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Dorsal hippocampus cannabinoid type


1 receptors modulate the expression
of contextual fear conditioning in rats:
Involvement of local glutamatergic/nitrergic
and GABAergic neurotransmissions
Gabriela B.L. Spiacci, Leandro S. Antero, Daniel G. Reis,
Sabrina F. Lisboan, Leonardo B. Ressteln

Pharmacology Department, Medical School of Ribeiro Preto, University of So Paulo, Av Bandeirantes


3900, Monte Alegre, 14049-900 Ribeiro Preto, So Paulo, Brazil

Received 14 January 2016; received in revised form 4 August 2016; accepted 22 August 2016

KEYWORDS Abstract
Endocannabinoid The cannabinoid receptor type 1 (CB1) is highly expressed in the dorsal portion of hippocampus
system; a brain region that has been involved in the control of conditioned emotional response (CER)
CB1 receptors; in the contextual fear conditioning (CFC) model. These responses are characterized by
NMDA receptors; increased freezing behavior and autonomic parameters. Moreover, CB1 receptors activation
Nitric oxide;
negatively modulate the release of several neurotransmitters, including glutamate and GABA,
Freezing behavior;
which also have been related to modulation of CER. Therefore, our aim was to investigate the
Autonomic responses
involvement of CB1 receptors in the dorsal hippocampus on CER expression. Independent groups
of male Wistar rats submitted to the contextual fear conditioning received bilateral intra-
hippocampal injections (500 nL/side) of the following drugs or vehicle before re-exposure to
the aversive context: AM251 (CB1 antagonist; 0.1, 0.3 and 1 nmol); AP7 (NMDA antagonist;
1nmol) +AM251 (0.3 nmol); NPLA (0.01 nmol; nNOS inhibitor) +AM251 (0.3 nmol); Bicuculline
(1.3 pmol; GABAA antagonist) +AM251 (0.1 and 1 nmol). In the present paper, AM251 (0.3 nmol)
increased CER, while this response was prevented by both AP7 and NPLA pretreatment. After
pretreatment with Bicuculline, the lower and higher ineffective doses of AM251 were able to
increase the CER, supporting the balance between GABAergic and glutamatergic mechanisms
controlling this response. Our results suggest that increased CER evoked by CB1 blockade in the

n
Corresponding authors.
E-mail addresses: sa_lisboa@hotmail.com (S.F. Lisboa),
leoresstel@yahoo.com.br (L.B. Resstel).

http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
0924-977X/& 2016 Elsevier B.V. and ECNP. All rights reserved.

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
2 G.B.L. Spiacci et al.

dorsal hippocampus depends on NMDA receptor activation and NO formation. Moreover, a ne-
tune control promoted by GABAergic and glutamatergic mechanisms in this brain area modulate
the CER after CB1 blockade.
& 2016 Elsevier B.V. and ECNP. All rights reserved.

1. Introduction composed by cannabinoid receptors type 1 (CB1) and type 2


(CB2); by endogenous ligands, the endocannabinoids (eCBs),
Hippocampus is a limbic brain structure situated medial to such as N-Arachidonoylethanolamine (Anandamide) and
the lateral ventricle (O'Mara et al., 2001). Cognitive pro- 2-arachidonoylglycerol; and synthesis and degradation
cesses, such as learning and memory (Bannerman et al., enzymes (Iannotti et al., 2016; Lu and Mackie, 2016).
1999; Clark et al., 2000; Fanselow and Dong, 2010; Holland The CB1 receptors are coupled to a Gi protein located
and Bouton, 1999; Kraus and Canlon, 2012), emotional primarily in presynaptic neurons (Iannotti et al., 2016),
processing (Siegel and Flynn, 1968), stress modulation and regulating, therefore, the release of several neurotransmit-
anxiety (Moraes-Neto et al., 2014; Padovan et al., 2000; ters (Howlett et al., 2002), including GABA (Katona and
Rosa et al., 2002; Scopinho et al., 2013) are strongly linked Freund, 2012) and glutamate (Katona et al., 1999; Katona
to hippocampus. Excitotoxic or electrolytic lesions of the et al., 2006; Wang, 2003). In addition, CB1 receptors are
dorsal portion of hippocampus (DH) in rats promoted highly expressed in brain regions involved with processing of
anxiolytic-like effect in the social interaction and elevated fear and anxiety, such as the hippocampus, amygdala and
plus maze (EPM) tests (Bannerman et al., 2002). Moreover, medial prefrontal cortex, indicating that these structures
DH temporary blockade after a fear conditioning procedure could underlie eCB effects on emotional processing
attenuated the expression of aversive memory in the (Busquets-Garcia et al., 2015; Fowler, 2015). In rats,
contextual fear conditioning model (CFC) (Resstel et al., activation of DH CB1 receptors by modulators of eCB system
2008b). induced anxiolytic-like effect in different animal models
The CFC model is characterized by an association (Lisboa et al., 2015), suggesting this signaling could be a
between a neutral (an environment, like a chamber) and target for pharmacological agents modulating anxiety.
an aversive, unconditioned stimulus (US, like footshocks). CB1 receptors are expressed both by axonal terminals of
After paired with the US, the previously neutral stimulus glutamatergic and GABAergic synapses in the DH, but at
will predict the footshock occurrence, becoming a condi- much higher levels in the GABAergic terminals (Katona
tioned stimulus (CS). The result is a state of fear, antici- et al., 2006). Administration of the cannabinoid agonist
patory anxiety (Fanselow, 2000; Fendt and Fanselow, 1999), WIN 55,212-2 to mice hippocampal slices reduced both
characterized by expression of freezing behavior (tense glutamatergic (Domenici et al., 2006) and GABAergic neu-
posture adopted by the animal, with no movements except rotransmissions (Katona et al., 1999) in a dose-dependent
those necessary to keep breathing (Fanselow, 1980), and manner. Therefore, eCBs could promote their effects by
autonomic changes, such as elevation in the mean arterial modulating both glutamatergic/nitrergic and GABAergic
pressure (MAP), heart rate (HR) and reduction of tail neurotransmissions (Lisboa et al., 2013), and the predomi-
cutaneous temperature (TCT) (Carrive, 2000, 2002; nance of effect would depend on the brain area, the drug
LeDoux et al., 1988; Resstel et al., 2006, 2008a, 2008b; and dose used as well as on the animal model.
Vianna and Carrive, 2005). These alterations are collec- Glutamatergic ionotropic N-methyl-D-aspartate (NMDA)
tively called conditioned emotional response (CER). receptors are expressed in the DH and evidence point out to
Several piece of evidence suggest the involvement of DH their involvement in the modulation of behavioral conse-
in the modulation of CER. The level of protein c-FOS, a quences of stress (Padovan et al., 2000) and anxiety
marker of neuronal activity, is increased in the DH of responses by DH (Fabri et al., 2014). Moreover, synthesis
animals after re-exposure to a conditioned aversive envir- of nitric oxide (NO) is closely related to activation of NMDA
onment (Beck and Fibiger, 1995). However, reversible receptors, since activation of the later induce activation of
blockade of the DH neurotransmission before re-exposing the neuronal NO synthase (nNOS) enzyme, resulting in NO
rats to the CS in the CFC model attenuated the cardiovas- formation (Garthwaite, 1991). nNOS is expressed by DH
cular, but not the behavioral response (Resstel et al., neurons (Blackshaw et al., 2003) and administration of a
2008b). Likewise, DH reversible blockade before submitting selective nNOS inhibitor into this region induced anxiolytic-
rats to an acute restraint-stress attenuated the autonomic like effects in different animal models (Spolidorio et al.,
response during stress, but not the later anxiogenic-like 2007).
behavior (Scopinho et al., 2013), suggesting distinct mod- The evidence showing that activation of CB1 receptors
ulation of the CER components by the DH. decrease anxiety-like behavior, probably by decreasing
In the DH, several neurotransmitter systems are related glutamate release and NO formation, along with the fact
to the modulation of defensive behaviors, such as GABAergic that DH is involved in the modulation of conditioned
(Rezayat et al., 2005; Zhang et al., 2014), glutamatergic emotional response and that CB1 receptors are highly
(Fabri et al., 2014; Kamprath et al., 2009) and endocanna- expressed in this region led us to hypothesize that inhibition
binoid (eCB) systems (Hill et al., 2006). The eCB system is of DH CB1 receptors increase contextual conditioned

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
Dorsal hippocampus CB1 receptors modulate conditioned emotional response in rats 3

Figure 1 Temporal representation of the experimental procedures, as described in the text. The experimental procedures started
one week after the stereotaxic surgery. 24 h after conditioning procedure, a catheter was inserted into the femoral artery in order
to register the cardiovascular activity through abdominal aorta 24 h later, during the test.

emotional response by activation of NMDA receptors and NO and a catheter (PE-10; a 45 cm segment heat-bound to a 13 cm PE-
pathway. In addition, considering that CB1 receptors also 50 1213 cm segment, Clay Adams, USA) was inserted into the
modulate GABA release, we tested the hypothesis that in abdominal aorta through the femoral artery. The catheter was
the case of absence of effect of the cannabinoid antagonist, tunneled under the skin and exteriorized on the animal's dorsum.
this would be due to activation of GABAA receptors.

2. Experimental procedures 2.4. Fear conditioning and testing

The procedure was conducted as depicted in the Figure 1. Habitua-


2.1. Subjects
tion, conditioning and testing were performed in a 25  22  22 cm
footshock chamber (Insight Instruments Ribeiro Preto, Brazil). The
Male Wistar rats (200220 g) were used. Animals were kept in the chamber had a grid oor composed of 18 stainless-steel rods (2 mm
Animal Care Unit of the Pharmacology Department, Medical School in diameter), spaced 1.5 cm apart and wired to a shock generator.
of Ribeiro Preto, University of So Paulo. Rats were housed in our The chamber was cleaned with 70% ethanol between each animal.
laboratory animal care in groups of 5 animals per home (polypro-
The habituation consisted of one 10-min-long pre-exposure to the
pylene box; 39  32  17 cm) under a 12-h light/dark cycle (lights chamber without shock presentation. Two hours after the habitua-
on 06: 30 hours) with food and water available ad libitum. The tion session, the conditioning shock session started. The animals
Institution's Animal Ethics Committee approved housing conditions were submitted to the conditioning session, which consisted of
and experimental procedures (process number: 127/2011). three electric footshocks of 0.85 mA/2 s delivered at 20-s to 1-min
intervals (Lisboa et al., 2010). Twenty-four hours later, a catheter
2.2. Drugs was implanted into the femoral artery for cardiovascular recording.
Cardiovascular and behavioral responses evoked by re-exposure
The CB1 receptor antagonist N-(piperidin-1 yl) -5 - (4-Iodophenyl) -1 to context previously paired with footshock were evaluated 2 days
(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide (AM251, after the conditioning session, in the test session. This consisted of
Tocris) was dissolved in 10% Dimethyl-sulfoxide (DMSO) in saline a 10-min-long re-exposure to the chamber without any shock
(0.9% NaCl). The GABAA receptor antagonist 1(S),9(R)-( )-Bicucul- delivery. Animals were transferred from the animal room to the
line methiodide (Bicuculline, Sigma - Aldrich), the antagonist of the procedure room (a different room from conditioning session) in
glutamatergic NMDA receptors DL-2-amino-7-phosphonoheptanoic their home box. The mean arterial pressure (MAP), heart rate (HR)
acid (AP7 DL, Tocris) and nNOS inhibitor N-propyl-L-arginine and tail cutaneous temperature (TCT) were recorded using an
(NPLA, Tocris) were dissolved in saline (0.9% NaCl). The solutions amplier (AECADE 04 P) connected to a signal acquisition board
were prepared immediately before use and were kept on ice and (Power Lab, USA) and processes by a computer. Rats were tested
protected from the light during the experimental sessions. Tribro- one at a time. After 10 min of habituation, animals received
moethanol (2.5%; Sigma, USA) and urethane (25%; Sigma) were bilateral microinjections into the DH. Two 33 G needles (Small
dissolved in distilled water. All the doses used in the present study Parts, USA) 1mm longer than the guide cannula and connected to a
were based on previous studies from our lab (Fabri et al., 2014; 10-ml syringe (7002-H, Hamilton Co., USA) through a PE-10 tubing
Lisboa and Guimaraes, 2012; Lisboa et al., 2010). were used. Needles were carefully inserted into the guide cannulae
and the solutions were infused over a 30-s period with a rate of
500 nL/min. They remained in place for an additional 20-s period to
2.3. Surgical procedures prevent reux. The interval between the rst and second micro-
injection was 5 min and the animals were tested 10 min after the
For implantation of guide cannulae in the DH, rats were rst last microinjection. Cardiovascular parameters recorded during the
anaesthetized with 2,2,2-tribromoethanol 2.5% (Sigma-Aldrich, 10 min following the last drug injection were used as baseline
USA, 10 ml/Kg, i.p), them a scalp anesthesia with 2% lidocaine measurements.
was performed and the skull was surgically exposed. Stainless steel Freezing was evaluated during the test by an experimenter
guide cannulae (33 G) were bilaterally implanted into the DH using blinded to the treatment groups, seated 50 cm from the footshock
a stereotaxic apparatus (Stoelting, USA). The coordinates for chamber. Freezing is dened as the complete absence of move-
cannulae implantation (AP= 4.0 mm from the bregma; L= ment, except for those necessary to breathe, while the animal
+2.8 mm from the medial suture; V= 2.1 mm in relation to the assumes a characteristic tense posture (Fanselow, 1980). During the
bone structure of the skull; incisive: 2.5 mm) were selected from experimental session, the percentage of the total freezing time was
the rat brain atlas of Paxinos and Watson (2007), except for the used represent the observed effects.
incisive parameter, which was adjusted to the weight of the animals
used, as described in previous work from our group (Fabri et al.,
2014; Moraes-Neto et al., 2014). Cannulae were xed to the skull
with dental cement and one metal screw. After surgery, the animals 2.5. Experimental design
received a poly-antibiotic injection (Pentabiotico, 0.2 ml, i.m.,
Fontoura-Wyeth, BR) to prevent infection and the non-steroidal Experimental groups are depicted in the Figure 2.
anti-inammatory drug unixin meglumine (Banamine 1, Brazil) for Independent group of animals received a rst bilaterally micro-
post-surgery analgesia. injection of vehicle (Veh) or drugs followed 5 min later by vehicle or
For the femoral artery cannulation for cardiovascular recording drugs. After 10 min, they were re-exposed to the chamber previous
during the test, rats were anaesthetized with tribromoethanol 2.5% paired with footshocks.

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
4 G.B.L. Spiacci et al.

Figure 2 Temporal representation of the treatment scheme


used. Dose-response curve in the dorsal hippocampus for the
CB1 antagonist, AM251 (I). NMDA-receptors antagonist, AP7 (II);
nNOS inhibitor, NPLA (III); or the GABAA antagonist, Bicuculline
(IV, V) were administered 5 minutes before the CB1 antagonist,
AM251. The test was performed 10 minutes after this last
injection. The doses used are represented in the gure.

2.6. Histological procedure

At the end of experiments, rats were anaesthetized with urethane


(1.25 g/kg i.p.) and 200 nL of 1% Evan's blue dye was bilaterally
injected into the DH as a site marker. The chest was surgically
opened, the descending aorta was occluded, the right atrium was
severed and the brain was perfused with 10% formalin through the
left ventricle. Brains were post xed for 24 h at 4 1C, and 40 mm
sections were cut using a cryostat (CM-1900, Germany). Serial brain
sections were stained with 1% neutral red and injection sites
Figure 3 Histological localization of injection sites located in
determined using the rat brain atlas of (Paxinos and Watson,
the DH (black circles), based on the atlas of Paxinos and Watson
2007) as reference. A representative photomicrography of a coronal
brain section and the microinjection sites in DH can be seen (2007). Due to overlap, the number of points represented is
in Figure 3. fewer than the real number of rats used in the experiments.
The open circles represent the injections outside the DH.

2.7. Data analysis


treatment (MAP, F56,350 = 4.66; po0.05; HR, F56,350 = 2.94;
po0.005; TCT, F56,350 = 2.94; po0.05) during re-exposure to
The MAP, HR and TCT values were continuously recorded during the
the conditioning chamber. Similar to behavioral effect,
5-min-period before (baseline) and during the 10 min-test period.
Data were expressed as means 7 SEM of changes from baseline ().
AM251 in the dose of 0.3 nmol, but not in the doses of
MAP (mmHg), HR (bpm) and TCT (1C) were obtained by 0.1 nmol and 1 nmol, accentuated the elevation of MAP
subtracting each value from the mean baseline value (the average (po0.05, Duncan; Figure 4B) and HR (po0.05, Duncan;
of the ve baseline observations that were taken). The data were Figure 4C) and the reduction of TCT (po0.05, Duncan,
evaluated by analysis of variance for repeated measures (MANOVA), Figure 4D). AM251 neither altered the baseline autonomic
with time as the repeated measure and treatment (vehicle or drug) parameters (MAP, p40.05; FC, p40.05; TCT, p40.05) nor
as the independent variable. Freezing behavior was expressed as induced any effect when microinjected in surrounding areas
percentage of test duration and analyzed by one-way ANOVA. of DH (freezing behavior, p40.05, n = 4; Figure 4A; MAP,
Duncan post-hoc test was used when signicant effects were p40.05; HR, p40.05; TCT, p40.05; Figure 4BD).
detected. Statistical signicance was considered when po0.05.

3.2. Evaluation of NMDA receptors involvement in


3. Results the CB1 antagonism by AM251 (0.3 nmol) in the DH
on the expression of the CER
3.1. Evaluation of the CB1 receptors antagonism
in the DH on the expression of CER Intra DH administration of AM251 (0.3 nmol), but not NMDA
antagonist, AP7, signicantly increased the percentage of
Intra-DH administration of the CB1 antagonist, AM251, time spent in freezing behavior (F4,23 =3.85; po0.005,
increased the percentage of time spent in freezing behavior Duncan, po0.05; n = 66/group; Figure 5A). Pre-treatment
only in the intermediate dose (0.3 nmol) (F4,25 = 4.14; with AP7, however, blocked AM251 effect (Figure 5A).
po0.005, Duncan, po0.05; n= 5/group; Figure 4A). In the In the autonomic evaluation, there was a signicant effect
autonomic evaluation, there was a signicant effect of time of time (MAP, F14,322 =51.36; po0.005; HR, F14,322 =30.85,
(MAP, F14,350 = 68.69; po0.005; HR, F14,350 = 53.89, po0.005; TCT, F14,322 =16.04, po0.005), treatment (MAP,
po0.005; TCT, F14,350 = 53.89, po0.005), treatment (MAP, F4,23 =3.74, po0.005; HR, F4,23 =5.61, po0.005; TCT,
F4,25 = 18.28, po0.005; HR, F4,25 = 8.16, po0.005; TCT, F4,23 =7.26 po0.05) and interaction between time and
F4,25 = 8.16 po0.05) and interaction between time and treatment (MAP, F56,322 =1.48; po0.05; HR, F56,322 =2.21;

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
Dorsal hippocampus CB1 receptors modulate conditioned emotional response in rats 5

Figure 4 The CB1 antagonist AM251 intra-DH increased contextual conditioned emotional response. Effects of bilateral
microinjection of vehicle (500 nL) or AM251 (0.11 nmol) intra-DH before re-exposure to the context aversive on A) freezing
behavior, B) mean arterial pressure variation (MAP), C) heart rate variation (HR) and D) on cutaneous temperature variation
(TCT). DMSO: Dimethyl-sulfoxide. The columns and symbols represent the mean and bars the SEM. *po0.05 compared to the
vehicle group, followed by post-hoc Duncan. n =59/group.

Figure 5 NMDA receptors are involved in the conditioned emotional response induced by AM251 (0.3 nmol). Effect of bilateral
microinjection of vehicle (500 nL) or AP7 (1 nmol) followed by vehicle or AM251 (0.3 nmol) intra-DH before re-exposure to aversive
context on freezing behavior on A) freezing behavior, B) mean arterial pressure variation (MAP), C) heart rate variation (HR) and
D) on cutaneous temperature variation (TCT). DMSO: Dimethyl-sulfoxide. The columns and symbols represent the mean and bars
the SEM *po0.05 compared to the vehicle group, #po0.05 compared to the vehicle and AM251 (0.3 nmol) group, followed by post-
hoc Duncan. N =56/group.

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
6 G.B.L. Spiacci et al.

po0.005; TCT, F56,322 =2.06; po0.05)) during re-exposure to 0.3 nmol accentuated the elevation of MAP (po0.05, Duncan;
the conditioning chamber to behavioral effect, AM251 in the Figure 6B) and HR (po0.05, Duncan; Figure 6C) and the
dose of 0.3 nmol, accentuated the elevation of MAP reduction of TCT (po0.05, Duncan, Figure 6D) and NPLA
(po0.05, Duncan; Figure 5B) and HR (po0.05, Duncan; prevented these effects (p40.05). AM251 and NPLA neither
Figure 5C) and the reduction of TCT (po0.05, Duncan, altered the baseline autonomic parameters (MAP, p40.05; FC,
Figure 5D) and pretreatment with AP7 prevented these p40.05; TCT, p40.05) nor induced any effect when micro-
effects (p40.05). AM251 and AP7 neither altered the base- injected in surrounding areas of DH (freezing behavior,
line autonomic parameters (MAP, p40.05; FC, p40.05; TCT, p40.05, n=4; Figure 6A; MAP, p40.05; HR, p40.05; TCT,
p40.05) nor induced any effect when microinjected in p40.05; Figure 6BD).
surrounding areas of DH (freezing behavior, p40.05, n=4;
Figure 5A; MAP, p40.05; HR, p40.05; TCT, p40.05;
Figure 5BD). 3.4. Pretreatment with the GABAA receptor
antagonist Bicuculline into the DH revealed a pro-
3.3. Evaluation of the involvement of nNOS in the aversive effect of previously ineffective doses of
CB1 antagonism by AM251 (0.3 nmol) in the DH on AM251 in the CER
the expression of the CER
Neither AM251 in the doses of 0.1 nmol or 1 nmol nor
Intra DH administration of AM251 (0.3 nmol), but not the Bicuculline (BIC; 1.3 pmol) increased freezing behavior
nNOS inhibitor, NPLA, signicantly increased the percentage (p40.05; Duncan; n = 5, 5, 6/group; Figure 7A). However,
of time spent in freezing behavior (F4,21 = 7.81; po0.005, after pre-treatment with BIC, increased freezing behavior
Duncan, po0.05; n = 56/group; Figure 6A). Pre-treatment was observed both with the lower (0.1 nmol) and the higher
with NPLA, however, blocked AM251 effect (Figure 6A). (1 nmol) doses of AM251 (F6,30 = 6.02; po0.05; Duncan;
In the autonomic evaluation, there was a signicant effect n = 55/ group; Figure 7A).
of time (MAP, F14,294 =42.42; po0.005; HR, F14,294 =9.06, In the autonomic evaluation, there was a signicant effect
po0.005; TCT, F14,294 =8.38, po0.005), treatment (MAP, of time (MAP, F14,420 =93.96; po0.005; HR, F14,420 =59.30,
F4,21 =8.54, po0.005; HR, F4,21 =4.73, po0.005; TCT, F4,21 = po0.005; TCT, F14,294 =8.38, po0.005), treatment (MAP,
10.79 po0.05) and interaction between time and treatment F6,30 =5.89, po0.005; HR, F6,30 =8.94, po0.005; TCT,
(MAP, F56,294 =2.67; po0.05; HR, F56,294 =1.40; po0.005; TCT, F6,30 =5.73 po0.05) and interaction between time and
F56,294 =1.31; po0.05) during re-exposure to the conditioning treatment (MAP, F84,420 =2.39; po0.05; HR, F84,420 =3.03;
chamber. Similar to behavioral effect, AM251 in the dose of po0.005; TCT, F84,420 =1.98; po0.05) during re-exposure to

Figure 6 nNOS is involved in the conditioned emotional response induced by AM251 (0.3 nmol). Effect of bilateral microinjection of
vehicle (500 nL) or NPLA(0.01 nmol) followed by vehicle or AM251 (0.3 nmol) intra-DH before re-exposure to aversive context on
freezing behavior on (A) freezing behavior, (B) mean arterial pressure variation (MAP), (C) heart rate variation (HR) and (D) on
cutaneous temperature variation (TCT). DMSO: Dimethyl-sulfoxide. The columns and symbols represent the mean and bars the SEM
*po0.05 compared to the vehicle group, #po0.05 compared to the vehicle and AM251 (0.3 nmol) group, followed by post-hoc
Duncan. N =56/group.

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
Dorsal hippocampus CB1 receptors modulate conditioned emotional response in rats 7

Figure 7 GABAA receptors are involved in the lack of effect of AM251 (0.1 nmol and 1 nmol) in the conditioned emotional response.
Effect of bilateral microinjection of vehicle (500 nL) or Bicuculline (1.3 pmol) followed by vehicle or AM251 (0.1 or 1 nmol) intra-DH
before re-exposure to aversive context on A) freezing behavior, B) mean arterial pressure variation (MAP), (C) heart rate variation
(HR) and D) on cutaneous temperature variation (TCT). The columns and symbols represent the mean and bars the SEM *po0.05
compared to the vehicle group, followed by post-hoc Duncan. N =5-6/group.

the conditioning chamber. Neither AM251 nor BIC altered the 2008b), the fact that the whole synaptic activity in the DH
autonomic basal values (MAP, p40.05; HR, p40.05; TCT, was blocked does not allows us to rule out the possibility
p40.05; Duncan). The combination of them, however, that specic neurotransmitters in the DH could differen-
accentuated the increase of MAP (po0.05; Duncan; tially modulate the behavioral response. Likewise, it was
Figure 7B), HR (po0.05; Duncan; Figure 7C) and the reduc- observed that administration of an NMDA antagonist or an
tion of TCT (po0.05; Duncan; Figure 7D). Neither of the nNOS inhibitor in the DH of rats attenuated both autonomic
drugs induced effect when microinjected in surrounding and behavioral components of the CER during re-exposure
areas of DH (freezing behavior, p40.05, n=4; Figure 7A; to the aversive context (Fabri et al., 2014). This suggests
MAP, p40.05; HR, p40.05; TCT, p40.05; Figure 7BD). that although during aversive situations the whole DH is
important to modulation of the autonomic activity, specic
4. Discussion neurotransmitters could differently drive modulation of
autonomic and behavioral responses.
In the present study we found that administration of the High freezing behavior is observed during re-exposure to
CB1 antagonist AM251 at the dose of 0.3 nmol in the dorsal a conditioned cue after pharmacological inhibition or
hippocampus (DH) increased the expression of contextual genetic deletion of CB1 receptors in mice (Kamprath
conditioned emotional response (CER). In addition, we also et al., 2006; Marsicano et al., 2002; Metna-Laurent et al.,
have shown that this effect depends on glutamatergic and 2012; Niyuhire et al., 2007). Conversely, systemic adminis-
nitrergic mechanisms, since AP7- an NMDA receptors tration of the CB1 agonist WIN-55,212-2 to rats was able to
antagonist, and NPLA- an nNOS inhibitor, prevented AM251 reduce the expression of contextual fear conditioning
effect. Finally, we found that lack of effect of the lowest (Pamplona et al., 2006). Moreover, administration of low
and the highest doses of AM251 used probably involves doses of THC (tetrahydrocannabinol) to mice was able to
GABAergic neurotransmission, considering that in the pre- attenuate the increased auditory fear conditioning (Metna-
sence of the GABAA receptor antagonist Bicuculline both Laurent et al., 2012). Recently we have shown that activa-
doses increased the CER. tion of CB1 receptors in the DH induces anxiolytic-like
Temporary inactivation of DH neurotransmission with a effect in the Vogel conict test and in the elevated plus
synaptic blocker before re-exposure to an aversive context maze (EPM), two animal models of anxiety (Lisboa et al.,
attenuated the increased cardiovascular activity, but not 2015). Conversely, in the present study we showed that
freezing behavior (Resstel et al., 2008b), suggesting that bilateral administration of the intermediate dose of AM251
this brain area participates only in the cardiovascular in DH was able to increase CER evoked by re-exposure to the
component of the CER. Although the behavioral component aversive context. Therefore, our results showing that block-
in that preliminary study was not altered (Resstel et al., ade of CB1 receptors in the DH increases expression of

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
8 G.B.L. Spiacci et al.

contextual fear conditioning suggest that this brain struc- It is well known that activation of NMDA receptors by
ture could be a potential site for cannabinoid drugs glutamate promotes an increase in NO synthesis via activa-
administered systemically, as previously suggested. tion of nNOS (Garthwaite and Boulton, 1995; Garthwaite
CB1 receptors are expressed in presynaptic terminals in et al., 1988). In addition, it was already showed that re-
the hippocampus (Kawamura et al., 2006) predominantly in exposure to an aversive context signicantly increases the
GABAergic terminals, where they modulate the GABA levels of NO metabolites in the DH, suggesting an increase in
release (Katona et al., 1999; Marsicano and Lutz, 1999). DH NO production during CER (Fabri et al., 2014). Conrm-
However, CB1 receptors are also found in other hippocampal ing the importance of DH NO system to the expression of
neuronal subpopulations, including glutamatergic terminals CER, inhibition of nitrergic neurotransmission in this area
(for review see Katona and Freund, 2008), controlling before re-exposure to the context previously paired with
glutamatergic synaptic neurotransmission (Domenici et al., footshocks attenuated that response (Fabri et al., 2014).
2006; Kawamura et al., 2006). In this way, administration of The involvement of NO in the effect induced by activation
a cannabinoid agonist to mice hippocampal slices containing of DH NMDA receptors on cardiovascular activity was also
axonal terminals decreased glutamatergic excitatory trans- previously described. Glutamate administration in the DH
mission only when CB1 receptors were absent in the promoted increased cardiovascular responses, similar to
GABAergic neurons, but not in the glutamatergic, indicating those observed during CER, in an NMDA- and nNOS-
that excitatory synaptic transmission in forebrain areas is dependent manner (Moraes-Neto et al., 2014). Moreover,
directly modulated by CB1 receptors expressed on glutama- data from electrophysiological study in the hippocampus
tergic presynaptic axon terminals (Domenici et al., 2006). suggest that NO is involved in depolarization-induced sup-
Based on that observation, we hypothesized that blockade pression of inhibition (DSI) induced by CB1 receptors, in a
of CB1 receptors in the DH could disinhibit glutamatergic cholinergic receptors activation dependent manner (Makara
transmission, probably accounting for the increased condi- et al., 2007). Therefore, our results showing that inhibition
tioned emotional response. of nNOS also prevented the increase of CER induced by the
Some contradictory data, however, have also been observed. CB1 antagonist suggest that CB1 receptors antagonism leads
Roohbakhsh and colleagues (2007) showed that AM251 admin- to activation of nNOS, probably after activation of NMDA
istration into the CA1 region of the DH of rats induced receptor.
anxiolytic-like effect, whereas a cannabinoid agonist induced The demonstration that ineffective doses of AM251 were
opposite effects. In addition to methodological differences, a able to increase the CER only after pretreatment with
possible explanation for the anxiolytic-like effect of AM251 on Bicuculline suggests that the contextual CER evoked by
their study could be due to a non-selective blockade of CB1 manipulating CB1 receptors in the DH results from CB1
receptors both on GABAergic and glutamatergic synapses, receptors control of both GABAergic and glutamatergic
indiscriminately (Roohbakhsh et al., 2007). Furthermore, con- neurotransmissions. This pattern of bell-shaped dose-
sidering that fear conditioning is a more stressful test than response curve is frequently observed after eCB system
elevated plus maze, we could argue that our protocol evokes a manipulation. Systemic administrations of increasing doses
greater engagement of glutamatergic neurotransmission in the of CB1 receptor agonists or antagonists in mice were able to
DH. Therefore, blocking CB1 receptors in this region could promote anxiolytic and anxiogenic-like effects, respectively
increase glutamate release, consequently increasing the CER. (Patel and Hillard, 2006). As mentioned before, this could
Corroborating this possibility, a recent study showed that be due to CB1 expression in axonal endings of glutamatergic
mice with genetic deletion of CB1 receptors on glutama- and GABAergic neurons, respectively. It was reported that
tergic neurons presented increased freezing behavior during low doses of cannabinoid compounds induced anxiolytic-like
re-exposure to an auditory cue (Metna-Laurent et al., effect only when CB1 receptors were normally expressed on
2012). Regarding the hippocampus, it has been shown that glutamatergic neurons, but could be absent in GABAergic
administration of the cannabinoid receptor agonist neurons, suggesting that at low doses activation of CB1
WIN55,212-2 to hippocampal slices signicantly reduced receptors decrease glutamate release (Ruehle et al., 2012).
the excitatory postsynaptic potential (EPSP), suggesting In contrast, the anxiogenic-like effect induced by higher
that CB1 receptors can modulate excitatory neurotransmis- doses of the same cannabinoid compound was only observed
sion in this brain structure (Takahashi and Castillo, 2006). when CB1 receptors were intact on GABAergic neurons, but
Furthermore, when WIN55,212-2 was administered to hip- not on glutamatergic. Therefore, higher doses could
pocampal slices derived from CB1 Glu-/- mice, but not from decrease GABA release and induce anxiogenic-like effect
CB1 GABA /- mice, this inhibitory response was abolished, (Rey et al., 2012). In addition, similar results were also
suggesting that excitatory transmission is modulate by CB1 obtained in fear conditioning model (Metna-Laurent et al.,
receptors located on presynaptic axon terminals of gluta- 2012).
matergic neurons (Domenici et al., 2006). Based on the present ndings, it is possible that inhibition
We have previously shown that NMDA activation in the DH of DH CB1 receptors increases the glutamate release,
is directly involved in the expression of the CER (Fabri leading to NMDA receptors activation followed by the
et al., 2014). Conrming NMDA involvement in the present induction of NO synthesis. The NO, in turn, by its action in
results, we showed that pretreatment with an antagonist of presynaptic terminals, activates sGC increasing cGMP
those receptors, AP7, blocked the increased CER induced by levels. As consequence, more glutamate is released and
CB1 receptors antagonism. Therefore, during re-exposure to the expression of contextual fear conditioning is increased.
the aversive context, the CB1 blockade might have induced On the other hand, GABA release is also increased, possibly
increases in glutamatergic NMDA signaling, resulting in the as a mechanism to counterbalance the glutamatergic system
observed increased conditioned emotional response. actions, resulting in biphasic effects of modulation of the

Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
Dorsal hippocampus CB1 receptors modulate conditioned emotional response in rats 9

eCB system. In conclusion, our results support the idea that Carrive, P., 2000. Conditioned fear to environmental context:
the DH eCB system, through CB1 receptors, regulates the cardiovascular and behavioral components in the rat. Brain
expression of contextual fear response by modulating a Res. 858 (2), 440445.
possible NMDA/NO/sGC pathway. Carrive, P., 2002. Cardiovascular and behavioural components of
conditioned fear to context after ganglionic and alpha-
adrenergic blockade. Auton. Neurosci. 98 (12), 9093.
Role of the funding sources Clark, R.E., Zola, S.M., Squire, L.R., 2000. Impaired recognition
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This work was supported by Grants from FAPESP, CAPES, Domenici, M.R., Azad, S.C., Marsicano, G., Schierloh, A., Wotjak, C.
CNPq. These funding sources, however, had no role on study T., Dodt, H.U., Zieglgansberger, W., Lutz, B., Rammes, G., 2006.
design; in the collection, analysis, and interpretation of Cannabinoid receptor type 1 located on presynaptic terminals of
data; in the writing of the report; and in the decision to principal neurons in the forebrain controls glutamatergic synap-
submit the paper for publication. tic transmission. J. Neurosci. 26 (21), 57945799.
Fabri, D.R., Hott, S.C., Reis, D.G., Biojone, C., Correa, F.M.,
Resstel, L.B., 2014. The expression of contextual fear condition-
Contributors ing involves activation of a NMDA receptor-nitric oxide-cGMP
pathway in the dorsal hippocampus of rats. Eur. Neuropsycho-
Sabrina F. Lisboa (SFL) and Leonardo B. Resstel (LBR) designed the pharmacol. 24 (10), 16761686.
study. Gabriela B.L. Spiacci (GBLS) conducted all the experiments. Fanselow, M.S., 1980. Conditioned and unconditional components
Leandro A. da Silva (LAS) helped with experimental procedure. of post-shock freezing. Pavlov J Biol. Sci. 15 (4), 177182.
Daniel G. Reis (DGR) helped obtaining cutaneous temperature data. Fanselow, M.S., 2000. Contextual fear, gestalt memories, and the
GBLS, SFL and LBR performed statistical analysis and wrote the hippocampus. Behav. Brain Res. 110 (12), 7381.
manuscript. All authors read and approved the nal version of the Fanselow, M.S., Dong, H.W., 2010. Are the dorsal and ventral
manuscript. hippocampus functionally distinct structures? Neuron 65 (1),
719.
Fendt, M., Fanselow, M.S., 1999. The neuroanatomical and neuro-
Conict of interests chemical basis of conditioned fear. Neurosci. Biobehav. Rev. 23
(5), 743760.
Fowler, C.J., 2015. The potential of inhibitors of endocannabinoid
The authors declare no conicts of interest.
metabolism as anxiolytic and antidepressive drugs a practical
view. Eur. Neuropsychopharmacol. 25 (6), 749762.
Garthwaite, J., 1991. Glutamate, nitric oxide and cellcell signal-
Acknowledgments ling in the nervous system. Trends Neurosci. 14 (2), 6067.
Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the
The authors thank L.H.A. Camargo and O. Mesquita Jr for technical central nervous system. Annu Rev. Physiol. 57, 683706.
support. This work was supported by Grants from So Paulo Garthwaite, J., Charles, S.L., Chess-Williams, R., 1988.
Research Foundation (FAPESP, 12/17626-7; 13/07470-2), Brazil; Endothelium-derived relaxing factor release on activation of
Coordination for the Improvement of Higher Education Personnel NMDA receptors suggests role as intercellular messenger in the
(CAPES), Brazil; and National Council for Scientic and Technologi- brain. Nature 336 (6197), 385388.
cal Development (CNPq). Hill, M.N., Kambo, J.S., Sun, J.C., Gorzalka, B.B., Galea, L.A.,
2006. Endocannabinoids modulate stress-induced suppression of
hippocampal cell proliferation and activation of defensive
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fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
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fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010
Dorsal hippocampus CB1 receptors modulate conditioned emotional response in rats 11

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Please cite this article as: Spiacci, G.B.L., et al., Dorsal hippocampus cannabinoid type 1 receptors modulate the expression of contextual
fear conditioning in rats: Involvement of.... European Neuropsychopharmacology (2016), http://dx.doi.org/10.1016/j.euroneuro.2016.08.010