Psychoneuroendocrinology (2014) 43, 114125

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Mineralocorticoid receptors in the ventral

tegmental area regulate dopamine efflux in
the basolateral amygdala during the
expression of conditioned fear
Amanda R. de Oliveira a,b,*, Adriano E. Reimer a,b,
Marcus L. Brandao a,b

a
Laboratorio de Psicobiologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto,
Universidade de Sao Paulo, Ribeirao Preto, SP, Brazil
b
Instituto de Neurociencias e Comportamento (INeC), Ribeirao Preto, SP, Brazil

Received 18 November 2013; received in revised form 20 January 2014; accepted 10 February 2014

KEYWORDS Summary Despite the recognized involvement of corticosteroids in the modulation of emotional
Fear conditioning; behavior, the specific role of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs) in
Mineralocorticoid the expression of conditioned fear responses is still open to investigation. The present study sought to
receptors; clarify the involvement of both types of corticosteroid receptors in two different brain regions the
Glucocorticoid receptors; ventral tegmental area (VTA) and the basolateral amygdala complex (BLA) on the expression of
Ventral tegmental area; conditioned fear. The first experiment assessed the effects of intra-VTA or intra-BLA administration of
Basolateral amygdala spironolactone (MR antagonist) or mifepristone (GR antagonist) on the expression of conditioned
complex; freezingtoalight-CSandonmotorperformanceintheopen-fieldtest.Intra-VTAspironolactone,butnot
Dopamine mifepristone, attenuated the expression of the conditioned freezing response whereas intra-BLA
spironolactone or mifepristone had no significant effects. These treatments did not affect motor
performance in the open-field test. Since dopamine is released in the BLA from the VTA during the
expressionofconditionedfear,theanxiolytic-likeeffectofdecreasedcorticosteroidactivityinthefirst
experiment could be associated with changes in dopaminergic neurotransmission. The second experi-
ment, using in vivo microdialysis, investigated the role of MRs in the VTA on dopamine levels in the BLA
during the expression of conditioned fear. Blocking MRs locally in the VTAwith spironolactone reduced
dopamine efflux in the BLA and decreased the expression of conditionedfreezing in response to the CS.
Taken together, the data indicate that corticosterone, acting locally on MRs in the VTA, stimulates
dopamine efflux in the BLA, which facilitates the expression of conditioned freezing to a light-CS.
# 2014 Elsevier Ltd. All rights reserved.

* Corresponding author at: Laboratorio de Psicobiologia, Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao
Paulo. Av. Bandeirantes, 3900, Ribeirao Preto. SP 14090-901, Brazil. Tel.: +55 16 3602 3838; fax: +55 16 3602 4830.
E-mail address: arobio@usp.br (A.R. de Oliveira).

http://dx.doi.org/10.1016/j.psyneuen.2014.02.010
0306-4530/# 2014 Elsevier Ltd. All rights reserved.
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear
1. Introduction
Considering the complexity of aversive information proces-
sing and defensive response expression, a combined action of
several stress mediators may be required for optimal per-
formance during threatening situations. With specific regard
to fear conditioning, much research has been performed
elucidating the involvement of distinct mediators during
its acquisition and consolidation phases, but comparatively
less is known about the retrieval and expression of condi-
tioned fear memories (Lupien and McEwen, 1997; Rodrigues
et al., 2009). Taking into account the adaptive importance of
previous experience retrieval for the expression of appro-
priate defensive responses, studying the neural substrates
and mediators involved in these processes is of great interest,
especially because of their relevance to different aspects of
human anxiety disorders.
The hypothalamic-pituitary-adrenocortical (HPA) axis
activity, which leads to the release into the bloodstream
of corticosteroids (cortisol in primates, corticosterone in
rodents), has been considered a key part of the stress reac-
tion and can be triggered either by innate or conditioned
aversive stimuli (Cordero et al., 1998; Reis et al., 2012).
Corticosteroids are hormones that can easily pass the blood
brain barrier, thus affecting a variety of fear-related brain
areas (McEwen et al., 1969; Stevens et al., 1971). In the
brain, corticosteroids bind to two types of receptors: miner-
alocorticoids (MRs) and glucocorticoids (GRs) (Reul and de
Kloet, 1986; Lu et al., 2006). Despite the recognized involve-
ment of corticosteroids in modulating emotional behavior,
the specific role of MRs and GRs in the expression of condi-
tioned fear responses is still open to investigation. The
present study sought to clarify the possible involvement of
both types of corticosteroid receptors in two different fear-
related brain regions the ventral tegmental area (VTA) and
the basolateral amygdala complex (BLA) on the expression
of conditioned fear. Previous studies confirmed that both MRs
and GRs are present in VTA and BLA neurons (Harfstrand
et al., 1986; Ronken et al., 1994; Johnson et al., 2005). So,
the first experiment assessed the effects of intra-VTA or
intra-BLA administration of spironolactone (MR antagonist)
or mifepristone (GR antagonist) on the expression of condi-
tioned freezing to a light-CS and on motor performance in the
open-field test.
Recently, several laboratories have shown great interest
in the interaction between the activation of the HPA axis and
dopaminergic neurotransmission during aversive states (Barr
et al., 2009; Sapolsky, 2009). Dopamine, although more
commonly associated with the reinforcing effects of various
stimuli, is one of the most active neuromodulators of fear and
anxiety (Reis et al., 2004; de Oliveira et al., 2006; Fadok et al.,
2009; Zweifel et al., 2011). In fact, dopamine is released in the
BLA (consisting of the lateral, basal and accessory basal
nuclei) from neurons of the VTA during the expression of
conditioned fear (de Oliveira et al., 2011, 2013). Further-
more, quinpirole (a dopamine D2 receptor agonist) targeting
autoreceptors in the VTA or microinjections of sulpiride (a
dopamine D2 receptor antagonist) into the BLA decrease the
expression of conditioned fear responses (de Oliveira et al.,
2009, 2011; de Souza Caetano et al., 2013). These findings
suggest that reducing the activity of dopaminergic neurons in
115
the VTA-BLA pathway reduces conditioned fear. However, the
neurohumoral mechanisms involved in regulating the dopa-
mine efflux in the VTA-BLA pathway triggered by a CS remain
to be clarified.
In an attempt to determine the extent to which the
combined action of the HPA axis and dopaminergic neuro-
transmission is important for the expression of conditioned
fear responses, we observed in a previous study that systemic
administration of metyrapone (a corticosterone synthesis
blocker) prevented enhanced dopamine release in the BLA
during a conditioned fear test and decreased the expression
of conditioned freezing (de Oliveira et al., 2013). Thus, HPA
axis activation seems to be an important step in an integrated
neuroendocrineneurochemicalbehavioral response when
the organism evaluates and interprets the threat associated
with a specific environmental stimulus and subsequently
triggers adaptive defense reactions to cope with this situa-
tion. To further clarify this issue, in the second part of the
present study using in vivo microdialysis, we examined the
influence of MRs in the VTA on modulating the release of
dopamine in the BLA during the expression of conditioned
fear.
2. Methods
2.1. Animals
One-hundred and forty naive male Wistar rats from the
animal facility of the Campus of the University of Sao Paulo
at Ribeirao Preto were used. The rats, weighing 270290 g at
the beginning of the experiments, were housed in groups of
four in plastic boxes (40 cm  33 cm  26 cm) and main-
tained under controlled conditions (23  1 8C; 12 h/12 h
light/dark cycle, lights on at 0700 h) with food and water
freely available. The experiments were carried out during
the light phase of the cycle. All the procedures were
approved by the Committee for Animal Care and Use of
the University of Sao Paulo at Ribeirao Preto (No.
10.1.595.53.7), and were performed in compliance with
the recommendations of the Brazilian Society of Neu-
roscience and Behavior, which are based on the National
Institutes of Health Guide for the Care and Use of Laboratory
Animals. All efforts were made to minimize animal suffering,
to reduce the number of animals used, and to utilize alter-
natives to in vivo techniques, if available.
2.2. Surgery
The rats were anesthetized with ketamine/xylazine (100/
7.5 mg/kg, intraperitoneal; Agener Uniao, Embu-Guacu, SP,
Brazil) and fixed in a stereotaxic frame (David Kopf Instru-
ments, Tujunga, CA, USA). The incisor bar was set at 3.0 mm
below the interaural line, such that the skull was horizontal
between bregma and lambda. For experiment 1, bilateral
guide cannulae for drug injections (0.6 mm outer diameter,
0.4 mm inner diameter) were stereotaxically implanted over
the VTA or the BLA. For experiment 2, unilateral VTA cannu-
lation for drug injection and unilateral BLA cannulation for
the microdialysis probe (CMA/12; CMA/Microdialysis AB,
Solna, Sweden) were performed in the right hemisphere.
In previous studies, we showed that microdialysis with a
116
probe implanted unilaterally into the BLA accurately mea-
sures changes in the extracellular concentrations of dopa-
mine in this structure (de Oliveira et al., 2011, 2013). Taking
bregma as the reference point, the following coordinates
were used (Paxinos and Watson, 2007): VTA (anterior/poster-
ior, 5.9 mm; medial/lateral, 0.7 mm; dorsal/ventral,
7.6 mm) and BLA (anterior/posterior, 2.3 mm; medial/
lateral, 5.5 mm; dorsal/ventral, 7.0 mm). The cannulae
were fixed to the skull with acrylic resin and stainless steel
screws. In addition, each guide cannula was sealed with a
stainless steel wire to protect it from blockage. At the end of
surgery, the rats received an intramuscular injection of a
polyvalent veterinary antibiotic (Pentabiotico, 0.2 ml; Fort
Dodge, Campinas, SP, Brazil) and a subcutaneous injection of
the anti-inflammatory and analgesic flunixin meglumine
(Banamine, 2.5 mg/kg; Schering-Plough, Cotia, SP, Brazil).
Afterwards, the rats were allowed 5 days to recover from the
surgical procedure.
2.3. Drugs
The following drugs were used: the corticosteroid MR antago-
nist spironolactone and the corticosteroid GR antagonist
mifepristone. Spironolactone and mifepristone (Sigma; St.
Louis, MO, USA) were dissolved initially in 100% DMSO, kept in
a stock solution at 70 8C, and then diluted in saline shortly
before use. The final DMSO concentration was 1%. Both drugs
were microinjected into the VTA or BLA (5 or 10 ng/0.2 ml/
site) 10 min before the test session. The drugs, doses, and
injection times were based on previous studies (Roozendaal
and McGaugh, 1997; Conrad et al., 2004; Yang et al., 2006).
Each treatment group had its own control group that received
an injection of saline + DMSO (1%) 10 min before the test
session.
2.4. Microinjection procedure
The injection needle was a thin dental needle (0.3 mm outer
diameter) connected to a 10 ml syringe (Hamilton Company,
Reno, NV, USA) by means of a polyethylene tube (PE-10;
BectonDickinson, Franklin Lakes, NJ, USA). The injection
needle was introduced through the guide cannula until its
lower end reached 1 mm below the cannula. The solutions
were injected into the VTA or BLA using an infusion pump
(Harvard Apparatus, South Natick, MA, USA). The displace-
ment of an air bubble inside the polyethylene catheter
connecting the syringe needle to the intracerebral needle
was used to monitor the microinjection. The microinjection
lasted 1 min and the needle was held in place for an addi-
tional 1 min to maximize diffusion from the needle tip.
2.5. Experiment 1: conditioned fear and open-
field tests
2.5.1. Conditioned fear
Training: The rats were conditioned to a light-CS in a cage
(20 cm  20 cm  25 cm) with stainless steel side and back
walls, a transparent Plexiglas ceiling and front door, and a grid
floor that consisted of stainless-steel rods spaced 1.0 cm apart.
This cage was located within a ventilated and sound-attenu-
ated chamber (45 cm  45 cm  45 cm). After recovery from
A.R. de Oliveira et al.
surgery, the rats were placed in the training cage and received
10 CS-US pairings after a habituation phase of 5 min using a 4 s,
6 W light-CS that coterminated with a 1 s, 0.6 mA footshock-
US. The footshocks were delivered through the training cage
floor by a constant current generator built with a scrambler
(Albarsh Instruments, Porto Alegre, RS, Brazil). The intertrial
interval varied randomly between 60 and 180 s. Stimulus
presentation was controlled by a microprocessor and an
input/output board (Insight Equipment, Ribeirao Preto, SP,
Brazil). Each animal was removed 2 min after the last foot-
shock and returned to its home cage. The duration of the
training session was approximately 25 min, including habitua-
tion time.
Testing: The conditioned fear test was conducted with-
out footshock presentation in a wire-grid cage (16.5 cm 
7.5 cm  7.5 cm) different from the training cage to avoid
conditioning to the context and located inside a ventilated,
sound-attenuating plywood chamber (64 cm  60 cm 
40 cm). Twenty-four hours after training, the rats received
intra-VTA or intra-BLA administration of spironolactone,
mifepristone or vehicle and, after 10 min, were placed in
the test cage. After a habituation phase of 5 min, the rats
received 10 CS presentations (4 s, 6 W light-CS). The dura-
tion of the test session, including habituation time, was
20 min. The behavior of the rats was recorded by a video
camera positioned behind the observation chamber with
the signal relayed to a monitor in another room via a closed
circuit. The behavioral measure that was used to assess
conditioned fear was the time that rats spent freezing
during this test session. Freezing was operationally defined
as the total absence of movement of the body and vibrissae,
except those required for respiration, for at least 6 s.
Freezing behavior was monitored during the test and sub-
sequently scored from video recordings by a person who
was blind with regard to the experimental condition of each
rat. The results are presented as total time rats spent
freezing during the test session. Each rat was subjected
to only one treatment and to a single conditioned fear test
session.
2.5.2. Open-field
The same rats used for the conditioned fear experiment were
also used to assess the effects of the drug treatments on
motor activity in the open-field test. This experiment was
conducted in an arena consisting of a circular enclosure made
of transparent Plexiglas (60 cm in diameter and 50 cm
height). The arena was located in a room with a controlled
and indirect illumination of 30 lux on the floor of the appa-
ratus. The rats behavior was recorded by a video camera
positioned above the arena. Two days after the conditioning
testing session, the rats received intra-VTA or intra-BLA
administration of spironolactone, mifepristone or vehicle.
Ten min after drug injection, rats were placed in the middle
of the arena and left for a 20 min period of free exploration.
The following behavioral responses were recorded over the
course of the session: total distance traveled, distance tra-
veled in the border and in the center of the arena, time spent
in the border and in the center of the arena, and total
immobility time. Motor activity was monitored during the
test and subsequently scored automatically from video
recordings using ANY-maze software (version 4.7; Stoelting,
Wood Dale, IL, USA).
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear
2.6. Experiment 2: conditioned fear and in vivo
microdialysis
Training: For this experiment, additional groups of rats were
subjected to fear conditioning to a light-CS as described for
experiment 1.
Testing: The next day, the rats were placed in a micro-
dialysis bowl (BAS, West Lafayette, IN, USA) and a probe
(2 mm membrane; CMA/Microdialysis AB) was inserted into
the BLA and perfused with Ringers solution (147.0 mM NaCl,
4.0 mM KCl, and 2.2 mM CaCl2) at a constant flow rate of
1.5 ml/min (Microinjection pump; BAS). Following a 2 h equi-
librium period, ten dialysate samples (four during baseline,
one during the test, five post-test) were collected every
30 min into vials that contained 3 ml of 0.05 M perchloric
acid solution. After the baseline samples were collected, the
rats received intra-VTA administration of spironolactone or
vehicle and were transferred to a Plexiglas cage
(20 cm  23 cm  31 cm) for the conditioned fear test. In
this cage, after a 10 min habituation period, over the course
of the 20 min test session, the 4 s light-CS was presented 10
times with an interstimulus interval of 60180 s. The beha-
vioral criterion used to assess conditioned fear was again the
time rats spent freezing. At the end of the test session, the
rats were placed back in the microdialysis bowl and dialy-
sates were collected for an additional 150 min. The mean of
the four baseline samples served as the reference for all
analyses, which were therefore expressed as percent
changes. Each rat was subjected to only one treatment
and to a single conditioned fear test session.
Dopamine assay: The amount of dopamine in the dialy-
sates was analyzed using high-performance liquid chroma-
tography (HPLC). The reverse-phase ODS column was a
HyperClone 150 mm  2.0 mm C-18 with a 3 mm particle
size (Phenomenex, Torrance, CA, USA). The HPLC device
consisted of a BAS Epsilon electrochemical detector with a
glass-carbon electrode and pump (PM-92e). The potential
was set at 650 mV (compared with the Ag-AgCl reference
electrode). The mobile phase, consisting of 50 mM NaH2PO4,
0.1 mM Na2-EDTA, 0.5 mM n-octyl sodium sulfate, and 10%
methanol (pH 5.5), was filtered and pumped through the
system at a flow rate of 200 ml/min. The injection volume
was 50 ml. This set-up allowed the dopamine levels in each
sample to be analyzed in a run that lasted approximately
12 min.
2.7. Histology
Upon the conclusion of the experiments, in order to confirm
the position of the microinjection and microdialysis sites, the
rats were given a lethal dose of urethane (3 g/kg; Sigma
Aldrich, St. Louis, MO, USA) and 0.2 ml of Evans Blue (2%) was
microinjected into the VTA or BLA. Afterwards, the rats were
perfused transcardially with 0.9% saline and 4% paraformal-
dehyde solutions. The brains were removed from the skulls,
maintained in paraformaldehyde for 2 h, and then cryopro-
tected in 30% sucrose until soaked. Coronal 60 mm slices were
cut, mounted on gelatin-coated slides, and stained with
cresyl violet (5%), allowing localization of the microinjection
and probe sites according to the atlas of Paxinos and Watson
(2007).
117
2.8. Statistical analysis
The injection and/or microdialysis sites of all animals con-
sidered for the statistical analysis in the present study were
located inside the VTA and/or BLA, depending on the experi-
ment. The data are reported as mean  S.E.M. For experi-
ment 1, the conditioned freezing response and activity in the
open-field test were subjected to one-way analysis of var-
iance (ANOVA). For experiment 2, the conditioned freezing
response was subjected to a Students t-test. Two-way
repeated-measures ANOVA was used for the microdialysis
data, with treatment (drug and its control) as the
between-subjects factor, and time of sample collection as
the within-subjects factor. Significant comparisons were fol-
lowed by the NewmanKeuls post hoc test. The significance
level was set at p < 0.05.
3. Results
3.1. Experiment 1: conditioned fear and open-
field tests
Only rats with microinjection sites located bilaterally within
the VTA were included in this part of the study. We had 17
animals with cannulae outside the VTA. Since these rats were
distributed in six distinct groups of the present experiment,
the number of rats with misplaced cannulae per group was
not sufficient for entering into the statistical analysis. Fig. 1A
shows a photomicrograph of representative microinjection
sites in the VTA. Fig. 1B shows the histological localization of
the injection sites in the VTA, depicted on diagrams from the
atlas of Paxinos and Watson (2007).
Fig. 2A depicts the timeline of the procedures looking at
VTA MR or GR involvement in the expression of conditioned
freezing response and motor activity. Fig. 2B shows the mean
freezing response for rats that received intra-VTA microin-
jection of vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR
antagonist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/
site spironolactone (Spiro10; n = 10). One-way ANOVA
revealed significant effects of the treatments on the condi-
tioned freezing response (F 2,27 = 3.98, p < 0.05). The New-
manKeuls post hoc test revealed that 10 ng/0.2 ml/site
spironolactone significantly reduced the duration of the
conditioned freezing compared with the control group
(p < 0.05). Fig. 2C and D displays the data obtained with
rats that received intra-VTA vehicle (control; n = 7), 5 ng/
0.2 ml/site spironolactone (Spiro5; n = 7) or 10 ng/0.2 ml/site
spironolactone (Spiro10; n = 7) in the open-field test. One-
way ANOVA did not reveal significant effects of the treat-
ments on the total distance traveled (F 2,18 = 1.27, p > 0.05)
or total time spent immobile (F 2,18 = 0.38, p > 0.05). One-
way ANOVA also showed no significant effects on the distance
traveled in the border (F 2,18 = 1.68, p > 0.05) or in the center
of the arena (F 2,18 = 0.4, p > 0.05), or on time spent in the
border (F 2,18 = 0.09, p > 0.05) or in the center of the arena
(F 2,18 = 0.72, p > 0.05) data not shown graphically. Fig. 2E
shows the mean freezing response for rats that received
intra-VTA microinjection of vehicle (control; n = 10), 5 ng/
0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10)
or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10). One-way
ANOVA revealed no significant effects of the treatments on
118
Figure 1 Target sites for microinjection into the ventral teg-
mental area (VTA). Photomicrograph showing representative
microinjection sites in the VTA (A). Outline of microinjection
locations on cross-sections from the Paxinos and Watson atlas
(2007) (B). The number of points in the figure is less than the
total number of rats used because of several overlaps. Scale
bar = 0.5 mm. DMPAG, dorsomedial periaqueductal gray; CLi,
caudal linear nucleus of the raphe; Pn, pontine nuclei.
the conditioned freezing response (F 2,27 = 2.04, p > 0.05).
Fig. 2F and G displays the data from the rats treated with
intra-VTA vehicle (control; n = 7), 5 ng/0.2 ml/site mifepris-
tone (Mifep5; n = 7) or 10 ng/0.2 ml/site mifepristone
(Mifep10; n = 7) in the open-field test. One-way ANOVA did
not reveal significant effects of the treatments on the total
distance traveled (F 2,18 = 2.12, p > 0.05) or total time spent
immobile (F 2,18 = 1.84, p > 0.05). One-way ANOVA also
revealed no significant effects on the distance traveled in
the border (F 2,18 = 2.75, p > 0.05) or in the center of the
arena (F 2,18 = 0.08, p > 0.05), or on time spent in the border
(F 2,18 = 2.21, p > 0.05) or in the center of the arena
(F 2,18 = 1.08, p > 0.05) data not shown graphically.
Only rats with microinjection sites located bilaterally
within the BLA were included in the next part of the study.
We had 20 animals with cannulae outside the BLA. Since these
rats were distributed in six distinct groups, the number of
rats with misplaced cannulae per group was not sufficient for
entering into the statistical analysis. Fig. 3A shows a photo-
micrograph of representative microinjection sites in the BLA.
Fig. 3B shows the histological localization of the injection
sites in the BLA, depicted on diagrams from the atlas of
Paxinos and Watson (2007).
Fig. 4A displays the timeline of the procedures looking at
BLA MR or GR involvement in the expression of conditioned
A.R. de Oliveira et al.
freezing and motor activity. Fig. 4B shows the mean freezing
response for rats that received intra-BLA microinjection of
vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antago-
nist spironolactone (Spiro5; n = 10) or 10 ng/0.2 ml/site spir-
onolactone (Spiro10; n = 10). One-way ANOVA revealed no
significant effects of the treatments on the conditioned
freezing response (F 2,27 = 0.16, p > 0.05). Fig. 4C and D
shows the data for the rats treated with intra-BLA vehicle
(control; n = 7), 5 ng/0.2 ml/site spironolactone (Spiro5;
n = 7) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 7) in
the open-field test. One-way ANOVA did not reveal significant
effects of the treatments on the total distance traveled
(F 2,18 = 0.09, p > 0.05) or total time spent immobile
(F 2,18 = 0.52, p > 0.05). One-way ANOVA also showed no
significant effects on the distance traveled in the border
(F 2,18 = 0.21, p > 0.05) or in the center of the arena
(F 2,18 = 0.22, p > 0.05), or on time spent in the border
(F 2,18 = 0.85, p > 0.05) or in the center of the arena
(F 2,18 = 1.05, p > 0.05) data not shown graphically.
Fig. 4E shows the mean freezing response for rats that
received intra-BLA microinjection of vehicle (control;
n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone
(Mifep5; n = 10) or 10 ng/0.2 ml/site mifepristone (Mifep10;
n = 10). One-way ANOVA revealed no significant effects of the
treatments on the conditioned freezing response
(F 2,27 = 0.19, p > 0.05). Fig. 4F and G shows the data from
the rats treated with intra-BLA vehicle (control; n = 7), 5 ng/
0.2 ml/site mifepristone (Mifep5; n = 7) or 10 ng/0.2 ml/site
mifepristone (Mifep10; n = 7) in the open-field test. One-way
ANOVA did not reveal significant effects of the treatments on
the total distance traveled (F 2,18 = 0.55, p > 0.05) or total
time spent immobile (F 2,18 = 1.80, p > 0.05). One-way
ANOVA also showed no significant effects on the distance
traveled in the border (F 2,18 = 0.02, p > 0.05) or in the center
of the arena (F 2,18 = 2.29, p > 0.05), or on time spent in the
border (F 2,18 = 2.44, p > 0.05) or in the center of the arena
(F 2,18 = 3.57, p > 0.05) data not shown graphically.
3.2. Experiment 2: conditioned fear and in vivo
microdialysis
Only rats with microinjection and probe sites located within
the VTA and BLA, respectively, were included in this experi-
ment. Five animals with misplaced cannulae were not con-
sidered for the statistical analysis. Photomicrographs of
representative microinjection and probe sites into the VTA
and BLA are shown in Fig. 5A and B. Fig. 5C and D displays the
histological localization of the injection sites in the VTA and
probe sites in the BLA, depicted on diagrams from the atlas of
Paxinos and Watson (2007).
Fig. 6A displays the procedures timeline for testing the
effects of intra-VTA administration of the MR antagonist
spironolactone on extracellular dopamine concentration in
the BLA and the conditioned freezing response. Fig. 6B shows
the time-course of extracellular dopamine concentration in
the BLA in rats subjected to the conditioned fear test and
treated with vehicle (control; n = 10) or 10 ng/0.2 ml spiro-
nolactone (Spiro10; n = 10). Two-way repeated-measures
ANOVA revealed no significant effect of treatments
(F 1,162 = 0.06, p > 0.05), but a significant effect of time
(F 10,199 = 3.78, p < 0.05) and a significant treatments  time
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear 119

A Conditioned Fear
Drugs Drugs
(intra-VTA) (intra-VTA)
Surgery Training Testing Open-Field Histology
5 days 24 h 2 days
25 min 20 min 20 min
400 30 900
Conditioned Freezing (s)

B C D

Distance Travelld (m)

300

Imobility (s)
20 600
200
10 300
100 *
0 0 0
Control Spiro5 Spiro10 Control Spiro5 Spiro10 Control Spiro5 Spiro10

400 30 900
G
Conditioned Freezing (s)

E F

Distance Travelld (m)

300

Imobility (s)
20 600
200
10 300
100

0 0 0
Control Mifep5 Mifep10 Control Mifep5 Mifep10 Control Mifep5 Mifep10

Figure 2 Mineralocorticoid (MRs) and glucocorticoid (GRs) receptors in the ventral tegmental area (VTA) and conditioned fear.
Timeline of the procedures looking at VTA MR and GR involvement in the expression of conditioned fear and motor activity (A). Time of
freezing in rats that received intra-VTA vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5; n = 10) or
10 ng/0.2 ml/site spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Motor activity of rats treated
with intra-VTA vehicle (control; n = 7), 5 ng spironolactone (Spiro5; n = 7) or 10 ng spironolactone (Spiro10; n = 7) (C and D). Time of
freezing in rats that received intra-VTA vehicle (control; n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10) or
10 ng/0.2 ml/site mifepristone (Mifep10; n = 10) and were subjected to the conditioned fear test (E). Motor activity of rats treated with
intra-VTA vehicle (control; n = 7), 5 ng mifepristone (Mifep5; n = 7) or 10 ng mifepristone (Mifep10; n = 7) (F and G). *p < 0.05, different
from control.

time interaction (F 10,199 = 2.41, p < 0.05). The Newman
Keuls post hoc test revealed an increase in dopamine con-
centration in the BLA of control rats during the conditioned
fear test compared with baseline (p < 0.05). NewmanKeuls
post hoc comparison also showed that intra-VTA spironolac-
tone blocked this increase in dopamine concentration in the
BLA (p < 0.05). Fig. 6C shows the conditioned freezing
response for the same rats for the 20 min test period that
corresponded to the fifth microdialysis sample. Students t-
test revealed that intra-VTA spironolactone significantly
decreased the conditioned freezing response (t18 = 2.42,
p < 0.05), as seen in our first experiment.
4. Discussion
Conditioned freezing is the main response to cues associated
with footshock and is a widely used index of conditioned fear
in rodents. In the present study, light-CS consistently induced
a conditioned freezing response during the test session in all
control rats. This behavioral result shows the efficacy of the
conditioning protocol used here.
The MR antagonist spironolactone decreased the condi-
tioned freezing response when administered intra-VTA
before the test session. Spironolactones effect cannot be
attributed to nonspecific effects since the same dose did not
affect the performance of rats in the open-field test. On the
other hand, intra-VTA microinjection of the GR antagonist
mifepristone, or intra-BLA administration of spironolactone
or mifepristone, caused no significant effects on freezing
behavior. The doses of spironolactone and mifepristone
injected into the VTA and BLA also did not affect motor
performance in the open-field test. In fact, spironolactone
and mifepristone did not alter either locomotor (e.g., total
distance traveled and distance traveled in the border) or
unconditioned fear behaviors, reflected in the distance tra-
veled and time spent in the center of the open-field. There-
fore, it may be suggested that the MR and GR antagonists did
not have a broad anxiolytic-like action when injected intra-
VTA or intra-BLA; however, intra-VTA administration of the
MR antagonist exhibited a specific anxiolytic-like effect in
the conditioned fear test used in the present study. This
difference in the effects of intra-VTA spironolactone may
reflect distinct modulatory roles of corticosteroids in differ-
ent defensive responses and it is in agreement with the idea
that corticosteroids may be more important for cognitive-
related emotional responses than for instinctive fear beha-
viors, as pointed out by previous studies from our group
120
Figure 3 Target sites for microinjection into the basolateral
amygdala complex (BLA). Photomicrograph of representative
microinjection sites in the BLA (A). Outline of microinjection
locations on cross-sections from the Paxinos and Watson atlas
(2007) (B). The number of points in the figure is less than the
total number of rats used because of several overlaps. Scale
bar = 1.0 mm. CA3, CA3 field of the hippocampus; VPM, ventral
posteromedial thalamic nucleus; ec, external capsule; MeP,
medial amygdaloid nuclei.
(Albrechet-Souza et al., 2007; Reis et al., 2012; de Oliveira
et al., 2013).
The intra-VTA spironolactone effect observed here is
consistent with other studies that showed anxiolytic-like
effects of systemic or intracerebroventricular injections of
this MR antagonist on the expression of contextual condi-
tioned freezing responses (Korte et al., 1995; Ninomiya et al.,
2010). As the concentration of corticosteroids may be
increased after spironolactone administration (Ratka
et al., 1989; Otte et al., 2007), we cannot exclude that an
enhanced activation of GRs might have contributed to the
present results. However, this seems unlikely since in our
previous work systemic administration of corticosterone did
not change the expression of conditioned freezing (de Oli-
veira et al., 2013). In fact, we showed in that same study that
blocking corticosterone synthesis with metyrapone impaired
A.R. de Oliveira et al.
the expression of conditioned freezing to a light-CS in rats.
Convergent with this, decreased HPA axis activity has also
been associated with impaired emotional memory in humans,
while no effects were observed on the recall of neutral
information (Marin et al., 2011; Rimmele et al., 2013).
Now, the present data suggest that VTA MRs are involved
in the expression of conditioned fear. The selectivity of the
effect produced by the MR antagonist is further attested by
the lack of effect of pre-testing intra-VTA microinjection of
the GR antagonist on the conditioned freezing response. The
present data also indicate that MRs and GRs in the BLA do not
appear to be important for the expression of conditioned fear
to a light-CS.
The above-mentioned general absence of effects of mife-
pristone has to be interpreted with caution since only two
doses of this GR antagonist were used in the present study.
Higher doses were not used because they could act at recep-
tors other than GRs; for instance, mifepristone may act as a
progesterone antagonist (Gaillard et al., 1984; Lu et al.,
2006). To avoid these side effects, the doses commonly used
in studies showing that mifepristone affected fear under
other experimental conditions do not go beyond the ones
used in the present study (Roozendaal and McGaugh, 1997;
Conrad et al., 2004; Yang et al., 2006). Also, when injected
systemically or intracerebroventricularly, mifepristone did
not affect the expression of conditioned freezing to the
context (Korte et al., 1995; Ninomiya et al., 2010). Another
important point that should be considered is that, although
the classical mechanism of corticosteroid action involves
intracellular receptors that regulate gene expression, the
rapidity with which corticosteroids exert some of their
effects indicates that they also act through nongenomic
mechanisms, possibly by binding to neuronal membrane
receptors (Schumacher, 1990; Orchinik et al., 1991; Lu
et al., 2006; Tasker et al., 2006). Since the GR antagonist
mifepristone alters the nuclear translocation of GRs (Tasker
et al., 2006; Spiga et al., 2011), one possibility is that it may
be specific for genomic mechanisms of receptor signaling,
leaving the membrane action of GRs still free to occur. The
rapidity of the anxiolytic-like effect of the MR antagonist in
the present study also argues against a genomic action,
suggesting that corticosterone, via high affinity MRs located
in the VTA membrane, modulates the expression of condi-
tioned freezing.
MRs and GRs may mediate different effects of corticos-
teroids in fear memory, since they differ in their function,
neuroanatomical distribution, and affinity for corticosterone
(Lupien and McEwen, 1997; Lu et al., 2006). For many years it
was thought that stress-induced changes were mediated via
the low-affinity GRs rather than the MRs. Recently, however,
it has become evident that stress-induced rises in corticos-
terone can also activate MRs located in the neuronal mem-
brane, which lead to rapid nongenomic effects (de Kloet
et al., 2008; Joels et al., 2008). According to this view,
activation of MRs appears to be involved in the fast/adaptive
behavioral response to environmental aversive cues, while
GR-mediated effects promote consolidation of recently
acquired information (Lupien and McEwen, 1997; de Kloet
et al., 2008; Joels et al., 2008). In this way, a blockade or
deficiency in MR activation would make it more difficult for
an individual to discriminate relevant cues from irrelevant
ones.
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear 121

Conditioned Fear
A Drugs Drugs
(intra-BLA) (intra-BLA)
Surgery Training Testing Open-Field Histology
5 days 24 h 2 days
25 min 20 min 20 min

400 30 900
B C D
Conditioned Freezing (s)

Distance Travelld (m)

300

Imobility (s)
20 600

200
10 300
100

0 0 0
Control Spiro5 Spiro10 Control Spiro5 Spiro10 Control Spiro5 Spiro10

400 30 900
Conditioned Freezing (s)

E F G

Distance Travelld (m)

300

Imobility (s)
20 600

200
10 300
100

0 0 0
Control Mifep5 Mifep10 Control Mifep5 Mifep10 Control Mifep5 Mifep10

Figure 4 Mineralocorticoid (MRs) and glucocorticoid (GRs) receptors in the basolateral amygdala complex (BLA) and conditioned
fear. Timeline of the procedures for looking at BLA MR and GR involvement in the expression of conditioned fear and motor activity (A).
Time of freezing in rats that received intra-BLA vehicle (control; n = 10), 5 ng/0.2 ml/site of the MR antagonist spironolactone (Spiro5;
n = 10) or 10 ng/0.2 ml/site spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Motor activity of rats
treated with intra-BLA vehicle (control; n = 7), 5 ng spironolactone (Spiro5; n = 7) or 10 ng spironolactone (Spiro10; n = 7) (CD). Time
of freezing of rats that received intra-BLA vehicle (control; n = 10), 5 ng/0.2 ml/site of the GR antagonist mifepristone (Mifep5; n = 10)
or 10 ng/0.2 ml/site mifepristone (Mifep10; n = 10) and were subjected to the conditioned fear test (E). Motor activity of rats treated
with intra-BLA vehicle (control; n = 7), 5 ng mifepristone (Mifep5; n = 7) or 10 ng mifepristone (Mifep10; n = 7) (F and G).

Altogether, the results of our first experiment suggest an
important role of MRs located in the VTA in modulating the
aversive memory retrieval and conditioned freezing expres-
sion. Since dopamine is released in the BLA from the VTA
during the expression of conditioned fear (de Oliveira et al.,
2011, 2013), the anxiolytic-like effect of decreased HPA axis
activity in the expression of conditioned freezing could be
associated to changes in dopamine levels subsequent to the
binding of corticosterone to MRs in the VTA. In our second
experiment, the role of MRs in the VTA on dopaminergic
neurotransmission in the BLA during the expression of con-
ditioned fear was examined in a combined pharmacological/
neurochemical study using in vivo microdialysis.
With the second experiment, we demonstrated that the
anxiolytic-like effect of intra-VTA spironolactone administra-
tion was associated with changes in dopaminergic efflux in
the BLA. Similar to what we observed in previous studies (de
Oliveira et al., 2011, 2013), rats that received microinjection
of vehicle and were subjected to the conditioned fear test
exhibited an anxiety-like profile, reflected by freezing beha-
vior and increased dopamine release in the BLA. This pattern,
however, was blocked by treatment with intra-VTA spirono-
lactone that prevented the increase in dopamine release in
the BLA during the conditioned fear test and also decreased
the expression of the conditioned freezing response. So,
these results confirm the influence of HPA axis mobilization
upon the expression of conditioned fear. Immediate corti-
costerone release in response to a stressful situation appears
to play a stimulating role in dopaminergic mechanisms in the
BLA via MRs in the VTA, facilitating the expression of adaptive
responses to cues that signal threatening conditions. Overall,
the present data suggest that interference with the ability of
the fear-evoking CS to activate VTA-BLA dopaminergic neu-
rons modulated by the action of corticosteroids on MRs in the
VTA is associated with a reduction in the expression of
conditioned fear.
A relationship between corticosteroids and dopamine has
already been demonstrated. For example, intraperitoneal
administration of metyrapone inhibited the increase in dopa-
mine levels in the BLA in response to an aversive light-CS (de
Oliveira et al., 2013). Also, adrenalectomy inhibited the
increase in dopamine in the prefrontal cortex induced by
restraint stress (Imperato et al., 1989) and prevented the
occurrence of behavioral sensitization to amphetamine
(Rivet et al., 1989). The VTA is a major source of dopamine
in the brain and it is known that aversive CS elicits an
activation of VTA forebrain projections to the amygdala,
but also to the nucleus accumbens core and shell subregions
122 A.R. de Oliveira et al.

Figure 5 Target sites for microinjections into the ventral tegmental area (VTA) and for microdialysis in the basolateral amygdala
complex (BLA). Photomicrographs showing a representative microinjection site in the VTA (A) and a representative probe site for
microdialysis in the BLA (B). Outline of microinjection sites in the VTA (C) and probe locations in the BLA (D) on cross-sections from the
Paxinos and Watson atlas (2007). The number of points in the figures is less than the total number of rats used because of several
overlaps. Scale bar = 0.5 mm. DMPAG, dorsomedial periaqueductal gray; CLi, caudal linear nucleus of the raphe; Pn, pontine nuclei;
CA3, CA3 field of the hippocampus; VPL, ventral posterolateral thalamic nucleus; MeP, medial amygdaloid nuclei.

and to different areas of the medial prefrontal cortex
(Yoshioka et al., 1996; Martinez et al., 2008; de Oliveira
et al., 2011, 2013). Although our results implicate a VTA-BLA
dopaminergic pathway in the expression of conditioned fear,
determining whether the above mentioned effect may also
be observed in other regions of the mesocorticolimbic system
after pharmacological manipulation of VTA MRs during con-
ditioned fear remains to be investigated. Further studies
using in vivo microdialysis targeting dopamine in these
regions will help to elucidate this point.
Altogether, the results obtained with the present study
show that intra-VTA injections of the MR antagonist spirono-
lactone inhibited the efflux of dopamine in the BLA in
response to the light-CS and also decreased the expression
of conditioned freezing response. These findings suggest that
corticosteroids, acting through MRs and together with dopa-
minergic neurotransmission, play an important role in the
expression of conditioned fear elaborated via the VTA-BLA
pathway. Once more, because of the existence of multiple
mediators with distinct but overlapping temporal and
mechanistic attributes, more studies are needed in the
attempt to clarify the circuits and mechanisms recruited
during the expression of conditioned fear. For example,
glutamatergic neurons from a variety of cortical, thalamic,
and brainstem structures provide excitatory input to the VTA,
which is also under the inhibitory control of GABAergic
interneurons (Johnson and North, 1992; Bonci and Malenka,
1999). Also, our findings are consistent with a model in which
the amygdala receives emotionally arousing inputs from
multiple sources and modulates fear expression elaborated
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear 123

A Microdialysis - BLA
Drugs
(intra-VTA)
Surgery Training Equilibrium Baseline Testing Post-Testing Histology
5 days 24 h
25 min 2h 2h 20 min 3h

B C
300 600
Control
250 Spiro10 # 500

Conditioned Freezing (s)

DA (% Baseline)

200 drug 400

150 300
*
100 200 *
50 100
testing
0 0
-90 -60 -30 0 30 60 90 120 150 180 Control Spiro10
Time (min)

Figure 6 Mineralocorticoid receptors (MRs) in the ventral tegmental area (VTA) and dopamine levels in the basolateral amygdala
complex (BLA) during conditioned fear. Timeline of the procedures for assessing the effects of intra-VTA administration of the MR
antagonist spironolactone on extracellular dopamine concentration in the BLA during the expression of conditioned fear (A).
Extracellular dopamine concentration in the BLA in rats that received intra-VTA microinjection of vehicle (control; n = 10) or
10 ng/0.2 ml of the MR antagonist spironolactone (Spiro10; n = 10) and were subjected to the conditioned fear test (B). Time of
conditioned freezing in the same rats during the 20 min test session (C). #p < 0.05, different from baseline; *p < 0.05, different from
control.

in other brain regions (Maren and Fanselow, 1996; LeDoux, Role of the funding source
2003; Pare et al., 2004).
Finally, studying the mediators and neural substrates This research was supported by FAPESP (Proc. no. 11/00041-
involved in the expression of conditioned fear responses is 3) and CNPq (Proc. no. 471325/2011-2). AR de Oliveira and AE
of great interest because they are thought to be important Reimer hold Post-Doctoral fellowships from FAPESP (Proc. no.
for different aspects of human anxiety disorders (Phelps and 10/50669-6 and Proc. no. 13/04741-5, respectively). FAPESP
LeDoux, 2005; Indovina et al., 2011). The present findings and CNPq funded this study but had no further role in the
may have clinical implications by enabling better under- study design, collection, analysis and interpretation of the
standing of the relationship between hormonal and neuro- data, writing of the report, and decision to submit the paper
chemical events that are important for fear-related for publication.
behaviors or, more precisely, by highlighting the interplay
between corticosteroids and dopamine in the expression of
conditioned fear responses. The use of agents targeting Conflict of interest
dopaminergic neurotransmission as adjunctive therapy in
anxiety disorders is beginning to be explored, at least in None declared.
the treatment of certain individuals that have not responded
adequately to current interventions (Houlihan, 2011; Katz-
Acknowledgements
man, 2011). The action of agents that decrease HPA axis
activation may also be useful in preventing subsequent
Research was supported by FAPESP (Proc. no. 11/00041-3)
stress-induced activation of BLA neurons and associated
and CNPq (Proc. no. 471325/2011-2). AR de Oliveira and AE
increases in emotionality. Since dopamine plays an important
Reimer hold Post-Doctoral fellowships from FAPESP (Proc. no.
role in the pathogenesis of several psychiatric disorders, such
10/50669-6 and Proc. no. 13/04741-5, respectively).
agents could be therapeutic alternatives not only for anxiety
but also for other dopamine related disorders. In summary,
the present study shows that corticosteroids, acting through References
MRs in the VTA, upregulate the dopaminergic system in the
BLA, enabling a better evaluation of threats associated with Albrechet-Souza, L., Carvalho, M.C., Franci, C.R., Brandao, M.L.,
aversive stimuli and the expression of appropriate defense 2007. Increases in plasma corticosterone and stretched-attend
reactions to cope with the situation. postures in rats naive and previously exposed to the elevated
124
plus-maze are sensitive to the anxiolytic-like effects of midazo-
lam. Horm. Behav. 52, 267273.
Barr, G.A., Moriceau, S., Shionoya, K., Muzny, K., Gao, P., Wang, S.,
Sullivan, R.M., 2009. Transitions in infant learning are modulated
by dopamine in the amygdala. Nat. Neurosci. 12, 13671369.
Bonci, A., Malenka, R.C., 1999. Properties and plasticity of excita-
tory synapses on dopaminergic and GABAergic cells in the ventral
tegmental area. J. Neurosci. 19, 37233730.
Conrad, C.D., MacMillan 2nd, D.D., Tsekhanov, S., Wright, R.L.,
Baran, S.E., Fuchs, R.A., 2004. Influence of chronic corticoster-
one and glucocorticoid receptor antagonism in the amygdala on
fear conditioning. Neurobiol. Learn. Mem. 81, 185199.
Cordero, M.I., Merino, J.J., Sandi, C., 1998. Correlational relation-
ship between shock intensity and corticosterone secretion on the
establishment and subsequent expression of contextual fear
conditioning. Behav. Neurosci. 112, 885891.
de Kloet, E.R., Karst, H., Joels, M., 2008. Corticosteroid hormones in
the central stress response: quick-and-slow. Front. Neuroendo-
crinol. 29, 268272.
de Oliveira, A.R., Reimer, A.E., Brandao, M.L., 2006. Dopamine D2
receptor mechanisms in the expression of conditioned fear.
Pharmacol. Biochem. Behav. 84, 102111.
de Oliveira, A.R., Reimer, A.E., Brandao, M.L., 2009. Role of dopa-
mine receptors in the ventral tegmental area in conditioned fear.
Behav. Brain. Res. 199, 271277.
de Oliveira, A.R., Reimer, A.E., de Macedo, C.E., de Carvalho, M.C.,
Silva, M.A., Brandao, M.L., 2011. Conditioned fear is modulated
by D2 receptor pathway connecting the ventral tegmental area
and basolateral amygdala. Neurobiol. Learn. Mem. 95, 3745.
de Oliveira, A.R., Reimer, A.E., Reis, F.M., Brandao, M.L., 2013.
Conditioned fear response is modulated by a combined action
of the hypothalamic-pituitary-adrenal axis and dopamine activity
in the basolateral amygdala. Eur. Neuropsychopharmacol. 23,
379389.
de Souza Caetano, K.A., de Oliveira, A.R., Brandao, M.L., 2013.
Dopamine D2 receptors modulate the expression of contextual
conditioned fear: role of the ventral tegmental area and the
basolateral amygdala. Behav. Pharmacol. 24, 264274.
Fadok, J.P., Dickerson, T.M., Palmiter, R.D., 2009. Dopamine is
necessary for cue-dependent fear conditioning. J. Neurosci.
29, 1108911097.
Gaillard, R.C., Riondel, A., Muller, A.F., Herrmann, W., Baulieu, E.E.,
1984. RU 486: a steroid with antiglucocorticosteroid activity that
only disinhibits the human pituitary-adrenal system at a specific
time of day. Proc. Natl. Acad. Sci. U. S. A. 81, 38793882.
Harfstrand, A., Fuxe, K., Cintra, A., Agnati, L.F., Zini, I., Wikstrom,
A.C., Okret, S., Yu, Z.Y., Goldstein, M., Steinbusch, H., et al., 1986.
Glucocorticoid receptor immunoreactivity in monoaminergic neu-
rons of rat brain. Proc. Natl. Acad. Sci. U. S. A. 83, 97799783.
Houlihan, D.J., 2011. Psychostimulant treatment of combat-related
posttraumatic stress disorder. J. Psychopharmacol. 25, 1568
1572.
Imperato, A., Puglisi-Allegra, S., Casolini, P., Zocchi, A., Angelucci,
L., 1989. Stress-induced enhancement of dopamine and acetyl-
choline release in limbic structures: role of corticosterone. Eur. J.
Pharmacol. 165, 337338.
Indovina, I., Robbins, T.W., Nunez-Elizalde, A.O., Dunn, B.D., Bishop,
S.J., 2011. Fear-conditioning mechanisms associated with trait
vulnerability to anxiety in humans. Neuron 69, 563571.
Joels, M., Karst, H., DeRijk, R., de Kloet, E.R., 2008. The coming out
of the brain mineralocorticoid receptor. Trends Neurosci. 31, 17.
Johnson, L.R., Farb, C., Morrison, J.H., McEwen, B.S., LeDoux, J.E.,
2005. Localization of glucocorticoid receptors at postsynaptic
membranes in the lateral amygdala. Neuroscience 136, 289299.
Johnson, S.W., North, R.A., 1992. Two types of neurone in the rat
ventral tegmental area and their synaptic inputs. J. Physiol. 450,
455468.
A.R. de Oliveira et al.
Katzman, M.A., 2011. Aripiprazole: a clinical review of its use for the
treatment of anxiety disorders and anxiety as a comorbidity in
mental illness. J. Affect. Disord. 128 (Suppl. 1) S11L 20.
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, 385394.
LeDoux, J., 2003. The emotional brain, fear, and the amygdala. Cell
Mol. Neurobiol. 23, 727738.
Lu, N.Z., Wardell, S.E., Burnstein, K.L., Defranco, D., Fuller, P.J.,
Giguere, V., Hochberg, R.B., McKay, L., Renoir, J.M., Weigel, N.L.,
Wilson, E.M., McDonnell, D.P., Cidlowski, J.A., 2006. Interna-
tional Union of Pharmacology. LXV. The pharmacology and classi-
fication of the nuclear receptor superfamily: glucocorticoid,
mineralocorticoid, progesterone, and androgen receptors. Phar-
macol. Rev. 58, 782797.
Lupien, S.J., McEwen, B.S., 1997. The acute effects of corticoster-
oids on cognition: integration of animal and human model studies.
Brain Res. Brain Res. Rev. 24, 127.
Maren, S., Fanselow, M.S., 1996. The amygdala and fear condition-
ing: has the nut been cracked? Neuron 16, 237240.
Marin, M.F., Hupbach, A., Maheu, F.S., Nader, K., Lupien, S.J., 2011.
Metyrapone administration reduces the strength of an emotional
memory trace in a long-lasting manner. J. Clin. Endocrinol.
Metab. 96, E1221E1227.
Martinez, R.C., Oliveira, A.R., Macedo, C.E., Molina, V.A., Brandao,
M.L., 2008. Involvement of dopaminergic mechanisms in the
nucleus accumbens core and shell subregions in the expression
of fear conditioning. Neurosci. Lett. 446, 112116.
McEwen, B.S., Weiss, J.M., Schwartz, L.S., 1969. Uptake of corti-
costerone by rat brain and its concentration by certain limbic
structures. Brain Res. 16, 227241.
Ninomiya, E.M., Martynhak, B.J., Zanoveli, J.M., Correia, D., da
Cunha, C., Andreatini, R., 2010. Spironolactone and low-dose
dexamethasone enhance extinction of contextual fear condition-
ing. Prog. Neuropsychopharmacol. Biol. Psychiatry 34, 1229
1235.
Orchinik, M., Murray, T.F., Moore, F.L., 1991. A corticosteroid recep-
tor in neuronal membranes. Science 252, 18481851.
Otte, C., Moritz, S., Yassouridis, A., Koop, M., Madrischewski, A.M.,
Wiedemann, K., Kellner, M., 2007. Blockade of the mineralocor-
ticoid receptor in healthy men: effects on experimentally
induced panic symptoms, stress hormones, and cognition. Neu-
ropsychopharmacology 32, 232238.
Pare, D., Quirk, G.J., Ledoux, J.E., 2004. New vistas on amygdala
networks in conditioned fear. J. Neurophysiol. 92, 19.
Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordi-
nates: Hard Cover Edition, 6th ed. Elsevier Science, Amsterdam.
Phelps, E.A., LeDoux, J.E., 2005. Contributions of the amygdala to
emotion processing: from animal models to human behavior.
Neuron 48, 175187.
Ratka, A., Sutanto, W., Bloemers, M., de Kloet, E.R., 1989. On the
role of brain mineralocorticoid (type I) and glucocorticoid (type II)
receptors in neuroendocrine regulation. Neuroendocrinology 50,
117123.
Reis, F.L., Masson, S., de Oliveira, A.R., Brandao, M.L., 2004. Dopa-
minergic mechanisms in the conditioned and unconditioned fear
as assessed by the two-way avoidance and light switch-off tests.
Pharmacol. Biochem. Behav. 79, 359365.
Reis, F.M., Albrechet-Souza, L., Franci, C.R., Brandao, M.L., 2012.
Risk assessment behaviors associated with corticosterone trigger
the defense reaction to social isolation in rats: role of the anterior
cingulate cortex. Stress 15, 318328.
Reul, J.M., de Kloet, E.R., 1986. Anatomical resolution of two types
of corticosterone receptor sites in rat brain with in vitro auto-
radiography and computerized image analysis. J. Steroid Bio-
chem. 24, 269272.
Ventral tegmental area mineralocorticoid receptors regulate the expression of conditioned fear
Rimmele, U., Besedovsky, L., Lange, T., Born, J., 2013. Blocking
mineralocorticoid receptors impairs, blocking glucocorticoid
receptors enhances memory retrieval in humans. Neuropsycho-
pharmacology 38, 884894.
Rivet, J.M., Stinus, L., LeMoal, M., Mormede, P., 1989. Behavioral
sensitization to amphetamine is dependent on corticosteroid
receptor activation. Brain Res. 498, 149153.
Rodrigues, S.M., LeDoux, J.E., Sapolsky, R.M., 2009. The influence of
stress hormones on fear circuitry. Annu. Rev. Neurosci. 32, 289
313.
Ronken, E., Mulder, A.H., Schoffelmeer, A.N., 1994. Glucocorticoid
and mineralocorticoid receptors differentially modulate cultured
dopaminergic neurons of rat ventral mesencephalon. Eur. J.
Pharmacol. 263, 149156.
Roozendaal, B., McGaugh, J.L., 1997. Glucocorticoid receptor ago-
nist and antagonist administration into the basolateral but not
central amygdala modulates memory storage. Neurobiol. Learn.
Mem. 67, 176179.
Sapolsky, R., 2009. Any kind of mother in a storm. Nat. Neurosci. 12,
13551356.
Schumacher, M., 1990. Rapid membrane effects of steroid hormones:
an emerging concept in neuroendocrinology. Trends Neurosci. 13,
359362.
125
Spiga, F., Knight, D.M., Droste, S.K., Conway-Campbell, B., Kershaw,
Y., MacSweeney, C.P., Thomson, F.J., Craighead, M., Peeters,
B.W., Lightman, S.L., 2011. Differential effect of glucocorticoid
receptor antagonists on glucocorticoid receptor nuclear translo-
cation and DNA binding. J. Psychopharmacol. 25, 211221.
Stevens, W., Grosser, B.I., Reed, D.J., 1971. Corticosterone-binding
molecules in rat brain cytosols: regional distribution. Brain Res.
35, 602607.
Tasker, J.G., Di, S., Malcher-Lopes, R., 2006. Minireview: rapid
glucocorticoid signaling via membrane-associated receptors.
Endocrinology 147, 55495556.
Yang, Y.L., Chao, P.K., Lu, K.T., 2006. Systemic and intra-amygdala
administration of glucocorticoid agonist and antagonist modulate
extinction of conditioned fear. Neuropsychopharmacology 31,
912924.
Yoshioka, M., Matsumoto, M., Togashi, H., Saito, H., 1996. Effect of
conditioned fear stress on dopamine release in the rat prefrontal
cortex. Neurosci. Lett. 209, 201203.
Zweifel, L.S., Fadok, J.P., Argilli, E., Garelick, M.G., Jones, G.L.,
Dickerson, T.M., Allen, J.M., Mizumori, S.J., Bonci, A., Palmiter,
R.D., 2011. Activation of dopamine neurons is critical for aversive
conditioning and prevention of generalized anxiety. Nat. Neu-
rosci. 14, 620626.