Neuropharmacology 113 (2017) 156e166

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

CB1 cannabinoid receptor-mediated anandamide signalling reduces

the defensive behaviour evoked through GABAA receptor blockade
in the dorsomedial division of the ventromedial hypothalamus
Tayllon dos Anjos-Garcia a, Farhad Ullah a, Luiz Luciano Falconi-Sobrinho a,
Norberto Cysne Coimbra a, b, *
a ~o Preto Medical School of the University of Sa
Laboratory of Neuroanatomy and Neuropsychobiology, Department of Pharmacology, Ribeira ~o Paulo (USP),
~o Preto, Sa
Av. Bandeirantes 3900, Ribeira ~o Paulo, 14049-900, Brazil
b ~o Preto, Sa
NAP-USP-Neurobiology of Emotions (NuPNE) Research Centre, FMRP-USP, Ribeira ~o Paulo, Brazil

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

Article history: The effects of cannabinoids in brain areas expressing cannabinoid receptors, such as hypothalamic nuclei,
Received 12 August 2015 are not yet well known. Several studies have demonstrated the role of hypothalamic nuclei in the
Received in revised form organisation of behavioural responses induced through innate fear and panic attacks. Panic-prone states
22 March 2016
are experimentally induced in laboratory animals through a reduction in the GABAergic activity. The aim
Accepted 4 April 2016
Available online 8 April 2016
of the present study was to examine panic-like elaborated defensive behaviour evoked by GABAA re-
ceptor blockade with bicuculline (BIC) in the dorsomedial division of the ventromedial hypothalamus
(VMHdm). We also aimed to characterise the involvement of endocannabinoids and the CB1 cannabinoid
Keywords:
Endocannabinoid neuromodulators
receptor in the modulation of elaborated defence behavioural responses organised with the VMHdm. The
GABAA receptor guide-cannula was stereotaxicaly implanted in VMHdm and the animals were treated with anandamide
Ventromedial hypothalamus (AEA) at different doses, and the effective dose was used after the pre-treatment with the CB1 receptor
Defensive behaviour antagonist AM251, followed by GABAA receptor blockade in VMHdm. The results showed that the intra-
CB1 cannabinoid receptor hypothalamic administration of AEA at an intermediate dose (5 pmol) attenuated defence responses
TRPV1 channel induced through the intra-VMHdm microinjection of bicuculline (40 ng). This effect, however, was
inhibited when applied central microinjection of the CB1 receptor antagonist AM251 in the VMHdm.
Moreover, AM251 potentiates de non-oriented escape induced by bicuculline, effect blocked by pre-
treatment with the TRPV1 channel antagonist 6-I-CPS. These results indicate that AEA modulates the
pro-aversive effects of intra-VMHdm-bicuculline treatment, recruiting CB1 cannabinoid receptors and
the TRPV1 channel is involved in the AM251-related potentiation of bicuculline effects on non-oriented
escape behaviour.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction neurotransmitters, endocannabinoids are not stored in vesicles,

The endocannabinoid system is one of several systems that
regulate panic-like defence responses. The term endocannabinoid
has been used to designate endogenous ligands, such as ananda-
mide (AEA) and 2-arachidonoylglycerol (2-AG). These substances
are endogenous lipids, synthesised from neuronal membranes in
response to elevated intracellular calcium. Unlike classical
* Corresponding author. Laboratorio de Neuroanatomia & Neuropsicobiologia,
~o Preto da Universidade de Sa
Faculdade de Medicina de Ribeira ~o Paulo (FMRP-USP),
Avenida dos Bandeirantes 3900, Ribeira~o Preto, Sa
~o Paulo, 14049-900, Brasil.
E-mail address: nccoimbr@fmrp.usp.br (N.C. Coimbra).
and these molecules activate cannabinoid receptor types 1 and 2
(CB1 and CB2, respectively) (Pertwee et al., 2010).
Agonist stimulation activates a number of signal transduction
pathways via Gi/o, a member of the G protein family. The activity of
anandamide is limited by the re-uptake via the anandamide
transporter and subsequent metabolic action of the fatty acid
amide hydrolase enzyme (FAAH). After activation, these receptors
inhibit adenylyl cyclase and activate mitogen-activated protein ki-
nase, thereby decreasing the excitability of the pre-synaptic ter-
minal through the inhibition of calcium influx currents and
inwardly rectifying potassium channels (Devane et al., 1988;
Demuth and Molleman, 2006).

http://dx.doi.org/10.1016/j.neuropharm.2016.04.003
0028-3908/© 2016 Elsevier Ltd. All rights reserved.
 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166
Unlike the CB2 receptors in the cells and tissues of immune
system, the CB1 receptor is commonly observed in the central
nervous system (Pertwee et al., 2010). A modest number of CB1
receptors have been identified in the hypothalamus (Pettit et al.,
1998), and immunoreactive staining was also detected in the par-
aventricular nucleus, lateral hypothalamic region, the infundibular
stem (Tsou et al., 1998) and suprachiasmatic, supraoptic, and par-
aventricular nuclei. Interestingly, slightly elevated ligand binding
forms a rim around the ventromedial nucleus of the hypothalamus
(Herkenham et al., 1991).
Studies have shown that the activation of the CB1 receptor, in-
hibition of FAAH or inhibition of endocannabinoid reuptake pro-
duces anxiolytic-like effects in animal models of anxiety (Micale
et al., 2009). Microinjections of the CB1 agonist in the medial pre-
frontal cortex (Rubino et al., 2008), central amygdala (Zarrindast
et al., 2008) and periaqueductal grey matter (Finn et al., 2003)
also produce anxiolytic-like responses.
On the other hand, microinjections of the GABAA receptor
antagonist bicuculline into structures with limbic functions, i.e.,
mesencephalic tectum (Branda ~o et al., 1988) and diencephalic
structures (Milani and Graeff, 1987) have been used to evoke
defensive reactions commonly related to anxiety and mainly panic
state in laboratory animals (bicuculline panic-like effects). How-
ever, there is evidence that the defensive behaviours were more
explosive when bicuculline was injected into mesencephalic
structures and more oriented when was injected into diencephalic
structures (Di Scala et al., 1984). These two kinds of escape are
typically considered panic-like behaviours, in contrast with
anxiety-like behaviours, such as defensive attention (alertness), flat
back approaching and stretch attend posture, or risk assessment
(Blanchard et al., 2001; Borelli et al., 2004, 2005; Shekhar, 1994).
Indeed, these behavioural responses are commonly displayed by
threatened preys in dangerous situations, like prey vs snake
confrontation paradigms (Almada et al., 2015; Almada and
Coimbra, 2015; Twardowschy et al., 2013; Uribe-Marin ~ o et al.,
2012).
In addition, it was recently demonstrated that bicuculline
treatment in dorsomedial and posterior hypothalamus (DMH and
PH, respectively) induces oriented escape expresses by organised
running towards a safe place, while the same treatment in dorsal
columns of periaqueductal grey matter (dPAG) induces non-
oriented escape expressed by explosive/non-oriented running
(Biagioni et al., 2016b). Similar results were obtained with micro-
injections of N-methyl-d-aspartic acid (NMDA) at different con-
centrations either in dorsal midbrain or in medial hypothalamus;
again, explosive/non-oriented reactions were elicited after the
chemical stimulation of the midbrain tectum, while more oriented
escape reactions were elicited by chemical stimulation of hypo-
thalamic nuclei (Ullah et al., 2015).
In this sense, several studies have shown the involvement of the
ventromedial hypothalamus in organisation of panic-like defensive
reactions in rodents (de Freitas et al., 2013; Freitas et al., 2009). In
fact, the deep brain stimulation of the VMH performed in an awake
patient was proved to be involved in the genesis of panic attacks
(Wilent et al., 2010). More specifically, the dorsomedial division of
the ventromedial hypothalamus (VMHdm) has been proposed as a
key diencephalic subnucleus responsible for the integration of
these responses (Canteras and Graeff, 2014). Despite these evi-
dence, it has been demonstrated repeatedly that the
endocannabinoid-signalling system modulates defensive re-
sponses in different structures of the brain aversion system,
without mentioning the role played by the endocannabinoid sys-
tem of hypothalamic nuclei in the control of defence-related
behaviours.
Thus, considering the presence of CB1 receptors in the VMHdm,
157
we hypothesised that the administration of the cannabinoid re-
ceptor agonist anandamide in the VMHdm will reduce the intensity
of defensive behaviours elicited by the intradiencephalic GABAA
receptor blockade, via CB1-cannabinoid receptor activation.
2. Material and methods
2.1. Animals and experimental apparatus
Male Wistar rats (Rattus norvegicus, Rodentia, Muridae),
weighing 230e270 g (n ¼ 6e9 per group), were bred in the animal
~o Preto Medical School of the University of Sa
facility at the Ribeira ~o
Paulo (FMRP-USP). The rats were housed in groups of ten animals
(habituation section) in a polygonal (rectangular) arena with
154 cm in length, 72 cm in width and 64 cm in height that consists
in a semi-transparent acrylic enclosure with a light-reflector film,
providing 80% light reflection (Biagioni et al., 2016b; Ullah et al.,
2015). The floor of the arena was constructed from a transparent
acrylic platform (divided into 20 equal rectangles) and was placed
on a rectangular stainless steel plaque beneath the arena. To
minimise vibration, the entire apparatus was placed on a granite
rock surface (170 cm in length, 85 cm in width and 2 cm in height)
elevated 83 cm above the floor. A burrow with black acrylic walls
(36 cm in length, 26 cm in width and 12.5 cm in height) inside the
rectangular arena was placed in one corner of the polygonal
enclosure. This burrow had two entries located on opposite sides,
thus allowing the rats to enter and exit from the burrow in two
different positions to the polygonal arena. We used a translucent
ceiling for the burrow to prevent place preference (Prus et al., 2009)
during each neuropharmacological experiment. During the three
days of habituation in this apparatus, the animals were maintained
in a 12 h light/dark cycle in an air-conditioned room (24 ± 1  C)
with open access to food and water, except during the experimental
procedures performed in the light cycle (Almada and Coimbra,
2015). The experimental procedures were performed in accor-
dance with the recommendations of the Commission of Ethics in
Animal Experimentation of the FMRP-USP (CEUA-FMRP-USP pro-
cess 107/2012). These recommendations are in agreement with the
ethical principles of animal research adopted by the Brazilian Col-
lege of Animal Experimentation (COBEA) and approved by the
FMRP-USP Commission of Ethics in Animal Research (CETEA).
2.2. Drugs
The following drugs were used: GABAA receptor antagonist
[R-(R*,S*)]-5-(6,8-Dihydro-8-oxofuro[3,4-e]-1,3-benzodioxol-6-yl)-
5,6,7,8-tetrahydro-6,6-dimethyl-1,3-dioxolo[4,5-g]isoquinolinium
iodide (Bicuculline; Tocris Bioscience, Bristol, UK) at 40 ng diluted
in 0.9% NaCl (Biagioni et al., 2013); the cannabinoid and
vanilloid receptor agonist, (2-Hydroxyethyl)-5Z,8Z,11Z,14Z-eicosa-
tetraenamide (Anandamide; Tocris Bioscience, Bristol, UK), at 0.5, 5
and 50 pmol was diluted in Tocrisolve TM100, a solvent containing a
1:4 ratio of soybean oil/water, emulsified with the block
co-polymer Pluronic F68 (Moreira et al., 2007); the CB1
cannabinoid antagonist N-(Piperidin-1-yl)-5-(4-iodophenyl)-
1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide
(AM251; Tocris Bioscience, Bristol, UK) at 100 pmol diluted in 10%
DMSO (Almeida-Santos et al., 2013); the TRPV1 (transient receptor
potential cation channel, subfamily V, member 1) channel antago-
nist 6-Iodonordihydrocapsaicin (6-I-CPS; Tocris Bioscience, Bristol,
UK) at 3 nmol diluted in 100% DMSO (Fogaça et al., 2012).
2.3. Surgical procedure
The animals were anaesthetised with 92 mg/kg ketamine
158 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166
(Ketamine Agener, Unia ~o Química Farmace ^utica Nacional, Brazil)
and 9.2 mg/kg xylazine (Dopaser®, Hertape/Calier, Juatuba, Minas
Gerais, Brazil) and secured in a stereotaxic frame (David Kopf,
Tujunga, California, USA). A stainless steel guide-cannula (0.6 mm
outer diameter and 0.4 mm inner diameter) was implanted in the
diencephalon to target the dorsomedial division of ventromedial
hypothalamus (VMHdm). The upper incisor bar was set 3 mm
below the interaural line, such that the skull was horizontal be-
tween the bregma and lambda. The guide-cannula was vertically
introduced using the coordinates AP ¼ 2.4 mm, ML ¼ 0.3 mm,
DV ¼ 8.1 mm, with the bregma as a reference, according to the
atlas of Paxinos and Watson (2007). The guide-cannula was fixed to
the skull using acrylic resin and one stainless steel screw. Each
guide-cannula was sealed with a stainless steel wire for protection
from obstruction. After surgery, each rodent was treated with an
intramuscular injection of penicillin G-benzatine (120.000UI/
0.2 mL) followed by an intramuscular injection of the analgesic and
anti-inflammatory flunixin meglumine (2.5 mg/kg). The animals
were maintained in post-operative recovery for five days.
2.4. Procedure
The experiment was performed using independent groups of
animals (n ¼ 6e9 per group) after five days of stereotaxic surgery
and three days of habituation. Habituation was conducted to
generate a non-aversive and safe environment for the rat (Almada
et al., 2015; Almada and Coimbra, 2015; Biagioni et al., 2016b;
Twardowschy et al., 2013; Ullah et al., 2015; Uribe-Marin ~ o et al.,
2012). On the day of the experiment, the animals were gently
shifted from the polygonal arena to the home cages and were
randomly assigned to one of the intradiencephalic treatments
(intra-hypothalamic microinjections). Counterbalanced microin-
jections (either at right or left brain hemisphere) were performed
using a dental needle (0.3 mm OD), at a length 1 mm longer than
the guide-cannula. The drugs were injected through polyethylene
tube (PE-10) for 15 s using a microsyringe (Hamilton, Reno, Nevada,
USA) connected to an infusion pump (Stoelting, Kiel, Wisconsin,
USA) through polyethylene tube (PE-10). To ensure the movement
of the drug, a bubble was created between the distilled water col-
umn and the drug. In all cases, the respective diluents were used as
controls in the same volume used for drugs.
In the first set of experiments, the animals initially received an
intra-VMHdm microinjection of anandamide (AEA 0.5, 5 or
50 pmol) or vehicle (veh), followed by another microinjection with
bicuculline (BIC 40 ng) or vehicle (veh) 5 min later. Immediately
after the last microinjection, the animals were placed inside the
rectangular arena to record the behavioural responses for 10 min. In
this experiment, a volume of 0.2 mL was injected for each drug and
respective diluents (veh) into the VMHdm comprising 0.4 mL per
animal.
In the second set of experiments, the animals initially received
an intra-VMHdm microinjection of AM251 (AM251 100 pmol) or
vehicle (veh), followed by another microinjection with anandamide
(AEA 5 pmol) or vehicle (veh) 5 min later. At 5 min after the second
microinjection, the animals received a final intra-VMHdm admin-
istration of bicuculline (BIC 40 ng) or vehicle (veh). Immediately
after the last microinjection, the animals were placed inside the
rectangular arena to record the behavioural responses for 10 min. In
this experiment, a volume of 0.1 mL for each drug and respective
diluents (veh) ware injected into the VMHdm comprising 0.3 mL per
animal.
In the third set of experiments, the animals initially received an
intra-VMHdm microinjection of 6-I-CPS (6-I-CPS 3 nmol) or vehicle
(veh), followed by another microinjection with AM251 (AM251
100 pmol) or vehicle (veh) 5 min later. Five minutes after the
second microinjection, the animals received a final intra-VMHdm
administration of bicuculline (BIC 40 ng) or vehicle (veh). Imme-
diately after the last microinjection, the animals were placed inside
the rectangular arena to record the behavioural responses for
10 min. In this approach, we used a volume of 0.1 mL for each drug
and its respective diluents (veh) microinjected into the VMHdm,
comprising 0.3 mL per animal.
Considering previous evidence suggesting that the diffusion of
substances microinjected in the central nervous system into the
tissue surrounding the site of injection is directly related to the
volume administered (Myers, 1966; Routtenberg, 1972), we
assumed that 0.5 mL causes an average spread of 1.04 mm, consis-
tent with Myers (1966), and volumes no larger than 0.4 mL were
microinjected into the VMHdm. Doses (Almeida-Santos et al., 2013;
Biagioni et al., 2013; Fogaça et al., 2012; Moreira et al., 2007) and
drug treatments (Batista et al., 2015; Lisboa and Guimara ~es, 2012)
were based on the literature.
2.5. Behavioural recordings
The behavioural responses of the rats were recorded for 10 min
using a camera (Sony Handcam HDR-CX350, Konan, Minato-ku,
Tokyo, Japan). Subsequently, the behavioural responses were ana-
lysed using the free software X-Plo-Rat version 1.1.0, developed at
the Laboratory of Exploratory Behaviour of Ribeira ~o Preto School of
Philosophy, Sciences and Literature of the University of S~ ao Paulo.
This software does not perform the automatic measurement of
behaviours, the experimenter evaluates the behaviour and uses the
software to help quantify the number and duration of each
behavioural responses.
The behavioural analysis included defensive attention (alert-
ness), defined as an interruption of on-going behaviour when the
rodents displays attentive posture with small head movements,
rearing and scan environment by smelling the surrounding air.
Defensive immobility (freezing) was considered when the rat
showed immobility during at least 5s followed by autonomic re-
actions, such as defecation, exophthalmia and/or micturition
(Biagioni et al., 2013, 2016a, 2016b).
The number of crossing (stepping with four legs within a
delimited rectangle in the arena after crossing the border of each
section line) was recorded for quantitatively measurement of escape
behaviour expressed by running. The escape behaviour was classi-
fied as either non-oriented or oriented escape. Non-oriented escape
was characterised by meaningless running in different directions of
the arena, whereas oriented escape was more elaborate, charac-
terised by running to the burrow and shifting the directions of the
running toward the burrow position (Almada et al., 2015; Almada
and Coimbra, 2015; Twardowschy et al., 2013; Uribe-Marin ~ o et al.,
2012). The climbing (to the border of the arena or burrow) and
vertical jumping behaviours were also considered as oriented
escape. According to those authors and supported by Blanchard
et al. (2001), Borelli et al. (2004, 2005) and Shekhar (1994), escape
and freezing behaviour are typically considered panic-like re-
sponses unlike alertness which is considered anxiety-like responses.
The time spent outside the burrow was also recorded.
2.6. Histology
Upon completion of the experiments, the animals were anaes-
thetised with pentobarbital (40 mg/kg) 1 mL/100 g of 5% chloral
hydrate (Vetec Química Fina®, Duque de Caxias, Rio de Janeiro,
Brasil) and perfused through the left ventricle using a perfusion
pump (Master Flex® L/S™ peristaltic tubing pump, East Bunker
Court Vernon Hills, Illinois, USA). The blood was washed out using
40 mL of cold, oxygenated, Caþþ -free Tyrod's buffer followed by 200
 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166 159

of ice-cold 4% (w/v) paraformaldehyde (LabSynth, Brazil) in 0.1 M
PBS (LabSynth, Brazil), pH 7.3, over 15 min, at a pressure of
50 mmHg. The brain was immediately removed and soaked for 4 h
in fresh fixative (4% paraformaldehyde) at 4  C. After fixation, the
brain was sectioned, and the diencephalon was immersed in 10%
and 20% sucrose dissolved in 0.1 M sodium phosphate buffer (pH
7.3) at 4  C for at least 12 h in each solution. The tissue pieces were
frozen in isopentane (Sigma Aldrich, St. Louis, Missouri, USA), stored
on dry ice, embedded in Tissue Tek and cut using a cryostat (CM
1950 Leica, Wetzlar, Germany). The slices were subsequently
mounted on glass slides (coated with chrome alum gelatine to
prevent detachment) and stained with haematoxylin-eosin using an
autostainer (CV 5030 Autostainer XL, Leica, Wetzlar, Germany). The
positions of the guide-cannula tips were defined according to the
brain stereotaxic atlas by Paxinos and Watson (2007) under a
motorised photomicroscope (AxioImager Z1; Zeiss, Oberkochen,
Germany). The data from rats with guide-cannula tips located
outside the VMHdm were not included in the statistical analyses, but
as an additional control, these animals were analysed together in an
OUT group and compared with groups only treated with vehicle.
2.7. Statistical analysis
The results, expressed as scatter dot plot showing the
mean ± standard error of the mean (S.E.M.), were analysed using
one- or three-way analysis of variance (ANOVA), depending on the
treatments compared, followed by Newman-Keuls post hoc test
when appropriated. Both experiments were analysed using treat-
ment as main factor. Results with p < 0.05 were considered sta-
tistically significant.
3. Results
3.1. Histologic confirmation of the microinjection sites
All rats used in the present work received microinjections in the
VMHdm, as shown in Fig. 1. Although counterbalanced microin-
jections were made, all sites of microinjection of drugs in the
diencephalon were represented in the same side of the brain.
3.2. Anandamide (AEA) attenuated the defensive behaviours
induced after microinjections of bicuculline (BIC) into the VMHdm
The antagonism of GABAergic receptors in the VMHdm after
microinjection with the GABAA receptor antagonist bicuculline
induced defensive behaviours associated with anxiety and fear,
characterised by alertness and escape. In the present study, defen-
sive immobility behaviour was not observed after any treatment.
The number of crossing reflected a quantitative measurement
of running behaviour displayed by rodents submitted to the

Fig. 1. Histologically confirmed localisation of the microinjection sites (filled circles) in modified diagrams of Paxinos and Watson's (2007). Injections sites outside (X) the dor-
somedial division of the ventromedial hypothalamus (VMHdm) are also displayed. The image presented on the lower right is an original photomicrograph of a diencephalic coronal
section at bregma 2.64 mm, showing a representative site (arrow) of the central microinjection of drugs into the VMHdm. Scale bar: 500 mm, stained with haematoxylin and eosin.
160 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166

pharmacological treatments. One-way ANOVA showed a significant
effect of the treatment on number of crossings (F(7,53) ¼ 12.44;
p < 0.001). Newman-Keuls post hoc test showed that, in compari-
son with the veh-veh treatment, the veh-BIC treatment signifi-
cantly increased the number of crossing (p < 0.001). Only pre-
treatment with 5 pmol of AEA decreased this behaviour
(p < 0.05). AEA (5 pmol) per se and microinjections outside VMHdm
had no effect compared with the veh-veh treatment (p > 0.05,
Fig. 2A).
One-way ANOVA showed a significant effect of treatment on
time spent outside the burrow (F(7,53) ¼ 15.71 p < 0.001). Newman-
Keuls post hoc tests showed that compared with the veh-veh
treatment, the veh-BIC treatment significantly increased the time
spent outside the burrow (p < 0.001). Only pre-treatment with
5 pmol of AEA decreased this time (p < 0.05). AEA (5 pmol) per se
and microinjections outside VMHdm had no effect compared with
the veh-veh treatment (p > 0.05, Fig. 2B). Regarding the alertness,
one-way ANOVA showed a significant effect of treatment on
number (F(7,53) ¼ 14.15; p < 0.001) and duration (F(7,53) ¼ 11.40;
p < 0.001). Newman-Keuls post hoc tests showed that compared
with the veh-veh treatment, the veh-BIC treatment significantly
increased the number (p < 0.001) and duration (p < 0.001) of
alertness. Pre-treatment with AEA (0.5, 5 and 50 pmol) decreased
the number (p < 0.001) and duration (p < 0.001) compared
with veh-BIC treatment. AEA (5 pmol) per se and microinjections
outside VMHdm had no effect compared with veh-veh treatment
(p > 0.05, Fig. 2C and D).
The escape behavioural responses were classified as non-
oriented (Fig. 3A and B) and oriented (Fig. 3C and D) escape
behaviour. The one-way ANOVA showed a significant effect of
treatment on both non-oriented escape (number, F(7,53) ¼ 10.21;
p < 0.001 and duration F(7,53) ¼ 7.59; p < 0.001) and oriented escape
(number, F(7,53) ¼ 16.75; p < 0.001 and duration, F(7,53) ¼ 12.27;
p < 0.001). In all cases, Newman-Keuls post hoc tests showed that
treatment with veh-BIC significantly increased all responses
(p < 0.001). Pre-treatment with AEA (0.5 and 5 pmol) decreased the
number and duration of non-oriented escape (p < 0.001) compared
with veh-BIC treatment. Regarding oriented escape, pre-treatment
with AEA (0.5 and 5 pmol) decreased the number (p < 0.05 and
p < 0.001, respectively), but only treatment with 5 pmol AEA
decreased the duration (p < 0.001) of oriented escape. In both cases,
AEA (5 pmol) per se and microinjections outside VMHdm had no
effect compared with veh-veh treatment (p > 0.05).
3.3. An intermediate dose of anandamide (AEA) attenuated the
defensive behaviour induced by bicuculline (BIC) microinjection into
the VMHdm via CB1 receptors
According to the results of first experiment, bicuculline-induced
defensive behaviours are characterised by alertness and escape,
and defensive immobility was not observed. Similarly, intra-
diencephalic pre-treatment with 5 pmol anandamide, followed
by bicuculline microinjection into the VMHdm, reduces defensive
responses.
Three-way ANOVA showed a significant interaction concerning
the number of crossing between the first and third injection
(F(7,53) ¼ 9.18; p < 0.05) and between the second and third injec-
tion (F(7,53) ¼ 10.33; p < 0.05). Newman-Keuls post hoc tests
showed that BIC treatment increased the number of crossing,
whereas AEA treatment significantly decreased this behaviour
(F(7,53) ¼ 44.64; p < 0.001, Fig. 4A). These effects were prevented
after pre-treatment with AM251 at 100 pmol (p > 0.05). The

Fig. 2. The effect of the central administration of anandamide (AEA 0.5, 5 or 50 pmol/0.2 mL), followed by the intra-VMHdm injection of bicuculline (BIC 40 ng/0.2 mL) or vehicle
(veh) on the number of crossing (A), time spent outside the burrow (B) and number and duration of alertness behaviours (C and D). The animals treated with microinjections of
drugs outside of the VMHdm were analysed and assembled into an OUT-group. The data are expressed as the mean ± S.E.M. (scatter dot plot; each dot representing one response
per animal); n ¼ 8, 8, 8, 9, 7, 7, 7 and 7, per group, respectively from left to right. *p < 0.05 and ***p < 0.001, compared with the veh-veh-treated group. #p < 0.05 and ##p < 0.01,
compared with the veh-BIC group (ANOVA, followed by Newman-Keuls post hoc test).
 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166 161

Fig. 3. The effect of the central administration of anandamide (AEA 0.5, 5 or 50 pmol/0.2 mL), followed by the intra-VMHdm injection of bicuculline (BIC 40 ng/0.2 mL) or vehicle
(veh) on the number and duration of non-oriented (A and B) or oriented (C and D) escape. The animals treated with microinjections outside of the VMHdm were analysed and
assembled into an OUT-group. The data are expressed as the mean ± S.E.M. (scatter dot plot; each dot representing one response per animal); n ¼ 8, 8, 8, 9, 7, 7, 7 and 7, per group,
respectively from left to right. **p < 0.01 and ***p < 0.001, compared with the veh-veh-treated group. #p < 0.05 and ###p < 0.001, compared with the veh-BIC-treated group
(ANOVA, followed by Newman-Keuls post hoc test).

Fig. 4. The effect of the central administration of the cannabinoid CB1 receptor antagonist AM251 (100 pmol/0.1 mL), followed by microinjections of anandamide (AEA 5 pmol/0.1 mL)
on the effect of the intra-VMHdm injection of bicuculline (BIC 40 ng/0.1 mL) on the number of crossing (A), time spent outside the burrow (B) and number and duration of alertness
(C and D). The data are expressed as the mean ± S.E.M. (scatter dot plot; each dot representing one response per animal); n ¼ 8, 6, 9, 8, 7, 7, 8 and 8 per group, respectively from left
to right. *p < 0.05 and ***p < 0.001, compared with veh-veh-veh-treated group. ##p < 0.01 and ###p < 0.001, compared with veh-veh-BIC-treated group (ANOVA, followed by
Newman-Keuls post hoc test).
162 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166

Fig. 5. The effect of the central administration of the cannabinoid CB1 receptor antagonist AM251 (100 pmol/0.1 mL), followed by microinjections of anandamide (AEA 5 pmol/0.1 mL)
on the effect of the intra-VMHdm injection of bicuculline (BIC 40 ng/0.1 mL) on the number and duration of non-oriented (A and B) or oriented escape (C and D). The data are
expressed as the mean ± S.E.M. (scatter dot plot; each dot representing one response per animal); n ¼ 8, 6, 9, 8, 7, 7, 8, and 8, per group, respectively from left to right. **p < 0.01 and
***p < 0.001, compared with veh-veh-veh-treated group. ##p < 0.01 and ###p < 0.001, compared with veh-veh-BIC-treated group (ANOVA, followed by Newman-Keuls post hoc
test).

AM251-veh-BIC group showed a tendency (p ¼ 0.614) in increase
the number of crossing compared with veh-veh-BIC group. A sig-
nificant interaction in the time spent outside the burrow was
observed between the first and second injection (F(7,53) ¼ 4.90;
p < 0.05). Newman-Keuls post hoc test showed that BIC treatment
increased the time spent outside the burrow, whereas AEA treat-
ment significantly decreased this time (F(7,53) ¼ 31.05; p < 0.001,
Fig. 4B), which was prevented by the VMHdm pre-treatment with
AM251 at 100 pmol (p > 0.05).
Three-way ANOVA showed a significant interaction only in
duration of alertness behaviour between the first and the second
injection (number, F(7,53) ¼ 3.44; p > 0.05 and duration
F(7,53) ¼ 4.42; p < 0.05). Newman-Keuls post hoc test showed that
AEA significantly attenuated the alertness behaviour induced by
VMHdm treatment with bicuculline (number, F(7,53) ¼ 8.46;
p < 0.001 and duration F(7,53) ¼ 6.48; p < 0.001, Fig. 4C and D). In all
cases, the VMHdm pre-treatment with AM251 (100 pmol) did not
change the observed responses compared with veh-veh-BIC
treatment (p > 0.05), and the respective control treatments had
no effect compared with veh-veh-veh treated-group (p > 0.05).
Regarding escape, three-way ANOVA showed a significant
interaction between the first and third injections (number,
F(7,53) ¼ 12.27; p < 0.05 and duration, F(7,53) ¼ 12.75; p < 0.05) and
second and third injections (number, F(7,53) ¼ 13.26; p < 0.05 and
duration, F(7,53) ¼ 13.51; p < 0.05) for non-oriented escape, whereas
a significant interaction was observed between the first and second
injections (number, F(7,53) ¼ 8.27; p < 0.05 and duration,
F(7,53) ¼ 17.47; p < 0.05), between the first and third injections
(number, F(7,53) ¼ 6.19; p < 0.05 and duration, F(7,53) ¼ 7.84;
p < 0.05), second and third injections (number, F(7,53) ¼ 12.57;
p < 0.05 and duration, F(7,53) ¼ 5.25; p < 0.05) and interaction be-
tween this three injections (number, F(7,53) ¼ 6.11; p < 0.05 and
duration, F(7,53) ¼ 9.66; p < 0.05) for oriented escape. Similar to the
first experiment, the Newman-Keuls post hoc tests showed that
pre-treatment with AEA significantly attenuated the non-oriented
(number, F(7,53) ¼ 16.46; p < 0.001 and duration, F(7,53) ¼ 16.36;
p < 0.001) and oriented (number, F(7,53) ¼ 24.97; p < 0.001 and
duration, F(7,53) ¼ 23.72; p < 0.001) escape induced with bicuculline
microinjection in the VMHdm. These effects were prevented after
VMHdm pre-treatment with AM251 at 100 pmol (p > 0.05). In
contrast to oriented escape, the VMHdm pre-treatment with
AM251 at 100 pmol (AM251-veh-BIC treatment) significantly
increased (p < 0.001) the effects of bicuculline (veh-veh-BIC
treatment) for non-oriented escape behaviour. No effects were
observed in the control groups compared with the veh-veh-veh
treatment group (p > 0.05, Fig. 5A, B, C and D).
3.4. TRPV1 (transient receptor potential cation channel, subfamily
V, member 1) channel is involved in the AM251-related potentiation
of bicuculline effects on non-oriented escape
According to the results from the second experiment, the
VMHdm treatment with bicuculline induced defensive responses
and AM251 potentiated the facilitatory effect of bicuculline on non-
oriented escape behaviour. However, this proaversive effect was
prevented by the blockade of TRPV1 channel.
One-way ANOVA showed a significant effect of the treatment on
number of crossings (F(3,27) ¼ 43.72; p < 0.001). Newman-Keuls
post hoc test showed that VMHdm treatment with bicuculline
elicited escape behaviour with enhancement of the number of
crossings, whereas AM251 treatment potentiated this response
(p < 0.01). These effects were, however, prevented by VMHdm pre-
treatment with 6-I-CPS at 3 nmol (p > 0.05, Fig. 6A). One-way
ANOVA showed a significant effect of treatment on time spent
outside the burrow (F(3,27) ¼ 57.09; p < 0.001). Newman-Keuls post
hoc tests showed that VMHdm treatment with bicuculline
 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166 163

Fig. 6. The effect of the central administration of the TRPV1 (transient receptor potential cation channel, subfamily V, member 1) vanilloid antagonist 6-Iodonordihydrocapsaicin (6-
I-CPS; 3 nmol/0.1 mL), followed by microinjections of the CB1 cannabinoid receptor antagonist AM251 (100 pmol/0.1 mL) on the effect of the dorsomedial division of the ventromedial
hypothalamus (VMHdm) treatment with bicuculline (BIC 40 ng/0.1 mL) on the number of crossing (A), time spent outside the burrow (B) and number and duration of alertness (C
and D). The data are expressed as the mean ± S.E.M. (scatter dot plot; each dot representing one response per animal); n ¼ 8, 8, 8 and 7 per group, respectively from left to right.
**p < 0.01 and ***p < 0.001, compared with veh-veh-veh-treated group. ##p < 0.01 compared with veh-veh-BIC-treated group (according to ANOVA, followed by Newman-Keuls
post hoc test).

significantly increased the time spent outside the burrow
(p < 0.001). Any VMHdm pre-treatment changed this response
(p > 0.05, Fig. 6B). One-way ANOVA showed a significant effect of
treatment on number (F(3,27) ¼ 11.25; p < 0.001) and duration
(F(3,27) ¼ 7.30; p < 0.01) of alertness. Newman-Keuls post hoc tests
showed that VMHdm treatment with bicuculline significantly
increased the number (p < 0.001) and duration (p < 0.01) of
alertness and any VMHdm pre-treatment changed this response
(p > 0.05; Fig. 6C and D).
Regarding the escape behaviour, one-way ANOVA showed a
significant effect of treatment on number (F(3,27) ¼ 11.38; p < 0.001)
and duration (F(3,27) ¼ 9.51; p < 0.001) of non-oriented escape
behaviour. Newman-Keuls post hoc tests showed that VMHdm
treatment with bicuculline (veh-veh-BIC treatment) significantly
increased the number (p < 0.01) and duration (p < 0.05) of non-
oriented escape behaviour and VMHdm pre-treatment with
AM251 at 100 pmol (veh-AM251-BIC treatment) potentiated the
proaversive effect of VMHdm treatment with bicuculline (number
and duration of non-oriented escape behaviour, p < 0.05 in both
cases). These effects were prevented by VMHdm pre-treatment
with 6-I-CPS at 3 nmol (p > 0.05; Fig. 7A and B), reflecting on the
increasing of crossings behaviour observed previously. There was a
significant effect of treatment on number (F(3,27) ¼ 9.42; p < 0.001)
and duration (F(3,27) ¼ 12.94; p < 0.001) of oriented escape, post hoc
tests showed that VMHdm treatment with bicuculline significantly
increased the number (p < 0.001) and duration (p < 0.001) of ori-
ented escape and any diencephalic pre-treatment changed this
behavioural responses (p > 0.05 in all cases; Fig. 7C and D).
4. Discussion
The polygonal arena comprises a complex experimental envi-
ronment with an artificial burrow for the study and discrimination
between the oriented and non-oriented escape behaviours
(Biagioni et al., 2016b; Ullah et al., 2015). In the present study, we
provided the first evidence that the intra-VMHdm administration
of bicuculline induced anxiety and panic-like responses attenuated
by an intermediate dose of anandamide, which was prevented after
microinjections of the CB1 receptor antagonist.
Studies have shown that the inhibition of GABAergic neuro-
transmission or administration of excitatory amino acids in the
mesencephalic tectum (Branda ~o et al., 1988; Ullah et al., 2015) or in
diencephalic structures, such as the hypothalamus (Biagioni et al.,
2013, 2016a, 2016b; de Freitas et al., 2014; Freitas et al., 2009;
Milani and Graeff, 1987; Ullah et al., 2015), induces panic-like
defence reactions in rodents. However, there is evidence that the
defensive behaviour organised through hypothalamic neurons is
rich in exploratory responses and vertical jumps compared with
more explosive reactions elaborated through dorsal midbrain
structures (Brand~ ao et al., 1986; Coimbra et al., 2006; Di Scala et al.,
1984; Schmitt et al., 1985). These studies suggest that the hypo-
thalamus is responsible for integrating more elaborate behavioural
responses compared with more explosive/non-oriented responses
organised through midbrain structures, as recently reported (Ullah
et al., 2015). These responses are characterised by less intense in-
crease in coordinated escape to a safe place, interspersed by
alertness behaviour (Biagioni et al., 2012; Branda ~o et al., 1986; Di
Scala et al., 1984; Freitas et al., 2009; Ullah et al., 2015).
Interestingly, regarding the escape response, we observed that
the number of oriented escape responses was two-fold higher than
the number of non-oriented escape responses. Oriented responses,
such as climbing the walls of the arena, climbing the roof of the
burrow, escaping to the burrow and vertical jumping, suggest
behavioural responses directed to a safe place. Indeed, the pattern
of responses evoked through the chemical stimulation of hypo-
thalamic areas are more elaborated (Branda ~o et al., 1986; Di Scala
164 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166

Fig. 7. The effect of the central administration of TRPV1 (transient receptor potential cation channel, subfamily V, member 1) vanilloid antagonist 6-Iodonordihydrocapsaicin (6-I-
CPS; 3 nmol/0.1 mL), followed by microinjections of cannabinoid CB1 receptor antagonist AM251 (100 pmol/0.1 mL) on the effect of the dorsomedial division of the ventromedial
hypothalamus (VMHdm) treatment with bicuculline (BIC 40 ng/0.1 mL) on the number and duration of non-oriented (A and B) or oriented escape (C and D). The data are expressed
as the mean ± S.E.M. (scatter dot plot; each dot representing one response per animal); n ¼ 8, 8, 8, and 7, per group, respectively from left to right. *p < 0.05, **p < 0.01 and
***p < 0.001, compared with veh-veh-veh-treated group. #p < 0.05 compared with veh-veh-BIC-treated group (according to ANOVA, followed by Newman-Keuls post hoc test).

et al., 1984; Freitas et al., 2009) compared with the chemical
stimulation of mesencephalic structures (Coimbra et al., 2006).
Escape and defensive immobility are susceptible to panicolytic
drugs (Blanchard et al., 1997; McNaughton and Corr, 2004).
Consistent with the data obtained in the present study, ananda-
mide promotes panicolytic- and anxiolytic-like effects or generally
anti-aversive effects, considering that this endocannabinoid
reduced both escape and alertness behavioural responses, respec-
tively, elicited after VMHdm treatment with bicuculline. Moreover,
the anti-aversive effect of anandamide followed a bell-shaped
dose-response curve, suggesting that intra-hypothalamic admin-
istrations of anandamide induce different responses at low, inter-
mediate and high doses. In contrast to the classical inverted U-
shaped dose-response curve of endocannabinoids, U-shaped dose-
response curves were generated in the present study. This differ-
ence might reflect differences in the aversion caused by central
bicuculline microinjection, which induced defensive responses,
compared with other studies using other stressors, such as natural
predators (Almada et al., 2015; Almada and Coimbra, 2015;
Twardowschy et al., 2013; Uribe-Marin ~ o et al., 2012) or unpro-
tected spaces (Moreira et al., 2007).
Anandamide promotes panicolytic and anxiolytic-like effects as
showed in the first experiment and replicated in the second set of
experiments. Pre-treatment of the VMHdm with the CB1 receptor
antagonist AM251 followed by administration of anandamide
(AM251-AEA-BIC treatment) restored the aversive effects of intra-
hypothalamic treatment with bicuculline. These findings suggest
that the activation of the CB1 receptor also modulates panic- and
anxiety-like responses in diencephalic areas.
Interestingly, previous studies have shown that anandamide
activates both CB1 and transient receptor potential cation channel,
subfamily V, member 1 (TRPV1) vanilloid receptors, resulting in
panicolytic and panicogenic-like effects associated with the inhi-
bition and facilitation of escape responses, respectively. The TRPV1
is an ion channel permeable to calcium ions, and this ion channel
has been implicated in panic-like responses (Chhatwal and Ressler,
2007; Xing and Li, 2007). Thus, the inhibition of TRPV1 receptors
potentiated the effects of high doses of anandamide and decreased
oxide nitric-induced defensive responses (Lisboa and Guimara ~es,
2012). Consistently, high doses of anandamide used in the pre-
sent study did not decrease the defensive responses induced
through bicuculline microinjection in the VMHdm. Accordingly,
some studies have shown that TRPV1 antagonists show anti-
aversive effects, likely reflecting the binding of endogenous anan-
damide to CB1 receptors. This hypothesis is supported by the in-
hibition of the anti-aversive effect of TRPV1 antagonists through
the pre-treatment with CB1 antagonists (Almeida-Santos et al.,
2013; Casarotto et al., 2012), thereby explaining the effects
observed after administering a high dose of anandamide in the
present study.
The present work showed for the first time that the endo-
cannabinoid neuromodulator anandamide is able to change
defensive responses elaborated by ventromedial hypothalamus
neurons once the previous studies focused on other limbic struc-
tures activity. Regarding the dorsolateral columns of the peri-
aqueductal grey matter (dPAG), there is evidence that
endocannabinoids can modulate defensive behaviour through CB1
receptors in animals submitted to the elevated plus-maze test, an
animal model of anxiety. In fact, the anandamide induced
anxiolytic-like effect through CB1 receptor since this anxiolytic-like
effect was prevented by the intramesencephalic pre-treatment
with the CB1 receptor antagonist AM251 (Moreira et al., 2007),
exactly the same treatment used in our study but microinjected in
diencephalic structures. Other investigation showed similar results
using dPAG electrical stimulation, since the pre-treatment of the
dorsal midbrain with the CB1 receptor antagonist AM251 was able
to prevent the panicolytic-like effect of the CB1 receptor agonist
ACEA (Casarotto et al., 2012). Still in relation to dPAG, a recent
 T. dos Anjos-Garcia et al. / Neuropharmacology 113 (2017) 156e166
report showed that the nitric oxide donor SIN-1 induces flight re-
sponses evaluated by increase in distance moved and speed of
motor responses displayed by rats in a circular arena of an open-
field test, and these responses were attenuated by anandamide,
whereas the blockade of CB1 receptors with AM251 was able to
prevent that proaversive effect (Lisboa and Guimara ~es, 2012). In the
present work, instead of only recording the distance travelled by
stimulated rodents and the speed of movements, we measured the
effect of CB1 cannabinoid receptor-mediated antiaversive effect of
anandamide microinjected in the medial hypothalamus on defen-
sive behavioural reactions, highlighting the differences between
oriented and non-oriented escape behaviour orchestrated by the
dorsomedial division of ventromedial hypothalamus.
Regarding the telencephalic structures, studies have shown that
the prefrontal cortex treatment with the CB1 receptor agonist
methanandamide (mAEA) induces anxiolytic-like effects compa-
rable in intensity to that caused by the referential benzodiazepine
compound midazolam tested in the elevated plus-maze test; an
effect also prevented by AM251 (Rubino et al., 2008). Similar
findings were found on the prelimbic medial prefrontal cortex
neuronal activity (Fogaça et al., 2012).
In addition, although it is difficult to compare the pattern of the
modulatory effect exerted by anandamide on defensive responses
evoked by either telencephalic, diencephalic or mesencephalic
structures activation in rodents submitted to different experi-
mental models of anxiety and instinctive fear, the CB1-canabinoid
signalled antiaversive effect of anandamide seems to be consistent.
In the present work, it was the first time in which the effect of
anandamide was investigated on diencephalic structures on both
oriented and non-oriented escape behavioural reactions elicited in
complex environments, as recently clearly described in literature
(Biagioni et al., 2016b; Ullah et al., 2015).
Interestingly, AM251-veh-BIC treatment increased the number
and duration of non-oriented escape evoked after bicuculline
treatment, a pattern of responses evoked through the stimulation
of mesencephalic structures (Biagioni et al., 2016b; Coimbra et al.,
2006; Ullah et al., 2015). The third set of experiments was per-
formed to investigate the hypothesis that the potentiation observed
with AM251-veh-BIC treatment depends on TRPV1 channel acti-
vation. Our results showed that VMHdm pre-treatment with a
TRPV1 channel selective antagonist (6-I-CPS-AM251-BIC treat-
ment) did not change the behavioural responses, except the non-
oriented escape behaviour which was restored by VMHdm treat-
ment with this selective TRPV1 channel antagonist. This effect can
be explained by the fact that the CB1 antagonist facilitated the
activation of TRPV1 channels through endogenous anandamide,
thereby increasing defensive behaviour possibly through the
recruitment of mesencephalic structures, considering the hypo-
thalamic projections to the periaqueductal grey matter (Canteras
et al., 1994), and because of this we observed a potentiation of
non-oriented escape behaviour whit veh-AM251-BIC treatment.
Although similar responses have previously been observed
(Lisboa and Guimara ~es, 2012), we cannot exclude the potential
motor intrinsic effect of AM251. Studies have shown that the
administration of the antagonist/inverse agonist of the CB1 receptor
rimonabant (20 mg) in humans increases the excitability of cortical
and spinal motor neurons (Oliviero et al., 2012). However, AM251-
veh-veh treatment did not change any of the motor behavioural
responses presently studied, excluding any non-specific effects on
motor activity caused by AM251 microinjections in VMHdm.
Morphological studies have shown that many axon terminals
express CB1 receptors, which inhibit the release of excitatory and
inhibitory neurotransmitters (Alger, 2002; Schlicker and Kathmann,
2001). CB1 receptors are present in both glutamatergic and
GABAergic projections (Freund et al., 2003). The immunoreactivity
165
of CB1 receptors has been previously demonstrated in the para-
ventricular and ventromedial nuclei in infundibular and lateral hy-
pothalamic areas. These receptors in the hypothalamus have been
identified on glutamatergic neurons (Marsicano and Lutz, 1999).
Additionally, other studies have showed the predominance of CB1
receptors on the axon terminals of GABAergic neurons (Katona et al.,
1999; Szabo and Schlicker, 2005).
In summary, the pharmacological treatments used in the pre-
sent study indicate a role for the VMHdm in the organisation of
defensive behaviour, suggesting a CB1 receptor-mediated modula-
tory role for anandamide in the organisation of the panic-attack-
like emotional responses elaborated through this structure. Addi-
tional neuropharmacological evidence suggests a critical role of
TRPV1 channel in the AM251-related potentiation of hypothalamic
facilitatory bicuculline effects on non-oriented panic-like escape
behaviour.
Conflict of interest
The authors declare that there are no conflicts of interest with
respect to the work presented herein.
Appendix A. Supplementary data
Supplementary video related to this article can be found at
http://dx.doi.org/10.1016/j.neuropharm.2016.04.003.
Acknowledgements
This research was financially supported through grants from
FAPESP (processes 2007/01174-1, 2009/54014-7, and 2012/03798-
0) and CNPq (processes 483763/2010-1 and 474853/2013-6). T. dos
Anjos-Garcia was financially supported through CNPq (M.Sc.
fellowship, process 130124/2012-5; Sc. D. fellowship, process
141124/2014-8). Each organisation had no further role in the study
design, the collection, analysis, and interpretation of the data,
writing the report, or the decision to submit the paper for publi-
cation. F. Ullah was financially supported through TWAS-CNPq
(Sc.D. fellowship; process 190316/2010-1). L.L. Falconi-Sobrinho
was supported by FAPESP (M.Sc. fellowships process 2013/10984-
8). N.C. Coimbra is a researcher (level 1A) from CNPq (processes
301905/2010-0 and 301341/2015-0). The authors would like to
thank Daoud Hibrahim Elias Filho for expert technical assistance.
D.H. Elias-Filho received technician scholarships from FAPESP (TT-2,
process 02/01497-1) and CNPq (processes 501858/2005-9, 372654/
2006-1, 372810/2008-0, 372877/2010-9, and 474853/2013-6).
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