Dilemmas

For the purpose of this study, we created a database of 16 moral dilemmas and 16 non-moral (control) dilemmas, each of which was edited both in French and in English. The English version of the dilemmas were obtained from the same database of 44 stimuli used by Greene and colleagues (2001): in particular, the moral dilemmas corresponded to the “moral-personal” scenarios in Greene’s study, whereas the “non-moral” controls corresponded to the “neutral” scenarios. These dilemmas were translated ad hoc by a native speaker proficient in English. The original scenarios were also modified to incorporate cultural differences (e.g., changing “$” to “CHF”).

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In a pilot study, we asked proficient speakers in either French (20 volunteers: 12 females; aged 19–50 mean 28.8 SD 6.82 years) or English (37 volunteers: 19 females; aged 20-43 mean 28.81 SD 4.89) to evaluate each of the 44 (25 moral and 19 non-moral) dilemmas in their corresponding language. In particular, individuals taking part to the pilot study rated each dilemma according to the following dimensions. (1) “How much the course of action described in the story is appropriate for you?”;

participants marked the point corresponding to their judgment on a visual analog scale ranging from

“extremely inappropriate” to “extremely appropriate”. (2) “How emotionally engaged were you when reading the vignette?”; participants responded using a visual analog scale ranging from “not engaged at all” to “extremely engaged”. (3) “How comprehensible was the vignette?”; participants responded using a scale ranging from “extremely incomprehensible” to “extremely comprehensible”. The ratings were divided in three blocks, one for each question during which all 44 dilemmas were evaluated. The order of the blocks, and the order of the dilemmas within each block, was randomized across participants.

The data from these rating tasks were used to select 16 moral and 16 non-moral dilemmas, who displayed, in both their English and French formulation, the following properties: (a) moral dilemmas were associated with the lowest appropriateness ratings and with the highest emotional engagement ratings; (b) non-moral dilemmas were associated with the highest appropriateness ratings and the lowest emotional engagement ratings; (c) all dilemmas were associated with high comprehensibility ratings. Supplementary information (SI) Figure 32 reports the data associated with the selected 32 dilemmas, which show a clear dissociation between moral and non-moral scenarios (but nevertheless exhibit considerable variability within each category).

3.3.5 Experimental Setup 3.3.5.1 Task Design

The main experimental task consisted of 76 trials (see Figure 24A-B). On each trial, a 1.5 s predictive cue was presented and followed by an inter-stimulus interval (ISI) with a fixation cross at the screen center. In Experiment 3, this ISI had a fixed duration of 2 s, whereas in Experiment 4 the ISI duration was jittered, ranging from 1.75 s to 6.25 s (average 4 s) with an incremental step of 0.25 s, to accommodate the standard requirements of MRI research. Next, a 3 s countdown period was presented with the instruction “Breathe-out” written on the screen. Once the countdown reached 0, a 1 s “Breathe-in” instruction appeared together with either a thermal or olfactory stimulation.

Olfactory stimuli lasted 2 s (Experiment 3) or 3 s (Experiment 4). The duration of the olfactory stimulus was longer in Experiment 4 following pilot testing in the MRI setting for the olfactometer. Thermal stimuli always lasted 2 s, although additional 3 s were necessary for the thermal stimulator to reach the target temperature. Participants were prompted to breathe according to the instructions. After

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the stimulation, participants rated its unpleasantness on a VAS using the keys at their right hand’s reach. The VAS remained on the screen until a response was delivered for a maximum time of 6 s. The scale was followed by an inter-trial interval (ITI) with a fixation cross at the screen center (Experiment 3: duration 4 s; Experiment 4: duration ranging from 1.75 s to 6.25 s [average 4 s] with an incremental step of 0.25 s).

The olfactory and thermal stimuli were selected in individual basis following the pretesting sessions (see above) so that the two modalities were matched in unpleasantness, and were organized as follows: high-pain (HP), low-pain (LP), high-disgust (HD), and low-disgust (LD). Each of these stimuli were presented following their corresponding cues, which were schematic representations of either a smelly sock (predicting low- and high-disgusting odor) or a flame (predicting low- and high-painful temperatures) (see Figure 24A).

Keep in mind that participants were informed that the cue would always predict correctly the upcoming stimulation. Furthermore, they were also informed that in some trials a dilemma would be presented prior to the stimulus period. Indeed, in 32 of the 76 trials, participants faced a moral or non-moral dilemma between the cue and the stimulus. The 16 non-moral and 16 non-non-moral dilemmas were randomly associated with each of the four cue/stimulus conditions. The association changed on a subject-by-subject basis to minimize putative idiosyncratic confounds of the dilemmas. This yielded four balanced conditions of interest: dilemmas (half moral and half non-moral) preceded by LP, HP, LD and HD cues. For Experiment 4 only, a graphical representation of the previously-presented cue was displayed on the top-left corner of the screen also during the presentation of the dilemmas (this was motivated to potentially enhance any expectancy effect from Experiment 3, also in a noisy environment such as the MRI). The dilemma remained on the screen until participant pressed a key, for a maximum duration of 60 s. Subsequently, participants rated how much a course of action associated with the story was appropriate on a VAS ranging from -50 (extremely inappropriate) to +50 (extremely appropriate). The VAS remained on the screen until a response was delivered for a maximum time of 10 s.

The structure of experiment is fully described in Figure 24B. Participants received 32 “reference” trials in which cues followed directly by their predicted stimuli (8 trials HP, 8 trials LP, 8 trials HD and 8 trials LD), and 32 trials in which dilemmas were presented between the cues and the predicted stimuli according to the 4 conditions described above (8 trials dHP, 8 trials dLP, 8 trials dHD, 8 trials dLD – each of these groups contained 4 moral and 4 non-moral dilemmas). These 32 dilemma trials were the main objective of the experiment. Finally, these 64 trials (32 reference trials + 32 dilemma trials) were further intermingled with 12 trials in which the positive odor was administered following a

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corresponding cue (schematic flower). Experiment 3 was organized in one unique block of about 40 minutes, in which all 76 trials were presented in random order. Experiment 4 was instead split in 4 independent blocks, each lasting about 12 minutes and comprising one fourth (19) of the overall trials, to minimize potential movement artifacts and signal drop in MRI data. The experiments were all run using Cogent 2000 (Wellcome Dept., London, UK), as implemented in Matlab R2012a (Mathworks, Natick, MA).

Figure 24 – Task of Experiments 3-4.

(A) Structure of a dilemma trial in Experiment 3 and 4. Each trial began with a pictorial cue presented and followed by an inter-stimulus interval (ISI). Next, participants were required to read a dilemma, and then judge a presented course of action on a visual analog scale (VAS) of appropriateness. Following the judgement period, participants were then instructed to

“breathe-out” during a countdown period. Then, a “breathe-in” instruction appeared together with the stimulus delivery – which could be either olfactory or thermal. Consequently, participants rated the unpleasnentes associated with the stimulus on a VAS, which was followed by an inter-trial interval (ITI). Four different kinds of cues were presented, predicting the unpleasantness (high/low) and modality (pain/disgust) of the upcoming stimulation (thermal pain/olfactory disgust). Please see Methods section duration details of each event in Experiment 3 and in Experiment 4. (B) Structure of the main experimental session consisting 64 trials (in Experiment 3, all trials were presented in one block; in Experiment 4, the trials were divided into four balanced blocks), half of which were critical or testing expectancy effects on morality (“Dilemma trials”). The remaining half of trials were control stimulation trials (“Reference trials”) in which a dilemma was not presented between the cue and the stimulus periods.

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B

124 3.3.5.2 Procedure and apparatus

Reminiscently to Experiment 1, prior to the main experiment, a battery of questionnaires (STAI, SPQ, PANAS, DS-R, and MFQ) was acquired to capture trait-related individual variability that might impact the performance in the expectancy task. Participants were then seated in a lab-chair in front of a computer screen (Experiment 3) or, laid supine in the MRI patient-table with their head fixated by firm foam pads (Experiment 4). They were then connected to both the olfactometer and the thermode, and underwent the olfactory and thermal stimuli-selection sessions (as described above), followed by the main experiment. After completion of the experiment, participants were debriefed through ad hoc questionnaires aimed at measuring task-related individual variability, such as the degree to which they relied on the cues. Such debrief allowed us to assess whether individuals who might distrust our experimental set-up would exhibit weaker expectancy effects.

In Experiment 3, visual stimuli were projected on a PC screen (Dell) on a screen (1024 × 768 resolution).

Key-presses were recorded on keyboard (Dell). In Experiment 4, the visual stimuli were projected inside the scanner bore with a LCD projector (CP-SX1350, Hitachi, Japan) on a screen (1024 × 768 resolution). Key-presses were recorded on an MRI-compatible bimanual response button box (HH-2 x 4-C, Current Designs Inc., USA).

3.3.6 Physiological measures

Electrodermal activity, finger pulse rate (Experiment 3) and respiration (both Experiment 3 and 4) were recorded throughout the experiment using the MP150 Biopac Systems (Santa Barbara, CA) with a 1000 Hz sampling rate. Pulse rate and electrodermal activity were measured as indices of autonomic arousal. Respiration was measured to monitor the participants’ engagement in sniffing the odorants.

Electrodermal activity was measured with Beckman Ag–AgCl electrodes (8-mm diameter active area) filled with a skin conductance paste (Biopac) attached to participants’ non-dominant hand on the palmar side of the middle phalanges of the second and third fingers. Data were low pass filtered (cut-off 1 Hz) before the analysis. Finger pulse frequency was measured using a photoplethysmographic probe (3.2 cm/1.8 cm, LED type photodetector) placed on the thumb of the non-dominant (i.e., left) hand. Data were reduced to pulse rate in beats per minute (BPM), with low pass filter of 30 Hz and high pass filter of 1 Hz. Finally, respiratory activity was recorded through a 2.5 mm tube (interior diameter) positioned at the entrance of the participants’ right nostril, on the nasal cannula used to deliver the odorants, which was connected to a differential pressure transducer (TSD160A; ± 2.5 cm H2O sensitivity range) to continuously record variations in the nostril airflow. This allowed acquisition of both inhalation and exhalation phases. The respiration data was processed with a low pass filter of 10 Hz.

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For each subject (and, in the case of Experiment 4 for each session), the time-course of each physiological measure was z-transformed, downsampled to 10 Hz, and fed into a first-level analysis using the general linear model (GLM) framework as implemented in PsPM 3.0.2 (http://pspm.sourceforge.net)⁵⁵³. In particular, we run a model using finite impulse response (FIR) as basis function, which poses no a priori assumption on the properties of the event-related response.

Dilemma epochs were modeled with 24 bins of 1 second each, ranging from the last 12 seconds in which the dilemma was read, to the first 12 seconds in which the appropriateness judgment was made. Please note that the duration of the dilemma and its subsequent response changed in a trial-by-trial basis, thus preventing us to use a fixed window to model the FIR; however, the time-window chosen for this GLM covered the majority of the trials in this experiment (only in 2% of the trials dilemmas were read in less than 12 seconds). In similar fashion, we modeled stimuli events with 12 bins of 1 second each, covering both the time in which stimuli were delivered and the subsequent rating. In particular, in line with the analysis of fMRI data (see below), for the electrodermal activity the onsets of olfactory stimuli corresponded to the estimated time in which odorants reached participants’ nose (synchronized with the cued inspiration occurring during the end of the countdown), whereas the onsets of thermal stimuli corresponded to the estimated time in which the thermode reached the plateau temperature (approximately 3 seconds following the end of the countdown). This was not the case of the analysis of the heart rate and respiration, in which stimuli events were always modeled to the onset of the cued-inspiration, as for these measures it is theoretically relevant to model the sniff-related response (see Experiments 1-2)^507,520.

This led, for each GLM, to an overall of 204 parameters, corresponding to 96 parameters associated with dilemma epochs (24 time-bins for LP, HP, LD and HD), 48 parameters associated with stimuli events following the dilemmas (12 time-bins for LP, HP, LD and HD) and 60 parameters associated with stimuli events which didn’t follow the dilemma (“reference trials” – 12 bins for LP, HP, LD, HD, Positive odor). Furthermore, to account for the moral content of each dilemma, we added additional regressors testing the parametrical modulation of the appropriateness rating associated to each story as measured in the pilot study (see SI Figure 32 – for English-speaking participants we used data of English-speaking pilot subjects, whereas for speaking participants we used data of French-speaking pilot subjects). This led to additional 96 regressors for the dilemma epochs and 48 additional regressors for stimuli events following the dilemmas.

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3.3.7.1 Data acquisition

Functional images were acquired using a 3T whole-body MRI scanner (Trio TIM, Siemens) with a 32-channel head coil. We used an EPI sequence with TR = 2100 ms, TE = 30 ms, flip angle = 50°, 36 interleaved slices, 64 x 64 pixels, 3 x 3 x 3 mm3 voxel size, and 3.9 mm slice spacing. Structural images were acquired with a T1 weighted 3D sequence (MPRAGE, TR/TI/TE = 1900/900/2.27 ms, flip angle = 9 degrees, PAT factor = 2, 192 sagittal slices, 1 x 1 x 1 mm3 voxel sizes, 256 x 256 pixels).

3.3.7.2 Preprocessing

Preprocessing of EPI volumes was carried out with the software SPM12 (Wellcome Department of Cognitive Neurology, London, UK). For each subject, the volumes were realigned, coregistered to the structural image, normalized to a template based on 152 brains from the Montreal Neurological Institute (MNI), and finally smoothed by convolution with an 8 mm full-width at half-maximum isotropic Gaussian kernel.

3.3.7.3 First-level analysis

Data were then fed into a first-level analysis using the general linear model framework⁵²² implemented in SPM12. Consistently with the analysis of physiological responses, also in the analysis of the BOLD signal we planned to modulate the appropriateness of each dilemma parametrically. However, as SPM models each block separately, first-level parametrical modulations are only informative of effects occurring within each block, and not across the whole experimental session. For this reason, we decided run a first-level model in which each dilemma epoch, and each subsequent stimulus, was modeled separately, so that their associated parameters can be subsequently fed on a second-level group analysis in which the parametrical effect of appropriateness is tested across all dilemmas (see⁵⁵⁴ for a similar approach).

For each experimental block, we fitted each dilemma reading period, and each dilemma rating period, with a boxcar function with a duration corresponding to the dilemma reading/rating time. In addition, we also modelled each kind of stimulus events following a dilemma as follows: olfactory stimuli were modeled as events of 3 seconds whose onsets corresponded to the estimated time in which odorants reached participants’ nose (synchronized with the cued inspiration occurring during the end of the countdown); thermal stimuli were modeled as events of 2 seconds whose onsets corresponded to the estimated time in which the thermode reached the plateau temperature (approximately 3 seconds following the end of the countdown). Finally, we modeled also the occurrence of thermal/olfactory events which didn’t follow the dilemma (“reference trials”). This led to 29 regressors on each block (8 separate dilemma reading epochs, 8 separate dilemma rating epochs, 8 separated stimuli events

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following the dilemmas, 5 kinds of “reference trials” [LP, HP, LP, HD, Positive odor]) which were convolved with a canonical hemodynamic response function and associated with regressors describing their first order time derivative. To account for movement-related variance and other sources of noise, we also included 8 additional covariates of no interest: these were the six differential realignment parameters, and the average BOLD time-courses extracted from anatomical masks of white-matter and cerebrospinal fluid (CSF). The overall 66 regressors (29 main regressors, 29 first-order time derivative regressors, 8 covariates of no interest) represented each of the four experimental blocks modeled in the first level analysis. Low-frequency signal drifts were filtered using a cutoff period of 128 s. Global scaling was applied, with each fMRI value rescaled to a percentage value of the average whole-brain signal for that scan.

3.3.7.4 Second-level analysis

The average parameter estimates from the first-level model were fed into 3 separate second-level group analyses testing the effects associated with “dilemma reading epochs”, “dilemma rating epochs” and “stimuli epochs following dilemma”, respectively. For each of these epochs, the corresponding parameters were fed into a second-level flexible factorial analyses with a factor

“condition” with 4 levels (LP, HP, LD, HD), “gender” as a factor with 2 levels (males, females), and

“subjects” as a random factor. In addition, we tested, separately for each of the four conditions, a parametric modulator “appropriateness”, which consisted the dilemma appropriateness’ ratings from the pilot study. In modeling the variance components, we allowed both the “condition” and the

“gender” factors to have unequal variance between their levels, whereas the factor “subjects” was modelled with an equal variance. Activations in these analyses were considered as significant if exceeding an extent threshold allowing p < 0.05 correction for multiple comparison for the whole brain (corresponding to 127 consecutive voxels - Friston et al., 1993), with an underlying height threshold corresponding to p < 0.001 uncorrected [t(504) ≥ 3.10, for all the activation contrasts].

3.4 Results

Following Experiments 1-2 we focused on the “reference trials” in which stimuli were delivered in absence of moral dilemmas (see SI Figure 33). In particular we excluded all subjects/sessions, in which the “reference trials” with high pain or high disgust stimuli were rated as neutral (HP ≥ -5 or HD ≥ -5), or considered as equally (or more) pleasant than the low pain or low disgust stimuli respectively (HP

≥ LP or HD ≥ LD). Furthermore, as the aim of the study was to inspect pain and disgust under equal unpleasantness, we excluded also those subjects/sessions in which HP and HD exhibited a difference in unpleasantness greater than 15. As a result, 33 out of 100 blocks (25 subjects * 4 blocks per subject) collected in Experiment 4 were excluded from the final analysis (all subjects in Experiment 3 fulfilled these criteria). Such procedure insures that, for the remaining part of the data, high pain/disgust

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stimuli were indeed experienced as more unpleasant than their corresponding control conditions, and matched with one another. Supplementary information shows all details about the Reference trials of the selected blocks, which are associated with physiological and neural responses consistent (although not identical) with that of previous studies (Experiments 1-2). Keep in mind, however, that sessions were never discarded on the basis of data from the stimulation trials following a dilemma, which were the real conditions of interest for the present study.

3.4.1 Behavioral Responses