Journal of Behavioral and Brain Science, 2011, 1, 181-187
doi:10.4236/jbbs.2011.13024 Published Online August 2011 (http://www.SciRP.org/journal/jbbs)
Anxiety-Behavior Modulated by Ventral Medial Prefrontal
Cortex of Rats Submitted to the Vogel Conflict Test
Involves a Local NMDA Receptor and Nitric Oxide
Sabrina F. Lisboa, Francisco S. Guimarães, Leonardo B. M. Resstel*
Department of Ph arm acology, School of Medicine of Ribeirão Preto,
University of São Paulo, Ribeirão Preto, Brazil
Received March 30, 2011; revised April 18, 2011; accepted April 25, 2011
Abstract
It was demonstrated in the Vogel conflict test (VCT) that the ventral portion of medial prefrontal cortex
(vMPFC) of rats is involved with anxiety behavior. Moreover, the vMPFC local glutamatergic and nitrergic
system interaction is involved in modulation of fear conditioning, a model of anxiety. To better understand
the role of the MPFC-glutamatergic and nitrergic system on the VTC behavior response, male Wistar rats
(250 g) were water deprived for 48 h before the VCT. After 24 h of water deprivation, they were subjected to
an initial 3-min non-punished (pre-test) drinking session. Twenty-four hours later bilateral microinjections of
NMDA-antagonist LY235959 (4 nmol/200 nL), the specific nNOS inhibitor N-Propyl-L-arginine (N-Propyl
–0.08 nmol/200 nL), the NO scavenger Carboxi-PTIO (C-PTIO, 2 nmol/200 nL) or 200nL of vehicle were
applied in the vMPFC. After 10 min, the animals were submitted to 3-min punished-licking session. LY235959
increased the number of punished licks. Similar to LY235959, both N-Propyl and C-PTIO also increased the
number of punished licks. No changes were observed when LY235959, N-Propyl and C-PTIO were micro-
injected into vMPFC surrounding structures such as the cingulate cortex area 1, the corpus callosum and the
tenia tecta. In control experiments these drugs did not change neither the number of unpunished licks nor had
any effect in the tail-flick test. The results show that NO signaling in the vMPFC can modulate anxiety-be-
havior in the VCT by control punished behavior. Moreover, this NO modulation could be associated with
local glutamatergic activation through NMDA receptors.
Keywords: Infra Limbic Cortex, Prelimbic Cortex, Anxiolytic-Like Effects, Defensive Behavior
1. Introduction
The medial prefrontal cortex (MPFC) of rats has been the
focus of considerable studies, owing in part to under-
standing the central importance of its dysfunction in a
wide array of psychopathological conditions in humans
[1-3]. In rats, the MPFC is activated by exposure to a vari-
ety of anxiety provoking challenges, and can be blocked
by anxiolytic benzodiazepine [4-8]. Moreover, in rodents
the MPFC presents an important role on neuroendocrine,
autonomic and behavioral modulation during defense
reactions [6,9-11].
The ventral portion of the MPFC (vMPFC), which is
composed of prelimbic cortex (PL) and infralimbic cor-
tex (IL) [10], is particularly responsive to threat stimuli
and the inhibition of its neurotransmission induces anx-
iolytic-like effect accompanied by attenuated cardiovas-
cular activity in a model of contextual fear conditioning
[4,10,12,13]. These data show the specific importance of
local vMPFC neurotransmission in responses evoked by
anxiety behavior in animal model.
The Vogel conflict test (VCT) is an animal model used
to study the anxiety response based on suppression of
punished responses, when water-deprived rats are ex-
posed to the conflict between licking the spout of a bottle
and receiving a mild shock on the tong [14-16]. Anxio-
lytic drugs, such as the benzodiazepines, are able to in-
crease the number of punished licks [14,15]. Moreover, it
has recently described that local vMPFC neurotransmis-
sion is involved with anxiety-like behavior response ob-
served in VCT [17].
It has been described that during defensive reactions,

S. F. LISBOA ET AL.
182
glutamate levels are increased in the vMPFC of rats [18].
Moreover, results from our laboratory has been demon-
strated that both NMDA glutamate receptors and nitric
oxide (NO) present in the vMPFC play an important role
in the expression of the behavioral and cardiovascular
responses observed during fear evoked by contextual con-
ditioning [19], showing an important role of these neuro-
transmitters in anxiety behaviour. Therefore, it is possi-
ble that, similar to previous results, NMDA receptors and
NO in the vMPFC also regulate the behavioral responses
observed in the VCT. Thus, the aims of the present
study were to investigate the effects of vMPFC NMDA
receptors and NO inactivation in rats submitted to the
VCT.
2. Material and Methods
2.1. Animal Preparation
Male Wistar rats weighing 230 - 270 g were used. Ani-
mals were kept in the Animal Care Unit of the Depart-
ment of Pharmacology, School of Medicine of Ribeirão
Preto, University of São Paulo. Rats were housed indi-
vidually in plastic cages with free access to food and
water and under a 12 h light/dark cycle (lights on at
06:30 h). The Institution’s Animal Ethics Committee
approved housing conditions and experimental proce-
dures (process number: 215 - 2005).
Seven days before the experiment rats were anesthe-
tized with tribromoethanol (250 mg/kg i.p.). After scalp
anesthesia with 2% lidocaine the skull was surgically
exposed and stainless steel guide cannulae (26G) were
implanted bilaterally in the vMPFC using a stereotaxic
apparatus (Stoelting, Wood Dale, Illinois, USA). Coor-
dinates for cannula implantation (AP = +2.2 mm; L = 2.8
mm from the medial suture, V = –3.3 mm from the skull
with a lateral inclination of 23˚) were selected from the
rat brain atlas of Paxinos and Watson (1997). A control
group of animals had stainless steel guide cannulas im-
planted bilaterally into surrounding structures of the
vMPFC such as the cingulate cortex area 1 (AP = +1.2
mm; L = 1.5 mm from the medial suture, V = –2.3 mm
from the skull), the corpus callosum (AP = +1.2 mm; L =
2.8 mm from the medial suture, V = –2.3 mm from the
skull) and the tenia tecta(AP = +1.2 mm; L = 3 mm from
the medial suture, V = –4.3 mm from the skull). Cannu-
lae were fixed to the skull with dental cement and one
metal screw.
2.2. Drugs
The following drugs were used: LY235959 (Tocris,
Westwoods Business Park Ellisville, MO, USA), Nω-
Propyl-L-arginine (Tocris, Westwoods Business Park
Ellisville, MO, USA) and Carboxy-PTIO ((S)-3-Carboxy-
4-hydroxyphenylglicine (c-PTIO, St. Louis, Missouri,
USA), morphine (Sigma, St. Louis, MO, USA), Tribro-
moethanol (Aldrich, St. Louis, MO, USA) and Urethane
(Sigma, St. Louis, MO, USA) were dissolved in sterile
artificial cerebrospinal fluid (aCSF - composition: NaCl
100 mM; Na3PO4 2 mM; KCl 2.5 mM; MgCl2 1 mM;
NaHCO3 27 mM; CaCl2 2.5 mM; pH = 7.4).
2.3. Vogel Conflict Test
The Vogel conflict test was performed in a Plexiglas box
(42 × 50 × 25 cm) with a stainless grid floor. The metal-
lic spout of a drinking bottle containing water projected
into the box. The contact of the animal with the spout
and the grid floor closed an electrical circuit controlled
by a sensor (Anxio-Meter model 102, Columbus, USA),
which produced 7 pulses/s whenever the animal was in
contact with both components. Each pulse was consid-
ered as a lick and every 20 licks the animal received a
0.5 mA shock on the metallic drinking spout for 2 s. The
sensor recorded the total number of licks and shocks de-
livered during the test period. The whole apparatus was
located inside a sound-attenuated cage [20].
The animals were water deprived for 48 h before the
test. After the first 24 h of deprivation they were allowed
to drink freely for 3 min in the test cage in order to find
the drinking bottle spout. Some animals did not find the
spout and were not included in the experiment. Twenty-
four hours later the drugs were injected into the vMPFC
and, after 10 min, the animals were placed into the test
box. The test period lasted for 3 min and the animals
received a 0.5 mA shock every 20 licks. The number of
licks and shocks delivered were registered. Although the
number of shocks delivered by the system was propor-
tional to the number of licks performed by the rat (every
20 licks, one shock), sometimes the end of the test oc-
curred when the animal was still licking but had not yet
received the next shock. So, the number of licks is usu-
ally slightly higher than one would expect considering
the number of shocks.
2.4. Water Consumption Evaluation
The apparatus was the same used in the test above;
however, the electric shock delivering system was inop-
erative.
2.5. Tail-Flick Test
The apparatus consisted of an acrylic platform with a
nichrome wire coil (Insight Instruments. Brazil) main-

S. F. LISBOA ET AL.
183
tained at room temperature (24 - 26˚C). The rats were
gently handled and their tails were laid across the coil.
The coil temperature was then raised at 9˚C/s by the
passage of electric current. The system had a cut-off time
of 6 s to prevent tissue damage when the coil tempera-
ture approached 80˚C. The time to withdraw the tail was
recorded as tail-flick latency. The electric current was
calibrated to provoke this reflex within 2.5 - 3.5 s in
non-treated animals [17,20].
The tail-flick test was conducted in independent groups
of animals receiving vehicle, LY235959, N-Propyl, c-
PTIO intra-vMPFC or morphine i.p.. The heating was
applied to a portion of the ventral surface of the tail lo-
cated between 4 and 6 cm from its end. The tail-flick
latency was measured at 5-min intervals until a stable
baseline (BL) was obtained over three consecutive trials.
The latency was measured again within 30 s after drug
administration and then at 10-min intervals for up to 40
min [17,20].
2.6. Procedures
Microinjections of 200 nL of vehicle or the specific
NMDA receptor antagonist LY235959 [21,22]; the neu-
ronal isoform nitric oxide synthase inhibitor N-Propyl
[19,23] or the NO scavenger Carboxi-PTIO [19,24] were
bilaterally injected into the vMPFC 10 min prior the test
session. As a control for drug spread, the drugs were
microinjected into vMPFC surrounding structures. All
drugs were administered 10 min before the test. Mor-
phine chloride (5 mg/kg), dissolved in saline, was used
as a positive control in the tail-flick test (see below) and
was administered 30 min before tail-flick evaluation.
2.7. Histological Procedure
At the end of the experiments the rats were anesthetized
with urethane (1.25 g/kg, i.p.) and 200 nL of 1% Evan’s
blue dye was bilaterally injected into the vMPFC as a
marker of the injection sites. The chest was surgically
opened; the descending aorta occluded; the right atrium
severed and the brain perfused with 10% formalin
through the left ventricle. The brains were post fixed for
24 h at 4˚C, and 40 μm sections were cut with a cryostat
(CM 1900, Leica, Germany). Brain sections were stained
with 1% neutral red. The actual placement of the inject-
tion needles was identified with the help of the rat brain
atlas of Paxinos and Watson (1997). Animals that re-
ceived drugs outside the vMPFC were joined in an OUT
group.
2.8. Data Analysis
The data were expressed as means ± SEM. The number
of licks and shocks were analyzed by one-way ANOVA
followed by the Dunnett’s post hoc test. The latency of
tail withdrawal was analyzed by two way-ANOVA with
treatment and time as the two factors. In case of signifi-
cant interaction between these factors the groups were
compared by the Bonferroni’s post hoc test. Results of
statistical tests with P < 0.05 were considered significant.
3. Results
The injection sites and a diagrammatic representation
indicating the injection sites of all drugs injection into
the vMPFC are presented in Figure 1.
3.1. Effect of Bilateral Microinjection of Vehicle,
LY, N-Propyl or c-PTIO into the vMPFC on
the VCT
No changes were observed in the number of licks at the
non-punished, first day of exposition to the apparatus
(F3,28 = 0.7, P > 0.05). On test day, bilateral vMPFC
injection of LY (n = 8), c-PTIO (n = 8) or N-Propyl (n = 8)
(a) (b)
Figure 1. (a) Photomicrograph of a coronal brain section showing bilateral microinjections sites in the vMPFC. (b)
Diagrammatic representation based on the rat brain atlas of Paxinos and Watson (1997) indicating the drugs sites into the
vMPFC (closed circles). Animals with drug injection sites outside the vMPFC were represented by opened circles. Cg1-
cingulate cortex area 1; PL - prelimbic cortex; IL - infralimbic cortex; DP - dorsal peduncular cortex; cc - corpus callosum
and TT- tenia tecta. IA- inter aural.

S. F. LISBOA ET AL.
184
Figure 2. Effects of bilateral microinjection of 200 nL of
vehicle, 4 nmol of LY235959 (LY), 0.04 nmol of N-Propyl or
1 nmol of c-PTIO (n = 8, each treatment) in the vMPFC on
the number of punished licks in the Vogel conflict test.
Columns represent the mean and bars the SEM, *P < 0.05
(compared to vehicle group), Dunnett’s post-test.
increased the number of punished licks (F3,22 = 9.7, P <
0.01) and the total number of licks (F3,20 = 9.7, P < 0.01)
when compared to the vehicle group (n = 8, Figure 2).
No changes were observed when LY, N-Propyl or c-PTIO
(n = 9 each treatment) were microinjected into vMPFC
surrounding structures such as the cingulate cortex area 1,
the corpus callosum and the tenia tecta (F3,16 = 9.7, P >
0.05, Fig ur e 3).
In the control test in which no shocks were delivered,
the number of licks were not different between the
groups (F3,32 = 0.9, P > 0.05, Figure 4), indicating that
vMPFC inhibition did not influence water consumption
(n = 5, each treatment).
3.2. Drug Effects in the Tail-Flick Test
The repeated measures ANOVA revealed a significant
drug × time interaction (F20,114 = 2.8, P < 0.001). More-
over, there was a significant drug effect (F4,114 = 19.6,
P < 0.001) and a time effect (F5,114 = 10.7, P < 0.001).
Post-hoc comparisons indicated that the withdrawal la-
tencies were significant greater than vehicle at 10, 20, 30
and 40 min after the injection in the group receiving
morphine (n = 5, P < 0.001). No effect was observed
after LY, N-Propyl or c-PTIO bilateral microinjection
into the vMPFC (n = 5, each treatment, Figure 5).
4. Discussion
The present study showed that NMDA receptors antago-
nism or reducing the NO synaptic concentration, inhibit-
ing NO synthesis or scavenging NO, in the vMPFC in-
duced an increase in the number of punished licks in the
VCT, an anxiolytic-like effect, supporting previous re-
sults suggesting that these neurotransmitters, glutamate
and NO, have an important role in control of anxiety by
Figure 3. Effects of bilateral microinjection of 200 nL of
vehicle, 4 nmol of LY235959 (LY), 0.04 nmol of N-Propyl or
1 nmol of c-PTIO (n = 9, each treatment) outside the
vMPFC on the number of punished licks in the Vogel con-
flict test. Columns represent the mean and bars the SEM.
V- vehicle; Cg1 - cingulate cortex area 1; cc - corpus callo-
sum and TT- tenia tecta.
Figure 4. Effects of bilateral microinjection of 200 nL of
vehicle, 4 nmol of LY235959 (LY), 0.04 nmol of N-Propyl or
1 nmol of c-PTIO (n = 5, each treatment) in the vMPFC of
the rats submitted to evaluation of water consumption.
Columns represent the means and bars the S.E.M. of num-
ber of licks measured for each group in a 3 min period after
24 h (Day 1) and 48h (Day 2) of water deprivation.
Figure 5. Time course of the effects of i.p. administration of
vehicle or morphine 5 mg/kg or bilateral microinjections
200 nL of vehicle, 4 nmol of LY235959 (LY), 0.04 nmol of
N-Propyl or 1 nmol of c-PTIO (n = 5, each treatment) in the
vMPFC on the tail flick test. Each point represents the
mean and bars the SEM for the latency of tail withdrawal,
*P < 0.05 compared to vehicle (ANOVA followed by Bon-
ferroni’s post hoc test).

S. F. LISBOA ET AL.
185
brain structures like the dorsolateral periaqueductal gray,
inferior colliculus [25] and vMPFC [19]. Since these
results could reflect non-specific interference with water
consumption and/or nociceptive threshold [15], we also
tested the effects of these drugs in these two parameters.
The drugs, however, failed to change the number of un-
punished licks and tail flick latency. Also, no effects
were observed when the drugs were bilaterally injected
into nearby structures (Cg1, TT or cc). These results,
therefore, suggest that inactivation of these neurotrans-
mitters in vMPFC induces effects to anxiety in the VCT.
The present results confirm our previous work, which
supported a possible existence of a NMDA/NO pathway
in the vMPFC involved with anxiety-responses observed
in the contextual fear conditioning, since its NMDA re-
ceptors or NO blockade impaired the fear response, cha-
racterized by increased freezing behavior and auto-
nomic activity [19]. Both glutamatergic terminals and
NMDA receptors are present in the vMPFC of rats
[26,27] and, during stress conditions, glutamate levels
are increased in this cortical structure [18]. It is well
known that in central nervous system acute activation of
NMDA receptors results in an increase in NO synthesis
[28] by the activation of neuronial isoforma of NO syn-
thase (nNOS) and, as suggested by these studies, these
interaction could also occur in the vMPFC. Thus, the
modulation of this pathway in the vMPFC can be in-
volved with control of anxiety behavior.
These results also agree with studies which employed
systemic administration of NMDA antagonists. In this
context, Plaznik and cols using noncompetitive or com-
petitive NMDA receptor antagonists showed anxiolytic-
like effect in the VCT [29]. In addition, the NMDA an-
tagonist, MK801, attenuated anxious-like behavior in-
duced by exposing rats to a live cat, suggesting the in-
volvement of these receptors in the neural alterations
mediating disrupting-behavior effect of severe stress [30].
Another NMDA antagonist, the CGP37849, retained its
anxiolytic-like effect in the VCT after repeated systemic
administration and it was also able to increase explora-
tory behavior not related to motor activity [31]. Together
with our findings, these results could suggest that
vMPFC is a brain site of action of NMDA antagonists
after systemic administration.
The anxiolytic-like effect observed after blocking
NMDA/NO in the vMPFC in the present study and in the
previous one [19] corroborate previous findings showing
that temporary inhibition of vMPFC with cobalt chloride
induced an anxiolytic-like effect in the VCT and contex-
tual fear conditioning [13,17], supporting the view that
the neurotransmitters glutamate and NO in the vMPFC
are important to the modulation of anxiety. It is sug-
gested that the anxiolytic-like effect induced by blocking
NMDA/NO in the vMPFC could be related only to anxi-
ety induced by associative learned fear, like in the VCT
and the contextual fear conditioning, since in animal
models of unlearned innate fear, the elevated plus maze
and the light-dark box, the reversible blockade of vMPFC
induced anxiogenic-like effects [32]. The exact mecha-
nism involved in the modulation of anxiety behavior by
the NMDA/NO in the vMPFC is not entirely know, but
could also involve other brain structures. The vMPFC
projects to several regions related to autonomic and be-
havioral responses to an aversive stimulus, including the
amygdaloid nuclei, hippocampus, dorsal raphe nuclei
and dorsal periaqueductal gray [33,34].
In agreement with the present and previous results,
anxiolytic-like effects of GABA potentiation or gluta-
mate antagonist have been reported after direct drug in-
jection into most of these structures (for review, see [35].
Thus, it is possible that the bloking of the NMDA/NO in
the vMPFC could also leads to inhibition of excitatory
projections or desinhibition of inhibitory projections to
some of that structures related to induction of behavioral
responses to an aversive stimulus. In addition to simply
inhibit/desinhibit brain structures linked to vMPFC, an-
other explanation could be based on vMPFC activity,
which has been proposed to reflect an interaction be-
tween cognitive processing and emotional state [14,36].
It is well known that new environments are sources of
ambiguous stimuli and potential dangers. In the VCT, as
in the contextual fear conditioning, where the source of
danger is well defined, drinking spout in the VCT, the
local vMPFC inhibition of NMDA/NO, with consequent
inhibition of glutamate and NO neurotransmission,
would reduces the emotional impact of the threatening
stimulus, leading to an anxiolytic effect. It is speculated,
therefore, that inhibition of this possible pathway in the
vMPFC of animals submitted to animal models of un-
learned innate fear could induce opposed effects, but this
still have to be addressed.
5. Conclusions
In conclusion, the findings of the present work support
the view that NMDA receptors and NO presents in the
vMPFC could participate in the modulation of anxiety
behavior elicited by associative learned fear.
6. Acknowledgements
The authors wish to thank to Laura H. A. de Camargo,
Ivanilda A.C. Fortunato and José Carlos de Aguiar for
technical support. S.F. Lisboa is recipients of a PhD fel-
lowship from FAPESP (07/06999-9). Research sup-
ported by a grant from FAPESP (2009/03187-9), CNPq

S. F. LISBOA ET AL.
186
(470042/2009-5 and 305996/2008-8) and FAEPA.
7. Conflicts of Interest
The authors state that there are no conflicts of interests
that relate to this research.
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