Contents:
1. Supplementary Methods
a. Prolonged Exposure Therapy [page 1]
b. Psychological Measures [page 1]
c. Neuropsychological Measures [page 2]
d. Stimulus and Apparatus [page 2]
e. Image Acquisition [page 2]
f. Functional Imaging Analysis [page 3]
g. Functional connectivity analysis [page 4]
2. Supplementary Results
a. Brain x Behavior [page 6]
3. Supplementary Tables and Figures [page 7]
4. References [page 11]
1. Supplementary Methods
Prolonged Exposure Therapy:
During PE, patients complete twelve 90-min individual treatment sessions. PE has three main
components 1) psycho-education and deep breathing, 2) in-vivo exposure, and 3) imaginal exposure.
Psycho-education involves providing information on general PTSD aspects and planning a course of
treatment. The goal of this phase is to help patients understand that people often have similar
experiences after a life-threatening combat experience and to allow them to be prepared to engage in a
challenging but efficacious intervention. Deep breathing involves training in a core technique for
physiological control and relaxation that can aid in the processes of working through exposure
experiences. In-vivo exposure involves creating a hierarchy of situations that patients fear and avoid and
then gradually exposing them to these situations to help patients habituate to them. Patients create the
hierarchy during the sessions and practice daily in between sessions, to perpetuate habituation.
Imaginal exposure involves having patients review the traumatic event that caused the PTSD in session
by mentally placing themselves back in the situation. Patients are instructed to close their eyes and
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speak in the present tense while undergoing imaginal exposure and listen to audio recordings of the
imaginal exposures daily in between sessions.
Psychological Measures:
PTSD diagnosis was determined by the Clinically Administered PTSD scale(CAPS)(1), a structured
interview with 17 items from the DSM-IV criteria B, C, and D. The Structured Clinical Interview for DSMIV Axis I Disorders(SCID)(2), without the PTSD module, was used to diagnose major Axis I disorders,
including depression and substance abuse/dependence. The Deployment Risk and Resilience
Inventory(DDRI)(3) was administered to determine relevant risk factors pre, during, and post trauma.
The DDRI is a collection of measures for studying deployment-related experiences of military personnel
and veterans validated on military serving in the Gulf War. In addition, all subjects completed the
Connor Davidson Resilience Scale(CD-RISC-10)(4) and the Toronto Alexithymia Scale(TAS)(5) to quantify
resilience and the ability to identify and describe emotions, respectively. Clinical interview occurred on
the same day as the scanning session and was conducted by trained staff and validated by a clinical
psychologist (ANS).
Neuropsychological Measures:
Neuropsychological battery, which took place immediately following the end of second imaging
session, consisted of the following tests covering a variety of cognitive domains with particular emphasis
on memory, executive function, and attention: Wechsler Adult Intelligence Scale-3rd Edition(WAIS-III)
subtests(6), Vocabulary, Letter Number Sequencing, Matrix Reasoning, Controlled Oral Word
Association Test(FAS)(7), DKEFS Stroop Color and Word Test(8), and The Trail Making Test(TMT), Parts A
and B(9).
Stimulus and Apparatus:
2
During fMRI, all subjects performed a cued anticipation task that combined a continuous
performance task(CPT) with the interspersed presentation of aversive affective stimuli(supplementary
Figure 1). During the CPT, subjects were instructed to press a LEFT mouse button whenever they saw a
blue circle and a RIGHT mouse button whenever they saw a blue square on the screen. Stimuli were
presented at a visual angle of 4 degrees at a rate of 0.5Hz. Simultaneously, a 250msec long 500Hz tone
was presented at a rate of 2Hz. Subjects were instructed prior to the task that a switch to a green shape
accompanied by a 250Hz tone would indicate that a positive image was going to appear, and that a
switch to a red shape accompanied by a 1000Hz tone would indicate that a negative image was going to
appear. Seventeen positive images were taken from the International Affective Picture System(IAPS).
Seventeen negative images were selected that displayed individuals in combat situations without
graphic violence or open wounds. The total duration of the task was 580 seconds. Behavioral data
related to performance of the CPT were collected and scored for accuracy and latency of response.
Response accuracy and response latency(RT) were obtained during:(1) performance of the CPT,(2)
anticipation of a positive image(PA), and(3) anticipation of a negative image(NA).
Image Acquisition:
During the task, an fMRI run sensitive to BOLD contrast was collected for each subject using a
Signa EXCITE(GE Healthcare, Milwaukee) 3.0T scanner(T2*weighted echo planar imaging, TR=2000ms,
TE=32ms, FOV=250x250mm3, 64×64 matrix, 30 2.6mm axial slices with a 1.4mm gap, 290 scans). fMRI
acquisitions were time-locked to the onset of each trial. During the same experimental session, a highresolution T1-weighted image(TI=450ms, TR=8ms, TE=4ms, flip angle=12°, FOV=250x250, 172 sagittal
slices, ~1mm3 voxels) was obtained for anatomical reference.
Functional Imaging Analysis:
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Functional data were preprocessed and analyzed with the Analysis of Functional
NeuroImages(AFNI) software package. Preprocessed time series data for each individual were analyzed
using a multiple regression model. Regressors of interest included: 1) the PA, i.e. what activated during
anticipation of a positive image and 2) the NA, i.e. what activated during anticipation of a negative
image. In addition, nuisance regressors were entered into the linear regression model: positive and
negative image phases, three movement-related regressors used to account for residual motion(in the
roll, pitch, and yaw direction), and regressors for baseline and linear trends used to eliminate slow signal
drifts. To reduce the false positives induced by cross correlations of the time series data was fit using the
AFNI program 3dREML. Subsequently, contrasts were constructed on an individual subject level for
differential anticipation of negative and positive(i.e. NA- PA) images. A Gaussian filter with full widthhalf maximum 6 mm was applied to the voxel-wise percent signal change data to account for individual
variations in the anatomical landmarks. Data of each subject were normalized to Talairach coordinates
as defined by AFNI’s built-in atlases.
Voxel-wise percent signal change data were entered into a linear mixed effects model with
Time(pre/post-therapy) and Group(remitted/non-remitted, i.e., R-PTSD/NR-PTSD) as fixed factors and
subjects entered as a random factor. Analysis was done with the AFNI function 3dLME.R which used the
statistical program R(www.cran.org) and the lm library. Results are displayed that differ significantly in
the interaction(GroupxTime), between remitting(R-PTSD) and non-remitting(NR-PTSD; Group effect),
and over time(pre/post; Time effect). These results were masked by those that showed a significant task
effect for the NA-PA contrast in both groups.
A Monte-Carlo simulation(iterations =10,000) using AlphaSim was used to determine that a
cluster size of 832mm3(13voxels) in the task-related areas was required to control for multiple
comparisons maintaining an alpha of .05. The cluster F-values were calculated by averaging the voxel
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based F-values in each cluster. Average percent signal difference was extracted for post-hoc
correlational analysis.
Functional connectivity analysis:
Functional connectivity analysis was performed on both R-PTSD and NR-PTSD groups. The
connectivity results are reported for the NA–PA contrast. We used functional connectivity method based
on the psychophysiological interaction method introduced by Friston et al.(1997) and adapted for
AFNI(http://afni.nimh.nih.gov/sscc/gangc/CD-CorrAna.html). Before conducting the functional
connectivity analysis the individual raw signal datasets were: 1) band-pass filtered(0.009<ƒ<0.08), 2)
corrected for slice-dependent time shifts, 3) corrected for interleaved acquisition, 4) corrected for rigid
body head motion, and 5) warped to conform to the Talairach atlas(23). Individual time courses in these
processed raw signal datasets were extracted for the seed ROI, namely left ventral anterior insula that
showed significant TimexGroup interaction(Figure 1). Data points were censored if they differed by
more than 2 standard deviation from the average echoplanar signal for the seed ROI. We then used
AFNI function 3dTfitter to remove hemodynamic delay from the insula timecourse. The resultant signal
was then multiplied by the differential anticipation regressor(i.e, NA-PA) thereby creating an interaction
timecourse, which was then convolved with gamma-variate hemodynamic using the AFNI function
“waver.” Interaction time course were used as regressor of interest together with nuisance regressors
controlling for all task regressors, baseline differences, linear drift, non-time interaction seed time
series, and head movement(roll, pitch, and yaw). REML fitting was performed to reduce false positives
due to time-dependent cross correlations. The resulting correlation coefficient for the timecourse in the
insula ROI was calculated for each voxel. This provided correlation maps for the time course in the insula
ROI and the time course from all other brain voxels as a function of differential anticipation. The Fisher Z
transforms of these correlation maps were then warped to conform to the Talairach atlas(23) and a
5
Gaussian blur of 6 mm FWHM was applied. Voxel-wise data were entered into a linear mixed effects
model with Time(pre/post-therapy) and Group(R-PTSD/NR-PTSD) entered as fixed factors and subjects
entered as a random factor. Results are displayed that differed significantly in the
interaction(GroupxTime), between remitting(R-PTSD) and non-remitting(NR-PTSD; Group effect), and
over time(pre/post; Time effect). The resulting data was cluster thresholded using Monte-Carlo
simulations as described above, resulting in a minimum cluster of 3072mm3(48voxels) in the whole
brain.
2. Supplementary Results:
Brain x Behavior Relationships:
To investigate how signal change(anterior insula) or strength of connectivity changes between pre
and post-treatment(cingulate, mid-posterior insula), related to psychological and neuropsychological
variables we performed exploratory post-hoc Pearson correlations between the pre/post signal change
and behavioral/neuropsychological measures. Within the group as a whole(i.e., R-PTSD and NR-PTSD
combined) only the WAIS vocabulary(r=.457, n=24, p=0.033), CD-RISC-10(r=.614, n=24, p=0.007), and
TAS-20: Difficulty Describing Feelings(r=-.564, n=24, p=0.015) correlated significantly with the change in
functional connectivity between the anterior insula and right cingulate. Within the non-remitting group
a similar correlation was found with the WAIS Vocabulary(r=.589, n=24, p=0.021), CD-RISC-10(r=.701,
n=24, p=0.011), and TAS-20: Difficulty Describing Feelings(r=-.765, n=24, p=0.004) scores. However,
several weak correlations existed between change in the mid-posterior insula connectivity with Trails A
time(r=.525, n=24, p=0.045) and the change in anterior insula with Stroop Color Naming(r=-.545, n=24,
p=0.044). In the remitting group, there was a correlation between change in insula activation and Trails
B time(r=.761, n=24, p=0.047).
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3. Supplementary Tables and Figures:
Supplementary Table 1. , Group Psychological and Neuropsychological Performance
Variable
Remitting
CI Non-Remitting
CI Withdrawn
Number(All Males)
9
15
Neuropsychological Battery
Vocabulary SS
12.2(2.9)
10-14.4
11.7(3.1)
10-13.4
1
Matrix Reasoning SS
11.8(2.9)
9.6-14
11.5(3.3)
9.7-13.3
Letter Number Sequencing SS1
10(3.6)
7.2-12.8
9.9(2.1)
8.7-11.1
Trails A time(sec)
26.4(6.7)
21.3-31.6
26.1(9.4)
20.9-31.3
Trails B time(sec)
68.9(18.8)
54.5-83.4
61.8(20.4)
50.5-73.1
Stroop –Color Naming(sec)1
31(4.9)
27.2-34.8
29.5(6.6)
25.9-33.2
1
Stroop –Word Reading(sec)
22.2(3.6)
19.4-25
21.9(3.7)
19.9-24
Stroop –Color X Word(sec)1
56(13)
46-66
50.2(9.6)
44.9-55.5
Stroop –Color X Word Reversal(sec) 1
65.9(21.5)
49.4-82.4
52.2(9.2)
47.1-57.3
1
FAS Total Correct
45.1(10.8)
36.8-53.4
40.9(13.3)
33.5-48.3
Psychiatric Measures2
CD-RISC 10
31.4(5)
27.6-35.2
21.5(10.6)
15.6-27.4
TAS-20:Difficulty Identifying Feelings
20(9.7)
12.5-27.5
24.2(5)
21.4-27
TAS-20:Difficulty Describing Feelings
17.8(3.7)
15-20.6
18.8(3.5)
16.9-20.7
TAS-20:Externally-Oriented Thinking
21.8(3.6)
19-24.6
26.3(2.4)
25-27.6
TAS-20:Total
59.5(11.8)
50.4-68.6
69.2(8.4)
64.6-73.9
DRRI:Childhood Stress
4.1(2.8)
2-6.3
4.7(2.9)
3.1-6.3
DRRI:Prior Stress
43.4(5.4)
39.3-47.6
43.6(4.5)
41.1-46.1
DRRI:Combat Exposure Stress
55.2(11.9)
46.1-64.4
47(9.8)
41.6-52.4
DRRI:Life Disruption
58.1(9.1)
51.1-65.1
59.4(8.7)
54.6-64.2
DRRI:Social Support
24.4(9.7)
16.9-31.9
25.7(13.4)
18.3-33.1
DRRI:Work Environment
44.4(13.8)
33.8-55
40.7(17.1)
31.2-50.2
DRRI:Post Battle Exposure
18.7(4.5)
15.2-22.2
22.9(7.5)
18.8-27.1
DRRI:Harassment
51.3(5.3)
47.2-55.4
51.9(8.8)
47-56.8
DRRI:Perceived Threat
8.9(3.6)
6.1-11.7
10.5(3.4)
8.6-12.4
DRRI:Toxic Exposure
12.4(3.5)
9.7-15.1
12(3.3)
10.2-13.8
DRRI:Preparedness
23.2(6.2)
18.4-28
22.9(7.2)
18.9-26.9
DRRI:Post Stressors
53.2(15.4)
41.4-65
56.9(6.8)
53.1-60.7
DRRI:Post Social Support
5(3.8)
2.1-7.9
5.5(3.2)
3.7-7.3
1
2
Note. =One Non-remitting subject did not complete test item; = Non-remitting subjects(n=10) and
remitting subjects(n=8 or 7 for DRRI) ; SS=Scaled Score; sec=seconds;
Supplementary Table 2. Whole brain Treatment (R-PTSD/NR-PTSD) by Time (Pre/Post) Effects
voxels x
y
z F-stat Within
BA
314 -6 -72 -15 8.025 Left Cerebellum
280 42 -17 19
7.568 Right Insula
13
100 14 23 40
6.287 Right Medial Frontal Gyrus
32
86 -31 -45 22
6.008 Left Insula
13
75 -43 28
6
7.422 Left Insula/Inferior Frontal Gyrus 13
8
75
74
71
68
54
45
42
-25
-14
18
15
19
-23
-35
-15 41
7.348
-46 -27 8.427
37
1
6.259
-35 14
7.564
-86 2 10.179
14 30
6.166
-43 -18 7.013
Left Middle Frontal Gyrus
Left Cerebellum
Right Anterior Cingulate
Right Thalamus
Right Lingual Gyrus
Left Cingulate Gyrus
Left Fusiform Gyrus
9
24
32
17
32
20
Supplementary Figure 1. Affective anticipation task. In this task, blue shapes are presented at baseline,
while green shapes cue positive images and red shapes cue negative images.
10
Supplementary Figure 2. Whole brain Treatment (R-PTSD/NR-PTSD) by Time (Pre/Post) Effects
11
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