Infusion of Cannabidiol Into Infralimbic Cortex Facilitates Fear Extinction Via

Behavioural Brain Research 250 (2013) 23–27

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Behavioural Brain Research

j ournal homepage: www.elsevier.com/locate/bbr

Short communication

Infusion of cannabidiol into infralimbic cortex facilitates fear

extinction via CB1 receptors

∗,1 1

Fabricio H. Do Monte , Rimenez R. Souza , Rafael M. Bitencourt, Juliana A. Kroon,

Reinaldo N. Takahashi

Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

h i g h l i g h t s

•

Intra-IL infusion of cannabidiol facilitates extinction of contextual fear.

•

Rimonabant blocks the facilitating effect of intra-IL cannabidiol on fear extinction.

•

Cannabidiol may have a therapeutical potential in fear regulation in humans.

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

Article history: Previous studies have implicated cannabinoids in extinction of conditioned fear. We have recently

Received 20 March 2013

showed that intraventricular infusion of the phytocannabinoid cannabidiol (CBD) facilitates fear extinc-

Received in revised form 21 April 2013

tion, but the brain regions underlying this effect remained unknown. Here we demonstrate that repeated

Accepted 25 April 2013

microinjections of CBD into the infralimbic cortex (IL) facilitated fear extinction, as indicated by reduced

Available online 1 May 2013

levels of freezing during extinction test. Systemic administration of the CB1 receptor antagonist rimon-

abant blocked the effects of intra-IL CBD, suggesting that CBD acts through CB1 receptors to facilitate

Keywords:

fear extinction. Our ﬁndings suggest a potential therapeutic use of CBD for extinction-based therapies of

Fear extinction

Infralimbic cortex Rimonabant

Conditioned freezing

The major effects of the marijuana plant (Cannabis sativa) have Numerous studies have shown that the eCB system plays a key

been attributed to two main constituents: the psychotropic com- role in fear extinction [5]. CB1 agonists or eCB uptake/hydrolysis

ponent -tetrahydrocannabinol (THC) and the non-psychotropic inhibitors facilitate fear extinction [6,7], whereas either pharma-

phytocannabinoid cannabidiol (CBD) [1]. Most of the pharmacological blockade or genetic deletion of CB1 receptors impairs fear

cological effects of THC are mediated by activation of CB1 and extinction [8,9]. A recent study from our laboratory demonstrated

CB2 cannabinoid receptors subtypes, the same targets activated that intraventricular administration of CBD facilitates contextual

by endogenous ligands such as the endocannabinoid (eCB) anan- fear extinction [10], however the brain circuits mediating this effect

damide [2]. In contrast, the pharmacology of CBD is not completely remained to be investigated.

understood [3]. Despite weakly binding to CB1 and CB2 receptors, The contribution of the eCB system to fear extinction is sup-

one of the potential mechanisms proposed for CBD is the inhibi- ported by the high concentration of CB1 receptors in brain regions

tion of anandamide uptake/hydrolysis, resulting in higher activity implicated in fear regulation, such as the amygdala [11], the hip-

of eCB receptors [4]. pocampus [12], and the medial prefrontal cortex (mPFC) [13].

Special interest in the infralimbic (IL) subregion of the mPFC

emerged from previous studies reporting the essential role of this

structure in fear extinction [14,15]. Thus, considering the recently

described role of CBD in fear extinction and the crucial involvement

∗

Corresponding author at: Laboratory of Fear Learning, Department of Psychiatry, of IL in this process, we investigated the effects of intra-IL injections

School of Medicine, University of Puerto Rico, UPR, PO Box 365067, San Juan 00936,

of CBD using a contextual fear extinction paradigm in rats.

Puerto Rico. Tel.: +1 787 9993057; fax: +1 787 9993057.

Male Long–Evans Hooded rats (n = 46) weighing 300–350 g

E-mail address: [email protected] (F.H. Do Monte).

These authors contributed equally to this work. at the time of experiments were obtained and maintained as

24 F.H. Do Monte et al. / Behavioural Brain Research 250 (2013) 23–27

previously described [16]. Each behavioral test was conducted

during the light phase of the cycle (9:00 am–5:00 pm) using

independent experimental groups. All procedures were approved

by the Institutional Ethics Committee on the Use of Animals

(23080.006118/2004-36/UFSC/BRASIL) in compliance with NIH

animal care guidelines [17].

Rats were bilaterally implanted with two stainless steel guide

cannulas (diameter 0.7 mm, length 11 mm) aimed at the IL. Coor-

dinates were: anteroposterior +3.0 mm from bregma; mediolateral

0.6 mm from midline and dorsoventral −4.0 mm from skull sur-

face [18]. The contextual fear conditioning was performed seven

days after surgery allowing rats to recover from the surgery. In

brief, rats were placed in the conditioning box for 3 min where they

received a single footshock (1.5 mA, 1 s). They were maintained for

an additional minute in the box before being returned to their home

cages. On the next day, rats were randomly assigned to groups

that received intra-IL infusions of CBD (0.1 ␮g or 0.4 ␮g/0.2 ␮l/side)

or vehicle 5 min before being re-exposed to the conditioning box

for the ﬁrst extinction session (9 min). The same treatment was

repeated for each animal on the following two days (extinction ses-

sions 2 and 3). On day 4, rats were returned to the chamber without

drug treatment for a contextual fear memory test (3 min).

Cannabidiol (Tocris, USA) was stored in DMSO stock solution

(50 mM) and diluted in 0.1 M PBS, pH = 7.4, yielding a ﬁnal concen-

tration of 10% DMSO. The same vehicle (VEH) was used as control

solution. Infusions were carried out by inserting two stainless steel

needles (31 gauge, 12 mm) through the guide cannulas while the rat

was gently restrained. The needles were connected to a 5 ␮l Hamil-

ton microsyringe with PE-10 tubing and joined to an infusion pump

(Insight, B12000 model). Drugs were infused at a rate of 0.2 ␮l/min

and the needles were kept in the place for an additional minute Fig. 1. (A) Photomicrograph of rat’s brain showing microinjection placements (black

arrows) in the infralimbic cortex (approximately 2.7 mm anterior to bregma). Scale

to allow drug diffusion. Drug dosage and administration schedule

bar equals 1 mm. (B) Schematic representation of microinjection sites in IL (an exam-

were selected based on previous studies [10,19]. All phases of the

ple from the CBD 0.4 group). Legend: PL = prelimbic cortex; IL = infralimbic cortex;

experiments were video recorded and subsequently analyzed by a

cc = corpus callosum.

trained observer blind to the experimental groups. The percentage

of time the rats spent freezing in each session was registered and

used as an index of fear memory retrieval.

Upon completion of experiments, rats were anesthetized, per- To study the lasting effects of CBD on contextual fear extinction,

fused and had their brains sectioned as previously mentioned [16]. rats were tested in a drug free condition during 3 min in the same

Fig. 1A shows a representative photomicrograph of cannula place- chamber on day 4. One-way ANOVA showed a signiﬁcant effect

ment in IL. Fig. 1B shows a schematic representation of injection of group (F(2,24) = 5.45, p < 0.05) in the mean percentage of freez-

sites adapted from [18]. Only rats with microinjections bilaterally ing during extinction test session. Tukey post hoc test conﬁrmed

located within the IL were included in the statistical analysis. The a signiﬁcant decrease in the percentage of freezing in the IL-CBD

␮

level of statistical signiﬁcance adopted was p < 0.05. All statistical 0.4 g group compared to IL-VEH group (Fig. 2B). This suggests that

␮

analyses were performed using the Statistica software package intra-IL infusions of CBD 0.4 g facilitate extinction of contextual

(version 8.0, Statsoft , Tulsa, USA). fear.

To evaluate the effects of CBD microinjected directly into IL Another possible explanation for the fear reduction induced by

during contextual fear extinction, rats were infused with two dif- intra-IL infusion of CBD 0.4 would be a disruptive process in fear

ferent doses of CBD (0.1 ␮g or 0.4 ␮g/0.2 ␮l/side) or VEH prior to reconsolidation. However, contrary to this assumption, previous

extinction sessions 1–3. Repeated-measures ANOVA of freezing studies have shown that long retrieval sessions (more than 3 min)

during extinction sessions revealed effects for group [F(2,24) = 4.55; trigger extinction mechanisms rather than reconsolidation [21],

p < 0.05], extinction session [F(2,48) = 45.21; p < 0.0001] and their excluding this possibility.

interaction [F(4,48) = 3.10, p < 0.05]. Tukey post hoc comparison An important issue regarding intracerebral microinjection is

revealed a significant reduction in freezing from the first to the drug backflow along the cannula track, which may result in non-

third extinction session in the VEH group, indicating successful speciﬁc targeting effects. For this reason, we analyzed the freezing

contextual fear extinction (Fig. 2A). Intra-IL injection of CBD 0.4 ␮g levels of three rats that were microinjected with CBD 0.4, but had

reduced the percentage of freezing during extinction sessions 2 and misplaced microinjections located dorsal to IL, within the prelimbic

3 when compared to IL-VEH group (all p values < 0.05), suggest- cortex (PL). When comparing to VEH control group, repeated-

ing facilitation of contextual fear extinction (Fig. 2A). This effect measures ANOVA of freezing during extinction sessions did not

was not a result of a reduction in fear expression, because rats reveal effects for group [F(1,10) = 0.56; p = 0.46], extinction session

showed similar levels of freezing during the ﬁrst 3 min of the ﬁrst [F(2,20) = 1.52; p = 0.24] and their interaction [F(2,20) = 1.02; p = 0.37].

extinction session (Unpaired Student’s t-test, IL-CBD 0.4 = 76.1%; IL- In addition, Unpaired Student’s t-test did not show any signiﬁcant

VEH = 77.6%, t = 0.09; p = 0.40). Also, the drug effect was not likely difference between groups in the mean percentage of freezing dur-

due to non-speciﬁc locomotor interference, since intra-IL infusion ing extinction test session on day 4 (VEH = 40.1%, PL–CBD = 35.1%,

of CBD even in doses higher than that used here, did not alter loco- t = 0.58; p = 0.57), suggesting that the facilitatory effect of CBD on

motion in rats tested in the open ﬁeld arena [20]. fear extinction was not mediated by PL.

F.H. Do Monte et al. / Behavioural Brain Research 250 (2013) 23–27 25

A Fear Extinction 60 IL-VEH IL-CBD 0.1 g IL-CBD 0.4 g 40 * 20

% Freezing *

0 Day 1 Day 2 Day 3 Day 4 (drug free test) B 60

20 * % Freezing

IL-VEH IL-CBD IL-CBD

0.1 g 0.4 g Fig. 3. (A) Effects of the repeated microinjection of CB1 antagonist rimonabant

(Rimo; 0.2 mg/kg i.p., 20 min before) plus intra-IL infusion of vehicle (Rimo + VEH;

n = 9); rimonabant plus intra-IL infusion of CBD 0.4 ␮g/side (Rimo + CBD 0.4; n = 10);

Fig. 2. (A) Effects of repeated microinjection of vehicle (n = 9), CBD 0.1 ␮g/side (n = 9)

or single intra-IL infusion of CBD 0.4 ␮g/side (CBD 0.4; n = 9, same showed in Fig. 2)

or CBD 0.4 ␮g/side (n = 9) during the acquisition of contextual fear extinction (5 min

during the acquisition of contextual fear extinction on the mean percentage of freez-

before) on the mean percentage of freezing in the extinction sessions (9 min each);

ing in the extinction sessions (9 min each); (B) the same groups of rats during a single

(B) the same groups of rats during a single 3 min exposure to the conditioning cham-

3 min exposure to the conditioning chamber in a drug-free state 24 h after the last

ber in a drug-free state 24 h after the last extinction session. Data are expressed as

extinction session. Data are expressed as the mean ± SEM. The arrows indicate the

the mean ± SEM. The arrows indicate the moment of infusion. *p < 0.05 compared

moment of infusion. *p < 0.05 compared to vehicle control group.

to vehicle group.

the percentage of freezing in the IL-CBD group when compared to

IL-CBD + i.p.-Rimo group (Fig. 3B).

To investigate the hypothesis that intra-IL infusion of CBD is Since previous studies have demonstrated that administration

facilitating extinction through CB1 receptor activation, two addi- of the solvent DMSO may induce a wide range of pharmacologi-

tional groups were injected with rimonabant (Rimo; 0.2 mg/kg, i.p.) cal effects [22], one could argue that the blockade of the extinction

20 min before the intra-IL infusions of CBD 0.4 ␮g or VEH prior to facilitating effects in the IL-CBD + i.p.-Rimo group was due to DMSO

extinction sessions 1–3. Rimonabant, a CB1 cannabinoid receptor effects. To investigate this possibility, a group of rats receiving

antagonist (Sanoﬁ-Aventis, France), was dissolved in 10% DMSO DMSO 10% prior to extinction sessions 1–3 was compared to a

plus 0.1% Tween 80 in saline (NaCl 0.9%). The dose of rimonabant saline-treated group. Repeated-measures ANOVA of freezing per-

was selected based on previous reports [10] and pilot studies. To centage during extinction sessions did not reveal any signiﬁcant

check if the administration of rimonabant alone induced changes in differences (F(2,36) = 0.003; p = 0.99) between the groups treated

freezing, we compared the percentage of freezing of IL-VEH + i.p.- with DMSO (Ext 1 = 46.3%, Ext 2 = 36.5%, Ext 3 = 25.5%) or saline

Rimo group with the IL-VEH group during the ﬁrst extinction (Ext 1 = 49.8%; Ext 2 = 40.3%; Ext 3 = 29.5%) along the extinction ses-

session. Unpaired Student’s t-test demonstrated that both groups sions. Unpaired Student’s t-test performed in day 4 did not reveal

presented similar levels of freezing (IL-VEH + i.p.-Rimo = 39.65%; IL- any signiﬁcant differences (t = 0.60; p = 0.51) in the mean percent-

VEH = 44.97%, t = 1.02; p = 0.40), indicating that the selected dose age of freezing during the extinction test session between DMSO

of rimonabant itself was not enough to change freezing behav- (30.2%) and saline (35.7%) groups, indicating that DMSO did not

ior. When comparing the different groups, repeated-measures affect the rate of extinction. Our ﬁndings suggest that blockade of

ANOVA of freezing during extinction sessions showed effects for CB1 receptors after rimonabant administration blocks the extinc-

extinction session [F(2,50) = 99.29; p < 0.0001] and the interaction tion facilitating effects of intra-IL CBD.

between group and extinction session [F(4,50) = 5.58; p < 0.001]. As Recent ﬁndings have shown that compounds with THC-like

illustrated in Fig. 3A, Tukey post hoc comparison revealed a sig- activity alleviate the symptoms of PTSD [23] and facilitate extinc-

niﬁcant reduction in freezing during extinction sessions 2 and 3 in tion of conditioned fear in humans [24]. However, the therapeutic

the IL-CBD group when compared to the IL-CBD + i.p.-Rimo group usefulness of these compounds for extinction-based therapy is

(all p values < 0.05). One-way ANOVA of freezing performed in limited by psychoactivity, tolerance and dependence induced by

day 4 revealed a signiﬁcant effect of group (F(2,25) = 5.26; p < 0.05) these drugs [25]. Here we demonstrated that CBD, a major non-

in the mean percentage of freezing during the extinction test psychoactive constituent of the C. sativa plant, which is devoid

session. Tukey post hoc test conﬁrmed a signiﬁcant decrease in of these adverse effects [3], also facilitates fear extinction. We

26 F.H. Do Monte et al. / Behavioural Brain Research 250 (2013) 23–27

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