Behavioural Brain Research 201 (2009) 338–342

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Behavioural Brain Research

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Short communication

Effects of medial prefrontal cortex lesions on anxiety-like behaviour in restrained

and non-restrained rats
Eduardo Blanco a,1 , Estela Castilla-Ortega a,1 , Rubén Miranda b , Azucena Begega c , José A. Aguirre d ,
Jorge L. Arias c , Luis J. Santín a,∗
a
Departamento de Psicobiología y Metodología de las CC, Facultad de Psicología, Universidad Málaga, Campus de Teatinos s/n, CP 29071 Málaga, Spain
b
Departamento de Psicología, Facultad de Psicología, Universidad de las Islas Baleares, Carretera de Valldemossa km 7.5, 07122 Palma de Mallorca, Spain
c
Departamento de Psicología, Facultad de Psicología, Universidad de Oviedo, Plaza Feijoo s/n, 33003 Oviedo, Spain
d
Departamento de Fisiología Humana, Facultad de Medicina, Universidad Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain

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

Article history: The medial prefrontal cortex has been associated with fear, anxiety and stress regulation, and has recently
Received 11 December 2008 been suggested to play a crucial role in the development of behavioural changes in response to stress. In
Received in revised form 1 March 2009 this study, we evaluated medial prefrontal cortex (mPFC) involvement in both anxiety-like behaviour and
Accepted 3 March 2009
increased anxiety-like responses induced by uncontrollable restraint. Rats with mPFC electrolytic lesions
Available online 17 March 2009
(n = 7) and sham-lesioned (n = 8) were tested in the elevated T-maze (ETM). Restrained rats with mPFC
lesions (n = 8) and sham-lesioned rats (n = 6) were tested in the elevated T-maze 24 h after restraint. Both
Keywords:
two-trial passive avoidance and one-trial escape behaviours were assessed. The results revealed that
Stress
Frontal cortex
mPFC lesions impair passive avoidance, but not escape behaviour. In addition, decreased anxiety-like
Elevated T-maze behaviour in both passive avoidance and escape behaviours were observed in restrained rats with mPFC
Anxiety lesions. Our results suggest that mPFC is important in mediating both anxiety-like behaviour expression
Restraint and long-term anxiogenic-like effects induced by acute restraint.
Rodent © 2009 Elsevier B.V. All rights reserved.

It is well known that stress interacts with fear and anxiety.
In this interaction, the degree of controllability for the stressors
is crucial, and only uncontrollable stressors are able to poten-
tiate fear conditioning and anxiety-like behaviours [21]. Thus,
prior stressful experiences modify the animal’s responses to new
aversive stimuli, which can increase their anxiety-like behaviours
[18,19]. The magnitude of the stress response depends on the
type, intensity and duration of the stressor. In this regard, acute
immobilization results in an immediate activation of the central
noradrenalin (NA) system [33], which leads to a serotonin (5-HT)
response in the hindbrain raphe nuclei [32] and parallel activation
of the hypothalamic-pituitary-adrenal (HPA) axis with a subse-
quent increase in serum corticosterone levels [31]. These hormonal
and neurochemical events underlie, at least in part, the behavioural
consequences of the acute stress [17,31]. In accord with those
reports, we demonstrated that prior exposure to a restraint ses-
sion induced anxiety-like behaviours when rats were assessed in
the elevated T-maze (ETM), where enhanced fear is expressed as
an increase in passive avoidance (PA) behaviour [31]. Recently, the
medial prefrontal cortex (mPFC) has been shown to be involved in
∗ Corresponding author. Fax: +34 952 13 2506.
E-mail address: luis@uma.es (L.J. Santín).
1
These authors contributed equally to this work.
the identification of whether or not a stressor is under the organ-
ism’s control. In the presence of acute controllable stressors, the
mPFC inhibited both the activation of the dorsal raphe nuclei and
the behavioural consequences that are induced by acute stress [1].
Although mPFC function is disturbed by uncontrollable acute stress
and has been implicated in the integration of the stress response
[30,37], the involvement of the mPFC in the behavioural conse-
quences of uncontrollable acute stress is still unclear.
In this study, we suggest that mPFC may be critically involved
in the emotional sequelae of a prior exposure to acute uncontrol-
lable stress. To address this hypothesis, we have studied whether
mPFC lesions in restrained and non-restrained rats modulate their
anxiety-like behaviours, such as PA and escape responses, in the
elevated T-maze.
Thirty-six male Sprague–Dawley rats (Charles River, Barcelona,
Spain) weighing 300–350 g were used. All rats were maintained
in a colony room at 21 ◦ C with a 12 h light–dark cycle. Food and
water were available ad libitum. The experiments were carried out
in accordance with the European Communities Council directive
(86/609/EEC) for animal care and experimental procedure, and the
experiments were approved by the Ethical Committee for Animal
Research of the University of Malaga. All rats underwent surgical
procedures, which included either an mPFC lesion or a sham-lesion.
Briefly, each subject was anesthetised with equithesin (3 ml/kg i.p.)
and the head was fixed on a stereotaxic instrument (Kopf instru-

0166-4328/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbr.2009.03.001
 E. Blanco et al. / Behavioural Brain Research 201 (2009) 338–342
ments, USA). An insulated 0.3 mm o.d. tungsten wire electrode with
only the tip being conductive was lowered into the mPFC. Ani-
mals were exposed to two bilateral lesions using the following
coordinates: anterior: 3.2 mm anterior to bregma, 0.4 mm lateral
to the midline and 3 mm ventral to dura; posterior: 1.7 mm ante-
rior to bregma, 0.5 mm lateral to the midline and 2 mm ventral to
dura [26]. Constant positive current (2 mA) was applied for 20 s.
The same procedure was performed in the sham-lesioned rats, but
no current was applied. After recovery, all rats were handled and
monitored for their weight. The rats were used for the behavioural
experiments 10 days after the surgery. Some of the animals were
restrained through immobilization (IMMO) for 3 h in a Plexiglas
tube (5.5 cm × 21 cm). Animals were divided into four experimental
groups: (1) mPFC lesion (n = 10); (2) mPFC sham-lesion (n = 8); (3)
mPFC lesion + IMMO (n = 10); (4) mPFC sham-lesion + IMMO (n = 8).
The animals were tested in the ETM 24 h after the immobiliza-
tion. The apparatus was made of black-painted wood and consisted
of two open arms (10 cm × 50 cm with 0.7 cm raised edges), one
closed arm (10 cm × 50 cm with 40 cm high side and end walls)
and a connecting central platform (10 cm × 10 cm). The maze was
raised 50 cm above the floor. ETM was used to study the effect of
the mPFC lesion alone and combined with the acute stress (immo-
bilization for 3 h) on anxiety-like behaviours, such as PA and escape
responses [11]. Animals were transported from the housing room
into the testing room 1 h prior to testing. Behavioural experiments
were carried out between 10:00 am and 14:00 pm. The PA response
was evaluated in two trials, with an intertrial interval of 30 s. The
rat was placed in the bottom of the closed arm and the time spent to
reach the open arm was registered (avoidance latency). The escape
response was assessed in the same animal 30 s after the second PA
trial and the time spent to reach the closed arm from the bottom
of the open arm was registered (escape latency). The maximum
duration for each trial was 120 s.
After completion of the behavioural testing, all the animals were
anesthetised with sodium pentobarbital (mebumal; 100 mg/kg i.p)
and intracardially perfused with 200 ml isotonic ice-cold saline
followed by 200 ml of fixative solution (4 ◦ C) during 10 min. The fix-
ative consisted of 4% paraformaldehyde (w/v) in 0.1 M PBS, pH 7.4.
The brains were removed and postfixed for 24 h in the same fixative.
The brains were then dehydrated, paraffin embedded, systemati-
cally cut with a microtome (Leica, Germany) into 10 ␮m-thick serial
coronal sections, stained with toluidine blue and coverslipped with
DPX mounting medium. Stained sections were then used to verify
placement and extent of the electrolytic lesion. Both the location
and the extent of the lesions were afterwards reconstructed for
each animal according to the rat brain atlas [26]. The effect of mPFC
lesion and acute restraint on PA behaviour were analysed via a
two-way analysis of variance (ANOVA) with one repeated mea-
sure (lesion and stress treatments as two factors and avoidance
trials as repeated measure), which was followed by post-hoc com-
parisons (LSD). The escape response was analysed via a two-way
ANOVA with lesion and acute stress as the two factors, which was
followed by post-hoc test (LSD). Results were expressed as group
mean ± S.E.M. Statistical significance was set at p < 0.05.
On the basis of anatomical and connectivity criteria, the mPFC
is comprised of several cortical regions, which include area 2 of
the frontal cortex, the anterior cingulate and both the prelimbic
and infralimbic cortices [12]. A representative reconstruction of
an mPFC lesion is shown in Fig. 1A. Twenty-nine animals were
included in the behavioural analysis. Two rats from the mPFC lesion
and two from the mPFC sham-lesion + IMMO groups died after the
surgery. Histological analysis revealed that one rat from the mPFC
lesion group and two from the mPFC lesion + IMMO group had an
incomplete unilateral lesion, and these animals were removed from
the behavioural study. In most animals, the lesion extended along
the anterior–posterior axis, 4.2 mm anterior and 0.8 mm posterior
339
to bregma. In 13 animals, the area of the lesion included the ante-
rior cingulate cortex (Cg1 and Cg2), the infralimbic and prelimbic
regions (IL and PL) and the most lateral part of the frontal cor-
tex, which is area 2 (Fr2) (see largest lesion, Fig. 1A). In the mPFC
lesioned group, two rats showed a lesion that was restricted to the
most medial part of Cg1 and Cg2 and the dorsolateral region of the
IL region (see smallest lesion, Fig. 1). Therefore, the final number of
animals included in each group for the behavioural analyses was:
seven in the mPFC lesion group, eight in the mPFC sham-lesion
group, eight in the mPFC lesion + IMMO group and six in the mPFC
sham-lesion + IMMO group.
With regard to the PA results (Fig. 1B), the ANOVA revealed sev-
eral main effects. The mPFC sham-lesion group spent more time in
the closed arm when compared to the mPFC lesion group (F = 34.04,
df 1/25, p < 0.001). The rats that endured acute stress spent more
time in the closed arm than those that were not stressed (F = 10.95,
df 1/25, p < 0.003), and their time spent in the closed arm was longer
during the second (PA2 ) rather than the first (PA1 ) avoidance trial
(F = 7.51, df 1/25, p < 0.01). Post-hoc comparisons showed that the
mPFC sham-lesion group spent more time in the closed arm when
compared to the mPFC lesion group only in the PA2 trial (p < 0.05).
There were differences between the mPFC sham-lesion + IMMO and
the mPFC lesion + IMMO groups in both avoidance trials (p < 0.05).
There were also significant differences between the first and second
avoidance trials (F = 7.51, df 1/25, p < 0.01). Post-hoc comparisons
showed that only the mPFC sham-lesion and mPFC lesion + IMMO
groups spent more time in the closed arm during the second
avoidance trial than the first avoidance trial (p < 0.05). During the
escape trial, the lesion effect and the interaction between the mPFC
lesion and immobilization were observed (F = 7.41, df 1/25, p < 0.01
and F = 4.6, df 1/25, p < 0.05, respectively). Post-hoc comparisons
showed that the mPFC lesion + IMMO group spent more time in
the open arm than the mPFC sham-lesion + IMMO group (p < 0.05)
(Fig. 1C).
These results show that PA and escape in the ETM were dif-
ferentially affected by the mPFC lesions in non-restrained and
restrained rats. In non-restrained animals, the mPFC modulates
anxiety-like behaviour by affecting the acquisition of PA without
interfering with the escape response (Fig. 1B and C). However,
the available literature does not support mPFC involvement in the
acquisition of conditioned fear [28]. It is likely that our data on PA
suggest an anxiolytic-like effect of mPFC lesion rather than mem-
ory impairment, because if the rats do not perceive a potential
threat during the first PA trial, then they will not increase avoid-
ance time in the second PA trial [11]. In line with this hypothesis,
when stress induced an anxiety-like behaviour during the first
avoidance trial, rats with mPFC lesions increased their avoidance
time in the second exposure to the closed arm (Fig. 1B). Sev-
eral studies that have shown that mPFC lesions in rodents have
anxiolytic-like effects in the elevated plus maze (EPM) [20], sup-
port the anxiolytic-like effect that is reported here. Furthermore,
rats that perform the PA but not the escape task in the ETM have
increased c-Fos immunoreactivity in the prefrontal cortex [36].
It is likely that the anxiolytic-like effects of the mPFC lesion in
the ETM may be explained by the regulation of subcortical brain
regions that are related to fear and anxiety [16,36,42]. Recently,
it has been reported that microinjection of a 5-HT1A agonist into
the median raphe nucleus (MRN) impaired PA acquisition, but not
the escape response [42]. This similar involvement of mPFC and
MRN in the avoidance response suggests that mPFC projections
to the MRN [41] may be necessary to exhibit a normal avoidance
response. However, we cannot discount additional involvement of
the amygdala, because similar results have been obtained after
GABAA receptor agonist (muscimol) microinjections into the baso-
lateral nucleus [8]. Furthermore, lesion and physiological studies
have suggested that the mPFC modulates conditioned fear via the
340 E. Blanco et al. / Behavioural Brain Research 201 (2009) 338–342

Fig. 1. (A) Reconstruction of the mPFC lesions in consecutive brain sections. Largest (light grey) and smallest (dark grey) mPFC lesions are shown. Numbers in each section
show AP level anterior to bregma. Cg1: cingulate cortex, area 1; Cg2: cingulate cortex, area 2; Fr2: frontal cortex, area 2; IL: infralimbic cortex; PL: prelimbic cortex. (B)
Effects of the mPFC lesion on the passive avoidance response in the elevated T-maze. (C) Effects of the mPFC lesion on the escape response in the elevated T-maze. (*) mPFC
sham-lesion vs. mPFC lesion (p < 0.05, LSD). Passive avoidance trials were defined as the time spent to reach the enclosed arm. PA1 : first passive avoidance trial; PA2 : second
passive avoidance trial, performed 30 s after PA1 ; ETM: elevated T-maze. Columns represent the mean, and bars represent the S.E.M.

amygdala [22,29]. Finally, it is important to note that the mPFC is
not functionally unified [12]. Although our lesions compromised
several mPFC subfields, the literature suggests that the anxiolytic
effect reported here could be explained by the damage to the ven-
tral part of the mPFC [38]. Therefore, selective disruption of the
infralimbic cortex is followed by anxiolytic-like behaviour and inhi-
bition of the hypothalamo–pituitary–adrenal axis in response to
stressors [13,38]. In contrast, anxiogenic or inexistent effects were
reported after selective manipulation of the anterior cingulate and
dorsal prelimbic cortices [5,13]. However, two rats in our study that
had dorsally located lesions (Fig. 1A) showed reduced anxiety. This
suggests that the dorsal mPFC might have a relevant role in our
results, but it is also possible that the dorsal lesions, with a notable
anterior–posterior extent, damaged important pathways that con-
nect the ventral mPFC with other brain structures [40]. Further
investigation would be needed to establish strong conclusions in
this regard.
Previous studies in rats have shown that stressors, which include
restraint, can facilitate fear and anxiety-like behaviours [25,31,35].
Our data do confirm that the ETM is able to detect behavioural
changes that are induced by restraint stress, which is a form
of uncontrollable stress. The ceiling effect that is observed dur-
ing the second avoidance trial in the mPFC sham-lesioned and
restrained animals prevents us from observing the acquisition of
the PA response that was previously described in the ETM after
acute restraint [31]. However anxiety-like effects that are induced
by restraint were clearly observed in this task (Fig. 1B). As has been
reported in other studies [9,31], the experience with restraint 24 h
before testing could increase the expression, rather than the con-
ditioning, of the anxiety-like response [9,31]. In addition, it has
been shown that exploratory impairments in both the elevated
plus maze [24,25] and the ETM [31] are due to the pre-exposure to
acute stress [24,25]. Electrolytic lesions in the mPFC decrease anx-
iety in rats that had been restrained 24 h before testing. As noted
above, the reduced anxiety-like responses in the mPFC lesioned rats
were evident after ETM testing in the absence of previous restraint
(Fig. 1B). There are, however, important differences in the effect
of the mPFC lesion on stressed versus non-stressed animals. For
instance, a mPFC lesion impairs PA acquisition but not the escape
response in non-restrained animals. Nevertheless, when the ani-
mals were restrained, the mPFC lesioned rats exhibited adequate PA
acquisition responses in the ETM, even when the degree of avoid-
ance was lower than in the mPFC sham-lesioned and restrained rats.
These results indicate that acute restraint is able to enhance the
anxiety-like behaviour in mPFC lesioned animals. This, therefore,
suggests that mPFC is important for the expression of anxiety but
not for the conditioning in rats that are pre-exposed to restraint.
In line with our results, experimental evidence further supports
that uncontrollable stress may increase the conditioned anxiety
responses by an mPFC-independent mechanism [1,4].
With regard to the behavioural consequences of stress on the
escape response, no effects were observed after restraint. Con-
 E. Blanco et al. / Behavioural Brain Research 201 (2009) 338–342
versely, other studies that used conditioning paradigms have found
altered escape responses after acute stress [4], although they failed
to reveal any alterations in the one-trial stress-induced escape
response in the ETM [31]. These observations are consistent with
the lack of anxiety-like effects that are induced by restraint in the
unconditioned escape response that is reported here. Interestingly,
only the mPFC lesioned rats that were restrained 24 h before testing
showed impaired escape responses, as demonstrated by more time
being spent in the open arm (Fig. 1C). Increased time in the open
arm in the mPFC lesioned rats may be caused by enhanced explo-
ration and locomotor activity [10,43]. However, the absence of a
similar effect on the mPFC lesioned but unrestrained rats does not
support this hypothesis (Fig. 1B). Taken together, these data sug-
gest that the mPFC is also important in mediating the behavioural
consequences of acute uncontrollable stress on unconditioned,
anxiety-like behaviour. In summary, our data support a role for
the involvement of the mPFC in the expression of anxiety-like
behaviours and demonstrate that uncontrollable stress can inten-
sify the expression of anxiety-like behaviours via activation of the
mPFC. Indeed, recent studies have demonstrated that acute uncon-
trollable stress can induce functional and morphological changes
in the mPFC that may be associated with cognitive impairment, as
observed in depression or post-traumatic stress disorder [7,23].
Although the neurobiological system that underlies this effect
is not fully understood, the available evidence suggests that a brain
system that is comprised of the mPFC and the serotoninergic and
noradrenergic brainstem nuclei [3,6] may be involved in the long-
lasting behavioural effects of stress. Accordingly, the raphe nuclei,
the locus coeruleus and the ventral mPFC have been identified as
regulatory regions that are involved in the cognitive processing
of the signs of stress [2,39]. In addition, a reduction of limbic 5-
HT/NA levels [15,27] and morphofunctional changes in the mPFC
[14] in animals that developed long-lasting behavioural alterations
after acute stress have been reported. Similarly, we have recently
shown that behavioural impairments in restrained rats were asso-
ciated with both a reduced density of 5-HT immunoreactivity in
the midbrain raphe nuclei and c-Fos immunoreactivity in raphe
and locus coeruleus nuclei [34]. These observations strongly sug-
gest that the anxiolytic-like effects that are seen in mPFC lesioned
and restrained animals may be associated with the lack of mPFC
control over the aminergic brainstem nuclei. However, additional
studies are required to better understand the brain system that
underlies stress-induced alterations and to provide new insights
into the pathophysiology and treatment of anxiety disorders.
Acknowledgements
Funding from MEC SEJ2007-61187 and CTS-195. E. Castilla-
Ortega was supported by a FPU-MEC (AP-2006-02582).
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