Behavioural Brain Research 286 (2015) 49–56
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Effects of intra-infralimbic prefrontal cortex injections of cannabidiol in the modulation of emotional behaviors in rats: Contribution of 5HT1A receptors and stressful experiences A.L.Z. Marinho a,∗ , C. Vila-Verde a , M.V. Fogac¸a a,b , F.S. Guimarães a,b a b
Department of Pharmacology, Medical School of Ribeirão Preto, University of São Paulo (FMRP-USP), Ribeirão Preto, São Paulo, Brazil Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo (USP), São Paulo, Brazil
h i g h l i g h t s • • • • •
CBD behavioral effects depend on previous stressful experience. CBD intra-IL injection induces anxiogenic-like effects in the fear conditioning test. CBD intra-IL injection promotes anxiolytic-like effects in the elevated plus maze. The anxiolytic-like CBD effect disappeared when the animals were previously stressed. Anxiolytic and anxiogenic CBD effects are blocked by a 5HT1A receptor antagonist.
a r t i c l e
i n f o
Article history: Received 18 December 2014 Received in revised form 6 February 2015 Accepted 10 February 2015 Available online 19 February 2015 Keywords: Anxiety Cannabidiol Stress Prefrontal cortex Infralimbic 5HT1A receptors
a b s t r a c t The infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex are involved in behavioral responses observed during defensive reactions. Intra-PL or IL injections of cannabidiol (CBD), a major non-psychotomimetic cannabinoid present in the Cannabis sativa plant, result in opposite behavioral effects in the contextual fear conditioning (CFC) paradigm. The intra-PL effects of CBD are mediated by 5HT1A receptors and depend on previous stressful experiences but the mechanisms and effects of intra-IL CBD injected are unknown. To this aim the present work veriﬁed the effects of intra-IL administration of CBD on two animal models of anxiety, the elevated plus maze (EPM) and CFC. We also investigated if these effects were mediated by 5HT1A receptors and depended on previous stressful experience. Male Wistar rats received bilateral microinjections of vehicle, WAY100635 (5HT1A receptor antagonist, 0.37 nmol) and/or CBD (15, 30 or 60 nmol) before being submitted to the behavioral tests. Intra-IL CBD induced anxiolytic and anxiogenic in the EPM and CFC, respectively. To verify if these effects are inﬂuenced by the previous stressful experience (footshocks) in the CFC model, we tested the animals in the EPM 24 h after a 2-h restraint period. The anxiolytic-like effect of CBD in the EPM disappeared when the animals were previously stressed. Both responses, i.e., anxiolytic and anxiogenic, were prevented by WAY100635, indicating that they involve local 5HT1A -mediated neurotransmission. Together these results indicate that CBD effects in the IL depend on the nature of the animal model, being inﬂuenced by previous stressful experiences and mediated by facilitation of 5HT1A receptors-mediated neurotransmission. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The prefrontal cortex (PFC) has been related to a several behavioral and cognitive disorders . Studies performed in rodents and
∗ Corresponding author at: Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, 3900, Bandeirantes Avenue, Monte Alegre, Ribeirão Preto, SP, Brazil. Tel.: +55 1633153325; fax: +55 1633153325. E-mail address: [email protected]
(A.L.Z. Marinho). http://dx.doi.org/10.1016/j.bbr.2015.02.023 0166-4328/© 2015 Elsevier B.V. All rights reserved.
non-human primates suggest that different cognitive and emotional processes are mediate by anatomically distinct subregions of the PFC [2,3,6]. The rodent PFC has been divided into medial, lateral and ventral/orbital [1,4]. Speciﬁcally, the medial PFC (mPFC) comprises the cingulated (AC), prelimbic (PL) and infralimbic (IL) areas [5–7], which present functional differences and distinct patterns of behavioral control [8–10]. The mPFC plays an important but complex role in the modulation of defensive-related behaviors that seems to depend on the nature of the threatening stimuli . For example, whereas CoCl2
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
(a selective inhibitor of synaptic activity) induces anxiolytic-like effects in the contextual fear conditioning (CFC) and Vogel conﬂict (VCT) tests, it enhances anxiety in rats submitted to the elevated plus-maze (EPM) and light-dark box. Regarding particularly the PL and IL areas, these adjacent cortical regions produce different responses in several aversive situations. This was demonstrated, for example, in cardiovascular response evoked by restraint stress in rats. Bilateral microinjection of CoCl2 (200 nl, 1 mM) into the PL increased heart rate (HR) response associated with restraint, without affecting the restraint-induced blood pressure (BP) response. However, when local synapses in the IL were inhibited by bilateral injection of CoCl2 , the restraint-induced HR increases were significantly reduced without a signiﬁcant effect on the concomitant BP response . Also in agreement with this proposal, Lemos et al.  showed opposite effects of intra-PL and -IL injections of the phytocannabinoid cannabidiol (CBD) in the CFC model. Direct CBD (30 nmol) microinjection into the PL reduced freezing induced by re-exposure to the aversively conditioned context. In the IL, however, CBD (15 and 30 nmol) produced an opposite results, increasing the expression of contextual fear conditioning. Preclinical and clinical studies demonstrated that CBD has antipsychotic, antidepressive, anxiolytic and neuroprotective properties [12–16]. The anxiolytic effects of this compound have been shown by several studies with different animal models. Systemic administration of CBD produced anxiolytic-like effects in the EPM, VCT and the CFC [14,17,18] whereas direct administration of this drug into the dorsal portions of periaqueductal gray matter (DPAG) or the bed nucleus of the stria terminallis (BNST) induced anxiolytic-like effects in the EPM [19,20], CFC  and VCT . In these studies the CBD effects were abolished by previous injection of WAY100635, a 5HT1A receptor antagonist. Recent results from our group showed that intra-PL CBD induces opposite effects in rats submitted to the EPM and CFC models, being anxiogenic and anxiolytic, respectively. The anxiogenic effect observed in the EPM turned into an anxiolytic response when the animals were submitted to 2 h of forced restraint 24 h before the test. This stress inﬂuence could help to explain the opposite effects of CBD observed in the CFC model, since in this test the animals were also submitted to a previous (24 h) stressor (the footshocks in the conditioning session). CBD opposite responses in the PL (anxiolytic or anxiogenic) were prevented by a 5HT1A receptor antagonist (WAY100635) . Based on these ﬁndings, the present work aimed at verifying if intra-IL CBD effects can also be inﬂuenced by the animal model used to assess anxiety and by previous stressful experiences. Additionally, we also investigated if they are mediated by 5HT1A receptors. 2. Experimental procedures 2.1. Animals Male Wistar rats (270–310 g) originated from the Central Animal Farm of the Medical School of Ribeirão Preto, University of São Paulo (FMRP-USP) were maintained in groups of ﬁve animals per cage (41 cm × 33 cm × 17 cm), with food and water ad libitum (41 × 33 × 17 cm) in a temperature controlled room (24 ± 2 ◦ C) with a 12 × 12 h light-dark cycle (lights on at 6 h 30 a.m. and off at 6 h 30 p.m.). All experimental procedures were submitted and approved by the local Ethics Committee (process number: 114/2013). 2.2. Drugs The following drugs were employed: cannabidiol (CBD, THC Pharma, Frankfurt, Germany), dissolved in 100% grape oil and used
at the doses of 15, 30 and 60 nmol (Lemos et al. ); the 5HT1A antagonist WAY100635 (Sigma, USA), dissolved in saline (NaCl 0.9%) and used at the dose of 0.37 nmol. This dose was based on a previous studies showing that it can antagonize CBD anxiolytic effects observed after intra-cerebral administration [19,22]. 2.3. Stereotaxic surgery Animals were submitted to a stereotaxic surgery procedure to bilaterally implant cannulae (9.0 mm, 0.6 mm OD) into the IL using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). Coordinates for cannula implantation were: anteroposterior: 3.2 mm; lateral: ±2.3 mm; depth: 3.8 mm; angle: 22◦ (Paxinos and Watson ). Cannulae were ﬁxed to the skull with dental cement and one metal screw. The surgeries were performed under deep anesthesia with tribromoethanol 2.5% (10.0 ml/kg, intraperitonneally, i.p.) and immediately after the animals received Veterinary Pentabiotic (0.2 ml, intramuscular) and analgesic (Banamine, 1.0 ml/kg, subcutaneous) to prevent infections and decrease post-surgical pain. After surgery, animals underwent a recovery period of 5–7 days before the behavioral tests. 2.4. Microinjection Animals received bilateral microinjections of vehicle, WAY100635 (0.37 nmol) or CBD (15, 30 or 60 nmol) into the IL before being submitted to the behavioral tests. To this aim, needles (10.0 mm, 0.3 mm OD) connected to a microliter syringe (Hamilton, USA, 10 L) through a segment of polyethylene (P10) were inserted into the guide cannulae. The solutions were injected using an infusion pump (KD Scientiﬁc, USA). A 0.2 L solution volume was injected over 30 s. After the injections, the needles remained in the cannulae for additional 30 s to prevent drug reﬂux. Except for the experiment that performed the dose–response curve of CBD in the EPM, animals received two injections of drug and/or vehicle with a 5 min interval between them. In all experiments, the time elapsed between the last microinjection and the beginning of the tests was 10 min. 2.5. Apparatus 2.5.1. Contextual fear conditioning test Animals were submitted to a fear conditioning box (37 × 25 × 25 cm) containing a grid ﬂoor with 18 stainless steel rods (3 mm diameter) spaced 1.5 cm apart and wired to a shock generator (Automatic Reﬂex Conditioner, model EP 101; Insight, Brazil). The chamber was cleaned with 70% ethanol before and after each experimental session. The experimental procedure started 5–7 days after stereotaxic surgery and consisted in an initial exposure of each animal to the box during 10 min in the morning period for initial familiarization session (habituation). In the afternoon the rats were placed again in the chamber for the contextual conditioning session. In this session, animals were separated into two experimental groups: non-conditioned and conditioned. The non-conditioned group was exposed to the footshock chamber for 10 min but no shock was delivered. The conditioned group was submitted to a shock session during which it received, after 2 min of habituation (pre-shock period), three 0.35 mA/1 s randomized electrical foot shocks delivered at 20–60 s intervals . The animal remained in this chamber for additional 2 min (post-shock period). The test session was performed 24 h after the conditioning session and consisted of a 10-min-long re-exposure to the footshock chamber without shock delivery. The rats were tested only once and one at a time. During the re-exposure to the aversively conditioned context the total time of freezing response, deﬁned as the complete absence of movement
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
Fig. 1. Microphotography and histological localization of injection sites located in the infralimbic medial prefrontal cortex in diagrams (mm from Bregma) based on the Atlas of Paxinos and Watson . Scale bar: 600 m.
(except respiration) while the animal assumed a characteristic tense posture , was measured. 2.5.2. Elevated plus-maze (EPM) The wooden-made EPM was located in a sound attenuated and temperature controlled room (23 ◦ C), with one incandescent light (40 W–60 lux) placed 1.3 m away from the maze. The apparatus consisted of two opposing open arms (50 × 10 cm) without sidewalls perpendicular to two enclosed arms (50 × 10 × 40 cm), with a central platform common to all arms (10 × 10 cm). It was elevated 50 cm above the ﬂoor and an acrylic edge (1 cm) in the open arms helped to prevent animal falls. In this model, rodents naturally avoid the open arms, exploring more extensively the enclosed arms. Anxiolytic drugs, in non-sedative doses, increase the exploration of the open arms without affecting the number of enclosed arms entries, which is usually used to assess general exploratory activity . Ten minutes after the last injection the rat was placed on the central platform of the maze with the head facing one of the enclosed arms. The test lasted for 5 min and was video recorded. The animal behavior was analyzed with the help of the Anymaze Software (version 4.5, Stoelting, Wood Dale, USA). This software indicates the location of the animal in the EPM and automatically calculates the percentage of entries (PeO) and time spent in the open arms (PtO) and the number of enclosed arms entries (EA). Animals were only considered to enter an open or enclosed arm when 85% of their bodies were inside the region. All experiments were performed in the morning period (8 a.m. to 12 p.m.). 2.6. Acute restraint stress procedure The experimental apparatus consisted of a metallic tube (6.3 × 19.3 cm) with an adjustable roof, ventilated by holes. Twenty-four hours before being submitted to the EPM test, an independent group of rats was exposed to a single restraint episode during 2 h. Immediately after stress, rats were individually housed until the day of the test. 2.7. Histology After the behavioral tests, animals were anesthetized with 4% chloral hydrate (Sigma–Aldrich, 10 ml/kg) and 0.2 L of 1% Fast Green dye was bilaterally injected into the IL as a marker of the injection sites. The animals were perfused with saline 0.9% and formalin 10%. The brains were removed and kept in 10% formalin solution for 2–5 days. Soon after, brains were cut into 50-m thick sections in a cryostat (Cryocut 1800, Leica, Heerbrugg, Switzerland). The injection sites were identiﬁed in diagrams from the Paxinos and Watson’s atlas . Rats receiving injections outside the IL were not included in the analysis.
Fig. 2. Effects of WAY100635 and/or CBD (30 nmol) in the contextual fear conditioning test. CBD increased total freezing time (%), indicating enhanced expression of conditioned fear compared to the vehicle-treated group. Pretreatment with the 5HT1A receptor antagonist WAY100635 (0.37 nmol) prevented this effect. Bars represent the mean ± standard error of the mean (S.E.M.). *Signiﬁcant difference from non-conditioned vehicle group (p < 0.05, Student-t test). #Signiﬁcant difference from all other groups (p < 0.05, ANOVA, Duncan test). Conditioned animals, n = 7–11/group; non-conditioned animals, n = 4/group. V = vehicle; CBD = cannabidiol; WAY = WAY100635.
2.8. Statistical analysis The percentages of open arm entries and time spent in these arms, the number of enclosed arm entries and the time spent in freezing were analyzed by one-way or, in experiments with combined injections, two-way Analysis of Variance (ANOVA). The Duncan posthoc test was used for multiple comparisons. Conditioned and non-conditioned control groups were compared using the t-Student test. In all cases, statistic signiﬁcance were assumed at p < 0.05. 3. Results 3.1. Injection sites Diagrams showing representative injection sites in the infralimbic medial prefrontal cortex and a representative microphotography are shown in Fig. 1. 3.2. Intra-IL injection of WAY100635 prevented the anxiogenic-like effect of CBD in the contextual fear conditioning test Non-conditioned vehicle animals showed less freezing compared to conditioned animals vehicle [Fig. 2 (t9 = 3.660, p = 0.005, Student-t test)]. In conditioned animals, the two-way ANOVA indicated an interaction between the ﬁrst and second
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
Fig. 3. Dose response curve of CBD (15, 30 and 60 nmol) in the elevated plus-maze test. CBD induced an anxiolytic-like effect at the doses of 15 and 30 nmol, represented by an increase in the percentage of entries and time spent in the open arms. No changes were observed in the number of enclosed arm entries. Bars represent the mean ± standard error of the mean (S.E.M.). *Signiﬁcant difference from vehicle group (ANOVA followed by Duncan test, p < 0.05, n = 7–8/group; Out: n = 7). V, vehicle; CBD, cannabidiol; WAY, WAY100635.
injection (F1,32 = 11.458; p = 0.002). Intra-IL injection of CBD (30 nmol) induced an anxiogenic-like effect in the CFC test, represented by an increase in total freezing time compared to vehicle-treated animals. This effect was prevented by prior injection of WAY100635 (F3,32 = 8.859, p = 0.0001, one-way ANOVA followed by Duncan’s test).
3.3. Intra-IL injection of CBD induces an anxiolytic-like effect in the elevated plus-maze test Intra-IL injection of CBD (15 and 30 nmol) induced an anxiolyticlike effect in the EPM test, represented by an increase in open arm exploration compared to vehicle-treated animals [Fig. 3 (PeO: F3,27 = 6.90, p = 0.001; PtO: F3,27 = 5.052, p = 0.007, one-way ANOVA followed by Duncan’s test)]. The higher dose (60 nmol) did not produce any effect. No changes in the number of enclosed arm entries were found. Finally, when CBD was administered into IL nearby structures (out group) no changes were observed in the PeO, PtO or the number of enclosed arms compared to vehicle group (Out: PeO: 19.43 ± 2.49 vs. 21.50 ± 3.36; PtO: 9.13 ± 1.66 vs. 8.90 ± 1.60; enclosed arms: 7.85 ± 0.62 vs. 9.44 ± 1.05).
Fig. 4. Effects of WAY100635 (0.37 nmol) and/or CBD (30 nmol) in rats submitted to the elevated plus-maze test. Pre-treatment with the 5HT1A receptor antagonist WAY100635 prevented CBD anxiolytic effect. No changes were observed in the number of enclosed arm entries. Bars represent the mean ± standard error of the mean (S.E.M.). *Signiﬁcant difference from vehicle group. #Signiﬁcant difference from WAY + CBD group (ANOVA followed by Duncan test, p < 0.05, n = 7–9/group; Out: n = 15). V, vehicle; CBD, cannabidiol.
3.4. Intra-IL injection of WAY100635 prevented CBD anxiolytic-like effect in the elevated plus-maze test Prior intra-IL injection of an ineffective dose of WAY100635 (0.37 nmol) prevented the anxiolytic-like effect of CBD (30 nmol) [Fig. 4 (Two-way ANOVA, interaction between ﬁrst vs. second injection: PeO: F1,26 = 6.767, p = 0.015; PtO: F1,26 = 5.553, p = 0.026)]. No changes were found in the number of enclosed arm entries. 3.5. Lack of intra-IL CBD effects in rats submitted to restraint stress 24 h before the elevated plus-maze test Acute restraint stress induced an anxiogenic-like effect in rats tested 24 h later in the EPM test, represented by a decrease in open arms exploration [Fig. 5 (PeO: F5,53 = 5.961, p = 0.0001, oneway ANOVA followed by Duncan’s test)] compared to non-stressed animals. Intra-IL CBD injected into non-stressed animals increased the PtO compared to all other groups (F5,53 = 4.886, p = 0.001, ANOVA followed by Duncan’s test). However, this anxiolytic-like effect disappeared when the animals had been previously restrained. Furthermore, there was no difference among restricted animals from the different experimental groups (PeO: F3,35 = 1.932,
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
Fig. 5. Effects of WAY100635 (0.37 nmol) and/or CBD (30 nmol) in rats submitted to the elevated plus-maze test 24 h after the acute restraint procedure. Restraint stress (2 h) induced an anxiogenic-like effect. CBD induced an anxiolytic-like effect in non-stressed animals. This effect was not seen in stressed animals. No changes were observed in the number of enclosed arm entries. Bars represent the mean ± standard error of the mean (S.E.M.). *Signiﬁcant difference from non-restricted vehicle group. #Signiﬁcant difference from all restricted groups. +Signiﬁcant difference from all other groups (ANOVA followed by Duncan test, p < 0.05, n = 7–14/group). V, vehicle; CBD, cannabidiol; WAY, WAY100635.
p = 0.142; PtO: F3,35 = 1.706, p = 0.184, one-way ANOVA). No changes were found in the number of enclosed arm entries. 4. Discussion The present study showed that intra-IL injection of CBD induced different effects depending on the animal model employed and previous stressful experiences (footshocks or acute restraint-stress). In the EPM, an anxiety test that is based on innate fear , CBD caused an anxiolytic-like effect represented by an increase in open arms exploration. On the other hand, the same compound produced an anxiogenic-like response in the CFC, an animal model that, unlike EPM, is based on associative (conditioned) learning  and involves previous stressful experience (footshocks) and memory. Both responses, i.e., anxiolytic and anxiogenic, were prevented by the 5HT1A antagonist WAY100635, indicating a recruitment of the serotoninergic system. Moreover, the anxiolytic effect of CBD in the EPM disappeared when the animals had been submitted to an acute
episode of restraint stress 24 h before the test, showing that CBD effects in the PL are inﬂuenced by previous stressful experiences. Animal models of fear/anxiety can be classiﬁed based on the nature of the threatening stimuli used, involving associative (conditioned) learning in the case of CFC and ethological, unconditioned threat, represented by the open arms of the EPM [26,76]. Even if learning mechanisms could also play a role in the EPM test, Lisboa et al.  showed that inactivation of the ventral medial PFC (vmPFC) induced anxiolytic-like effects in the CFC and VCT tests but enhanced anxiety in rats submitted to the EPM and light-dark box. These results indicate that the vmPFC plays an important but complex role in the modulation of defensive related behaviors, which seems to depend on the nature of the anxiety/fear inducing stimuli. The mechanisms and structures responsible for CBD effects are not fully understood. Several pieces of evidence show that this drug can produce multiple pharmacological effects by different mechanisms . For example, CBD shows low afﬁnity for CB1 and CB2 receptors [28,29] but can facilitate endocannabinoid signaling by inhibition of FAAH, an enzyme responsible for anandamide degradation. Furthermore, CBD can activate TRPV1 and facilitate the activation of 5HT1A receptors, probably as an allosteric modulator [30–34]. Accordingly, direct administration of CBD into the DPAG or BNST produced anxiolytic-like effects in different animal models through 5HT1A receptors activation [19–21]. In relation to the PL and IL areas, previous work from our group reported that direct CBD (30 nmol) microinjection into the PL reduced freezing induced by re-exposure to an aversively conditioned context. On the other hand, intra-IL CBD (15 and 30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning . Our study conﬁrmed the latter result and, additionally, showed that it depends on local 5HT1A -mediated neurotransmission. The PL and IL are associated with different brain structures important for the expression of conditioned emotional response (CER), including the amygdala . The involvement of this latter structure with conditioned responses was ﬁrst shown by Blanchard and Blanchard . They demonstrated that lesions in the amygdala abolished the freezing response to aversively conditioned context. The PL activates the basolateral complex of the amygdala (BLA) that sends glutamatergic projections to the medial sector of the central nucleus (CeM) [36–38]. The CeM is the main source of amygdala outputs to the brainstem and hypothalamic sites  that mediate the behavioral and autonomic correlates of fear [40–42]. PL inactivation reduced freezing both to a tone and a context that had been previously paired with footshock (learned fear), suggesting that this region integrates information from auditory and contextual inputs and regulates expression of fear memories via projections to the BLA . Differently, the IL inhibits CeM projection neurons by activating GABAergic intercalated cells (ITC), thereby reducing CeM responses to inputs from the BLA, resulting in decreased freezing response [10,44,45]. In the present work intra-IL CBD produced an anxiogenic-like effect in the CFC that was prevented by WAY100635. Since activation of 5HT1A receptors causes neuronal hyperpolarization through the G-protein-coupled opening of K+ channels [46,77], CBD could have inhibited, via 5HT1A receptors, pyramidal projecting neurons located in IL. As a consequence, glutamatergic inputs from the IL to ITC would decrease, resulting in CeM disinhibition. This would allow for the expression of defensive behaviors, including the freezing response . Moreover, Ji and Neugebauer  demonstrated an inverse interaction between IL and PL regions. They reported that IL activation can inhibit PL output, suggesting that IL-mediated extinction mechanisms may not only involve direct interactions with the amygdala but also control of PL-facilitatory inﬂuences on fear expression. Corroborating this idea, PL inactivation reduces
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
fear expression [43,49,50] and microstimulation of this structure increases CER . Regarding the EPM test, the present study showed that intraIL CBD produced an anxiolytic-like effect that depended on 5HT1A receptors activation, once the selective antagonist abolished this effect. This response was opposite to that observed in the CFC test. Similar to our study, Fogac¸a et al.  showed that intra-PL CBD induced opposite effects in the CFC and EPM tests. However, they went on in an opposite direction, being anxiolytic and anxiogenic, respectively. Both responses were also prevented by WAY100635. The role of 5HT1A receptors in the modulation of anxiety states appears to be complex  and depends on their pre- and postsynaptic locations. They are present as inhibitory autoreceptors in the dendrites of serotonergic cell bodies in raphe nuclei but are also localized postsynaptically in the hippocampus, septum, amygdala, PAG, entorhinal cortex and mPFC [26,51,52]. Concerning the latter structure, there are reciprocal projections between the dorsal and median raphe nuclei and cingulated, PL and IL cortices [53–57], suggesting that the mPFC can directly inﬂuence serotonergic neurons in the raphe nuclei. How this inﬂuence would explain the present results, however, is still unclear. 5-HT1A receptors exert an inhibitory inﬂuence in limbic and cortical release of glutamate [58–60] and a disinhibition of glutamatergic activity may contribute to the behavior changes observed in mice genetically deﬁcient in these receptors. These animals usually show enhanced anxiety and sensitivity to stress [61–65] that could involve interactions between postsynaptic 5-HT1A receptors and glutamatergic transmission in pyramidal cells of the frontal cortex and, possibly, subcortical structures . In line with the present ﬁndings, Lil-Lin Bi et al.  showed that IL activation produced by infusion of the GABAA receptor antagonist bicuculline induced anxiogenic-like effects reﬂected by decreased time spent in the center area of an open ﬁeld arena and open arm exploration in the EPM test. It also increased the latency to eat in the novelty suppressed feeding test. On the other hand, IL cortex inactivation with the AMPA receptor antagonist CNQX produced opposite, anxiolytic-like effects. Similarly, CBD, by acting on 5HT1A receptors in the IL and hyperpolarizing local glutamatergic neurons, could have inhibited this region. This inactivation would lead to reduction in the activation of glutamatergic projections from IL to encephalic regions associated with anxiety such as the dorsolateral periaqueductal gray (dlPAG). Electrical and chemical stimulation of this region causes defensive responses [68,69,70] whereas its inhibition induces anxiolytic effects [71–75]. Therefore, intra-IL CBD could lead, depending on the animal model used, to preferential activation/inactivation of different structures such as the amygdala in CFC and the dlPAG in the EPM. Another factor that could be involved in the opposite effects observed in the EPM and CFC was stress controllability. Even if exposure to the EPM increases plasma corticosterone concentrations [78,79], there is a component of controllable stress in this model since the animal can avoid the open, more aversive arms. The concept of stress control is based on the capacity of the animal to manipulate the intensity, termination, duration, pattern or the onset of a given stressor . Behavior changes following presentation of an incontrollable acute stressor persist for days, indicating the participation of plastic alterations in the central nervous system . Unlike the EPM, the CFC, besides being based on learning and memory paradigms, is a model that involves the forced re-exposure to a context in which the animal had received uncontrollable electrical footshocks 24 h before the test . Therefore, the opposite results obtained in the CFC and EPM could also be due to the previous uncontrollable stress experience. To test this possibility the animals were initially submitted to an uncontrollable stressor, restraint stress, 24 h before being tested in the EPM. Interestingly, using this protocol the anxiolytic-like effect of CBD in the
EPM disappeared. Since restraint stress causes an anxiogenic effect by itself [83,84] a bottom effect could have prevented the disclosure of an inversion of CBD effects by the previous stress experience as observed in the PL . Restraint stress produces long-lasting behavioral changes, such as reduced exploratory activity, facilitation of inhibitory avoidance in the T-maze test and reduced open arm exploration in the EPM [83,85–87]. Furthermore, stress can cause plastic changes in mPFC sub regions, including the PL and IL [88,89]. For example, exposure to an uncontrollable stressor causes a retraction of apical dendrites of IL pyramidal neurons . Thus, the two stressors employed in the present study, restraint and footshocks, could have caused molecular changes in the mPFC that would inﬂuence the effects of intra-IL CBD. As reported in the introduction, Fogac¸a et al.  have also demonstrated that intra-PL CBD effects depend on previous stressful experiences. In the opposite direction to the present ﬁndings with the IL, restraint stress 24 h before testing turned the anxiogenic effect of CBD intra-PL into an anxiolytic one in the EPM. The molecular basis of this stress interference on intra-PL and intra-IL administered CBD is still unknown and needs further investigation. 5. Conclusion Our results show that acute intra-IL injection of CBD modulates anxiety responses by facilitation of 5HT1A receptor-mediated neurotransmission. They also indicate that this modulation is complex and depends on the animal model used to evaluate anxiety-like behaviors and on previous stressful experiences. Conﬂict of interests The authors declare that they have no conﬂict of interests. Funding This study was supported by grants from CAPES, CNPq and FAPESP. Acknowledgements We thank J. C. Aguiar and E.T. Gomes for the technical support. References  Dalley JW, Cardinal RN, Robbins TW. Prefrontal executive and cognitive functions in rodents: neural and neurochemical substrates. Neurosci Biobehav Rev 2004;28:771–84.  Dias R, Robbins TW, Roberts AC. Dissociation in prefrontal cortex of affective and attentional shifts. Nature 2006;380:69–72.  Bussey TJ, Everitt BJ, Robbins TW. Dissociable effects of cingulated and medial frontal lesions on stimulus-reward learning using a novel Pavlovian autoshaping procedure for the rat: implications for the neurobiology of emotion. Behav Neurosci 1997;111:908–19.  Ongur D, Price JL. The organization of networks within the orbital and medial prefrontal cortex of rats, monkeys and humans. Cereb Cortex 2000;10:206–19.  Berendse HW, Groenewegen HJ. Restricted cortical termination ﬁelds of the midline and intralaminar thalamic nuclei in the rat. Neuroscience 1991;42:73–102.  Ray JP, Price JL. The organization of the thalamocortical connections of the mediodorsal thalamic nucleus in the rat, related to the ventral forebrain prefrontal cortex topography. J Comp Neurol 1992;323:167–97.  Heidbreder CA, Groenewegen HJ. The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci Biobehav Rev 2003;27:555–79.  Tavares RF, Correa FM, Resstel LB. Opposite role of infralimbic and prelimbic cortex in the tachycardiac response evoked by acute restraint stress in rats. J Neurosci Res 2009;87(11):2601–7.  Lemos JI, Resstel LB, Guimarães FS. Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats. Behav Brain Res 2010;207:105–11.
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56  Vidal-Gonzalez I, Vidal-Gonzalez B, Rauch SL, Quirk GJ. Microstimulation reveals opposing inﬂuences of prelimbic and infralimbic cortex on the expression of conditioned fear. Learn Mem 2006;13:728–33.  Lisboa SF, Stecchini MF, Correa FM, Guimaraes FS, Resstel LB. Different role of the ventral medial prefrontal cortex on modulation of innate and associative learned fear. Neuroscience 2010;171:760–8.  Bergamaschi MM, Queiroz RH, Zuardi AW, Crippa JA. Safety and side effects of cannabidiol, a Cannabis sativa constituent. Curr Drug Saf 2011;6:237–49.  Campos AC, Moreira FA, Gomes FV, Del Bel EA, Guimarães FS. Multiples mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders. Philos Trans R Soc Lond B: Biol Sci 2012;367:3364–78.  Guimarães FS, Chiaretti TM, Graeff FG, Zuardi AW. Antianxiety effect of cannabidiol in the elevated plus-maze. Psychopharmacology 1990;100:558–9.  Zuardi AW, Cosme RA, Graeff FG, Guimarães FS. Effects of ipsapirone and cannabidiol on human experimental anxiety. J Psychopharmacol 1993;7(1):82–8.  Zanelati TV, Biojone C, Moreira FA, Guimarães FS, Joca SR. Antidepressant like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors. Br J Pharmacol 2010;159:122–8.  Moreira FA, Aguiar DC, Guimarães FS. Anxiolytic-like effect of cannabidiol in the rat Vogel conﬂict test. Prog Neuropsychopharmacol Biol Psychiatry 2006;30:1466–71.  Resstel LB, Joca SR, Moreira FA, Correa FM, Guimarães FS. Effects of cannabidiol and diazepam on behavioral and cardiovascular responses induced by contextual conditioned fear in rats. Behav Brain Res 2006;172:294–8.  Campos AC, Guimarães FS. Involvement of 5HT1A receptors in the anxiolyticlike effects of cannabidiol injected into the dorsolateral periaqueductal gray of rats. Psychopharmacology (Berlin) 2008;199:223–30.  Gomes FV, Resstel LB, Guimarães FS. The anxiolytic-like effects of cannabidiol injected into the bed nucleus of the stria terminalis are mediated by 5-HT1A receptors. Psychopharmacology (Berlin) 2011;213:465–73.  Gomes FV, Reis DG, Alves FH, Corrêa FM, Guimarães FS, Resstel LB. Cannabidiol injected into the bed nucleus of the stria terminalis reduces the expression of contextual fear conditioning via 5-HT1A receptors. J Psychopharmacol 2012;26:104–13.  Fogac¸a MV, Reis FMCV, Campos AC, Guimarães FS. Effects of intraprelimbic prefrontal cortex injection of cannabidiol on anxiety-like behavior: involvement of 5HT1A receptors and previous stressful experience. Eur Neuropharmacol 2013;24(3):410–9.  Inoue T, Tsuchiya K, Koyama T. Serotonergic activation reduces defensive freezing in the conditioned fear paradigm. Pharmacol Biochem Behav 1996;53:825–31.  File SE, Mabbutt PS, Hitchcott PK. Characterisation of the phenomenon of “onetrial tolerance” to the anxiolytic effect of chlordiazepoxide in the elevated plusmaze. Psychopharmacology (Berlin) 1990;102:98–101.  Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Amsterdam: Elsevier Academic Press; 2005.  Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70:83–244.  Izzo AA, Borreli F, Capasso R, Di Marzo V, Mechoulam R. Non-psychotropic plant cannabinoids: new therapeutic opportunities from an ancient herb. Trends Pharmacol Sci 2009;30:515–52.  Petitet F, Jeantaud B, Reibaud M, Imperato A, Dubroeucq MC. Complex pharmacology of natural cannabinoids: evidence for partial agonist activity of delta9-tetrahydrocannabinol and antagonist activity of cannabidiol on rat brain cannabinoid receptors. Life Sci 1998;63:1–6.  Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reﬂects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berlin) 2009;204:361–73.  Bisogno T, Hanus L, De Petrocellis L, Tchilibon S, Ponde DE, Brandi I. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. Braz J Pharmacol 2001;134:845–52.  Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of cannabidiol at 5-HT1a receptors. Neurochem Res 2005;30:1037–43.  Russo EB. Taming THC: potential cannabis synergy and phytocannabinoidterpenoid entourage effects. Br J Pharmacol 2011;163:1344–64.  Carrier EJ, Auchampach JA, Hillard CJ. Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc Natl Acad Sci U S A 2006;103:7895–900.  Campos AC, Guimarães FS. Evidence for a potential role for TRPV1 receptors in the dorsolateral periaqueductal gray in the attenuation of the anxiolytic effects of cannabinoids. Prog Neuro-Psychopharmacol Biol Psychiatry 2009;33(8):1517–21.  Blanchard DC, Blanchard RJ. Innate and conditioned reactions to threat in rats with amygdaloid lesions. J Comp Physiol Psychol 1972;81:281–90.  Smith Y, Paré D. Intra-amygdaloid projections of the lateral nucleus in the cat: PHA-L anterograde labeling combined with postembedding GABA and glutamate immunocytochemistry. J Comp Neurol 1994;342:232–48.  Paré D, Smith Y, Paré JF. Intra-amygdaloid projections of the basolateral and basomedial nuclei in the cat: Phaseolus vulgaris-leucoagglutinin anterograde tracing at the light and electron microscopic level. Neuroscience 1995;69:567–83.  Pitkanen A, Stefanacci L, Farb CR, Go GG, LeDoux JE, Amaral DG. Intrinsic connections of the rat amygdaloid complex: projections originating in the lateral nucleus. J Comp Neurol 1995;356:288–310.
 Holstege G, Bandler R, Saper CB. The emotional motor system. Prog Brain Res 1996;107:3–6.  LeDoux JE, Iwata J, Cicchetti P, Reis DJ. Different projections of the central amygdaloid nucleus mediate autonomic and behavioral correlates of conditioned fear. J Neurosci 1998;8:2517–29.  De Oca BM, DeCola JP, Maren S, Fanselow MS. Distinct regions of the periaqueductal gray are involved in the acquisition and expression of defensive responses. J Neurosci 1998;18(9):3426–32.  Davis M, Whalen PJ. The amygdala: vigilance and emotion. Mol Psychiatry 2001;6:13–34.  Corcoran KA, Quirk GJ. Activity in prelimbic cortex is necessary for the expression of learned, but not innate, fears. J Neurosci 2007;24:840–4.  Quirk GJ, Likhtik E, Pelletier JG, Pare D. Stimulation of medial prefrontal cortex decreases the responsiveness of central amygdala output neurons. J Neurosci 2003;23:8800–7.  Milad MR, Vidal-Gonzalez I, Quirk G. Electrical stimulation of medial prefrontal cortex reduces conditioned fear in a temporally speciﬁc manner. Behav Neurosci 2004;118:389–94.  Barners NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacology 1999;38:1083–152.  Ciocchi S, Herry C, Grenier F, Wolff SB, Letzkus JJ, Vlachos I, Ehrlich I, Sprengel R, Deisseroth K, Stadler MB, Müller C, Lüthi A. Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 2010;468:277–82.  Ji G, Neugebauer V. Modulation of medial prefrontal cortical activity using in vivo recordings and optogenetics. Mol Brain 2012;5:2–10.  Blum S, Hebert AE, Dash PK. A role for the prefrontal cortex in recall of recent and remote memories. Neuroreport 2006;17:341–4.  Laurent V, Westrook RF. Inactivation of the infralimbic but not the prelimbic cortex impairs consolidation and retrieval of fear extinction. Learn Mem 2009;16:520–9.  Pratt JA. The neuroanatomical basis of anxiety. Pharmacol Ther 1992;55:149–81.  Ishida Y, Hashiguchi H, Takeda R, Ishizuka Y, Mitsuyama Y, Kannan H, Nishimori T, Nakahara D. Conditioned-fear stress increases Fos expression in monoaminergic and GABAergic neurons of the locus coeruleus and dorsal raphe nuclei. Synapse 2002;45:46–51.  Sesack SR, Deutc AY, Roth RH, Bunney BS. Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract-tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 1989;290:213–42.  Hurley KM, Herbert H, Moga MM, Saper CB. Efferent projections of the infralimbic cortex of the rat. J Comp Neurol 1991;308:249–76.  Takagishi M, Chiba T. Efferent projections of the infralimbic (area 25) region of the medial prefrontal cortex in the rat: an anterograde tracer PHA-L study. Brain Res 1991;566:26–39.  Groenewegen HJ, Uylings HBM. The prefrontal cortex and the integration of sensory, limbic and autonomic information. Prog Brain Res 2000;126:3–28.  Hajós M, Richards CD, Székely AD, Sharp T. An electrophysiological and neuroanatomical study of the medial prefrontal cortical projection to the midbrain raphe nuclei in the rat. Neuroscience 1998;87:95–108.  Cheng LL, Wang SJ, Gean PW. Serotonin depresses excitatory synaptic transmission and depolarization-evoked Ca2+ inﬂux in rat basolateral amygdala via 5-HT1A receptors. Eur J Neurosci 1998;10:2163–72.  Lin CH, Huang YC, Tsai JJ, Gean PW. Modulation of voltage-dependent calcium currents by serotonin in acutely isolated rat amygdala neurons. Synapse 2001;41:351–9.  Wang SJ, Coutinho V, Sivra TS. Presynaptic cross-talk of adrenoceptor and 5-hydroxytryptamine receptor signalling in the modulation of glutamate release from cerebrocortical nerve terminals. Br J Pharmacol 2002;137: 1371–9.  Heisler LK, Chu HM, Brennan TJ, Danao JA, Bajwa P, Parsons LH, Tecott LH. Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Aca Sci U S A 1998;95:15049–54.  Sibille E, Hen R. Serotonin1A receptors in mood disorders: a combined genetic and genomic approach. Behav Pharmacol 2001;12:429–38.  Pattij T, Groenink L, Hijzen TH, Oosting RS, Maes RAA, Van Der Gugten J, Olivier B. Autonomic changes associated with enhanced anxiety in 5-HT1A receptor knock-out mice. Neuropsychopharmacology 2002;27:380–90.  Groenink L, Pattij TD, Jongh R, Van Der Gugten J, Oosting RS, Dirks A, Olivier B. 5-HT1A receptor knock-out mice and mice overexpressing corticotrophinreleasing hormone in models of anxiety. Eur J Pharmacol 2003;463:185–97.  Toth M. 5-HT1A receptor knock-out mouse as a genetic model of anxiety. Eur J Pharmacol 2003;463:177–84.  Cai X, Gu Z, Zhong P, Ren Y, Yan Z. Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+ /calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons. J Biol Chem 2002;277:36553–62.  Bi LL, Wang J, Luo ZY, Chen SP, Geng F, Chen YH, Li SJ, Yuan CH, Lin S, Gao TM. Enhanced excitability in the infralimbic cortex produces anxiety-like behaviors. Neuropharmacology 2013;72:148–56.  Krieger JE, Graeff FG. Defensive behavior and hypertension induced by glutamate in the midbrain central gray of the rat. Braz J Med Biol Res 1985;18:61–7.  Bandler R, Carrive P. Integrated defence reaction elicited by excitatory amino acid microinjection in the midbrain periaqueductal gray region of the unrestrained cat. Brain Res 1998;439:95–106.  Gerhardt CC, Heerikhuizen H. Functional characteristics of heterologously expressed 5-HT receptors. Eur J Pharmacol 1997;334:1–23.
A.L.Z. Marinho et al. / Behavioural Brain Research 286 (2015) 49–56
 Guimarães FS, Carobrez AP, De Aguiar JC, Graeff FG. Anxiolytic effect in the elevated plus-maze of the NMDA receptor antagonist AP7 microinjected into the dorsal periaqueductal gray. Psychopharmacology (Berlin) 1991;103:91–4.  Molchanov ML, Guimarães FS. Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats. Braz J Med Biol Res 1999;32:1533–7.  Matheus MG, Guimarães FS. Antagonism of non-NMDA receptors in the dorsal periaqueductal gray induces anxiolytic effect in the elevated plus maze. Psychopharmacology (Berlin) 1997;132:14–8.  Russo AS, Guimarães FS, De Aguiar JC, Graeff FG. Role of benzodiazepine receptors located in the dorsal periaqueductal grey of rats in anxiety. Psychopharmacology (Berlin) 1993;110:198–202.  Russo AS, Guimarães FS, De Aguiar JC, Graeff FG. Anxiolytic effect of midazolam microinjected into the dorsal periaqueductal grey area of rats. Braz J Med Biol Res 1991;24:607–9.  Campos AC, Fogac¸a MV, Aguiar DC, Guimaraes FS. Animal models of anxiety disorders and stress. Rev Bras Psiquiatr 2013;35:101–11.  Raymond JR, Mukhin YV, Gettys TW, Garnovsakaya MN. The recombinant 5HT1A receptor: G-protein coupling and signaling pathways. Brit J Pharmacol 1999;127:1751–64.  Rodgers RJ, Cole JC, Aboualfa K, Stephenson LH. Ethopharmacological analysis of the effects of putative ‘anxiogenic’ agents in the mouse elevated plus-maze. Pharmacol Biochem Behav 1995;52:805–13.  Reis FM, Albrechet-Souza L, Franci CR, Brandão ML. Risk assessment behaviors associated with corticosterone trigger the defense reaction to social isolation in rats: role of the anterior cingulate cortex. Stress 2012;15:318–28.
 Maier SF, Watkins LR. Role of the medial prefrontal cortex in coping and resilience. Brain Res 2010;1355:52–60.  Maier SF, Watkins LR. Stressor controllability, anxiety, and serotonin. Cogn Therapy Res 1998;6:595–613.  Resstel LB, Souza RF, Guimarães FS. Anxiolytic-like effects induced by medial prefrontal cortex inhibition in rats submitted to the Vogel conﬂict test. Physiol Behav 2008;93:200–5.  Padovan CM, Guimarães FS. Restraint-induced hypoactivity in an elevated plusmaze. Braz J Med Res 2000;33:79–83.  Campos AC, Ferreira FR, Guimarães FS, Lemos JI. Facilitation of endocannabinoid effects in the ventral hippocampus modulates anxiety-like behaviors depending on previous stress experience. Neuroscience 2010;167:238–46.  Albonetti ME, Farabollini F. Behavioural responses to single and repeated restraint in male and female rats. Behav Process 1992;28:97–110.  Mendonc¸a FH, Félix FH, Del-Bel EA, Guimarães FS. Intraventricular cycloheximide attenuates the restraint-induced long-lasting effect on plus maze exploration. Braz J Med Biol Res 1996;29:501–5.  De Paula Soares V, Vicente MA, Biojone C, Zangrossi HJR, Guimarães FS, Joca SR. Distinct behavioral consequences of stress models of depression in the elevated T-maze. Behav Brain Res 2011;225:590–5.  Radley JJ, Arias CM, Sawchenko PE. Regional differentiation of the medial prefrontal cortex in regulating adaptive responses to acute emotional stress. J Neurosci 2006;26:12967–76.  Izquierdo A, Wellman CL, Holmes A. Brief uncontrollable stress causes dendritic retraction in infralimbic cortex and resistance to fear extinction in mice. J Neurosci 2006;23:4406–9.