Participants

In total, 376 healthy individuals (mean age: 24.5 years, SD: 3.3; 258 females) participated in the study that was part of a large behavioral test-battery. 349 participants (242 females, 22% smokers) had valid data on both consecutive days of the experiment and were included in the analyses. As such, the participants were not matched regarding smoking status and other individual differences, but rather to represent an age-specific proportion of smokers and non-smokers in Germany (Bundeszentrale für gesundheitliche Aufklärung, 2015). The effects of gender and trait anxiety in this sample have been published elsewhere (Haaker et al, 2015b; Lonsdorf et al, 2015) and current analyses controlled for their influence. Participants gave written informed consent. The study was approved by the local ethics committee in Hamburg (Ärztekammer Hamburg). Overall, 22% of the participants were smokers and were allowed to smoke freely before the experiment on both days (see Table 1 for sample description and (Haaker et al, 2015b) for sample and task details).

Table 1 Sample Description of Smoking and Non-Smoking Participants Full size table

Stimuli

Two geometric symbols (circle and triangle) served as CSs presented on either a blue or yellow screen background, which served as contexts. Assignment of the symbols and the background colors were counterbalanced across participants. Stimuli were presented using Presentation Software (NeuroBehavioral Systems, Albany California, USA). The US consisted of a train of three square-wave pulses of 2 ms each (DS7A, Digitimer, Welwyn Garden City, UK) delivered on the right dorsal hand and was individually adjusted to a level of maximum tolerable pain (subjective ratings from 0: ‘I feel nothing’ to 10: ‘maximally unpleasant’, see Table 1) before the experiment. There were no difference between smoking and non-smoking participants with regard to the US current amplitude on day 1 (non-smokers mean: 4.3 mA, SD: 5.7; smokers mean: 4.4 mA, SD: 3.70, p>0.97) or day 2 (non-smokers mean: 4.4 mA, SD 5.71; smokers mean: 4.5 mA SD: 3.68, p>0.81), and importantly there were no differences between groups in the subjective rating of the painfulness of the US on either day (day 1: non-smokers mean: 6.12, SD: 1.30; smokers mean: 6.16, SD: 1.23, p>0.81; day 2: non-smokers mean: 6.01, SD: 1.39; smokers mean: 6.03, SD: 1.23, p>0.92).

Paradigm

Fear conditioning and extinction learning was employed as a within-subject design (Figure 1a). The protocol consisted of an A 1 B 1 A 2 B 2 design, with two interleaved blocks of fear conditioning in one context (A 1 and A 2 : danger context) and extinction in another context (B 1 and B 2 : safe context) on day 1 (learning). Each block consisted of 12 presentations of the CS+ and the CS−, and each CS had a duration of 3 s. The intertrial interval (ITI) was jittered in duration between 2.5 and 4 s. During conditioning in the danger context, the CS+ was paired in 50% of trials with the aversive, electrotactile US (onset 2.5 s after CS+ onset), whereas the CS− was never paired with the US. During extinction in the safe context, both CSs were never paired with a US. Hence, on day 1, the CR (differential response to the CS+>CS−) in the danger context on day 1 can be used to index learned fear, whereas the CR in the safe context provides an inverse measurement of fear inhibition, a high CR indicating a deficit in inhibition (in particular, inhibition of fear to the CS+).

On day 2 (retrieval), US intensity was individually adjusted again. This was followed by 10 interleaved blocks of each context employing an A 1 B 1 A 2 B 2 …A 10 B 10 design. Each block consisted of one presentation of both the CS+ and the CS−, but without any US presentation in both contexts. Producing a CR to a CS+ in the danger context (‘renewal’, Bouton, 2002; Vervliet et al, 2013) can be considered adaptive, given the danger context has been associated with threat on day 1. However, in the safe context, a high CR would indicate a deficit in the retrieval or expression of the fear-inhibitory memory formed during extinction in this context on day 1. Hence, an adaptive response profile on day 1 would consist in a marked differentiation of CRs as a function of context (high in danger, low in safe). We term this ‘contextual fear inhibition’.

Physiological Measurements

Skin conductance responses (SCRs) were recorded as previously described (Haaker et al, 2015b). In short, skin conductance was recorded using a BIOPAC MP35 amplifier (BIOPAC Systems, California, USA) and self-adhesive Ag/AgCl electrodes placed on the palmar side of the left hand on the distal and proximal hypothenar eminence. Data were down-sampled to 10 Hz, phasic SCRs were automatically scored as an increase in skin conductance within 1–3.5 s after stimulus onset and raw SCR amplitudes were then logarithmized and range-corrected [(SCR/SCRmax)+1]. Non-responders were defined as participants not showing any US response during the first half of the experiment (day 1) and excluded from all SCR analyses (N=32). Additional 24 participants were excluded from the analyses, due to missing data on one of the days. In total 290 participants were included in the SCR analyses (61 smokers). Several control analyses (see previous publication, Haaker et al, 2015b) confirmed that USs that preceded or followed the CS presentations did not bias SCRs on day 1.

Subjective Measurements

In addition, participants rated their stress/fear/tension evoked by the CSs as well as their US expectancy after each block (4CS+ and 4CS− trials) on day 1 and after each block (1CS+ and 1CS− trials) on day 2, by answering the questions ‘When you saw the symbol, how strong was your stress/fear/tension’ (for fear) and ‘When you saw the symbol, to what extent did you expect the painful stimulus?’ (for US expectancy). Ratings were given on a visual analog scale ranging from 0 (none) to 100 (maximum).

Statistical Analyses

In general, only participants with valid data-points on day 1 and day 2 were included in our analyses (Fear ratings and US expectancy: N=349, 242 females, 22% smokers; SCR: N=290, 203 females, 21% smokers). CRs were analyzed using repeated-measures ANOVAs with stimulus (CS+/CS−) and context (danger/safety) as within-subject factors and smoking status as between-subject factor, separately for each day. Repeated-measures ANOVAs controlling for individual differences in novelty seeking, consumption of alcohol and coffee, as well as age and gender and anxious temperament (STAI trait anxiety) were performed separately. Pack-years approximation was calculated as the product of self-reported daily cigarette consumption (1–10 cigarettes=0.5 packs per day; 11–20 cigarettes=1 pack per day; 21–30 cigarettes=1.5 packs per day) and the smoking duration (in years). Correlation between pack-years and CRs in both contexts were calculated as Pearson correlation coefficients and tested for statistical difference (Lee and Preacher, n.d.). Analyses controlling for the influence of individual differences in novelty seeking, consumption of alcohol and coffee, as well as age, gender and anxious temperament (STAI trait anxiety) were performed as linear regression models (step-wise inclusion) for day 1 and day 2. In addition, analyses exploring the moderation of smoking effects by trait anxiety were performed as linear regression models including pack-years (for day 1) or smoking status (for day 2), trait anxiety as well as the interaction of both (mean centered).

If appropriate, Greenhouse–Geisser corrections were adopted and p<0.05 was considered significant.