Participants

Sample size was determined using G*Power38, which indicated that N = 40 was required to detect all relevant physiological effects at a medium effect size (f = 0.25, α error = 0.05, power = 0.8, and assumed correlation of repeated measures = 0.4). This stop rule for data collection was also in line with previous startle studies using emotional facial expression and threat-of-shock instructions3,24,30. Forty healthy volunteers (10 males) were recruited from the students of the psychology department at the University of Mannheim. Participants’ age ranged between 17 and 52 (M = 22.7, SD = 7.1) and the sample was within the normal range of state and trait anxiety (STAI, M = 35.3 and 35.1, SD = 5.8 and 8.8), social anxiety (SPIN, M = 10.9, SD = 8.2), and depression (BDI, M = 5.9, SD = 6.5). All participants were informed about the general study procedure before informed consent was obtained. The ethical review committee of the University of Mannheim approved all utilized procedures and methods. Participants received course credits for their participation.

Stimulus materials and presentation

Face pictures were selected from the Karolinska Directed Emotional Faces (KDEF39), a well-established stimulus set providing pictures of human facial expressions of emotion. Sixteen actors (eight females) displaying happy, neutral, and angry facial expressions, were selected based on visual inspection (i.e., seven raters agreed upon the clarity and recognizability of facial expressions). The KDEF face identifiers were af01, af07, af09, af11, af19, af20, af22, af29, am02, am03, am07, am08, am10, am13, am14, and am25.

All 48 pictures (1024 × 768 pixels) were presented for 6 s separated by variable inter-trial intervals (ITI) ranging from 10 to 15 s to allow response recovery (see Fig. 1). To provoke the defensive startle reflex, auditory startle probes (white noise, 105 dB, 50 ms) were presented during half of the picture trials. The 48 pictures (including the 24 picture-startle trials) were evenly distributed across two experimental blocks (instantiation, reversal) and three facial expressions (happy, neutral, angry), resulting in four picture-startle trials for each experimental condition per participant. To prevent the predictability of auditory stimulation, startle probes were presented at either 4, 4.5, 5 or 5.5 s after picture onset (i.e., while the picture was still visible), and six additional startle probes (three per block) were presented during the ITI. Startle probes were presented binaurally using headphones (AKG K44 Perception) and the average lag between probes was 28.8 s.

Figure 1 Schematic illustration of the experimental procedures and stimulus presentation. (A) After a brief practice run and shock work-up procedure, participants were verbally instructed that one particular emotional facial expression serves as a cue for threat-of-shock (e.g., happy) or safety (e.g., angry) and the first experimental block started (instantiation). Preceding the second experimental block (reversal), a verbal reversal instruction stated that now threat and safety contingencies are reversed (e.g., now angry faces cue threat and happy cue safety). The order in which facial expressions cued threat or safety was tested in two groups (each N = 20 completed the happy-angry or angry-happy threat order). Please note, neutral faces always cued safety. Following each block, threat and safety cues were rated regarding valence, arousal, and perceived threat. (B) Within each block, face pictures displaying happy, neutral, and angry facial expressions were presented (each 6 s) with a variable intertrial interval (ITI, 10 to 15 s). Auditory startle probes were presented occasionally during pictures and ITIs, no shocks were presented during the experiment. Example pictures are taken from the KDEF (identifiers: af01has, am08ans, am10nes, and af20ans). Full size image

Presentation software (Neurobehavioral Systems, Inc., Albany, CA, USA) served to control the stimulus presentation, which was pseudorandom regarding picture sequence (no immediate repetition of the same face actor, no more than three pictures of the same facial expression in a row) and regarding startle presentation (no more than two picture-startle trials in a row). Electric stimuli for the shock work-up procedure were presented using a Digitimer Stimulator DS-5 (up to 10 shocks, with maximal 10 mA, 100 ms).

Experimental task and instructions

Participants’ task was to look at all pictures, which were presented during the two experimental blocks (instantiation and reversal; see Fig. 1). Immediately before the first block started (instantiation), participants were verbally instructed that they might receive up to three electric shocks under specific conditions. One group of participants (N = 20) was told that electric shocks might be administered whenever an angry face is presented (angry = threat) but not when they see a happy face (happy = safety). The other group (N = 20) received the opposite instruction, stating that happy facial expressions cued threat-of-shock (happy = threat), and safety condition being signaled by angry faces (angry = safety). For the second experimental block (reversal), all participants were verbally instructed that now threat and safety contingencies were reversed. Specifically, the previous threat cue becomes safe, and the previous safety cue becomes threatening. Thus, across both experimental groups, happy and angry facial expressions served equally often as instructed and reversed threat and safety cue; neutral faces always signaled safety.

Procedure

Participants completed questionnaires on general and social anxiety and depression (State-Trait Anxiety Inventory [STAI-state/trait], Social Phobia Inventory [SPIN], Social Interaction Anxiety Scale [SIAS], Beck Depression Inventory [BDI]). Sensors for physiological recordings were attached, and an electric stimulation electrode was placed at the right upper arm. Next, a brief shock work-up procedure (without picture presentation) was carried out to ensure the credibility of the threat instruction22,40. To set the shock intensity individually at a level rated as “maximally unpleasant but not yet painful”, participants received up to 10 shocks with increasing intensity. Participants were then instructed that the intensity of the electric shocks given during the experiment would be equal to the most unpleasant test stimulus.

Practice trials served to familiarize participants with the picture and startle presentation procedure and to allow for initial habituation of the startle reflex. Afterward, verbal instructions regarding threat and safety contingencies were given (i.e., which facial expression signals threat-of-shock and which signals safety) and the first experimental block started (instantiation). Following this block, participants rated the hedonic valence and arousal using the Self-Assessment Manikin (SAM)41, and perceived threat of the facial expressions using a visual analog scale ranging from not at all to highly threatening (1 to 10). Then all participants received the instruction that threat/safety contingencies were now reversed (e.g., the threat cue becomes safe, and safety cue becomes threatening), and the second block started (reversal). Facial expressions were rated again after the reversal block. Finally, participants were debriefed. No shocks were presented during the experiment. Thus, results reflect physiological responding during the anticipation (but not experience) of electric shocks.

Data recording and reduction

Psychophysiological measures were recorded continuously with a vAmp amplifier (BrainProducts, Munich, Germany). Startle amplitudes were derived from the electromyogram of the orbicularis muscle using two miniature Ag/AgCl electrodes. The raw signal was recorded at a 1000 Hz sampling rate and frequencies below 28 Hz and above 500 Hz were filtered out with a band-pass filter (24 dB/octave roll-off). Raw electromyogram (EMG) data were rectified and smoothed with a moving average procedure (50 ms) in VisionAnalyzer 2.0 (BrainProducts). Startle responses were scored with an automated procedure and defined as the maximum peak in the 21–150 ms time window following each startle probe. Peak amplitudes were calculated relative to a mean baseline period (50 ms preceding startle response time window)28,42.

As an index of phasic autonomic activation, skin conductance responses (SCRs) were recorded with Ag/AgCl electrodes (constant voltage of 0.5 V; 20 Hz sampling rate) placed at the hypothenar eminence of the non-dominant hand. SCRs to picture onset were calculated as the maximum increase in skin conductance in the interval of 1 to 6 s (relative to a 1 s pre-stimulus period). A minimum threshold of 0.02 µS was used for zero-response detection, and range and distribution corrections were applied. Phasic heart rate changes to picture onset was derived from the electrocardiogram recorded at lead II. The electrocardiogram signal was recorded at 1000 Hz, and frequencies below 0.1 and above 13 Hz were filtered. The weighted HR averages every half second were expressed in terms of differential scores with respect to a 2 s baseline period24.

Data analysis and statistical design

Self-report (valence, arousal, and threat ratings) and physiological data (startle-EMG and SCR) were submitted to (2 × 2) × 2 repeated measures ANOVA, including the within-subject factors Instruction (threat vs. safety) and Block (instantiation Block 1 vs. reversal Block 2), as well as the between-group factor Order (happy-angry vs. angry-happy). The Order referred to the sequence in which emotional facial expression cued threat or safety in which experimental block. Specifically, for the happy-angry order, happy faces served as threat cue during instantiation block (Block 1), and angry faces cued threat during the following reversal block (Block 2). This was reversed for the angry-happy order, in which angry faces during Block 1 and happy faces in Block 2 cued threat-of-shock. Regarding phasic changes in heart rate, an additional factor, Time (12), was implemented to compare half-second changes after picture onset.

To examine the impact of emotional facial expression on the instantiation and reversal of threat instructions, planned comparisons focused separately on each Order (happy-angry vs. angry-happy). Please note that for reasons of brevity and to reduce the complexity of the overall design, neutral faces cued safety in both blocks and were thus excluded from the analyses of instructed and reversed threat. However, supplementary analyses were conducted to compare Facial expression (happy vs. neutral vs. angry) when serving as a safety cue (see Supplemental Material). Covariation analyses were conducted to test the impact of inter-individual differences in reported social- and trait-anxiety on defense activation.

Greenhouse-Geisser corrections were used where relevant, and the partial ƞ2 is reported as a measure of effect size. To control for Type 1 error, Bonferroni correction was applied for post-hoc t-tests.