Central amygdala nucleus (Ce) gene expression linked to increased trait-like Ce metabolism and anxious temperament in young primates.
ABSTRACT Children with anxious temperament (AT) are particularly sensitive to new social experiences and have increased risk for developing anxiety and depression. The young rhesus monkey is optimal for studying the origin of human AT because it shares with humans the genetic, neural, and phenotypic underpinnings of complex social and emotional functioning. In vivo imaging in young monkeys demonstrated that central nucleus of the amygdala (Ce) metabolism is relatively stable across development and predicts AT. Transcriptome-wide gene expression, which reflects combined genetic and environmental influences, was assessed within the Ce. Results support a maladaptive neurodevelopmental hypothesis linking decreased amygdala neuroplasticity to early-life dispositional anxiety. For example, high AT individuals had decreased mRNA expression of neurotrophic tyrosine kinase, receptor, type 3 (NTRK3). Moreover, variation in Ce NTRK3 expression was inversely correlated with Ce metabolism and other AT-substrates. These data suggest that altered amygdala neuroplasticity may play a role the early dispositional risk to develop anxiety and depression.
- SourceAvailable from: Alexander Joseph Shackman[Show abstract] [Hide abstract]
ABSTRACT: Recent years have witnessed the emergence of powerful new tools for assaying the brain and a remarkable acceleration of research focused on the interplay of emotion and cognition. This work has begun to yield new insights into fundamental questions about the nature of the mind and important clues about the origins of mental illness. In particular, this research demonstrates that stress, anxiety, and other kinds of emotion can profoundly influence key elements of cognition, including selective attention, working memory, and cognitive control. Often, this influence persists beyond the duration of transient emotional challenges, partially reflecting the slower molecular dynamics of catecholamine and hormonal neurochemistry. In turn, circuits involved in attention, executive control, and working memory contribute to the regulation of emotion. The distinction between the ‘emotional’ and the ‘cognitive’ brain is fuzzy and context-dependent. Indeed, there is compelling evidence that brain territories and psychological processes commonly associated with cognition, such as the dorsolateral prefrontal cortex and working memory, play a central role in emotion. Furthermore, putatively emotional and cognitive regions influence one another via a complex web of connections in ways that jointly contribute to adaptive and maladaptive behavior. This work demonstrates that emotion and cognition are deeply interwoven in the fabric of the brain, suggesting that widely held beliefs about the key constituents of ‘the emotional brain’ and ‘the cognitive brain’ are fundamentally flawed. We conclude by outlining several strategies for enhancing future research. Developing a deeper understanding of the emotional-cognitive brain is important, not just for understanding the mind but also for elucidating the root causes of its disorders.Frontiers in Human Neuroscience 01/2015; 9:58. · 2.90 Impact Factor
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ABSTRACT: This review brings together recent research from molecular, neural circuit, animal model, and human studies to help understand the neurodevelopmental mechanisms underlying social anxiety disorder. Social anxiety disorder is common and debilitating, and it often leads to further psychopathology. Numerous studies have demonstrated that extremely behaviorally inhibited and temperamentally anxious young children are at marked risk of developing social anxiety disorder. Recent work in human and nonhuman primates has identified a distributed brain network that underlies early-life anxiety including the central nucleus of the amygdala, the anterior hippocampus, and the orbitofrontal cortex. Studies in nonhuman primates have demonstrated that alterations in this circuit are trait-like in that they are stable over time and across contexts. Notably, the components of this circuit are differentially influenced by heritable and environmental factors, and specific lesion studies have demonstrated a causal role for multiple components of the circuit. Molecular studies in rodents and primates point to disrupted neurodevelopmental and neuroplastic processes within critical components of the early-life dispositional anxiety neural circuit. The possibility of identifying an early-life at-risk phenotype, along with an understanding of its neurobiology, provides an unusual opportunity to conceptualize novel preventive intervention strategies aimed at reducing the suffering of anxious children and preventing them from developing further psychopathology.American Journal of Psychiatry 08/2014; · 13.56 Impact Factor
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ABSTRACT: Behavioral inhibition (BI) is a temperament identified early in life that is associated with increased risk for anxiety disorders. Amygdala hyperresponsivity, found both in behaviorally inhibited and anxious individuals, suggests that amygdala dysfunction may represent a marker of anxiety risk. However, broader amygdala networks have not been examined in individuals with a history of childhood BI. This study uses resting state fMRI to assess amygdala intrinsic functional connectivity (iFC) in 38 healthy young adults (19 with a history of BI, 19 with no history of BI) selected from a longitudinal study. Centromedial, basolateral, and superficial amygdala iFCs were compared between groups and examined in relation to self-report measures of anxiety. Group differences were observed in amygdala iFC with prefrontal cortex, striatum, anterior insula, and cerebellum. Adults characterized with BI in childhood endorsed greater state anxiety prior to entering the scanner, which was associated with several of the group differences. Findings support enduring effects of BI on amygdala circuitry, even in the absence of current psychopathology.Biological Psychology 09/2014; · 3.47 Impact Factor
Central amygdala nucleus (Ce) gene expression
linked to increased trait-like Ce metabolism and
anxious temperament in young primates
Andrew S. Foxa,b,c,1, Jonathan A. Olerb,d, Steven E. Sheltonb,d, Steven A. Nandad, Richard J. Davidsona,b,c,d,
Patrick H. Roseboomd, and Ned H. Kalina,b,c,d,1
Departments ofaPsychology anddPsychiatry andbHealthEmotions Research Institute, University of Wisconsin, Madison, WI 53719; andcWaisman Laboratory
for Brain Imaging and Behavior, University of Wisconsin, Madison, WI 53705
Edited by Marcus E. Raichle, Washington University in St. Louis, St. Louis, MO, and approved September 11, 2012 (received for review April 23, 2012)
Children with anxious temperament (AT) are particularly sensitive
to new social experiences and have increased risk for developing
anxiety and depression. The young rhesus monkey is optimal for
studying the origin of human AT because it shares with humans
the genetic, neural, and phenotypic underpinnings of complex social
and emotional functioning. In vivo imaging in young monkeys
demonstrated that central nucleus of the amygdala (Ce) metabo-
lism is relatively stable across development and predicts AT. Tran-
scriptome-wide gene expression, which reflects combined genetic
and environmental influences, was assessed within the Ce. Results
support a maladaptive neurodevelopmental hypothesis linking
decreased amygdala neuroplasticity to early-life dispositional
anxiety. For example, high AT individuals had decreased mRNA
expression of neurotrophic tyrosine kinase, receptor, type 3 (NTRK3).
Moreover, variation in Ce NTRK3 expression was inversely corre-
lated with Ce metabolism and other AT-substrates. These data sug-
gest that altered amygdala neuroplasticity may play a role the early
dispositional risk to develop anxiety and depression.
positron-emission tomography|microarray|brain imaging
critical for establishing novel early-life interventions aimed at
preventing the chronic and debilitating outcomes associated with
these common illnesses. To this end, we have optimized a model
of anxious temperament (AT), the conserved at-risk phenotype,
in young developing rhesus monkeys (1–4). The rhesus monkey is
ideal for studying the origin of human AT because these species
share the genetic, neural, and phenotypic underpinnings of com-
plex social and emotional functioning (5–10). Importantly, the
rhesus developmental model bridges the critical gap between
human psychopathology and rodent models, allowing for trans-
lation to humans by using in vivo imaging measures and trans-
lation to rodents by using ex vivo molecular methods. Thus, the
unique hypotheses that can be generated from the rhesus model
are invaluable in guiding both imaging studies in children and
mechanistic efforts in rodents.
Of particular relevance to the AT rhesus model is the rela-
tively recent evolutionary divergence between rhesus monkeys
and humans (25 million years) compared with rodents and humans
(70 million years) (5). This evolutionary closeness is reflected in
the species’ similarities in social and emotional behaviors. These
homologies, instantiated in their conserved genetic and neural
systems, underlie the ability of both humans and rhesus monkeys
to form and maintain the relationships necessary for living in
complex social environments. In this regard, the experience of
anxiety has evolved in primates to motivate the formation of
long-lasting attachment bonds that serve to increase security and
group cohesion. The comparable rearing practices shared by
these species (e.g., close mother–infant bonding) promote early
social/emotional learning, which serves to adaptively regulate
anxiety and promote survival (7).
he ability to identify brain mechanisms underlying the risk
during childhood for developing anxiety and depression is
Although periods of marked anxiety and fear are common
during early childhood, most children overcome these anxieties
through learning associated with experience and maturation. As
they develop, typical children learn to discern real threats from
distorted fears and, in concert, effectively regulate their behavior
to adaptively cope. However, a subset of children with extreme
AT do not develop this capacity, maintaining a stable anxious
disposition that confers increased risk for the development of
anxiety and mood disorders (11–13). AT begins as early shyness
and is later characterized by chronic anxiety, negative affect, and
worry (14). AT is also associated with increased activity of stress-
sensitive peripheral systems, including increased pituitary–adre-
nal tone and heightened sympathetic activity (11).
Because early social-emotional learning is critical for the
adaptive regulation of anxiety, we have been especially interested
in processes demonstrated to underlie learning during develop-
ment. Furthermore, recent preclinical and clinical research has
identified neurotrophic factors (15) and other neuroplastic pro-
cesses as critical for overcoming adult psychopathology (16–18).
Therefore, we hypothesized that altered neurotrophic processes
in the young brain would lead to the emergence and mainte-
nance of childhood AT. In particular, we theorize that deficits in
the ability to modify the connections and composition of AT’s
neural substrate could result in a failure to learn how to adap-
tively regulate anxiety, which can manifest as a tendency to gen-
eralize perceptions of threat to neutral stimuli. Because AT can
be identified early in life, characterizing the biological factors
that promote the maintenance of stable AT can potentially lead
to targeted early-life interventions aimed at decreasing the risk
for developing psychopathology.
Similar to anxious children, young monkeys with high levels of
AT are those that show increased freezing, decreased vocal-
izations, and increased cortisol when exposed to the no-eye
contact condition (NEC) of the human-intruder paradigm, an
ethologically relevant mild social threat. Our studies demonstrated
that, like human AT, monkey AT is trait-like and heritable (19).
Using functional brain imaging in conjunction with ex vivo mo-
lecular analyses of relevant brain regions, the monkey model
allows for the longitudinal study of AT and its underlying neural
substrates. With functional brain imaging, we identified the central
nucleus of the amygdala (Ce) and anterior hippocampus as
components of the neural circuit underlying AT (2, 19). More-
Author contributions: S.E.S., R.J.D., P.H.R., and N.H.K. designed research; A.S.F., S.E.S.,
S.A.N., P.H.R., and N.H.K. performed research; A.S.F. contributed new reagents/analytic
tools; A.S.F., J.A.O., P.H.R., and N.H.K. analyzed data; and A.S.F., J.A.O., and N.H.K. wrote
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 30, 2012
| vol. 109
| no. 44www.pnas.org/cgi/doi/10.1073/pnas.1206723109
over, we found that young primates with high AT have in-
creased metabolism in these regions when studied in both
stressful and nonstressful contexts (2). These data set the stage
for in-depth molecular studies in primates focused on under-
standing the mechanisms mediating the function of the brain
regions underlying AT.
The Ce is of interest because its efferent projections coor-
dinate autonomic, hormonal, behavioral, and emotional responses
to stress (20), and Ce lesions in monkeys are sufficient to reduce
AT (21). Furthermore, rodent studies demonstrate that direct Ce
manipulations markedly alter unconditioned anxiety responses
(22), similar to those elicited by novel or potentially threatening
situations in children with high AT. The prefrontal cortex and
other amygdala nuclei primarily influence fear and anxiety-re-
lated responding via the Ce, where intra-Ce microcircuits play a
critical role dynamically gating these inputs (23–26). Recent work
in rhesus monkeys demonstrates that, unlike most amygdala
nuclei, the Ce continues to mature from the first year of life into
early adulthood (27). This protracted developmental period sug-
gests that Ce maturation may be particularly susceptible to envi-
ronmental influences. A causal relation between social group size
and dorsal amygdala volume demonstrates the importance of
social influences on the primate amygdala (28). These findings
are consistent with our data highlighting the importance of en-
vironmental contributions to Ce metabolism as it relates to early-
life AT (19). For these reasons, we selected the Ce for in depth
molecular analyses, with a particular focus on processes within
the Ce that underlie learning. Although learning-related re-
search has generally focused on the hippocampus (e.g., refs. 29
and 30) and basal/lateral amygdala regions (31), recent rodent
studies highlight the role of plasticity and emotional learning in
the Ce (32, 33). In addition to its role in anxiety, the Ce has re-
cently been linked to habit formation (34) and, at a cellular and
neurochemical level, it has much in common with the striatum
(35), a structure known to mediate the development of long-term
ingrained response patterns (36). Although Ce microcircuits are
ideally suited to perform the childhood learning that results in
adaptive anxiety, when Ce learning is disrupted, it could result in
trait-like habitual fear and anxiety responding.
Building on our finding that individual differences in Ce me-
tabolism predict AT, we performed mRNA expression studies in
Ce tissue collected from young monkeys repeatedly phenotyped
for AT and its associated brain metabolism. This unique, mul-
tilevel approach combines the power of functional brain imaging
with the potential of gene expression studies to characterize the
mRNAs that could underlie the risk for developing anxiety and
depression. We hypothesized that high-AT individuals would
have alterations in mRNAs within the Ce that reflect the influ-
ences of experience on the persistent expression of anxiety. Spe-
cifically, we predicted a role for mRNAs encoding molecules with
the potential to facilitate habitual anxiety and developmentally
appropriate adaptive fear learning. Such alterations are of par-
ticular interest, because manipulations of these substrates could
result in treatments for high-AT children that would facilitate
their ability to modify and adaptively regulate their anxiety.
Accordingly, a subset of 24 animals was selected from 238
rhesus monkeys that were initially characterized for behavior and
brain metabolism. The 238 monkeys were injected with [18F]-
fluoro-2-deoxyglucose (FDG) and exposed for 30 min to the
NEC condition that elicits the AT phenotype (19). During NEC,
a human (“the intruder”) enters the test room and presents her
profile to the monkey, avoiding eye contact (1). Following NEC
exposure, animals were anesthetized, and high-resolution posi-
tron-emission tomography (PET) scans were performed to ex-
amine the integrated brain metabolism that occurred during the
preceding 30-min NEC exposure. FDG-PET is optimal for si-
multaneously assessing sustained neural activity and natural be-
havior in unconstrained individuals because FDG is taken up
into metabolically active cells over the course of ∼30 min and
remains trapped for the duration of its ∼110-min half-life. This
extended assessment of brain metabolism is ideal for studying
the neural underpinnings of AT, which are sustained over time.
Results and Discussion
The subset of 24 males underwent further testing to characterize
the trait-like components of AT and its neural substrate across
development [Fig. 1A; animals noted in pink constituted the
subset that was further tested; age at first assessment: mean, 2.1
(range, 0.85–3.5 y); age at last assessment: mean, 3.2 (range, 1.8–
4.2)]. The age span of this sample is similar to childhood through
early adolescence in humans because 1-y-old monkeys are similar
to 3- to 4-y-old children, and male monkeys enter adolescence
around 3–4 y of age. The 24 monkeys were phenotyped for brain
and behavior on two additional occasions, 6–18 mo after their
initial assessment. Between the second and third assessment, half
of the animals were relocated every 5 d over a period of 3 wk.
Relocation did not have any significant effects on behavior or
physiology (SI Methods). To examine the stability of AT, we tested
the interrelations among the three repeated measurements, con-
trolling for relocation, age, and interval between assessments.
Relations among the AT measures were significant [time 1 to
time 2: t = 6.83, P < 0.0001; time 1 to time 3: t = 3.80, P = 0.001;
time 2 to time 3: t = 5.16, P < 0.0001; accounting for between
44% and 72% of the variance in AT across time points and cor-
responding to an interclass correlation coefficient (ICC)3,1= 0.72],
confirming AT’s relative stability over this developmental period.
We hypothesized that the stability of AT would be reflected by
similar consistency in the function of its neural substrates. There-
fore, we intercorrelated the three measures of NEC-induced brain
metabolism in the regions predictive of AT, controlling for re-
location, age, and interval between assessments. AT-predictive
regions were defined from our previous study and included bi-
lateral anterior temporal lobe clusters, bilateral occipital lobe
clusters, and a midline parietal lobe cluster (19). Results dem-
onstrated relative stability as denoted by significant interrelations
among the three NEC-induced metabolism measurements within
each of these clusters [corrected for multiple comparisons using
false discovery rate (FDR); q < 0.05 FDR across maps in AT-
related regions, with voxel-wise ICC3,1coefficients ranging from
0.30 to 0.70 (median, 0.57); Fig. 1B and Table S1]. Voxels within
every cluster we tested, including dorsal and ventral regions of
amygdala, anterior portions of hippocampus, superior temporal
sulcus (STS), agranular insula, temporal and insular proisocortices,
claustrum, visual cortex, and precuneus, demonstrated signifi-
cantly stable NEC-induced metabolism. These data demonstrate
that individual differences in metabolism within the neural circuit
underlying AT are relatively stable across juvenile development.
We next examined covariation between the stable components
of AT and its neural substrates. For each animal, the stable
components of AT and regional brain metabolism were esti-
mated by computing their means across each of the three lon-
gitudinal assessments. Mean AT was regressed against mean
voxel-wise metabolism in significantly stable AT-related regions,
while controlling for relocation, age, the interval between scans,
and voxel-wise gray matter probability (GMP). Results demon-
strated that mean metabolism in amygdalar regions (including
Ce and the basolateral complex), anterior hippocampus, and
visual cortex predicted mean AT (q < 0.05 FDR in stable AT-
related regions; Fig. 1 C and D and Table S2). To confirm in-
volvement of the Ce within the larger amygdalar cluster we
assessed the overlap between this cluster and a map of in vivo
serotonin transporter binding derived from separate animals
(37). Precise localization of the Ce can be determined with this
method because this amygdalar nucleus has the highest density
of serotonin transporter binding compared with neighboring
structures (38) (SI Methods). Results confirmed that mean
Fox et al.PNAS
| October 30, 2012
| vol. 109
| no. 44
metabolism in the Ce region predicted mean AT across assess-
ments. This longitudinal assessment of brain and behavior extends
prior work characterizing the neural substrates of AT by dem-
onstrating that the trait-like nature of AT is reflected in trait-like
metabolism within the Ce and other AT-related brain regions.
To characterize the molecular underpinnings of AT and its
neural substrate, animals were killed and brain tissue was col-
lected for assessment of gene expression. Because of our interest
in the trait-like nature of AT and its context-independent brain
metabolism, we collected brain tissue from animals in their
baseline state, 4–5 d following final NEC exposure. In contrast to
studies of stimulus-evoked gene expression, this approach allows
for optimal characterization of temperament-related transcripts.
mRNA was extracted from the Ce region most predictive of AT
(n = 238; Figs. 1A and 2). mRNA expression levels were assessed
using the Affymetrix Rhesus Monkey microarray (SI Methods).
Brain samples collected were counterbalanced for hemisphere
because bilateral Ce metabolism was associated with AT. To
quantify the association between dispositional AT and Ce mRNA
levels, mean AT was correlated with mRNA expression levels.
Microarray data were analyzed using bioconductor (39) for
microarray analysis in R (see SI Methods for details). Microarray
data were preprocessed using robust multichip average (RMA)
background correction, constant normalized across chips, and
summarized across probes using the median-polish technique
(40). Resulting gene expression levels were visually inspected
using MA plots (Fig. S1). Gene expression was also assessed using
Plier background correction and quantile normalization to verify
that gene distribution patterns did not arise from specific pre-
processing techniques (i.e., Plier and quantile normalization).
Robust regression analyses between mean AT- and RMA-de-
termined mRNA levels (controlling for relocation, biopsy hemi-
sphere, and age) were performed on annotated transcripts (http://
www.unmc.edu/rhesusgenechip/) that had at least moderate ex-
pression levels [>log2(100)]. Covarying for age ensures that
significant AT-related transcripts, although assessed during
development, are not reflective of age-related changes. The
empirical Bayes method was used to determine levels of signifi-
cance and the FDR was used to account for multiple comparisons.
Results revealed 139 RMA-determined transcripts that pre-
dicted AT (FDR q < 0.05, two-tailed; Table S3; to demonstrate
these correlations are not attributable to the normalization tech-
nique, Table S3 also includes the correlations between AT and
Plier background corrected and quantile normalized transcripts).
Consistent with the concept that multiple systems underlie stress-
related psychopathology, both manual inspection and gene-on-
tology enrichment analyses revealed that the 139 FDR-corrected
AT-related transcripts reflect diverse biological systems (SI
Methods and Table S4). Within the Biological Processes ontol-
ogy, 35 significantly overrepresented terms were identified. Three
of these terms are of particular interest in relation to our mal-
adaptive neurodevelopmental hypothesis of AT. These include
response to hormone stimulus (GO:0009725) and related terms,
positive regulation of axon extension (GO:0045773), and positive
regulation of developmental growth (GO:0048639). Of note, the
neurotrophic tyrosine kinase, receptor, type 3 (NTRK3) and the
leucine-rich repeat protein ISLR2 (Ig superfamily containing
leucine-rich repeat 2; also known as Linx) are the only con-
stituents from the FDR corrected list of 139 genes that are
represented across all 3 terms. Because multiple comparison
correction of AT-related transcripts likely results in false neg-
atives and the possibility that the exclusion of these genes can
alter ontology term representation, we also performed gene-
ontology enrichment analyses on significantly (P < 0.05) un-
corrected AT-related genes. Although not identical, the results
support the inferences of the FDR-corrected ontology analyses
(Table S5). Significantly overrepresented terms included regu-
lation of axon regeneration (GO:0048679) and cell morphogenesis
(GO:0000902), both of which included NTRK3. Although future
research would benefit from exploring other significantly AT-re-
lated genes and overrepresented ontology terms, because of our
theoretical interest in the mechanisms of Ce-learning during
positional AT. (A) In a large cohort of monkeys (n =
238), we identified regions in which metabolic ac-
tivity predicted AT [yellow; see Oler et al. (19)]. In
yellow is the anterior temporal lobe cluster, which
encompasses the Ce region (red) most predictive of
AT. As can be seen in the regression plot, 24 animals
(pink dots) were selected to represent the full range
of variability in AT and Ce metabolism. (B) Longi-
tudinal assessments of brain metabolism in the 24
animals demonstrate that portions of the temporal
lobe clusters are stable over time (purple) [FDR-
corrected within AT-related regions defined by Oler
et al. (19) (yellow) for three pairwise stability tests,
i.e., T1-T2, T2-T3, T1-T3]. (C) Mean longitudinal
assessments of brain metabolism and AT were sig-
nificantly correlated (pink) within the region where
metabolism was stable (FDR-corrected within the
purple region of stability from B). (D) Scatterplot
depicting relations between metabolism within the
amygdala region (pink) depicted in C and mean AT
residualized for relocation, age and interval be-
tween assessments (r = 0.47; P = 0.0377).
Stable amygdala metabolism predicts dis-
| www.pnas.org/cgi/doi/10.1073/pnas.1206723109Fox et al.
development, we focused our analyses on NTKR3 and related
transcripts. Quantitative real-time PCR (qRT-PCR) was performed
on select transcripts including NTRK3, IRS2, and ISLR2 (P values,
<0.05) to confirm the relations between gene expression and AT.
NTRK3 and insulin receptor substrate 2 (IRS2) are of partic-
ular interest because their activation by the endogenous growth
factor neurotrophin-3 (NTF-3) can initiate synaptogenesis and
neurogenesis (41, 42) (Fig. 3). The NTRK3 gene encodes a
membrane-bound receptor that phosphorylates intracellular
transducers, including IRS2 (43). The downstream influences
of IRS2 can manifest via activation of the phosphoinositide 3-
kinase/protein kinase B (PI3K/AKT) pathway (44). AKT signaling
affects synaptic plasticity, axonal development, amygdala-depen-
dent learning, and behavioral responses to stress (45, 46). Ad-
ditionally, ISLR2 sits on the cell membrane, aids in the guiding
of axons, and can facilitate the activation of trk receptors, in-
cluding NTRK3 (47, 48). In addition to its involvement in our
theoretically motivated focus on neuroplasticity, NTRK3 genetic
variation has been linked to human psychopathology, including
childhood-onset mood disorders (49–52), and via its extracellular
domain may provide an accessible drug target. Taken together,
these findings implicate NTRK3 as a prominent target for future
mechanistic studies examining childhood AT.
Using the prospectively acquired trait-like measures of brain
metabolism, we investigated the neural systems by which altered
Ce NTRK3 expression influences AT. A voxel-wise search, con-
trolling for age, hemisphere of biopsy, relocation, and GMP, was
performed to identify AT-related brain regions in which Ce
NTRK3 mRNA levels predicted mean metabolic activity. Results
demonstrated significant negative relationships between Ce NTRK3
expression and glucose metabolism within the right amygdala
(including the Ce region) and anterior hippocampus, as well as
significant positive relations within visual cortex (Fig. 4; FDR q <
0.05 in stable AT-predictive regions; Table S6). Although the left
Ce region did not survive multiple comparison correction, the
relationship between NTRK3 expression and left Ce metabolism
was significant at an uncorrected P = 0.02, which was not sig-
nificantly weaker than the relationship between NTRK3 ex-
pression and right Ce metabolism (t = 0.86; P = 0.40) (53).
These primate data combine the use of brain imaging and micro-
array technology in the same animals and suggest that Ce NTRK3
is a key molecular mediator of AT via its influences on the neural
circuit underlying AT.
To examine the unique relation between Ce NTRK3 expres-
sion and AT, we assessed motor cortex NTRK3 mRNA. Results
demonstrated that motor cortex NTRK3 mRNA levels were un-
related to Ce NTRK3 mRNA levels (t = 0.09; P = 0.928). More-
over, motor cortex NTRK3 mRNA levels were not related to AT
(t = 0.24; P = 0.812) nor metabolism in AT-related regions (no
amygdala biopsy. (A–C) Brains from the 24 animals were sectioned into 4.5-
mm coronal slabs centered on the functionally defined amygdala region (∼0.9
mm [orange] to ∼5.4 mm [cyan], posterior to anterior commissure), shown as
a 3D rendering (A), a 2D slice through the functionally defined amygdala
region (B), and a representative single-subject slab with amygdala biopsy site
(magenta arrow) (C). (D) The biopsied region corresponds to the location of
Ce (pink), as shown in the series of atlas slices [adapted with permission (69)].
Slices are arranged from anterior (orange) to posterior (cyan).
Stable Ce regions predictive of dispositional AT were used to guide
(green). Microarray data showed that individuals with higher levels of Ce NTRK3 mRNA expression exhibited lower AT. A similar pattern was found for
a downstream target of NTRK3, IRS2 (yellow). Other molecules in the NTRK3 pathway are depicted in gray. (B) qRT-PCR confirmed the negative relationship
between Ce NTRK3 mRNA expression levels and AT, controlling for relocation, biopsy hemisphere, and age (r = 0.49; P = 0.029).
Ce expression of the neurotrophic receptor NTRK3 predicts AT. (A) Schematic of the pathway for NTRK3, a neuroplasticity-associated molecule
Fox et al. PNAS
| October 30, 2012
| vol. 109
| no. 44
significant results at FDR q < 0.05). Although motor cortex me-
tabolism was highly predictive of levels of locomotion (P < 0.05,
Sidak-corrected; SI Methods and Table S7), motor cortex NTRK3
mRNA expression was not significantly correlated with either
locomotion (t = −1.31; P = 0.190) or motor cortex metabolism
(no significant results at FDR q < 0.05; SI Methods). These
findings demonstrate that regional NTRK3 mRNA expression is
not a general marker for brain metabolism nor is it a nonspecific
reflection of behaviors dependent on the region in which NTRK3
mRNA is assessed (e.g., locomotion for motor cortex). This fur-
ther underscores the specificity of Ce NTRK3 in relation to AT
and highlights the importance of site-specific, differential regu-
lation of the NTRK3 gene.
Our results highlight the role of NTRK3 in AT during devel-
opment but do not implicate NTRK3 mechanistically. Rather,
these findings provide an initial rationale for exploring behav-
ioral or pharmacological interventions aimed at up-regulating
regional NTRK3 expression early in the lives of individuals likely
to develop anxiety and depressive disorders. Recent studies in
rodents demonstrate the feasibility of performing early targeted
interventions that have long-term impacts on anxiety and adap-
tive responses to stress (54–56). For example, neonatal injections
of the neuroplasticity-related growth factor fibroblast growth
factor 2 (FGF2) altered the developmental trajectory of high-
anxious rodents, resulting in decreased adult anxiety (55). Im-
portantly, early-life FGF2 treatment also enhanced adult neu-
rogenesis, which was accompanied by increased hippocampal
expression of NTRK3 (55). Moreover, primate research has
demonstrated that neurodevelopmentally relevant gene expres-
sion in the amygdala is altered by prolonged maternal separation
(57). Taken together, these data are consistent with our dem-
onstration that monkeys with lower levels of AT show greater Ce
expression of NTRK3 and further motivate mechanistic research
into the role for neurodevelopmentally important transcripts in
the development of AT.
Although we focus on NTRK3, it is important to clarify that
other genes are also of interest. For example, in addition to its
importance in neurotrophic signaling, the involvement of IRS2,
which is also critical for insulin signaling, is interesting in its own
right. Because of its multiple functions, regulation of IRS2 may
be important in the linkages between stress, psychopathology,
and the development of associated physiological alterations such
as metabolic syndrome and type 2 diabetes. In the periphery,
IRS2 regulates insulin sensitivity and in the brain IRS2 impacts
multiple functions including reward (58), memory (59, 60), and
energy homeostasis (61, 62). Moreover, type 2 diabetes has been
associated with amygdala atrophy (63), and bidirectional asso-
ciations between insulin resistance and affective disorders have
been reported (64–66). The possibility that altered IRS2 function
may play a role in the associations between insulin-related dis-
orders and alterations in stress-related psychopathology via its
effects on the amygdala is intriguing. Of particular interest to
stress and AT, alterations in cortisol could be important in
modulating IRS2 function because the synthetic glucocorticoid
dexamethasone prevents phosphorylation of IRS2 (67, 68).
Recent work demonstrating the ongoing development of the
primate Ce suggests this nucleus to be particularly susceptible to
environmental influences throughout childhood and adolescence
(19, 27). Early in childhood, as young children first extend be-
yond their parents’ reach, they must approach novelty with trep-
idation. By way of experience and maturation, most children learn
to regulate their anxieties and see the world as an opportunity for
exploration. This experience-dependent learning results in refined
discrimination between threatening and nonthreatening stimuli
and likely involves sculpting of intra-Ce connections that dynam-
ically gate the sensory and prefrontal triggers of fear. We hy-
pothesize that decreased capacity for learning and modification
within the Ce microcircuit could explain why some children fail
to regulate their anxieties and develop an extreme anxious
temperament. The findings presented here begin to link specific
experience-dependent molecular pathways within the Ce to
chronically elevated Ce metabolism and extreme tempera-
mental anxiety. Although there are likely many mediators of AT,
these gene expression data are consistent with a maladaptive neu-
rodevelopmental hypothesis as a basis for AT. Future work should
aim to extend these data in support of a molecular-neuroscien-
tific rationale for conceptualizing new treatment strategies aimed
at normalizing Ce function in vulnerable children before the
development of the detrimental behavioral, emotional, and brain
sequelae associated with the long-term consequences of chronic
anxiety and depression.
Twenty-four male rhesus monkeys were selected from subjects used by Oler
et al. (19) to undergo longitudinal AT and FDG-PET assessments during expo-
sure to potential threat (NEC). Brain tissue was biopsied from the Ce region
most predictive of AT, and transcriptome wide RNA expression levels were
assessed using Affymetrix GeneChip Rhesus Macaque Genome arrays. All
preprocessing and statistical analyses were performed using standard meth-
ods. See SI Methods for a detailed description of the procedures used.
ACKNOWLEDGMENTS. We thank the staff at the Wisconsin National Primate
Center, the Harlow Center for Biological Psychology, the HealthEmotions Re-
search Institute, and The Waisman Laboratory for Brain Imaging and Behavior
for facilitating our research. We also thank A. Shackman, A. Alexander,
B. Christian, L. Ahlers, A. Converse, T. Oakes, H. Van Valkenberg, K. Myer,
Acetylcholinesterase (AChE) stain (Left) was used to definitively identify Ce
in the brain of one subject. In situ hybridization of NTRK3 was performed on
an adjacent slice from the same subject (Right). Magnified insets (Center)
reveal that the AChE-defined Ce (dashed-pink) expresses NTRK3 mRNA. (B)
Individuals showing higher levels of NTRK3 mRNA expression, indexed by
qRT-PCR, show reduced Ce metabolism in vivo (green) [FDR-corrected within
the stable AT-related region (pink)].
NTRK3 is expressed in Ce and negatively predicts Ce metabolism. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.1206723109Fox et al.
J. Spears, E. Larson, and D. French for their technical and analytic assistance in
running subjects and analyzing data. This work was supported by National
Institutes of Health (NIH) Grants MH91550, MH046729, MH081884, MH084051,
HD008352, and HD003352; NIH Training Grant MH018931; the Wisconsin
National Primate Research Center (through NIH Grants P51OD011106 and
P51RR000167); and the HealthEmotions Research Institute.
1. Kalin NH, Shelton SE (1989) Defensive behaviors in infant rhesus monkeys: Environ-
mental cues and neurochemical regulation. Science 243:1718–1721.
2. Fox AS, Shelton SE, Oakes TR, Davidson RJ, Kalin NH (2008) Trait-like brain activity
during adolescence predicts anxious temperament in primates. PLoS ONE 3:e2570.
3. Fox AS, et al. (2005) Calling for help is independently modulated by brain systems
underlying goal-directed behavior and threat perception. Proc Natl Acad Sci USA 102:
4. Kalin NH, Shelton SE, Fox AS, Oakes TR, Davidson RJ (2005) Brain regions associated
with the expression and contextual regulation of anxiety in primates. Biol Psychiatry
5. Gibbs RA, et al.; Rhesus Macaque Genome Sequencing and Analysis Consortium
(2007) Evolutionary and biomedical insights from the rhesus macaque genome. Sci-
6. Kalin NH, Shelton SE (2003) Nonhuman primate models to study anxiety, emotion
regulation, and psychopathology. Ann N Y Acad Sci 1008:189–200.
7. Harlow HF (1958) The nature of love. Am Psychol 13:673–685.
8. Adolphs R (2010) What does the amygdala contribute to social cognition? Ann N Y
Acad Sci 1191:42–61.
9. Wallis JD (2012) Cross-species studies of orbitofrontal cortex and value-based de-
cision-making. Nat Neurosci 15:13–19.
10. Chareyron LJ, Banta Lavenex P, Amaral DG, Lavenex P (2011) Stereological analysis of
the rat and monkey amygdala. J Comp Neurol 519:3218–3239.
11. Fox NA, Henderson HA, Marshall PJ, Nichols KE, Ghera MM (2005) Behavioral in-
hibition: Linking biology and behavior within a developmental framework. Annu Rev
12. Biederman J, et al. (2001) Further evidence of association between behavioral in-
hibition and social anxiety in children. Am J Psychiatry 158:1673–1679.
13. Essex MJ, Klein MH, Slattery MJ, Goldsmith HH, Kalin NH (2010) Early risk factors and
developmental pathways to chronic high inhibition and social anxiety disorder in
adolescence. Am J Psychiatry 167:40–46.
14. Kagan J, Reznick JS, Snidman N (1988) Biological bases of childhood shyness. Science
15. Martinowich K, Manji H, Lu B (2007) New insights into BDNF function in depression
and anxiety. Nat Neurosci 10:1089–1093.
16. Krystal JH, et al. (2009) Neuroplasticity as a target for the pharmacotherapy of anxiety
disorders, mood disorders, and schizophrenia. Drug Discov Today 14:690–697.
17. Manji HK, Moore GJ, Rajkowska G, Chen G (2000) Neuroplasticity and cellular resil-
ience in mood disorders. Mol Psychiatry 5:578–593.
18. Pittenger C, Duman RS (2008) Stress, depression, and neuroplasticity: A convergence
of mechanisms. Neuropsychopharmacology 33:88–109.
19. Oler JA, et al. (2010) Amygdalar and hippocampal substrates of anxious temperament
differ in their heritability. Nature 466:864–868.
20. Davis M, Whalen PJ (2001) The amygdala: Vigilance and emotion. Mol Psychiatry 6:
21. Kalin NH, Shelton SE, Davidson RJ (2004) The role of the central nucleus of the
amygdala in mediating fear and anxiety in the primate. J Neurosci 24:5506–5515.
22. Tye KM, et al. (2011) Amygdala circuitry mediating reversible and bidirectional con-
trol of anxiety. Nature 471:358–362.
23. Ehrlich I, et al. (2009) Amygdala inhibitory circuits and the control of fear memory.
24. Ciocchi S, et al. (2010) Encoding of conditioned fear in central amygdala inhibitory
circuits. Nature 468:277–282.
25. Haubensak W, et al. (2010) Genetic dissection of an amygdala microcircuit that gates
conditioned fear. Nature 468:270–276.
26. Pare D, Duvarci S (2012) Amygdala microcircuits mediating fear expression and ex-
tinction. Curr Opin Neurobiol 22:717–723.
27. Chareyron LJ, Lavenex PB, Amaral DG, Lavenex P (2012) Postnatal development of the
amygdala: A stereological study in macaque monkeys. J Comp Neurol 520:1965–1984.
28. Sallet J, et al. (2011) Social network size affects neural circuits in macaques. Science
29. Kim JJ, Diamond DM (2002) The stressed hippocampus, synaptic plasticity and lost
memories. Nat Rev Neurosci 3:453–462.
30. Ming G-L, Song H (2011) Adult neurogenesis in the mammalian brain: Significant
answers and significant questions. Neuron 70:687–702.
31. Fanselow MS, LeDoux JE (1999) Why we think plasticity underlying Pavlovian fear
conditioning occurs in the basolateral amygdala. Neuron 23:229–232.
32. Paré D, Quirk GJ, Ledoux JE (2004) New vistas on amygdala networks in conditioned
fear. J Neurophysiol 92:1–9.
33. Samson RD, Duvarci S, Paré D (2005) Synaptic plasticity in the central nucleus of the
amygdala. Rev Neurosci 16:287–302.
34. Lingawi NW, Balleine BW (2012) Amygdala central nucleus interacts with dorsolateral
striatum to regulate the acquisition of habits. J Neurosci 32:1073–1081.
35. Swanson LW, Petrovich GD (1998) What is the amygdala? Trends Neurosci 21:
36. Packard MG, Knowlton BJ (2002) Learning and memory functions of the Basal Gan-
glia. Annu Rev Neurosci 25:563–593.
37. Christian BT, et al. (2009) Serotonin transporter binding and genotype in the non-
human primate brain using [C-11]DASB PET. Neuroimage 47:1230–1236.
38. O’Rourke H, Fudge JL (2006) Distribution of serotonin transporter labeled fibers in
amygdaloid subregions: Implications for mood disorders. Biol Psychiatry 60:479–490.
39. Gentleman RC, et al. (2004) Bioconductor: Open software development for compu-
tational biology and bioinformatics. Genome Biol 5:R80.
40. Gautier L, Cope L, Bolstad BM, Irizarry RA (2004) affy—analysis of Affymetrix Gen-
eChip data at the probe level. Bioinformatics 20:307–315.
41. Lamballe F, Klein R, Barbacid M (1991) trkC, a new member of the trk family of ty-
rosine protein kinases, is a receptor for neurotrophin-3. Cell 66:967–979.
42. Duman CH, Duman RS (2005) Neurobiology and treatment of anxiety: Signal trans-
duction and neural plasticity. Handb Exp Pharmacol 169:305–334.
43. Yamada M, et al. (1997) Insulin receptor substrate (IRS)-1 and IRS-2 are tyrosine-
phosphorylated and associated with phosphatidylinositol 3-kinase in response to
brain-derived neurotrophic factor in cultured cerebral cortical neurons. J Biol Chem
44. van der Heide LP, Ramakers GMJ, Smidt MP (2006) Insulin signaling in the central
nervous system: Learning to survive. Prog Neurobiol 79:205–221.
45. Lin CH, et al. (2001) A role for the PI-3 kinase signaling pathway in fear conditioning
and synaptic plasticity in the amygdala. Neuron 31:841–851.
46. Ou L-C, Gean P-W (2006) Regulation of amygdala-dependent learning by brain-de-
rived neurotrophic factor is mediated by extracellular signal-regulated kinase and
phosphatidylinositol-3-kinase. Neuropsychopharmacology 31:287–296.
47. Mandai K, et al. (2009) LIG family receptor tyrosine kinase-associated proteins mod-
ulate growth factor signals during neural development. Neuron 63:614–627.
48. de Wit J, Hong W, Luo L, Ghosh A (2011) Role of leucine-rich repeat proteins in the
development and function of neural circuits. Annu Rev Cell Dev Biol 27:697–729.
49. Feng Y, et al.; International Consortium for Childhood-Onset Mood Disorders (2008)
Association of the neurotrophic tyrosine kinase receptor 3 (NTRK3) gene and child-
hood-onset mood disorders. Am J Psychiatry 165:610–616.
50. Armengol L, et al. (2002) 5′ UTR-region SNP in the NTRK3 gene is associated with
panic disorder. Mol Psychiatry 7:928–930.
51. Alonso P, et al. (2008) Genetic susceptibility to obsessive-compulsive hoarding: The
contribution of neurotrophic tyrosine kinase receptor type 3 gene. Genes Brain Behav
52. Athanasiu L, et al. (2011) Intron 12 in NTRK3 is associated with bipolar disorder.
Psychiatry Res 185:358–362.
53. Williams EJ (1959) The Comparison of Regression Variables. J R Stat Soc Series B 21:
54. Zhang T-Y, Parent C, Weaver I, Meaney MJ (2004) Maternal programming of in-
dividual differences in defensive responses in the rat. Ann N Y Acad Sci 1032:85–103.
55. Turner CA, Clinton SM, Thompson RC, Watson SJ, Jr., Akil H (2011) Fibroblast growth
factor-2 (FGF2) augmentation early in life alters hippocampal development and res-
cues the anxiety phenotype in vulnerable animals. Proc Natl Acad Sci USA 108:
56. Upton KJ, Sullivan RM (2010) Defining age limits of the sensitive period for attach-
ment learning in rat pups. Dev Psychobiol 52:453–464.
57. Sabatini MJ, et al. (2007) Amygdala gene expression correlates of social behavior in
monkeys experiencing maternal separation. J Neurosci 27:3295–3304.
58. Russo SJ, et al. (2007) IRS2-Akt pathway in midbrain dopamine neurons regulates
behavioral and cellular responses to opiates. Nat Neurosci 10:93–99.
59. Irvine EE, et al. (2011) Insulin receptor substrate 2 is a negative regulator of memory
formation. Learn Mem 18:375–383.
60. Martín ED, et al. (2012) IRS-2 Deficiency impairs NMDA receptor-dependent long-
term potentiation. Cereb Cortex 22:1717–1727.
61. White MF (2003) Insulin signaling in health and disease. Science 302:1710–1711.
62. Taguchi A, Wartschow LM, White MF (2007) Brain IRS2 signaling coordinates life span
and nutrient homeostasis. Science 317:369–372.
63. den Heijer T, et al. (2003) Type 2 diabetes and atrophy of medial temporal lobe
structures on brain MRI. Diabetologia 46:1604–1610.
64. Li C, et al. (2008) Diabetes and anxiety in US adults: Findings from the 2006 Behavioral
Risk Factor Surveillance System. Diabet Med 25:878–881.
65. Peyrot M, Rubin RR (1997) Levels and risks of depression and anxiety symptomatology
among diabetic adults. Diabetes Care 20:585–590.
66. Anderson RJ, Freedland KE, Clouse RE, Lustman PJ (2001) The prevalence of comorbid
depression in adults with diabetes: A meta-analysis. Diabetes Care 24:1069–1078.
67. Caperuto LC, et al. (2006) Distinct regulation of IRS proteins in adipose tissue from
obese aged and dexamethasone-treated rats. Endocrine 29:391–398.
68. Rojas FA, Hirata AE, Saad MJA (2003) Regulation of insulin receptor substrate-2 ty-
rosine phosphorylation in animal models of insulin resistance. Endocrine 21:115–122.
69. Paxinos G (2009) The Rhesus Monkey Brain in Stereotaxic Coordinates (Academic,
Amsterdam, Boston, London), 2nd Ed. Copyright Academic Press (2009).
Fox et al.PNAS
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| vol. 109
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