Chapter 5 – Experimental fear 157 before the heat pain application. Pain-related fear was reported on a 010 NRS from no fear to worst fear imaginable. These measurements were similar to previous studies 30. The startle eyeblink reflex was measured as an indicator of conditioned fear, as it is modulated by fear-evoking stimuli and by brain areas responsible for affective processing such as the amygdala and the anterior cingulate cortex 31. Eyeblink startle response modulation was measured during the presentation of nocebo and control visual cues. Orbicularis oculi EMG activity was recorded with 3 square EL504 BIOPAC electrodes (2.5x2.5 cm diameter; BIOPAC Systems, Goleta, CA) filled with electrolyte gel. To reduce interelectrode resistance, participants' skin was scrubbed with an exfoliating gel and cleaned with an alcohol wipe. Subsequently, electrodes were placed on the right side of the face according to the specifications proposed by 32. The raw signal was amplified by an isolated EMG100c amplifier module (BIOPAC Systems, Goleta, CA). EMG recordings were acquired through AcqKnowledge (AcqKnowledge software, Biopac Systems, Goleta, CA) at a sampling rate of 2000Hz, with a low-pass filter of 500Hz and a highpass filter of 10Hz. The eyeblink startle response was elicited by use of a white-noise burst of 100 ms duration (i.e. startle probe), with instantaneous rise time, presented binaurally via earphones (Samsung Headset Stereo, model EHS64). The noise was calibrated at approximately 90 dBA, which is safe for hearing 32. These auditory startle probes were delivered within a random 1 s window, 7 s after visual cue presentation and 1 s before heat pain application. The startle probes were presented in two thirds of the acquisition and extinction trials (trials during which participants were not asked to provide a fear rating), including the first and last two extinction trials, which were used to calculate the magnitude of nocebo hyperalgesia at the end of acquisition and extinction, respectively.
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