Behavioural Brain Research 286 (2015) 49–56

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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 [1]. 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 [11]. For example, whereas CoCl2

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(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 [8]. Also in agreement with this proposal, Lemos et al. [9] 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 [21] and VCT [20]. 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) [22]. 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. [9]); 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 [25]). 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 [9]. 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

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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 [25]. Scale bar: 600 ␮m.

(except respiration) while the animal assumed a characteristic tense posture [23], 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 [24]. 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 [25]. 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

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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,

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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 [26], 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 [26] 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

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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. [11] 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 [27]. 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 [9]. 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 [10]. The involvement of this latter structure with conditioned responses was first shown by Blanchard and Blanchard [35]. 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 [39] 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 [43]. 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 [47]. Moreover, Ji and Neugebauer [48] 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

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fear expression [43,49,50] and microstimulation of this structure increases CER [10]. 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. [22] 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 [23] 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 [66]. In line with the present findings, Lil-Lin Bi et al. [67] 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 [80]. Behavior changes following presentation of an incontrollable acute stressor persist for days, indicating the participation of plastic alterations in the central nervous system [81]. 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 [82]. 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 [22]. 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 [89]. 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. [22] 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 [1] Dalley JW, Cardinal RN, Robbins TW. Prefrontal executive and cognitive functions in rodents: neural and neurochemical substrates. Neurosci Biobehav Rev 2004;28:771–84. [2] Dias R, Robbins TW, Roberts AC. Dissociation in prefrontal cortex of affective and attentional shifts. Nature 2006;380:69–72. [3] 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. [4] 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. [5] Berendse HW, Groenewegen HJ. Restricted cortical termination fields of the midline and intralaminar thalamic nuclei in the rat. Neuroscience 1991;42:73–102. [6] 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. [7] 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. [8] 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. [9] 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.

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