Evidence for coordinated functional activity within the extended amygdala of
non-human and human primates
Jonathan A. Olera,⁎, Rasmus M. Birna,c, Rémi Patriatc, Andrew S. Foxb, Steven E. Sheltona, Cory A. Burghyb,
Diane E. Stodolab, Marilyn J. Essexa, Richard J. Davidsonb,a, Ned H. Kalina,b
aDepartment of Psychiatry, University of Wisconsin-Madison, Madison, WI, USA
bDepartment of Psychology, University of Wisconsin-Madison, Madison, WI, USA
cDepartment of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
a b s t r a c ta r t i c l e i n f o
Accepted 11 March 2012
Available online 23 March 2012
Bed nucleus of the stria terminalis
Neuroanatomists posit that the central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis
(BST) comprise two major nodes of a macrostructural forebrain entity termed the extended amygdala. The
extended amygdala is thought to play a critical role in adaptive motivational behavior and is implicated in
the pathophysiology of maladaptive fear and anxiety. Resting functional connectivity of the Ce was examined
in 107 young anesthetized rhesus monkeys and 105 young humans using standard resting-state functional
magnetic resonance imaging (fMRI) methods to assess temporal correlations across the brain. The data
expand the neuroanatomical concept of the extended amygdala by finding, in both species, highly significant
functional coupling between the Ce and the BST. These results support the use of in vivo functional imaging
methods in nonhuman and human primates to probe the functional anatomy of major brain networks such as
the extended amygdala.
© 2012 Elsevier Inc. All rights reserved.
The amygdala occupies an important position in contemporary
neural models of emotion, and mounting evidence suggests that
amygdalar circuits play a key role in the pathophysiology of anxiety,
mood disorders and substance abuse (Aggleton, 2000; LeDoux,
2007; Shinnick-Gallagher et al., 2003; Whalen and Phelps, 2009). As
research into the function, hodology and clinical significance of
the amygdala progresses, the organization of this forebrain region
continues to be redefined (Roy et al., 2009; Solano-Castiella et al.,
2010) and some have called into question whether the conceptualiza-
tion of the amygdala as a unitary entity remains relevant (Cassell,
1998;McDonald, 2003;SwansonandPetrovitch,1998). The amygdala
is hypothesized to have four major supranuclear divisions: a super-
ficial cortical-like nuclear group, a basolateral nuclear complex, an
unclassified cell group (e.g. the intercalated cell islands), and an
siderable focus has recently been placed on the clinical relevance of
the extended amygdala (Luyten et al., 2012; Somerville et al., 2012),
an anatomical construct that was originally described by Johnson
(1923) in the early part of the last century and elaborated more
recently by Alheid and Heimer (1988), De Olmos and Ingram (1972)
and Heimer (2003). The extended amygdala is a basal forebrain con-
tinuum of striatal-like medium spiny neurons that run from the dorsal
amygdala, through the substantia innominata(SI), to the bed nuclei of
the stria terminalis (BST) and the shell of the nucleus accumbens
(Alheid, 2003; de Olmos and Heimer, 1999; Martin et al., 1991;
McDonald, 1992). Heimer et al. have proposed that two parallel col-
umns of extended amygdala neurons exist within the basal forebrain,
a central extended amygdala that includes the central nucleus of the
amygdala (Ce) and lateral portions of the BST (BSTL), and a medial
extended amygdala including the medial nucleus of the amygdala
(Me) and medial BST (see Figs. 1A, B).
Neuroanatomical studies in rodents and nonhuman primates
support the central extended amygdala concept by demonstrating
that the Ce and BSTL are strongly connected and share many efferent
targets (de Olmos and Heimer, 1999; Dong et al., 2001; Heimer and
Van Hoesen, 2006; Nagy and Pare, 2008; see Fig. 1C). Additionally,
although first noted by Johnson (1923), recent developmental data
suggest that some of the constituent neurons of the Ce and BSTL
are derived from similar embryological origins (Bupesh et al.,
2011). Despite the fact that these neuroanatomical data support
the concept of the extended amygdala, little data addresses the
degree to which activity in the Ce and BST regions comprises a func-
Components of the extended amygdala play pivotal roles mediat-
ing diverse behavioral, emotional, and physiological responses associ-
ated with stress, anxiety, reproduction and other motivational states
(Duvarci et al., 2009; Park et al., 2012; Regev et al., 2011). Animal
NeuroImage 61 (2012) 1059–1066
⁎ Corresponding author at: Wisconsin Psychiatric Institute and Clinics, HealthEmotions
Research Institute, 6001 Research Park Blvd., Madison, WI 53719, USA.
E-mail address: email@example.com (J.A. Oler).
1053-8119/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
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Fig. 1. The extended amygdala. (A) The human basal forebrain depicted in a series of coronal sections through the right hemisphere from the level of the ventral striatum (1) to
the level of the caudal amygdala (4). The central extended amygdala is depicted in yellow, the medial extended amygdala in green. The images were modified (with permission)
from Heimer et al. (1999), art by Medical Scientific Illustration, Crozet, Virginia. See original reference for details. (B) Schematic pseudo-3D illustration of the right amygdala
within the medial temporal lobe (modified with permission). For continuity, the central extended amygdala is depicted in yellow, the medial extended amygdala in green.
The extended amygdala is depicted as a continuous bridge of neurons running from the Ce and Me, through the SI region, to the BST and the VS (shell of the nucleus accumbens).
(C) 3D rendering of the rhesus monkey brain showing the approximate location and angle of the enlarged photo on the right. The autoradiograph (dark-field illumination)
depicts axon transport in the rhesus monkey following an injection of3H-amino acids into the region of the Ce. Note the dense labeling of axons and terminals through the
sublenticular area of the extended amygdala (reprinted with permission). Abbreviations: ac: anterior commissure; BLA: basolateral complex; BSTL: lateral bed nucleus of stria
terminalis; BSTM: medial bed nucleus of stria terminalis; BSTS/st: bed nucleus of stria terminalis, supra-capsular part/stria terminalis; Ce: central nucleus of the amygdala;
f: fornix; ic: internal capsule; LV: lateral ventricle; Me: medial nucleus of the amygdala; opt: optic tract; ox: optic chiasm; SI; substantia innominata; SLEA: sublenticular part
of extended amygdala; VS: ventral striatum.
J.A. Oler et al. / NeuroImage 61 (2012) 1059–1066
work suggests that altered amygdala and BST function may be impor-
tant in the pathophysiology of psychiatric disorders (Davis et al.,
2010; Fox et al., 2008; Heimer, 2003; Oler et al., 2010; Sink et al.,
2012). To examine whether the neuroanatomical concept of the
extended amygdala pertains to measures of temporal functional
connectivity, we used well-established functional magnetic reso-
nance imaging (fMRI) methods in a large group of young anesthe-
tized rhesus monkeys to assess whether intrinsic activity in the Ce
region is linked and coordinated with that in the BST. Resting-
state fMRI reflects synchronized variations in the neuronal activity
of a network, providing a window into the interaction, or connec-
tion, between brain areas. Rhesus monkeys were used since the
cytoarchitecture of the macaque amygdala is similar to that in
the human (Freese and Amaral, 2009), and this species provides
optimal translational models for understanding brain mechanisms
that underlie human psychopathology (Kalin and Shelton, 2003;
Machado and Bachevalier, 2003).
fMRI scans were performed in a large sample of peri-adolescent
monkeys using methods modified from prior work demonstrating
the reliability of collecting resting fMRI data in anesthetized rhesus
monkeys (Vincent et al., 2007). Blood oxygenation level dependent
(BOLD) time-series data were extracted from a right hemisphere Ce
region of interest (ROI) that served as a seed cluster for later connec-
tivity analyses. To assess the relevance of monkey Ce resting-state
connectivity to humans, we used a similar strategy to examine the
functional connectivity of the dorsal amygdala in an equivalently
sized sample of children and adolescents in which resting-state
fMRI data were collected.
Non-human primate imaging analyses
107 rhesus macaque monkeys (57 female; mean (±s.d.) age=
2.59 (±1.02) years) were used in this study. All animals were
mother-reared, and pair-housed at the Harlow Primate Laboratory or
the Wisconsin National Primate Research Center. All study procedures
were performed in accordance with the guidelines set forth by the
University of Wisconsin-Madison Animal Care and Use Committee
MRI data acquisition
Magnetic resonance imaging (MRI) data were collected using a
General Electric Signa 3T scanner (General Electric Medical Systems,
Waukesha, WI) with a quadrature birdcage extremity coil. Structural
MRI data were acquired using an axial 3D T1-weighted inversion-
recovery fast gradient echo sequence (repetition time, 9.4 ms; echo
time, 2.1 ms; field of view, 14 cm; flip angle, 10°; number of excita-
tions, 2; in-plane resolution, 0.2734 mm; number of slices, 248; slice
thickness, 1 mm; −0.05 mm interslice gap). Before undergoing MRI
acquisition, the monkeys were anesthetized with an intramuscular
injection of ketamine (15 mg/kg). Structural MRI data were collected
immediately prior to functional scans.
Functional MRI data acquisition
Functional data were acquired using a series of coronal T2*-
weighted echo-planar images (EPI) with the following parameters:
TR=2.5 s; TE=25 ms; flip angle=90°; FOV=140 mm; 26 slices;
matrix 64×64, voxel size 2.19×2.19×3.1 mm; 360 volumes; and
Resting state data analysis — preprocessing
All preprocessing steps were carried out in AFNI (Cox, 1996). EPI
data were corrected for slice timing differences and volumes were
registered using rigid-body image registration to correct for subject
motion. Field-map correction was performed to correct for echo-
planar image warping induced by B0-field inhomogeneities. For
each subject, EPI volumes were aligned to that subject's anatomical
image (using AFNI's 3dAllineate) and then aligned to a standard an-
atomical template (derived from the average of 238 high-resolution
anatomical images, see below) using a 12-parameter affine trans-
formation, and re-sampled to 0.625×0.625×0.625 mm resolution.
Data were then spatially smoothed (using a Gaussian kernel with
FWHM 3 mm), and temporally band-pass filtered to 0.01–0.1 Hz
(using 3dFourier). The first three imaging volumes for each resting
run were ignored to allow the magnetization to reach equilibrium.
Rhesus MRI brain template
The anatomical template used in this study was created using
standard methods, and was transformed to the stereotaxic space of
Paxinos et al. (2009). First, each subject's T1-MRI image was manually
stripped of non-brain tissue using SPAMALIZE; http://dx.doi.org/
htm. Brain extracted MRI images were originally registered to a 34-
brain template in standard space, using a 12-parameter linear trans-
formation with FMRIB Software Library's “flirt” tool (FSL; http://dx.
doi.org/http://www.fmrib.ox.ac.uk/fsl/) (Jenkinson et al., 2002). Im-
ages were manually verified, and averaged to create a study-specific
238-brain template in standard space. The brain-extracted MRI im-
ages in original space were then transformed to match this study-
specific template using non-linear transformation tool in FSL (“fnirt”).
5-HTT availability map and region of interest (ROI) creation
The [11C]DASB-PET methods are detailed elsewhere (Christian et
al., 2009), and are only briefly described here. DASB is a high-affinity
ligand of the serotonin transporter (5-HTT), and the carbon-11 for
the radiolabeling was produced with a National Electrostatics
9SDH 6 MeV Van de Graff tandem accelerator (Middleton, WI).
[11C]DASB-PET data were acquired in an independent sample of 34
rhesus monkeys (mean age=4.4 years; 12 male, 22 female) using a
Concorde microPET P4 scanner (Tai et al., 2001). The dynamic PET
time series were transformed into parametric images with each
voxel representing the distribution volume ratio (DVR) serving as
an index of receptor binding (Innis et al., 2007). The cerebellum
was used as a reference region, and all voxels were divided by
the mean cerebellar binding values. Each subject's DVR image
was transformed (based on the corresponding MRI transformation)
into the same standard space that the fMRI data were. This 5-HTT
availability map, thresholded at 250× background binding, precisely
localizes the Ce because compared with other dorsal amygdalar
regions the lateral division of the Ce has the highest levels of 5-HTT
binding (Bauman and Amaral, 2005; Freedman and Shi, 2001;
O'Rourke and Fudge, 2006) (Fig. 2B). The right Ce ROI used in the
present study was 40 mm3and can be seen in Fig. 2A.
Resting state connectivity analysis
Functional connectivity was computed using a seed-region based
approach. The amygdala ROI was defined using the 5-HTT availability,
as described in the section above. The resting-state EPI signalintensity
time courses were averaged over the seed ROI. Single-subject connec-
tivity maps were generated by computing a voxelwise general linear
model (GLM) with the mean seed time-series as the explanatory
variable. The resulting single-subject correlation coefficient (“connec-
tivity”) maps were normalized using Fisher's r-to-z transformation. In
order to account for physiological noise, average signal intensity time
courses from the white matter (WM) and cerebral spinal fluid (CSF),
were included as nuisance regressors (Jo et al., 2010). These nuisance
regressors wereobtainedin the followingmanner.First,each subject's
T1-weighted anatomical image was segmented into WM, gray matter,
and CSF using the FAST routine from the FMRIB Software Library (FSL)
(Zhang et al., 2001). WM masks were eroded by 2 voxels in each
J.A. Oler et al. / NeuroImage 61 (2012) 1059–1066
dimension in order to avoid partial volume contributions from gray
matter and CSF. To isolate the ventricular CSF, the CSF mask was
multiplied by a template-defined mask of the lateral ventricles. The
ventricular CSF and WM masks were then transformed to template
space and the average EPI signals over the ventricular CSF and white
matter were extracted. For group level analyses, a main effect t-test
(against zero) was performed on the Fisher-Z transformed correlation
coefficients for the seed time-series (using 3dttest).
Calculating the 95% spatial confidence intervals
Intrinsic fluctuations in the Ce signal were significantly correlated
with a number of brain regions (see Table 1). Connectivity maps were
stringently thresholded (t=10.0, and a minimum spatial extent
of 10.0 mm3), allowing us to isolate the regions with the strongest
functional connectivity with the Ce seed. Only clusters>10 mm3that
were located outside of the Ce seed region were further examined.
Within clusters, local maxima were identified using code adapted
Fig. 2. Serotonin transporter (5-HTT) labeling in the central nucleus (Ce) region of rhesus monkey amygdala used to delineate the seed region for monkey fMRI analysis. (A) In vivo
PET image demonstrating 5-HTT binding availability in the Ce and dorsomedially adjacent substantia innominata (SI) region, adapted from data first reported in Christian et al.
(2009). The image is thresholded at 250× the background binding level, and the circles indicate the regions of interest (ROIs) used in the present study as seed clusters for
analysis of the monkey functional connectivity data. (B) Comparable low-power photomicrograph showing the dense and selective expression of 5-HTT in the lateral division
of the Ce (image provided by Dr. Julie Fudge, University of Rochester School of Medicine and reprinted with permission of the publisher).
Locations of peaks in the functional connectivity data are presented above with the hemisphere, brain region and the volume of each Ce-connected cluster. All correlations were
positive and only clusters larger than 10 mm3are reported. Also presented are the t-values and the location (in millimeters relative to the anterior commissure, ac) of the peak
voxel within each macroscopic region in the cluster.
Coordinate of peak in mm relative to
Hemisphere ClusterVolume (mm3) Local maximatxyz
Anterior temporal lobe
Lateral occipital cortex
Anterior temporal lobe
Lateral occipital cortex
Inferotemporal cortex (TE)
Coordinate of peak in mm relative to
HemisphereCluster Volume (mm3)Local maximatxyz
L Anterior temporal lobe/striatum/basal forebrain 10,963Amygdala
Nucleus accumbens/BST region
Dorsal temporopolar region
Superior temporal sulcus
Lateral temporopolar region
Posterior midcingulate cortex
−15R Anterior temporal lobe/striatum/basal
J.A. Oler et al. / NeuroImage 61 (2012) 1059–1066
from FMRISTAT (http://dx.doi.org/http://www.math.mcgill.ca/keith/
fmristat/). Spatial confidence intervals (CI) around local maxima
in the connectivity maps were calculated using FMRISTAT (95%
2(.05))1/2) (Ma et al., 1999).
Human imaging analyses
To generate a comparable sample size, subjects from three indepen-
dent datasets were combined. Data from two different publicly avail-
able datasets were downloaded from the NITRC (http://dx.doi.org/
http://www.nitrc.org/projects/fcon_1000/) and combined with fMRI
datasets collected in our lab. The two sets of images downloaded from
the online database came from New York University (NYU, Milham,
M.P./Castellanos, F.X.) and the Nathan Kline Institute (NKI/Rockland
sample, Castellanos, F.X., Leventhal, B., Milham, M.P., Nooner, K.). Thus
for the present study, we analyzed data from 105 subjects. Sixty-six of
Study of Familyand Work, a prospective longitudinal study of child and
adolescent development (Essex et al., 2011). The mean (±s.d.) age of
the 66 adolescents was 18.44 (±0.19) years, 34 were male, and 10%
were racial or ethnic minorities. All study procedures were performed
in accordance with the guidelines set forth by the University of
the NYU data available onlineon theNITRC database. The mean (±s.d.)
age was 12.14 (±2.49) years, with the youngest participant being
7.88 years of age and the oldest 15.58 years old. More details on the
subjects are available in previously published studies (Di Martino et
al., 2008; Kelly et al., 2010; Margulies et al., 2007; Shehzad et al.,
2009). Finally, twenty-one subjects (11 males, 20% of the total number
of participants) with a mean (±s.d.) age of 12.25 (±3.67) years, were
selected from the NKI dataset, the youngest participant being 4 years
of age and the oldest 17 years old.
fMRI data collection
For the NYU dataset, a 3-Tesla Siemens Allegra scanner was used
to obtain the EPI and anatomical images. A gradient echo sequence
with the following parameters was used for the EPI: TR=2 s;
TE=25 ms; flip angle=90°; FOV=192 mm; 39 axial slices; matrix
64×64, voxel size 3×3×3 mm; 197 volumes; and duration=6 min
38 s. The anatomical images were acquired using a T1-weighted
MP-RAGE sequence with the following parameters: TR=2.5 s; TE=
4.35 ms; TI=900 ms; flip angle=8°; FOV=256 mm; 176 slices; and
voxel size 1×1×1 mm. For the NKI dataset a 3T Siemens Magnetom
Trio Tim scanner was used. The parameters for the EPI scans were as
follows: TR=2.5 s; TE=30 ms; flip angle=80°; FOV=216 mm; 38
axial slices; voxel size 3×3×3 mm; and duration=10 min 55 s. The
anatomical images were acquired with the following properties:
T1-weighted; MP-RAGE; TR=2.5 s; TE=3.5 ms; TI=1200 ms; flip
angle=8°; FOV=256 mm; 192 slices; and voxel size 1×1×1 mm.
Finally, the brain data collected at the Waisman Laboratory for Brain
Imaging and Behavior were acquired on a 3-Tesla Discovery MR750
with the following EPI scan parameters: TR=2 s; TE=25 ms; flip
angle=60°; FOV=24 cm; slice thickness=5 mm; matrix=64×64;
30 sagittal slices.
Resting state data analysis: preprocessing
All preprocessing steps were similar to those performed on the
monkey data, and were carried out in AFNI (Cox, 1996). Every subject
from each dataset was analyzed separately with the same steps. Slice
timing correction, motion correction (using Fourier interpolation),
spatial smoothing (using a Gaussian kernel with FWHM 6 mm), and
temporal band-pass filtering to 0.01–0.1 Hz (using 3dFourier) were
performed. The first three imaging volumes for each resting run
were ignored to allow the magnetization to reach equilibrium.
Image alignment and registration
Each anatomical MR image was skull-stripped and aligned to the
EPI in native space using AFNI. Functional and structural brain data
were then registered using affine transformation to a standard
template in AFNI that was centered relative to the posterior edge of
the anterior commissure. Taking the average of all 105 brain scans
generated a mean anatomical image.
Resting-state connectivity analysis
Functional connectivity was computed using a seed-region based
approach. An ROI corresponding to the Ce region was manually created
in the right amygdala on a standard 152-brain MRI template (see
Fig. 3D, inset). This was accomplished using the ROI drawing tool in
AFNI and was based on the detailed human brain atlas of Mai (Mai
et al., 2003), which is in the same standard space to which the
brain data were aligned. The right Ce seed ROI was then inflated by
2 mm in each cardinal (A–P, R–L, I–S) direction in order to account
for slight differences in registration across subjects, resulting in a Ce
ROI cluster of 944 mm3. The seed ROIs were mapped back to each
subject's EPI data, and the resting-state signal intensity time courses
were averaged over the ROI. Connectivity maps were generated for
each subject by regressing the seed time-series against all other
voxels in the brain. In order to account for physiological processes and
motion, average signal intensity time courses from the white matter
and CSF, along with six parameters of estimated subject movement (3
translations, 3 rotations), were included as nuisance regressors in this
menting each subject's T1-weighted anatomical image (using FSL's
FAST routine). These masks were multiplied by a priori tissue ROIs to
localize CSF signal to the lateral ventricles and to reduce partial volume
contributions of gray matter and CSF to the white matter. The CSF and
white matter masks were resampled to the EPI resolution, and the EPI
data within the masks were averaged. For group level analyses, a
main effect t-test (against zero) was performed on the Fisher-Z trans-
formed correlation coefficients for the seed time-series (using 3dttest).
A t=16.0 was used to stringently threshold the human connectivity
map, allowing us to isolate regions with the strongest functional con-
nectivity with the Ce seed.
In anesthetized peri-adolescent monkeys, spontaneous fluctua-
tions in the right Ce BOLD signal were highly significantly correlated
with BOLD signal fluctuations within six clusters identified using a
stringent statistical threshold (t>10.0; pb6.0e−17). The six clusters
consisted of bilateral anterior temporal lobe, bilateral basal forebrain
regions corresponding to the BST (Fig. 3A) and bilateral parieto-
occipital cortex (V4/V5). The locations of the peaks functionally con-
nected to the Ce (Table 1) were determined within each cluster by
calculating the 95% spatial confidence intervals around the voxels
containing the maximum t-values. Outside of the right temporal
lobe cluster containing the seeded region, the cluster demonstrating
the most significant functional connectivity with the right Ce seed
was in the contralateral (left-hemisphere) amygdala, while the clus-
ters displaying the third and fourth highest temporal correlations
with the Ce time-series contained the right and left BST, respectively.
We formally tested the extent to which connectivity with the BST was
greater than connectivity with V4/V5, the region exhibiting the next
highest peak correlated with Ce (see Table 1). Connectivity data
were extracted from the BST containing clusters, and connectivity
data were also extracted from the V4/V5 cluster in the right hemi-
sphere. To compare the extracted correlation coefficients, Pearson's
r-values were converted to standardized scores with Fisher's Z-
transform, and a paired-sample t-test of the difference between Ce–
BST and Ce–V4/V5 connectivity was conducted. This analysis revealed
J.A. Oler et al. / NeuroImage 61 (2012) 1059–1066
that Ce–BST connectivity was significantly greater than Ce–V4/V5
connectivity (t=4.629, pb0.00001).
To understand the extent to which Ce function is linked to compo-
nents of the extended amygdala other than BST, we examined Ce–SI
connectivity. While significant connectivity was observed between
Ce and SI (t=6.373, pb1.0e−8), it is important to note that the
level of connectivity between these regions was below the statistical
threshold that was used to identify the initial six Ce connected clus-
ters reported in Table 1. To examine whether the Ce–BST connectivity
was significantly greater than that observed for Ce–SI, we tested the
difference between Ce–BST and Ce–SI connectivity. As performed
previously to identify the Ce seed, the [11C]-DASB 5-HTT map was
used to generate a SI seed. This SI seed region can be seen in
Fig. 2A, dorsal and medial to the Ce seed. Ce connectivity data were
extracted from the SI region in the right hemisphere, the correlation
coefficients were standardized with Fisher's Z transform, and a
paired-sample t-test of the difference between Ce–BST and Ce–SI
connectivity was conducted. The analysis revealed that Ce–BST con-
nectivity was significantly greater than Ce–SI connectivity (t=2.213,
We next sought to assess whether the tight functional coupling
between Ce and BST we observed in anesthetized adolescent mon-
keys is present in awake young humans. As can be seen in Fig. 3C,
the thresholded Ce-connectivity map of the human brain revealed
discrete clusters of voxels with significant functional connectivity in
the BST region (t>16.0; pb8.0e−30). The locations of the peaks in
the human Ce functional connectivity data are presented in Table 1.
While differences in Ce functional connectivity exist between the
monkey and human, these potential disparities should be interpreted
with caution due to necessary methodological differences in acquiring
data from the two species (e.g., anesthesia). The possible confound to
cross-species comparison posed by the use of the 5-HTT map in the
Fig. 3. Ce and BST are functionally connected in the monkey and human. (A) Monkey and (C) human brain regions where the fMRI time-series were highly correlated (monkey:
dark purple: t=10.0; dark green: t=20; light green: t=30; human: dark purple: t=16.0; dark green: t=20; light green: t=24). The Ce seed region is depicted in the adjacent
section for both monkey and human (light purple). Red with yellow outline represents the 95% spatial confidence intervals around the peaks of the Ce-correlated activity within
each cluster. Sections modified (and reprinted with permission) from stereotaxic atlases of the (B) rhesus monkey brain (Paxinos et al., 2009) and (D) human brain (Mai et al.,
2003) corresponding to the MRI slices shown in A and C, respectively. The BST is depicted in light blue.
J.A. Oler et al. / NeuroImage 61 (2012) 1059–1066
monkeys versus manually drawn Ce ROI for the humans, should also
The present study expands the anatomical concept of the
extended amygdala to a functional level by providing evidence in 2
primate species for evolutionarily conserved strong temporal coupling
ical and neurochemical data linking the Ce and BST, and supports the
hypothesis that these structures are components of a coordinated func-
tional network. Rodent studies suggest an important dissociation
between the Ce and BST with respect to defensive behaviors, such
that the Ce is involved in acute fear-related responding, and the BST is
thought to mediate anxiety-like responses to sustained or ambiguous
threats (Walker and Davis, 2008). In previous rhesus monkey studies,
we reported that individual differences in BST (Fox et al., 2008; Kalin
et al., 2005) and Ce (Oler et al., 2010) metabolic activity, as assessed
with PET imaging, were predictive of individual differences in trait-
like anxiety in young monkeys. Additionally, recent human imaging
studies have associated these regions with vigilance, threat monitoring
and anticipatory anxiety (Alvarez et al., 2010; Mobbs et al., 2010;
Somerville et al., 2010; Straube et al., 2007). The current findings
present novel evidence that Ce and BST functional activations, as
assessed with the BOLD signal, are highly coordinated. These data sup-
port the hypothesis that coordinated actions between key components
of the extended amygdala are the substrate for the adaptive interplay
between immediate responses to threat and longer-term, sustained
vigilance for potential future threats.
The present fMRI data do not address the directionality of con-
nectivity between these structures. It is important to note that the
monkey and human amygdala share a similar pattern of connections
with cortical and sub-cortical regions (Amaral et al., 1992; Freese and
Amaral, 2009). In primates the BST receives inputs from almost all of
the amygdalar nuclei (Price and Amaral, 1981; Price et al., 1987),
including the Ce (see Fig. 1C). Preliminary work in rhesus monkeys has
examined the extent to which neurons in the BST project to the
Ce. Using retrograde tracer injections into the Ce, few retrogradely
labeled cells are observed in the BST (personal communication,
Julie Fudge, University of Rochester). It is noteworthy that injections
of retrograde tracers into the Ce reveal dense projections originating
in other amygdala nuclei (Fudge and Tucker, 2009). In the rat, the
species most extensively studied with tract tracing techniques, only
a few reports describe light structural connections projecting from
BST to Ce (Pitkänen, 2000). Thus, the marked preponderance of
efferent projections from Ce to BST supports similar directionality
underlying the functional connectivity observed in the present study
between Ce and BST. It is also unclear whether the source of the Ce–
BST functional connectivity documented here is via monosynaptic or
polysynaptic pathways. It is possible that the temporal correlation
between Ce and BST signal is due to direct monosynaptic Ce→BST
connections via the stria terminalis and/or the ventral amygdalofugal
pathway/sublenticular bundle (Amaral et al., 1992; Klingler and Gloor,
1960). Alternatively, it is possible that Ce–BST functional connectivity
could result from shared afferent input (i.e., from the basolateral com-
plex) driving activity in both the Ce and BST.
It is important to emphasize that the extended amygdala concept
involves regions other than the Ce and BST. The SI, which is inter-
posed between the Ce and BST, is hypothesized to contain extended
amygdala neurons, as extensive histochemical studies demonstrate
islands of cell groups in the SI region that appear to be continuous
with cells in the Ce and BST (Heimer et al., 1997, 1999). To under-
stand the extent to which Ce function is linked to components of
the extended amygdala other than the BST, Ce–SI connectivity was
examined. Although temporal fluctuations in the SI signal were
significantly correlated with the Ce time-series, analyses revealed
that Ce–BST connectivity was significantly greater than Ce–SI connec-
tivity (pb0.03). Perhaps, the relatively decreased connectivity found
between Ce and SI is secondary to the fact that SI extended amygdala
cells are interdigitated with other cell types such as magnocellular
cholinergic cells. Within the extended amygdala, the data support a
particularly robust functional connectivity between Ce and BST.
Taken together the data support the use of in vivo functional
imaging methods in nonhuman and human primates to quantify the
degree of coupling between components of the extended amygdala.
Future studies using functional connectivity to assess the degree of
spontaneous and task-evoked coupling between the Ce and BST
have the potential to provide new insights into the pathophysiology
of anxiety, affective, and stress-related disorders. Finally, these data
provide an fMRI-based demonstration that the extended amygdala
is conserved across primate species, and that, at rest, the Ce and BST
function as a coordinated circuit, supporting further investigation of
the role of the BST in anxiety-like processes in primates.
This work was supported by the HealthEmotions Research Institute
and the National Institutes of Mental Health: R01-MH046729 (to
N.H.K.), R01-MH081884 (to N.H.K.), P50-MH084051 (to R.J.D, M.J.E.
N.H.K.) and P30-HD03352. We thank the staff of the Harlow Center
for Biological Psychology, HealthEmotions Research Institute, Waisman
Laboratory for Brain Imaging and Behavior, Wisconsin National Primate
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