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Psychopharmacology ISSN 0033-3158 PsychopharmacologyDOI 10.1007/s00213-012-2927-2

Effects of glutamate NMDA and TRPV1receptor antagonists on the biphasicresponses to anandamide injected into thedorsolateral periaqueductal grey of WistarratsManoela V. Fogaça, Felipe V. Gomes,Fabrício A. Moreira, FranciscoS. Guimarães & Daniele C. Aguiar

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ORIGINAL INVESTIGATION

Effects of glutamate NMDA and TRPV1 receptor antagonistson the biphasic responses to anandamide injectedinto the dorsolateral periaqueductal grey of Wistar rats

Manoela V. Fogaça & Felipe V. Gomes &

Fabrício A. Moreira & Francisco S. Guimarães &

Daniele C. Aguiar

Received: 13 March 2012 /Accepted: 13 November 2012# Springer-Verlag Berlin Heidelberg 2012

AbstractRationale The endocannabinoid and endovanniloid ananda-mide (AEA) exerts biphasic effects when injected into thedorsolateral periaqueductal grey (dlPAG) in rats submittedto threatening situations. Whereas lower doses of AEAinduce anxiolytic-like effects by activating cannabinoidCB1 receptors, no effects are observed with higher doses,possibly due to the simultaneous activation of transientreceptor potential vanilloid type 1 (TRPV1) receptors. Thisactivation would facilitate glutamatergic neurotransmission.Objective Considering that the blockade of TRPV1 orNMDA receptors in the dlPAG induces anxiolytic-like effects,we tested the hypothesis that facilitation of glutamate trans-mission through TRPV1 is responsible for the lack ofanxiolytic-like effect observed with high AEA doses.Methods Male Wistar rats with a unilateral cannula aimed atthe dlPAG received injections of an ineffective dose of AP7(an NMDA antagonist, 1 nmol) or capsazepine (CPZ, aTRPV1 antagonist, 10 nmol), followed by a high dose ofAEA (50 and 200 pmol) and were exposed to the elevatedplus maze (EPM) or the Vogel conflict test (VCT).

Results AP7, CPZ, or AEA did not induce any significanteffects when administered alone. However, AP7 or CPZ priorto AEA significantly increased the percentage of entries andtime spent in the open arms of EPM and the number ofpunished licks in the VCT suggesting an anxiolytic-like effect.Conclusions These results suggest that the lack of anxiolytic-like effect of higher AEA doses is due to facilitation ofglutamate release in the dlPAG, probably via activation ofTRPV1 receptors in this structure.

Keywords Glutamate . Endovanilloids . Anxiety .

Defensive behavior

Introduction

The dorsolateral periaqueductal grey (dlPAG) is responsiblefor elaborating active defensive responses displayed by ani-mals in dangerous situations (Bandler et al. 2000; Carrive1993). Under natural conditions, these responses are proba-bly mediated by local glutamate release (Guimaraes et al.2005). Accordingly, intra-dlPAG administration of gluta-mate ionotropic receptor (NMDA or non-NMDA) agonistsinduces escape reactions (Aguiar et al. 2006; Bittencourt etal. 2004), whereas the blockade of these receptors causesanxiolytic-like and anti-aversive effects in rodents (Aguiarand Guimaraes 2009; Guimaraes et al. 1991; Matheus andGuimaraes 1997; Molchanov and Guimaraes 2002).

In addition to glutamate, other neurotransmitters havealso been shown to modulate anxiety-like behaviors in thedlPAG, including GABA, nitric oxide (NO), endocannabi-noids and, more recently, endovanilloids (Casarotto et al.2012; Finn et al. 2003; McGregor et al. 2004; Moreira et al.2007; Terzian et al. 2009). Endovanilloids are putative en-dogenous agonists of the transient receptor potential

Fogaça M.V. and Gomes F.V. contributed equally to this work.

M. V. Fogaça : F. V. Gomes : F. S. GuimarãesDepartment of Pharmacology, School of Medicine of Ribeirão Preto,University of São Paulo, Av. Bandeirantes 3900,Ribeirão Preto, São Paulo 14049900, Brazil

F. A. Moreira :D. C. Aguiar (*)Department of Pharmacology, Institute of Biological Sciences,Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627,Belo Horizonte, Minas Gerais 31270-901, Brazile-mail: danieleaguiar@ufmg.br

M. V. Fogaça : F. V. Gomes : F. S. GuimarãesCenter for Interdisciplinary Research on Applied Neurosciences(NAPNA), University of São Paulo, São Paulo, Brazil

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vanilloid type 1 (TRPV1), a nonselective cation channel(Caterina et al. 1997; Clapham et al. 2005). The first endo-vanilloid described was anandamide (N-arachidonoyletha-nolamine, AEA), an arachidonic acid-derived compound,which is also an endocannabinoid and an agonist of canna-binoid receptor type 1 (CB1) (Di Marzo et al. 2001; Smartand Jerman 2000; Zygmunt et al. 2000). Although AEA isthe endogenous ligand of both cannabinoid and vanilloidsystem, its affinity for TRPV1 is lower than that needed toactivate CB1 receptors (Ross 2003).

Several results have suggested that endocannabinoids andendovanilloids modulate emotional states in opposite ways(for review see Moreira et al. 2012; Viveros et al. 2007). Inline with these pieces of evidence, previous studies from ourgroup showed that lower doses of AEA injected into thedlPAG caused anxiolytic-like effects in distinct animal modelsof anxiety. These effects were blocked by previous treatmentwith AM251, a CB1 receptor antagonist (Moreira et al. 2007).AEA, however, induced a bell-shaped dose–response curve,with higher doses being ineffective (Moreira et al. 2007).

Studies employing PAG slices have shown that activationof CB1 receptors inhibits GABAergic and glutamatergicsynaptic transmission (Vaughan et al. 2000), which may berelated to the biphasic effects on anxiety-like responsesobserved with CB1 receptor agonists in the PAG (Moreiraet al. 2007). However, PAG neurons also express TRPV1receptors (Cavanaugh et al. 2011; Cristino et al. 2006; Maioneet al. 2006; Toth et al. 2005) and, similar to glutamate, TRPV1activation has been suggested to facilitate anxiety-likeresponses (Marsch et al. 2007; Rubino et al. 2008; Terzian etal. 2009; Toth et al. 2005). In the central nervous system(CNS), TRPV1 receptors are proposed to modulate neuronalactivity mainly through potentiation of glutamatergic signal-ing (Kawahara et al. 2011; McGaraughty et al. 2003;Starowicz et al. 2007; Xing and Li 2007), an effect that hasalso been shown in PAG (Kawahara et al. 2011; Palazzo et al.2002; Starowicz et al. 2007).

Since high doses of cannabinoids such as AEA, ACEA,and WIN 55,212-2 can also activate TRPV1 receptors(Casarotto et al. 2012; Di Marzo et al. 2001; Fogaca et al.2012; Pertwee 2006), we hypothesized that the bell-shapeddose–response curves observed in anxiety models after ad-ministration of AEA in the dlPAG, with higher doses beingineffective, depend on increased glutamate release via acti-vation of TRPV1 receptors.

Materials and methods

Subjects

Male Wistar rats were obtained from the colony of ratsmaintained by the Campus of the University of São Paulo-

Ribeirão Preto. The rats weighing 220–240 g at the begin-ning of each experiment were housed in groups of four in atemperature-controlled room (24±1 °C) under standard lab-oratory conditions with free access to food and water and a12-h light/12-h dark cycle (lights on at 6:30 a.m.). All theexperiments were conducted between 8 a.m. and 12 a.m. Toavoid interference from different batches of animals, eachexperiment always included control and treated rats.Procedures were conformed within the Brazilian Society ofNeuroscience and Behavior guidelines for the care and use oflaboratory animals. The experimental protocols were ap-proved by the Brazilian College of Animal Experimentationand by the School of Medicine of Ribeirao Preto of theUniversity of Sao Paulo Ethical Commission of Ethics inAnimal Research (CETEA; no. 083/2008).

Drugs

The CB1/TRPV1 agonist anandamide (AEA) (50 and200 pmol/0.2 μL; Tocris, USA) was dissolved inTocrisolveTM® 100 (a solvent that contains a 1:4 ratio ofsoya oil/water, emulsified with the block co-polymerPluronic F68). The TRPV1 antagonist capsazepine (CPZ;10 nmol/0.2 μL; Tocris, USA) was dissolved in dimethylsulfoxide (DMSO) 100 %. The glutamate NMDA receptorantagonist, 2-amino-7-phosphonoheptanoic acid (AP7;1 nmol/0.2 μL; Tocris, USA) was dissolved in sterile saline.The solutions were prepared immediately before use. Thechosen doses of AEA were those that did not induceanxiolytic-like effects in a previous work from our group(Moreira et al. 2007). The doses of the antagonists used inthis study were chosen based on the dose–responsecurves from previous studies. Since the blockade ofTRPV1 and NMDA receptors in the dlPAG inducesanxiolytic-like effects per se, doses that have been prov-en ineffective in these studies were chosen (Guimaraeset al. 1991; Molchanov and Guimaraes 2002; Terzian etal. 2009).

Apparatus

Elevated plus maze

The elevated plus maze (EPM) consisted of two oppositewooden open arms (50×10 cm) crossed at a right angle bytwo arms of the same dimensions enclosed by 40-cm-highwalls (Pellow and File 1986). The maze was located 50 cmabove the floor and the ANY-maze Software (version 4.5,Stoelting, USA) was employed for behavioral analysis in theapparatus. The EPM was located in a sound attenuated,temperature controlled (23 °C) room, and the environmentwas illuminated by a 40-W fluorescent light placed 1.3 maway from the maze. The sessions lasted for 5 min, and after

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each trial, the maze was cleaned with an alcohol solution(Aguiar et al. 2009).

Vogel conflict test

The Vogel conflict test (VCT) was performed in a Plexiglasbox (42×25×20 cm) with a stainless grid floor. The metallicspout of a drinking bottle containing water projected into thebox. The contact of the animal with the spout and the gridfloor closed an electrical circuit controlled by a sensor(Anxiometer model 102, Columbus, USA), which recordedthe total number of licks and shocks delivered during the testperiod (3 min). The animals received a 0.5-mA shock for 2 s,after 20 licks (Geller and Seifter 1960; Moreira et al. 2006).

Water consummatory evaluation

To assess whether the treatments could have affected theVCT by altering water consumption, independent groups ofanimals were exposed to the same experimental conditions(3 min each/section) described above except that the electricshock delivering system was rendered inoperative.

Tail flick test

To verify if the drugs interfered with nociceptive threshold,rats were submitted to the tail flick test. The apparatusconsisted of an acrylic platform with a nichrome wire coilmaintained at room temperature (24–26 °C). The rats weregently handled, and their tails were laid across the coil wherethe temperature was raised at 9 °C/s by the passage of electriccurrent. The system had a cutoff time of 6 s to prevent tissuedamage when the coil temperature approached 80 °C(Moreira et al. 2006).

Surgery

The rats were anesthetized with tribromoethanol (250 mg/kgi.p., Sigma-Aldrich, USA) and were fixed in a stereotaxicframe. A stainless steel guide cannula (11 mm; 0.6 mm,outer diameter (OD)) was implanted unilaterally on the rightside aimed at the dlPAG (coordinates: AP00 from lambda,L0−1.9 mm at an angle of 16°, D0−4.0 mm) according tothe atlas published by Paxinos and Watson (Paxinos andWatson 1997). The cannula was attached to the boneswith stainless steel screws and acrylic cement. An ob-turator inside the guide cannulae prevented obstruction.Immediately after the surgery, the animals received ben-zylpenicillin (0.27 g/kg i.m.; Pentabiotico®, Fort Dodge,Brazil) to prevent infection and a nonsteroidal anti-inflammatory drug, flunixin meglumine (0.025 g/kgs.c.;Banamine®, Schering Plough, Brazil) for postoperativeanalgesia.

Procedure

Intra-dlPAG injection

Seven days after surgery, the animals were randomlyassigned to one of the treatment groups. Intracerebral injec-tions were performed with a thin dental needle (0.3 mm,OD) introduced through the guide cannula until its tip was1.0 mm below the cannula end. A volume of 0.2 μL wasinjected in 30 s using a microsyringe (Hamilton, USA)connected to an infusion pump (KD Scientific, USA). Inorder to prevent reflux, the guide cannula was left in placefor 30 s after the end of each injection. A polyethylenecatheter (PE 10) was interposed between the upper end ofthe dental needle and the syringe.

Experiment 1

The rats received intra-dlPAG injections of vehicle (saline)or AP7 (1 nmol) followed, 5 min later, by vehicle(Tocrisolve® in saline) or AEA (50 or 200 pmol). Tenminutes later, theywere placed in the center of the EPM facingan enclosed arm. The percentage of entries and time spent inthe open arms and the number of entries into the enclosedarms of the maze was recorded for 5 min. The experimentalgroups were vehicle (veh) + veh, veh + AEA 50, veh + AEA200, AP7 + veh, AP7 + AEA 50, AP7 + AEA 200.

Experiment 2

The procedure was similar to experiment 1 except that theanimals received vehicle (DMSO 100 %) or capsazepine(CPZ, 10 nmol) before vehicle (Tocrisolve® in saline) orAEA (50 or 200 pmol) injection. No additional tissue dam-age was observed in DMSO-treated animals compared tothe other vehicles. As previously described, 10 min after thesecond injection the animals were submitted to the EPM testfor 5 min. The experimental groups were veh + veh, veh +AEA50, veh + AEA 200, CPZ + veh, CPZ + AEA 50, CPZ +AEA 200.

Experiment 3

The animals were water-deprived for 48 h before the test.After the first 24 h of deprivation, they were allowed todrink freely for 3 min in the test cage in order to find thedrinking bottle spout. Twenty-four hours later, they receivedintra-dlPAG injections of vehicle, capsazepine (10 nmol) orAP7 (1 nmol) followed, 5 min later, by vehicle or AEAinjection (200 pmol). Ten minutes after the second injection,they were placed in the apparatus. The test period lasted for3 min, and the animals received a 0.5-mA shock through thebottle spout every 20 licks. The number of punished licks was

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registered. The procedure was similar to the one already usedand validated in the laboratory (Aguiar et al. 2009; Lisboa etal. 2008). The experimental groups were veh + veh, CPZ +veh, AP7 + veh, veh + AEA, CPZ + AEA, AP7 + AEA.Results from the vehicles used for CPZ and AP7 were notstatistically different. Therefore, they were grouped togetherin the vehicle + vehicle group.

Experiment 4

The tail flick test was conducted in independent groups ofanimals receiving vehicle + vehicle, capsazepine + AEA, orAP7 + AEA into the dlPAG. Morphine was administeredsystemically (5 mg/kgi.p.) as a positive control. The animalswere gently handled, and their tails were laid across the coil.The heating was applied to a portion of the ventral surface ofthe tail between 4 and 6 cm from its end. The time towithdraw the tail was recorded as tail flick latency. Theelectric current was calibrated to provoke this reflex within2.5–3.5 s in nontreated animals. The tail flick latency wasmeasured at 5-min intervals until a stable baseline wasobtained over three consecutive trials. After drug adminis-tration, the tail withdrawal latencies were measured again at10-min intervals for up to 30 min.

Experiment 5

The procedure was the same used in the punished lickingtest; however, the electric shock delivering system wasrender inoperative. Ten minutes after vehicle + vehicle,CPZ + AEA, or AP7 + AEA injections, rats were allowedto drink freely for 3 min. The number of licks during thisperiod was registered.

Histology

After the behavioral tests, the rats were sacrificed under deepchloral hydrate 5 % (Sigma-Aldrich, USA) anesthesia andperfused through the left ventricle of the heart with isotonicsaline followed by 10 % formalin solution. After that, thebrains were removed and, after a minimum period of 5 days,immersed in a 10 % formalin solution, 50 μm sections wereobtained in a Cryostat (Cryocut 1800, Leica, Germany). Theinjection sites were identified in diagrams from the PaxinosandWatson atlas (Paxinos and Watson 2007) and are illustrat-ed in Fig. 1. Rats receiving drug injections outside the aimedarea were included in an additional (OUT) group.

Statistical analysis

The percentages of entries and time spent in the open arms[(open/open + enclosed)×100] during the 5-min sessions inthe EPM were calculated for each animal. Since the data

from animals submitted to experiment 1 did not follow anormal distribution, the EPM data from experiment 1 and 2,percentage of entries and time spent in the open arms andnumber of enclosed arms, were analyzed by Kruskal–Wallisfollowed by Mann–Whitney test. The number of punishedlicks (experiment 3) was analyzed by two-way ANOVAusing the first and the second injections as main factors.Data from the tail flick (experiment 4) were analyzed byrepeated measures ANOVAwith treatment as the independentfactor and time as the repeated measure. In the case of signif-icant interactions, a one-way ANOVA was performed fol-lowed by the Duncan test for multiple comparisons. Resultsfrom the water consumption test (experiment 5) were analyzedby one-way ANOVA followed by Duncan test. Differenceswere considered significant at p<0.05 level.

Fig. 1 a Photomicrography of a representative site injection in thedlPAG. b Histological localization of injection sites in diagrams basedon the atlas of Paxinos and Watson (2007). The black and the greycircles represent the injection sites inside and outside of the dlPAG,respectively

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Results

Experiment 1 There were significant effects of treatment onthe percentage of entries (X2

(5)016.96, p<0.05) and timespent in the open arms of EPM (X2

(5)012.33, p<0.05). AP7(1 nmol) or AEA (50 and 200 pmol) did not induce anysignificant effects when administered alone. However, AP7prior to AEA significantly increased the percentage ofentries (50 and 200 nmol) and time spent (only the 200 nmoldose) in the open arms of the EPM in comparison to controlgroup (Mann–Whitney test, p<0.05; Fig. 2), suggesting ananxiolytic-like effect. No effect was found in the number ofenclosed arms entries (X2(5)08.615, p00.125), indicating thatthis effect is not affecting basal motor activity. Additionally,animals that received AP7 + AEA (200 pmol) outside thedlPAG (percentage of open arm entries026.2±1.5, percentageof time spent in open arms08.8±2.2, n06) were not differentfrom the vehicle-treated group.

Experiment 2 There were significant effects of the percent-age of time (X2

(5)011.74, p00.038) and in the percentage ofentries (X2

(5)016.26, p00.006) in the open arms of EPM. Aspreviously observed, AEA (50 and 200 pmol) did not induceany behavioral change (Mann–Whitney test, p>0.05).Moreover, no effect was detected with capsazepine(10 nmol) alone (Mann–Whitney test, p>0.05). However,capsazepine prior to AEA (50 and 200 pmol) significantlyincreased the percentage of entries in the open arms of EPMcompared to the control group (Mann–Whitney test, p<0.05; Fig. 3). No effect was observed in the number ofenclosed arms entries (X2

(5)06.09, p00.296). Animals thatreceived CPZ+AEA (200 pmol) outside the dlPAG ((per-centage of open arm entries022.3±1.5, percentage of timespent in open arms06.3±1.8, n06) were not different fromvehicle-treated rats.

Experiment 3 Since no difference was found between ani-mals that received saline (n04) or DMSO (n03) followedby Tocrisolve as vehicles, they were joined together in acontrol group. Two-way ANOVA showed significant effectsof the first (F2,3804,36; p00.019) and second (F1,38012.38;p00.001) microinjections and a significant interaction(F2,3803.28; p00.048). Post hoc analysis indicated thatAP7 and capsazepine prior to AEA injection increased thenumber of punished licks in the VCT (Duncan test, p<0.05;Fig. 4). These drugs did not induce any effect when admin-istered alone. Moreover, animals that received AP7 + AEAor CPZ + AEA (punished licks092±10.6, n08) outside thedlPAG were not different from controls.

Experiment 4 A repeated measure ANOVA showed sig-nificant effects of time (F5,84032.0, p<0.05), treatment(F3,84074.8, p<0.05) and interaction (F15,84015.1, p<

0.05) on the withdrawal latency in the tail flick test.Morphine, the positive control, increased the latency of tailwithdrawal 10, 20, and 30 min after drug administration(Duncan test, p<0.05; Fig. 5). Moreover, there was an

Fig. 2 Effects of vehicle or AP7 (1 nmol) followed by vehicle oranandamide (AEA; 50 or 200 pmol) administered into the dlPAG of ratstested in the EPM. The bars represent the mean ± S.E.M. of the numberof entries into the enclosed arms (a), the percentage of entries in theopen arms (b), and percentage of time in the open arms (c). The datawere analyzed by Kruskal–Wallis followed by Mann–Whitney test(asterisks indicate the significant difference from vehicle–vehiclegroup, p<0.05, n05–7)

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increase in the latency of tail withdrawal 20 min after druginjection in animals that received CPZ or AP7 prior to AEA(Duncan test, p<0.05; Fig. 5). This latency, however, was notdifferent from baseline values (p>0.05).

Experiment 5 The effects in VCT were not due an in-crease of water consumption since there was no differ-ence in the number of unpunished licks among thegroups (veh + veh n04; mean number of licks, 964.3±53.1; CPZ + AEA n05; mean number of licks, 991.6±52.4; AP7 + AEA n05; mean number of licks, 876±29.1; F2,1101.9, Duncan test, p00.19).

Discussion

The present results showed that high, ineffective doses ofAEA in the dlPAG can become anxiolytic when animalswere pretreated with TRPV1 or NMDA antagonists. Thisanxiolytic-like effect was observed in two distinct animalmodels of anxiety, the EPM and VCT, and depended ondrug effects in the dlPAG, since no difference was observedwhen the injections were performed near, but not into, this

Fig. 3 Effects of vehicle or capsazepine (CPZ, 10 nmol) followed byvehicle or AEA (50 or 200 pmol) administered into the dlPAG of ratstested in the EPM. Bars represent the mean ± S.E.M. of the number ofentries into the enclosed arms (a), the percentage of entries in the openarms (b), and percentage of time in the open arms (c). The data wereanalyzed by Kruskal–Wallis followed by Mann–Whitney test (asterisksindicate the significant difference from vehicle–vehicle group, p<0.05,n07–9)

Fig. 4 Effects of CPZ (10 nmol) and AP7 (1 nmol) followed by AEA(200 pmol) administered into the dlPAG of rats submitted to the Vogelconflict test. Bars represent the mean ± S.E.M. total number of pun-ished licks in the 3-min session. Asterisks indicate the significantdifference from vehicle–vehicle group. The data were analyzed bytwo-way ANOVA followed by the Duncan test (*p<0.05 from vehi-cle–vehicle group; n07π11)

Fig. 5 Time course of the effects of intra-dlPAG administration ofvehicle + vehicle (Veh + Veh), capsazepine (CPZ, 10 nmol) + AEA 200pmol, AP7 (1 nmol) + AEA (200 pmol), or systemically injectedmorphine (5 mg/kg, i.p.) in the tail flick test. Each point representsthe mean ± S.E.M. for the latency of tail withdrawal. The data wereanalyzed by two-way ANOVA followed by the Duncan test (asterisksindicate the significant difference p<0.05, from vehicle group, n04–5)

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region. The drug-induced decrease in anxiety was notaccompanied by changes in the number of enclosedarms entries in the EPM, discarding motor effect as apossible confounding factor (File 1992). Moreover, con-trol experiments for the VCT showed that neither AEAplus AP7 nor AEA plus CPZ altered water consump-tion. Both treatments, however, did increase the latencyof tail withdrawal 20 min after injection, suggesting anantinociceptive effect at this time point. Nevertheless, inthe present study the VCT was performed 10 min afterinjections, a time point where no differences were foundin the tail flick test. In addition, the antinociceptiveeffect was small and did not differ from the baselinemeasures. Therefore, it seems unlikely that the anticon-flict results obtained in the VCT were due to antinoci-ceptive effects. In previous studies of our group, similarresults (anxiolytic with a small antinociceptive effect) wereobtained after intra-dlPAG injection of the inhibitor of anan-damide uptake AM404 or the phytocannabinoid cannabidiol(Campos and Guimaraes 2008; Lisboa et al. 2008). This ishardly surprising, since cannabinoid-, vanilloid-, andglutamatergic-mediated neurotransmissions are involved innociceptive control in this brain area (Finn et al. 2003;Maione et al. 2006, 2009; Palazzo et al. 2001, 2002; Walkeret al. 1999).

Together, the present results indicate that at highdoses, such as those used in the present study (Moreiraet al. 2007), the anxiolytic-like effects of AEA in thedlPAG disappear due to its ability to activate TRPV1receptors. In agreement with this possibility, the affinityof AEA at TRPV1 is relatively low compared to CB1receptors (Ross 2003).

Different from CB1 receptors, TRPV1 seems to mediatepro-aversive effects. TRPV1 knockout mice showed reducedanxiety-like behavior in the EPM and light–dark box, as wellas reduced fear conditioning (Marsch et al. 2007). Moreover,either systemic or local injection of TRPV1 antagonists intospecific brain structures, such as the dlPAG (Terzian et al.2009), prefrontal cortex (Aguiar et al. 2009; Fogaca et al.2012; Rubino et al. 2008), and ventral hippocampus (Santoset al. 2008), induces anxiolytic-like effects in rats.

In the CNS, the main effect resulting from activation ofTRPV1 receptors is the facilitation of glutamatergic neuro-transmission (Starowicz et al. 2007; Urban and Dray 1992;Urban et al. 1985; Xing and Li 2007). Neurochemical andelectrophysiological data showed that activation of TRPV1induced glutamate release through a mechanism dependenton Ca+2, which was inhibited by TRPV1 antagonism(Palazzo et al. 2002).

Increased glutamate release in the dlPAG induced byactivation of TRPV1 receptors after high doses of somecannabinoids such as AEA (Palazzos et al. 2006) couldfacilitate defensive responses (Guimaraes et al. 2005).

Accordingly, in the presence of an NMDA antagonist, inef-fective high doses of AEA became anxiolytic in animalsexposed to both EPM and VCT. Even if we have notmeasured local glutamate release, several studies corrobo-rate this possibility. For example, injection of capsaicin intothe dlPAG induced nociception and local release of gluta-mate, an effect reversed by pretreatment with the TRPV1antagonist capsazepine (Palazzo et al. 2002). Similar effectswere described in vitro for other regions such as the hypo-thalamus, substantia nigra, and locus coeruleus (Marinelli etal. 2002, 2003; Palazzo et al. 2002). Furthermore, support-ing the hypothesis of interaction between glutamatergic andvanilloid systems, Starowicz et al. (2007) showed that neu-rons in the ventrolateral portion of the PAG, which alsoexpress TRPV1 receptors, are surrounded by glutamatergicneurons. Additionally, these glutamatergic neurons respondto TRPV1 stimulation by releasing glutamate, an effectblocked by pretreatment with a vanilloid antagonist(Starowicz et al. 2007). The expression of TRPV1 in gluta-matergic neurons was also detected in the dorsal horn of thespinal cord (Zhou et al. 2009).

In order to verify which glutamate receptors are related tothe nociceptive responses induced by capsaicin, Jin et al.(2012) observed that capsaicin-evoked c-Fos expression inthe spinal cord dorsal horn was prevented by NMDA recep-tor antagonists, suggesting that activation of ionotropic glu-tamate receptors are involved in nociceptive responsesinduced by capsaicin (Jin et al. 2012). Moreover, theantidepressant and anxiolytic-like behavioral profiles ob-served in TRPV1 knockout mice were associated withchanges in the expression of NMDA receptors in thestriatum and CA1 region of the hippocampus (You etal. 2012). Altogether, these results suggest that NMDAreceptors are involved in the effects mediated by TRPV1activation.

The atypical neurotransmitters nitric oxide has also beensuggested to facilitate defensive responses in the dlPAG byfacilitating glutamate release (Guimaraes et al. 2005).Further supporting an interaction between NMDA andTRPV1 receptors, AEA increased the pro-aversive effectsof nitric oxide in the dlPAG through activation of latterreceptors (Lisboa and Guimaraes 2012).

In conclusion, the present study showed that localdlPAG pretreatment with TRPV1 or NMDA receptorantagonists unveiled the anxiolytic-like effect of high,ineffective doses of AEA, suggesting that at higherdoses, facilitation of glutamate release in this region byTRPV1 activation prevents the anxiolytic-like effects ofthis compound.

Acknowledgments This research was supported by grants fromCAPES, CNPq, FAPESP, and FAPEMIG (APQ-01883-10). We thankJ.C. de Aguiar and E.T. Gomes for the excellent technical support.

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