Variation in mouse basolateral amygdala volume is associated with differences in stress reactivity and fear learning.
ABSTRACT A wealth of research identifies the amygdala as a key brain region mediating negative affect, and implicates amygdala dysfunction in the pathophysiology of anxiety disorders. Although there is a strong genetic component to anxiety disorders such as posttraumatic stress disorder (PTSD) there remains debate about whether abnormalities in amygdala function predispose to these disorders. In the present study, groups of C57BL/6 x DBA/2 (B x D) recombinant inbred strains of mice were selected for differences in volume of the basolateral amygdala complex (BLA). Strains with relatively small, medium, or large BLA volumes were compared for Pavlovian fear learning and memory, anxiety-related behaviors, depression-related behavior, and glucocorticoid responses to stress. Strains with relatively small BLA exhibited stronger conditioned fear responses to both auditory tone and contextual stimuli, as compared to groups with larger BLA. The small BLA group also showed significantly greater corticosterone responses to stress than the larger BLA groups. BLA volume did not predict clear differences in measures of anxiety-like behavior or depression-related behavior, other than greater locomotor inhibition to novelty in strains with smaller BLA. Neither striatal, hippocampal nor cerebellar volumes correlated significantly with any behavioral measure. The present data demonstrate a phenotype of enhanced fear conditioning and exaggerated glucocorticoid responses to stress associated with small BLA volume. This profile is reminiscent of the increased fear processing and stress reactivity that is associated with amygdala excitability and reduced amygdala volume in humans carrying loss of function polymorphisms in the serotonin transporter and monoamine oxidase A genes. Our study provides a unique example of how natural variation in amygdala volume associates with specific fear- and stress-related phenotypes in rodents, and further supports the role of amygdala dysfunction in anxiety disorders such as PTSD.
- SourceAvailable from: PubMed Central[Show abstract] [Hide abstract]
ABSTRACT: Aberrations in the elaboration of both aversive and rewarding stimuli characterize several psychopathologies including anxiety, depression and addiction. Several studies suggest that different neurotrasmitters, within the corticolimbic system, are critically involved in the processing of positive and negative stimuli. Individual differences in this system, depending on genotype, have been shown to act as a liability factor for different psychopathologies. Inbred mouse strains are commonly used in preclinical studies of normal and pathological behaviors. In particular, C57BL/6J (C57) and DBA/2J (DBA) strains have permitted to disclose the impact of different genetic backgrounds over the corticolimbic system functions. Here, we summarize the main findings collected over the years in our laboratory, showing how the genetic background plays a critical role in modulating amminergic and GABAergic neurotransmission in prefrontal-accumbal-amygdala system response to different rewarding and aversive experiences, as well as to stress response. Finally, we propose a top-down model for the response to rewarding and aversive stimuli in which amminergic transmission in prefrontal cortex (PFC) controls accumbal and amygdala neurotransmitter response.Frontiers in Systems Neuroscience 02/2015; 8.
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ABSTRACT: Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95(GK)), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95(GK) mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.Molecular Psychiatry advance online publication, 16 December 2014; doi:10.1038/mp.2014.161.Molecular Psychiatry 12/2014; · 15.15 Impact Factor
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ABSTRACT: Major neuropsychiatric disorders are highly heritable, with mounting evidence suggesting that these disorders share overlapping sets of molecular and cellular underpinnings. In the current article we systematically test the degree of genetic commonality across six major neuropsychiatric disorders-attention deficit hyperactivity disorder (ADHD), anxiety disorders (Anx), autistic spectrum disorders (ASD), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ). We curated a well-vetted list of genes based on large-scale human genetic studies based on the NHGRI catalog of published genome-wide association studies (GWAS). A total of 180 genes were accepted into the analysis on the basis of low but liberal GWAS p-values (<10(-5)). 22% of genes overlapped two or more disorders. The most widely shared subset of genes-common to five of six disorders-included ANK3, AS3MT, CACNA1C, CACNB2, CNNM2, CSMD1, DPCR1, ITIH3, NT5C2, PPP1R11, SYNE1, TCF4, TENM4, TRIM26, and ZNRD1. Using a suite of neuroinformatic resources, we showed that many of the shared genes are implicated in the postsynaptic density (PSD), expressed in immune tissues and co-expressed in developing human brain. Using a translational cross-species approach, we detected two distinct genetic components that were both shared by each of the six disorders; the 1st component is involved in CNS development, neural projections and synaptic transmission, while the 2nd is implicated in various cytoplasmic organelles and cellular processes. Combined, these genetic components account for 20-30% of the genetic load. The remaining risk is conferred by distinct, disorder-specific variants. Our systematic comparative analysis of shared and unique genetic factors highlights key gene sets and molecular processes that may ultimately translate into improved diagnosis and treatment of these debilitating disorders.Frontiers in Neuroscience 11/2014; 8:331.
Variation in Mouse Basolateral Amygdala Volume is
Associated With Differences in Stress Reactivity and Fear
Rebecca J Yang1, Khyobeni Mozhui2, Rose-Marie Karlsson1, Heather A Cameron3, Robert W Williams2and
1Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, NIH,
Rockville, MD, USA;2Department of Anatomy and Neurobiology, University of Tennessee Health Science Center, Memphis, TN, USA;3Unit on
Neuroplasticity, Mood and Anxiety Disorders Program, National Institute of Mental Health, NIH, Bethesda, MD, USA
A wealth of research identifies the amygdala as a key brain region mediating negative affect, and implicates amygdala dysfunction in the
pathophysiology of anxiety disorders. Although there is a strong genetic component to anxiety disorders such as posttraumatic stress
disorder (PTSD) there remains debate about whether abnormalities in amygdala function predispose to these disorders. In the present
study, groups of C57BL/6?DBA/2 (B?D) recombinant inbred strains of mice were selected for differences in volume of the basolateral
amygdala complex (BLA). Strains with relatively small, medium, or large BLA volumes were compared for Pavlovian fear learning and
memory, anxiety-related behaviors, depression-related behavior, and glucocorticoid responses to stress. Strains with relatively small BLA
exhibited stronger conditioned fear responses to both auditory tone and contextual stimuli, as compared to groups with larger BLA. The
small BLA group also showed significantly greater corticosterone responses to stress than the larger BLA groups. BLA volume did not
predict clear differences in measures of anxiety-like behavior or depression-related behavior, other than greater locomotor inhibition to
novelty in strains with smaller BLA. Neither striatal, hippocampal nor cerebellar volumes correlated significantly with any behavioral
measure. The present data demonstrate a phenotype of enhanced fear conditioning and exaggerated glucocorticoid responses to stress
associated with small BLA volume. This profile is reminiscent of the increased fear processing and stress reactivity that is associated with
amygdala excitability and reduced amygdala volume in humans carrying loss of function polymorphisms in the serotonin transporter and
monoamine oxidase A genes. Our study provides a unique example of how natural variation in amygdala volume associates with specific
fear- and stress-related phenotypes in rodents, and further supports the role of amygdala dysfunction in anxiety disorders such as PTSD.
Neuropsychopharmacology (2008) 33, 2595–2604; doi:10.1038/sj.npp.1301665; published online 9 January 2008
Keywords: amygdala; gene; fear; memory; PTSD; stress
The past decade has seen great advances in understanding
how genetically driven individual differences in the ana-
tomy and function of specific brain regions confer risk for a
variety of neuropsychiatric diseases (Caspi and Moffitt,
2006; Hariri et al, 2006). This literature is based upon the
assumption that associations between genes, neural func-
tion, and clinical end points are causative in nature. How-
ever, associations between clinical symptoms and measures
of neural activation are invariably made a posteriori: that
is after the phenotype is observed. By contrast, there has
been little demonstration that variation in a brain region
of interest can successfully predict, a priori, the likely
prevalence of a clinical or behavioral trait.
A pertinent case in point is posttraumatic stress disorder
(PTSD). There is a significant genetic component to risk for
PTSD (Kendler, 2001), as well as neurological abnormalities
in unaffected probands that are suggestive of antecedent
genetic or familial risk factors (Gurvits et al, 2006).
Functional neuroimaging studies have identified hyper-
activity of the amygdala as a neural correlate for the clinical
symptoms seen in PTSD (Rauch et al, 2006; Shin et al,
2006). In addition, some but not all structural imaging
and postmortem studies have found a trend for smaller left
amygdala volume in patients with the disorder (Bremner et al,
1997; Bonne et al, 2001; De Bellis et al, 2001, 2002; Fennema-
Notestine et al, 2002; Lindauer et al, 2004; Wignall et al, 2004;
Karl et al, 2006). Taken together, these findings suggest that
amygdala dysfunction may predispose individuals to PTSD
and other stress-related disorders. However, while there are
data showing that smaller hippocampal volume can precede
the development of PTSD (Gilbertson et al, 2002), the
Received 15 October 2007; revised 5 November 2007; accepted 7
*Correspondence: Dr A Holmes, Section on Behavioral Science and
Genetics, Laboratory for Integrative Neuroscience, National Institute
on Alcohol Abuse and Alcoholism, 5625 Fishers Lane Room 2N09,
Rockville, MD 20852-9411, USA, Tel: +1 301 402 3519, Fax: +1 301
480 1952, E-mail: email@example.com
Neuropsychopharmacology (2008) 33, 2595–2604
& 2008 Nature Publishing GroupAll rights reserved 0893-133X/08 $30.00
hypothesis that variation in amygdala anatomy can lead to
PTSD has not been directly tested.
A major role for the amygdala in PTSD is consistent with
a large corpus of data from animal models demonstrating
amygdala mediation of a spectrum of behavioral and
mnemonic functions that include, but are not limited to,
the regulation of emotion and negative affect (Davis and
Whalen, 2001; Robbins and Everitt, 2002; Fanselow and
Poulos, 2005; Phelps and LeDoux, 2005). Ablation of
specific subnuclei of the rat amygdala produces alterations
in certain fear-, anxiety-, and stress-related behaviors.
Specifically, the basolateral amygdala complex (BLA)
appears necessary for the formation and/or expression of
associative fear memories. This has led to a model in which
the lateral nucleus within BLA serves as a convergence site
for sensory and aversive information that is relayed either
directly or via the basal nucleus to the major output center
of the amygdala, the central nucleus (CeA), to drive fear-
related behaviors (Fanselow and LeDoux, 1999; Maren and
Quirk, 2004). Thus, rodents receiving pre-training lesions of
the lateral, but not basal, nucleus fail to form fear memories
(Goosens and Maren, 2001; Nader et al, 2001; Koo et al,
2004), while post-training lesions of the basal nucleus
prevent the expression of fear memory but not the ability to
acquire new memories (Anglada-Figueroa and Quirk, 2005).
These findings provide clear evidence that the BLA is
necessary for certain forms of learned fear behavior. They
do not, however, address the issue of whether natural
variation in amygdala anatomy and function is sufficient to
predict differences in these behaviors.
In the present study, we sought to address this issue by
utilizing a genetically defined panel of C57BL/6?DBA/2
(BXD) recombinant inbred strains that we have recently
shown to exhibit up to twofold variation in BLA volume in
large sample of 37 RI lines (Mozhui et al, 2007). BXD RI mice
are inbred strains derived from C57BL/6 and DBA/2 (Taylor,
1978; Chesler et al, 2003) that have been used to identify
chromosomal loci underlying fear- and anxiety-related
phenotypes (Caldarone et al, 1997; Owen et al, 1997; Wehner
et al, 1997; Valentinuzzi et al, 1998), as well as volume and
cell number variation in various brain regions (Neumann
et al, 1993; Airey et al, 2001, 2002; Lu et al, 2001; Rosen and
Williams, 2001; Williams et al, 2001; Seecharan et al, 2003).
We selected 17 BXD RI lines and assigned them to subgroups
with relatively small, medium, and large BLA volume. Our
objective was to test whether a priori categorization of
mice according to BLA volume differences within a natural
rangewas sufficient to predict differences in fear-,
anxiety-, and stress-related phenotypes. The results obtained
provide some of the strongest evidence to date that
genetically driven variation in amygdala volume is sufficient
to predict the magnitude of fear- and stress-related
MATERIALS AND METHODS
Histology and BXD RI Strain Selection
Histological analysis of the BXD RI lines was performed
previously at the Department of Anatomy and Neuro-
biology, University of Tennessee Health Science Center,
and is described in detail by Mozhui et al (2007). Briefly,
the BLA complex was defined as the portion between the
external capsule and the amygdalar capsule, as described by
Swanson and Petrovich (1998). This includes the lateral and
basolateral, but not basomedial, nuclei (Figure 1a). Images
from bilateral 30mm thick serial sections were captured
from a Zeiss light microscope to a computer and the BLA
was manually traced to determine cross-sectional area
(mm2) along the entire rostrocaudal thickness of the BLA
(B6–9 sections for each brain). Volume was calculated by
multiplying the interval thickness between serial sections
(which was 300mm) and correcting for volumetric shrink-
age caused by the histological process ((measured values/
total brain volume)?expected brain volume for given brain
weight). Post-processing total brain volume was determined
by the point-counting method described by Williams
(2000). These data can be viewed at an anatomical database
Selection of BXD RI Lines and Assignment to BLA
In an earlier study, we conducted a comprehensive analysis
of 35 BXD RI lines (plus the C57BL/6J and DBA/2J parental
lines) for BLA volume (Mozhui et al, 2007). This revealed
up to twofold variation in BLA volume across this panel of
RI lines. In the current study we obtained a subset of this
larger panel. We selected lines on the basis of a desire to
obtain a range of relatively small, medium, and large BLA
volumes; but this was limited by the availability of lines
from a single commercial vendor (The Jackson Laboratory,
Bar Harbor, ME), which was necessary to avoid a potentially
BXD RI lines. (a) Neuroanatomical definition of the BLA complex. (b) BLA
volume differences in mice selected for relatively small (S), medium (M),
and large (L) BLA. (c) Dorsal striatal, total hippocampal, and total cerebellar
volume differences in mice selected for BLA volume differences (n¼24–38
per volume group). **Po0.01 vs L,##Po0.01 vs M. Data in Figures 1–4
Basolateral amygdala (BLA) volume differences in a panel of
BLA volume and fear
RJ Yang et al
confounding source of variability (ie supplier). Of the 15
BXD RI lines (plus the C57BL/6J and DBA/2J parental lines)
obtained, we cross-referenced with the dataset (Mozhui
et al, 2007) to assign an approximately equal number of
lines to ‘small,’ ‘medium,’ and ‘large’ groups. This resulted
in five lines in the small group (BXD13, BXD21, BXD28,
BXD38, and BXD39), seven lines in the medium group
(BXD8, BXD11, BXD12, BXD23, BXD31, BXD34, and DBA/
2J), and five lines in the large group (BXD16, BXD19,
BXD22, BXD32, and C57BL/6J). Next, we took the BLA
volume data for each line (an average of n¼4–8 mice per
line) from the dataset (Mozhui et al, 2007) and calculated the
mean average and within-group variance of BLA volume
within each of our three volume groupings. Volume
differences between the subgroups were then analyzed using
one-way analysis of variance (ANOVA) followed by New-
man–Keuls post hoc analysis. This showed that the subgroups
differed significantly from one another (main effect of
volume group: F2,14¼18.58, post hoc comparisons: all
Po0.01). For depiction of mean differences and within-
group variance as measured by standard error, see Figure 1b.
For depiction of mean differences and within-group variance
as measured by standard deviation, see Supplementary
Figure 1a. For depiction of mean differences and individual
line values, see Supplementary Figure 1b.
To determine whether BLA volume differences in our
groups generalized to other brain regions, we obtained the
volume values for dorsal striatum (caudate putamen), total
hippocampus, and total cerebellum in each of the RI lines
we tested from www.genenetwork.org. These data were
originally described in the following sources: for dorsal
striatum, Rosen and Williams (2001) and Lu et al, 2001, for
hippocampus, including dentate gyrus, excluding subicu-
lum, Peirce et al (2003); and for cerebellum, Airey et al
(2001). For neuroanatomical definitions of these regions
see Supplementary Figure 2. One-way ANOVA and post hoc
tests showed that the subset of RI lines we designated as
the large BLA groups also had significantly greater striatal
volume than the group designated as the small BLA
group (main effect of group: F2,14¼3.78, Po0.05, post hoc
comparison: Po0.05; Figure 1c). ANOVA showed that the
BLA subgroups did not significantly differ in either total
hippocampal or cerebellar volume (Figure 1c).
Animal Husbandry and Behavioral Phenotyping
Mice were bred at The Jackson Laboratory and transported
to the NIH at B8 weeks of age and housed 1–4 per cage in
same-strain groupings in a temperature- and humidity-
controlled vivarium under a 12h light–dark cycle (lights on
0600 hours). There were a total of 190 male mice at the start
of the study, with at least n¼8–12/RI line except for BXD28
(n¼5). This resulted in a total of 47 mice in the small BLA
group, 82 in the medium BLA group, and 61 in the large
BLA group. Behavioral testing was conducted during the
light phase between 0900 and 1700 hours. Given evidence of
qualitative differences between rodent tests for anxiety-like
behavior (Holmes et al, 2003), three separate tests were
employed. Mice were tested on a battery of tests in the
following order with the putatively more stressful assays at
the end of the sequence and at least 1 week between tests:
exploratory and anxiety-like behavior (novel open-field,
elevated plus-maze, light–dark exploration tests), Pavlovian
fear conditioning, depression-related behavior (forced swim
test, FST), hot plate (as a control for pain perception in
fear conditioning), and glucocorticoid responses to stress
(corticosterone levels following swim stress). All experi-
mental procedures were performed in accordance with the
National Institutes of Health Guide for Care and Use of
Laboratory Animals and were approved by the local Animal
Care and Use Committee.
Pavlovian Fear Conditioning
Pavlovian fear conditioning (Kim and Fanselow, 1992) was
conducted in a chamber with transparent walls and a metal
rod floor based on methods described previously (Wellman
et al, 2007). To provide a distinctive olfactory environment,
the chamber was cleaned between subjects with a 79.5%
water/19.5% ethanol/1% vanilla extract solution. After an
initial 120s acclimation period, the mouse received three
pairings (60–120s interval after each pairing) between
the conditioned stimulus (CS; 30s, 80dB, 3kHz tone) and
the unconditioned stimulus (US; 2s, 0.6mA scrambled
footshock), in which the US was presented during the last
2s of the CS. The presentation of stimuli was controlled by a
San Diego Instruments (San Diego Instruments, San Diego,
CA) or Med Associates fear conditioning system (Med
Associates, Burlington, VT).
After 24h, expression of the fear memory for the CS was
tested in a novel context, in a different room from training.
The novel context had black/white checkered walls and a
solid Plexiglas, opaque floor. This chamber was cleaned
between subjects with a 50% ethanol/50% water solution.
After an initial 180s acclimation period, the CS was
continuously presented for 180s.
After 24h, expression of the fear memory for the condi-
tioned context was tested in the same room as conditioning
by placing the mouse in the training chamber for 5min.
Freezing was defined as absence of any visible movement
except that required for respiration, and was scored at 5s
intervals by an observer who was blind to BXD line. The
number of observations scored as freezing was converted to
a percentage ((number of freezing observations/total
number of observations)?100) for analysis.
The hot plate test was conducted as a control for group
differences in pain perception (Boyce-Rustay and Holmes,
2006). The apparatus was a flat plate (Columbus Instru-
ments, Columbus, OH) heated to 551C on which the mouse
was placed. The latency to show a hind paw shake or lick
was timed by an observer, with a maximum response
latency of 30s.
Depression-Related Behavior and Glucocorticoid
Responses to Stress
The FST was conducted as previously described (Porsolt
et al, 1978; Hefner and Holmes, 2007). The apparatus was a
transparent Plexiglas cylinder (25cm high, 20cm diameter)
filled halfway with water (24±11C) into which the mouse
was gently lowered for a 10min trial. The presence/absence
of immobility (cessation of limb movements except minor
involuntary movements of the hind limbs) was manually
observed using an instantaneous sampling technique every
BLA volume and fear
RJ Yang et al
5s during the last 8min and expressed as a percentage of
total observations. The result was converted to a percentage
((number of immobility observations/total number of
observations)?100) for analysis.
Glucocorticoid responses to stress were measured after a
4-week rest period following the FST described above. The
mouse was subjected to a 10min forced swim stress trial
and then returned to the home cage. After 30min, mice
were killed via rapid cervical dislocation and decapitation
to collect trunk blood. Nonstressed controls were killed
within 30s of removal from the home cage. To minimize
disturbance of nonstressed controls, all mice were indivi-
dually housed 24h prior to killing. Blood samples were
centrifuged at 13000r.p.m. for 30s. Serum was extracted
and assayed for total corticosterone (bound and free) using
the Coat-a-Count RIA TKRC1 kit (limit of detection 5.7ng/
ml; Diagnostic Products Corp, Los Angeles) as previously
described (Boyce-Rustay et al, 2007).
The novel open-field test was conducted as previously des-
cribed (Wiedholz et al, 2007). The apparatus was a square
arena (39cm?39cm?35cm) with opaque white Plexiglas
walls and floor that was evenly illuminated to B20 lux. The
mouse was placed in a corner and allowed to freely explore
for 30min. Distance traveled, time spent moving, time spent
in the (20cm?20cm) center, and entries into the center were
measured using the Ethovision videotracking system (Noldus
Information Technology Inc., Leesburg, VA). The apparatus
was cleaned with 70% EtOH (v/v) and dried between subjects.
The elevated plus-maze test was conducted as previously
described (Handley and Mithani, 1984; Boyce-Rustay and
Holmes, 2006). The apparatus consisted of two open arms
(30cm?5cm; 55 lux) and two closed arms (30cm?
5cm?15cm; 5 lux) extending from a 5cm?5cm central
area and elevated 20cm from the ground (San Diego
Instruments). The walls were made from black ABS plastic
and the floor from white ABS plastic. A 0.5cm raised lip
around the perimeter of the open arms prevented mice from
falling off the maze. The mouse was placed in the center
facing an open arm and allowed to explore the apparatus for
5min. Time spent in the open arms, and entries into the
open and closed arms were measured by the Ethovision
videotracking system (Noldus Information Technology
Inc.). The apparatus was cleaned with 70% EtOH (v/v)
and dried between subjects.
The light–dark exploration test was conducted as pre-
viously described (Crawley, 1981; Boyce-Rustay and Holmes,
2006). The apparatus consisted of two compartments (each
17cm?13cm?13cm), one with white Plexiglas walls
and clear Plexiglas floor (40 lux) (‘light’ compartment) and
the other with black Plexiglas walls and clear Plexiglas floor
(0 lux) (‘dark’ compartment), which were connected by
a partition at floor level with a small opening (5cm) (Med
Associates, Georgia, VT, Model ENV-3013). The mouse was
placed into the dark compartment facing away from the
aperture and allowed to explore the apparatus for 10min.
Time spent and full-body transitions into the light
compartment, and total full-body transitions between the
light and dark compartments were measured by photocells
connected to Med Associates software. The apparatus was
cleaned with 70% EtOH (v/v) and dried between subjects.
BLA group effects were analyzed using ANOVA and
Newman–Keuls post hoc tests where appropriate. One
mouse was an extreme outlier (45 standard deviations
from the mean) for preconditioning freezing and was exclu-
ded from the study. Genetic correlations were performed
(treating each of the 17 strains as an individual data point)
between BLA, dorsal striatal, total hippocampal, and
cerebellar volume, and behavioral and neuroendocrine
measures using Spearman’s test. The threshold for statis-
tical significance was set at Po0.05. DATA ACCESSAll
phenotype data sets that are part of this paper are available
at www.genenetwork.org in the mouse BXD phenotypes
database. Twenty-four records can be retrieved by entering
the term3yang rj2in the ALL field. Alternatively, individual
traits can be returned using GeneNetwork trait ID numbers
such as3108732for baseline plasma corticosterone titers.
Data include strain means and s.e.m values, but not data for
Pavlovian Fear Conditioning
There was no significant difference between BLA groups
for percent freezing during the (2-min) preconditioning or
(2-min) postconditioning periods (Figure 2a). BLA groups
significantly differed in percent freezing to the CS (F2,158¼
7.74, Po0.01) and context (F2,159¼5.37, Po0.01). The
small BLA group froze more in response to the CS
(Figure 2b) and to the context (Figure 2c) than the medium
or large groups. There was a significant genetic correlation
between freezing to CS and volume of BLA, but not
striatum, hippocampus, or cerebellum (Table 1).
There were no group differences in the hot plate test
for nociception (mean±SEM latency to respond: small¼
6.42±0.47s, medium¼7.39±0.48s and large¼6.14±0.37s).
Depression-Related Behavior and Glucocorticoid
Responses to Stress
There was no effect of BLA group on percent immobility in
the FST (Figure 3a). Corticosterone levels were affected by
stress in a BLA group-dependent manner (stress?group
interaction: F2,41¼3.41, Po0.05). While there were no
group differences in corticosterone in nonstressed mice,
corticosterone levels following stress were significantly
greater in the small BLA group than the medium or large
groups (Figure 3b). There was a significant baseline
correlation between nonstressed corticosterone and striatal,
but not BLA, hippocampus, or cerebellar, volume (Table 1).
Tests for Anxiety-Related Behavior
There was a significant effect of BLA group on distance
traveled (F2,186¼8.21, Po0.01), but not percent time in
the center in the novel open-field test. The small BLA
volume group traveled less than the medium or large BLA
BLA volume and fear
RJ Yang et al
volume groups (Figure 4a), while percent time in the center
was no different between groups (Figure 4b). There was a
significant genetic correlation between distance traveled
and volume of BLA, but not striatum, hippocampus or
cerebellum (Table 1).
There was a significant effect of BLA group on total
(F2,183¼4.24, Po0.05) and closed arm entries (F2,183¼
6.93, Po0.01) and percent time in the center square
(F2,183¼8.28, Po0.01), but not percent open arm time or
open arm entries in the elevated plus-maze. The medium
and small BLA groups spent less time in the center square
(Figure 4c) and made fewer total and closed arm entries
(Figure 4d) than the large BLA volume group. There was a
significant genetic correlation between percent center time
and volume of BLA, but not striatum, hippocampus, or
cerebellum (Table 1).
There was a significant effect of BLA group on percent
time in the light compartment (F2,175¼4.14, Po0.05) and
light–dark transitions (F2,175¼13.16, Po0.01) in the light–
dark exploration test. The medium and small BLA groups
spent less time in the light compartment (Figure 4e) and
made fewer light–dark transitions (Figure 4f) than the large
BLA volume group, while the medium group made fewer
transitions than the large group. Neither measure signifi-
cantly correlated with volume of BLA, striatum, hippocam-
pus, or cerebellum (Table 1).
The major finding of the present study was that variation in
volume of the BLA complex in a panel of BXD RI inbred strains
amygdala (BLA) volume. There were no differences in freezing during the
preconditioning or immediate postconditioning periods between mice with
relatively small (S), medium (M), or large (L) BLA volume. The small BLA
group froze more than medium or large BLA groups during exposure to
the conditioned stimulus (CS) and conditioned context (n¼35–75 per
volume group). **Po0.01, *Po0.05 vs L,##Po0.01 vs M.
Pavlovian fear conditioning in mice differing in basolateral
Table 1 Genetic Correlations Between Volume of BLA, Striatum,
Hippocampus, or Cerebellum and Fear, Anxiety- and Stress-
BLA Striatum Hippocampus Cerebellum
Pavlovian fear conditioning
Forced swim test
Novel open field
Distance traveled 0.50*0.040.30
?0.12Center time 0.340.180.39
Time in open arms
Time in center square
Total arm entries0.380.21
Open arm entries 0.02
0.42 Closed arm entries0.43
Time in light0.37
?0.24 Light–dark transitions0.340.11
Abbreviation: BLA, basolateral amygdala complex.
BLA volume and fear
RJ Yang et al
was associated with differences in specific measures of mouse
fear-, anxiety-, depression-, and stress-related phenotypes.
There is compelling evidence that the BLA is necessary for
the formation and expression of associative fear memories,
as typically evidenced by the effects of lesions on the type of
Pavlovian fear conditioning task employed in our study
(Davis and Whalen, 2001; Fanselow and Poulos, 2005;
Phelps and LeDoux, 2005). The present findings reinforce
and extend this literature by showing that mice with
naturally occurring variation in BLA volume exhibit
significant differences in fear conditioning. BXD RI strains
selected for BLA volumes that were relatively small showed
significantly greater levels of conditioned freezing than
strains with a larger BLA. There were no differences in levels
of freezing immediately prior to or immediately after
conditioning, demonstrating that differences between BLA
groups in the recall/expression of fear memory were not due
to differences in unconditioned freezing or in capacity to
express short-term fear memory. Moreover, pain perception
measured in the hot plate test did not differ between
the groups, excluding variation in pain perception as a
Differences in fear expression between volume BLA
groups were found both for conditioned fear to discrete
(auditory cue) and compound (context) stimuli, which is
consistent with the ability of BLA lesions (Goosens and
Maren, 2001) and gene knockout-induced gross disruption
of BLA (Lin et al, 2005) to impair both forms of associative
fear memory. Interestingly however, there was a significant
genetic correlation between BLA volume-varying BXD lines
and cued, but not contextual, conditioned fear. A relatively
greater correlation for cued conditioning is in agreement
with evidence from lesion studies demonstrating the impor-
tance of the BLA for cued relative to contextual fear
conditioning, the latter involving greater recruitment of
additional structures, notably the hippocampus (Maren and
Quirk, 2004). These data also concur with gene-mapping
studies in BXD RIs indicating that cued and contextual
conditioning are under partly dissociable genetic control
(Owen et al, 1997; Wehner et al, 1997). Finally, the fact that
we were able to detect two correlating phenotypic traits
(BLA volume and fear memory) across two laboratories
(University of Tennessee, NIH) speaks both to the strength
and replicability of the underlying genetic component
(Wahlsten et al, 2006).
BLA volume-related variation in fear conditioning was
dissociated from differences in anxiety-like behavior. The
small BLA volume group showed less locomotor exploration
in the novel open-field and elevated plus-maze than the
larger BLA groups, but there were no differences in the
principal measures of anxiety-like behavior (open-field
center time, elevated plus-maze open arm exploration). In a
third test of anxiety-like behavior, the light–dark explora-
tion test, the small and medium BLA groups spent less time
in the light compartment and made fewer light–dark inter-
compartment transitions than the large BLA group.
Different mouse tests for anxiety likely measure different
forms of behavior controlled by distinct genetic factors
(Turri et al, 1999; Holmes et al, 2003; Kliethermes and
Crabbe, 2006) and it is possible that the profile of the small
and medium BLA groups in the light–dark exploration test
reflects a high anxiety-like phenotype that is not seen in the
other tests. Taken together however, the differences between
BLA volume groups on the anxiety-related assays (particu-
larly the open-field and light–dark exploration tests)
were marginal and are most parsimoniously interpreted as
modest locomotor inhibition in response to novelty rather
than a clear high anxiety-like phenotype in mice with
smaller BLA volumes. Indeed, the absence of anxiety-related
differences would be consistent with the lack of effects
of BLA lesions or temporary inactivation on anxiety-like
behaviors in rats tested under the standard task conditions
used in the current study (Decker et al, 1995; Moller et al,
1997; Herry et al, 2007; Moreira et al, 2007).
To our knowledge, there has been little investigation
of the role of BLA in rodent depression-related behaviors;
with one study finding no effect of BLA lesions in the rat
FST for depression-related behavior (Shimazoe et al, 1988).
The present data found no link between variation in BLA
volume and immobility in the mouse FST. By contrast, BLA
groups differed markedly in their glucocorticoid responses
to swim stress. Thus, while baseline levels of corticosterone
were no different between the groups, corticosterone levels
following swim stress were significantly greater in small
BLA mice than in the larger BLA groups.
A pathway from the main output nucleus of the amygdala,
the CeA, to the paraventricular nucleus of the hypothalamus
(Swanson and Petrovich, 1998) is thought to modulate
hypothalamic–pituitary–adrenal (HPA) axis responses to
stress (reviewed in Herman et al, 2005). Lesions encom-
passing the CeA can inhibit HPA-axis responses to at least
some forms of stress (Beaulieu et al, 1986; Van de Kar
et al, 1991), whereas amygdala stimulation can increase
stress in mice differing in basolateral amygdala (BLA) volume. BLA volume
was not associated with differences in percent immobility in the forced
swim test (a) (n¼25–49 per volume group). Mice with relatively small (S)
BLA showed normal baseline corticosterone, but higher corticosterone
responses to swim stress than medium (M) or large (L) BLA groups (b)
(n¼5–11 per volume group per stress condition). **Po0.01, *Po0.05 vs L.
Depression-related behavior and glucocorticoid responses to
BLA volume and fear
RJ Yang et al
glucocorticoid release (Redgate and Fahringer, 1973; Dunn
and Whitener, 1986). As such, the relatively greater
HPA-axis response in small BLA mice could be another
manifestation of abnormal amygdala function. Corticoster-
one also exerts effects within the amygdala via actions at
glucocorticoid receptors (Aronsson et al, 1988). Of parti-
cular interest in this context, BLA has been identified as a
critical site of action through which glucocorticoids
modulate associative fear memory, (for example) gluco-
corticoid receptor agonists administered systemically or
directly into the BLA enhance fear conditioning in rats
(Zorawski and Killcross, 2002; Hui et al, 2004; Roozendaal
et al, 2006). This raises the possibility that increased
glucocorticoid receptor activity in smaller BLA mice may be
a factor driving enhanced fear conditioning, although this
remains to be tested. A related issue is whether BLA
variation is a consequence rather than cause of differences
in stress reactivity. While we do not discount this possibility
it seems unlikely given evidence that chronic stress
increases rather than decreases spine density and dendritic
arborization in rats and mice (Vyas et al, 2002; Mitra et al,
2005; Govindarajan et al, 2006). On the other hand, because
stress causes dendritic shrinkage in rodent ventromedial
prefrontal cortex (vmPFC) (Wellman, 2001; Izquierdo et al,
2006; Radley et al, 2006), it would be of interest to examine
vmPFC and fear-related behaviors mediated by vmPFC such
as fear extinction (Quirk and Mueller, 2007) in mice with
different BLA volumes.
BLA groups were selected for differences in BLA volume;
and the fact that groups comprised multiple RI strains, each
with a unique genotype, reduces the likelihood that
differences in fear conditioning and stress reactivity were
due to spurious idiosyncrasies (for example poor vision,
hearing, nociception) of specific strains. However, this
approach cannot exclude the potential contribution of other
phenotypic traits that covary with BLA volume. We have
recently shown that a common genetic locus may underlie
the structures of multiple forebrain regions including
amygdala, cerebellum, dorsal striatum, and portions of the
hippocampus (Mozhui et al, 2007). Analysis of the volume
of these three structures in our three BLA volume groups
indicated volume differences in dorsal striatal but not
hippocampal or cerebellar volume, raising the possibility
that striatal volume variation contributed to behavioral
distance than mice with medium (M) or large (L) BLA (a) while groups showed equivalent percent center time (b). Small and medium BLA groups spent less
time in the center square but not open arms (c) and made fewer total and closed but not open arm entries (d) in the elevated plus-maze than the large BLA
group. Small and medium BLA groups spent less time in the light compartment (e) and made fewer light–dark transitions (f) in the light–dark exploration test
than the large BLA group (n¼35–84 per volume group). **Po0.01, *Po.05 vs L,##Po0.01,#Po0.05 vs M.
Exploratory- and anxiety-like behaviors in mice differing in basolateral amygdala (BLA) volume. Mice with relatively small (S) BLA traveled less
BLA volume and fear
RJ Yang et al
differences between BLA groups, (for example via affects on
motor function. Arguing against this possibility, however,
there were no significant correlations between striatal
volume and any behavioral measure (c.f. baseline cortico-
sterone), while BLA volume significantly correlated with
novel open-field locomotion. Moreover, BLA volume
predicted corticosterone responses to stress, unlikely to be
influenced by locomotion, while other measures predicted
to be affected by locomotor differences, such as FST immo-
bility, were unrelated to BLA volume. Nonetheless, although
present data suggest a specific influence of amygdala func-
tion on fear conditioning and stress reactivity, we cannot
rule out the possible contribution of differences in other
brain regions or other, as yet undetermined, traits gene-
tically correlated with BLA volume. In fact, given the
highly integrated nature of the neural circuitry subserving
stress, fear, and anxiety behaviors (Amat et al, 2005; Hariri
and Holmes, 2006; Quirk and Mueller, 2007; Ressler and
Mayberg, 2007; Wellman et al, 2007), it is unlikely that the
amygdala would act in isolation to mediate these complex
The finding that relatively smaller BLA is associated with
increased fear and stress reactivity may at first appear
counterintuitive given the effects of amygdala lesions,
but this is in fact entirely consonant with recent findings
from genetic studies in humans. For example, functional
neuroimaging studies of two gene variants implicated in
stress-related disorders, the low-expressing forms of the
serotonin transporter and monoamine oxidase A, demons-
trate that these variants produce exaggerated amygdala
reactivity during fear processing and that this is coupled
with lesser amygdala volume (Pezawas et al, 2005; Meyer-
Lindenberg et al, 2006). The volume differences underlying
these fear-related phenotypes were modest, as they were in
our mouse sample. Thus, it appears that relatively modest
variation in BLA volume is sufficient to determine fear- and
Elucidating the molecular and physiological factors
associated with volume differences is likely to be key to
understanding this relationship between BLA volume and
behavior. A pertinent observation in this regard is that the
total number of neurons (and non-neuronal cells) in BLA
was equivalent across BLA volume groups (Mozhui et al,
2007), indicating that rather than having a relative loss of
neurons, the smaller BLA mice had a slightly denser packing
of neurons (see Figure 4d in Mozhui et al, 2007). An impor-
tant question for future studies will be how variation in this
and other intrinsic properties of BLA, such as neuronal
morphology and the ratio of excitatory over inhibitory cells,
might contribute to the behavioral differences between BLA
volume groups. Another interesting question is whether
phenotypic differences between BLA groups are localized to
specific subnuclei within the BLA complex. For example,
akin to the small BLA phenotype observed in our study,
lesions of the lateral nucleus prior to fear conditioning
block acquisition (Goosens and Maren, 2001; Nader et al,
2001; Koo et al, 2004), postconditioning lesions of the basal
nucleus (corresponding to basolateral nucleus in Figure 1a)
impair the expression, but not acquisition, of fear memory
(Anglada-Figueroa and Quirk, 2005).
In summary, the present study has shown that variation
in BLA volume is associated with differences in fear
memory, glucocorticoid responses to stress, and locomotor
inhibition in response to novelty. The present findings also
provide parallels with clinical data, to the extent that
(American Psychiatric Association, 1994; Peri et al, 2000)
and exaggerated glucocorticoid responses to stress (Yehuda,
2002; de Kloet et al, 2006), as well as amygdala hyperactivity
and possibly smaller amygdala volume (Bremner et al, 1997;
Wignall et al, 2004; Karl et al, 2006; Rauch et al, 2006; Shin
et al, 2006), relative to controls. Thus, the present study not
only provides a novel extension to the literature linking the
amygdala with fear- and stress-related processing with
possible implications for understanding amygdala dysfunc-
tion in PTSD, but also provides one of the first examples of
how structural variation in a specific brain region can
successfully predict differences in specific phenotypic traits
in the rodent.
We thank Dr. Glenn D. Rosen for access to a new
striatal volume data set on the BXD strains available in
GeneNetwork (BXD Phenotype GNID 10710). This research
was supported by the NIAAA (Z01-AA000411) and NIMH
(Z01-MH002784) intramural research programs, and by
NIDA, NIMH, and NIAAA HPG grant P20-DA 21131, NCRR
BIRN grant U24 RR021760 (BIRN), and NIAAA INIA grants
U01AA13499 and U24AA13513.
DISCLOSURE/CONFLICT OF INTEREST
The authors have no competing interests.
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