Physiological Responses

3.4.2.1 Moral Processing

For both electrodermal activity and heart rate, we fed the parameter estimates from the first level FIR into a Linear Mixed Model with Unpleasantness (high vs. low), Modality (pain vs. disgust), subjects’

Gender (male vs. females), and Time-Bins (including the last 12 seconds in which the dilemma was read, to the first 12 seconds in which the appropriateness was rated) as fixed factors, and subjects’

identity as random factor (see SI Figure 34A-B). The analysis of electrodermal activity revealed a significant Unpleasantness main effect (t(1900) = -2.53, p = 0.01), and a significant Unpleasantness * Modality interaction (t(1900) = 2.16, p = 0.03), reflecting decreased electrodermal activity when expecting HP relative to LP, HD and LD (see SI Figure 34A). No other effects were found to be significant (│t│s ≤ 1.51, [n.s.]). The analysis of heart rate revealed a main effect of Time-Bins (see SI Figure 34B), reflecting cardiac acceleration from 3 to 5 seconds from the onset of the judgment (0 vs. 2 seconds:

t(1900) = 2.93, p = 0.003; 0 vs. 3 seconds: t(1900) = 4.03, p < 0.001; 0 vs. 4 seconds: t(1900) = 3.17, p

= 0.002) and also during the reading period, approximately 10-11 seconds before the onset of the judgment (-11 vs. 0 seconds: t(1900) = 2.34, p = 0.02; -10 vs. 0 seconds: t(1900) = 2.05, p = 0.04). This modulation, however, was not influenced by any expectancy condition (all other tests: │t│s ≤ 1.57, n.s.). We then repeated the same analysis for the parameters in the first level GLM testing the parametrical effect of the appropriateness in the preceding dilemma. This analysis led to no significant effect, neither for the electrodermal activity, nor for the heart rate (│t│s ≤ 1.82, n.s.).

132 3.4.2.2 Pain & Disgust Processing

Figures 27 and 28 illustrate the physiological responses associated with the stimuli trials following a dilemma event, as estimated in the first level model with FIR basis function (see Methods section). In the analysis of the electrodermal activity (from Experiment 3), we considered as signal of interest those time-bins occurring at 4 or later seconds from the stimulus onset (skin conductance response is expected to raise between 1-4 seconds from the stimulus onset⁵²⁰, and fed them to a Linear Mixed Model with Unpleasantness (high vs. low), Modality (pain vs. disgust), subjects’ Gender (male vs.

females), and Time-Bins (from 4 to 12 seconds) as fixed factors, and subjects’ identity as random factor. This analysis revealed a significant modulation of electrodermal activity 11 and 12 seconds following the stimulus onset in the shape of both Unpleasantness * Time-Bin (4 vs. 11 seconds: t(700) = 2.18, p = 0.03; 4 vs. 12 seconds: t(700) = 2.51, p = 0.01) and Unpleasantness * Modality * Time-Bin interactions (4 vs. 11 seconds: t(700) = -1.99, p = 0.05; 4 vs. 12 seconds: t(700) = -2.27, p = 0.01). As visible in Figure 27A, these effects reflect significant modulation of electrodermal activity 11-12 seconds following a HP (relative to LP) stimulations, but no reliable effect associated to HD relative to LD. No other effect was found to be significant (│t│s ≤ 1.80, [n.s.]).

We then repeated the same analysis for the parameters in the first level model testing the parametrical effect of the appropriateness (as measured in the pilot study) in the preceding dilemma.

This analysis revealed only a Modality * Unpleasantness interaction (t(700) = -2.14, p = 0.03). No other effects were found to be significant (│t│s ≤ 1.12, [n.s.]). To further interpret this interaction effect, we repeated the model separately for each modality: when focusing only on pain, no effects were found to be significant (│t│s ≤ 1.24, [n.s.]), whereas when focusing on disgust an Unpleasantness main effect was found (t(340) = -2.32, p = 0.01 – no other effects significant │t│s ≤ 1.06, [n.s.]) reflecting HD (relative to LD) negatively modulated by the appropriateness of the preceding dilemma. Indeed, as visible in Figure 27B the difference in electrodermal activity between HD and LD is stronger following an inappropriate dilemma (appropriateness < 0), rather than an appropriate dilemma (appropriateness > 0). This is not the case for pain.

133

Figure 27 – Electrodermal activity of stimulation epochs following dilemmas.

(A) Electrodermal activity (as GLM β-estimates) for thermal pain (left side) and olfactory disgust (right side) stimulations following dilemmas, as estimated in the first level model with FIR basis function (see Methods section) for Experiment 3.

(B) Effect of the appropriateness in the preceding dilemma on the electrodermal activity (as GLM β-estimates) of subsequent (high vs. low) stimulations: thermal pain (left side) and olfactory disgust (right side). Stimulus delivery occurs at the first time bin.

As for the analysis of heart rate (from Experiment 3), consistently with previous studies^507,520, we observed significant cardiac acceleration induced by the cued inspiration (in the case of the present study between 1-6 seconds – see Figure 28A). Interestingly, previous studies showed that the heart rate following this respiration-induced acceleration was modulated by the unpleasantness of the olfactory^127,507 and thermal (see Experiment 1) stimulus. Therefore, for each subject, we calculated the average parameter estimate from the general linear model in the time-bins following 6 seconds from the stimulus onset, and fed them to a Linear Mixed model with Unpleasantness (high vs. low), Modality (pain vs. disgust) and subjects’ Gender (male vs. females) as fixed factors, and subjects’

identity as random factor. This analysis revealed a significant main effect of Unpleasantness (t(60) = 2.51, p = 0.015), reflecting higher heart rate for unpleasant as opposed to control stimuli, regardless of the Modality. No other main effect or interaction was found to be significant (│t│s ≤ 1.79, [n.s.]).

We then repeated the same analysis for the parameters in the first level model testing the parametrical effect of the appropriateness in the preceding dilemma. This analysis led to no significant effect (│t│s ≤ 1.82, [n.s.]).

A

B

134

In the analysis of respiration (both Experiments 3 and 4), we focused on the first-three time-bins which reflect the period in which the cued inspiration took place (Figure 28B-C). Our cued-sniffing paradigm was designed to stabilize as much as possible the amount of air inspiration across conditions; however, previous studies documented that, even under this paradigm, highly unpleasant odors elicit (compared to their tailored control) less inspiration of air volume^507,520. Consistently, also in our experiment, we found a marginal Unpleasantness*Modality interaction in Experiment 3 (t(60) = 1.92, p=0.059), but not in Experiment 4 (t(60) = 0.18, [n.s.]). However, for Experiment 4 we found main effect of Modality (Experiment 3: t(60) = 1.15 [n.s.]; Experiment 4: t(60) = 2.61, p = 0.011). No other main effect or interaction was found to be significant (│t│s ≤ 1.91, [n.s.]). To better understand the direction of these effects, we then compared three contrast of interest: HD>LD, HP>LP, and HD>HP. In both experiments, the inspired air volume was lower when the stimulus was of HD relative to HP (Experiment 3: t(60) = 2.46 , p = 0.016; Experiment 4: t(60) = 2.51, p = 0.014). Lower inspiration volume between HD and LD was also observed in Experiment 3 (t(60) = 2.03 , p = 0.046) but not in Experiment 4 (t(60) = 0.88, [n.s.]) – see Figure 28B-C). As for the case of heart rate, the analysis of the parameters testing the parametrical effect of the appropriateness revealed no significant effect (│t│s ≤ 1.25, [n.s.]).

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Figure 28 - Heart rate and respiration pattern (as GLM β-estimates) of stimulation epochs following dilemmas, as estimated in the first level model with FIR basis function (see Methods section).

(A) Cardiac response during thermal pain (left side) and olfactory disgust (right side) stimulations in Experiment 3. (B) Respiration pattern during thermal pain (left side) and olfactory disgust (right side) stimulations in Experiment 3 and (C) in Experiment 4. Respiration values below 0 refer to inspiration of air, whereas values above 0 refer to expiration of air. In all the subplots, stimulus delivery occurs at the first time bin. The different expectancy conditions preceding the dilemmas are labelled as follows: LP – expectancy for low pain, HP – expectancy for high pain, LD – expectancy for low disgust, and HD – expectancy for high disgust.