, realizing content-related associations with the painful sensations elicited by heat for both test stimulus (TS) and CS.In addition to pain ratings, we also recorded contact-heat evoked potentials (CHEPs) as physiological measure without cognitive biases to ensure capturing a CPM effect.We hypothesized that the CPM effect would be diminished or even abolished in the high-threat block; that is, emotional threat was assumed to minimize the CPM effect.

| Participants
Thirty-seven healthy volunteers of both genders and within an age range between 20 and 60 years were recruited by advertisement at the University of Bamberg and on social media.The age range was intentionally raised above the level of student population to also include people with an age that is already associated with the first onset of certain forms of chronic pain.We think that this age span is informative because certain risk factors for the development of chronic pain start to play their role at this time, the consideration of which has therefore become a general routine in our lab.
No participant had taken any analgesic medication or alcohol at least 24 h prior to the test session.Exclusion criteria (assessed by a telephone interview) included all acute or chronic diseases, especially those associated with acute and chronic pain.In addition, participants were screened for psychiatric disorders by the Mini-DIPS diagnostic interview based on DSM-IV and ICD-10 (Margraf et al., 2017) at the beginning of the experimental session and excluded from participation in the case of any current diagnosis (except for minor anxiety disorders, e.g., specific phobia).All subjects provided written informed consent and received either course credits or monetary compensation for their participation.The experimental procedure was approved by the ethics committee of the University of Bamberg (06/11/2013).

| Materials and procedures
All experimental sessions took place in the morning (always starting at 9 a.m.).Participants sat upright in a comfortable chair in front of a computer screen.The whole experiment lasted about 3 h and consisted of two experimental blocks ('low threat', 'high threat').The sequence of these two blocks was balanced across participants in a randomization protocol with the boundary condition of both sequences being equally frequent.Within each block, participants completed a standard CPM paradigm consisting of a baseline condition (test stimuli alone) and a CPM condition (test stimuli + CS).The baseline condition was always applied prior to the CPM condition (see Figure 1).There was a 10-min break between the two blocks where participants filled in a set of questionnaires.The present study was part of a larger study, in which the inhibition of startle EMG responses to loud tones during hand immersion in hot water were assessed in addition (Metzger et al., 2023).

| Conditioned pain modulation
Test stimuli (TS): TS were applied at the participant's left volar forearm by a computer-controlled contact-heat evoked potential stimulator (CHEPS, Medoc, Israel) with a round 27 mm-diameter surface thermode.A pair of thermocouples is embedded in the thermode lamination, which provides an assessment of the skin temperature at the stimulated area.
All TS had a peak temperature of 52°C and a baseline temperature of 40°C, which was held constant between stimuli.Temperature increased with a rate of 70°C/s and decreased with a rate of 40°C/s.Plateau duration of all stimuli was 10 ms.We applied three TS within each stimulation interval, that is, 15 TS per condition (see Figure 1).
The thermode was handheld by the experimenter, who slightly changed thermode position after each TS to prevent receptor fatigue (Granovsky et al., 2008).The timing of TS application was determined in a pseudo-random fashion with the following three limitations: (a) TS should not be presented within the first 2 seconds after picture appearance or in the rating period; (b) the inter-stimulus interval should be at least 10 s; (c) one TS should be applied during each of the three pictures.The exact timing of the TS within the 45 s stimulation interval was as follows: 8 s, 20 s, 37 s (stimulation interval 1); 5 s, 22 s, 33 s (stimulation interval 2); 7 s, 19 s, 34 s (stimulation interval 3); 8 s, 20 s, 37 s (stimulation interval 4); 2 s, 19 s, 36 s (stimulation interval 5).
Conditioning stimuli (CS): A heat stimulus was administered as CS in the CPM conditions by using a circulating water bath (Witeg GmbH, WiseCircu WCB-22, Wertheim, Germany), containing 46°C hot water.The temperature of 46°C was selected as the painful intensity of the CS based on the results of previous studies (Horn-Hofmann et al., 2016;Karmann et al., 2018;Lautenbacher et al., 2008), which leads to moderate levels of pain with small inter-individual variance and a low risk of reaching intolerable pain levels; the latter would make a premature ending of stimulation necessary.The participants immersed their right hands up to 2 cm above the wrist in this water bath (during baseline the hand laid on an armrest in a distance to the water bath far enough to avoid any heating by convection).The water temperature was controlled by a thermostat, and the water was stirred with a force and suction pump to avoid layers of lower temperature around the hand.The CS was always applied to the right hand.
Conditioning stimulation was applied during the five stimulation intervals when programmed for a CPM condition (see Figure 1).Participants were instructed to immerse their hand as soon as the fixation cross-appeared on the screen and to remove the hand upon appearance of the rating scales until the next trial started by appearance of the fixation cross.The message 'Please immerse hand now' was additionally displayed on the screen below the fixation cross.

Affective pictures
As stated in the introduction, experimental threat was manipulated by presenting either aversive pictures showing burn wounds (high-threat condition) or neutral pictures (low-threat condition).Our experimental protocol required 30 pictures per category.These pictures were selected in a two-step procedure which will be described in detail below: (a) pre-selection from the International Affective Picture System (IAPS; Lang, 2005) or the internet based on picture content and reference to normative data (in the case of IAPS pictures); (b) final selection based on affective ratings collected in a pilot study.
Pre-selection of pictures: Neutral pictures were selected from the IAPS based on normative data regarding valence and arousal ratings and on picture content.As described in the IAPS technical manual, pictures are classified as neutral in the case of valence ratings at or around the midline of the Self-Assessment Manikin scale which ranges from 1 to 9 (SAM; Lang, 1980); it is also stated that neutral pictures are commonly rated as less arousing than pleasant or unpleasant pictures (Lang, 2005).Accordingly, our selection criteria for neutral pictures were (a) valence ratings ≥4 and ≤6, (b) arousal ratings ≤4, and (c) neutral contents (e.g., household objects, vehicles; no people).
Aversive pictures were selected from the internet based on picture content; we decided to use pictures depicting burn injuries at the hand due to their associative relation to the pain stimulation used in our experiment (hot water immersion of the right hand as CS).Pictures were retrieved using Google image search with key words such as 'burn' or 'burn wound' in various languages (e.g., German 'Brandwunde ', Dutch 'Brandwond', French 'Brûlure', etc.).All pictures were proportionally altered to be approximately of the same size (about 13 × 9 cm).
Final selection of pictures -pilot study: The pre-selection procedure resulted in 40 neutral and 60 aversive pictures, which were used in randomized order as stimulus material in a pilot study.Thirty participants (female: N = 19) were recruited among psychology students of the University of Bamberg.Participants were asked to download the complete picture set and detailed instructions at home.Ratings should be given on SAM scales, ranging from 1 to 9, with 9 corresponding to positive valence and high arousal, respectively.Participants were asked to return the completed SAM ratings to us per e-mail.
The final set of pictures (30 aversive, 30 neutral i ) were then selected according to the following criteria: Neutral pictures were required to have mean valence ratings of 5 ± 1 and mean arousal ratings ≤2.5; aversive pictures were required to have mean valence ratings ≤2.5 and mean arousal ratings ≥5.5.Outliers from the respective picture category (neutral or aversive) were excluded.
Picture presentation: Each condition (Baseline low threat , CPM low threat ; baseline high threat , CPM high threat ) consisted of five 75 s trials, which were separated each time by a 40 s break, resulting in a total duration of about 9 min per condition (see Figure 1).Each trial started with a fixation cross, which was shown for 5 s, followed by a 45 s stimulation interval where pictures were presented and noxious stimulation was applied and ended with a 25 s rating period.Within each stimulation interval, three pictures were presented for 15 s each.Thus, 15 pictures were shown in each condition.For this purpose, the 30 pictures of each category (neutral, aversive) were randomly split in two subsets containing 15 pictures; the sequence of the 15 pictures within the subset was randomized once by a randomization program also simulating lottery draws and then set.The sequence of the two picture sets within each block (neutral 1 , neutral 2 ; aversive 1 , aversive 2 ) was balanced across participants.

| Ratings
Participants verbally provided pain and threat intensity ratings during the last 25 s of each trial.For this purpose, we used three numerical pain rating scales (NRS) each ranging from 0 to 10; participants were asked to rate the intensity of the TS (0 = not painful, 10 = extremely painful), the threat of the situation (0 = not threatening, 10 = extremely threatening) and (in the CPM conditions) the intensity of the CS (0 = not painful, 10 = extremely painful).The use of these rating scales was explained to the participants at the beginning of the experiment and practiced in a familiarization interval.In addition, rating scales were displayed on the screen during the rating period combined with a visual instruction.
For each of the four conditions (Baseline low threat , CPM low threat ; baseline high threat , CPM high threat ), TS ratings and threat ratings were averaged across the five rating assessments and then subjected to further analysis.Similarly, CS ratings were averaged across the five rating assessments for each of the two CPM conditions (CPM low threat , CPM high threat ) before subjecting them to further analysis.

| EEG recording and parametrization (CEPS)
EEG recording was accomplished by a DC Brain Amp amplifier (Brain Products GmbH, Germany) with a sampling rate of 1024 Hz and a recording bandwidth from 0.1 Hz to 300 Hz.For electrode placement, a commercial CI Electro-Cap Electrode system realizing the international 10-20 system was used (Electro-Cap International, USA).Cz served as reference.The impedances of all electrodes were kept below 5 kΩ.Furthermore, tin electrodes were placed on the mastoids for offline re-referencing the data to regain Cz.In addition, an electro-oculogram (EOG) was recorded.The EOG-biosignal was sampled at a rate of 512 Hz.
EEG data from Cz were analysed offline (Brain Vision analyser, Brain Products, Germany) to determine N2 and P2 latencies as well as N2P2 peak-to-peak amplitudes following mostly the protocol described by Granovsky et al. (2008), which have appeared to be valid indicators of the intensity dimension of central nociception.The 15 potentials evoked by the TS were averaged within each of the four conditions (Baseline low threat , CPM low threat ; baseline high threat , CPM high threat ).The averaged signals were used to determine two components: N2 was defined as the most negative peak in a time window from 200 to 500 ms, P2 was defined as the most positive peak in a time window from 400 to 650 ms (Priebe et al., 2016).For further analysis, the peak-to-peak N2P2 amplitude, that is, the absolute difference between the voltage of the N2 and the P2 was calculated.Consequently, four N2P2-complex amplitude scores resulted for each participant, one for the baseline condition and one for the CPM condition both in the low threat and high-threat block.Additionally, the same number of N2 and P2 latencies for the baseline and CPM conditions both in the low-threat and high-threat block was kept for further analysis.The ASI-3 is a 16-item self-report questionnaire designed to measure anxiety sensitivity, that is, the fear of anxiety-related sensations due to the belief that they might have harmful consequences.It is composed of 3 subscales: Fear of somatic sensations, fear of cognitive dyscontrol, and fear of socially observable anxiety reactions.The items (e. g., 'It scares me when my heart beats rapidly') are rated on a 5-point scale.In the present study, only the sum score was used, which can range from 0 to 64 (min = 0, max = 64; high scores indicating high anxiety sensitivity).Similar to the original English version, the German version (Kemper et al., 2009) demonstrated good internal consistency, with Cronbach's α for the three subscales ranging from 0.83 to 0.92 in two samples (Kemper et al., 2012).
The HADS (Zigmond et al., 1983) was developed as a screening instrument for depression and anxiety, which is applicable in patients treated in non-psychiatric hospital clinics.This is also true for persons with chronic pain, who are often affected by depression and increased anxiety levels.Thus, this questionnaire was selected to ensure the applicability of the complete study design in a chronic pain sample, which we are planning to investigate next.It consists of two subscales (depression and anxiety), each containing seven items.The items are rated on a 4-point scale.The score can range from 0 to 42 (depression: min = 0, max = 21; anxiety: min = 0, max = 21; high scores indicating high levels).Commonly, a cut-off score of 8+ on each scale is used to identify 'possible cases' (Zigmond et al., 1983).In accordance with the original English version, the German version (Herrmann et al., 1995) demonstrated good internal consistency: Cronbach's α = 0.80 for the anxiety scale and Cronbach's α = 0.81 for the depression scale.
The PCS (Sullivan et al., 1995) is designed to measure catastrophizing related to pain.It contains 13 items that can be divided into 3 subscales, namely rumination, magnification, and helplessness.The items (e.g., 'I worry all the time about whether the pain will end') are rated on a 5-point scale ('not at all' = 0, 'all the time'= 4).In the present study, only the sum score was used, which can range from 0 to 52 (min = 0, max = 52; higher scores indicating higher pain catastrophizing).The PCS showed good internal consistency (English version: Cronbach's α = 0.95 [Sullivan et al., 1995]; German version: Cronbach's α = 0.92 [Meyer et al., 2008]).
The FPQ-III (McNeil & Rainwater, 1998) was developed as a comprehensive measure of fear of pain.Participants are instructed to rate the degree of fear they would likely experience if confronted with a variety of potentially painful situations.The FPQ-III contains 30 items that can be divided into three subscales regarding the fear of three types of pain: severe pain ('breaking an arm'), minor pain ('paper cut on the finger') and medical pain ('receiving an injection in the mouth').The items are rated on a 5-point scale.In the present study, only the sum score was used, which can range from 30 to 150 (min = 30, max = 150; higher scores indicating higher fear of pain).The FPQ-III demonstrated good internal consistency: Cronbach's α = 0.92 (McNeil & Rainwater, 1998).
The FPQ-III was translated into German by our workgroup using a standard 'forward-backward-procedure'; translation to German was improved until the original English version and the final German version were sufficiently similar.This German version of the FPQ-III was successfully used in previous studies (Baum, Kappesser, et al., 2013;Baum, Schneider, et al., 2013;Priebe et al., 2015) and demonstrated good internal consistency (Cronbach's α = 0 0.90; Baum, Schneider, et al., 2013).

| Data reduction and analysis
We computed CPM scores (difference CPM -baseline) as a measure of CPM directionality, that is, inhibition versus facilitation, for each of the two blocks (low threat, high threat) both for TS ratings and N2P2-complex amplitudes.Thus, negative CPM scores were indicative of inhibition (pain ratings and N2P2-complex amplitudes reduced in the CPM conditions compared with baseline), whereas positive CPM scores were indicative of facilitation (pain ratings and N2P2-complex amplitudes increased in the CPM conditions compared with baseline).The descriptive values are presented in Table S1 of the supplementary material.In addition to the absolute CPM difference scores, CPM percent change scores were computed and are included in Table S1.
To investigate the effects of the CPM test and the threat manipulation on TS ratings and EEG parameters (N2P2complex amplitudes as well as N2 and P2 latencies), we computed separate repeated measurement ANOVAs with 'condition' (baseline, CPM) and 'block' (low threat, high threat) as within-subject factors.Effects of threat on CS ratings were tested using a paired samples t-test (low threat vs. high-threat block).
As manipulation check, threat ratings were analysed by a repeated measurement ANOVA with 'block' (low threat, high threat) and 'condition' (CPM, baseline) as within-subject factors.
Post-hoc t-tests were computed for detailed analyses in case of significant ANOVA results.Adjusting degrees of freedom with Greenhouse-Geisser correction was necessary in case of violation of sphericity.For F-tests, partial eta squared (η 2 ) is reported as an estimate of effect size; Cohen's s d is reported to describe effect size for paired comparisons.
To test for the stability of responses across conditions and for associations between the two methods of pain assessment (EEG and subjective ratings), we computed Pearson correlations among and between N2P2 peak-topeak amplitudes and TS ratings in the four experimental conditions.Additionally, correlations between the four CPM scores (CPM rating low threat , CPMrating high threat , CPM EEG low threat , CPM EEG high threat ) were computed to evaluate the reliability of CPM across measures and conditions.
Bonferroni correction was applied to correct for multiple testing.The alpha-level was set to 5% for significance testing.SPSS 25 (IBM) was used for all calculations.

| Sample characteristics
The gender ratio of the 37 participants was female = 21 to male = 16.The age range was between 22 and 54 years (mean age 34.9 years; SD = 11.0).Four women took oral contraceptives; of the remaining 17, three were in the first, five in the second, and six in the third phase of their menstrual cycle; two were postmenopausal and information was missing from one participant.Descriptive statistics of questionnaire scores are reported in Table 1.

| Manipulation check: Threat ratings
Descriptive statistics of threat ratings in both blocks and conditions are displayed in Table 1.The ANOVA yielded a significant main effect of 'block' on threat ratings (F(1,36) = 31.302,p < 0.001, η 2 = 0.465); as intended, ratings were higher in the high-threat block compared with the low-threat block (see Table 1).There was no significant main effect of 'condition', but a significant interaction 'condition' × 'block' (F(1,36) = 4.422, p = 0.043, η 2 = 0.109): Within the low-threat block, threat ratings were descriptively higher in the CPM condition compared with baseline whereas the opposite was the case for the high-threat block.However, paired comparisons failed to reach significance (baseline low threat vs. CPM low threat : p = 0.248; baseline high threat vs. CPM high threat : p = 0.211).Thus, according to these findings, our experimental threat manipulation was with only higher ratings in the threat blocks and no other unwanted differences.

| CS pain ratings
We detected no effect of 'block' (low threat vs. high threat) on CS ratings: t(36) = 0.180, p = 0.858, d = 0.014).Overall, the CS (hot water immersion) was rated as moderately painful (M = 3.7, SD = 2.3) and provided by the latter ideal conditions for CPM modulation.

| Association between CHEPs and subjective ratings
Pearson correlations among and between psycho-physiological and indicators of pain responses (N2P2 peak-to-peak amplitudes and TS ratings) are displayed in Table 3. Within each of the two methods, we obtained significant high correlations between the four conditions (see Table 3).However, there were no significant correlations across the two methods (EEG and TS pain ratings; Bonferroni-corrected α = 0.001), suggesting that these two indicators of pain processing were only weakly related in the present study.
F I G U R E 3 N2P2 peak-to-peak amplitudes (mean, and individual scores) in the baseline and CPM conditions for the two experimental blocks.Asterisks indicate significant differences (p < 0.05).
T A B L E 3 Pearson correlations among and between N2P2 peak-to-peak amplitudes and TS ratings in the four experimental conditions.

| of CPM
Pearson correlations among the four CPM scores (CPM rating low threat , CPM rating high threat , CPM EEG low threat , CPM EEG high threat ), indicating the absolute CPM effects, are displayed in Table 4. None of the correlation coefficients passed the level of significance.These findings do not allow for assuming high reliability of the CPM effects, neither across nor within the method of pain assessment (EEG and TS pain ratings; Bonferroni-corrected α = 0.008).Similar non-significant outcomes were obtained when using percent change CPM scores (see Table S2 in the supplementary material).

| DISCUSSION
To our knowledge, our study is one of the few, which have investigated the effect of experimentally induced threat on CPM and the first to use symbolic threat.Symbolic, that is, content-related threat was induced by presenting photographs of burn injuries while participants underwent a CPM paradigm with contact heat as TS and hot water immersion of the contralateral hand as CS.The success of our threat manipulation was confirmed by increased subjective threat ratings.Furthermore, we could demonstrate CPM inhibition by nociceptive evoked brain potentials (CHEPs).However, despite of positive threat and CPM effects, their interaction did not become significant.Thus, contrary to our hypotheses, there was no effect of threat on CPM.The key findings and their implications will be discussed in more detail in the following paragraphs.

| Threat effects on CPM
Our analyses showed a clear inhibitory CPM effect (main effect of 'condition') as regards the CHEPs amplitudes (N2P2) and a smaller CPM effect as regards P2 latency.However, there was no interaction with 'block', indicating no differences in CPM between the low-threat block (neutral pictures) and the high-threat block (burn injury pictures) although subjective ratings confirmed a significant difference in perceived threat between the two blocks.
Thus, we could not demonstrate an effect of subjective threat on CPM.This is in line with the one preceding study investigating threat effects on CPM (Bernaba et al., 2014).The authors used cold water immersion as CS and manipulated threat by asking participants to either imagine that the CS might have harmful consequences like frostbite or to focus on the safety of this procedure.This threat manipulation was adapted from a study by Jackson et al. (2005) where it led to lower pain tolerance and increased pain catastrophizing.However, Bernaba and colleagues found that the CPM effect was unaffected by threat, with both experimental conditions (instructed threat and instructed safety) not differing from a neutral control condition.Taken together, these findings of CPM being immune to two established threat manipulation procedures with proven effects on pain measures (instructed threat and affective pictures) suggest that negative affective states might have a negligible influence on the CPM effect.Combined with the also predominantly negative evidence concerning an association between affect-related personality traits like trait anxiety or pain catastrophizing and CPM (Bouhassira et al., 2013;Granot et al., 2008;Grosen et al., 2014;Horn-Hofmann et al., 2016;Ibancos-Losada et al., 2020;Lee et al., 2013;Marouf et al., 2014;Martel et al., 2013;Nir et al., 2012), the assumption that CPM might be a pain inhibitory circuit, which operates widely independent from emotional influences seems plausible.

| Threat effects on pain perception in general
Our observation that aversive pictures did not even modulate TS and CS ratings considered separately and independently from their CPM interaction is definitely surprising because a wealth of studies suggests effects of emotional pictures viewing on pain perception (Bartley & Rhudy, 2008;De Wied & Verbaten, 2001;Kamping et al., 2013;Kenntner-Mabiala et al., 2007;Meagher et al., 2001;Rhudy et al., 2005Rhudy et al., , 2013;;Roy et al., 2009Roy et al., , 2011;;Zunhammer et al., 2016).One possible reason for this finding is that our study did not include positive pictures and some studies suggest that emotional pain modulation is driven more strongly by pain reduction induced by pictures than by pain enhancement by negative pictures (De Wied & Verbaten, 2001;Kenntner-Mabiala & Pauli, 2005, Kenntner-Mabiala et al., 2007;Rhudy et al., 2005).In addition, our pictures might have induced disgust instead of or additionally to fear/anxiety in some of the participants, and disgustevoking pictures have been shown to be less efficient in modulating pain than fear-evoking pictures (Meagher et al., 2001).Furthermore, picture presentation over 15 s was long compared with other studies (De Wied & Verbaten, 2001;Kenntner-Mabiala & Pauli, 2005, Kenntner-Mabiala et al., 2007;Rhudy et al., 2005), which may have reduced its emotional efficiency in the course of presentation.Since pain is very imperative and definitely efficient also in our experiment in the forms of TS and CS, it may also be that the participants may have been distracted from the pictures.Also, it has been previously stressed that effects of affective pain modulation are possibly overestimated due to publication bias (Zunhammer et al., 2016).

| Dissociation between electrophysiological and subjective measures
An additional interesting finding of our study is the discrepancy between a clear inhibitory CPM effect in CHEPs amplitudes, but no CPM effect in TS pain ratings.This dissociation between subjective and electrophysiological measures is in line with a previous study (Albu & Meagher, 2019), detecting a facilitatory CPM effect for pain ratings which was accompanied by (non-significant) the suppression of CHEPs amplitudes.Similar results have been obtained by two studies using electrical stimulation (Goffaux et al., 2007;Piché et al., 2014), which observed inhibitory CPM effects on evoked potentials that were not mirrored by changes in ratings and nociceptive flexion reflex responses.In addition, two other recent studies found an inhibitory CPM effect for both pain ratings and evoked potentials, but no correlation between the respective change scores (Do et al., 2020;Squintani et al., 2021).Hence, there is quite some evidence for a dissociation between brain activity and subjective or spinal measures of pain perception in CPM paradigms that calls for further investigation.Factors like the subjective painfulness of the CS should be considered as a few studies have shown effects on CPM as regards the TS pain ratings (Nir et al., 2011(Nir et al., , 2012) ) if CS ratings, which were low in the present study, are more substantial as in previous studies from our lab using the same physical stimulus and intensity (Horn-Hofmann et al., 2016;Lautenbacher et al., 2008).The neutral influence of rather weak CS stimuli might have preferentially affected the CPM effects indicated by TS pain ratings because evoked brain potentials (EBP) have shown to be very sensitive to detect subtle CPM effects (Höffken et al., 2017;Jutzeler et al., 2017;Kunz et al., 2014).Worth mentioning is that our CS also suppressed the intensity ratings and startle reflexes evoked by aversive loud tones (Metzger et al., 2023).

| Strengths and weaknesses
There was no evidence given by our questionnaires for heightened general or pain-related anxiety or depressiveness in our non-clinical sample (Baum, Kappesser, et al., 2013;Bocéréan & Dupret, 2014;Horn-Hofmann et al., 2016;Peterson & Reiss, 1992).This is not very surprising given that we excluded participants with mental disorders and pain problems.Thus, we obtained a sample allowing experimental threat manipulation without ethical risk.
To our knowledge, our study is the first to investigate the effects of experimentally induced threat on CPM using both CHEPs and pain ratings as two separate indicators of CPM.The observed dissociation between these two measures which has already been reported before (Albu & Meagher, 2019;Goffaux et al., 2007;Piché et al., 2014) stresses the importance of not solely relying on pain ratings in CPM studies as CPM effects might not be sufficiently captured by the subjective measures.CHEPs have been shown in several studies to be valid indicators of central nociception (e.g., Granovsky et al., 2008;Priebe et al., 2016); however, not completely excluding artefacts from other physiological sources.The correlations between CHEPs and subjective pain rating were low in the present study, which is -as just stated -not the regular but also no exceptional finding.We applied our CPM paradigm in the standard order with a baseline condition first including only TS and thereafter the treatment condition including concurrently both TS and CS.This frequently used design, however, does not control for order effects.
We intentionally selected burn injury pictures as stimulus material as we aimed to maximize the threat value by the symbolic, that is, content-related association between the noxious heat stimulation and the pictures.However, these pictures might have partly induced other emotions as intended (e.g., disgust) and our findings cannot be generalized to aversive pictures with a different content.Threat ratings clearly differentiated between the two blocks but were generally low even in the high-threat block.However, this might be a problem inherent to most experimental threat inductions as assuring the participants of the safety of all procedures is a due ethical concerns.It should also be stressed that the correlations between CPM scores across conditions were low, indicating insufficient reliability of CPM effects.The problem of CPM reliability has been discussed repeatedly (Kennedy et al., 2016;Lewis et al., 2012;Martel et al., 2013;Valencia et al., 2013) and still needs to be addressed in future research.

| CONCLUSIONS
Our study showed no modulation of the CPM effect by pictures of burn injuries that were used as an experimental threat induction.Taken together with a previous study, which found no modulation of CPM by threatening instructions (Bernaba et al., 2014), there is now evidence against CPM being counteracted by situational threat.Future studies should investigate the effects of other threat induction methods on CPM (the introduction of unpredictable variations in CS intensity might be particularly promising) before finally assuming that CPM is a pain inhibitory circuit without major emotional influences due to threat induction.

F
Illustration of the experimental protocol.Each condition consisted of five 75 s trials, containing stimulation intervals with three heat pulses (TS, indicated by the flash) and three pictures (neutral pictures in the low-threat block and aversive pictures in the highthreat block).Hot water immersion (CS, indicated by the wave line) was applied only in the CPM conditions.
2.2.5 | QuestionnairesParticipants filled in a set of questionnaires assessing affective processing in general and relating to pain.This set consisted of German versions of the Anxiety Sensitivity Index (ASI-3;Taylor et al., 2007; German version: Kemper  et al., 2009), the Hospital Anxiety and Depression Scale (HADS;Zigmond & Snaith, 1983; German version  HADS-D: Herrmann et al., 1995), the Pain Catastrophizing Scale (PCS;Sullivan et al., 1995; German version: Meyer  et al., 2008)  and the Fear of Pain Questionnaire (FPQ-III;McNeil & Rainwater, 1998).

T A B L E 1
Descriptive statistics of questionnaire sum scores (mean, standard deviation [SD]).F I G U R E 2 TS ratings (mean, and individual scores) in the baseline and CPM conditions for the two experimental blocks.T A B L E 2 Descriptive statistics of N2 and P2 latencies (mean, standard deviation [SD]) in the four experimental conditions.