Hypnotic modulation of pain perception and of brain activity triggered by nociceptive laser stimuli

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1 cortex xxx (2012) 1e17 Available online at Journal homepage: Research report Hypnotic modulation of pain perception and of brain activity triggered by nociceptive laser stimuli Elia Valentini a,b, *, Viviana Betti b,c,lihu d,e and Salvatore M. Aglioti a,b a Sapienza Università di Roma, Dipartimento di Psicologia, Italy b Fondazione Santa Lucia, Istituto di Ricovero e Cura a Carattere Scientifico, Italy c Department of Neuroscience and Imaging and Institute for Advanced Biomedical Technology, D Annunzio University Foundation, Chieti, Italy d Key Laboratory of Cognition and Personality, Ministry of Education, Chongqing, China e School of Psychology, Southwest University, Chongqing, China article info Article history: Received 18 August 2011 Reviewed 2 January 2012 Revised 9 January 2012 Accepted 15 February 2012 Action editor Sergio Della Sala Published online xxx Keywords: Cingulate cortex Hypnosis Laser-evoked potentials (LEPs) Nociception Posterior parietal cortex abstract Introduction: Neuroimaging studies indicate that hypnotic suggestions of increased and decreased pain intensity and unpleasantness may modulate somatosensory and cingulate cortex activity, respectively. Methods: Using a within subject design and a strict subject selection procedure, we tested in High (Highs) and Low (Lows) hypnotically suggestible individuals whether hypnotic suggestions of sensory and affective hypoalgesia or hyperalgesia differentially affected subjective ratings of laser-induced pain and nociceptive-related brain activity in the timeand time-frequency domain. Results: Hypnotic modulation of pain intensity and unpleasantness affected subjective ratings of laser-induced pain only in Highs. Such modulation was more specific for unpleasantness manipulation and more evident for suggestions of hyperalgesia. Importantly, Highs and Lows showed increase and decrease of P2a and P2b wave amplitudes and gamma band power, respectively. Conclusions: Hypnotic suggestions exerted a topedown modulatory effect on both evoked and induced-cortical brain responses triggered by selective nociceptive laser inputs. Furthermore, correlation analyses indicated that gamma power modulation and suggestions of hyperalgesia may reflect the process of allocating control resources to salient and threatening sensory-affective dimensions of pain. ª 2012 Elsevier Srl. All rights reserved. 1. Introduction Pain perception can be influenced by a number of purely cognitive manipulations such as attentional focusing (Bantick et al., 2002), meditation (Perlman et al., 2010), pain expectation and anticipation (Ploghaus et al., 1999; Porro et al., 2002), placebo/nocebo experience (Wager et al., 2004; Kong et al., 2008) and, relevant to the present study, hypnotic suggestion (Rainville et al., 1999; Faymonville et al., 2006; Derbyshire et al., 2009). Positron emission tomography (PET) studies indicate that hypnotic suggestions of decreased and increased unpleasantness of thermal pain caused decrease and increase * Corresponding author. Dipartimento di Psicologia, Sapienza Università di Roma, Via dei Marsi 78, Roma, Italy. address: elia.valentini@uniroma1.it (E. Valentini) /$ e see front matter ª 2012 Elsevier Srl. All rights reserved. doi: /j.cortex

2 2 cortex xxx (2012) 1e17 of neural activity in the anterior cingulate cortex (ACC), respectively. No such effect was found in the primary and secondary somatosensory cortices (SI-SII) (Rainville et al., 1997). In contrast, suggestions of decreased or increased intensity of pain brought about decrease and increase of neural activity in SI and SII but not in the ACC (Hofbauer et al., 2001). However, the notion of a neat double dissociation has been questioned by studies indicating that not only SI and SII, but also the mid-cingulate (MCC), the posterior (PIC), and anterior insular cortex (AIC) may code for the intensity of pain (e.g., Coghill et al., 1999; Buchel et al., 2002), and for its perceived threat value (Wiech et al., 2010). Moreover, differences in amplitude of event-related brain potentials (ERPs) induced by hypnotic suggestions concerning pain perception are inconsistent, as absence of modulation (Halliday and Mason, 1964; Meier et al., 1993), increased amplitude (Friederich et al., 2001), or reduction of middle and late latency potentials following focused hypnotic hypoalgesia (De Pascalis et al., 2001, 2008) were reported. Here, we expand previous knowledge by testing, for the first time in the same subjects, whether hypnotic suggestions of unpleasantness or intensity of pain may differentially modulate the affective and sensory dimensions of pain experience as well as the brain activity triggered by selective nociceptive stimuli. We used laser stimuli to specifically activate Ad and C nociceptors in the most superficial skin layers (Bromm and Treede, 1984). Such stimuli elicit a number of transient brain responses (laser-evoked potentials e LEPs) in the electroencephalogram (EEG) (Carmon et al., 1976). The magnitude of these brain responses can be modulated by attention (e.g., Legrain et al., 2002, 2003a) and stimulus predictability (Brown et al., 2008; Clark et al., 2008), or by crossmodal integration (Longo et al., 2009). It has been suggested that the early N1 potential may represent an index of bottomeup processing of the ascending nociceptive input, whereas the later N2 and P2 waves may be considered as indices of topedown perceptual integration of the somatosensory input (Lee et al., 2009). It is relevant that the laser-evoked P2 component can be functionally differentiated in two sub-components (the P2a and P2b waves) that are susceptible to bottomeup attentional capture by novel/deviant nociceptive stimuli (Legrain et al., 2009b). Importantly, in addition to the analysis of time- and phaselocked deflections (i.e., ERPs), we measured also transient modulations of the ongoing oscillatory EEG activity. These modulations may be expressed either as an increase (eventrelated synchronization e ERS) or as a decrease (event-related desynchronization e ERD) of EEG power, not phase-locked to the stimulus onset. To date, an integral description of phase and non-phase-locked time-frequency representation of cortical activity triggered by selective nociceptive input during hypnosis is lacking (see Croft et al., 2002; De Pascalis et al., 2004). 2. Materials and methods 2.1. Participants EEG data were collected from twenty-four healthy female subjects, aged (mean standard deviation (SD)) who were selected from a sample of a hundred and forty-six female subjects tested for their hypnotic suggestibility (Braffman and Kirsch, 1999). All participants were righthanded (Handedness Edinburgh Inventory: M ¼ 16.2, SD ¼ 2.1; Oldfield, 1971). None of the subjects had a history of neurological or psychiatric disease. None of them used drugs potentially interfering with pain sensitivity. Participants gave written informed consent and were paid for their participation. The procedures were approved by the ethics committee at the IRCCS Fondazione Santa Lucia, and conformed to the standards required by the Declaration of Helsinki Hypnotic suggestibility assessment and group selection To control for the reliability of the hypnotic scores, and for any difference related to the hypnotist s gender and voice timbre, two psychologists trained as hypnotists, one male (E.V.) and one female (V.B.), administered the Italian version (Traina, 1976) of the standardized Stanford Hypnotic Susceptibility Scale, Form C (SHSS: C; Weitzenhoffer and Hilgard, 1962; De Pascalis et al., 2000). All participants were thus preliminary tested with the SHSS: C on two sessions, once by each of the hypnotists according to a testeretest procedure. The possible scores ranged between 0 (no hypnotic suggestibility) and 12 (maximal suggestibility). Subjects who scored 8 were assigned to the High hypnotically suggestible group (Highs: N ¼ 12, M ¼ 10, SD ¼ 1.1, range ¼ 8e12). Subjects who scored 4 were assigned to the Low hypnotically suggestible group (Lows: N ¼ 12, M ¼ 1.5, SD ¼ 1.0, range ¼ 0e4). The order of the hypnotists was counterbalanced and each hypnotist was kept blind to the colleague s assessment until the two sessions were completed. In addition, it is worth noting that this procedure also served as a control for the possible instability of the hypnotisability trait (Fassler et al., 2008). In fact, subjects in whom the scores provided by two hypnotists differed more than two points, were not included in the study. Hypnotic induction was obtained using the induction by eye closure item of the SHSS: C (unscored item 0 of the scale). This induction procedure invites the subject to focus his own attention on a small black pin on the wall while listening to the hypnotist delivering suggestions of relaxation and absorption (e.g., What I would like you to do is to relax in the chair, look steadily at the black point, and listen to my voice. Meanwhile, I ll give you some instructions that will help you to relax and focus your attention even more. [.] ), progressively becoming suggestions of drowsiness and sleepiness (e.g., [.] You are going to get much more drowsy and sleepy. Soon you will be deep asleep but you will have no trouble hearing me. You will not wake up until I ask you to.), and finally guide the subject to deepen such state of relaxation and absorption through a count-up from one to twenty (e.g., [.] Five.Six.Seven.You are sinking into a deep, deep sleep. Nothing will disturb you.. I would like you to hold your thoughts on my voice and those things I ask you to think of. [.]) Nociceptive stimulation Radiant heat stimuli were delivered to the dorsum of the right hand with an infrared neodymium yttrium aluminum

3 cortex xxx (2012) 1e17 3 perovskite (Nd:Yap) laser with a wavelength of 1.34 mm (El.En, Florence, Italy). At this wavelength, the laser pulses activate directly the Ad and C-fiber nociceptive terminals located in the superficial layers of the skin (Iannetti et al., 2006). The laser beam was transmitted via an optic fiber and its diameter was set at 6 mm. Laser pulses were directed to the dorsum of the right hand on a 5 5cm 2 area, defined prior to the beginning of the experimental session. To avoid nociceptors fatigue and sensitization, the location of the irradiated spot was manually shifted after each stimulus. The inter-stimulus interval (ISI) was set between 7 and 14 sec. Overall mean intensity was 2.5 Joules (J) and the duration 3 msec Determination of pain perception before and after hypnosis induction To assess possible changes of stimulus perception due to relaxation and hypnosis induction (Langlade et al., 2002; Sharav and Tal, 2004; Emery et al., 2008) the following procedure was used: subjects were asked to report the intensity of laser stimuli with a numerical description (0 ¼ no sensation, 1 ¼ low warmth, 2 ¼ moderate warmth, 3 ¼ high warmth, 4 ¼ non-painful pinprick sensation, 5 ¼ mild pain, 6 ¼ moderate pain, 7 ¼ high pain; 8 ¼ unbearable pain). The energy of the laser stimulus was individually adjusted using the method of limits (step size ¼.25 J) until a value of 6 was reached. At this stimulation intensity no withdrawal reflexes and motor contractions were observed. The assessment was performed before and after hypnosis induction (see Fig. 1) Suggestion protocols and experimental procedure Fig. 1 provides a schematic representation of the experimental design and procedure. Each subject was submitted to two experimental sessions, where the hypnotist manipulated, in different blocks, Intensity (INT) or Unpleasantness (UNP) of the laser pain using specific hypnotic suggestions. The interval between the two experimental sessions was at least one week. Within each experimental session the selected twenty-four subjects underwent LEPs recording during three different suggestion protocols derived by Rainville et al. (1999; see this study for exact verbatim of hypnotic suggestions) and adapted to the specific circumstance by a native English speaker who is highly proficient in Italian: (1) increase of perceived Intensity or Unpleasantness of the pain sensation ( Up direction e e.g., suggestions of increased pain Unpleasantness: [.] Although you will continue to experience normal sensation, your experience will seem surprisingly more unpleasant, surprisingly more uncomfortable, surprisingly more disturbing than you might have expected [.]); or (2) decrease of perceived Intensity or Unpleasantness of the pain sensation ( Down direction e e.g., suggestions of decreased pain Intensity: When you feel the unpleasant stimulus on your hand dorsum, you may be surprised to notice how much less intense the sensation is than you might have expected it to be, how it tends to feel only warm [.] ). The effects of these modulatory directions were compared to the effect observed during (3) a control relaxation suggestion protocol ( Control suggestion), derived from the hypnosis induction protocol of the SHSS: C, and characterized by deep muscle relaxation (e.g., You are relaxed, you are deeply relaxed. When you think of relaxing, your muscles will relax. You can reach a state of deeper, more complete relaxation [.] ), increased sleepiness and pleasant warmth sensation (e.g., You feel a pleasant warm tingling throughout your body as you get more and more tired and sleepy.sleepy.drowsy.drowsy and sleepy [.] ), numbness (e.g., You feel numb or heavy in your legs and feet.in your hands and arms.throughout all your body.as though you were settling deep into the chair [.] ), breathing function amelioration (e.g., You are breathing freely and deeply.freely and deeply. You can feel your breath flowing slowly and deeply.slowly and deeply [.] ). The order of the two experimental sessions and of the three suggestion protocols was counterbalanced across subjects. For each experimental block, thirty laser stimuli were delivered to the right hand dorsum. After every five stimuli, subjects rated pain intensity and unpleasantness by imagining a 101-point visual analog scale (VAS). At the bottom of this scale (no color e blank) zero represented no pricking/ burning/itching for the intensity assessment and not at all unpleasant for the unpleasantness assessment. At the top of this scale, a hundred (dark red) represented intolerable intensity or intolerable unpleasantness. Intensity and unpleasantness ratings were asked according to a pseudorandom order (no more than twice the same order within each block). Subjects were instructed to refer the ratings specifically to the experimental stimulus context and to avoid comparing the ongoing painful sensation with prior, different experiences of pain. The distinction between pain intensity (i.e., how strong was the sensation?) and pain unpleasantness (i.e., how bothering and concerning was the sensation?), and between the two scales, was defined before the experiment started. To maintain the subjects attention and expectation high, the suggestion protocol relative to each block was repeated every ten laser stimuli. To check that the hypnotic state was kept stable, the control relaxation protocol was repeated between the stimulation blocks and the subjects were tested with three randomly chosen items of the SHSS: C. This between-blocks procedure allowed us to minimize possible carry over effects (Hofbauer et al., 2001) due to the counterbalanced design (Up direction before than Down and vice versa). In fact, the procedure allowed us to reset the subject to the hypnotic relaxation baseline condition, before the beginning of each recording block EEG recording EEG recordings were obtained from fifty-six electrodes placed according to the positions of the 10e20 International System (Fp1, Fp2, Fpz, AF3, AF4, AF7, AF8, F3, F4, F5, F6, F7, F8, FC1, FC2, FC3, FC4, FC5, FC6, FT7, FT8, Cz, FCz, Fz, Oz, POz, Pz, C3, C4, C5, C6, T7, T8, TP7, TP8, CP1, CP2, CP3, CP4, CP5, CP6, CPz, P3, P4, P5, P6, P7, P8, PO3, PO4, PO7, PO8, O1, O2); the remaining two surface electrodes were positioned for the vertical and horizontal electro-oculographic (EOG) recording. The reference was at the nose and the ground at AFz. Electrodes impedance was kept below 5 KU. The EEG signal was amplified and digitized at 1000 Hz.

4 4 cortex xxx (2012) 1e17 Fig. 1 e Schematic representation of experimental design and procedure. Low (Lows) and High (Highs) hypnotically suggestible individuals underwent two experimental sessions. In each session either intensity or unpleasantness of the laser-induced pain perception was manipulated. In each session, blocks of decrease and increase of the manipulated pain property were suggested according to formal protocols, repeated at constant intervals. Moreover, general relaxation suggestions were used as control suggestions. For each block, thirty laser stimuli were delivered. Subjective ratings were collected every five stimuli while suggestion protocols were repeated each ten laser stimuli. Between each stimulation block, hypnosis depth was checked by scoring in randomly chosen items from the SHSS: C. Pre-post hypnotic induction changes of pain sensitivity were measured so as to check the efficacy of the procedure in the two groups of subjects. The spiral image is used here only as a symbol to represent the hypnotic induction and suggestions EEG data analysis Preprocessing EEG data were analyzed using Letswave ( webnode.com) (Mouraux and Iannetti, 2008) and EEGLAB (Delorme and Makeig, 2004). EEG signal passed through an offline.1e90 Hz band-pass filter. Pre-stimulus (500 msec) and post-stimulus (1000 msec) segments were extracted from the EEG, and the whole epoch was baseline corrected by the prestimulus. Preliminary analysis included visual inspection of epoched data. Epochs with amplitude values exceeding 100 mv (i.e., epochs likely to be contaminated by an artifact) were excluded from additional analysis. At least 92% of the trials were used for averaging Analysis in the time domain Epochs belonging to the same experimental condition were further low-pass filtered (high edge: 30 Hz) and averaged together, time-locked to the onset of each stimulus in a recording block. This procedure yielded six average waveforms (one for each experimental condition: intensity control e INT CTRL, Unpleasantness control e UNP CTRL, Intensity down e INT DWN, Unpleasantness down e UNP DWN, Intensity up e INT UP, Unpleasantness up e UNP UP ) for each subject. For each experimental condition, single-trial latency and baselineto-peak amplitude of the evoked potential were measured. The N1 wave was measured at the central electrode controlateral to the stimulated right hand side (left hemisphere, C3), referenced to Fz and defined as the negative deflection (peaking at approximately 160 msec) preceding the N2 wave (Hu et al., 2010). The N2 was measured at the Cz electrode and was defined as the most negative deflection after stimulus onset (peaking at about 200 msec). According to Legrain et al. (2009b; see also Bastuji et al., 2008) the positivity showed two overlapping sub-components with different latencies and scalp topographies. The P2a wave peak was measured at FCz in the 260e360 msec post-stimulus interval (peaking at about 330 msec at centro-frontal electrodes), while P2b wave was measured at Pz in the 340e440 msec post-stimulus (peaking at about 380 msec at centro-parietal electrodes) Analysis in the time-frequency domain An estimate of the magnitude of oscillatory activity as a function of time and frequency was obtained for each EEG

5 cortex xxx (2012) 1e17 5 epoch. Because this estimate is a time-varying expression of oscillation magnitude regardless of its phase, averaging these estimates across trials discloses both phase-locked and nonphase-locked modulations of signal magnitude, provided that these modulations are both time-locked to the onset of the event and consistent in frequency (i.e., the latency and frequency at which they occur are reproducible across trials). The estimate of each single EEG epoch was obtained using the windowed Fourier transform (WFT). In this study, a fixed Hanning window with a duration of 200 msec was used in the WFT to explore both phase-locked and non-phase-locked EEG information. For each single trial, the WFT translated EEG responses into a complex time-frequency spectral estimate with the explored frequencies ranging from 1 to 90 Hz in steps of 1 Hz. For each estimated frequency, results were displayed as an event-related percentage (ER 100%) increase or decrease of oscillation amplitude relative to a pre-stimulus reference interval (e.4 to e.1 sec before the onset of laser stimuli), according to the following formula: ER(t,f ) ¼ [F(t,f ) R( f )]/ R( f ), where F(t,f ) is the signal spectrum at a given time t and frequency f, while R( f ) is the signal spectrum at the frequency f averaged within the reference interval (Pfurtscheller and Lopes da Silva, 1999). Across-trial averaging of time-frequency representations produced six average EEG spectrograms of the oscillatory amplitude as a function of time and frequency (one for each experimental condition: Intensity control e INT CTRL, Unpleasantness control e UNP CTRL, Intensity down e INT DWN, Unpleasantness down e UNP DWN, Intensity up e INT UP, Unpleasantness up e UNP UP ) for each subject. Four time-frequency regions of interest (ROIs) were defined in the spectrograms obtained at Cz, where the main spectral events maximally express their magnitude (Iannetti et al., 2008). The time-frequency limits of these time-frequency ROIs, defined on the basis of previous studies (Mouraux et al., 2003; Ploner et al., 2006), were as follows: LEP (1e8 Hz and 100e500 msec), Laser-induced beta band synchronization (ERS; 10e20 Hz and 100e500 msec), Laser-induced alpha band desynchronization (ERD; 7e13 Hz and 400e900 msec), Laser-induced gamma band oscillations (GBO; 30e90 Hz and 250e450 msec). Within each time-frequency ROI, ER 100% power values were extracted by computing the mean of the 10% pixels displaying the highest increase (LEP, ERS, GBO) or decrease (ERD). This top 10% summary measure reflects the higher ER 100% values within each window of interest, with the aim of reducing the noise introduced by including near-to-zero values. This approach, which was successfully used to analyze both EEG (Iannetti et al., 2008) and fmri data (Iannetti et al., 2005; Mitsis et al., 2008), proved suitable to disclose condition-specific effects (for a review see Mouraux and Iannetti, 2008) Statistical analysis Analysis of variance (ANOVA) Subjective ratings of laser-induced pain, LEPs (raw N1, N2, P2a and P2b latencies and amplitudes), time-frequency ER 100% power (LEP, ERS, ERD, and GBO), obtained during suggestion of increase (Up) or decrease (Down) of pain were expressed as the difference (Δ) from hypnotic relaxation condition (Control). Normalized subjective ratings of laser pain were entered in two separate three-way mixed-model ANOVAs (one for each Manipulated pain dimension, Intensity, Unpleasantness) with Rated pain dimension (intensity, unpleasantness) and Pain manipulation direction (Down, Up) as within subject variables, and Group (Lows, Highs) as between subjects variable. Both time- and time-frequency domain values were entered in two separate two-way mixed-model ANOVAs (one for each Manipulated pain dimension, Intensity, Unpleasantness) with Pain manipulation direction (Down, Up) as within subject variables and Group (Lows, Highs) as between subjects variable. Post-hoc comparisons were computed by means of NewmaneKeuls range test ( p <.05). We also tested whether the time-frequency spectral values within the post-stimulus time interval were significantly different from those within the pre-stimulus time interval by means of a bootstrapping method (Delorme and Makeig, 2004; Durka et al., 2004). This approach provided further evidence for robustness and reliability of the nociceptive-related signal in the time-frequency spectrum (details about the method are provided as Supplemental material) Correlation analysis To test any functional relationship between electrophysiological dependent variables significantly modulated by hypnotic suggestion and subjective ratings in a given condition (e.g., P2a amplitude observed during UNP UP and unpleasantness ratings during this condition in Highs), we computed Pearson s r correlation coefficients. The Bonferroni-corrected p value was.012. This value was obtained correcting the standard p ¼.05 by the number of comparisons for each direction (Down and Up), each rating type (intensity and unpleasantness), separately for each group (Lows and Highs) and Manipulated pain dimension (i.e., four comparisons). 3. Results Means and standard errors (mean SE) of subjective and electrophysiological data which resulted significantly different among conditions and groups in the ANOVA are reported in Table Subjective reports Hypnotic manipulation of pain Intensity Results of the three-way ANOVA indicate a significant main effect of Pain manipulation direction (F 1,22 ¼ 9.87, p ¼.005) with higher subjective pain ratings during Up than Down direction (1.89 vs e.48). Importantly, the significance of the Pain manipulation direction and Group interaction (F 1,22 ¼ 5.19, p ¼.03) is explained by significantly higher pain ratings during suggestion of increased than decreased pain Intensity in Highs (for both intensity and unpleasantness) (2.78 vs 1.00, p ¼.005). No such effect was found in Lows (.99 vs.04, p ¼.93) (Fig. 2, leftpanel) Hypnotic manipulation of pain Unpleasantness Results of the three-way ANOVA revealed a significant main effect of Pain manipulation direction (F 1,22 ¼ 7.43, p ¼.01) with

6 6 cortex xxx (2012) 1e17 Table 1 e Means and standard errors (mean ± SE) of subjective and electrophysiological data which resulted significantly different among conditions and groups in the ANOVA (ratings, P2a and P2b LEP amplitudes, GBO power). Data obtained during suggestion of increase (Up) or decrease (Down) of pain are expressed as the difference from hypnotic relaxation condition (Control). Normalized data Subjective ratings LEP P2a (mv) LEP P2b (mv) GBO (ER100%) Manipulation of Unpleasantness Manipulation of Intensity Manipulation of Unpleasantness Manipulation of Intensity Manipulation of Unpleasantness Manipulation of Intensity Manipulation of Unpleasantness Manipulation of Intensity unpleasantness ratings intensity ratings unpleasantness ratings intensity ratings.08 (.43).01 (.43).37 (.32).53 (.35) 1.51 (1.60) 1.03 (1.48).06 (1.14) 2.03 (1.23).18 (.12).30 (.18) Lows Down direction Up direction 1.13 (.43).85 (.40).10 (.43).17 (.41) 1.28 (1.28) 2.38 (1.74).04 (1.12) 1.45 (1.36).07 (.25).66 (.32) Highs Down 1.24 (.69).78 (.81).02 (.68) 1.08 (.40).42 (2.68).77 (1.29).63 (1.42) 1.90 (1.09).30 (.35).14 (.23) direction Up direction 2.73 (.67) 2.83 (.67) 1.63 (.72) 2.71 (.84) 1.67 (2.22) 3.78 (1.50).93 (1.46) 2.50 (1.27).98 (.34).86 (.41) higher subjective pain ratings during Up than Down direction (1.00 vs.49). Importantly, the Pain manipulation direction and Group interaction was significant (F 1,22 ¼ 5.61, p ¼.03). Post-hoc tests indicate that intensity and unpleasantness pain ratings in Highs were significantly higher during Up than Down direction (2.23 vs.38, p ¼.008) and with respect to Up (2.23 vs.45, p ¼.02) and Down (2.23 vs.14, p ¼.03) manipulation direction in the Lows. The significance of the Rated pain dimension Pain manipulation direction Group interaction (F 1,22 ¼ 9.24, p ¼.006) indicated that the modulatory effect of hypnosis had a specific effect on subjective ratings in the Highs only. As indicated by the post-hoc tests, Unpleasantness manipulation induced higher subjective ratings of unpleasantness during Up direction with respect to unpleasantness ratings during Down direction (2.71 vs 1.08; p <.001), and to intensity ratings during Up (2.71 vs 1.63; p <.001) and Down (2.71 vs.02, p <.001) direction. In addition, ratings of intensity during Up direction were significantly higher than ratings of intensity (1.63 vs.02, p ¼.007) and unpleasantness (1.63 vs 1.08, p ¼.003) during Down direction. No such effects were found in the Lows ( p s >.05), in which no difference between intensity ratings during Up (.10) and Down (.37) direction, and unpleasantness ratings during Up (.17) and Down (.53) direction was found (Fig. 2, right panel) Electrophysiological analysis in the time domain Grand average LEP waveforms along with their relative scalp topographies (N1 as measured at C3 referenced to Fz; N2 as measured at Cz referenced to the nose) during hypnotic suggestion blocks concerning pain Intensity or Unpleasantness, and during the three manipulation directions (Control, Down, Up) in Lows and Highs, are shown in Fig. 3. The difference between late latency positivity in P2a (as measured at FCz referenced to nose) and P2b (as measured at Pz referenced to the nose) waves, along with their relative scalp topography, is represented in Fig Hypnotic manipulation of pain Intensity No significant main effects or interactions with Group ( p s >.05) for the normalized N1, N2, P2a and P2b amplitudes were found Hypnotic manipulation of pain Unpleasantness Analysis of P2a peak amplitudes showed a significant main effect of Group (F 1,22 ¼ 4.27, p ¼.05), which was accounted for by higher values in Highs than in Lows (1.45 vs.15 mv). The interaction Pain manipulation direction and Group was significant (F 1,22 ¼ 5.52, p ¼.03). Post-hoc comparisons disclosed higher amplitude modulation in Highs with respect to Lows during Up direction (3.78 vs 2.38 mv, p ¼.03), and in Up versus Down manipulation direction (3.78 vs.76 mv, p ¼.03) within Highs (Fig. 5, top right panel). Also the analysis of P2b peak amplitudes showed a significant main effect of Group (F 1,22 ¼ 6.39, p ¼.02) for the P2b peak amplitudes which was entirely explained by higher amplitude in Highs than in Lows (1.49 vs.88 mv) (see Fig. 5, bottom right panel). No significant main effects or interactions for N1 and N2 amplitudes were found. Moreover, no significant modulation for any of the LEP latencies was found.

7 cortex xxx (2012) 1e17 7 Fig. 2 e Hypnotic modulation of subjective pain ratings in the different experimental conditions. y axis represents means and SE (error bars) of the D index (Up or Down directions e control hypnosis). Hypnotic manipulations were effective in the Highs group and especially during Unpleasantness manipulation (* indicates p <.05). Left panel: Intensity manipulation. The interaction of Pain manipulation direction and Group shows how higher pain (regardless of the reported rating concerned intensity or unpleasantness of pain) was felt during Up direction, and lower pain during Down direction of manipulation. Right panel: Unpleasantness manipulation. The Rated pain dimension 3 Pain manipulation direction 3 Group interaction shows how a more selective effect on pain unpleasantness than intensity was exerted by the hypnotic manipulation of Unpleasantness in Highs. In sum, the analysis of time-domain data revealed that the hypnotically-induced effect was specific for the amplitude of the late P2a and P2b waves during the Unpleasantness manipulation blocks. In particular, when suggestions of pain increase and decrease were focused on pain Unpleasantness, an opposite modulation of the P2a and P2b amplitudes took place in Highs and Lows. Importantly, a strong between group dissociation was detected following hypnotic suggestion of increased pain Unpleasantness only for P2a amplitudes. No group-specific effect of modulation direction was observed on P2b amplitudes Electrophysiological analysis in the time-frequency domain Grand average spectrograms of nociceptive-related brain activity (as measured at Cz referenced to the nose) during both Intensity and Unpleasantness manipulations, and during the three manipulation directions (Control, Down, Up) in Lows and Highs, are shown in Fig. 6 (A, B panels). Nociceptive-related brain activity obtained through WFT was substantiated by the bootstrapping analysis which clearly shows a consistent post-stimulus significance (with respect to pre-stimulus) of evoked and induced activity in both groups. This additional analysis revealed also a significant between group difference in the GBO ROI, due to a larger post-stimulus significant activity in Highs than in Lows (see Supplemental material) Hypnotic manipulation of pain Intensity No significant main effects or interactions with Group ( p s >.05) for the normalized LEP, ERS, ERD were found. The analysis of GBO ROI ER 100% showed an interaction of Pain manipulation direction and Group (F 1,22 ¼ 5.12, p ¼.03). Posthoc test revealed that the modulation was mainly accounted for by higher amplitude during Up direction in Highs with respect to Lows during Up direction (.98 vs.07, p ¼.03) and Down direction (.98 vs.18, p ¼.05), and a trend to significant higher amplitude in Highs during Up direction with respect to Down direction in the same group (.98 vs.30, p ¼.06) (see Fig. 6C panel, top graph) Hypnotic manipulation of pain Unpleasantness Manipulated pain dimension showed no significant main effects or interactions with Group ( p s >.05) for the normalized

8 8 cortex xxx (2012) 1e17 Fig. 3 e Group-level average LEP waveforms in the different experimental conditions: Negativities. Waves and topographies of peak amplitude are shown for each experimental condition. Hypnotic relaxation (control condition) is represented in blue color scale. Down and Up Pain manipulation directions are represented in green and red color scale, respectively. Top panel shows signals recorded at electrode C3 re-referenced to Fz. Bottom panel shows signals recorded at electrode Cz referenced to the nose (N2eP2 biphasic complex) with scalp topography of N2 wave. x axis, time (s); y axis, amplitude (mv). Lows: low hypnotically suggestible individuals. Highs: high hypnotically suggestible individuals. Manipulation stands for the factor Manipulated pain dimension : Intensity (INT); Unpleasantness (UNP). Pain manipulation directions : DWN; CTRL; UP.

9 cortex xxx (2012) 1e17 9 Fig. 4 e Group-level average LEP waveforms in the different experimental conditions: Positivities. Waves and topographies of peak amplitude are shown for each experimental condition. Hypnotic relaxation (control condition) is represented in blue color scale. Down and Up Pain manipulation directions are represented in green and red color scale, respectively. Top panel shows signals recorded at electrode FCz (P2a wave detected between 260 and 360 msec) and its scalp topographies. Bottom panel shows signals recorded at electrode Pz (P2b wave detected between 340 and 440 msec) and its scalp topographies. x axis, time (s); y axis, amplitude (mv). Lows: low hypnotically suggestible individuals. Highs: high hypnotically suggestible individuals. Manipulation stands for the factor Manipulated pain dimension : Intensity (INT); Unpleasantness (UNP). Pain manipulation directions : DWN; CTRL; UP.

10 10 cortex xxx (2012) 1e17 Fig. 5 e Hypnotic modulation of LEP P2a and P2b amplitude in the different experimental conditions. y axis shows means of peak amplitude (mv) and SE (error bars) of the D index (Up or Down modulation direction e control hypnosis). The significant two-way interaction (* indicates p <.05) indicates that hypnosis functionally dissociated between Highs and Lows only during Unpleasantness manipulation. Top panel: P2a wave amplitudes. The interaction of Pain manipulation direction and Group shows that while P2a amplitude increased during Up manipulation direction in Highs, it decreased when the suggestions focused on perceived Unpleasantness in Lows. In addition, P2a amplitude was higher during Up manipulation direction than during Down direction in Highs. Bottom panel: P2b wave amplitudes. The main effect of Group shows that while higher P2b amplitudes were observed following hypnotic manipulation of Unpleasantness in Highs, reduced modulation of P2b amplitude was found in Lows. LEP, ERS and ERD. A significant main effect of Group was detected on GBO ROI (F 1,22 ¼ 5.71, p ¼.03) which was explained by higher gamma band ER 100% amplitudes in Highs than Lows (.36 vs.48). The significance of interaction of Pain manipulation direction and Group (F 1,22 ¼ 8.70, p ¼.007) is accounted for by higher amplitudes during Up direction in Highs with respect to Lows during both Down (.86 vs.30, p ¼.02) and Up (.86 vs.66, p ¼.005) direction. Moreover, the GBO amplitude was higher during Up than Down direction in Highs (.86 vs.14, p ¼.006) (Fig. 6, panel C, bottom graph). In sum, GBO was

11 cortex xxx (2012) 1e17 11 Fig. 6 e Time-frequency decomposition in the different experimental conditions. Grand average time-frequency representation of nociceptive-related oscillatory activity (as measured at Cz) during both Intensity (panel A) and Unpleasantness (panel B) manipulation, in both Lows and Highs (left and right side in panel A and B, respectively), and during the three manipulation directions (Control e blue, Down e green, Up e red). Four time-frequency ROIs were defined. For each ROI, top 10% (ER 100%) increase (LEP, ERS, GBO) or decrease (ERD) of signal power relative to the pre-stimulus interval (L.4 to L.1 sec before the onset of laser stimuli) was extracted (color scale). Note the increase of gamma amplitude in Highs during Up direction of both Intensity and Unpleasantness Manipulated pain dimension. Panel C: the y axis shows means of top 10% oscillatory amplitude (ER 100%) and SE (error bars) of the D index (Up or Down modulation direction e control hypnosis) during both Intensity (top graph) and Unpleasantness (bottom graph) manipulation. The significant two-way interaction (* indicates p <.05) indicates that hypnotic suggestions functionally dissociated between Highs and Lows, and both during Intensity and Unpleasantness manipulation in the GBO ROI. Top graph: Intensity manipulation. The interaction of Pain manipulation direction and Group shows that the main group difference is determined by increased amplitude during Up manipulation direction in Highs with respect to Lows. Bottom graph: Unpleasantness manipulation. GBO magnitude was higher during Up manipulation direction with respect to Down direction in Highs, and higher with respect to both Up and Down direction in Lows. affected during both Manipulated pain dimensions but only for Up direction and mainly for Unpleasantness manipulation Correlations between subjective ratings and neurophysiological variables The correlation between GBO power and ratings of pain intensity during suggestions of increased Unpleasantness was significant only in Highs (r ¼.77, p ¼.003) (Fig. 7). In a similar vein, the correlation between GBO power and pain unpleasantness during suggestions of increased Unpleasantness showed a trend toward a significant positive correlation only in Highs (r ¼.65, p ¼.02). All the remaining correlation coefficients were not significant ( p s >.05) Pre-post hypnotic induction changes of pain sensitivity The laser intensity values (joules) ensuing in ratings of 6 (moderate pain perception) were significantly different before and after hypnotic induction in the two groups (Highs: M ¼ vs , t ¼ 2.46, df ¼ 11, p ¼.03; Lows: M ¼ vs , t ¼ 2.15, df ¼ 11, p ¼.05).

12 12 cortex xxx (2012) 1e17 Intensity and Unpleasantness (Fig. 6, panel A and B). It is worth noting that during manipulation of Unpleasantness, GBO magnitude was increased in Highs and decreased in Lows (Fig. 6, panel C, bottom graph). (d) The modulation of the subjective experience of pain was paralleled by the variation of GBO magnitude during suggestions of increased Unpleasantness in Highs Asymmetric hypnotic modulation of affective and sensory subjective ratings of pain Fig. 7 e Correlation analysis between subjective reports and electrophysiological variables. Pearson s r correlation between subjective ratings of pain intensity during suggestions of increased Unpleasantness ( y axis) and GBO power (x axis) in Highs, expressed as difference with control hypnosis during Unpleasantness manipulation (see paragraph 2.8 Statistical analysis). The scatterplot shows how the higher the rating of pain intensity, the higher the magnitude of GBOs (r [.77, p [.003). In other words, when the hypnotist suggested an increase of the unpleasant sensation Highs perceived the nociceptive laser stimulus as increasingly more intense. Such heightened perception was paralleled by the higher increase in gamma band synchronization. 4. Discussion Unlike previous studies where the modulation of sensory and affective dimensions of pain was explored separately in different groups of subjects (Rainville et al., 1997; Hofbauer et al., 2001), here we used a more robust within subject design and demonstrate that hypnotic suggestions may modulate both sensory and affective dimensions of the subjective experience of pain. The effect of hypnotic manipulation was found only in Highs and it was more specific for Unpleasantness than Intensity manipulation (Fig. 2). Moreover, one main point of novelty of the present study is that, for the first time, we characterize the modulatory effect of hypnotic suggestions on the time- and time-frequency brain responses triggered by selective nociceptive laser stimuli. The neurophysiological effects of hypnotic manipulations can be summarized as follows: (a) no early-stage sensory processing activity (N1 wave) was significantly affected by modulatory hypnotic suggestions (Fig. 3); (b) conversely, a clear modulation of the late P2a and P2b sub-components amplitudes revealed both between groups (P2a and P2b) and within Highs group differences (P2a) during Unpleasantness manipulation (Figs. 4 and 5); (c) an increase of oscillatory gamma band ERS (30e90 Hz) activity was detected only in Highs; interestingly, the effect was significant only during hyperalgesia suggestions (Up direction) and involved manipulation of both pain The influence of specific hypnotic modulation on subjective ratings of tonic heat-pain induced by 1-min hand immersion in hot water has been previously demonstrated in between subjects design studies (Rainville et al., 1999; Hofbauer et al., 2001). Our study significantly expands previous evidence by testing both affective and sensory qualities of phasic thermal pain in the same subject. Moreover, our strict subject selection procedure allowed us to highlight the difference in interindividual reactivity to the hypnotic manipulation of the two fundamental dimensions of pain experience. In the Highs group manipulation of both Intensity and Unpleasantness proved effective in changing subjective reports of pain. However, suggestions of Unpleasantness had a more selective influence than suggestions of Intensity, a result in keeping with what observed in long-term meditation practitioners (Perlman et al., 2010). Our findings replicate the observation that hypnotic manipulation of affective pain dimension modulates self-reported unpleasantness rather than intensity reports (cf. Rainville et al., 1999). In contrast, manipulation of Intensity modulated both affective and sensory qualities of the pain experience (see Fig. 2). It is also relevant that hypnotic suggestions of hyperalgesia (Up) were more effective than suggestions of hypoalgesia (Down) in both groups. This result may have to do with the saliency and motivational relevance of hyperalgesic sensory events, which typically trigger rapid escape reactions to avoid harmful situations or reduce the detrimental effects of potentially damaging stimuli (Melzack and Casey, 1968; Loeser and Melzack, 1999). Information on the actual sensory magnitude (intensity) of painful stimuli is crucial when individuals try to statistically interpret their feelings and define the quality of their perception as requested in our experiment. Thus, self-mitigating pain perception seems more difficult than self-increasing it Hypnotic modulations of LEP waveforms In our study, N1 and N2 amplitudes were not modulated by hypnotic suggestions (as compared to a control hypnotic relaxation condition). In contrast, P2a and P2b amplitudes were significantly modulated during suggestions of pain Unpleasantness but not of pain Intensity (Figs. 3 and 4). Moreover, while the modulation of P2b amplitude was nonspecific (higher in Highs independently of the suggestion direction), P2a amplitude in Highs reflected the suggestion direction (higher during Up suggestion) (Fig. 5). Interestingly, a significant reduction of laser N2 and P2 amplitudes with respect to a control relaxation condition took place in Highs during non-hypnotic distraction from pain, whereas a tendency to an increase of amplitude of the

13 cortex xxx (2012) 1e17 13 same components was found during hypnotic hypoalgesia (Friederich et al., 2001). Our findings are consistent with studies reporting that attending to the incoming laser stimulus could induce a strong enhancement of the vertex N2eP2 complex (Siedenberg and Treede, 1996; Garcia-Larrea et al., 1997; Yamasaki et al., 1999), whereas no significant variations of the earlier N1 wave amplitude can be appreciated (see Garcia-Larrea et al., 1997). Similarly, our results are in agreement with the increase of P2 wave amplitude exerted by high expectation of impending threat (Hauck et al., 2007; Clark et al., 2008). Relevant to the present study is that the laser P2 wave consists of two morphologically and topographically different sub-components (P2a and P2b) (Bastuji et al., 2008; Legrain et al., 2009b). Interestingly, the conditions that bring about selective enhancement of laser P2a amplitude are reminiscent of those modulating the P3a component evoked by deviant/ novel auditory and visual stimuli (Squires et al., 1975; Escera et al., 1998; Katayama and Polich, 1998). It has been suggested that the laser P2a and auditory or visual P3a may share common brain mechanisms involved in orienting attention to new salient events (Legrain et al., 2002, 2003a, 2003b, 2005). Principal component analysis isolates the Supplementary Motor Area (SMA) or ACCeMCC as generators for the P3a (Dien et al., 2003), while P3b has been associated to temporaleparietal cortices generators (Bledowski et al., 2004). Tellingly, the latency of ACCeMCC activation is compatible with the time window of the laser-evoked P2a, while the parietal topography reflected by the P2b wave may be associated to a major involvement of the PPC (Legrain et al., 2009b). In keeping with general models of selective attention (e.g., Miller and Cohen, 2001) and their application in hypnosis (Egner et al., 2005), the observation that hypnotic suggestions for focused attention to pain Unpleasantness modulate the magnitude of the P2a and P2b waves (Fig. 5) may be interpreted as the product of the interplay of excitatory and inhibitory fronto-parietal processes (Corbetta and Shulman, 2002) during monitoring and orienting of attention toward noxious events (e.g., Duncan and Albanese, 2003; Villemure and Bushnell, 2009). In this vein, the increased P2a in Highs during suggestions of increased pain Unpleasantness (Up direction) may reflect neural processes involved in involuntary shifts of attention toward the expected unpleasant sensory event. Similar results have been obtained when processing unexpected events during oddball tasks (Legrain et al., 2003a, 2009b). That P2b was equally affected by suggestions of reduction and increase of pain Unpleasantness may imply the activation of neural processes related to the detection of the attended events regardless of their relative saliency (Legrain et al., 2002; Polich, 2007). In addition, the observation that the P3b amplitude increases gradually with task relevance (Sawaki and Katayama, 2006; Fogelson et al., 2009) is compatible with the P2b relative increase (Highs) or decrease (Lows) of amplitude during either modulation direction Hypnotic modulations of nociceptive oscillatory activity By adopting the approach described in previous studies (Iannetti et al., 2008; Valentini et al., 2011), here we observed that sensory and affective hypnotic manipulations determined the appearance of a laser-induced gamma band ERS (30e90 Hz) in Highs during suggestions of hyperalgesia (Up direction) (Fig. 6, panel A and B). A higher between group difference was observed during the Unpleasantness manipulation, where suggestions of a more unpleasant and uncomfortable pain sensation resulted in a significant increase of GBO magnitude in Highs. No similar effect was found in Lows (Fig. 6, panel B and C, bottom graph). It is worth noting that modulation of induced gamma activity, particularly when occurring 200 msec after stimulus onset, may reflect topedown processing (e.g., Tallon-Baudry et al., 1997; Engel et al., 2001; Karakas et al., 2001). Modulations of gamma band neuronal oscillations clearly appear integral to higher order mental functions as attention and meditation (Lutz et al., 2008; Cahn et al., 2010), crossmodal (Kanayama et al., 2009), sensorimotor integration (Bauer et al., 2006; Szurhaj and Derambure, 2006), empathy for pain (Betti et al., 2009), and working memory (Herrmann et al., 2010). Our result may suggest that hypnosis in Highs reflects a state of highly focused attention (Barber, 1960; Hilgard, 1965; Tellegen and Atkinson, 1974) rather than an impairment of executive functions (Crawford, 1994; Gruzelier, 1998). This suggestion is in keeping with a study indicating that highly focused attention ( mental absorption ) and full sensory relaxation ( mental relaxation ) are the core features of consciousness during hypnosis (Rainville et al., 2002). Although the relationship between hypnotizability and attention may be more complex then what suggested to date (e.g., Egner et al., 2005; Varga et al., 2011), we posit that the effect observed on GBO power may be explained by the higher allocation of control resources recruited by suggestions of hyperalgesia. In addition, the allocation of neural resources may rather be associated to the negative valence emotional state that would follow the suggestions of hyperalgesia, which affects perceptual and executive functions (e.g., Pessoa, 2009 for a useful discussion). It is worth noting that the higher homeostatic relevance of negative valence is supported by the observation that nocebo hyperalgesia can be induced by nonhypnotic verbal suggestion while placebo hypoalgesia strongly relies on conditioned learning (Colloca et al., 2008) A neurocognitive model of hypnotic modulation of pain processing Our study does not confirm the phenomenological and neural double dissociation between manipulated pain feature and sensory or affective regions of the pain matrix (Rainville et al., 1997; Hofbauer et al., 2001). One may observe that the discrepancy between our data and the two above studies may concern the higher spatial resolution of the PET (Rainville et al., 1997; Hofbauer et al., 2001) versus LEPs technique. However, we suggest that hypnotic modulation mainly influences pain unpleasantness and neural processing in the socalled affective node of the pain matrix. Indeed, nociceptive laser stimuli induce a fast increase of activity in the posterior mid-cingulate cortex (pmcc) (Frot et al., 2008), but they influence much less SII activity (Yamasaki et al., 1999; Nakamura et al., 2002). Moreover, the latency of ACCeMCC activation is compatible with the time window of the laser-