Author’s Accepted Manuscript

Intrinsic Functional Connectivity of the Central

Nucleus of the Amygdala and Bed Nucleus of the
Stria Terminalis

Adam X. Gorka, Salvatore Torrisi, Alexander J.

Shackman, Christian Grillon, Monique Ernst
www.elsevier.com

PII: S1053-8119(17)30215-X
DOI: http://dx.doi.org/10.1016/j.neuroimage.2017.03.007
Reference: YNIMG13882
To appear in: NeuroImage
Received date: 21 November 2016
Revised date: 2 March 2017
Accepted date: 3 March 2017
Cite this article as: Adam X. Gorka, Salvatore Torrisi, Alexander J. Shackman,
Christian Grillon and Monique Ernst, Intrinsic Functional Connectivity of the
Central Nucleus of the Amygdala and Bed Nucleus of the Stria Terminalis,
NeuroImage, http://dx.doi.org/10.1016/j.neuroimage.2017.03.007
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Intrinsic Functional Connectivity of the Central Nucleus of the Amygdala and Bed
Nucleus of the Stria Terminalis

Adam X. Gorka1*, Salvatore Torrisi1, Alexander J. Shackman2, Christian Grillon1,

Monique Ernst1

1
Section on the Neurobiology of Fear & Anxiety, National Institute of Mental Health,
Bethesda, MD 20892 USA,

2
Department of Psychology and Neuroscience and Cognitive Science Program,
University of Maryland, College Park, MD 20742 USA

*Corresponding author: Adam X. Gorka axgorka@gmail.com; 15K North Drive Rm 300-

F, Bethesda, MD 20892

Abstract
The central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis
(BNST), two nuclei within the central extended amygdala, function as critical relays
within the distributed neural networks that coordinate sensory, emotional, and cognitive
responses to threat. These structures have overlapping anatomical projections to
downstream targets that initiate defensive responses. Despite these commonalities,
researchers have also proposed a functional dissociation between the CeA and BNST,
with the CeA promoting responses to discrete stimuli and the BNST promoting
responses to diffuse threat. Intrinsic functional connectivity (iFC) provides a means to
investigate the functional architecture of the brain, unbiased by task demands. Using
ultra-high field neuroimaging (7-Tesla fMRI), which provides increased spatial
resolution, this study compared the iFC networks of the CeA and BNST in 27 healthy
individuals. Both structures were coupled with areas of the medial prefrontal cortex,
hippocampus, thalamus, and periaqueductal gray matter. Compared to the BNST, the
bilateral CeA was more strongly coupled with the insula and regions that support
sensory processing, including thalamus and fusiform gyrus. In contrast, the bilateral
BNST was more strongly coupled with regions involved in cognitive and motivational
processes, including the dorsal paracingulate gyrus, posterior cingulate cortex, and
striatum. Collectively, these findings suggest that responses to sensory stimulation are
preferentially coordinated by the CeA and cognitive and motivational responses are
preferentially coordinated by the BNST.

Keywords:

Central nucleus of the amygdala; bed nucleus of the stria terminalis BNST; resting state
networks; intrinsic functional connectivity; 7-Tesla fMRI

1. Introduction
The central nucleus of the amygdala (CeA) and the lateral bed nucleus of stria
terminalis (BNST) are two small, highly interconnected structures, which are the key
components of the central extended amygdala (Olmos & Heimer, 1999). Anatomical
tracing studies in animals demonstrate that the CeA and BNST are both ideally
positioned to contribute to the behavioral and physiological responses to threat (Davis &
Shi, 1999) and a large animal literature suggests that these structures function to
maintain defensive responses to threat in the environment (Davis, Walker, Miles, &
Grillon, 2010). Researchers have suggested that the central extended amygdala plays a
critical role in the development and maintenance of anxiety disorders, depression, and
substance abuse (Avery, Clauss, & Blackford, 2016; Koob & Volkow, 2010; Shin &
Liberzon, 2009) which highlights the importance of developing a deeper understanding
of the functional role played by the CeA and BNST. Previously, we reported on the
intrinsic connectivity of the BNST using ultra-high field neuroimaging (7-Tesla fMRI),
which provides increased spatial resolution and provides an opportunity to examine the
functional architecture of small neural structures (Torrisi et al., 2015). Here, we set out
to address the novel question of whether patterns of functional connections differentiate
the BNST from the CeA, within these same participants.

In addition to their shared efferent projections to effector centers for defensive

responses, such as the PAG, the CeA and BNST both receive afferents from structures
of the emotion processing network such as the hippocampus, insula, and medial
prefrontal cortex (mPFC) (Bienkowski & Rinaman, 2012; Cullinan, Herman, & Watson,
1993; Hurley, Herbert, Moga, & Saper, 1991; Shi & Cassell, 1998). Despite widespread
commonalities in CeA and BNST patterns of anatomical connectivity, notable
differences exist. Recent research in rodents has demonstrated that the CeA receives a
higher number of projections from cortical and sensory-related regions relative to the
BNST, whereas the BNST showed preferential inputs from areas of the striatum
(Bienkowski & Rinaman, 2012). Additionally, lesions of the CeA block the expression of
threat-related responses to conditioned cues during aversive Pavlovian conditioning,
whereas lesions of the BNST block threat-related responses more diffuse aversive
contextual stimuli (Sullivan et al., 2004; Waddell, Morris, & Bouton, 2006). This
behavioral dissociation has led researchers to suggest that the CeA is preferentially
involved in “fear” responses to imminent threat, whereas the BNST orchestrates
”anxiety-like” responses to more diffuse and uncertain threat (Davis et al., 2010).
However, this specialization has recently been questioned, based on observations that
the CeA and BNST both respond to discrete, as well as diffuse, threat (Shackman &
Fox, 2016). An important way to contribute to this debate is to compare the functional
networks associated with these two structures.

iFC reflects the coherence of low frequency activity among brain regions, and provides
maps of brain functional networks (Fox & Raichle, 2007) which can shed light on the
potential similarities and differences between these two nuclei. Critically, previous
research has demonstrated that patterns of iFC can characterize functional
heterogeneity within neural structures (Cohen et al., 2008; Deen, Pitskel, & Pelphrey,
2011; Shen, Papademetris, & Constable, 2010), including the amygdala (Roy et al.,
2009). Yet to date, no research has directly examined the functional heterogeneity
within the dorsal extended amygdala (i.e., the BNST and CeA) using measures of iFC.
Although resting-state functional connectivity partly reflects the strength of anatomical
projections (Deco, Jirsa, & McIntosh, 2011), it is not limited to monosynaptic
connections and can also reveal more distributed networks of regions working together
(Raichle, 2009). Importantly, neural structures that lack direct anatomical projections to
the CeA and BNST, such as the dorsolateral prefrontal cortex or posterior cingulate, are
involved with the cognitive processing of negative information (Berman et al., 2010;
Cooney, Joormann, Eugène, Dennis, & Gotlib, 2010), and may be highly relevant to
subjective feelings of fear and anxiety. Comparing the iFC networks of the CeA and
BNST can clarify whether and how these structures interact with higher-order cognitive
systems.

Previous research has demonstrated that the central/medial region of the amygdala,
including the CeA, is functionally connected to multiple nodes of the emotional
processing network, such as the hippocampus, mPFC, anterior insula, and striatum
(Roy et al., 2009). Studies that have assessed the iFC networks of the CeA specifically,
demonstrate functional connections to regions that support cued aversive Pavlovian
conditioning (i.e. the dorsal anterior cingulate and thalamus) and sensory processing
(i.e. occipital cortex, temporal area TE, and the superior temporal sulcus) (Birn et al.,
2014; Oler et al., 2012, 2016). Although previous research suggests that the BNST is
functionally connected to the mPFC, hippocampus, thalamus, and striatum (Avery et al.,
2014; Torrisi et al., 2015), these studies do not report functional connections with the
anterior insula. This raises the possibility that the CeA and BNST differ in their
functional connections throughout the brain. However, direct comparisons of the iFC
networks of the CeA and BNST cannot be based on previous literature due to variability
in the sample, data collection, and analysis methods used to determine iFC. Here, we
set out to address this gap in the literature by using ultra-high field neuroimaging (7-
Tesla fMRI) to directly contrast the relative iFC strength of the BNST, which has
previously been reported for this sample (Torrisi et al., 2015), to the relative iFC
strength of the CeA

Based on the known anatomical projections and previously reported iFC networks of
these structures, we predicted that both the CeA and BNST would exhibit strong iFC
patterns with the PAG, thalamus, and hypothalamus, all of which receive afferents from
both the CeA and BNST. Furthermore, we predicted that the CeA would exhibit
significantly stronger iFC with the neural structures that support fear-related processes
(e.g., cued aversive Pavlovian learning) and that the BNST would exhibit significantly
stronger iFC with the neural structures that support anxiety-related processes (e.g.,
contextual aversive conditioning). Accordingly, the CeA would be expected to be
coupled more strongly with the dACC, anterior insula, thalamus, and areas of sensory
cortex (Fullana et al., 2016), whereas the BNST would be more strongly coupled with
the anterior hippocampus (Avery et al., 2014; Maren, Phan, & Liberzon, 2013; Strange,
Witter, Lein, & Moser, 2014; Torrisi et al., 2015), and regions associated with cognitive
processes (e.g., worry) recruited in response to anxiety (mPFC and medial posterior
cingulate cortex (Cooney et al., 2010; Paulesu et al., 2010).Third, based on a recent
tract-tracing report (Bienkowski & Rinaman, 2012), the BNST would be relatively more
strongly coupled with the striatum, whereas the CeA would be relatively more strongly
coupled with somatosensory information processing regions.

Finally, iFC patterns of the amygdala have been shown to be functionally lateralized
(Makovac et al., 2016; Roy et al., 2009). Previous research suggests that the left
amygdala habituates more slowly than the right, which has led researchers to suggest
that the left amygdala is specialized for sustained attention to discrete stimuli, whereas
the right amygdala is specialized for the dynamic detection of emotional cues (Wright et
al., 2001). Regarding the BNST, lesions to the medial frontal lobe impact the
metabolism of the right, but not left BNST (Motzkin et al., 2015). Taken together, these
reports suggest that both the CeA and BNST may exhibit different laterality effects
across left and right hemispheres. Although we make no a priori hypotheses concerning
the location or directionality of these effects, we expect that our exploratory analyses
might reveal similar hemispheric asymmetries when comparing the CeA to the BNST,
as those reported regarding iFC patterns of the amygdala (Makovac et al., 2016; Roy et
al., 2009).

2. Methods
 2.1. Participants
The sample reported here is identical to that reported in Torrisi et al., 2015. Twenty-nine
right-handed volunteers were recruited via flyers, print advertisements, and internet
listservs for participation in this study. Informed written consent was obtained from all
participants and approved by the National Institute of Mental Health (NIMH) Combined
Neuroscience Institutional Review Board. All participants were free from the following
exclusionary criteria: (a) current or past Axis I psychiatric disorder as assessed through
a clinician administered SCID-I/NP (First, Spitzer, Gibbon, & Williams, 2001), (b) first-
degree relative with a psychotic disorder, (c) a medical condition conflicting with safety
or design of the study, (d) brain abnormality on MRI as assessed by a radiologist, (e)
positive toxicology screen, or (f) MRI contraindication. Two participants were omitted
from analyses due to excessive head motion (i.e., more than 15% of acquired TRs with
a frame-to-frame Euclidean norm motion derivative greater than 0.3 mm) leaving a final
sample of 27 participants (M = 27.3 ± 6 SD years; 14 women).

2.2. Physiological Measures

To correct the fMRI data for nuisance physiological signals, respiration was measured
using a pneumatic belt placed around the diaphragm, and cardiac rhythm was
measured using a pulse oximeter affixed to the right index finger. Such correction has
been shown to greatly improve temporal signal-to-noise and BOLD sensitivity at high
spatial resolution (Hutton et al., 2011). Physiological data were sampled at 500 Hz using
a BioPac MP150 system (www.biopac.com).

2.3. BOLD fMRI Data Acquisition

MRI data were acquired using a Siemens Magnetom 7T scanner and 32-chanel head
coil. Third-order shimming was used to correct for field inhomogeneities. Blood oxygen
level-dependent (BOLD) signal was acquired using T2*-weighted echo-planar imaging
(EPI) across 49 ascending interleaved axial slices using the following parameters: TR =
2,500 ms, TE = 27 ms, flip angle = 70°, 1.3 mm3 voxels, acquisition matrix = 154 × 154;
240 volumes (10 min). A partial-brain field-of-view (FOV) was selected to capture dorsal
ACC/mPFC, BNST, amygdala, and hippocampus, while avoiding the eyes, which can
contribute to artifact (see Supplementary Figure 1 for illustration of brain coverage).
During the scan, participants were instructed to remain awake and fixate on a white
cross presented on a black background. All participants reported keeping their eyes
open during the resting-state scan. A 0.7 mm isotropic T1-weighted anatomical image
was also collected using a gradient-echo sequence (TR = 2,200 ms, TE = 3.01, flip angle
7°, acquisition matrix = 320 × 320).

2.4. CeA and BNST Seed ROIs

As shown in Figure 1A, the left and right CeA seed regions of interest (ROIs) were
defined using the publicly available probabilistic atlas of Tyska and Pauli (Tyszka &
Pauli, 2016), and thresholded at 20%.

Left and right BNST seed ROIs (Figure 1B) were manually prescribed on each
participants' T1-weighted image in native space as described previously (Torrisi et al.,
2015). Briefly, three authors independently demarcated the boundaries of the BNST for
each participant based on anatomical criteria. The three binary masks were averaged
and final ROIs were determined by majority consensus (i.e., thresholded at .667).

The volume of CeA seeds (left = 58 voxels; right = 46 voxels) and BNST seeds was
similar (left = 35.41 ± 9.7 voxels; right = 38.44 ± 8.4 voxels). However, follow-up
analyses demonstrate that our results are largely unchanged when the CeA seed is
thresholded to best reflect a priori neuroanatomical estimates of volume (for details, see
Supplemental Materials).

2.5. BOLD fMRI Data Preprocessing and Analysis

Resting state preprocessing began with FreeSurfer tissue segmentation (Fischl et al.,
2002). Ventricular images were resampled to 1.0 mm isotropic voxel resolution and
eroded by one voxel in each direction to ensure that these areas did not overlap with
gray or white matter. The rest of preprocessing and analyses then continued using AFNI
(Cox, 1996). The first three volumes of the functional run were discarded to allow for
steady-state equilibrium. Functional volumes were slice-time corrected using 3dTshift,
motion corrected and realigned using 3dvolreg, and co-registered to corresponding T1-
weighted image using 3dAllineate. T1-weighted images were normalized using
3dQwarp to the “ICBM 2009b Nonlinear Asymmetric” template in Montreal Neurological
Institute (MNI) space (http://www.bic.mni.mcgill.ca/ServicesAtlases/ICBM152NLin2009)
(Fonov et al., 2011; Fonov, Evans, McKinstry, Almli, & Collins, 2009). The resulting
affine plus nonlinear parameters were then applied to the functional volumes, which
were then spatially smoothed (2.6-mm FWHM Gaussian kernel). The final nominal
resolution of our functional data remained at 1.3 mm3.

The following nuisance signals were regressed from the functional volumes: 1) six head
motion parameters and their first derivatives; 2) eight slice-based cardiac regressors
(Glover, Li, & Ress, 2000); 3) five slice-based respiration regressors (Birn, Smith,
Jones, & Bandettini, 2008; Glover et al., 2000); 4) time series extracted from the lateral,
3rd, and 4th ventricles; 5) 0.01–0.1 Hz bandpass filter regressors; and 6) time-series from
local white matter within a 13-mm radius sphere surrounding each voxel using the
ANATICOR approach (Jo, Saad, Simmons, Milbury, & Cox, 2010). Average time series
were extracted for both bilateral seed ROIs, and separate seeds for each hemisphere
from the residualized functional images within the CeA and BNST. Pearson correlations
between each time series and all other voxels in the brain were calculated and Fisher r-
to-z transformed for group analyses.

2.6. Statistics and Analytic Strategy

The Fisher-transformed statistical images resulting from our preprocessing stream
represent estimates of iFC. Statistical images reflecting the iFC of the bilateral CeA and
of the bilateral BNST were entered into separate one-sample t-tests to determine which
voxels exhibited iFC that was significantly different from zero.

Paired t-tests were used to contrast CeA-iFC to BNST-iFC. The variable used was the
Fisher-transformed statistical variable reflecting the iFC of the bilateral CeA and bilateral
BNST.
Differences between CeA and BNST iFC can be driven by two patterns: 1) the iFC for
one seed is significantly greater than zero, whereas the iFC for the second seed is not
significantly different from zero, leading to significant greater iFC for the first seed
compared to the second; and 2) the iFC is significantly greater than zero for both seeds,
yet the mean iFC for one seed is nonetheless significantly greater than the other. In
order to distinguish between these two patterns, clusters identified as exhibiting different
iFC between CeA and BNST were characterized as being located within areas that
exhibited iFC with each seed, or areas in which the iFC for the two seeds overlapped.

Following the identification of the voxels which exhibited significant iFC with the CeA
and BNST respectively, results from each one sample t-test were binarized, such that
all voxels that survived correction for multiple comparisons were assigned a value of 1,
whereas all other voxels were assigned a value of zero. The resulting binarized images
were then multiplied together to reveal the conjunction of CeA and BNST iFC patterns.
Here, the term conjunction refers to the logical operator “AND” (Nichols, Brett,
Andersson, Wager, & Poline, 2005). Specifically, the logical conjunction of A and B is
true, when both A is true and B is true.

Finally, an exploratory analysis was conducted to assess hemispheric differences of iFC

between CeA and BNST. Statistical time-series for the left and right CeA, and for left
and right BNST were entered into a repeated-measures ANOVA model to test for
interactions of seeds (CeA vs. BNST) with hemisphere (left vs. right).

All group-level statistical maps were thresholded (p<0.05, whole-brain corrected) based
on cluster extent, using a cluster-forming threshold of (p < 0.0005, k = 21 (p = 5 × 10^−4))
via updated versions of 3dFWHMx and 3dClustSim. These updates incorporate a mixed
autocorrelation function (acf) that better models non-Gaussian noise structure (Cox,
Reynolds, & Taylor, 2016; Eklund, Nichols, & Knutsson, 2016).
3. Results
3.1. Bilateral CeA and BNST iFC
The CeA showed significant iFC with cortical and subcortical regions, including
distributed regions of the cortex, medial temporal lobes, BNST, striatum, PAG, and
cerebellum (Supplemental Figure 2).

The BNST iFC has been previously reported for these participants (Torrisi et al., 2015).
In brief, the BNST was intrinsically coupled with cortical and subcortical regions, notably
the CeA, striatum, PAG, and cerebellum (Supplemental Figure 3).

No negative associations (i.e. anti-correlations) were detected at corrected thresholds

for either the CeA or BNST seed.

3.2. Target regions shared by bilateral CeA and BNST iFC

As expected, a number of regions exhibited significant iFC with both the CeA and BNST
(Figure 2 and Table 1) as indexed by a conjunction analysis. These shared target
regions were distributed across subcortical and cortical areas. Subcortically, they
included midline thalamus (including areas consistent with the nucleus reuniens and
medial dorsal thalamus), anterior and posterior hippocampus (Figure 2A), amygdala
(including areas consistent with the lateral and basal amygdala nuclei) (Figure 2B),
striatal regions (caudate, ventral striatum, putamen) (Figure 2C), and multiple areas of
the midbrain, notably PAG (Figure 2D). Cortically, they consisted of clusters in the
frontal, parietal, and temporal lobes. Frontal cortical regions included an mPFC cluster,
extending ventrally into orbitofrontal cortex (BA11) and dorsally into dorsal mPFC
(BA9/10), and a rostral mPFC cluster within the paracingulate gyrus (Figure 2E).
Parietal cortical regions were in the precuneus (Figure 2E). The temporal cortical cluster
was within the superior temporal sulcus (Figure 2F). Finally, the posterior insula was
also coupled with both CeA and BNST.

3.3. Target regions that differ between bilateral CeA and BNST iFC
3.3.1. Regions more strongly connected to CeA

As shown in Figure 3, a paired t-test revealed significant differences in regional iFC

between the CeA and BNST (Table 2). Compared to the BNST, the CeA was more
strongly coupled with the middle insula (Figure 3A), ventral posterior thalamus (Figure
3B), supramarginal gyrus (BA40) (Figure 3C), post-central gyrus (BA43) (Figure 3C),
fusiform cortex (Figure 3D) and areas of the amygdala. These amygdala regions
included areas which were consistent with the basal and lateral nuclei.

To fully characterize differences between CeA and BNST iFCs, the location of each
significant cluster was carefully assessed in relation to the map of shared iFC
(conjunction analysis). The cluster located within the post-central gyrus did not overlap
with either the conjunction analysis or the areas identified as exhibiting iFC with CeA via
one-sample t-test. All clusters not centered on the right or left amygdala, including all
clusters in the insula, thalamus, supramarginal gyrus, and fusiform gyrus (Table 2)
existed outside of the areas identified through the conjunction analysis. The majority of
the voxels in these clusters overlapped with areas identified as exhibiting iFC with CeA
via one-sample t-test (average cluster overlap = 86.8% of voxels, range = 68.8 – 100%).
Meanwhile, of the voxels centered on the amygdala which were more strongly coupled
with the CeA, approximately 30 percent were located within the conjunction analysis
(left: 203 voxels, right: 222 voxels) and mostly centered within the dorsal amygdala. The
remaining voxels within the amygdala which were more strongly coupled with the CeA
existed outside of the conjunction mask (left: 488 voxels, right: 534 voxels) and
centered mostly within the ventral and lateral aspects of the amygdala.

3.3.2. Regions more strongly connected to BNST

Compared to the CeA, the BNST was more strongly coupled with the paracingulate
gyrus (on the border of BA9/32) and posterior cingulate cortex (BA23) (Figure 3E). The
BNST was also more strongly coupled with a cluster in the head and body of the
caudate nucleus bilaterally, extending into the ventral striatum (Figure 3F).

Notably, the clusters located within the paracingulate gyrus and posterior cingulate
cortex did not fall within the conjunction mask. The voxels within these clusters partially
overlapped with areas identified as exhibiting iFC with BNST via one-sample t-test for
both the paracingulate gyrus (63.5%) and the posterior cingulate cortex (36.4%).
Meanwhile, approximately 12 percent of the voxels within the contiguous clusters
centered on the BNST fall within the conjunction mask (left: 103 voxels, right: 135
voxels) centered on the BNST and ventral striatum, whereas the remaining voxels in the
cluster (left: 860 voxels, right: 988 voxels) fall outside of the conjunction analysis within
the dorsal striatum and caudate.

3.4. Effects of Hemisphere

Critically, repeated measure ANOVA analyses revealed interactions between seed and
hemisphere, such that differences in the iFC patterns between the CeA and BNST were
significantly different in the left and right hemispheres (Table 3). Four clusters which
exhibited an interaction between seed and hemisphere were centered on the right and
left CeA and BNST. Additional clusters exhibiting interactions were detected within the
middle temporal gyrus (BA 38), the inferior frontal gyrus (BA 44), and vmPFC (BA 11).
Post-hoc comparisons revealed that these three clusters were driven by a similar
pattern of results with the CeA exhibiting significantly stronger iFC compared to the
BNST in the left hemisphere (middle temporal gyrus: T = 4.33, p < 0.001; inferior gyrus:
T = 3.00, p < 0.01; vmPFC: T = 3.53, p < 0.005), whereas the BNST exhibited
significantly stronger iFC compared to the CeA in the right hemisphere (middle temporal
gyrus: T = -3.50, p < 0.005; inferior gyrus: T = -2.78, p < 0.05; vmPFC: T = -2.25, p <
0.05). Further, iFC of the left CeA was significantly stronger than the connectivity of the
right CeA (middle temporal gyrus: T = 5.00, p < 0.001; inferior gyrus: T = 4.71, p <
0.001; vmPFC: T = 4.81, p < 0.001), whereas for the BNST the right hemisphere
consistently demonstrated enhanced connectivity (middle temporal gyrus: T = -3.71, p <
0.001; inferior gyrus: T = -2.34, p < 0.05; vmPFC: T = -3.63, p < 0.001). No post-hoc
comparisons between left CeA and right BNST, or between right CeA and left BNST
reached statistical significance (all p’s > 0.05).

Notably, the vmPFC (Figure 4) cluster exhibiting an interaction between seed ROI and
hemisphere was located within the conjunction mask and was also located within an
area that demonstrated an effect of hemisphere, specifically left CeA > right CeA, after
correction for multiple comparisons (for details see, Supplemental Tables).
For completeness, we report results from paired t-tests assessing differences between
left and right hemispheres, for both CeA and BNST seeds separately, within
Supplemental Materials (for details, see Supplemental Table 2 and 3).

4. Discussion
In line with expectations, the whole-brain iFC maps of the CeA and BNST exhibited
broad overlaps, particularly regarding connectivity with downstream neural effectors for
defensive responses, such as the PAG and areas of the midbrain, and with upstream
structures involved in threat-related information processing, such as the mPFC,
hippocampus, and thalamus. The main and novel findings concern the regional
differences in whole-brain iFC of these two structures. The CeA was more strongly
connected to the insula, a region involved in internal-state information processing
(Craig, 2002), and to regions supporting sensory processing, i.e., thalamus and fusiform
gyrus (Alitto & Usrey, 2003; Tyler et al., 2013). Conversely, the BNST was more
strongly connected to regions involved in the conscious appraisal of threat and decision-
making, i.e., dorsal paracingulate gyrus (Mechias, Etkin, & Kalisch, 2010; Venkatraman,
Rosati, Taren, & Huettel, 2009), motivated behavior, i.e., striatum (Liljeholm &
O’Doherty, 2012), and self-referential processes, i.e., posterior cingulate cortex (pCC)
(Northoff et al., 2006). Additionally, our results further demonstrate that the relative iFC
strength of the CeA compared to the BNST within the vmPFC, inferior frontal gyrus, and
middle temporal gyrus, vary between left and right hemispheres of the brain. These
differences in iFC between the CeA and BNST will be discussed within the framework of
the ongoing debate about the relative role of these structures in defensive responses
(e.g. Davis et al., 2010; Shackman & Fox, 2016).

4.1. CeA, BNST, and defensive responses to threat:

In agreement with findings from anatomical studies, both the CeA and the BNST were
intrinsically connected to the PAG and areas of the midbrain. The PAG mediates
physiological and behavioral defensive responses (Bandler, Keay, Floyd, & Price, 2000;
LeDoux, Iwata, Cicchetti, & Reis, 1988) and the overlapping intrinsic connectivity of the
CeA and the BNST with this structure may reflect their common role in initiating
defensive responses to environmental threat.

Additionally, both the CeA and BNST exhibited intrinsic connectivity with multiple areas
of the midline thalamus which previous research has suggested are important for the
processing of threatening information. Both the CeA and BNST were intrinsically
connected to an area of the thalamus consistent with the interthalamic adhesion. The
interthalamic adhesion contains the nucleus reuniens, which is known from non-human
research to receive afferents from both the CeA and BNST (Aggleton & Mishkin, 1984;
Dong & Swanson, 2004). Additionally, areas of the medial dorsal thalamus likewise
exhibit functional connections with both the CeA and BNST. Stimulation of the medial
dorsal thalamus results in defensive behavioral responses (Roberts, 1962), and
previous research suggests that the medial dorsal thalamus is involved with cued and
context conditioning (Antoniadis & McDonald, 2006; Sarter & Markowitsch, 1985).
Although our observed results do not extend into the lateral geniculate nucleus, the
anatomical specificity of our findings suggests that patterns of iFC likely reflect the
functional role of these thalamic nuclei in processing threatening emotional information.

Lastly, the CeA and BNST were both intrinsically connected to the caudate and regions
of the ventral striatum, hippocampus, and large regions of the medial prefrontal cortex.
Specifically, areas of the medial prefrontal cortex included regions of vmPFC (BA 11)
and more dorsal areas of the medial frontal lobe (BA 9/10), but few clusters were
observed more caudally (i.e. BA 8/32). Exposure to threat can impact operant
responding (Jaisinghani & Rosenkranz, 2015), in addition to influencing sensory
(Grosso, Cambiaghi, Concina, Sacco, & Sacchetti, 2015) and cognitive processes
(Okon-Singer, Hendler, Pessoa, & Shackman, 2015). The present results suggest that
the CeA and BNST both interact with the neural networks that support instrumental,
sensory, and cognitive responses to threat.
4.2. CeA: preferential coupling with somatic and sensory networks

The CeA, relative to the BNST, exhibited stronger coupling with targets involved in
sensory processing. These regions consisted of the ventral posterior thalamus, the mid-
insula, the fusiform gyrus, and areas of the post-central gyrus.

The thalamus is a complex multi-nuclei structure, and the ventral-posterior region of the
thalamus processes noxious somatosensory information (Casey & Morrow, 1983).
Fibers from the ventral-posterior thalamic region also co-terminate in the amygdala
together with with input from other perceptual systems (Paré, Quirk, & Ledoux, 2004).
The co-occurrence of action potentials from neurons responding to noxious stimuli and
other neurons responding to perceptual qualities of originally neutral stimuli may form
the basis of aversive stimulus-stimulus associations (Blair, Schafe, Bauer, Rodrigues, &
LeDoux, 2001), and the greater iFC between the CeA and the ventral-posterior thalamic
region may reflect the role of this thalamic subregion in facilitating fear conditioning.

The insula is a primary hub for processing interoceptive information (Craig, 2002). The
iFC networks of the insula can be divided into three functional subdivisions (Deen et al.,
2011). The dorsal anterior insula is functionally connected to the dACC and cognitive
control network, the ventral anterior insula is connected to the pregenual cingulate
cortex, whereas the posterior insula is functionally connected to the primary and
secondary somatomotor cortices. Notably, the clusters more strongly connected to the
CeA, compared to the BNST, were distributed broadly throughout the insula and
appeared to be located in the posterior as well as the dorsal and ventral anterior
subdivisions. This differential coupling suggests that, compared to the BNST, the CeA is
more strongly communicates more strongly with regions involved with interoceptive
processing within multiple functional domains.

The fusiform gyrus is part of the ventral visual processing stream and forms the core
system for face perception and decoding, essential for social interactions (Dziobek I,
Bahnemann M, Convit A, & Heekeren HR, 2010; Tyler et al., 2013). Previous research
suggests that the amygdala interacts with the ventral visual stream during the
processing of emotional information (Vuilleumier & Driver, 2007) and the enhanced
coupled of the CeA and fusiform may reflect the well-established role of the amygdala in
responding to discrete biologically relevant stimuli, such as human faces.

Finally, the post-central gyrus processes somatosensory related information (Shergill et

al., 2013). Unfortunately, our functional imaging field of view did not extend into primary
somatosensory cortex (see Supplemental Figure 1), and as such, the full extent to
which the CeA is differentially coupled with the somatosensory system remains unclear.

Collectively, these results suggest that the CeA is functionally connected to regions
which process visual, interoceptive, and somatosensory information more strongly than
the BNST. This finding may reflect the particularly important generic role of the
amygdala in integrating salience and valence of stimuli for adaptive behavioral
responses. Although speculative, this relatively stronger functional association with the
coding of sensory characteristics of stimuli and their associated affective value might
reflect a closer link with the building blocks of fear learning. However, it should also be
noted that the CeA was also more strongly connected to the right supramarginal gyrus,
which is involved with a wide array of visuo-spatial processes including attention,
saccade eye movements, and grasping (Simon, Mangin, Cohen, Le Bihan, & Dehaene,
2002). As such, it is unlikely that the preferential role of the CeA, compared to the
BNST, be limited to strictly sensory domains.

4.3. BNST: preferential coupling with attentional, motivational, and self-referential

networks

The BNST, relative to the CeA, exhibited stronger coupling with regions involved in the
anticipation of negative events and in the integration of self-referential values to
behavior. These targets consisted of clusters in the ventral striatum, paracingulate
gyrus, and posterior cingulate cortex (pCC).

The nucleus accumbens and caudate nucleus are critical for the acquisition and
performance of instrumental actions (Berridge, Robinson, & Aldridge, 2009). Although,
there is no clear evidence that the BNST has direct anatomical connections with the
caudate nucleus, recent research from independent samples has demonstrated that
these two structures are strongly coupled at rest (Avery et al., 2014), and that fiber
paths from the BNST to the medial prefrontal cortex travel through the head of the
caudate (Krüger, Shiozawa, Kreifelts, Scheffler, & Ethofer, 2015). Taken together, the
strong coupling of the BNST with the striatum might represent one mechanism
underpinning how anxiety can impact motivated behavior (Avery, Clauss, & Blackford,
2016; Stamatakis et al., 2014).

The paracingulate region more coupled with the BNST than CeA was located within the
rostral dorsal mPFC. Specifically, this cluster was located on the border of BA 8/32,
caudally to areas identified through the conjunction analysis to be coupled with both the
CeA and BNST. Meta-analyses have suggested that a similar area of the rostral dorsal
mPFC is the most consistently activated region during the conscious appraisal of threat
in instructed-fear conditioning paradigms (Mechias et al., 2010). This region has also
recently received much attention with regards to negative affect, including anxiety,
linking it to the generation and maintenance of threat appraisal (Bijsterbosch, Smith, &
Bishop, 2015; Kim, Gee, Loucks, Davis, & Whalen, 2011; Shackman et al., 2011).

Meanwhile, the pCC, is more strongly coupled with the BNST than CeA. The pCC is
involved in self-referential mental processes and mind-wandering (Northoff et al., 2006).
Taken together, this differential connectivity pattern with structures involved in self-
generated thoughts, typical of worry, may suggest a preferential involvement of the
BNST with the cognitive aspects of anxiety, relative to the amygdala. However, pCC
activity has been linked to other mental activities, including autobiographical memory
(Spreng, Mar, & Kim, 2008) and researchers have suggested that the pCC may
promote internally-directed cognition or may serve to regulate the focus of attention
(Leech & Sharp, 2014). As such, our interpretation that iFC between the BNST and pCC
reflects self-referential activity is speculative, and this enhanced connectivity may reflect
other internally directed mental processes.

Finally, contrary to our expectation, the hippocampus was not found to be more strongly
connected with the BNST than the CeA. The hippocampus is critical for contextual
conditioning (Maren et al., 2013), which serves as a model for anxiety (defensive
response to diffuse threat) (Davis et al., 2010), and therefore, was expected to be more
closely associated with the BNST than the CeA. Although the BNST is strongly coupled
with the anterior hippocampus, our results do not support the hypothesis that, compared
to the CeA, the BNST is more strongly connected with the neural systems that support
contextual threat learning at rest.

4.4. Laterality Effects

We hypothesized that differences in iFC between the CeA and BNST would vary across
hemispheres, however, we made no a priori hypotheses concerning the location or
directionality of such effect. Although previous research has addressed differences
between the iFC of the left and right amygdala, the majority of studies focus on
associations between iFC and between subject factors such as gender or mood
diagnosis (Hahn et al., 2011; Kilpatrick, Zald, Pardo, & Cahill, 2006; Makovac et al.,
2016). Further, prior work which has formally tested for differences between amygdala
subregions do not report whole brain analyses testing for effects of CeA laterality (Roy
et al., 2009). Nonetheless, findings from animal models and human neuroscience can
provide insights into our results.

Research in animals suggests that dopamine and serotonin signaling within the left and
right amygdala differentially impact behavior, and that pain processing within CeA,
specifically, is lateralized (Andersen & Teicher, 1999; Bradbury, Costall, Domeney, &
Naylor, 1985; Ji & Neugebauer, 2009) Additionally, meta-analyses have demonstrated
that the left amygdala is more strongly activated than the right amygdala regardless of
paradigm and stimulus type (Baas, Aleman, & Kahn, 2004). During the repeated
presentation of emotional stimuli, the left amygdala habituates more slowly than the
right amygdala which has led researchers to suggest that the left amygdala is
specialized for sustained attention to discrete stimuli, whereas the right amygdala is
specialized for the dynamic detection of emotional cues (Wright et al., 2001).
Meanwhile, research has consistently demonstrated that the vmPFC is critically
involved with the extinction of discrete conditioned stimuli during cued aversive
Pavlovian learning (Ahs, Kragel, Zielinski, Brady, & LaBar, 2015; Milad et al., 2007;
Phelps, Delgado, Nearing, & LeDoux, 2004), and it is possible that the enhanced
connectivity between the left CeA and vmPFC reflects the need for enhanced regulation
of sustained CeA responses in the left hemisphere.
Alternatively, lesions of the orbitofrontal cortex in primates reduce BNST metabolism
and decrease freezing behavior (Fox et al., 2010). In line with this evidence, humans
who have sustained damage to the ventromedial prefrontal cortex exhibit reduced
resting metabolism within the BNST (Motzkin et al., 2015), suggesting that in contrast to
its role in down-regulating amygdala responses, the vmPFC may serve to up-regulate
activity within the BNST. Intriguingly, research suggests that vmPFC lesions impact the
metabolism of the right, but not left BNST (Motzkin et al., 2015), and it is possible that
the role of the vmPFC in generating BNST responses is reflected in the preferential iFC
between the vmPFC and right BNST.

Nonetheless, interactions between seed ROI and hemisphere were also observed
within the inferior frontal gyrus and middle temporal gyrus, and as such it is unclear
whether this pattern necessarily reflects the differential impact of regulatory structures
on CeA and BNST responses. Future research will be necessary to confirm these
patterns and shed light on their functional significance.

4.5. Future Challenges

This study is not without limitations. First, the field of view (FOV) used for functional
data collection did not include the most superior aspects of the frontal and parietal lobes
(see Supplement F1). As such, these data cannot address whether the CeA and BNST
are preferentially coupled with primary somatosensory and motor cortices, respectively.
This is important as anatomical tract tracing studies in rodents indicate that the CeA
receives a higher number of projections from cortical and sensory-related regions,
whereas the BNST shows preferential inputs from motor-related regions (Bienkowski &
Rinaman, 2012). Future research will need to fill this gap. Secondly, BNST seeds were
hand drawn resulting in a unique seed region for each participant. CeA seeds
meanwhile, were based on a standardized template and it is possible that this lack of
anatomical specificity impacted the signal to noise ratio in our measurements.
Additionally, caution should be used when interpreting clusters which exhibit significant
iFC, yet are in close proximity to seed ROIs. Although the Euclidean distance between
the BNST and caudate, and the distance between the CeA and lateral nucleus of the
amygdala, are large enough that we would not expect these results to be due to
smoothing (See Supplementary Results for full details), future research will be needed
to replicate the pattern of findings reported here.

Moreover, it is important to keep in mind that the resting-state fMRI approach only
informs connectivity patterns at rest. Patterns of connectivity during a task may
complement these resting-state maps by challenging the networks of these maps and
by providing functional significance. For example, the relative strength of the functional
connectivity of the BNST and CeA may appear different during acute threat vs.
sustained threat. The present work will help guide future experiments that test functional
probes of the BNST and CeA networks. Lastly, findings in healthy participants will need
to be contrasted with findings in patients suffering from anxiety or mood disorders, given
the central role of the CeA and BNST in emotion perturbations.

4.6. Conclusion
Collectively, the findings of this study suggest that the human CeA is most strongly
coupled with the neural systems that coordinate responses to sensory stimuli, whereas
BNST is preferentially coupled with regions that support self-representational processes
and goal-directed behavior. Additionally, these findings provide initial evidence that the
relative strength of CeA and BNST iFC differ across left and right hemispheres, which
may suggest differences in the lateralization of function between these nodes of the
central extended amygdala. The relative strengths of iFC networks are thought to reflect
the history of usage of these networks as well as structural connections between brain
regions (Deco et al., 2011; Raichle, 2009). As such, these results can inform
hypotheses about the organization of functional resting state networks and may shed
light on the relative roles of the CeA and BNST within distributed neural networks that
coordinate sensory, emotional, and cognitive responses to threat exposure.

5. Acknowledgements
Special thanks to Katherine O’Connell, Andrew Davis, and Brendon Nacewicz. This
work utilized the computational resources of the NIH HPC Biowulf cluster
(http://hpc.nih.gov). This work was supported by the Intramural Research Program of
the National Institutes of Mental Health, project number ZIAMH002798 (clinical protocol
02-M-0321, NCT00047853) to CG, and research grants from the National Institutes of
Health (DA040717 and MH107444) to AJS. The authors report no competing interest.
The author(s) declare that, except for income received from the primary employer, no
financial support or compensation has been received from any individual or corporate
entity over the past 3 years for research or professional service and there are no
personal financial holdings that could be perceived as constituting a potential conflict of
interest.
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Table 1. Spatial coordinates from the center of mass of clusters exhibiting intrinsic
connectivity with both the CeA and BNST as determined by the overlap between one-
sample t-tests.

# of
Coordinates voxels
Subcortical X Y Z
Right medial temporal lobe 20.7 -9.8 -11.3 1660
-contiguous cluster including the
hippocampus, amygdala, basal
forebrain, striatum, and thalamus

Left medial temporal lobe -15.8 -8.4 -7.1 2353

- contiguous cluster including the
hippocampus, amygdala, basal
forebrain, striatum, and thalamus

Right caudate (body) 10.4 3.6 13.1 32

8.2 15.7 6.7 21

Left insula -38.7 -22.4 4 46

Right parahippocampal gyrus 28.7 -35.6 -13.8 54

24 -29.8 -18.3 40

Left parahippocampal gyrus -29.2 -42.6 -7.1 30

-22.6 -38.8 -15.9 25
-17.7 -33.3 -11.7 21
 Right thalamus 11.7 -25 -10.1 31
3.2 -10.9 -10.4 30

Left thalamus -13.9 -26.7 -7 76

-1.6 -15.1 -9.8 29

Left claustrum -33.2 -4 -4.4 26

PAG 0.3 -31.8 -11.3 187

Midbrain -0.2 -21 -19.7 29

Frontal Lobe
mPFC -1.5 49.4 5.9 1312
-1.4 30 -23.2 23

Right inferior frontal gyrus 28.6 15.8 -18.9 59

Left inferior frontal gyrus -30.6 15.2 -18.2 25

Right precentral Gyrus 55.7 -6.6 37.8 25

Left precentral Gyrus -58.2 -10.3 36.7 37

Right middle Frontal Gyrus 38.4 35.6 -15.5 34

Temporal
Lobe
Right superior temporal
sulcus 55.9 -10.4 -11 114

Left superior temporal sulcus -61.9 -7.5 -9.3 128

-64.2 -20.7 -8.3 40

Left superior temporal gyrus -60.7 7.3 -5.6 31

-49.8 -13.2 6.8 26
 -67.1 -44.4 16.8 23

Left transverse temporal gyrus -50.3 -24 12.4 48

-41.9 -32.2 11.4 37

Right transverse temporal

gyrus 46.6 -20.6 10.1 22

Middle Temporal Gyrus (BA39) -51.1 -68.1 9.7 46

Left Fusiform Gyrus -46.6 -57.1 -21.5 29

Parietal Lobe Precuneus 3.2 -59.7 18 359

Posterior Cingulate -15.7 -54 4.7 33

6.7 -59.6 27.6 28
-3.8 -57.3 19.9 23

Occipital Lobe Left middle Occipital Gyrus -50.6 -78.9 -2.2 77

Cerebellum Right culmen 15.1 -54.9 -15.2 107

Table 2. Spatial coordinates and statistics from peak voxels exhibiting significantly
different intrinsic connectivity (CeA vs. BNST) as determined by paired t-tests

# of
Coordinates T statistic voxels
BNST >
CeA X Y Z
 Right BNST, caudate 6.8 4.3 1.4 18.33 1123

Left BNST, caudate -7.6 4.3 -1.2 20.87 963

Paracingulate gyrus -3.7 34.2 33.9 5.83 74

Posterior cingulate
cortex 4.1 -26.8 31.3 5.4 33

CeA >
BNST
Right amygdala 26.2 -10 -11.5 16.54 756

Left amygdala -24.5 -10 -11.5 21.16 691

Left insula -41.4 -2.2 9.2 7.74 188

-40.1 0.4 -9 5.66 60

Right insula 36.6 0.4 15.8 6.2 127

43.1 9.6 -7.7 7.09 23
36.6 4.3 -12.9 4.91 21

Right thalamus 15.8 -17.8 -1.2 5.89 47

Left thalamus -15.4 -19.1 1.4 5.16 26

Post-central gyrus 58.8 -21.7 28.8 5.29 37

Right supramarginal
gyrus 62.6 -13.8 14.4 5.68 32

Right fusiform gyrus 32.8 -35.9 -18 5.4 26

Table 3. Spatial coordinates and statistics from peak voxels exhibiting a significant seed
ROI (CeA vs. BNST) x Hemisphere (left vs. right) interaction as determined by repeated
measure ANOVA.

# of
Coordinates F statistic voxels

Seed ROI x
Hemisphere
Right BNST 6.8 5.6 -1.2 225.31 204

Left CeA -23.2 -8.7 -11.5 327.00 181

Right CeA 26.2 -8.7 -11.5 327.00 149

Left BNST -6.2 3.1 0.2 174.42 102

Inferior frontal gyrus -54.4 22.6 23.5 39.92 35

Ventromedial
prefrontal cortex -1.1 35.6 -23.2 30.46 31

Middle temporal gyrus -57.0 5.6 -22.0 27.43 21

Figures

Figure 1. Seed ROIs for intrinsic connectivity analyses. A): central nucleus of the
amygdala (CeA); B): bed nucleus of the stria terminalis (BNST).
Figure 2. Shared coupling. Regions showing significant iFC with both the CeA and
BNST are depicted in red (p<.05, whole-brain corrected). A) Thalamus and
hippocampus. B) Amygdala, including regions of the lateral and basal nuclei. C)
Striatum, including the caudate, putamen, and ventral striatum. D) Midbrain, including
the PAG. E) mPFC. F) Superior temporal sulcus.

Figure 3. Distinct connections. Regions with greater CeA iFC are depicted in red-yellow,
whereas regions with greater BNST iFC are depicted in blue (p<.05, whole-brain
corrected). CeA-preferring regions include the (A) middle insula, (B) ventral posterior
thalamus, (C) postcentral and supramarginal gyri, and (D) and fusiform gyrus. BNST-
preferring regions include the (E) paracingulate gyrus and posterior cingulate and (F)
head and body of the caudate nucleus bilaterally, extending into ventral striatum.

Figure 4. Seed ROI x Hemisphere Effects within Ventromedial Prefrontal Cortex. (A)
Statistical image depicting results within the ventromedial prefrontal cortex (vmPFC)
that exhibit a significant interaction between seed ROI (CeA vs. BNST) and hemisphere
(left vs. right). (B) Bar graphs depicting mean iFC values from the vmPFC as a function
of ROI and hemisphere. iFC values are reported in arbitrary units (A.U.). Error bars
reflect 1 standard error.

Highlights

• The CeA and BNST have overlapping functional connections

• The CeA is more strongly connected to regions involved in sensory processing
• The BNST is more strongly connected to regions involved in motivational processing
Supplemental Materials

Supplemental Methods:

Our rationale for thresholding the amygdala ROI to match the size of the BNST was based on
the idea that different numbers of voxels within an ROI may lead to changes in the signal to
noise ratio of the ROI as a whole, which could potentially impact our reported findings. To
ensure that this decision did not impact our pattern of results, we conducted follow up analyses
wherein the CeA ROI was thresholded to best match a priori neuroanatomical estimates.
Previous post-mortem research has suggested that the volume of the CeA is approximately 50
ml in volume (Herzog & Kemper, 1980; Tyszka & Pauli, 2016). We therefore thresholded the
probabilistic CeA ROI at 60% resulting in CeA ROIs that closely approximate previous estimates
(left CeA: 30 voxels, 65.9 ml; right CeA: 22 voxels, 48.3 ml). These CeA ROIs were used to
calculate iFC, using steps identical to those reported in our Methods (for full details, see 2.5.
BOLD fMRI Data Preprocessing and Analysis). Paired t-tests subsequently demonstrated that
the decision to use a relatively larger CeA ROI did not meaningfully impact our results (Table
S1.)

Supplemental Results:

The average intrinsic smoothness of our iFC images was calculated to be 4.48 mm, using the
“acf” calculation in 3dFWHM. Subsequently, we calculated the Euclidean distance between our
seed ROIs, and clusters observed within the caudate and basolateral amygdala. For the
caudate, coordinates were selected to occupy the most rostral extent of the cluster, while for the
basolateral amygdala, coordinates were selected based on the most inferior extent of the
cluster. For the coordinates of the CeA and BNST seed ROIs, we calculated distance based on
both the center of mass, and for the most rostral edge for the BNST, and the most inferior edge
for the CeA. All distances were greater than 12 mm (range= 12.64 – 22.07 mm; mean = 17.77
mm), suggesting that the observed clusters were not due to smoothing, or the spatial proximity
to the seed region.

Supplemental Tables:

Table S1.

# of
Coordinates T statistic voxels
BNST > X Y Z
CeA
Right BNST, caudate 6.8 4.3 1.4 18.57 1235

Left BNST, caudate -7.6 4.3 -1.2 21.17 1023

Paracingulate gyrus -3.7 34.2 33.9 6.05 91

Posterior cingulate
cortex 4.1 -26.8 31.3 5.43 104

  -3.7 47.7 33 4.97 55

CeA >
BNST
Right amygdala 24.9 -8.7 -11.5 17.08 584

Left amygdala -24.5 -10 -11.5 24.50 516

Left insula -41.4 -2.2 9.2 7.31 151

-40.1 -0.9 -9.0 5.77 47
-36.2 6.9 5.3 5.97 24

  -29.7 23.8 4.0 4.86 23

           
Right insula 36.6 0.4 15.8 5.60 48
40.5 6.9 10.5 4.94 41

Right thalamus  14.5  -20.3  -1.2  5.90  44 

Left thalamus  -15.4  -19.1  1.4  5.20  25 

Post-central gyrus  58.8  -21.7  28.8  5.19  34 

         

Right fusiform gyrus 30.1 -33.3 -20.7 5.21 24

Table S1. Spatial coordinates and statistics from peak voxels exhibiting significantly different
intrinsic connectivity (CeA vs. BNST), stemming from the follow up analysis where CeA volume
was matched to a prior anatomical estimates.
Table S2.

# of
Coordinates T statistic voxels
Left CeA
> Right
CeA X Y Z
Left amygdala
-23.2  -8.7  -10.2  24.36  270 

Posterior cingulate
cortex   -7.6  -52.8  -31.3  6.10  62 

Frontal pole   -5.0  62.8  11.8  5.86  53 

Middle temporal
gyrus   -71.2 -17.8 -10.2 5.91 47
-59.6 -32.1 -6.3 5.89 45
 

Middle frontal gyrus  53.5  4.3  40.4  5.92  32 

Ventromedial
prefrontal cortex  -2.4 39.4 -24.5 5.83 21

 
Right
CeA >
Left CeA  
Right amygdala
24.9  -8.7  -10.2  32.97  185 

Table S2. Spatial coordinates and statistics from peak voxels exhibiting significantly
different intrinsic connectivity between left and right CeA as determined by paired t-test

Table S3.
 Coordinates T statistic # of voxels
Left
BNST >

Right
BNST X Y Z
Left BNST
-6.2  -3.1  0.2  18.51  298 

Inferior parietal lobule  54.8  -42.4  28.8  5.23  38 

Right
BNST >

Left
BNST 
Right BNST – extending
into caudate   6.8 4.3 -1.2 16.21 298

Putamen  15.8  9.6  -5.1  5.38  21 

 
Table S3.. Spatial coordinates and statistics from peak voxels exhibiting significantly
different intrinsic connectivity between left and right BNST as determined by paired t-
test

Supplemental Figures:

Supplemental Figure 1. Group level results of (CeA vs. BNST) paired t-test at statistical
threshold of p=1.0, to display the EPI voxel coverage of 95% or more of the subjects.
Supplemental Figure 2. Voxels exhibiting intrinsic connectivity with the CeA as determined by a
one-sample t-test
Supplemental Figure 3. Voxels exhibiting intrinsic connectivity with the BNST as determined by
a one-sample t-test

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