Introduction

The dorsolateral periaqueductal grey (dlPAG) is responsible for elaborating active defensive responses displayed by animals in dangerous situations (Bandler et al. 2000; Carrive 1993). Under natural conditions, these responses are probably mediated by local glutamate release (Guimaraes et al. 2005). Accordingly, intra-dlPAG administration of glutamate ionotropic receptor (NMDA or non-NMDA) agonists induces escape reactions (Aguiar et al. 2006; Bittencourt et al. 2004), whereas the blockade of these receptors causes anxiolytic-like and anti-aversive effects in rodents (Aguiar and Guimaraes 2009; Guimaraes et al. 1991; Matheus and Guimaraes 1997; Molchanov and Guimaraes 2002).

In addition to glutamate, other neurotransmitters have also been shown to modulate anxiety-like behaviors in the dlPAG, including GABA, nitric oxide (NO), endocannabinoids 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 endogenous agonists of the transient receptor potential vanilloid type 1 (TRPV1), a nonselective cation channel (Caterina et al. 1997; Clapham et al. 2005). The first endovanilloid described was anandamide (N-arachidonoylethanolamine, AEA), an arachidonic acid-derived compound, which is also an endocannabinoid and an agonist of cannabinoid receptor type 1 (CB1) (Di Marzo et al. 2001; Smart and Jerman 2000; Zygmunt et al. 2000). Although AEA is the endogenous ligand of both cannabinoid and vanilloid system, its affinity for TRPV1 is lower than that needed to activate CB1 receptors (Ross 2003).

Several results have suggested that endocannabinoids and endovanilloids modulate emotional states in opposite ways (for review see Moreira et al. 2012; Viveros et al. 2007). In line with these pieces of evidence, previous studies from our group showed that lower doses of AEA injected into the dlPAG caused anxiolytic-like effects in distinct animal models of anxiety. These effects were blocked by previous treatment with 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 activation of CB1 receptors inhibits GABAergic and glutamatergic synaptic transmission (Vaughan et al. 2000), which may be related to the biphasic effects on anxiety-like responses observed with CB1 receptor agonists in the PAG (Moreira et al. 2007). However, PAG neurons also express TRPV1 receptors (Cavanaugh et al. 2011; Cristino et al. 2006; Maione et al. 2006; Toth et al. 2005) and, similar to glutamate, TRPV1 activation has been suggested to facilitate anxiety-like responses (Marsch et al. 2007; Rubino et al. 2008; Terzian et al. 2009; Toth et al. 2005). In the central nervous system (CNS), TRPV1 receptors are proposed to modulate neuronal activity mainly through potentiation of glutamatergic signaling (Kawahara et al. 2011; McGaraughty et al. 2003; Starowicz et al. 2007; Xing and Li 2007), an effect that has also 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-shaped dose–response curves observed in anxiety models after administration of AEA in the dlPAG, with higher doses being ineffective, depend on increased glutamate release via activation of TRPV1 receptors.

Materials and methods

Subjects

Male Wistar rats were obtained from the colony of rats maintained by the Campus of the University of São Paulo-Ribeirão Preto. The rats weighing 220–240 g at the beginning of each experiment were housed in groups of four in a temperature-controlled room (24 ± 1 °C) under standard laboratory conditions with free access to food and water and a 12-h light/12-h dark cycle (lights on at 6:30 a.m.). All the experiments were conducted between 8 a.m. and 12 a.m. To avoid interference from different batches of animals, each experiment always included control and treated rats. Procedures were conformed within the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals. The experimental protocols were approved by the Brazilian College of Animal Experimentation and by the School of Medicine of Ribeirao Preto of the University of Sao Paulo Ethical Commission of Ethics in Animal Research (CETEA; no. 083/2008).

Drugs

The CB1/TRPV1 agonist anandamide (AEA) (50 and 200 pmol/0.2 μL; Tocris, USA) was dissolved in TocrisolveTM® 100 (a solvent that contains a 1:4 ratio of soya oil/water, emulsified with the block co-polymer Pluronic F68). The TRPV1 antagonist capsazepine (CPZ; 10 nmol/0.2 μL; Tocris, USA) was dissolved in dimethyl sulfoxide (DMSO) 100 %. The glutamate NMDA receptor antagonist, 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. The chosen doses of AEA were those that did not induce anxiolytic-like effects in a previous work from our group (Moreira et al. 2007). The doses of the antagonists used in this study were chosen based on the dose–response curves from previous studies. Since the blockade of TRPV1 and NMDA receptors in the dlPAG induces anxiolytic-like effects per se, doses that have been proven ineffective in these studies were chosen (Guimaraes et al. 1991; Molchanov and Guimaraes 2002; Terzian et al. 2009).

Apparatus

Elevated plus maze

The elevated plus maze (EPM) consisted of two opposite wooden open arms (50 × 10 cm) crossed at a right angle by two arms of the same dimensions enclosed by 40-cm-high walls (Pellow and File 1986). The maze was located 50 cm above the floor and the ANY-maze Software (version 4.5, Stoelting, USA) was employed for behavioral analysis in the apparatus. The EPM was located in a sound attenuated, temperature controlled (23 °C) room, and the environment was illuminated by a 40-W fluorescent light placed 1.3 m away from the maze. The sessions lasted for 5 min, and after 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 Plexiglas box (42 × 25 × 20 cm) with a stainless grid floor. The metallic spout of a drinking bottle containing water projected into the box. The contact of the animal with the spout and the grid floor closed an electrical circuit controlled by a sensor (Anxiometer model 102, Columbus, USA), which recorded the total number of licks and shocks delivered during the test period (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 the VCT by altering water consumption, independent groups of animals were exposed to the same experimental conditions (3 min each/section) described above except that the electric shock 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 apparatus consisted of an acrylic platform with a nichrome wire coil maintained at room temperature (24–26 °C). The rats were gently handled, and their tails were laid across the coil where the temperature was raised at 9 °C/s by the passage of electric current. The system had a cutoff time of 6 s to prevent tissue damage when the coil temperature approached 80 °C (Moreira et al. 2006).

Surgery

The rats were anesthetized with tribromoethanol (250 mg/kg i.p., Sigma-Aldrich, USA) and were fixed in a stereotaxic frame. A stainless steel guide cannula (11 mm; 0.6 mm, outer diameter (OD)) was implanted unilaterally on the right side aimed at the dlPAG (coordinates: AP = 0 from lambda, L = −1.9 mm at an angle of 16°, D = −4.0 mm) according to the atlas published by Paxinos and Watson (Paxinos and Watson 1997). The cannula was attached to the bones with stainless steel screws and acrylic cement. An obturator inside the guide cannulae prevented obstruction. Immediately after the surgery, the animals received benzylpenicillin (0.27 g/kg i.m.; Pentabiotico®, Fort Dodge, Brazil) to prevent infection and a nonsteroidal anti-inflammatory drug, flunixin meglumine (0.025 g/kg s.c.; Banamine®, Schering Plough, Brazil) for postoperative analgesia.

Procedure

Intra-dlPAG injection

Seven days after surgery, the animals were randomly assigned to one of the treatment groups. Intracerebral injections were performed with a thin dental needle (0.3 mm, OD) introduced through the guide cannula until its tip was 1.0 mm below the cannula end. A volume of 0.2 μL was injected in 30 s using a microsyringe (Hamilton, USA) connected to an infusion pump (KD Scientific, USA). In order to prevent reflux, the guide cannula was left in place for 30 s after the end of each injection. A polyethylene catheter (PE 10) was interposed between the upper end of the 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). Ten minutes later, they were placed in the center of the EPM facing an enclosed arm. The percentage of entries and time spent in the open arms and the number of entries into the enclosed arms of the maze was recorded for 5 min. The experimental groups were vehicle (veh) + veh, veh + AEA 50, veh + AEA 200, AP7 + veh, AP7 + AEA 50, AP7 + AEA 200.

Experiment 2

The procedure was similar to experiment 1 except that the animals received vehicle (DMSO 100 %) or capsazepine (CPZ, 10 nmol) before vehicle (Tocrisolve® in saline) or AEA (50 or 200 pmol) injection. No additional tissue damage was observed in DMSO-treated animals compared to the other vehicles. As previously described, 10 min after the second injection the animals were submitted to the EPM test for 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 to drink freely for 3 min in the test cage in order to find the drinking bottle spout. Twenty-four hours later, they received intra-dlPAG injections of vehicle, capsazepine (10 nmol) or AP7 (1 nmol) followed, 5 min later, by vehicle or AEA injection (200 pmol). Ten minutes after the second injection, they were placed in the apparatus. The test period lasted for 3 min, and the animals received a 0.5-mA shock through the bottle spout every 20 licks. The number of punished licks was registered. The procedure was similar to the one already used and validated in the laboratory (Aguiar et al. 2009; Lisboa et al. 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 not statistically different. Therefore, they were grouped together in the vehicle + vehicle group.

Experiment 4

The tail flick test was conducted in independent groups of animals receiving vehicle + vehicle, capsazepine + AEA, or AP7 + AEA into the dlPAG. Morphine was administered systemically (5 mg/kg i.p.) as a positive control. The animals were gently handled, and their tails were laid across the coil. The heating was applied to a portion of the ventral surface of the tail between 4 and 6 cm from its end. The time to withdraw the tail was recorded as tail flick latency. The electric current was calibrated to provoke this reflex within 2.5–3.5 s in nontreated animals. The tail flick latency was measured at 5-min intervals until a stable baseline was obtained over three consecutive trials. After drug administration, the tail withdrawal latencies were measured again at 10-min intervals for up to 30 min.

Experiment 5

The procedure was the same used in the punished licking test; however, the electric shock delivering system was render inoperative. Ten minutes after vehicle + vehicle, CPZ + AEA, or AP7 + AEA injections, rats were allowed to drink freely for 3 min. The number of licks during this period was registered.

Histology

After the behavioral tests, the rats were sacrificed under deep chloral hydrate 5 % (Sigma-Aldrich, USA) anesthesia and perfused through the left ventricle of the heart with isotonic saline followed by 10 % formalin solution. After that, the brains were removed and, after a minimum period of 5 days, immersed in a 10 % formalin solution, 50 μm sections were obtained in a Cryostat (Cryocut 1800, Leica, Germany). The injection sites were identified in diagrams from the Paxinos and Watson atlas (Paxinos and Watson 2007) and are illustrated in Fig. 1. Rats receiving drug injections outside the aimed area were included in an additional (OUT) group.

Fig. 1
figure 1

a Photomicrography of a representative site injection in the dlPAG. b Histological localization of injection sites in diagrams based on the atlas of Paxinos and Watson (2007). The black and the grey circles represent the injection sites inside and outside of the dlPAG, respectively

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Statistical analysis

The percentages of entries and time spent in the open arms [(open/open + enclosed) × 100] during the 5-min sessions in the EPM were calculated for each animal. Since the data from animals submitted to experiment 1 did not follow a normal distribution, the EPM data from experiment 1 and 2, percentage of entries and time spent in the open arms and number of enclosed arms, were analyzed by Kruskal–Wallis followed by Mann–Whitney test. The number of punished licks (experiment 3) was analyzed by two-way ANOVA using the first and the second injections as main factors. Data from the tail flick (experiment 4) were analyzed by repeated measures ANOVA with treatment as the independent factor and time as the repeated measure. In the case of significant interactions, a one-way ANOVA was performed followed by the Duncan test for multiple comparisons. Results from the water consumption test (experiment 5) were analyzed by one-way ANOVA followed by Duncan test. Differences were considered significant at p < 0.05 level.

Results

Experiment 1

There were significant effects of treatment on the percentage of entries (X 2 (5) = 16.96, p < 0.05) and time spent in the open arms of EPM (X 2 (5) = 12.33, p < 0.05). AP7 (1 nmol) or AEA (50 and 200 pmol) did not induce any significant effects when administered alone. However, AP7 prior to AEA significantly increased the percentage of entries (50 and 200 nmol) and time spent (only the 200 nmol dose) in the open arms of the EPM in comparison to control group (Mann–Whitney test, p < 0.05; Fig. 2), suggesting an anxiolytic-like effect. No effect was found in the number of enclosed arms entries (X 2 (5) = 8.615, p = 0.125), indicating that this effect is not affecting basal motor activity. Additionally, animals that received AP7 + AEA (200 pmol) outside the dlPAG (percentage of open arm entries = 26.2 ± 1.5, percentage of time spent in open arms = 8.8 ± 2.2, n = 6) were not different from the vehicle-treated group.

Fig. 2
figure 2

Effects of vehicle or AP7 (1 nmol) followed by vehicle or anandamide (AEA; 50 or 200 pmol) administered into the dlPAG of rats tested in the EPM. The bars represent the mean ± S.E.M. of the number of entries into the enclosed arms (a), the percentage of entries in the open arms (b), and percentage of time in the open arms (c). The data were analyzed by Kruskal–Wallis followed by Mann–Whitney test (asterisks indicate the significant difference from vehicle–vehicle group, p < 0.05, n = 5–7)

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Experiment 2

There were significant effects of the percentage of time (X 2 (5) = 11.74, p = 0.038) and in the percentage of entries (X 2 (5) = 16.26, p = 0.006) in the open arms of EPM. As previously observed, AEA (50 and 200 pmol) did not induce any 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) significantly increased the percentage of entries in the open arms of EPM compared to the control group (Mann–Whitney test, p < 0.05; Fig. 3). No effect was observed in the number of enclosed arms entries (X 2 (5) = 6.09, p = 0.296). Animals that received CPZ + AEA (200 pmol) outside the dlPAG ((percentage of open arm entries = 22.3 ± 1.5, percentage of time spent in open arms = 6.3 ± 1.8, n = 6) were not different from vehicle-treated rats.

Fig. 3
figure 3

Effects of vehicle or capsazepine (CPZ, 10 nmol) followed by vehicle or AEA (50 or 200 pmol) administered into the dlPAG of rats tested in the EPM. Bars represent the mean ± S.E.M. of the number of entries into the enclosed arms (a), the percentage of entries in the open arms (b), and percentage of time in the open arms (c). The data were analyzed by Kruskal–Wallis followed by Mann–Whitney test (asterisks indicate the significant difference from vehicle–vehicle group, p < 0.05, n = 7–9)

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Experiment 3

Since no difference was found between animals that received saline (n = 4) or DMSO (n = 3) followed by Tocrisolve as vehicles, they were joined together in a control group. Two-way ANOVA showed significant effects of the first (F 2,38 = 4,36; p = 0.019) and second (F 1,38 = 12.38; p = 0.001) microinjections and a significant interaction (F 2,38 = 3.28; p = 0.048). Post hoc analysis indicated that AP7 and capsazepine prior to AEA injection increased the number of punished licks in the VCT (Duncan test, p < 0.05; Fig. 4). These drugs did not induce any effect when administered alone. Moreover, animals that received AP7 + AEA or CPZ + AEA (punished licks = 92 ± 10.6, n = 8) outside the dlPAG were not different from controls.

Fig. 4
figure 4

Effects of CPZ (10 nmol) and AP7 (1 nmol) followed by AEA (200 pmol) administered into the dlPAG of rats submitted to the Vogel conflict test. Bars represent the mean ± S.E.M. total number of punished licks in the 3-min session. Asterisks indicate the significant difference from vehicle–vehicle group. The data were analyzed by two-way ANOVA followed by the Duncan test (*p < 0.05 from vehicle–vehicle group; n = 7π11)

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Experiment 4

A repeated measure ANOVA showed significant effects of time (F 5,84 = 32.0, p < 0.05), treatment (F 3,84 = 74.8, p < 0.05) and interaction (F 15,84 = 15.1, p < 0.05) on the withdrawal latency in the tail flick test. Morphine, the positive control, increased the latency of tail withdrawal 10, 20, and 30 min after drug administration (Duncan test, p < 0.05; Fig. 5). Moreover, there was an increase in the latency of tail withdrawal 20 min after drug injection in animals that received CPZ or AP7 prior to AEA (Duncan test, p < 0.05; Fig. 5). This latency, however, was not different from baseline values (p > 0.05).

Fig. 5
figure 5

Time course of the effects of intra-dlPAG administration of vehicle + vehicle (Veh + Veh), capsazepine (CPZ, 10 nmol) + AEA 200 pmol, AP7 (1 nmol) + AEA (200 pmol), or systemically injected morphine (5 mg/kg, i.p.) in the tail flick test. Each point represents the mean ± S.E.M. for the latency of tail withdrawal. The data were analyzed by two-way ANOVA followed by the Duncan test (asterisks indicate the significant difference p < 0.05, from vehicle group, n = 4–5)

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Experiment 5

The effects in VCT were not due an increase of water consumption since there was no difference in the number of unpunished licks among the groups (veh + veh n = 4; mean number of licks, 964.3 ± 53.1; CPZ + AEA n = 5; mean number of licks, 991.6 ± 52.4; AP7 + AEA n = 5; mean number of licks, 876 ± 29.1; F 2,11 = 1.9, Duncan test, p = 0.19).

Discussion

The present results showed that high, ineffective doses of AEA in the dlPAG can become anxiolytic when animals were pretreated with TRPV1 or NMDA antagonists. This anxiolytic-like effect was observed in two distinct animal models of anxiety, the EPM and VCT, and depended on drug effects in the dlPAG, since no difference was observed when the injections were performed near, but not into, this region. The drug-induced decrease in anxiety was not accompanied by changes in the number of enclosed arms entries in the EPM, discarding motor effect as a possible confounding factor (File 1992). Moreover, control experiments for the VCT showed that neither AEA plus AP7 nor AEA plus CPZ altered water consumption. Both treatments, however, did increase the latency of tail withdrawal 20 min after injection, suggesting an antinociceptive effect at this time point. Nevertheless, in the present study the VCT was performed 10 min after injections, a time point where no differences were found in the tail flick test. In addition, the antinociceptive effect was small and did not differ from the baseline measures. Therefore, it seems unlikely that the anticonflict results obtained in the VCT were due to antinociceptive effects. In previous studies of our group, similar results (anxiolytic with a small antinociceptive effect) were obtained after intra-dlPAG injection of the inhibitor of anandamide uptake AM404 or the phytocannabinoid cannabidiol (Campos and Guimaraes 2008; Lisboa et al. 2008). This is hardly surprising, since cannabinoid-, vanilloid-, and glutamatergic-mediated neurotransmissions are involved in nociceptive control in this brain area (Finn et al. 2003; Maione et al. 2006, 2009; Palazzo et al. 2001, 2002; Walker et al. 1999).

Together, the present results indicate that at high doses, such as those used in the present study (Moreira et al. 2007), the anxiolytic-like effects of AEA in the dlPAG disappear due to its ability to activate TRPV1 receptors. In agreement with this possibility, the affinity of AEA at TRPV1 is relatively low compared to CB1 receptors (Ross 2003).

Different from CB1 receptors, TRPV1 seems to mediate pro-aversive effects. TRPV1 knockout mice showed reduced anxiety-like behavior in the EPM and light–dark box, as well as reduced fear conditioning (Marsch et al. 2007). Moreover, either systemic or local injection of TRPV1 antagonists into specific 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 (Santos et al. 2008), induces anxiolytic-like effects in rats.

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

Increased glutamate release in the dlPAG induced by activation of TRPV1 receptors after high doses of some cannabinoids such as AEA (Palazzos et al. 2006) could facilitate defensive responses (Guimaraes et al. 2005). Accordingly, in the presence of an NMDA antagonist, ineffective high doses of AEA became anxiolytic in animals exposed to both EPM and VCT. Even if we have not measured local glutamate release, several studies corroborate this possibility. For example, injection of capsaicin into the dlPAG induced nociception and local release of glutamate, an effect reversed by pretreatment with the TRPV1 antagonist capsazepine (Palazzo et al. 2002). Similar effects were described in vitro for other regions such as the hypothalamus, substantia nigra, and locus coeruleus (Marinelli et al. 2002, 2003; Palazzo et al. 2002). Furthermore, supporting the hypothesis of interaction between glutamatergic and vanilloid systems, Starowicz et al. (2007) showed that neurons in the ventrolateral portion of the PAG, which also express TRPV1 receptors, are surrounded by glutamatergic neurons. Additionally, these glutamatergic neurons respond to TRPV1 stimulation by releasing glutamate, an effect blocked by pretreatment with a vanilloid antagonist (Starowicz et al. 2007). The expression of TRPV1 in glutamatergic neurons was also detected in the dorsal horn of the spinal cord (Zhou et al. 2009).

In order to verify which glutamate receptors are related to the nociceptive responses induced by capsaicin, Jin et al. (2012) observed that capsaicin-evoked c-Fos expression in the spinal cord dorsal horn was prevented by NMDA receptor antagonists, suggesting that activation of ionotropic glutamate receptors are involved in nociceptive responses induced by capsaicin (Jin et al. 2012). Moreover, the antidepressant and anxiolytic-like behavioral profiles observed in TRPV1 knockout mice were associated with changes in the expression of NMDA receptors in the striatum and CA1 region of the hippocampus (You et al. 2012). Altogether, these results suggest that NMDA receptors are involved in the effects mediated by TRPV1 activation.

The atypical neurotransmitters nitric oxide has also been suggested to facilitate defensive responses in the dlPAG by facilitating glutamate release (Guimaraes et al. 2005). Further supporting an interaction between NMDA and TRPV1 receptors, AEA increased the pro-aversive effects of nitric oxide in the dlPAG through activation of latter receptors (Lisboa and Guimaraes 2012).

In conclusion, the present study showed that local dlPAG pretreatment with TRPV1 or NMDA receptor antagonists unveiled the anxiolytic-like effect of high, ineffective doses of AEA, suggesting that at higher doses, facilitation of glutamate release in this region by TRPV1 activation prevents the anxiolytic-like effects of this compound.