related stimuli in posttraumatic stress disorder

Psychophysiology, 53 (2016), 1460–1471. Wiley Periodicals, Inc. Printed in the USA. C 2016 Society for Psychophysiological Research V

DOI: 10.1111/psyp.12699

Reduced amygdala responsivity during conditioning to traumarelated stimuli in posttraumatic stress disorder

SLAWOMIRA J. DIENER,a FRAUKE NEES,a MICHELE WESSA,a GUSTAV WIRTZ,b c a ULRICH FROMMBERGER, TINA PENGA, MICHAELA RUTTORF,d MATTHIAS RUF,e CHRISTIAN SCHMAHL,f AND HERTA FLORa a Department of Cognitive and Clinical Neuroscience, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany b Department of Psychiatry and Psychotherapy, SRH Klinikum Karlsbad-Langensteinbach GmbH, Karlsbad, Germany c Department of Psychiatry, Psychotherapy and Psychosomatics, MediClin Klinik an der Lindenh€ ohe, Offenburg, Germany d Computer Assisted Clinical Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany e Department of Neuroimaging, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany f Department of Psychosomatic Medicine and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany

Abstract Exaggerated conditioned fear responses and impaired extinction along with amygdala overactivation have been observed in posttraumatic stress disorder (PTSD). These fear responses might be triggered by cues related to the trauma through higher-order conditioning, where reminders of the trauma may serve as unconditioned stimuli (US) and could maintain the fear response. We compared arousal, valence, and US expectancy ratings and BOLD brain responses using fMRI in 14 traumatized persons with PTSD and 14 without PTSD (NPTSD) and 13 matched healthy controls (HC) in a differential aversive conditioning paradigm. The US were trauma-specific pictures for the PTSD and NPTSD group and equally aversive and arousing for the HC; the conditioned stimuli (CS) were graphic displays. During conditioning, the PTSD patients compared to the NPTSD and HC indicated higher arousal to the conditioned stimulus that was paired with the trauma picture (CS1) compared to the unpaired (CS2), increased dissociation during acquisition and extinction, and failure to extinguish the CS/US-association compared to NPTSD. During early and late acquisition, the PTSD patients showed a significantly lower amygdala activation to CS1 versus CS2 and a negative interaction between activation in the amygdala and dorsolateral prefrontal cortex (PFC), while NPTSD and HC displayed a negative interaction between amygdala and medial PFC. These findings suggest maladaptive anticipatory coping with trauma-related stimuli in patients with PTSD, indicated by enhanced conditioning, with related abnormal amygdala reactivity and connectivity, and delayed extinction. Descriptors: Fear conditioning, PTSD, Trauma, Anxiety, fMRI

Pavlovian fear conditioning is viewed as an important mechanism in the development of anxiety disorders (Pitman & Orr, 1986). In posttraumatic stress disorder (PTSD), the traumatic event is thought to serve as an unconditioned stimulus (US), which triggers an intense fear response that becomes associated with neutral conditioned stimuli (CS) present during the traumatic event. The CS then elicit a conditioned fear response (CR) without presence of the US. Research on PTSD often revealed enhanced conditioning and delayed extinction of fear responses (e.g, Blechert, Michael, Vriends, Margraf, & Wilhelm, 2007; Bremner et al., 2005; Peri et al., 2000). However, there is little research on the role of trauma

reminders as US, which might have a special role in the maintenance of the continued fear and stress response. Second-order conditioning could be a complementary process to the well-described overgeneralization (e.g., Levy-Gigy, Szabo, Richter-Levin, & Keri, 2015; Lissek & van Meurs, 2015, for a review) and deficient contextual conditioning (e.g., Garfinkel et al., 2014; Steiger, Nees, Wicking, Lang, & Flor, 2015) in PTSD patients that might especially maintain intrusions. For patients, heightened emotional and physiological reactivity to stimuli that resemble the original trauma was often reported (e.g., Ehlers & Clark, 2000; Orr, Metzger, & Pitman, 2002). These responses to trauma reminders were shown to correlate with reexperiencing and dissociation, and reported to predict PTSD symptoms (Kleim, Ehring, & Ehlers, 2011). Responsivity to trauma reminders is characterized by increased limbic and decreased medial prefrontal neural activation, specifically in the anterior cingulate cortex (ACC) and ventromedial prefrontal cortex (vmPFC; e.g., Lanius et al., 2005). A deficient

This work was supported by the Deutsche Forschungsgemeinschaft: grant SFB636/C1 to HF and grant SFB636/Z3 to LRS. SJD and FN contributed equally to this manuscript. Address correspondence to: Herta Flor, Ph.D., Department of Cognitive and Clinical Neuroscience, Central Institute of Mental Health, J 5, 68159 Mannheim, Germany. E-mail: [email protected] 1460

Conditioning to trauma-related stimuli in PTSD inhibition of subcortical limbic reactivity, specifically in the amygdala, by prefrontal regions could explain the lack of extinction and continued reexperiencing in PTSD patients (e.g., Milad, Rauch, Pitman, & Quirk, 2006; Giustino & Maren, 2015). Pendyam and colleagues (2013) highlighted a significant role of the interaction between the prelimbic PFC and the basolateral amygdala on the expression of fear, and discussed variation in this microcircuit as an effective target for anxiety disorders. We have previously shown that a visual trauma-related US (picture of the traumatic event) yielded enhanced conditioning and delayed extinction in patients with PTSD as indicated by skin conductance, verbal ratings, and electroencephalogram measures (Wessa & Flor, 2007). Thus, the mechanism of second-order conditioning (Rescorla, 1980) could be important in PTSD, where trauma-related stimuli that can be viewed as conditioned stimuli in fear learning can themselves take on the quality of an unconditioned stimulus and can create new fear responses to trauma reminders. In persons with phobias, Schweckendiek et al. (2011) showed enhanced amygdala activation during second-order conditioning, where a phobia-relevant aversive picture was used as US. Enhanced fear conditioning and delayed fear extinction in PTSD are assumed to result from insufficient inhibitory control from higher cortical areas like the medial PFC (mPFC) or ventral PFC regions (e.g., Bishop, 2007; Etkin, Egner, & Kalisch, 2011) on the limbic fear pathway, specifically the amygdala. Patients with PTSD, compared to trauma-exposed individuals without PTSD and also compared to control subjects, showed enhanced emotional responsiveness to negative distractors in an affective Stroop task (Blair et al., 2013), with disrupted recruitment of frontal and parietal cortical regions implicated in top-down attentional control. Moreover, activation in the dorsal PFC was shown to be positively correlated with activation in the amygdala, which might form the neural basis of adaptive anxiety processes (e.g., Etkin et al., 2011; Milad et al., 2007) that increase the ability to detect and avoid danger (e.g., Indovina, Robbins, Nu~nez-Elizalde, Dunn, & Bishop, 2011). An efficient interplay between the amygdala and prefrontal regions may thus be crucial for adaptive versus pathological anxiety (Giustino & Maren, 2015; Kim et al., 2011; Linnman et al., 2012). Interestingly, resting amygdala metabolism negatively predicted activation in the dorsal ACC, but was positively related to activation in the vmPFC during fear extinction and vice versa during extinction recall (Linnman et al., 2012). Robinson, Charney, Overstreet, Vytal, and Grillon (2012) reported that positive amygdala-dorsomedial PFC (dmPFC) connectivity during fearful, but not happy, face processing was significantly positively associated with the level of trait anxiety in healthy subjects and could thus constitute a vulnerability factor for the development of anxiety disorders. However, whether such neural response patterns serve as critical correlates in higher-order conditioning, where the traumarelated CS can take on the qualities of the original trauma and can serve as US, still needs to be investigated. The goal of the present study was to examine behavioral and functional brain correlates of fear conditioning to trauma-related stimuli in traumatized individuals with PTSD compared to those without a PTSD diagnosis and never-traumatized controls who viewed these stimuli as equally arousing and aversive. We expected to find enhanced fear conditioning and delayed extinction indicated by verbal reports, specifically, arousal and US expectancy, along with enhanced activation in the amygdala (Stevens et al., 2013) and deficient activation in medial prefrontal regions and a negative interaction of the amygdala and the mPFC during extinction (e.g., Marek, Strobel, Bredy, & Sah, 2013; Sehlmeyer

1461 et al., 2009) in PTSD patients compared to similarly traumatized persons without PTSD (NPTSD) and the never-traumatized controls. Method Participants Fourteen PTSD patients with Type I trauma, 14 NPTSD without a lifetime history of PTSD, and 13 nontraumatized HC were investigated. Two additional PTSD patients were excluded from the analysis based on functional magnetic resonance imaging (fMRI) movement artifacts. Four PTSD patients had taken part in our previous study more than 5 years prior to the investigation (Wessa & Flor, 2007). The participants did not significantly differ in sex, age, or education (Table 1). They were recruited at outpatient clinics and by public announcements. All NPTSD met the A1 and A2 criterion for PTSD of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV-TR; American Psychiatric Association, 2000), but had never met criteria for PTSD or any other disorder, except for one subject who also met criteria for specific phobia. The PTSD patients also fulfilled DSM-V criteria (American Psychiatric Association, 2013) for chronic PTSD and had comorbid disorders such as a major depressive episode (n 5 2), minor depression (n 5 7), panic disorder (n 5 1) and generalized anxiety disorder (n 5 2). The HC did not meet the PTSD A criteria or any DSM-V criterion for mental disorder. PTSD patients were unmedicated for at least 1 month prior to participation and were comparable to NPTSD in the self-reported severity of the traumatic event and the time period between trauma and study participation, except for higher feelings that their life was threatened during the trauma and higher depression scores, as assessed with the CES-D Depression Scale (Radloff, 1977; German Version: Hautzinger & Bailer, 1993; Table 1). Exclusion criteria were chronic and current substance abuse, borderline personality disorder, schizophrenia, cardiovascular or neurological disorders, brain injury, pregnancy, and metal implants. The study was approved by the local ethics committee. The participants gave written informed consent after a detailed description of the study. All participants were interviewed by a trained clinical psychologist using the Structured Clinical Interview for DSM-IV Axis I/II (First, Gibbon, Spitzer, Williams, & Benjamin, 1997; First, Spitzer, Gibbon, & Williams, 1997; Wittchen, Wunderlich, Gruschwitz, & Zaudig, 1997; Fydrich, Renneberg, Schmitz, & Wittchen, 1997). Experimental Procedure Picture-viewing experiment in the laboratory. All participants underwent a picture-viewing experiment before conditioning to identify the most aversive, and for the PTSD patients and NPTSD also trauma-relevant, pictures that were then used as US during conditioning. In this picture-viewing experiment, 25 pictures related to the individual trauma for the traumatized groups were presented. HC watched pictures from the same trauma category as the individual PTSD patient they were matched with as control. The trauma pictures were selected from the Internet and the International Affective Picture System (Lang, Bradley, & Cuthbert, 2008) and matched the specific trauma. All pictures were rated for arousal (later coded from 1 5 not arousing to 9 5 extremely

S.J. Diener et al.

1462 Table 1. Descriptive Statistics for the 14 Patients with PTSD, 14 Traumatized NPTSD Subjects, and 13 HC

Age: mean (SD) Gender: male:female Education 8 years 10 years 13 years Traumatic event

Depression scale (CES-D): sum (SD) Clinician Administered PTSD Scale (CAPS): sum (SD) Time period since the traumatic event in years; mean (SD) Trauma severity ; mean (SD): % amount of loss of control, 0 5 no loss of control; 100 5 complete loss of control % amount of helplessness, 0 5 no helplessness; 100 5 complete helplessness % amount of fear, 0 5 no fear; 100 5 extreme fear % amount of the feeling to die, 0 5 no feeling to die; 100 5 completely sure to die Dissociation during conditioning: 1 5 no dissociation; 9 5 extreme dissociation; mean (SD) After habituation After early acquisition After late acquisition After extinction Trauma relevance of the unconditioned stimuli; mean (SD) 0 5 does not at all remind me of my trauma; 10 5 reminds me very much of my trauma Unconditioned stimulus 1 (US1) Unconditioned stimulus 2 (US2) Ability to watch the unconditioned stimuli during conditioning: 1 5 could not look at them; 9 5 could look at them; mean (SD) After habituation After early acquisition After late acquisition After extinction Arousal rating of the US (mean of US1 and US2) during conditioning; mean (SD) After habituation After early acquisition After late acquisition After extinction Valence rating of the US (mean of US1 and US2) during conditioning; mean (SD) After habituation After early acquisition After late acquisition After extinction

PTSD N 5 14

NPTSD N 5 14

HC N 5 13

Group statistics

43. 29 (14.54) 8:6

39.64 (13.26) 9:5

42.46 (13.69) 7:6

F(2,38) 5 .27, p 5 .77 v2(2) 5 .32; p 5 .85

4 4 6 Air crash (n 5 5), car accident (n 5 3), violence (n 5 5), hospital stay (n 5 1)

2 3 8

v2(4) 5 5.54; p 5 .24

23.71 (10.74)

0 3 11 Car accident (n 5 6), violence (n 5 5), suicide witness(n 5 1), rape (n 5 1), fire (n 5 1) 6.14 (6.00)

7.25 (6.85)

F(2,37) 5 19.8, p < .001

58.14 (20.71)

3.15 (4.61)

–

t(25) 5 29.35, p < .001

10.50 (8.35)

8.64 (9.95)

75.71 (41.32)

67.69 (38.76)

– –

t(25) 5 2.52, p 5 .61

82.79 (34.01)

68.75 (33.18)

–

t(24) 5 21.06, p 5 .30

47.86 (44.92)

53.08 (35.91)

–

t(25) 5 .33, p 5 .74

59.64 (47.57)

15.00 (29.16)

–

t(25) 5 22.91, p < .01

t(26) 5 2.54, p 5 .60

-

3.86 3.71 4.07 4.43

(3.08) (3.12) (2.89) (2.82)

2.07 2.79 2.07 1.79

(2.16) (2.15) (2.13) (1.89)

1.46 1.46 1.38 1.38

(1.20) (1.13) (.96) (.96)

F(2,38) 5 3.99; F(2,38) 5 3.23; F(2,38) 5 5.66; F(2,38) 5 8.89;

– –

t(26) 5 22.16, p < .05 t(26) 5 21.88, p 5 .07

p < .05 p 5 .05 p < .01 p < .01

8.50 (2.59) 7.93 (2.81)

5.93 (3.62) 5.71 (3.38)

7.00 5.93 6.07 7.00

(2.11) (3.15) (3.05) (2.94)

8.50 8.71 8.86 8.79

(.94) (.61) (.36) (.58)

9.00 8.85 8.86 9.00

(.00) (.55) (.36) (.00)

F(2,38) 5 8.10; F(2,38) 5 10.36; F(2,38) 5 11.66; F(2,38) 5 5.42;

6.50 6.71 6.96 6.64

(2.13) (2.42) (2.00) (2.41)

6.43 6.43 6.18 6.00

(2.23) (2.25) (2.21) (2.08)

5.85 6.04 6.15 6.31

(1.70) (2.04) (2.22) (2.16)

F(2,38) 5 .41; F(2,38) 5 .31; F(2,38) 5 .64; F(2,38) 5 .29;

7.43 (2.43) 7.82 (1.58) 7.82( 1.45) 7.96 (1.13)

6.79 6.93 6.64 6.96

(2.15) (1.77) (2.02) (1.43)

7.58 7.54 7.54 7.46

(.81) (.97) (1.03) (1.09)

F(2,38) 5 .64; p 5 .53 F(2,38) 5 .131; p 5 .28 F(2,38) 5 2.15; p 5 .13 F(2,38) 5 2.31; p 5 .11

Note. PTSD 5 posttraumatic stress disorder; NPTSD 5 without lifetime history of PTSD; HC 5 nontraumatized healthy controls.

p < .01 p < .001 p < .001 p < .01

p 5 .66 p 5 .74 p 5 .53 p 5 .75

Conditioning to trauma-related stimuli in PTSD

1463

Figure 1. Ratings of (a) arousal, (b) valence, and (c) expectancy of the unconditioned stimulus in patients with posttraumatic stress disorder (PTSD), traumatized subjects without lifetime history of PTSD (NPTSD), and nontraumatized healthy controls (HC) after habituation, early and late acquisition, and extinction. The red lines indicate the CS1 and the blue lines the CS2. *p < .05; **p < .01.

arousing) and valence (later coded from 1 5 very pleasant to 9 5 very unpleasant) on the Self-Assessment Manikin (SAM; Bradley & Lang, 1994), and in patients with PTSD and NPTSD also for relevance for the trauma on a scale ranging from 0 5 does not at all remind me of my trauma to 10 5 reminds me very much of my trauma. Of these 25 pictures, the two most arousing and unpleasant and, for PTSD patients and NPTSD, additionally most trauma-relevant pictures were selected as US for the higher-order conditioning experiment. Conditioning experiment during fMRI. We used a picturepicture differential aversive delay conditioning with geometric symbols as CS and aversive, trauma-related pictures as US. One of the symbols later predicted the occurrence (CS1), one the absence of the US (CS2). Online supporting information Figure S1 presents the conditioning design and the timing of the CS1/CS2. The experiment consisted of habituation, early and late acquisition, and extinction. Habituation consisted of 10 3-s presentations of each of the two graphic symbols and two presentations of each of the US (also presented for 3 s) in a random (unpaired) order. During both acquisition phases, CS1 and CS2 were each presented 18 times, with CS1 followed directly by one of the two US in 50% of the trials. The two US were presented in random order. In the

extinction phase only, the CS were presented 18 times each. The intertrial interval varied from 10–15 s. SAM ratings of valence and arousal were used to obtain picture rating during conditioning, and US expectancy was rated on a visual analogue scale with the end points very unlikely that the US will follow to very likely that the US will follow (later recoded to a numeric scale ranging from 1–9). This expectancy rating indicates the degree of perceived CS/US association, both the CS1 presentation together with the US occurrence and the CS2 presentation together with the US nonoccurrence. This is an indicator of a participant’s perception of the CS1 as danger and the CS2 as safety signal. Moreover, we assessed dissociation and ability to watch the pictures using visual analogue scales (“How strong was your feeling to dissociate?” ranging from no dissociation to strong dissociation and “Was it possible for you to look at the unpleasant pictures?” ranging from could not at all look at them to could look at them very well; all later rescored from 1 to 9). All ratings were implemented after each of the four conditioning phases. To control if the participants looked at all at the stimuli, the participant’s eye gaze was manually monitored (online) during the entire experiment based on the visual output from an MR compatible eyetracking system (Resonance Technology Inc., Los Angeles, CA).

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S.J. Diener et al.

Figure 2. Amygdala activation (depicted as mean beta values) in response to CS1unpaired versus CS2 during early and late acquisition in (a) posttraumatic stress disorder (PTSD), (b) traumatized subjects without lifetime history of PTSD (NPTSD), and (c) nontraumatized healthy controls (HC). *p < .05, familywise error-corrected.

Image Acquisition Whole-brain fMRI images were acquired using a 3T Magnetom Trio whole-body MR scanner (Siemens Medical Solutions, Erlangen, Germany) with a 12-channel head coil. A gradient-echo echo planar imaging (EPI) sequence (TR 5 2,700 ms; TE 5 27 ms; matrix size 5 96 3 96; field of view 5 220 3 220 mm2; flip angle 5 90 8; GRAPPA with PAT 5 2) was used to record 785 functional volumes: 125 for habituation, and 220 each for early and late acquisition and extinction. The first four volumes were discarded to account for T1-saturation effects. Each volume consisted of 40 axial slices (slice thickness 5 2.3 mm; gap 5 0.7 mm) measured in descending order and positioned along the anterior-posterior commissure. For structural images, we used a high-resolution T1 magnetization-prepared rapid gradient-echo sequence MPRAGE (TR 5 2,300 ms; TE 5 2.98 ms; TI 5 900 ms; field of view 240 3 256 mm2; 160 sagittal slices, voxel size 1.0 3 1.0 3 1.1mm3).

Data Analysis Functional imaging data. Single subject and group fMRI analyses were performed using SPM5 (http://www.fil.ion.ucl.ac.uk/spm/ software/spm5) implemented in MATLAB 7.1 (The MathWorks Inc., Natick, MA). Functional volumes were realigned to the fifth volume by minimizing the mean square error. Two PTSD patients with motion estimates exceeding 3 mm and 3 8 were excluded. Images were slice-time corrected to reference slice 20 and normalized to the Montreal Neurological Institute (MNI) standard space using the EPI template of SPM5. The voxel size was set to 3 3 3 3 3 mm3. To reduce spatial noise, the volumes were smoothed with a 9 3 9 3 9 mm3 Gaussian kernel. The fMRI time series were high-pass filtered (temporal cutoff: 128 s) and corrected for serial autocorrelations using first-order autoregressive functions, AR(1). We performed individual statistical analyses by convoluting the stimulation time course with a standard hemodynamic response function to model blood oxygen level-dependent (BOLD) signal changes to the CS1unpaired, CS2, and CS1paired in each conditioning phase. For each subject, images for each stimulus presentation alone, as well as the contrast between CS1unpaired/CS2 was calculated with movement regressors included in the first-level statistical model. These individual statistical parametric maps were entered in second-level, random-effects analyses to generate groupspecific voxelwise t statistics to assess significant activations within each group. Analyses of variance (ANOVAs) were conducted for between-groups differences in the CS1unpaired and CS2

activations and in the CS1unpaired/CS2 contrast at whole brain level as well as using a region of interest (ROI) approach. ROI analysis. Since we were interested in the role and interaction of the amygdala and frontal cortical regions, we used the following set of predefined ROIs: amygdala, mPFC (ventral ACC: BA24, dorsal ACC: BA32, pregenual ACC: BA33), dorsolateral PFC (dlPFC; BA46, BA9), anterior PFC (BA10), and orbitofrontal cortex (OFC; medial area: BA11, rostral area: BA12) and the left and right insula. The Wake Forest University PickAtlas version 2.0 was used for ROI definition and a familywise error (FWE) threshold of p < .05. To directly relate our findings with those from previous studies, we additionally focused on the role of specific prefrontal areas that were identified in previous studies as significant target regions in traumatic imagery and instructed fear (Etkin & Wager, 2007; Mechias, Etkin, & Kalisch, 2010; Shin et al., 2004) using a sphere around the peak coordinates from these studies: the mPFC (peak: 10/52/2, sphere: 4 mm) and the rostral dmPFC (peak: 6/38/38, sphere: 5 mm). Psychophysiological interaction analysis (PPI). PPI was conducted with the activation in the amygdala as source region for all contrasts, but yielded significant results only for CS1unpaired > CS2 during early and late acquisition as well as extinction. Individual time series of the amygdala were extracted using anatomical ROIs and then multiplied with the modeled hemodynamic response for CS1unpaired > CS2, resulting in an interaction term. For these analyses, a threshold of FWE correction at p < .05 was applied. Ratings. For the ratings of arousal, valence, and US expectancy, repeated measures ANOVAs (p < .05) were conducted with phase (habituation, early/late acquisition, extinction), CS (CS1, CS2), or US (US1, US2) as within-subject and group as between-subjects factor. In Results, we only report significant effects involving group and follow-up t tests between the groups. The trauma relevance ratings of both US pictures were compared using a t test for independent samples. The ability to watch the US pictures and the feeling of dissociation were compared using univariate ANOVAs. Post hoc within- and between-groups t tests were Bonferronicorrected. Associations between brain activation and ratings. Correlational analyses included the ROIs during early and late acquisition and extinction, as well as valence, arousal, and US expectancy ratings, PTSD symptoms, and dissociation.


1465

Table 2. Differential Brain Activation to the CS in Acquisition and Extinction Group

contrast

Early acquisition PTSD

CS1 unpaired > CS2

NPTSD > PTSD

CS1 unpaired > CS2

Brain region

BA

Amygdala

HC > PTSD

CS1 unpaired > CS2

HC > NPTSD

CS1 unpaired

Late acquisition PTSD

Extinction HC

z

t value

p value

L

-18

-3

-21

-3.63

< .05

BA10 BA46

R L

42 245

54 42

3 18

4.31 3.40

Dorsolateral prefrontal cortex

BA46

Anterior insula

BA47

L R R

242 57 33

45 30 21

12 15 23

3.45 3.10 5.13

< .05 < .05 < .05 < .05 < .05 < .05

Insula

BA22

L

245

0

26

3.74

< .05

Ventral anterior cingulate cortex

BA24

R L

6 212

23 3

36 39

4.13 3.62

< .05 < .05

Anterior prefrontal cortex

BA10

Orbitofrontal cortex

BA11


BA24


BA9

L R R L R L L R

242 21 27 242 3 23 254 48

48 63 36 39 26 29 3 23

0 12 218 12 36 36 33 24

4.28 3.94 3.71 3.12 3.76 3.51 4.43 3.64

< .05 < .05 < .05 < .05 < .05 < .05 CS2

PTSD

CS1 unpaired

NPTSD

CS1 unpaired

HC

y

Anterior prefrontal cortex Dorsolateral prefrontal cortex

Amygdala HC

x

CS1 unpaired > CS2

HC > NPTSD

HC > PTSD

H

CS1 unpaired

PTSD

CS2

NPTSD

CS2

HC

CS2

Note. The values refer to CS1unpaired versus CS2, CS1unpaired alone, and CS2 alone during early and late acquisition and extinction in 14 PTSD patients, 14 NPTSD, and 13 HC. The p values refer to familywise-error rate (FWE) corrected based on a ROI analysis approach.

Results Responses to the Trauma-Related Stimuli During Picture Viewing and Higher-Order Conditioning Picture-viewing experiment before the conditioning procedure during fMRI. In both traumatized groups, the US pictures were relevant for the trauma (Table 1). PTSD patients were significantly more reminded of the traumatic event (t(26) 5 22.16; p 5 .04; effect size: 0.36; 95% CI of the difference: lower.12, upper 4.67) than NPTSD. The three groups did not significantly differ in

arousal or valence ratings of the US pictures before the learning phase. Higher-order conditioning. PTSD patients had significantly higher ratings of dissociation than the NPTSD and HC (habituation, early acquisition, late acquisition, extinction: t 2.63, p < .05, effect sizes: lowest .07, highest 1.04; 95% CI of the difference: lower 2.11, upper 24.86). In addition, PTSD patients rated themselves as less able to watch the US pictures compared to NPTSD and HC (all phases: t 2.7, p < .05, effect sizes: lowest 0.64,

S.J. Diener et al.

1466 highest 1.13; 95% CI of the difference: lower 2.46, upper 4.4; Table 1). During habituation, the two trauma pictures (US1, US2) for patients with PTSD and NPTSD and the matched pictures for the HC resulted in significant neural activation in fear-relevant brain regions such as the amygdala, insula, or dlPFC (Table S1). There were no significant correlations between dissociation and ability to look at the pictures with the neural activation in response to the trauma-related stimuli. Responses to Conditioned Stimuli During Higher-Order Conditioning Arousal ratings. For arousal, Phase 3 Group (F(6,74) 5 5.22; p < .001, g2p 5 .2), and Phase 3 CS 3 Group, (F(6,74) 5 3.39; p < .01, g2p 5 .06) were significant. Only PTSD rated the CS1 as significantly more arousing than the CS2 in both early (t(13) 5 2.20; p < .05, effect size;.38 95% CI: lower 2.15, upper 2.05), and late acquisition (t(13) 5 3.40; p < .01, effect size: .39; 95% CI of the difference: lower 2.13, upper 2.12). For extinction, we did not find any significant effects. Valence ratings. For emotional valence, we found no significant group-related effects for either acquisition or extinction. US expectancy ratings. US expectancy was significant for Phase 3 Group (F(6,114) 5 2.6; p < .05). Whereas CS1 and CS2 were significantly differentiated in late acquisition in all groups (PTSD: t(13) 5 2.9; p < .05, effect size; 1.32 95% CI of the difference: lower.86, upper 5.71; NPTSD: t(13) 5 4.4; p < .01, effect size: 3; 95% CI of the difference: lower 2.08, upper 6.06; HC: t(12) 5 3.1; p < .05, effect size: 1.55; 95% CI of the difference: lower 1.1, upper 6.59, in early acquisition only), PTSD patients and HC (PTSD: t(13) 5 2.9; p < .05; HC: t(12) 5 3.7; p < .01) showed this differentiation. During extinction, patients with PTSD continued to display a significant differentiation between CS1 and CS2 (t(13) 5 2.7; p < .05, effect size: 1.12, 95% CI of the difference: lower.44, upper 3.99), with significantly higher values compared to NPTSD (t(26) 5 22.49; p < .05, effect size: .49; 95% CI of the difference: lower.49, upper 4.79), but not HC (Figure 1). Brain activation: ROI analyses. During early acquisition, PTSD patients showed significantly lower left amygdala activation in response to CS1unpaired versus CS2 (Figure 2). During late acquisition, they displayed a significantly lower activation in the right amygdala in response to CS1unpaired versus CS2. During extinction, the HC group, but neither PTSD patients nor NPTSD, showed significant activation in the dlPFC (BA46) to CS1unpaired versus CS2. Between the groups, we did not find any significant effects during habituation. NPTSD and HC compared to patients with PTSD showed significantly more activation in the dlPFC (BA46) to CS1unpaired versus CS2. Moreover, HC activated the BA9 region of the dlPFC and the right anterior insula significantly more than PTSD patients and the left insula significantly more than NPTSD. Brain activation to CS1unpaired and CS2 alone showed that, during acquisition, and especially early acquisition, frontal regions were activated in response to the CS1unpaired in all groups, but with stronger responses in regions including the ACC, dlPFC, and OFC in the HC compared to PTSD patients and NPTSD. For CS2, we did not find any significant differences between the groups. During extinction, CS1unpaired presentation also resulted in frontal

activation in all three groups (HC: BA11, BA9, BA10, BA46; PTSD patients: BA11; NPTSD: BA33, BA24), and, additionally, also in response to the CS2 (HC: BA46; PTSD patients: BA46; NPTSD: BA33). An overview of the brain responses to CS1unpaired and CS2 alone as well as the differential brain activation (CS1unpaired vs. CS2) during early and late acquisition and extinction are given in Table 2. Brain activation: PPI analyses. For early acquisition, we found a significant negative interaction of the amygdala with the dlPFC (BA9) for CS1unpaired versus CS2 in the patients with PTSD, while NPTSD showed a significant positive interaction of the amygdala with the left ventral ACC (BA24), and a significant negative interaction with the dACC (BA32; see Figure 3). In the HC, we observed a significant negative interaction of the amygdala with the left and right ventral ACC (BA24; see Figure 3). During late acquisition and extinction, we did not find significant interactions of the amygdala and PFC or OFC in any of the three groups. Between-groups analyses revealed a significantly stronger negative interaction of the left amygdala with the left dlPFC (BA9) during early acquisition in PTSD patients compared to HC. During late acquisition and extinction, we did not find significant interactions of the amygdala and PFC or OFC between groups. An overview of the results of the PPI is shown in Table 3. Role of PTSD Symptoms, Dissociation, and Depression in Conditioning to Trauma-Related Stimuli We found no significant correlations between PTSD-related symptoms, dissociation, and ratings with the neural response patterns in the group of PTSD patients and NPTSD. Since the groups showed significant differences in depression (see Table 1), we repeated all the analyses using the CES-D depression scores as a covariate. This did not result in different findings, and depression was also not significantly correlated with ratings or brain responses. Discussion The goal of the present study was to examine behavioral and brain correlates of higher-order fear conditioning using trauma-related stimuli as US in PTSD. PTSD patients showed enhanced arousal ratings to the CS1 during acquisition and delayed extinction of the US-expectancy. This pattern was accompanied by reduced amygdala activation and negative amygdala-dorsolateral prefrontal connectivity during acquisition and may represent maladaptive anticipatory coping behavior for trauma-related stimuli in PTSD. The amygdala is a brain region that triggers emotional processes and activates prefrontal regions important for behavioral control. Deficient activation related to CS1 versus CS2 and negative connectivity might indicate insufficient preparation for the confrontation with the trauma-related stimuli as also evidenced by the higher arousal ratings and stronger perpetuation of US expectancy—a pattern that reflects an imbalance in psychophysiological responding in the context of exposure to trauma-related stimuli. These results refer to within-group effects, which did not become significant in modeling group (PTSD vs. NPTSD vs. HC) and CS (CS1 vs. CS2). During habituation, the presentation of trauma cues (US) led to robust responses in fear-related brain areas such as prefrontal regions and amygdala, which were consistently reported to be active during mental representations of emotion in healthy individuals (Barrett, Mesquita, Ochsner, & Gross, 2007). Moreover, we


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Figure 3. Psychophysiological interaction (PPI) analysis during CS1unpaired > CS2 during early acquisition in patients with posttraumatic stress disorder (PTSD), traumatized subjects without lifetime history of PTSD (NPTSD), and nontraumatized healthy controls (HC). Seed region: amygdala; p < .05, familywise error-corrected.

also found significant responses in the anterior insula, a region that is not only activated during fear processing, but also has a significant role in bodily and pain awareness (Craig, 2009). Together with high ratings of arousal and unpleasantness across all participants, these findings suggest that the picture US were appropriate and similar to first-order aversive stimuli. PTSD patients did not report significantly higher arousal or aversiveness ratings compared to NPTSD or healthy controls, thus ensuring that the picture stimuli used as US were comparable across the three groups. The dissociation present in the PTSD group might, however, have prevented even higher ratings. All participants learned the CS/US association, indicating explicit knowledge of the associations between CS1 and US occurrence and the CS2 and US nonoccurrence. These findings support the assumption that trauma-related stimuli can serve as unconditioned stimuli. During acquisition, we could not detect significant activation in the amygdala or the ACC in the HC, although these regions were active in previous fear conditioning studies (Sehlmeyer et al., 2009), including picture-picture conditioning (Klucken et al., 2008; Schweckendiek et al., 2011). However, in these studies, the authors merged patients with controls in their analyses with marginal results (Mechias et al., 2010; Schweckendiek et al., 2011), suggesting less stable and robust activations in general with pictures as US and higher-order conditioning designs. The HC in our study activated conditioning-relevant regions like the anterior insula and the dlPFC during early acquisition more than the patients with PTSD. The anterior insula was repeatedly reported to be involved in fear conditioning (Birbaumer et al., 2005; Sehlmeyer et al., 2009) and is

assumed to convey a cortical representation of fear to the amygdala (Phelps et al., 2001). The dlPFC was found to be activated during the anticipation of and exposure to aversive pictures (Nitschke, Sarinopoulos, Mackiewicz, Schaefer, & Davidson, 2006). Surprisingly, HC revealed increased dlPFC activation in response to CS1unpaired versus CS2 during extinction. However, additional analyses showed that this response was time-dependent—it was present in the first half, but not at the end of extinction—and may thus be explained by the strong response in this region during acquisition in HC. In contrast to our previous study (Wessa & Flor, 2007), the NPTSD failed to differentiate between CS1 and CS2 in arousal and valence, despite the adequate perception of the CS/US association. A major difference between the two studies was the MR environment, which was part of our present study but not of the study by Wessa & Flor (2007), which is known to increase arousal and aversiveness independent of specific stimuli and may thus provide a different baseline (Lang & Bradley, 2010). PTSD showed a different brain activation pattern, with lower activation in the amygdala in response to CS1unpaired versus CS2 during acquisition. Previously, an overactivation of the amygdala, the dlPFC, and the vmPFC was found in response to fearful emotional stimuli, partly additionally associated with hyperarousal (Stevens et al., 2013), and during the presentation of aversive or traumarelevant stimuli or during symptom induction in PTSD patients (Bryant et al., 2008; Shin, Rauch, & Pitman, 2006; Stevens et al., 2013). Insufficient inhibitory control from the mPFC on the limbic fear pathway was assumed to be responsible for failure to recall extinction in PTSD (Bremner et al., 2005; Milad et al., 2009). Blair

S.J. Diener et al.

1468 Table 3. Results from the Psychophysiological Interaction Analysis Group

Contrast

Brain region

BA

H

x

y

z

t value

p value


BA24

L R

23 3

0 3

36 33

5.65 5.12

< .05 < .05


BA9

R L

12 236

48 30

33 42*

5.71 4.68

< .05 < .05

Dorsal anterior cingulate cortex

BA32

R

15

33

21

4.64

< .05


BA24

R

9

33

18

2.93

< .05

Dorsolateral prefrontal cortex*

BA9

R

6

36

36

3.47

< .05

Positive interaction—Early acquisition NPTSD CS1unpaired > CS2 Negative interaction—Early acquisition PTSD CS1unpaired > CS2 NPTSD

CS1unpaired > CS2

HC

CS1unpaired > CS2

PTSD > HC

CS1unpaired > CS2

Note. The psychophysiological interaction analysis used the left amygdala as seed region in 14 patients with posttraumatic stress disorder (PTSD), 14 traumatized subjects without PTSD (NPTSD), and 13 non-traumatized healthy controls (HC) in the early acquisition phase. *ROI analysis based on sphere (Mechias, Etkin, Kalisch, 2010).

et al. (2013) found such disrupted recruitment of the dorsolateral, and inferior and superior frontal cortex also in response to trauma-related stimuli in PTSD patients compared to NPTSD and HC. Moreover, the dlPFC was previously found to be implicated in the regulation of negative (sadness and decease; Freed, Yanagihara, Hirsch, & Mann, 2009) and positive (happiness) emotions (Habel, Klein, Kellermann, Shah, & Schneider, 2005), and plays a role in conflict processing in an emotional context (K€uhn et al., 2011). In addition, high dlPFCamygdala connectivity was found to be associated with reduced attentional bias toward negatively valenced (decease-related) words (Freed et al., 2009). In the present study, we observed increased depression scores in PTSD patients compared to both control groups. However, depression was not significantly related to the neural activation patterns found in PTSD patients. Since higher-order conditioning presumes that the CS1 gains the emotional value of the US, which in our case was a trauma-related stimulus, and most probably causes reexperiencing symptoms in the patients, it was unexpected to observe a lower amygdala activity in patients with PTSD. However, the assumed exaggerated amygdala response to aversive stimuli has been reported to be less robust specifically in PTSD patients (Etkin & Wager, 2007). Based on a vigilance-avoidance hypothesis, after rapid response to aversive stimuli, anxious subjects tend to subsequently develop an attentional avoidance pattern to alleviate the fear reaction (Koster, Verschuere, Crombez, & Van Damme, 2005; Mogg, Baldwin, Brodrick, & Bradley, 2004; Pflugshaupt et al., 2005). Moreover, amygdala activation has recently been discussed as tracking associability rather than prediction error in associative fear learning, thereby representing a role in vigilance and attentional gating in learning (Li, Schiller, Schoenbaum, Phelps, & Daw, 2011). High associability and thus higher activation in the amygdala may reflect an increase in learning about cues with low predictability and a decrease in learning when predictions become reliable. The formation of new aversive associations, and thus new learning, is an important aspect in PTSD, being related to the generalization of conditioned fear, which was shown to be biased toward stimuli of higher fear intensity in PTSD patients and thus in particular to cues that merely resemble the trauma (Morey et al., 2015). This was found to be related to brain activation patterns including the amygdala, with amygdala-calcerine and amygdala-thalamic connectivity being increased in PTSD patients in this context, and amygdala-vmPFC connectivity being increased in traumatized non-PTSD individuals, thus representing better safety learning (Morey et al., 2015). The

lower amygdalar activation to CS1 versus CS2 that we found in the patients with PTSD could thus be interpreted as premature suppression of limbic regions in order to avoid trauma-related emotions, such as fear. This might be related to a reduced capacity to profit from the associability of a cue that gates future learning. To further understand this lower amygdalar activation to CS1 versus CS2 in PTSD patients, PPI analyses with amygdala as source area were conducted. They showed an interaction between decreased amygdala activation and enhanced dlPFC activity during CS1unpaired versus CS2 in the patients. In both control groups, less activation of the amygdala during CS1unpaired versus CS2 was linked to enhanced mPFC and ACC activity. Functional and anatomical connectivity between the amygdala and the mPFC is well documented (Carmichael & Price, 1995). Das et al. (2005) found an inverse relationship between left amygdala and ACC activation during fear versus baseline in HC. Sarinopoulos et al. (2010) reported ACC activation after an uncertain cue that was comparable to the 50% reinforced CS1 in our study, to be inversely related to later and concurrent amygdalar activity to aversive pictures. Etkin and Wager (2007) concluded that activation in the rostral ACC and vmPFC mediates emotional reflexive coping processes to regulate the affective state. This connectivity pattern was present in our NPTSD and HC groups, suggesting successful automatic emotional coping with the situation. Such reflexive emotional coping was also found to be disturbed in anxiety disorders, for example, generalized anxiety disorder (Etkin, Prater, Hoeft, Menon, & Schatzberg, 2010). In line with these findings, our PTSD patients failed to show an inverse amygdala-vmPFC connectivity pattern. The region that interacted inversely with the amygdala in our sample was the dlPFC, which is involved in voluntary sadness regulation (Levesque et al., 2003) and voluntary downregulation of anticipatory emotion during the expectation of aversive stimuli (Herwig et al., 2007). Two mechanisms were recently highlighted as underlying vulnerability to anxiety disorders (Robinson et al., 2012): positive amygdala-dmPFC hyperactivity was discussed as a key neural mechanism in maladaptive anxiety in terms of aversive top-down amplification and increased vigilance toward aversive stimuli and negative amygdala-vmPFC connectivity in terms of failed top-down inhibition and extinction. Although we did not observe increased amygdala-dmPFC connectivity, our findings of a negative interaction between amygdala and the dlPFC during acquisition in patients with PTSD might also be interpreted as a neural link to maladaptive anxiety. This


1469

overactivation of the dlPFC along with a lower amygdala response might be a consequence rather than a predisposing factor of PTSD, which might result in maladaptive anticipatory coping behavior for trauma-related stimuli. Herwig et al. (2007) found a similar neural pattern of increased left mPFC and dlPFC activation along with lower left amygdalar activation in HC that explicitly used a cognitive control strategy to attenuate the emotional response to an aversive picture. This also indicates that the dlPFC-amygdala circuit may constitute both an adaptive and a maladaptive anxiety response as discussed by Robinson and colleagues (2012) for the dmPFC and the amygdala. In a study of Phan, Britton, Taylor, Fig, and Liberzon (2006), left amygdalar deactivation in response to aversive pictures was reported in PTSD in a block design, which might also have led to voluntary emotional coping processes permitting the formation of expectations toward later stimuli. However, due to the crosssectional nature of the present study, these interpretations need to be viewed with caution; and, for clarification, further research, in particular using a longitudinal design, is needed. In line with previous conditioning studies (e.g., Peri, Ben-Shakhar, Orr, & Shalev, 2000; Wessa & Flor, 2007), PTSD patients maintained their expectation that the CS1 would be followed by the US at the end of the extinction phase, indicating a lack of learning about the switch in significance of the CS1 Arousal ratings revealed stronger CS1/CS2 differentiation in the PTSD compared to both control groups during acquisition, but not extinction, indicating successful extinction in this measure in PTSD. However, the CS2 was rated as significantly more arousing throughout the experiment in patients with PTSD compared to the control groups. This suggests a sensitization to the visual stimuli in the arousal ratings, as the CS2 was the stimulus that was never paired with the trauma-related stimuli. It might also be indicative of insufficient safety signal learning. Although we did not find significant brain (de)activation in response to the CS2 or the CS1 alone, we did observe higher amygdala activation to the CS2 compared to the CS1 in PTSD patients in the present study. This lower activation of the amygdala to the danger stimulus along with higher activation of the amygdala to the safety stimulus might be indicative of maladaptive coping with traumarelevant stimuli, as indicated above, and deficient mobilization of amygdala-driven inhibitory control mechanisms in response to adverse stimuli. This is in line with other studies in which patients showed difficulties in conditioned discrimination of danger and safety cues and thus inhibitory control processes (for review, Lissek & van Meurs, 2015). A lack of inhibitory responses to the CS2 in PTSD patients was also reported by Peri et al. (2000), who, along with larger autonomic responses to the CS1, observed elevated skin conductance responses to the CS2 during acquisition and extinction. The present study should be seen in the light of several limitations. We did not include a peripheral measure of conditioning such as skin conductance responses, which may differ from the verbal rating results (e.g., Birbaumer et al., 2005), and we did not assess contingency awareness in a postexperimental interview, which needs to be considered in future studies, as CS2US contingency learning was observed to be reduced in PTSD (e.g., Blechert et al., 2007). The small sample size is another limiting factor and might have underestimated some effects and may lead to nonreplicable significant findings, which is considered particularly problematic in fMRI research

(cf. Guo et al., 2014). Due to a technical problem, we could not synchronize the eye tracker recordings and the fMRI assessments. However, careful instruction and visual inspection of the online eyetracking data ensured that the participants did not close their eyes, and it is therefore unlikely that differences in the deployment of attention between the groups might explain the findings. In addition, we investigated gaze fixation in a subset of 10 participants per group in a laboratory setting and found that the PTSD patients actually showed a trend toward significantly longer gaze fixations (in seconds) on the trauma pictures (PTSD: M 5 3.933, SD 5 .807, NPTSD: M 5 3.422, SD 5 .584, HC: M 5 3.385, SD 5 .973, PTSD vs. NPTSD: p 5 .06, PTSD vs. HC: p < .05) than the NPTSD and HC, although they verbally indicated that they were less able to watch the US on a subjective level. This was substantiated by the verbal ratings of the PTSD patients who clearly recognized the CS2US association and thus cognitively processed the stimuli. Our finding of reduced activity in the amygdala during late acquisition in PTSD was inferred from a significant t test comparing the neural response in the amygdala to CS1 versus CS2 in the patients, but not in controls or in NPTSD, and not from a significant group effect. One could argue that this significant finding in one group but not the other groups is insufficient evidence for different task-related activity between groups, as mean activity in one group may be just below the critical threshold and in the other group just above. However, while we found a significantly reduced response in the amygdala to CS1 versus CS2 in PTSD patients, the amygdala response was increased to CS1 versus CS2 in the other two groups, although not significant. This speaks against a possible near-threshold effect. The absence of a significant group effect for the amygdala response might be due to the small sample size. The assessment of dissociation, based on only one question, is also problematic, although increased dissociation in PTSD patients may indicate an inadequate coping response. A lack of correlation between self-reported dissociation and the observed aberrant amygdala responses may be due to the low reliability of a one-item measure and the low sample size. Finally, the conditioning process may not have been entirely comparable between the groups, as the US was of different personal value to the traumatized versus nontraumatized participants. However, whereas the nontraumatized controls showed successful conditioning, the traumatized control (NPTSD) group barely showed any emotional learning, which might be indicative of resilience in this group. The aim of the present study was to identify the involvement of brain regions during higher-order fear conditioning that might be important for the maintenance of fear responses after trauma exposure. The present study supports the assumption that trauma-related stimuli serve as unconditioned stimuli. Our results reveal that PTSD patients showed enhanced acquisition and delayed extinction, which is partly in line with our previous findings of amplified conditionability and failure to extinguish the conditioned response in higher-order conditioning. Lower amygdala activation as well as negative interaction of amygdala and dlPFC during early acquisition may represent maladaptive anticipatory coping behavior for trauma-related stimuli in PTSD. This study highlights the importance of trauma-specific cues in our understanding of mechanisms related to PTSD risk and resilience and specifically to maintenance and generalization of the symptoms.

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Supporting Information Additional supporting information may be found in the online version of this article Table S1: Brain activation during US presentation in the habituation phase. Table S2: Ratings for CS1 and CS2 alone during conditioning. Figure S1: Design of the second-order conditioning procedure.

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