tmp3F13

ARCHIVAL REPORT
Overexpression of Corticotropin-Releasing Factor
Receptor Type 2 in the Bed Nucleus of Stria
Terminalis Improves Posttraumatic Stress Disorder-
like Symptoms in a Model of Incubation of Fear
Einat Elharrar, Gal Warhaftig, Orna Issler, Yehezkel Sztainberg, Yahav Dikshtein, Roy Zahut,
Lior Redlus, Alon Chen, and Gal Yadid
Background: Posttraumatic stress disorder (PTSD) is a severe, persistent psychiatric disorder in response to a traumatic event, causing
intense anxiety and fear. These responses may increase over time upon conditioning with fear-associated cues, a phenomenon termed
fear incubation. Corticotropin-releasing factor receptor type 1 (CRFR1) is involved in activation of the central stress response, while
corticotropin-releasing factor receptor type 2 (CRFR2) has been suggested to mediate termination of this response. Corticotropin-
releasing factor (CRF) receptors are found in stress-related regions, including the bed nucleus of stria terminalis (BNST), which is
implicated in sustained fear.
Methods: Fear-related behaviors were analyzed in rats exposed to predator-associated cues, a model of psychological trauma, over
10 weeks. Rats were classified as susceptible (PTSD-like) or resilient. Expression levels of CRF receptors were measured in the amygdala
nuclei and BNST of the two groups. In addition, lentiviruses overexpressing CRFR2 were injected into the medial division,
posterointermediate part of the BNST (BSTMPI) of susceptible and resilient rats and response to stress cues was measured.
Results: We found that exposure to stress and stress-associated cues induced a progressive increase in fear response of susceptible rats.
The behavioral manifestations of these rats were correlated both with sustained elevation in CRFR1 expression and long-term
downregulation in CRFR2 expression in the BSTMPI. Intra-BSTMPI injection of CRFR2 overexpressing lentiviruses attenuated behavioral
impairments of susceptible rats.
Conclusions: These results implicate the BNST CRF receptors in the mechanism of coping with stress. Our findings suggest increase of
CRFR2 levels as a new approach for understanding stress-related atypical psychiatric syndromes such as PTSD.
Key Words: Anxiety, BNST, CRFR1, CRFR2, fear, PTSD
P
osttraumatic stress disorder (PTSD) is a chronic and inca-
pacitating anxiety disorder that develops in some survivors
of a traumatic event and can cause intense fear and a
feeling of helplessness (1). Fear and anxiety may increase over
time, upon conditioning with fear-associated cues and in the
absence of further stress exposure (2). This phenomenon, termed
incubation of fear, has been demonstrated in humans (3) and
laboratory animals (4). However, the neural pathways involved in
generating and regulating incubation of fear and persistent PTSD-
like behavior remain unclear (5).
Posttraumatic stress disorder and other psychiatric disorders,
such as general anxiety disorder and panic disorder, are charac-
terized by dysfunctional regulation of the central stress response
(6,7). The corticotropin-releasing factor (CRF) family of ligands and
receptors are important regulators of the neuroendocrine and
behavioral responses to stressful challenges (8). Corticotropin-
releasing factor receptor type 1 (CRFR1) and corticotropin-
releasing factor receptor type 2 (CRFR2), encoded by different
genes and exhibiting unique central and peripheral distribution,
display distinct specificities for the CRF/urocortin (Ucn) family of
ligands (8–10). It has been suggested that the two receptor
subtypes have opposing roles, wherein CRFR1 participates in
initiation of the stress response, inducing fear and anxiety-like
responses, while CRFR2 activation re-establishes homeostasis (9).
However, results from several studies provide evidence that does
not support this notion (11).
Corticotropin-releasing factor receptor type 1 is distributed
widely in the brain, including regions that play central roles in the
neural circuit of fear, such as the central amygdala (CeA) and
basolateral amygdala (BLA) (12–16). Corticotropin-releasing factor
receptor type 2, however, is less prevalent, concentrated in
specific stress-related brain areas such as the bed nucleus of the
stria terminalis (BNST), dorsal raphe, and lateral septum (17,18).
As a subregion of the extended amygdala, the BNST is an integral
regulator of the hypothalamic-pituitary-adrenal stress axis. It acts
as a critical intermediary, receiving inputs from the corticolimbic
system and sending projections to the paraventricular nucleus of
hypothalamus, where CRF is released. This induces pituitary
activation and initiates the peripheral stress response (19).
Initial attempts to link the BNST with conditioned fear
responses were mostly unsuccessful; yet, subsequent studies
have shown that this region, and in particular the BNST CRF
receptors, play a role in some types of anxiety and stress
Authors EE and GW have contributed equally to this work.
From The Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain
Research Center (EE, GW, RZ, LR, GY), The Mina & Everard Goodman
Faculty of Life Sciences (EE, GW, YD, GY), Bar-Ilan University, Ramat-
Gan; and Department of Neurobiology (OI, YS, AC), Weizmann Institute
of Science, Rehovot, Israel.
Address correspondence to Gal Yadid, Ph.D., Bar-Ilan University, Multi-
disciplinary Brain Research Center, Geha Street, Ramat-Gan 52900,
Israel; E-mail: [email protected]; [email protected].
Received Nov 15, 2012; revised May 21, 2013; accepted May 24, 2013.
0006-3223/$36.00 BIOL PSYCHIATRY 2013;74:827–836
http://dx.doi.org/10.1016/j.biopsych.2013.05.039 & 2013 Society of Biological Psychiatry
responses (20). Walker et al. (13) have suggested that the BNST
mediates long-duration, but not short-duration, responses, i.e.,
sustained fear or anxiety, as opposed to phasic fear. More
recently, it was suggested that CRF neurons within the lateral
BNST modulate conditioned anxiety-like behaviors (21). Chronic
stress increases CRF concentrations and CRFR1 immunoreactivity
in the BNST (22,23). Corticotropin-releasing factor infusion into
the medial and dorsolateral BNST of rats enhances fear-
potentiated startle response, an effect blocked by nonselective
CRF receptor antagonism (24). Moreover, Arnold et al. (25)
showed that intracerebroventricular (ICV) injection of CRF induces
c-fos expression in the BNST, in a pattern similar to that found
after restraint stress. This effect is blocked by ICV injection of
a nonselective CRFR1 and CRFR2 receptor antagonist (25).
Bittencourt and Sawchenko (26) found a strong general corre-
spondence between the distribution of CRFR1-expressing cells
and CRF-stimulated Fos-immunoreactivity, revealing extensive
overlap in many brain regions. Although unique sites of CRFR2
expression, including the BNST, were relatively unresponsive to
CRF, probably due to the low affinity of CRF to CRFR2 (27,28),
these sites were more responsive after ICV administration of Ucn,
which interacts preferentially with CRFR2 (26). Taken together,
these findings suggest that stress, anxiety, and fear affect the CRF
system in the BNST, and notably the bed nucleus of the stria
terminalis, medial division, posterointermediate part (BSTMPI).
Indeed, CRFR1 is found in both medial and lateral aspects of the
BNST, and CRFR2 is predominantly localized in the BSTMPI and
the medial division, posteromedial part (18).
In this article, we examined the role of the BSTMPI CRF system in
development of fear incubation. We used an established rat model
for PTSD, which simulates several types of prevalent PTSD symp-
toms, including re-experiencing, avoidance, and hyperarousal (29),
after exposure to predator-associated stress. The analysis criteria
determine individual PTSD-like behavior, by categorizing the rats
into distinct groups according to their magnitude of response to the
stressful stimuli (30). Using our model, we found that exposure of
rats to stress and stress-associated cues induced a fear response that
incubates over time. In addition, the findings revealed an association
between fear incubation and impaired function of the CRF system in
the BSTMPI. Susceptible (PTSD-like) rats demonstrated both an
inability to suppress CRFR1 messenger RNA (mRNA) levels and a
marked decrease in CRFR2 mRNA levels in this region over the long
term. Next, we examined a possible interference in susceptible
behavior, by application of a viral construct designed to overexpress
CRFR2 into the BSTMPI, an approach that to our knowledge has not
yet been examined for its beneficial effect on fear symptoms or
anxiety disorders in general. We found that this treatment signifi-
cantly attenuated the conditioned fear response in susceptible rats.
Methods and Materials
Behavioral Procedure
Adult male Sprague–Dawley rats (250–300 g; Harlan, Rehovot,
Israel) were used. The model described herein is based on Kesner
et al. (30). This model consists of several stages (habituation, initial
exposure, first reminder [Re
1
], second reminder [Re
2
], and third
reminder [Re
3
]) encompassing 10 weeks, as depicted in Figure 1A
and detailed in Supplement 1.
Site-Specific Overexpression of CRFR2 Using Lentiviruses
After conclusion of the first testing stage, an additional,
separate group of rats were exposed to the same protocol
(habituation, initial exposure, Re
1
, Re
2
, and Re
3
). Rats were divided
into susceptible and resilient sets as above, based on their
behavioral response. Each subpopulation received a bilateral
injection of either CRFR2-overexpressing lentiviruses (treatment)
or control lentiviruses into the BSTMPI.
Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)
Analysis
For quantification of receptor levels in the BNST, CeA, and BLA,
total RNA was isolated from each region by the single-step
method (TriReagent, Sigma, Rehovot, Israel), using the manufac-
turer’s recommended procedure. RT-PCR reactions were carried
out using primers for CRFR1 and CRFR2 according to reaction
protocols (Supplement 1).
Corticosterone Assay
After decapitation, trunk blood was collected into chilled tubes
containing ethylenediaminetetraacetic acid solution. Blood sam-
ples were collected carefully. Samples were centrifuged for
10 minutes at 41C at 2500 rpm, and plasma was stored at 801C
until determination. On the day of assay, frozen plasma samples
were thawed, and corticosterone levels were measured with a
commercial radioimmunoassay kit (Diagnostic Products Corpora-
tion, Los Angeles, California). Each group was composed of 13 rats.
Design and Construction of Lentiviral Vectors for Site-Specific
Overexpression of CRFR2
Vector plasmids were constructed for the production of lentivi-
ruses expressing complementary DNA of mouse corticotropin-
releasing factor receptor type 2α (CRFR2α) using a minimal
promoter. The constructed viral plasmid pCSC-minimal promoter
tetracycline responsive element-CRFR2α-internal ribosome entry site
(IRES)-enhanced red fluorescent protein was generated by subclon-
ing the CRFR2α complementary DNA sequence upstream to an
IRES-enhanced red fluorescent protein site. The promoter sequence
used to drive the expression of CRFR2 in the viruses used in this
study was initially designed for conditional overexpression of CRFR2
using the TetOn system. However, due to the leakiness of the target
tetracycline responsive element viral construct, we used these
viruses (without the effector virus) to obtain constitutive expression
of CRFR2 in infected cells. Orientation was confirmed by diagnostic
cut and subsequent sequencing. pCSC-cytomegalovirus-IRES-green
fluorescent protein (GFP) plasmid was used for generating control
viruses.
Production of Lentiviral Vectors
Recombinant lentiviruses are produced by transient trans-
fection of HEK293T cells with the plasmid, as described previously
(31,32). Infectious lentiviruses are harvested at 48 hours and
72 hours posttransfection, filtered through .45μm-pore cellulose
acetate filters, and concentrated by ultracentrifugation.
In Vitro and In Vivo Validation of CRFR2 Lentiviral Vector
Activity
To determine the ability of the designed constructs to over-
express functional CRFR2, first an in vitro luciferase reporter assay
was performed in HEK293T cells. For this assessment, the CRFR2-
expressing plasmid was co-transfected with cyclic adenosine
monophosphate responsive element luciferase plasmid. The ability
of CRFR2 to activate cyclic adenosine monophosphate signaling in
response to different doses of the urocortin-2 (Ucn-2) ligand was
measured by luciferase activity, as previously described (33). Next,
to determine the ability of the designed construct to overexpress
828 BIOL PSYCHIATRY 2013;74:827–836 E. Elharrar et al.
www.sobp.org/journal
functional CRFR2 in brain tissue, it was microinjected into the
BSTMPI. Control animals received intra-BNST injection of the pCSC-
cytomegalovirus-IRES-GFP plasmid. Three days later, rats were
decapitated, brains were removed and sliced on ice-chilled
glass, and the BSTMPI was isolated. mRNA extraction and
RT-PCR were performed according to the above protocol, using
specific primers for CRFR2 and hypoxanthine-guanine phos-
phoribosyltransferase.
Intracerebral Injection of Lentiviral Vectors and Analysis
of Susceptible Rat Response
After Re
2
, susceptible and resilient sets were intraperitoneally
anesthetized with ketamine hydrochloride (100 mg/kg) and
xylazine (10 mg/kg). Each set was randomly divided into two
groups, each receiving a bilateral microinjection (.7 μL) of either
CRFR2 overexpressing lentivirus or control lentivirus into the
BSTMPI. Injection coordinates were as follows: α ¼ 14
o
, anterior
−.72 mm; lateral −2.9 mm, and ventral −7.0 mm from bregma.
Lentiviral injections were conducted using a computer-guided
stereotaxic instrument and a motorized nanoinjector (Angle Two
Stereotaxic Instrument, St. Louis, Missouri), which is fully inte-
grated with the Paxinos Rat Brain Atlas via a control panel. Rats
were given a 2-week recovery period, and Re
3
was then
performed followed by open field assessment of performance
under the three behavioral conditions. To verify site specificity of
this treatment, we examined CRFR2 mRNA levels also in the
lateral septum (LS), a stress-related region adjacent to the BNST,
in which CRFR2 is also concentrated. Experimental rats in which
CRFR2 expression levels in the LS were higher than those in the
LS of control rats (due to inaccurate lentiviral injection) were
excluded from the analysis.
Statistical Analysis
See Supplement 1.
Figure 1. Behavioral measurements. (A) Schematic description of the experimental design. After 2 weeks of habituation to the open field (5 min/day), the
rats began the experimental procedure. Their baseline response to the three behavioral conditions was monitored on day 1 (1d). Day 7 (7d): stress
exposure (initial exposure). Day 14 (14d): first re-exposure (Re
1
) to a cue reminder. Day 35 (35e): second re-exposure (Re
2
). One week later, a group of
susceptible and resilient rats received intra-bed nucleus of the stria terminalis, medial division, posterointermediate part lentivirus injection (virus
injection). Day 50 (50d): third re-exposure (Re
3
). (B) Upper figure: The entire distribution of the freezing data in each of the three conditions at Re
2
. The
dotted line is the upper level for excluding outliers in each condition. The gray line is the mean of each subpopulation. Lower figure: Correlations between
the three conditions for the entire population. There is a strong correlation between exploration versus social interaction, exploration versus hyperarousal,
and social interaction versus hyperarousal (R ¼ .37, .42, and .59, respectively; p Ͻ .0001).
E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827–836 829
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Results
Incubation of Fear in Rats Exposed to Stress
A total of 180 rats were exposed to the traumatic event (litter
with cat odor) and stress-associated cues. Posttraumatic stress
disorder-like behavior (measured by duration of freezing
response) of each rat was compared both with its own baseline
and with the range of the population. This ensured that two
unambiguous subpopulations could be significantly identified,
albeit the stressful component of the measurements. The inci-
dence of susceptible rats was approximately 20% (36 susceptible
rats and 144 resilient). Analysis of all baseline behavioral samples
revealed that the upper level for excluding outliers (95%
confidence) in the exploration and hyperarousal conditions was
1.5 times the interquartile range and for the social interaction
condition it was 2 times the interquartile range. Rats expressing
behavioral parameters that were two standard deviations from
the mean were considered outliers and excluded from the study.
The entire distribution of the freezing data is presented for
each of the three behavioral conditions at Re
2
(Figure 1B, top).
The distribution data show that the majority of rats were
concentrated in the resilient subpopulation. Moreover, it reveals
a greater increase from baseline in PTSD-like behavior of the
susceptible group, for all three behavioral conditions, as com-
pared with resilient rats. Pearson product-moment correlations
between exploration, social interaction, and hyperarousal were
.37, .42, and .59, respectively (Figure 1B, bottom). Thus, while
there was a degree of common variance between the three
conditions, most of the variance was unique to each condition.
Rats were then chosen from the susceptible and resilient sets,
based on extreme behavior (n ¼ 10 for each subpopulation).
A three-way analysis of variance (ANOVA) (with a Greenhouse-
Geisser correction) comparing freezing behavior over the four
time points (within subjects), the three behavioral conditions
(within subjects), and the two groups (susceptible vs. resilient)
revealed a statistically significant three-way interaction (F
3.25,55.16
¼ 3.79, p Ͻ .05). The group  time point (F
3,51
¼ 7.72, p Ͻ .001)
and the behavioral condition  group (F
1.21,20.56
¼ 6.21, p Ͻ .05)
interactions, as well as the main effects (which were not a focus of
the current investigation), were significant. The group  time
point interaction reveals that beyond the particular test, the
susceptible group increased its freezing duration over time,
significantly more than the resilient group. The three-way
interaction supports this finding, suggesting a different pattern
between the different conditions, for each group. The freezing
data for the three tests are presented in Figure 2.
To clarify the three-way interaction, follow-up one-way
ANOVAs compared the different time points in each group. Rats
from the susceptible subpopulation showed clear incubation of
fear during all three behavioral conditions (Figure 2B). During the
exploration conditioning, freezing behavior of susceptible rats
was significantly elevated from baseline after initial exposure, Re
1
,
and Re
2
(one-way ANOVA: F
3,8
¼ 32.285, p Ͻ .001; Tukey’s post
hoc: p Ͻ .01 [initial exposure and Re
1
], p Ͻ .001 [Re
2
]). For
resilient rats, freezing behavior was significantly elevated after
initial exposure and Re
1
and returned to baseline levels at a third
reminder (Re
3
) (F
3,8
¼ 5.918, p Ͻ .05; p Ͻ .01 [initial exposure],
p Ͻ .05 [Re
1
]).
During social interaction conditioning, freezing behavior of
susceptible rats after the Re
1
and the Re
2
were significantly
elevated from baseline (F
3,8
¼ 11.659, p Ͻ .01; p Ͻ .05 for both
time points), while no significant differences were detected for
resilient rats (p Ͼ .05). During hyperarousal conditioning, freezing
behavior of susceptible rats was significantly elevated after Re
2
, as
compared with baseline (F
3,8
¼ 2.833, p Ͼ .05; p Ͻ .05), while no
significant changes were detected for resilient rats (p Ͼ .05;
Figure 1B). Endocrine data also mimicked clinical observation. We
measured corticosterone levels and found the following results
for corticosterone: susceptible: 74 Ϯ 5 ng/mL, resilient: 107 Ϯ
14 ng/mL, naive rats: 102 Ϯ 10 ng/mL (one-way ANOVA, p Ͻ .05;
n ¼ 13 per group).
Low CRFR2 mRNA Expression in the BSTMPI of Susceptible
Rats
Next, we examined whether the behavioral manifestations of
susceptible rats were associated with changes in corticotropin-
Figure 2. Behavioral measurements. Freezing behavior during explora-
tion, social interaction, and hyperarousal conditions. Freezing time was
calibrated to the percentage of total time in the open field. After exposure
to stress and the stress-associated cue, susceptible rats showed an
increase from baseline in anxiety-like behavior, i.e., gradual intensification
(or incubation) of fear over time for all three behavioral conditions, as
opposed to resilient rats (exploration: **p Ͻ .01, ***p Ͻ .001,
##
p Ͻ .01,
#
p Ͻ .05; social interaction: *p Ͻ .05; and hyperarousal: *p Ͻ .05 compared
with baseline). Bars represent mean Ϯ SEM; n ¼ 10 per group
(¼subpopulation). Division between susceptible and resilient groups
was performed after second reminder (Re
2
); therefore, figures were
constructed based on retrospect data. Re
1
, first reminder.
830 BIOL PSYCHIATRY 2013;74:827–836 E. Elharrar et al.
www.sobp.org/journal
releasing factor receptor expression in regions linked with
anxiety-like behavior, i.e., the CeA, BLA, and BSTMPI. For
the CeA and BLA, we found no significant differences in
CRFR1 mRNA levels between control rats versus resilient and
susceptible rats after Re
2
(p Ͼ .05; n ¼ 9 for each group;
Figure 3B).
In the BSTMPI, however, a significant increase in CRFR1,
but not CRFR2, mRNA expression was observed in rats initially
exposed to stress (stress group), as compared with control
rats (one-way ANOVA: F
2,28
¼ 69.16, p Ͻ .0001; Tukey’s post
hoc: p Ͻ .001; n ¼ 10 for each group; Figure 3D). Over the longer
period, significantly decreased CRFR1 mRNA levels were found
after Re
2
in resilient rats, as compared with susceptible
and control rats (one-way ANOVA: F
2,28
¼ 69.16, p Ͻ .0001;
Tukey’s post hoc: p Ͻ .01, p Ͻ .05, respectively; n ¼ 10 for each
group; Figure 3D). Conversely, a significant decrease in CRFR2
mRNA expression was demonstrated after Re
2
, in susceptible
rats as compared with resilient and control rats (one-way ANOVA:
F
2,28
¼ 4.004, p Ͻ .01; Tukey’s post hoc: p Ͻ .05; n ¼ 10;
Figure 3E).
Establishment of a Lentiviral-Based System for Site-Specific
Overexpression of CRFR2
Because susceptibility to PTSD-like symptoms coincides with
robust changes in BNST CRFR2 levels, we subsequently examined
the effect of manipulating levels of this receptor specifically within
the BNST of susceptible rats. First, we generated a lentiviral system
for overexpression of CRFR2 at physiological levels (Figure 3A). We
then assessed the ability of this system to express a functional
receptor that responds in vitro to its specific ligand, Ucn2 (Figure 3C).
Using a cyclic adenosine monophosphate responsive element
luciferase reporter assay in HEK293T cells, we found significantly
higher levels of CRFR2 signaling following treatment with 1 or 100
μmol/L Ucn2, as compared with 0 and .01 μmol/L Ucn2 (one-way
ANOVA: F
3,24
¼ 17.658, p Ͻ .0001, Dunnett's post hoc: p Ͻ .0001 vs.
corresponding doses; n ¼ 7 for each group). Next, we examined the
ability of CRFR2 lentiviruses to overexpress the receptor in vivo in
naïve rats. Rats that received bilateral, intra-BSTMPI injections of the
lenti-CRFR2 showed significantly higher CRFR2 expression levels in
this region than rats that received control virus injection (Student t
test, p Ͻ .05; n ¼ 4 per group; Figure 4D).
Figure 3. Quantitative real-time polymer-
ase chain reaction analysis of corticotro-
pin-releasing factor receptor type 1
(CRFR1) and corticotropin-releasing factor
receptor type 2 (CRFR2) messenger RNA
(mRNA) expression in the central amyg-
dala (CeA), basolateral amygdala (BLA),
and bed nucleus of the stria terminalis.
(A) Rat coronal sections illustrating loca-
tion of punches taken from the CeA
(upper plate; bregma −2.52) or BLA (lower
plate; bregma −2.52). (B) Corticotropin-
releasing factor receptor type 1 mRNA
levels in the CeA and BLA following the
second reminder (Re
2
) (n ¼ 9 for each
group) showing no significant differences
between control vs. resilient and suscep-
tible rats (p Ͼ .05). (C) Simplified rat
coronal sections illustrating the location
of a punch taken from the bed nucleus of
the stria terminalis, medial division, pos-
terointermediate part (bregma −.72). (D)
Elevated CRFR1 levels were shown follow-
ing initial stressor exposure (stress). Fol-
lowing Re
2
, CRFR1 mRNA expression levels
were significantly decreased in resilient as
compared with susceptible and control
rats (***p Ͻ .001, **p Ͻ .01,
#
p Ͻ .05,
respectively; n ¼ 10 for each group). (E)
Corticotropin-releasing factor receptor
type 2 levels showed no change after
initial exposure (p Ͼ .05; n ¼ 10 for each
group). After Re
2
, significantly decreased
CRFR2 mRNA levels were found in suscep-
tible rats, compared with resilient and
control rats (*p Ͻ .05,
#
p Ͻ .05, respec-
tively; n ¼ 10 for each group). Bars
represent mean Ϯ SEM (A and C reprinted
from (77) with permission from Elsevier,
copyright 2008).
E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827–836 831
www.sobp.org/journal
Attenuation of Susceptible Behavior by Overexpression of
CRFR2 in the BSTMPI
Next, to assess the lentiviral effect on susceptible behavior, an
additional, separate group of rats were subjected to the behav-
ioral protocol. Rats were divided into susceptible and resilient sets
as above, and subpopulations were then extracted from these
sets (chosen based on extreme response or lack thereof; n ¼ 13 for
susceptible, n ¼ 14 for resilient). Each subpopulation received
bilateral, intra-BSTMPI injections of either high-titer CRFR2-express-
ing lentivirus (n ¼ 7 per group) or control lentivirus (n ¼ 6/7).
We found that overexpression of CRFR2 in the BSTMPI
significantly attenuated freezing behavior in the exploration,
social interaction, and hyperarousal conditions of susceptible rats
after Re
3
, as compared with susceptible rats that received the
control virus. A three-way ANOVA showed a significant group Â
treatment  time interaction (F
1,20
¼ 6.81, p Ͻ .05) over the three
different conditions, i.e., exploration, social interaction, and hyper-
arousal. In a four-way ANOVA, the condition  group  treatment
 time interaction was not significant, suggesting that the effect
was not significantly different between tests. As shown in
Figure 5, the susceptible overexpression group froze significantly
less than before CRFR2-expressing lentivirus administration (Re
2
)
(paired t test; n ¼ 6/7 for each group; p Ͻ .05, p Ͻ .01, p Ͻ .05,
p Ͻ .01 for exploration, social interaction, and hyperarousal,
respectively). To verify the site specificity of lentiviral system, mRNA
levels in the LS of infected susceptible rats was examined. We found
that rats with CRFR2 overexpressing lentiviruses in the BSTMPI
expressed higher mRNA levels as compared with susceptible rats
that received the control virus or with susceptible rats with no
injection (one-way ANOVA: F
3,5
¼ 120.3, p Ͻ .0001; n ¼ 5 for each
group; Figure 6A). Rats with CRFR2 overexpressing lentiviruses in
the LS expressed similar mRNA levels as compared with susceptible
rats that received the control virus or with susceptible rats with no
injection (one-way ANOVA: F
3,3
¼ .555, p Ͼ .5; n ¼ 5 for each group;
Figure 6B). Figure 6C shows GFP staining of the control lentiviruses.
Discussion
We used a rat model for PTSD to demonstrate a progressive
increase in fear response of susceptible (PTSD-like) rats after
exposure to stress and stress-associated cues. Moreover, fear
incubation in susceptible rats was associated with long-lasting
impairment of BNST function, demonstrated as prolonged upre-
gulation of CRFR1 expression levels, combined with a prominent
decrease in CRFR2 expression levels. Application of a CRFR2-
overexpressing lentivirus into the BSTMPI significantly attenuated
the fear response of susceptible rats.
Although incubation of fear over a period of 24 hours is well
established in rodents (34–36), results for longer durations are less
conclusive (5). Several reports have shown that conditioned fear
responses either remain constant over time or decrease to some
extent (37–40). Others have shown time-dependent intensification
of fear in response to shock-paired contexts over 1 month (41) and
2 months (4) in rats or over 2 weeks in mice (42,43). Yet, this
incubation phenomenon was inconsistent across age groups in
rats (4), and in mice it diminished after 2 weeks and showed
inconsistencies among strains (42,43). Our findings showed
gradual, time-dependent intensification of fear in response to
recurring stress cues over a period of 28 days. The maladaptive,
PTSD-like symptoms observed in our model correspond with
clinical manifestations of PTSD, including anxiety and defensive
behavior, social withdrawal and avoidance behaviors, hyperar-
ousal, and re-experiencing (5,29,30). Several studies have reported
that lower cortisol levels in the acute aftermath of trauma are
predictors for subsequent PTSD symptoms (44–46). Moreover,
lowered circulating cortisol was suggested as a major influence on
the formation and processing of traumatic memories and may be
associated with the underlying pathology of PTSD (47,48). In
addition, exogenous stressors, such as exposure to a live predator
(49–51), psychological stress (52), and the predatory cue used
herein (53–56), correspond well with natural stressors. Therefore,
the stressful stimuli used herein may have greater etiological
relevance than application of physical stressors, e.g., electric shock
(57–60), underwater trauma (61), and restraint stress (62,63).
Between 5% and 30% of trauma victims develop PTSD,
whereas others experiencing the same trauma appear to be
resilient (29). Our results, showing that 20% of rats developed
maladapted symptoms following exposure to stress and stress
cues, correlate well with the above statistics. This also empha-
sizes both the importance of individual analysis of behavior and
comparison of each exposed animal both with its own baseline
and with the entire population. Although this analysis yields a
lower percentage of PTSD-like rats, it more accurately simulates
Figure 4. Lentiviral system for overexpression of corticotropin-releasing factor receptor type 2 (CRFR2). Schematic illustration of (A) vectors designed to
overexpress CRFR2 and (B) its lentivirus control. (C) In vitro biological activity validation of CRFR2-expressing lentiviral construct using a cyclic adenosine
monophosphate-dependent luciferase assay. Higher levels of CRFR2 signaling was observed following treatment with 1 or 100 μmol/L of urocortin-2 (Ucn-2)
(***p Ͻ .0001 vs. corresponding doses; n ¼ 7 per group). (D) In vivo functionality assessment of CRFR2-expressing lentiviral construct. Corticotropin-releasing
factor receptor type 2 messenger RNA (mRNA) levels in the bed nucleus of the stria terminalis, medial division, posterointermediate part of rats infected with
lenti-CRFR2 were significantly higher than those of control-infected rats. Mean Ϯ SEM (*p Ͻ .05; n ¼ 4 per group). CMV, cytomegalovirus; CRFR2α,
corticotropin-releasing factor receptor type 2α; eRFP, enhanced red fluorescent protein; GFP, green fluorescent protein; IRES, internal ribosome entry site.
832 BIOL PSYCHIATRY 2013;74:827–836 E. Elharrar et al.
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the heterogeneous pattern of response to stressful events in
humans. Thus, the combination of the model components, i.e.,
exogenous stimuli, simulation of clinical manifestations, and
individual categorization, can produce more reliable results for
modeling the incubation of fear phenomenon.
Recent findings have demonstrated that the CeA and BNST are
functionally complementary, with the CeA mediating short-
duration threat responses (i.e., phasic fear) and the BNST
mediating long-duration responses (sustained fear or anxiety).
Moreover, CRF selectively participates in the BNST-dependent
response (13,64). Our results, showing dysfunction of the CRFR2
expressing neurons in BSTMPI, but not CeA or BLA, of susceptible
rats provide further support for involvement of this region in
sustained fear.
Figure 5. Effect of overexpression of corticotropin-releasing factor recep-
tor type 2 (CRFR2) in the bed nucleus of the stria terminalis, medial
division, posterointermediate part on behavioral manifestation. Suscep-
tible and resilient rats received injection of lenti-CRFR2 or control virus
into the bed nucleus of the stria terminalis, medial division, poster-
ointermediate part. Freezing behavior was assessed in exploration (A),
social interaction (B), and hyperarousal (C) conditions 2 weeks (third
reminder [Re
3
]) after lenti-CRFR2 injection. The line represents second
reminder (Re
2
) and the bars represent Re
3
. A significant reduction in
freezing behavior of susceptible rats was detected (Re
3
vs. Re
2
for
exploration, social interaction, and hyperarousal respectively; **p Ͻ .01,
*p Ͻ .05, **p Ͻ .01; n ¼ 6 per group) Mean Ϯ SEM.
Figure 6. Validation of corticotropin-releasing factor receptor type 2 (CRFR2)
overexpression in the bed nucleus of the stria terminalis, medial division,
posterointermediate part (BSTMPI). Corticotropin-releasing factor receptor
type 2 messenger RNA (mRNA) levels were quantified in susceptible rats
injected with lenti-CRFR2 (overexpression vector [O.E.]) into (A) the BNSTMPI
or (B) lateral septum (LS) in comparison with uninfected or control-infected
(control vector [C.V.]) rats (***p Ͻ .001 and p Ͼ .05, respectively); mean Ϯ
SEM, n ¼ 5 per group. (C) Image of the injection site that confirms bed
nucleus of the stria terminalis specific infected neurons via green fluorescent
protein immunostaining. Dots in a rat coronal illustration indicate site of
injection (reprinted from [78] with permission from Elsevier, copyright 2008).
E. Elharrar et al. BIOL PSYCHIATRY 2013;74:827–836 833
www.sobp.org/journal
It has been suggested that CRFR1 activation is critical for
initiating stress responses (65), while CRFR2 may be involved in
termination of stress responses and restoring homeostasis (66,67).
Thus, we postulate that the significant downregulation of CRFR1
mRNA levels in resilient rats may mediate their behavioral
resilience to stress. In contrast, the inability to lower CRFR1 mRNA
levels and the dramatic reduction in CRFR2 mRNA levels of
susceptible rats may mediate their long-lasting fear response and
incompetence. These rats show an exaggerated stress response
that remains active over the long term and shutting down of the
stress response fails to occur up to 1 month after exposure to
stress. In other words, the natural ability to adapt to stress may be
damaged in susceptible rats, thereby promoting sustained fear.
This hypothesis is further supported by studies showing that
CRFR2 knockout mice are hypersensitive to stress and show
increased anxiety-related behavior (68–70) and that ICV injection
of a selective CRFR2 antagonist dose-dependently enhances
conditioned fear response (71). Other findings implicate CRFR2
in mediating stress coping mechanisms (72). Corticotropin-
releasing factor receptor type 2, along with its specific ligands,
Ucn-2 and urocortin-3 (Ucn-3), are hypothesized to be involved in
dampening the stress response (8,28,73–75). In addition, Ucn-2
and Ucn-3 are essential for recovery from stressful challenges (75).
Thus, our finding showing significantly low levels of CRFR2 in the
BNST of susceptible rats probably affects Ucn-2 and Ucn-3
activity. Thus, in their own right, Ucns may be involved in the
pathogenesis and control of various disorders (76).
The BNST-CRFR2 system has already been implicated in
PTSD-like behavior in a mouse animal model (72). However,
this model did not use reminders; rather, mice experienced
stress and CRFR2 levels were then examined 2 weeks after rats
displayed long-term, steady state PTSD-like behavior. Findings
showed that the exposure to stress increased levels of CRFR2
mRNA in the BNST and that knockdown of BNST-CRFR2 reduced
the percentage of mice categorized as PTSD-like. In contrast,
our model showed a decrease in CRFR2 mRNA levels after
incubation of fear and that CRFR2 overexpression modified
susceptible behavior. Thus, the decrease in CRFR2 levels
following stress -associated cues may mimic physiological re-
experiencing of the original stressor after the pathology has
been established. Yet, taking together the findings of both
models indicates that balanced levels of CRFR2 are essential for
an appropriate response to stressful events.
In conclusion, we show a bi-directional association between CRF
receptor levels in the BSTMPI and incubation of fear in a rat model
for PTSD. The sustained upregulation in CRFR1 expression, combined
with reduced CRFR2 expression in the BNST, may have promoted
long-term conditioned fear response. Corticotropin-releasing factor
receptor type 2 is involved in mechanisms of coping with stressors
and challenging situations; thus, we hypothesize that PTSD mainly
represents malfunctioning of the ability to contend with stressful
stimuli. Even after prolonged dissociation from stress, the susceptible
individual may inappropriately react to imminent stress. This
emphasizes the need for long-term evaluation of symptoms follow-
ing exposure to stress. Moreover, further research focusing on the
beneficial effect of CRFR2 augmentation in the BNST may provide
new insights for the development of PTSD treatments.
We thank Dr. Tamar Green-Sadan for critically reviewing the
manuscript.
Professor Yadid received support for this study by the Rector
Award for Scientific Excellence (Bar Ilan University).
All the other authors reported no biomedical financial interests or
potential conflicts of interest.
Supplementary material cited in this article is available online at
http://dx.doi.org/10.1016/j.biopsych.2013.05.039.
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