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Susceptibility to the long-term anxiogenic

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activation of the glucocorticoid rece....

Article in Neuropharmacology · July 2011

DOI: 10.1016/j.neuropharm.2011.07.034 · Source: PubMed

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 Neuropharmacology 61 (2011) 1297e1305

Contents lists available at SciVerse ScienceDirect

Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Susceptibility to the long-term anxiogenic effects of an acute stressor is mediated

by the activation of the glucocorticoid receptors
Mira Jakovcevski a, b, Melitta Schachner a, c, Fabio Morellini a, d, *
a
Universitätsklinikum Hamburg-Eppendorf, Zentrum für Molekulare Neurobiologie Hamburg, Hamburg, Germany
b
University of Massachusetts Medical School, Department of Psychiatry, Worcester, MA, USA
c
Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA
d
Experimentelle Neuropaediatrie, Universitaetsklinikum Hamburg-Eppendorf, Hamburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The specificity of the response of an organism is an important variable influencing stress-related
Received 26 April 2011 parameters and psychopathological states. We have shown that trait anxiety in C57BL/6 mice, deter-
Received in revised form mined by their emergence latencies in the free choice open field test, positively correlates with the long-
1 July 2011
term behavioral and neuroendocrinological changes induced by a stressor. Here, we show that this
Accepted 21 July 2011
interindividual variability is caused by a different reactivity of the hypothalamusepituitaryeadrenal
(HPA) axis upon exposure to a stressor. Mice with high trait anxiety (long emergence latency, LEL) display
Keywords:
a more pronounced stress-induced activation of the HPA axis than mice with low trait anxiety (short
Addiction
c-fos
emergence latency, SEL). Moreover, stress-induced activation of tyrosine hydroxylase and corticotropin-
Hippocampus releasing hormone occurred in LEL but not SEL mice. In search of the molecular mechanisms underlying
HPA axis these differences, we found that under non-stressed conditions mRNA and protein levels of the gluco-
Paraventricular nucleus corticoid receptor in the hippocampus were higher in LEL mice compared to SEL mice. Also, systemic
Tyrosine hydroxylase injection of the glucocorticoid receptor antagonist RU486 decreased the stress-induced activation of the
HPA axis and the long-term anxiogenic effects of stress observed in LEL mice. Finally, the rewarding
properties of cocaine were enhanced in LEL mice compared to SEL mice, suggesting a causal link between
trait anxiety, stress activity and the behavioral responses to drugs of addiction.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Despite its genetic homogeneity, the inbred strain C57BL/6

The coping strategy of an animal (i.e., the modality by which it
responds at the neuroendocrinological and behavioral levels to envi-
ronmental stimuli and stressors) has an important influence on its
adaptive capacities. In humans, variability in the activity of the stress
response accounts for different coping styles, and correlates with the
probability of an individual to develop psychosomatic symptoms
caused by chronic stress (for review, see Ellis et al., 2006; Zozulya et al.,
2008). Therefore, understanding the determinants of interindividual
variability in the regulation of the hypothalamusepituitaryeadrenal
(HPA) axis and the mechanisms underlying pathologically relevant
dysregulation of the stress response is a key topic in psychobiological
stress research.
* Corresponding author. Experimentelle Neuropaediatrie, Universitaetsklinikum
Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany, Tel.: þ49 40
7410 56650; fax: þ49 40 7410 56263.
E-mail address: fabio.morellini@enp.org (F. Morellini).
displays a wide spectrum of coping strategies between individual
mice (Krishnan et al., 2007). We have described that within the
inbred mouse strain C57BL/6 a marked interindividual variability
exists in the behavioral and endocrinological response to acute
stress that correlates with individual levels of trait anxiety occur-
ring in this mouse line. Specifically, we observed that two different
behavioral responses can be identified in a homogeneous cohort of
C57BL/6 mice undergoing the free choice open field (FCOF) test,
a behavioral paradigm that is considered to measure trait anxiety,
i.e. the intrinsic anxiety level that is not elicited by external stimuli
and that is typical for an individual (Belzung and Berton, 1997; Goes
et al., 2009; Griebel et al., 1993; Teixeira-Silva et al., 2009). In this
test, mice have the choice between staying in their home cage and
entering an unfamiliar arena. We observed that on average about
one third of mice entered the arena within 300 s (defined as short
emergence latency, SEL, mice), whereas about half of mice did not
enter the arena within the maximal given time of 600 s (defined as
long emergence latency, LEL, mice) (Jakovcevski et al., 2008).
Further characterization of the C57BL/6 mice classified as having

0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuropharm.2011.07.034
1298 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305
low (SEL) and high (LEL) levels of trait anxiety indicated that they
diverge in their response to stress: An acute stressor led to long-
lasting anxiogenic effects in LEL mice, whereas it did not affect
the behavior of SEL mice (Jakovcevski et al., 2008).
Here, we tested the hypothesis that the described differences in
the long-term effects of an acute stressor are caused by enhanced
stress-induced activity of the HPA axis in LEL mice when compared to
SEL mice. Because anxiety disorders and stress have been reported to
be risk factors in development and escalation of substance abuse
(Redila and Chavkin, 2008; McLaughlin et al., 2006), we investigated
whether the two trait anxiety phenotypes of SEL and LEL mice
correlate with the behavioral responses to cocaine.
2. Material and methods
2.1. Animals
Ten- to twelve-week-old male mice were taken from the third generation of
a colony established with C57BL/6J mice purchased from Charles River Deutschland
(Sulzfeld, Germany) and maintained under pathogen-free conditions at the Univer-
sity Medical Center Hamburg Eppendorf. Mice were kept under an inverted 12:12 h
light:dark cycle (light off at 0700 h) and standard housing conditions (23  1  C; 50%
humidity; food and water ad libitum). Behavioral tests were performed in an exper-
imental room adjacent to the animal facility and illuminated with dim red light.
Experiments were performed in the middle of the dark phase of the mice when this
species is active and likely to be exposed to unfamiliar stimuli and stressors (Morellini
and Schachner, 2006; Schwartz and Zimmerman, 1990). All materials were cleaned
with soaped water, water and ethanol (75%) between mice. Experiments were carried
out in accordance with the European Community Council Directive (86/609/EEC), and
the procedures used were approved by the State of Hamburg. Care was taken to
minimize pain or discomfort for the animals.
2.2. General experimental design
To identify LEL/SEL mice used in our experiments, several cohorts of C57BL/6
mice underwent the FCOF test and only those characterized as LEL (no emergence
within 600 s) or SEL (emergence latency < 300 s) were then used for further
analyses and or treatments that were performed 5 days after the FCOF test. For
instance, in the experiment testing the stress-induced activity of the HPA axis of LEL
and SEL mice (which results are described in 3.1), we tested a cohort of 50 C57BL/6
mice in the FCOF and, based on their emergence latencies, identified the LEL and SEL
mice among them: Five days after the FCOF, LEL and SEL mice underwent either the
stress paradigm or were left undisturbed in their home cage and were then
immediately sacrificed for further molecular and endocrinological analyses. No
differences in body weight were detected between LEL and SEL mice
(LEL ¼ 30.4  0.4 g; SEL ¼ 29.9  0.8 g).
2.3. FCOF test
The test was used to characterize mice with regard to their levels of trait anxiety as
determined by their latencies to emerge from their home cage into an unfamiliar open
field (Belzung and Berton,1997; Griebel et al.,1993; Teixeira-Silva et al., 2009). Mice were
single-housed in transparent Plexiglas cages (23  17 cm and 14 cm high) five days
before the test was performed to ensure that mice perceived the cage as their home cage.
At the bottom of one of the small walls of the cage was a hole (diameter: 4 cm) occluded
by a removable door. On the test day, the cage with the mouse was placed next to
a rectangular open field (75  90 cm) surrounded by 30 cm high walls. The center of one
of the two 75 cm long walls of the open field had a gap where the small side of the cage
merged into, allowing direct access of the mouse from its cage to the open field when the
door occluding the hole in the cage was opened. The maze was surrounded by a black
curtain and illuminated with a vertical white bulb (the light density in the open field was
5 lux). The test started when the door was opened. After the mouse had introduced its
snout in the opening or displayed a stretched attend posture towards the opening for the
first time, it was given a maximum of 600 s to enter the open field with four paws. The
test was terminated either when a mouse had entered the open field or at the end of the
600 s if the mouse did not enter the open field. The latency to enter the open field
(emergence latency) was calculated as the time that a mouse needed to step with all four
paws into the open field starting from the moment it had recognized that the door was
open. Mice that did not enter the open field within 600 s were assigned to the LEL group,
while mice with emergence latencies below 300 s were assigned to the SEL group
(Jakovcevski et al., 2008).
2.4. Stress paradigm
Rat exposure has been shown to be a potent stressor for mice (Linthorst et al.,
2000). SEL and LEL mice were stressed by exposure to a rat as described
(Jakovcevski et al., 2008). Briefly, the exposure was carried out in Plexiglas cages
(42  26 cm and 16 cm high) subdivided by a vertical metal grid into two equally
sized compartments and closed by a horizontal metal grid on the top. Mice were
placed into one compartment having an adult male Wistar rat (Charles River) in the
adjacent compartment. Control mice were left undisturbed in their home cage.
2.5. Elevated plus maze
The maze was elevated 75 cm above the floor and consisted of two opposing
open arms (30  5 cm) and two opposing closed arms (same size of the open arms
but with 15 cm high walls) extending from a central squared platform (5  5 cm).
The test was performed in darkness and video-recorded with an infrared camera.
Animals were placed in the center of the maze facing one open arm and returned to
their home cage after 5 min. The behavior was analyzed with the software The
Observer (Noldus, Wageningen, The Netherlands) as described (Morellini and
Schachner, 2006).
2.6. Conditioned place preference
This test assesses rewarding properties of psychostimulants in rodents
(Tzschentke, 1998). Also, it allows the evaluation of sensitization defined by further
increase of drug-evoked locomotor activity after repeated administration of the drug
over consecutive experimental days (Druhan and Wilent, 1999). The conditioned
place preference test was performed in a 50  50  40 cm box, laminated with
rough, matted, light-gray resin. The box was subdivided into two compartments
(50  25  40 cm) by a white plastic wall with a hole in the middle allowing
transition of the mice between the two compartments during the pre- and post-
conditioning trials, whereas it was occluded with a plastic door during the condi-
tioning trials. Both compartments were illuminated by a white bulb (10 lux). The two
compartments differed in visual appearance of the walls and floor texture. The
experiment was carried out over five consecutive days. The protocol consisted of one
pre-conditioning trial (day 1), three conditioning sessions with two trials per day
(days 2e4) and a post-conditioning trial (day 5). For the pre-conditioning trial on
day 1, mice were placed at the hole connecting the two compartments and left
exploring both compartments for 20 min. The compartment in which mice spent
less time was chosen as the conditioned compartment (paired with the cocaine).
During the conditioning sessions on days 2e4 all mice underwent a first trial during
which they were first injected with vehicle (0.9% saline) and then placed into the
non-conditioned compartment for 20 min. Four hours later, all mice underwent the
second trial during which they were injected with 10 mg/kg cocaine hydrochloride
and placed in the conditioned compartment for 20 min. During all conditioning
trials, the door between the two compartments was closed to confine mice in the
compartment into which they had been placed. On day five, mice underwent the
post-conditioning trial, to determine the time spent in the conditioned compart-
ment as compared to chance level (50%). Mice were placed at the hole connecting
the two compartments and were free to explore both compartments for 20 min. All
trials were videotaped. The software Ethovision (Noldus) was used to track the
position of the mice and calculate distance moved and time spent in each
compartment.
2.7. Drugs
Diazepam (Ratiopharm, Ulm, Germany; 2 mg/kg) and 8-Hydroxy-dipropyl-
aminotetralin (8-OH-DPAT, SigmaeAldrich, Schnelldorf, Germany; 0.05 and 0.2 mg/
kg) were dissolved in tocopherol-stripped corn oil (MP Biomedicals, Eschwege,
Germany). Both drugs were delivered via oral self-administration using modified
1 ml syringes, allowing stress-free drug delivery since mice had been trained to
spontaneously drink 200 ml corn oil in less than 5 min from the modified syringe
introduced inside their cage. The glucocorticoid receptor (GR) antagonist RU486
(Mifipristone, SigmaeAldrich, Germany) was dissolved in 0.9% saline (B. Braun,
Melsungen, Germany) containing 5% DMSO (SigmaeAldrich) and 3 drops of Tween
20 (Merck Schuchardt, Hohenbrunn, Germany) per 1 ml solution. For the control
groups appropriate vehicles were used. RU486 (15 mg/kg) and vehicle were injected
intraperitoneally.
2.8. Tissue preparation
Mice were killed by decapitation. Trunk blood was collected in heparinized
collection tubes (Sarstedt, Nürnbrecht, Germany), centrifuged at 1700 g for 20 min at
4 C. Plasma was then collected and stored at 20  C until measurement of plasma

corticosterone. Brains were removed and pituitary glands, hippocampi and the
hypothalamic paraventricular nucleus (PVN) were dissected. The PVN was dissected
using a coronal mouse brain matrix (spacing 1 mm; World Precision Instruments,
Berlin, Germany). A 1 mm thick slice was cut out, setting 0 mm at the middle of the
optic chiasm (corresponding to: 0 mm to 1 mm from Bregma; according to
Franklin and Paxinos, 1997). A 0.5  0.5 cm slice containing the PVN was dissected
from this slice. Tissues were immediately frozen in liquid nitrogen within 5 min after
decapitation and stored at 80  C until RNA isolation.
 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305
2.9. Radioimmunoassay
Plasma corticosterone levels were determined using a corticosterone 125I
radioimmunoassay kit for rats and mice (MP Biomedicals, Eschwege, Germany). For
quantification precipitated samples were diluted in 500 ml 0.1 M NaOH, dissolved in
liquid scintillation cocktail (Optiphase ‘HiSafe’ 3, Perkin Elmer, Cologne, Germany)
and counted in a liquid scintillation counter (Wallac 1409, Wallac-Perkin Elmer,
Cologne, Germany) for 180 s. Values for each mouse were evaluated from duplicates
of the original sample relative to the standard curve using an analysis template for
radioimmunoassays provided by the GraphPad PRISM 3.0 software (GraphPad
Software, San Diego, CA, USA).
2.10. Quantitative real-time reverse transcriptionepolymerase chain reaction
Total RNA of all tissues was isolated with the RNeasy Lipid Tissue Kit (Qiagen,
Hilden, Germany) in conjunction with DNAse I (Qiagen) treatment by “on column”
digestion as described in the manufactures’ handbook. All RNA samples were stored
at 80  C until processing. RNA, 0.65 mge2.5 mg per sample, was reverse transcribed
into complementary DNA (cDNA) using Superscript II Reverse Transcriptase Kit
(Invitrogen, Karlsruhe, Germany) and random hexamer primers. The following
primers were used: GR (forward 50 -CAG CAT GCC GCT ATC GAA A-30 ; reverse 50 -CGC
GGC AGG AAC TAT TGT TTT-30 ; Genebank accession number: NM_008173.2), c-fos
(forward 50 -TTC CAC CCC AGA GTC TGA GGA-30 ; reverse 50 -GCT CCA CGT TGC TGA
TGC TC-30 ; Genebank accession number: NM_010234), tyrosine hydroxylase (TH;
forward 50 -ACA TTT GCC CAG TTC TCC CAG-30 ; reverse 50 -CCC AAA CTC CAC AGT GAA
CCA-30 ; Genebank accession number: NM_009377), corticotropin-releasing
hormone (CRH; forward 50 -CCG GGC AGA GCA GTT AGC-30 ; reverse 50 -CAA CAT
TTC ATT TCC CGA TAA TCT C-30 ; Genebank accession number: NM_205769) and, for
reference, hypoxanthine guanine phosphoribosyltransferase (HPRT; forward 50 -GTT
CTT TGC TGA CCT GCT GGA-30; reverse 50 -TCC CCC GTT GAC TGA TCA TT-30 ; Gen-
ebank accession number: NM_013556.1). Oligonucleotides were synthesized by
Metabion (Martinsried, Germany). Amplicons for all primers were sequence
analyzed to verify specific amplification. Quantitative PCR was performed in a total
reaction volume of 20 ml using a SYBR green master mix (qPCR Core Kit for SYB-
RGreenI; Eurogentec, Seraing, Belgium) with 1 ml (5 pmol) each of forward and
reverse primer and 1 ml of the 1:10 diluted cDNA sample. Duplicates of each sample
were measured and analyzed on an ABI 7900 HT sequencer (Applied Biosystems,
Darmstadt, Germany). Amplification conditions were as follows: 45 cycles of 15 s at
95  C and 60 s at 60  C. Real-time PCR data analysis was performed using the
comparative 2DDC(T) method (Livak and Schmittgen, 2001) with HPRT as an
endogenous reference. For graphical presentation and statistical analysis, the mRNA
level for each sample was expressed as percentage of the mean value of the control
(LEL) group, which was set to 100%.
2.11. Quantitative Western blot analysis
Hippocampi were homogenized in 160 ml ice-cold 0.5 M TriseHCl, pH 7.5,
containing 1.6 mg/ml protease inhibitor cocktail (Roche Applied Science, Indian-
apolis, IN, USA) using a 2 ml glass homogenizer (Wheaton, Millville, NJ, USA). To
allow complete isolation of nuclear GR protein all samples were incubated for
30 min at room temperature (20e25  C) with 40 ml lysis buffer (Promega, Man-
nheim, Germany). Aliquots from each sample were diluted 1:10 with PBS for protein
estimation using a BCA Protein Assay Reagent Kit (Pierce, Rockford, USA). Equal
amounts of protein (15 mg) of each sample were denatured in Laemmli buffer at
100  C for 10 min, electrophoretically separated on 8% Tris/HCl polyacrylamide gels
and transferred to nitrocellulose membranes (Protran, Schleicher & Schuell, Dassel,
Germany). All samples from LEL and SEL mice were run together on one gel.
Membranes were blocked with 4% milk powder (Frema Reform, DE-VAU-GE,
Lüneburg, Germany) in PBS/0.05% Tween 20 for 2 h at room temperature
(20e25  C) and incubated with polyclonal rabbit anti-mouse GR antibody (M-20;
1:2000; Santa Cruz Biotechnology, Heidelberg, Germany) in blocking buffer for 2 h
at room temperature (20e25  C). Membranes were washed 4 times with PBS/0.05%
Tween 20 and incubated with anti-rabbit HRP conjugated secondary antibody
(1:20 000; Dianova, Hamburg, Germany) for 1.5 h at room temperature (20e25  C).
Membranes were washed four times with PBS/0.05% Tween 20 and blots were
developed using chemiluminescence detection reagents (GE Healthcare, Munich,
Germany). All membranes were stripped and re-probed with polyclonal rabbit anti-
bIII tubulin antibody (1:5000; Covance, Munich, Germany) overnight at 4  C to
control for the amount of loaded protein. Western blots were scanned and densi-
tometric analysis was performed using TINA Image software (Version 2.0; Raytest
Isotopengeräte, Straubenhardt, Germany). Optical densities for the GR protein were
normalized by optical densities for the loading control. For graphical presentation
and statistical analysis, the normalized optical densities were expressed as
percentage of the mean value of the LEL group.
2.12. Statistical analysis
Data were analyzed with the non-parametric tests ManneWhitney and Kruskal
Wallis followed by Dunn’s comparisons (for unpaired data) or Wilcoxon matched pairs
1299
(for paired data). Preference for the conditioned compartment was assessed by testing
the difference to the chance level of 50% with the non-parametric Wilcoxon signed rank
test. Because there are no non-parametric tests for multifactorial analysis, data on the
short-term effects of stress on SEL and LEL mice were analyzed with the two-way
ANOVA test having ‘anxiety’ (SEL vs. LEL) and ‘stress’ (control vs. stressed) as between
groups factors, followed by NewmaneKeuls post-hoc analysis when appropriate. The
results from the conditioned place preference were analyzed with mixed ANOVA having
‘anxiety’ as between group factor and, depending on the experimental design, ‘time’
(the 5-min intervals of the pre-conditioning trial) or ‘trial’ (3 conditioning trials on
consecutive days) and ‘treatment’ (saline vs. cocaine) as within group factors, followed
by NewmaneKeuls post-hoc analysis when appropriate. In the case of the data analyzed
with the ANOVA, all the variables fulfilled the criteria for parametric analysis as tested
with the KolmogoroveSmirnov normality test for normal distribution and the Levine
test for equal variances between groups. Nevertheless, due to the fact that relatively
small sample sizes may not allow an appropriate valuation of the normal distribution of
the data, appropriate non-parametric tests were performed to confirm the results from
the ANOVA (the results from the non-parametric analyses are mentioned only when in
disagreement with the ANOVA). The data from the FCOF test after diazepam treatment
did not fulfill the criteria for parametric analysis and were therefore evaluated with non-
parametric tests.
3. Results
3.1. Stress-induced activity of the HPA axis is enhanced in LEL mice
compared to SEL mice
We have shown that acute stress has long-term effects on the
HPA axis activity and behavior of LEL but not SEL mice (Jakovcevski
et al., 2008). Two hypotheses can be put forward to explain these
results: 1. the short-term activation of the stress response after an
acute stressor is similar between SEL and LEL mice but leads to
plastic changes in the HPA axis and brain regions controlling
anxiety-like behavior in LEL mice but not in SEL mice; 2. the acti-
vation of the stress response immediately after an acute stressor is
higher in LEL mice compared to SEL mice, thus leading to long-term
effects only in the LEL group. Therefore, we tested the activation of
the HPA axis of SEL and LEL mice immediately after application of
a single acute stressor (30 min exposure to a rat). Control SEL and
LEL mice were left undisturbed in their home cages. Activation of the
HPA axis was evaluated by analyzing: concentration of plasma
corticosterone and levels of mRNA for corticotropin-releasing
hormone (CRH) in the PVN, tyrosine hydroxylase (TH) in the
adrenal gland, and mRNA levels of the immediate-early gene c-fos in
the hippocampus, PVN, pituitary and adrenal gland. The two-way
ANOVA having ‘stress’ and ‘anxiety’ as between group factors
detected a significant effect of ‘stress’ and of the interaction between
‘stress’ and ‘anxiety’ on almost all parameters analyzed (Table 1).
Levels of plasma corticosterone, TH mRNA in the adrenal gland, CRH
mRNA in the PVN and c-fos mRNA in any of the investigated regions
did not differ between unstressed control LEL and SEL mice. After
exposure to the stressor, LEL mice displayed higher values for all
parameters compared to stressed SEL mice (Fig. 1AeG). When
compared to the respective unstressed control groups, plasma
corticosterone concentrations and mRNA levels of c-fos in the
hippocampus, PVN and adrenal gland were upregulated in LEL and
SEL mice, indicating that the stress response was activated upon
stress exposure in both, SEL and LEL, groups. In the case of c-fos
mRNA levels in the pituitary gland, and mRNA levels of TH in the
adrenal gland and CRH in the PVN, stress induced an upregulation in
LEL but not SEL mice when compared to the respective unstressed
control groups. In LEL mice stress-induced levels of all other
investigated parameters were significantly higher than in SEL mice.
3.2. Stress-induced alterations in LEL mice are inhibited by the GR
antagonist RU486, whereas trait anxiety is reduced by diazepam
Since GR activity has been described as a modulator of the
HPA axis and anxiety-like behavior (de Kloet et al., 1998; Korte,
1300 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305

Table 1
Two-way ANOVA.

Stress Anxiety Stress  Anxiety

F P F P F P
Plasma corticosterone (F1,22) 95.85 <0.001 5.26 <0.05 2.88 n.s.
c-fos mRNA in the PVN (F1,17) 37.16 <0.001 2.7 n.s. 2.93 n.s.
c-fos mRNA in the pituitary glands (F1,12) 9.22 <0.05 1.54 n.s. 3.99 n.s.
c-fos mRNA in the adrenal glands (F1,17) 47.26 <0.001 6.06 <0.05 6.27 <0.05
c-fos mRNA in the hippocampus (F1,18) 78.48 <0.001 6.9 <0.05 6.78 <0.05
CRH mRNA in the PVN (F1,16) 4.81 <0.05 4.61 <0.05 5.17 <0.05
TH mRNA in the adrenal glands (F1,16) 5.38 <0.05 4.83 <0.05 3.77 n.s.

The degrees of freedom for F are indicated in brakets. n.s. ¼ not significant; PVN ¼ paraventricular nucleus; CRH ¼ corticotropin-releasing hormone; TH ¼ thyrosine
hydroxylase.

2001; Rochford et al., 1997; Tronche et al., 1999), we wondered induced hippocampal c-fos mRNA levels were reduced compared
whether GR expression differs between SEL and LEL mice and to vehicle-treated LEL mice and did not differ from those
measured protein and mRNA levels for GR in the hippocampus measured in stressed vehicle-treated SEL mice (Fig. 3B). More-
(one of the brain regions implicated in HPA axis regulation and over, RU486 treatment before the exposure to the rat rescued the
anxiety-like behavior) under non-stressed conditions. Indeed, long-term anxiogenic effects of the stressor: When tested in the
glucocorticoid and mineralocorticoid receptors in the hippo- elevated plus maze 24 h after exposure to the stressor, RU486-
campus are considered to regulate the negative feedback system treated LEL mice entered more promptly the open arms
of the HPA axis (Sapolsky et al., 1991; de Kloet et al., 2007). (Fig. 4A), had a higher percentage of open arm entries (Fig. 4B),
Hippocampal levels for mRNA and protein were significantly spent more time in the open arms (Fig. 4C), showed more head
higher in LEL mice compared to SEL mice (Fig. 2). We then tested dipping on the open arms (Fig. 4D), and displayed more rearing
whether the higher GR levels in the hippocampus of LEL mice bouts (Fig. 4E) and total transitions (Fig. 4F) than vehicle-treated
account for their enhanced trait anxiety and stress response by LEL mice. Moreover, whereas vehicle-treated LEL mice differed
treating the mice with the GR antagonist RU486. In the FCOF test for all parameters when compared to vehicle-treated SEL mice
the emergence latencies of LEL mice were not affected by (Fig. 4), LEL mice treated with RU486 did not behave differently
administration of RU486 90 min before the FCOF test (Fig. 3A) than SEL mice treated with vehicle, with the exception of closed
suggesting that trait anxiety of LEL mice is not modulated by GR. arm entries (Fig. 4G).
By contrast, RU486 injected 90 min before a 60 min exposure to Since trait anxiety of LEL mice was not affected by the GR
a rat reduced the effects of the stressor on the behavior and HPA antagonist RU486, we tested the effects of two anxiolytics, the
axis activity of LEL mice: in LEL mice treated with RU486 stress- benzodiazepine diazepam and the non-selective agonist of the

A B PVN
C p itu itary g lan d s D ad re n al g lan d s
300 300 250 800
*** ***
c-fos mRNA (% of control)

c-fos mRNA (% of control)

c-fos mRNA (% of control)

* ***
corticosterone (ng/ml)

700
250 200
+ 600
+ **
200 *** 200
150 + 500 ++
150 400 **
100 300
100 100
200
50 50
100
0 0 0 0
LEL SEL LEL SEL LEL SEL LEL SEL

E h ip p o camp u s F PVN G ad re n al g lan d s

800 200 150
***
c-fos mRNA (% of control)

*
CRH mRNA (% of control)

*
TH mRNA (% of control)

700
125
150
+
600
500 ++ 100
** ++
400 100 75
300
50
200 50
25 control
100
stressed
0 0 0
LEL SEL LEL SEL LEL SEL

Fig. 1. HPA axis activation after acute stress is increased in LEL mice. SEL and LEL mice were exposed to a rat for 30 min (“stressed”, white bars) or left undisturbed in their home
cages (“control”, black bars). (A) Plasma corticosterone levels in stressed and control LEL and SEL mice (N ¼ 6e8). (BeE) C-fos mRNA levels in the PVN (B) pituitary gland (C) adrenal
glands (D) and hippocampus (E) of stressed and control LEL and SEL mice (control, N ¼ 3e6; stressed, N ¼ 6e8). (F) CRH mRNA levels in the PVN and (G) TH mRNA levels in the
adrenal glands of stressed and control LEL and SEL mice (control, N ¼ 3; stressed, N ¼ 5e8). *, ** and ***p < 0.05, 0.01 and 0.001, respectively, compared with control mice within the
LEL or SEL groups. þ and þþp < 0.05 and 0.01, respectively, compared with the stressed LEL group (NewmaneKeuls post-hoc after two-way ANOVA). Diagrams represent mean
values þ SEM. Mean values are shown as percentage of the control LEL group that is set to 100%.
 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305 1301

A 125 B 125 C

G R mRNA (% of LEL)
100 100

GR (% of LEL)
* *
75 75 GR
100 kDa -
50 50
50 kDa - β III- tubulin
25 25 LEL SEL

0 0
LEL SEL LEL SEL
Fig. 2. Higher hippocampal GR mRNA and protein levels in LEL mice under non-stressed conditions. Levels of GR mRNA (A) and protein (B) in the hippocampus of LEL and SEL mice
were measured by quantitative real-time PCR and quantitative Western blot analysis, respectively. (C) Representative Western blot using antibodies against GR and b III-tubulin as
loading control in LEL (left) and SEL (right) mice. For mRNA (LEL, N ¼ 10; SEL, N ¼ 11) and protein levels (LEL, N ¼ 5; SEL, N ¼ 5) mean values þ SEM are indicated as percentage of
the LEL group, which is set to 100%. *p < 0.05 compared with the LEL group (ManneWhitney test).

serotonin receptor 1A (5-HT1A) 8-OH-DPAT, on the behavioral paradigm. The rewarding effects of cocaine on SEL and LEL mice
response of LEL mice to the FCOF test. LEL mice had reduced were determined by means of preference for the cocaine-paired
emergence latencies 15 min after administration of diazepam compartment, whereas sensitization was calculated by measuring
(2 mg/kg) compared to vehicle-treated LEL mice. One week after cocaine-induced hyperlocomotion after repeated cocaine injection
washout of the drug, the emergence latencies of LEL mice in the during the three conditioning trials.
diazepam-treated group were significantly longer than 15 min after The two-way mixed ANOVA detected a significant effect of the
diazepam exposure and did not differ from those of the vehicle- interaction between ‘anxiety’ and ‘time’ on distance moved during
treated LEL mice (Fig. 3C). The fact that LEL mice maintained their the pre-conditioning trial (F3,42 ¼ 9.46; p < 0.001): Post-hoc anal-
trait anxiety even after they had explored the FCOF under the yses indicated that SEL mice traveled longer distances compared to
anxiolytic effects of diazepam, supports the idea that emergence LEL mice during the first 10 min of the pre-conditioning trial,
latencies in this test are indicative of trait anxiety of a mouse suggesting that novelty-induced locomotion is higher in SEL than
(Belzung and Berton, 1997; Goes et al., 2009; Griebel et al., 1993; LEL mice (Fig. 5A). During the conditioning sessions, 3-way mixed
Teixeira-Silva et al., 2009). 8-OH-DPAT had no effect on LEL mice ANOVA (having ‘anxiety’ as between groups factor and ‘treatment’
15 min after administration of the drug (data not shown). and ‘trial’ as within group factor) detected a significant effect of
‘treatment’ on distance moved (F1,13 ¼ 104.4; p < 0.001) indicating
3.3. Cocaine-induced conditioned place preference is enhanced that mice covered longer distances after being injected with
in LEL vs. SEL mice cocaine than after being injected with saline (Fig. 5B). Distance
moved was also affected by the interaction between ‘treatment’
Response to stress has been proposed as potential risk factor in and ‘trial’ (F2,26 ¼ 6.7; p < 0.01): Post-hoc analyses revealed that
the development of substance abuse (McLaughlin et al., 2006; distance moved after cocaine injection was higher during trials 2
Redila and Chavkin, 2008). We, thus, tested whether SEL and LEL and 3 compared to trial 1, indicating that sensitization occurred. No
mice differ in the cocaine-induced conditioned place preference effect of ‘anxiety’ or of the interaction between ‘anxiety’ and

A B C ++
600 125 600
c-fos mRNA (% of vehicle)

*
Emergence latency (s)

Emergence latency (s)

500 100
450
400
75 **
300 300
50
200
150
25 Diaze p am
100
Ve h icle
0 0 0
V R SEL R V 15 min 7 d ays

Fig. 3. The GR antagonist RU486 reduces the stress-induced activation of the hippocampus, but not the trait anxiety, of LEL mice. (A) LEL mice were analyzed for their emergence
latencies in the FCOF test 1.5 h after application of the vehicle (“V“; white bar; N ¼ 10) or the GR antagonist RU486 (“R”; black bar; N ¼ 8). (B) Stress-induced c-fos mRNA expression
in the hippocampus in LEL mice subjected to acute stress (30 min olfactory contact to a rat) 1.5 h after application of the vehicle (“V”, white bar; N ¼ 6) or the GR antagonist RU486
(“R” black bar; N ¼ 6) and in vehicle-treated stressed SEL mice (“SEL”, gray bars; N ¼ 4). Diagrams represent mean values þ SEM. Data are indicated as percentage of the vehicle
group, which is set to 100%. *p < 0.05 compared to the vehicle-treated LEL group (Dunn’s comparisons after a significant effect detected by the KruskaleWallis test). (C) Diazepam
transiently reduces emergence latencies of LEL mice in the FCOF test. Naïve mice were classified as LEL mice in a first FCOF test. Five days after, mice were assigned to two groups
that underwent a FCOF test 15 min after having been treated with either vehicle (N ¼ 15) or diazepam (N ¼ 15). After 7 days, mice were analyzed again for their emergence latency
in the FCOF test without receiving any treatment. **p < 0.001 comparison between the vehicle and Diazepam groups at time point “15 min” after treatment (ManneWhitney
test); þþp < 0.001 comparison between the two time points (“15 min” and “7 days”) within the group of mice treated with Diazepam 15 min after the treatment (Wilcoxon
matched pairs test).
1302 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305

A B C D
40 20 20
Latency to enter the OA (s) 300
**

Unprotected head dip (n)

** **

Time on open arms (%)

O pen arm entries (%)
250 +++ **
30 15 15
200

150 20 10 10
+ + +
100 +
10 5 5
50

0 0 0 0
SEL R V SEL R V SEL R V SEL R V

E F G
20 20 15

Closed arm entries (n)

* **
Total transitions (n)

+
15 15 ++
Rearing (n)

++ +++ 10

10 10

5
5 5

0 0 0
SEL R V SEL R V SEL R V

Fig. 4. The GR antagonist RU486 inhibits the anxiogenic effects of stress on LEL mice. LEL mice were subjected to acute stress (1 h olfactory contact to a rat) 1.5 h after application of
the vehicle (V, white bars; N ¼ 15) or the GR antagonist RU486 (R, black bars; N ¼ 15). As reference for low anxiety-related behavior, vehicle-treated SEL mice (SEL, gray bars; N ¼ 11)
were included in the study. Stress-induced state anxiety was determined in the elevated plus maze 24 h after exposure to the stressor. (A) Latency to enter one of the two open arms
(OA). (B) Entries into the open arms calculated as percentage of total entries in the closed and open arms. (C) Percentage of total time spent on the open arms. (D) Number of head
dipping performed while the mouse was on the open arms. (E) Number of rearing bouts. (F) Number of total entries in the closed and open arms. (G) Number of entries into the
closed arms. * and **p < 0.05 and 0.01, respectively, as compared to RU486-treated LEL mice. þ, þþ and þþþp < 0.05, p < 0.01 and p < 0.001, respectively, compared to vehicle-
treated SEL mice (Dunn’s comparisons after a significant effect detected by the KruskaleWallis test).

‘treatment’ and between ‘anxiety’, ‘treatment’ and ‘trial’ on supported by several studies indicating that activation of the HPA
distance moved was detected, indicating that SEL and LEL mice axis is enhanced in animals with high levels of anxiety. One of the
showed similar cocaine-induced hyperlocomotion and sensitiza- most extensively investigated genetic models for high and low
tion (Fig. 5B). During the post-conditioning trial, LEL mice devel- anxiety are the HAB/LAB rats: These strains originate from Wistar
oped a preference for the cocaine-paired compartment, as rats selectively bred for their avoidance of (HAB) or preference for
indicated by the fact that the time spent in this compartment was (LAB) the open arms of the elevated plus maze (Landgraf and
significantly higher than the chance level of 50% (Fig. 5C). Wigger, 2002). Recently, HAB/LAB mice have also been generated
A tendency for a preference for the conditioned compartment was and characterized (Krömer et al., 2005). Similarly to what we
also detected for the SEL group (p < 0.06), although it was signifi- observe in LEL vs. SEL mice, HAB rats display enhanced plasma
cantly lower than in LEL mice (Fig. 5C) suggesting that cocaine was corticosterone concentrations compared to LAB rats after being
more rewarding for LEL than for SEL mice. exposed to a stressor (i.e., 5-min forced exposure to an elevated
open arm; Landgraf et al., 1999). Also, the stress-induced activation
4. Discussion of hypothalamic neurons, that we found to be enhanced in LEL mice
compared to SEL mice, was reported to be higher in HAB than in
We show that LEL mice, when compared to SEL mice, have LAB rats (Frank et al., 2006; Salchner et al., 2006; Salomé et al.,
a stronger central response to stress, that results in an enhanced 2004) and mice (Muigg et al., 2009). Thus, the fact that a positive
peripheral activation, as indicated by higher levels of stress- correlation between anxiety-related behaviors and activation of the
induced plasma corticosterone, c-fos mRNA in the core structures HPA axis was observed independently of the source of variability
of the HPA axis, TH mRNA in the adrenal glands, and CRH mRNA in (genetic in the case of the different breeding lines HAB/LAB, and
the PVN. These findings correlate with the higher stress-induced epigenetic in the case of the genetically identical LEL/SEL C57BL/6
anxiety-like behavior observed 24 h after stress in LEL, but not mice) strongly supports the idea of a functional link between the
SEL mice (Jakovcevski et al., 2008; present study). The different stress response and the anxiety trait of an individual.
activation of brain structures regulating stress responses (i.e., Based on the observation that more anxious individuals
hippocampus and PVN) indicates that SEL and LEL mice differ in respond to a stressor with an enhanced activation of the HPA axis
their perception or processing of stressors. (present study; Frank et al., 2006; Landgraf et al., 1999; Muigg
Our results suggest that the positive correlation which is et al., 2009; Salchner et al., 2006), we tested whether the stress-
supposed to exist between trait and state anxiety depends on the induced neuroendocrinological changes that we observe in LEL
activation of the HPA axis. Specifically, individuals with high trait mice could be inhibited by pharmacological blockade of the
anxiety have, compared to individuals with low trait anxiety, more corticosteroid receptors. Due to their involvement in hormonal
pronounced (or sensitive) stress-induced activations of the HPA stress response (de Kloet et al., 1998; Korte, 2001) and their impact
axis that in turn induce enhanced levels of state anxiety. This idea is on anxiety (Rochford et al., 1997; Tronche et al., 1999) the function
 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305
A 1000
Distance moved (cm)
**
750
**
500
250 LEL
SEL
0 0
0 51 102 15
3 420 5
Time (min )
Fig. 5. Cocaine-induced conditioned place preference is enhanced in LEL mice. LEL mice (black squares/bars, N ¼ 8) and SEL mice (white squares/bars, N ¼ 7) were subjected to the
conditioned place preference test. (A) Locomotor activity in the pre-conditioning trial (ManneWhitney test) and (B) during the saline (sal) and cocaine (coc) conditioning trials on
conditioning days 1e3. (C) Time spent in the cocaine compartment during the pre- (PRE) and post-conditioning (POST) trials. ** and ***p < 0.01 and p < 0.001, respectively,
compared to the pre-conditioning trial within the SEL or LEL groups. þþp < 0.01 compared to LEL mice during the post-conditioning trial (NewmaneKeuls post-hoc after two-way
ANOVA).
of the GR is of high interest for the interpretation of the enhanced
stress response of LEL mice, particularly since polymorphisms in
the GR gene have been associated with increased stress responses
(Xu et al., 2006; Wust et al., 2004). Moreover, we found that both
mRNA and protein levels for GR were higher in LEL than in SEL
mice under basal unstressed conditions. We thus treated LEL mice
with the GR antagonist RU486 shortly before they underwent the
stress paradigm. When compared to LEL mice injected with the
vehicle, RU486-treated LEL mice displayed reduced hippocampal
c-fos mRNA expression and decreased anxiety-like behavior on the
elevated plus maze, reaching levels similar to those observed in
stressed SEL mice. These observations suggest that the long-term
anxiogenic effects that a stressor has on LEL mice are caused by
their enhanced neuroendocrinological stress response that seems
to be regulated by elevated amounts of GR. These results are in line
with previous studies showing that RU486 reduced the anxiogenic
effects of an acute stressor, but did not affect anxiety-like behavior
under “basal” unstressed conditions (Korte et al., 1996, 1995). The
idea that stress-responses can be dampened by blockade of GRs is
supported by several animal and human studies. An upregulation
of the GR in the hippocampus has been reported for rats exposed
to a single-prolonged stressor. These rats showed post-traumatic
stress disorder-like behavioral and physiological alterations that
could be normalized by administration of a GR antagonist prior
exposure to the single-prolonged stressor (Kohda et al., 2007).
Similarly, anxiety-like behavior induced by chronic corticosterone
treatment (Myers and Greenwood-Van Meerveld, 2007) or by
restrained stress (Calfa et al., 2007; Calvo and Volosin, 2001; Korte
et al., 1995) could be reversed by application of a GR antagonist.
Consistently, clinical studies indicate that pharmacological GR
antagonism may be a useful treatment of major depression in
humans (DeBattista and Belanoff, 2006; Flores et al., 2006). The
fact that RU486 has been shown to reduce the anxiogenic effects of
an acute stressor but not the anxiety-like behavior under “basal”
unstressed conditions (Bitran et al., 1998; Smythe et al., 1997;
Korte et al., 1996) suggests that the effects of RU486 that we
observed on LEL mice are not caused by a general anxiolytic action
of RU486, but most likely by dampening the effects of the acute
activation of the stress response. In support of this idea, acute
injection of RU486 at concentrations similar to those used in our
study inhibited the stress-induced regulation of blood glucose
levels (Sim et al., 2010) and increase in glutamate release in the
prefrontal cortex (Musazzi et al., 2010) in rats, as well as the
depressive-like behavior of maternally separated rats (Aisa et al.,
2009). It should be mentioned that because RU486 was systemi-
cally applied, it cannot be excluded that the anxiolytic effects
1303
B 3500 C
LEL (sal) 70
++
LEL
***
Distance moved (cm)
LEL (coc)
3000 SEL
compartment (%)
60
Time in cocaine
SEL (sal) **
2500 SEL (coc) 50
2000 40
30
1500
20
1000
500 10
0
0 1 2 3 PRE POST
Day
observed in stressed LEL mice were mediated by its activity as
antagonist of the progesterone receptor (Andréen et al., 2009; but
see Beckley et al., 2011).
Our finding that hippocampal expression of the GR is higher in
mice with enhanced trait anxiety is counterintuitive to the general
assumption that GR expression should be reduced in depressed
patients (Webster et al., 2002), although this idea is based mainly on
indirect evidence from the dexamethasone suppression test (Barden
et al., 1995). Unfortunately, inconsistent results were obtained in
studies on genetically modified mice with altered GR expression
aiming at the elucidation of the role of GR on anxiety-related
disorders. Depending on the mouse model, both positive and
negative correlations between GR expression and anxiety have been
reported. For example, enhanced depressive-like behavior under
basal unstressed conditions has been described in mice with
forebrain-specific ablation of GR (Boyle et al., 2005). Conversely,
positive correlations between GR expression and anxiety (as we
observed in SEL and LEL mice), have been reported in several
transgenic mice (Tronche et al., 1999; Rochford et al., 1997).
Reversely, mice with about 75% GR overexpression in the forebrain
have been reported to express enhanced anxiety-like behavior and
an altered pattern of HPA responsiveness characterized by a delayed
termination of the stress response (Wei et al., 2004). Contradictory
results have also been reported for mice with GR ablation specifi-
cally in the forebrain that displayed slightly enhanced depressive-
like behavior in the forced swimming test (Boyle et al., 2005), but
reduced anxiety-like behavior in the light/dark and elevated plus
maze tests (Boyle et al., 2006), whereas exposure to restraint stress
affected the behavior of wild-type mice but not of the transgenic
mice (Boyle et al., 2006), similar to what we observe in SEL mice that
express reduced GR in the hippocampus when compared to LEL
mice. Thus, on the one hand the results obtained in mice with
transgenic manipulation of GR expression support the idea that GR
is an important regulator of the stress response and anxiety-related
behaviors. On the other hand, the variability and inconsistencies
between studies with different animal models indicate that the
relationships between GR function, anxiety and stress responses are
not linear and other factors must also play an important role, as for
instance the ratio between the expression of GR and mineralocor-
ticoid receptors (Joëls and de Kloet, 1994).
Treatment with RU486 did not decrease trait anxiety behavior
(i.e., emergence latencies in the FCOF test), possibly because under
non-stressed conditions most GRs are not bound to corticosterone
(de Kloet et al., 1998). Alternatively, it is possible that the concen-
tration used (15 mg/kg) was too low to reduce trait anxiety in the
free choice open field test although it was sufficient to inhibit the
1304 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305
long-term anxiogenic effects of stress as measured in the elevated
plus maze test. On the other hand, diazepam, that increases the
sensitivity of GABAA receptors for GABA (Bormann, 1991), reduced
the levels of trait anxiety in LEL mice. Our observation is in line with
a study showing that trait anxiety is increased in mice deficient for
the g2 GABAA receptor subunit (Earnheart et al., 2007). Of note, GR
has been reported to bind to the promoter region of the GABAA
receptor gene (Gerner et al., 1997) and thus to regulate the tran-
scription of this gene, suggesting that GR activity can modify GABAA
receptor expression. It is therefore plausible that the described
differences between LEL/SEL mice in GR expression levels and
activation of the HPA axis impinge differentially on GABAA trans-
mission leading to differences in trait anxiety expressed by these
mice.
In humans, comorbidity of psychostimulant-induced addiction
and anxiety or depression-related disorders has been reported
(Smith and Book, 2010). It has also been shown that certain
personality traits such as enhanced levels of trait anxiety correlate
with duration of abstinence and treatment outcome (O’Leary et al.,
2000). We thus investigated whether LEL/SEL mice have distinct
behavioral responses to cocaine. Our results from the cocaine-
induced conditioned place preference test indicate that LEL mice
display a higher preference for the cocaine compartment, although
it cannot be excluded that the enhanced preference of LEL mice for
the cocaine-paired compartment is caused by improved learning
and memory abilities and not by an increased susceptibility to the
rewarding effects of cocaine. Thus, it would be interesting to
analyze LEL/SEL mice in paradigms of drug self-administration to
test the idea that higher sensitivity of the HPA axis or enhanced
levels of trait anxiety correlate with enhanced vulnerability to the
neurological effects of drugs of abuse.
The fact that we regularly observe LEL/SEL mice within all
cohorts of C57BL/6 mice that we have analyzed (e.g., Jakovcevski
et al., 2008; present study; Ia Labadze, personal communication)
suggests that the LEL/SEL phenotypes indeed represent different
coping strategies present in mice or at least in the C57BL/6 strain.
The idea that LEL/SEL mice comprise two distinct phenotypes
rather than two extremes of a continuum is supported by the fact
that the emergence latencies of C57BL/6 mice in the free choice
open field test show a trimodal frequency distribution rather than
a Gaussian curve, with 34% of mice showing a SEL phenotype and
54% showing a LEL phenotype (Jakovcevski et al., 2008). This
distribution is also reflected in the expression of stress-induced
anxiety-related behaviors that were observed only in LEL mice
and not in SEL mice, thus showing a dichotomy rather than
a continuum (Jakovcevski et al., 2008; present study). Regarding
the origin of such remarkable interindividual variability, it is known
that different phenotypes in a genetically homogeneous population
can originate from several environmental factors. Interestingly, the
strong regulation of epigenetic factors on the coping strategy of an
individual is considered an important adaptive mechanism not only
in the mouse (Berry and Scriven 2005; Bronson, 1984), but also in
humans (Ellis et al. 2006; Nesse 1999; Troisi, 2005). In this context,
the LEL/SEL mice can be a valuable animal model, in addition to
those already available and described, to investigate the activity of
the stress axis because the source of variability is not induced by
selective breeding (with possible sources of spontaneous mutations
and genetic drift between the different lines), but rather represent
different adaptive phenotypes that have been selected during
evolution. Moreover, LEL/SEL mice could be used to investigate the
environmental factors and epigenetic mechanisms determining the
coping strategy of an animal. Finally, our study indicate that
interindividual variability in coping strategies, even within inbred
strains, should be carefully taken into account when studying the
ultimate and proximate causes of the stress response.
Acknowledgments
M. J. was supported by an individual grant from the German
Research Foundation (DFG) during the analysis of the experiments
and writing of the paper.
References
Aisa, B., Gil-Bea, F.J., Marcos, B., Tordera, R., Lasheras, B., Del Río, J., Ramírez, M.J., 2009.
Neonatal stress affects vulnerability of cholinergic neurons and cognition in the
rat: involvement of the HPA axis. Psychoneuroendocrinology 34, 1495e1505.
Andréen, L., Nyberg, S., Turkmen, S., van Wingen, G., Fernández, G., Bäckström, T., 2009.
Sex steroid induced negative mood may be explained by the paradoxical effect
mediated by GABAA modulators. Psychoneuroendocrinology 34, 1121e1132.
Barden, N., Reul, J.M., Holsboer, F., 1995. Do antidepressants stabilize mood through
actions on the hypothalamic-pituitary-adrenocortical system? Trends Neurosci.
18, 6e11.
Beckley, E.H., Scibelli, A.C., Finn, D.A., 2011. Progesterone receptor antagonist CDB-
4124 increases depression-like behavior in mice without affecting locomotor
ability. Psychoneuroendocrinology 36, 824e833.
Belzung, C., Berton, F., 1997. Further pharmacological validation of the BALB/c
neophobia in the free exploratory paradigm as an animal model of trait anxiety.
Behav. Pharmacol. 8, 541e548.
Berry, R.J., Scriven, P.N., 2005. The house mouse: a model and motor for evolu-
tionary understanding. Biol. J. Linn. Soc. 84, 335e347.
Bitran, D., Shiekh, M., Dowd, J.A., Dugan, M.M., Renda, P., 1998. Corticosterone is
permissive to the anxiolytic effect that results from the blockade of hippo-
campal mineralocorticoid receptors. Pharmacol. Biochem. Behav. 60, 879e887.
Bormann, J., 1991. Electrophysiological characterization of diazepam binding
inhibitor (DBI) on GABAA receptors. Neuropharmacology 30, 1387e1389.
Boyle, M.P., Brewer, J.A., Funatsu, M., Wozniak, D.F., Tsien, J.Z., Izumi, Y., Muglia, L.J.,
2005. Acquired deficit of forebrain glucocorticoid receptor produces
depression-like changes in adrenal axis regulation and behavior. Proc. Natl.
Acad. Sci. U S A 102, 473e478.
Boyle, M.P., Kolber, B.J., Vogt, S.K., Wozniak, D.F., Muglia, L.J., 2006. Forebrain
glucocorticoid receptors modulate anxiety-associated locomotor activation and
adrenal responsiveness. J. Neurosci. 26, 1971e1978.
Bronson, F.H., 1984. The adaptability of the house mouse. Sci. Am. 250, 116e125.
Calfa, G., Bussolino, D., Molina, V.A., 2007. Involvement of lateral septum and the
ventral hippocampus in the emotional sequelae induced by social defeat:role of
the glucocorticoid receptors. Behav. Brain Res. 181, 23e34.
Calvo, N., Volosin, M., 2001. Glucocorticoid and mineralocorticoid receptors are
involved in the facilitation of anxiety-like response induced by restraint.
Neuroendocrinology 73, 261e271.
DeBattista, C., Belanoff, J., 2006. The use of mifepristone in the treatment of
neuropsychiatric disorders. Trends Endocrinol. Metab. 17, 117e121.
Druhan, J.P., Wilent, W.B., 1999. Effects of the competitive N-methyl-D-aspartate
receptor antagonist, CPP, on the development and expression of conditioned
hyperactivity and sensitization induced by cocaine. Behav. Brain Res. 102,
195e210.
Earnheart, J.C., Schweizer, C., Crestani, F., Iwasato, T., Itohara, S., Mohler, H.,
Lüscher, B., 2007. GABAergic control of adult hippocampal neurogenesis in
relation to behavior indicative of trait anxiety and depression states. J. Neurosci.
27, 3845e3854.
Ellis, B.J., Jackson, J.J., Boyce, W.T., 2006. The stress response systems: universality
and adaptive individual differences. Dev. Rev. 26, 175e212.
Flores, B.H., Kenna, H., Keller, J., Solvason, H.B., Schatzberg, A.F., 2006. Clinical and
biological effects of mifepristone treatment for psychotic depression. Neuro-
psychopharmacology 31, 628e636.
Frank, E., Salchner, P., Aldag, J.M., Salomé, N., Singewald, N., Landgraf, R., Wigger, A.,
2006. Genetic predisposition to anxiety-related behavior determines coping
style, neuroendocrine responses, and neuronal activation during social defeat.
Behav. Neurosci. 120, 60e71.1. 628e636.
Franklin, K.B.J., Paxinos, G. (Eds.), 1997. The Mouse Brain in Stereotaxic Coordinates.
Academic Press, San Diego.
Gerner, F.M., Trapp, T., Hauser, C.A., 1997. A classic cAMP responsive element in the
promoter region of the alpha 1-GABAA receptor subunit has non-classic
properties. Funct. Neurol. 12, 55e61.
Goes, T.C., Antunes, F.D., Teixeira-Silva, F., 2009. Trait and state anxiety in animal
models: is there correlation? Neurosci. Lett. 450, 266e269.
Griebel, G., Belzung, C., Misslin, R., Vogel, E., 1993. The free-exploratory paradigm:
an effective method for measuring neophobic behaviour in mice and testing
potential neophobia-reducing drugs. Behav. Pharmacol. 4, 637e644.
Jakovcevski, M., Schachner, M., Morellini, F., 2008. Individual variability in the stress
response of C57BL/6J male mice correlates with trait anxiety. Genes Brain
Behav. 7, 235e243.
Joëls, M., de Kloet, E.R., 1994. Mineralocorticoid and glucocorticoid receptors in the
brain. Implications for ion permeability and transmitter systems. Prog. Neuro-
biol. 43, 1e36.
de Kloet, E.R., Vreugdenhil, E., Oitzl, M.S., Joels, M., 1998. Brain corticosteroid
receptor balance in health and disease. Endocr. Rev. 19, 269e301.
 M. Jakovcevski et al. / Neuropharmacology 61 (2011) 1297e1305
de Kloet, E.R., Derijk, R.H., Meijer, O.C., 2007. Therapy insight: is there an imbal-
anced response of mineralocorticoid and glucocorticoid receptors in depres-
sion? Nat. Clin. Pract. Endocrinol. Metab. 3, 168e179.
Kohda, K., Harada, K., Kato, K., Hoshino, A., Motohashi, J., Yamaji, T., Morinobu, S.,
Matsuoka, N., Kato, N., 2007. Glucocorticoid receptor activation is involved in
producing abnormal phenotypes of single-prolonged stress rats: a putative
post-traumatic stress disorder model. Neuroscience 148, 22e33.
Korte, S.M., 2001. Corticosteroids in relation to fear, anxiety and psychopathology.
Neurosci. Biobehav. Rev. 25, 117e142.
Korte, S.M., de Boer, S.F., de Kloet, E.R., Bohus, B., 1995. Anxiolytic-like effects of
selective mineralocorticoid and glucocorticoid antagonists on fear-enhanced
behavior in the elevated plus-maze. Psychoneuroendocrinology 20, 385e394.
Korte, S.M., Korte-Bouws, G.A., Koobm, G.F., De Kloet, E.R., Bohus, B., 1996. Miner-
alocorticoid and glucocorticoid receptor antagonists in animal models of
anxiety. Pharmacol. Biochem. Behav. 54, 261e267.
Krishnan, V., Han, M.H., Graham, D.L., Berton, O., Renthal, W., Russo, S.J., Laplant, Q.,
Graham, A., Lutter, M., Lagace, D.C., Ghose, S., Reister, R., Tannous, P., Green, T.A.,
Neve, R.L., Chakravarty, S., Kumar, A., Eisch, A.J., Self, D.W., Lee, F.S.,
Tamminga, C.A., Cooper, D.C., Gershenfeld, H.K., Nestler, E.J., 2007. Molecular
adaptations underlying susceptibility and resistance to social defeat in brain
reward regions. Cell 131, 391e404.
Krömer, S.A., Kessler, M.S., Milfay, D., Birg, I.N., Bunck, M., Czibere, L., Panhuysen, M.,
Pütz, B., Deussing, J.M., Holsboer, F., Landgraf, R., Turck, C.W., 2005. Identifica-
tion of glyoxalase-I as a protein marker in a mouse model of extremes in trait
anxiety. J. Neurosci. 25, 4375e4384.
Landgraf, R., Wigger, A., 2002. High vs low anxiety-related behavior rats: an animal
model of extremes in trait anxiety. Behav. Genet. 32, 301e314.
Landgraf, R., Wigger, A., Holsboer, F., Neumann, I.D., 1999. Hyper-reactive
hypothalamo-pituitary-adrenocortical axis in rats bred for high anxiety-related
behaviour. J. Neuroendocrinol. 11, 405e407.
Linthorst, A.C., Flachskamm, C., Barden, N., Holsboer, F., Reul, J.M., 2000. Gluco-
corticoid receptor impairment alters CNS responses to psychological stressor:
an in vivo microdialysis study in transgenic mice. Eur. J. Neurosci. 12, 283e291.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-
time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402e428.
McLaughlin, J.P., Land, B.B., Li, S., Pintar, J.E., Chavkin, C., 2006. Prior activation of
kappa opioid receptors by U50,488 mimics repeated forced swim stress to
potentiate cocaine place preference conditioning. Neuropsychopharmacology
31, 787e794.
Morellini, F., Schachner, M., 2006. Enhanced novelty-induced activity, reduced
anxiety, delayed resynchronization to daylight reversal and weaker muscle
strength in tenascin-C-deficient mice. Eur. J. Neurosci. 23, 1255e1268.
Muigg, P., Scheiber, S., Salchner, P., Bunck, M., Landgraf, R., Singewald, N., 2009.
Differential stress-induced neuronal activation patterns in mouse lines selec-
tively bred for high, normal or low anxiety. PLoS One 4 e5346.
Musazzi, L., Milanese, M., Farisello, P., Zappettini, S., Tardito, D., Barbiero, V.S.,
Bonifacino, T., Mallei, A., Baldelli, P., Racagni, G., Raiteri, M., Benfenati, F.,
Bonanno, G., Popoli, M., 2010. Acute stress increases depolarization-evoked
glutamate release in the rat prefrontal/frontal cortex: the dampening action
of antidepressants. PLoS One 5 e8566.
Myers, B., Greenwood-Van Meerveld, B., 2007. Corticosteroid receptor-mediated
mechanisms in the amygdala regulate anxiety and colonic sensitivity. Am. J.
Physiol. Gastrointest. Liver. Physiol. 292, G1622eG1629.
Nesse, R.M., 1999. Proximate and evolutionary studies of anxiety, stress and
depression: synergy at the interface. Neurosci. Biobehav. Rev. 23, 895e903.
O’Leary, T.A., Rohsenow, D.J., Martin, R., Colby, S.M., Eaton, C.A., Monti, P.M., 2000.
The relationship between anxiety levels and outcome of cocaine abuse treat-
ment. Am. J. Drug Alcohol Abuse 26, 179e194.
View publication stats
1305
Redila, V.A., Chavkin, C., 2008. Stress-induced reinstatement of cocaine seeking is
mediated by kappa opioid system. Psychopharmacology 200, 59e70.
Rochford, J., Beaulieu, S., Rousse, I., Glowa, J.R., Barden, N., 1997. Behavioral reactivity
to aversive stimuli in a transgenic mouse model of impaired glucocorticoid
(type II) receptor function: effects of diazepam and FG-7142. Psychopharma-
cology (Berl) 132, 145e152.
Salchner, P., Sartori, S.B., Sinner, C., Wigger, A., Frank, E., Landgraf, R., Singewald, N.,
2006. Airjet and FG-7142-induced fos expression differs in rats selectively
bred for high and low anxiety-related behavior. Neuropharmacology 50,
1048e1058.
Salomé, N., Salchner, P., Viltart, O., Sequeira, H., Wigger, A., Landgraf, R.,
Singewald, N., 2004. Neurobiological correlates of high (HAB) versus low
anxiety-related behavior (LAB): differential Fos expression in HAB and LAB rats.
Biol. Psychiatry 55, 715e723.
Sapolsky, R.M., Zola-Morgan, S., Squire, L.R., 1991. Inhibition of glucocorticoid
secretion by the hippocampal formation in the primate. J. Neurosci. 11,
3695e3704.
Schwartz, W.J., Zimmerman, P., 1990. Circadian timekeeping in BALB/c and C57BL/6
inbred mouse strains. J. Neurosci. 10, 3685e3694.
Sim, Y.B., Park, S.H., Kang, Y.J., Kim, S.M., Lee, J.K., Jung, J.S., Suh, H.W., 2010. The
regulation of blood glucose level in physical and emotional stress models:
possible involvement of adrenergic and glucocorticoid systems. Arch. Pharm.
Res. 33, 1679e1683.
Smith, J.P., Book, S.W., 2010. Comorbidity of generalized anxiety disorder and
alcohol use disorders among individuals seeking outpatient substance abuse
treatment. Addict. Behav. 35, 42e45.
Smythe, J.W., Murphy, D., Timothy, C., Costall, B., 1997. Hippocampal mineralocor-
ticoid, but not glucocorticoid, receptors modulate anxiety-like behavior in rats.
Pharmacol. Biochem. Behav. 56, 507e513.
Teixeira-Silva, F., Dias Antunes, F., Santos Silva, P.R., Goes, T.C., Dantas, E.C.,
Santiago, M.F., Machado de Andrade, R., 2009. The free-exploratory paradigm as
a model of trait anxiety in rats: test-retest reliability. Physiol. Behav. 96,
729e734.
Troisi, A., 2005. The concept of alternative strategies and its relevance to psychiatry
and clinical psychology. Neurosci. Biobehav. Rev. 29, 159e168.
Tronche, F., Kellendonk, C., Kretz, O., Gass, P., Anlag, K., Orban, P.C., Bock, R., Klein, R.,
Schütz, G., 1999. Disruption of the glucocorticoid receptor gene in the nervous
system results in reduced anxiety. Nat. Genet. 23, 99e103.
Tzschentke, T.M., 1998. Measuring reward with the conditioned place preference
paradigm: a comprehensive review of drug effects, recent progress and new
issues. Prog. Neurobiol. 56, 613e672.
Webster, M.J., Knable, M.B., O’Grady, J., Orthmann, J., Weickert, C.S., 2002. Regional
specificity of brain glucocorticoid receptor mRNA alterations in subjects with
schizophrenia and mood disorders. Mol. Psychiatry 7, 985e994.
Wei, Q., Lu, X.Y., Liu, L., Schafer, G., Shieh, K.R., Burke, S., Robinson, T.E., Watson, S.J.,
Seasholtz, A.F., Akil, H., 2004. Glucocorticoid receptor overexpression in fore-
brain: a mouse model of increased emotional lability. Proc. Natl. Acad. Sci. U S A
101, 11851e11856.
Wust, S., Van Rossum, E.F., Federenko, I.S., Koper, J.W., Kumsta, R., Hellhammer, D.H.,
2004. Common polymorphisms in the glucocorticoid receptor gene are asso-
ciated with adrenocorticalresponses to psychosocial stress. J. Clin. Endocrinol.
Metab. 89, 565e573.
Xu, D., Buehner, A., Xu, J., Lambert, T., Nekl, C., Nielsen, M.K., Zhou, Y., 2006.
A polymorphic glucocorticoid receptor in a mouse population may explain
inherited altered stress response and increased anxiety-type behaviors. FASEB J.
20, 2414e2416.
Zozulya, A.A., Gabaeva, M.V., Sokolov, O.Y., Surkina, I.D., Kost, N.V., 2008. Personality,
coping style, and constitutional neuroimmunology. J. Immunotoxicol. 5, 221e225.