Supplementary Material
Cold pressor test improves fear extinction in healthy men
Martin I. Antov*, Ursula Melicherová, and Ursula Stockhorst
Experimental Psychology II and Biological Psychology, Institute of Psychology,
University of Osnabrück, Seminarstraße 20, 49074 Osnabrück
Email addresses:
[email protected] (M. I. A.),
[email protected] (U. M.),
[email protected] (U. S.)
Running title: Cold pressor stress improves fear extinction
*Corresponding author at:
University of Osnabrück, Institute of Psychology,
Experimental Psychology II and Biological Psychology,
Seminarstrasse 20, D-49074 Osnabrück, Germany.
Phone +49 541 969 4530; Fax +49 541 969 4922
Email: [email protected]
Supplementary Material: Antov, Melicherová, & Stockhorst
1. Rationale
The supplementary material complements the main paper by adding:
(A) the analysis of the responses to the CS+ and CS- stimuli separately, and on a trial-by-trial basis,
and
(B) additional computation of the extinction retention index following the original version introduced by
Milad et al. (2006).
Ad (A): In the main paper, we reported results for skin conductance responses as differential skin
conductance responses (diffSCR). This type of analysis which is frequently used in fear-conditioning
studies, was selected in order to allow comparability to a recent study of our group (Antov and
Stockhorst, 2014) and it reflects individual differentiation between CS+ and CS-. In addition to
calculating a differential score, we aggregated responses over trials in our previous analyses,
concretely over the first four vs. the last four trials of each experimental phase, to provide a more
stable measure.
However, it may be also informative to complement data analysis by displaying responses to the CS+
and CS- separately. This also allows us to trace back group differences in SCR to responses to the
threat cue (CS+) vs. the safety cue (CS-).
Ad (B): In the main paper, we report an extinction retention index that was based on Milad et al.
(2006) but which was adapted to the features of our conditioning procedure. Our index computation
was modified to account for the fact that we used more trials than Milad et al. (2006) during all
conditioning phases, also in extinction retrieval on Day 2 (12 instead of 5 trials in Milad et al., 2006).
Moreover, we included a range-correction in our SCR data. To improve comparability between
studies, we here present data using the original computation as reported by Milad et al. (2006).
2. Supplementary Methods: Additional data analyses
2.1 Trial-by-trial analyses with CS-type as a within-subject factor
In order to accomplish the trial-by-trial analysis, we analyzed the three phases of the experiment (Day
1: acquisition and extinction, Day 2: extinction retrieval) using mixed ANOVAs with CS-type (CS+ vs.
CS-) and trial (1 to 12) as the within-subject factors, and – as previously – treatment (CPT vs. control)
as the between-subjects factor. We also conducted polynomial contrasts looking for a linear trend for
the CS-type x Trial interaction, indicating an increase vs. decrease in CS+/CS- differentiation, for
acquisition and extinction, respectively. ANOVA for acquisition also included the habituation trial (one
for each CS-type). Thus, the factor trial had the levels 1 to 13 for acquisition.
As in the main manuscript, we repeated Day 1 extinction analysis separately for the CPT and the
control group. Using post hoc paired-samples t-tests (2-tailed), we also compared CS+ vs. CSresponses during the last 4 trials of extinction on Day 1 for each treatment group. Because there were
significant baseline differences in diastolic blood pressure (diaBP) and salivary cortisol level on Day 2,
we included these variables as covariates in the analysis of SCRs on Day 2.
2.2 Tracing back group differences to responses to the CS+ or to the CSIn the main paper, we report results from the differential SCR, averaged over 4 trials. For descriptive
purpose, we here further compared SCRs to the CS+ in the CPT vs. control group using independent
samples t-tests (two-tailed) for each of the last 4 trials of extinction on Day 1, and for each of the first
4 trials of extinction retrieval on Day 2. Using the same approach, we compared SCRs to the CS-.
This additional analysis allows us to trace group differences back to differences in CS+ or CSresponses and to identify, which of the four trials contribute to the group difference reported in the
main paper.
2.3 Alternative computation of the extinction retention index and statistical analysis
The original extinction index introduced by Milad et al., (2006) was computed as follows: For each
participant mean diffSCRs for the first 2 trials of extinction retrieval on Day 2 was divided by his
largest diffSCR during acquisition on Day 1 and multiplied by 100. The result was then subtracted
from 100%. Calculating this extinction retention index according to Milad et al. (2006), we compared
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Supplementary Material: Antov, Melicherová, & Stockhorst
groups by an independent-samples t-test. As in the main paper, we then repeated analysis including
the level of extinction on Day 1, and baseline levels of diaBP and salivary cortisol (CORT) on Day 2
as covariates in an ANCOVA.
3. Supplementary Results
3.1 Day 1 Fear Acquisition
The 2 (Treatment) x 2 (CS-type) x 13 (trial) ANOVA revealed a significant main effect of CS-type (F(1,
2
38) = 55.09, p < .001, part. η = .592) with higher responses to the CS+ than to the CS- (Figure S1, left
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panel). There was a significant CS-type x Trial interaction (F(8.2, 310.2) = 5.67, p < .001, part. η = .130).
Polynomial contrasts showed a significant linear trend for CS-type x Trial (F(1, 38) = 24.83, p < .001,
part. η2 = .395), both indicating an increase of differentiation between CS+ and CS- over trials. We
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also found a Treatment x Trial interaction (F(7.7, 291.4) = 2.01, p = .049, part. η = .050), indicating a
different time-course of responding in the two groups. Importantly, there were no CS-type x Treatment
and no CS-type x Trial x Treatment interactions (both F < 1), indicating that both groups acquired the
differential conditioning to a similar level (Figure S1, left panel).
3.2 Day 1 Extinction Training
Here, the 2 (Treatment) x 2 (CS-type) x 12 (trial) ANOVA showed a significant main effect of CS-type
(F(1, 38) = 30.01, p < .001, part. η2 = .441) and a significant main effect of trial with decreasing
responses (F(6.9, 261.2) = 4.63, p < .001, part. η2 = .109) over the course of extinction. There were no
other significant main effects or interactions in this analysis (all F < 1). The linear trend for CS-type x
Trial was not significant (p = .109). Thus, there was no evidence for extinction learning in the entire
group (Figure S1, middle panel). When repeating the analysis per group, there was no evidence for a
reduction in differential responding in the control group (CS-type x Trial interaction & CS-type x Trial
linear trend: both F < 1). However, there was evidence for a significant reduction in the CPT group
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(CS-type x Trial interaction: F = 1.18; CS-type x Trial linear trend: F(1, 19) = 5.86, p = .026, part. η =
.236, Figure S1 A, middle panel). Paired-samples t-test per group indicated that the control group
continued to show higher responses to the CS+ than to the CS- during the last 4 trials of extinction
training (p = .004, p = .064, p = .020, and p = .038, respectively). This was not the case in the CPT
group (p = .328, p = .063, p = .069, and p = .224).
3.3 Day 2 Extinction retrieval
Here, we included baseline levels of diaBP and CORT on Day 2 as covariates in a 2 (Treatment) x 2
(CS-type) x 12 (trial) ANCOVA. Results showed a significant main effect of CS-type (F(1, 34) = 19.11, p
2
< .001, part. η = .360) and a significant Treatment x Trial interaction (F(6.6, 225.1) = 2.136, p = .044,
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part. η = .059). Importantly, there was a significant Treatment x CS-Type x Trial interaction (F(11, 374) =
1.94, p = .034, part. η2 = .054) indicating that groups showed a distinct time course of CS+/CSdifferentiation (Figure S1, right panel). Extinction recall is supposed to be best reflected during the
first trials of re-extinction. Post hoc paired t-tests per group showed no difference between CS+ and
CS- responses during 3 of the first 4 trials in the CPT group (p = .960, p = .279, p = .925, p = .049, for
trials 1 to 4, respectively). In the control group however, responses to the CS+ were higher than the
responses to the CS- for 3 of the first 4 trials (p = .030, p = .001, p = .001, p = .218, for trials 1 to 4,
respectively).
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Supplementary Material: Antov, Melicherová, & Stockhorst
Figure S1
Fear conditioning and extinction results from Day 1 and Day 2. Mean skin conductance
responses to the CS+ and CS− over trials for Day 1 acquisition phase (left panel), extinction phase (middle
panel), and Day 2 extinction retrieval (right panel) are displayed separately for the CPT group (A) and the control
group (B). Error bars show ±1 SEM; h denotes responses to habituation trials (one for CS+ and one CS-).
3.4 Tracing back group differences to differences in CS+ or CS- responses
Table S1 shows results of t-tests comparing CS+ and CS- responses separately for the CPT vs.
control group. Figure S2 shows the data already displayed in Figure S1, but now rearranged to allow
the direct comparison of responses to CS+ in the CPT and the control group (Figure S2, A) and,
comparably, for the responses to CS- in the CPT and the control group (Figure S2, B). As evident
from both, Table S1, and Figure S2, the lack of extinction in the control group on Day 1 can be traced
back (descriptively) to higher responses to the CS+ (trials 9 & descriptively 12). During extinction
retrieval on Day 2 there is evidence for both higher responses to the CS+ (trial 1) and lower
responses to the CS- (trial 3) in the control group.
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Supplementary Material: Antov, Melicherová, & Stockhorst
Table S1
Results from independent sample t-Tests comparing the CPT vs. the control group for
responses to the CS+ (left side) and CS- (right side) for the last four trials of extinction on Day 1 and the first four
trials of extinction retrieval on Day 2.
CS+ Responses
Day 1 Extinction
CS- Responses
t
df
p
d
t
Trial 09
2.17
34
.037
0.69
-0.04
Trial 10
0.77
38
.447
0.24
1.13
Trial 11
0.52
38
.604
0.17
Trial 12
1.58
34
.124
Trial 01
1.97
33.8
Trial 02
0.89
Trial 03
0.89
Trial 04
-1.16
df
p
d
38
.969
0.01
37.2
.265
0.36
-0.69
38
.493
0.22
0.50
0.15
38
.884
0.05
.057
0.62
0.28
38
.778
0.09
35.5
.380
0.28
-1.11
38
.272
0.35
38
.379
0.28
-2.14
38
.039
0.68
33.6
.253
0.37
-0.35
38
.729
0.11
Day 2 Extinction Retrieval
Note. t is the t-statistic for each CPT vs. control comparison; df, p, and d represent the associated degrees of freedom, p-value,
and Cohen’s effect size estimate d, respectively. All p-values are two-sided, but not corrected for multiple comparisons.
Figure S2
Fear conditioning by stimulus type (CS+ and CS-) comparing treatment groups (CPT vs
control). Data and annotations are identical to that displayed in Figure S 1, however grouped by stimulus type
CS+ (A) and CS- (B). Solid lines with black symbols represent the CPT-group; dashed lines with white symbols
represent the control group.
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Supplementary Material: Antov, Melicherová, & Stockhorst
3.5 Alternative computation of the extinction retention index
Comparing the CPT vs. control group for the extinction retention index computed according to the
original description (Milad et al., 2006) revealed a trend for higher extinction retention in the CPT
group (t(38) = 1.61, p = .058, Figure S3). Repeating the analyses with late extinction on Day 1 (mean
diffSCR for the last 4 trials), baseline diaBP and CORT as covariates rendered the group difference
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non-significant (F(1, 33) = 1.37, p = .251, part. η = .040).
Alternative computation of the extinction retention index as described in Milad et al., (2006).
Figure S3
Data are means and error bars show ±1 SEM
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Supplementary Material: Antov, Melicherová, & Stockhorst
3.6 Correlations with treatment validation variables
Table S2 summarizes all relevant correlations.
Table S2
Pearson correlations and significance levels per group for diffSCRs for early (trials 1-4) and late
(trials 9-12) phases of extinction on Day 1 and extinction retrieval on Day 2 with differences to baseline for
systolic (sysBP), diastolic blood pressure (diaBP), number of spontaneous skin conductance response (nsSCR),
and heart rate (HR) during hand immersion and difference to baseline for CORT at t2, and t3 after treatment.
4. Conclusion
The additional analyses reported here are largely in agreement with the results and conclusions of the
main paper. Including all trials in statistical analysis of CS+ and CS- responses led to similar results,
as already reported for differential SCRs. Concretely, we showed successful fear acquisition in both
groups, but the control group showed no fear extinction on Day 1 and this difference was maintained
on Day 2. Computing the extinction retention index based on fewer trials and without range correction
(Milad et al., 2006) now yielded only a trend compared to the significant group effect reported in the
main paper. The trend however, had the same direction: i.e. higher extinction retention values in the
CPT-group. We were able trace back group differences in extinction on Day 1 to higher responses to
the CS+ in the control group. Group differences on Day 2 seem to reflect both higher responses to the
CS+ in the control group (trial 1) but also lower responses to the CS- (trial 3). In sum, the additional
results reported here support the interpretation that the acute CPT (without group differences in
cortisol) placed immediately before extinction training can enhance extinction in humans.
Supplementary References
Antov, M.I., Stockhorst, U., 2014. Stress exposure prior to fear acquisition interacts with estradiol
status to alter recall of fear extinction in humans. Psychoneuroendocrinology 49, 106–118.
Milad, M.R., Goldstein, J.M., Orr, S.P., Wedig, M.M., Klibanski, A., Pitman, R.K., Rauch, S.L., 2006.
Fear conditioning and extinction: influence of sex and menstrual cycle in healthy humans.
Behav. Neurosci. 120, 1196–1203.
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