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Modulation of anxiety-like behavior by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray
Behavioural Brain Research 252 (2013) 10–17
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Modulation of anxiety-like behavior by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray A.F. Almeida-Santos a,1 , P.H. Gobira a,1 , L.C. Rosa a,b , F.S. Guimaraes b,c,2 , F.A. Moreira a , D.C. Aguiar a,∗ a Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Pres. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil b Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049900, Brazil c Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, Brazil
h i g h l i g h t s • • • •
We verified the role of the endocannabinoid 2AG on anxiety behaviors mediated by dlPAG. The augmentation of 2AG signaling induces anxiolytic-like effects in the E.P.M. test. These effects were also observed with the MGL inhibitor. This anxiolytic-like effect in the EPM was dependent on activation of CB1 and CB2 receptors.
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Article history: Received 24 April 2013 Received in revised form 16 May 2013 Accepted 18 May 2013 Available online 25 May 2013 Keywords: 2-Arachidonoylglycerol (2-AG) Endocannabinoids Anxiety Elevated plus maze Dorsolateral periaqueductal gray
a b s t r a c t Anandamide and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type 2 (CB2r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). 2-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing 2-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of 2-AG, URB602 (inhibitor of the 2-AG hydrolyzing enzyme, mono-acylglycerol lipase - MGL), AM251 (CB1r antagonist) and AM630 (CB2r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or 2-AG (5 pmol, 50 pmol, and 500 pmol). Exp. 2: veh or URB602 (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM251 (100 pmol) followed by veh or 2-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or 2-AG. Exp. 5: veh or AM251 followed by veh or URB602 (100 pmol). Exp. 6: veh or AM630 followed by veh or URB602. 2-AG (50 pmol) and URB602 (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM251) or CB2r (AM630) antagonists. Our results showed that the augmentation of 2-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB2r receptors. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Endocannabinoids are a class of bioactive lipids synthesized on demand from membrane phospholipids in postsynaptic neurons, acting as retrograde messengers (for review, see [1]).
∗ Corresponding author. Tel.: +55 31 3409 2718. E-mail addresses: [email protected], [email protected] (D.C. Aguiar). 1 These authors contributed equally to this work. 2 Tel.: +55 16 36023209; fax: +55 16 36332301. 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.05.027
The main representatives are the arachidonic acid derivates arachidonoyl ethanolamide, (AEA, also termed anandamide), and 2-arachidonoylglicerol (2-AG [2–4]). These substances exert their effects mainly through two Gi protein-coupled receptors, named by their order of discovery as cannabinoid receptors type 1 (CB1) and type 2 (CB2) [5]. Both AEA and 2-AG have slightly less affinity for CB2 as compared to CB1 receptor [2,3,6]. The mechanism of inactivation of these compounds involves a transport into cells followed by enzymatic hydrolysis through specific enzymes. AEA is preferentially degraded by fatty acid amide hydrolase (FAAH), whereas the mono-acylglycerol lipase (MGL) is the main responsible for 2-AG [7–11].
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Emerging evidence implicates endocannabinoids in a wide range of physiological and pathological processes, including the control of emotional states [9,12–15]. In line with this is the extensively expression of CB1 receptors in brain regions responsible for defensive reactions such as the hypothalamus, pre-frontal cortex (PFC), amygdala and periaqueductal gray (PAG, [16–19]). Moreover, systemic administration of the endocannabinoid transport inhibitor AM404 induced CB1-mediated anxiolytic-like effect associated with increased levels of AEA [20]. Further, genetic or pharmacological inhibition of FAAH, which increases the levels of AEA, also induces anxiolytic-like effects via CB1 mechanisms [9,21]. In order to verify the brain sites responsible for AEA effects, previous work from our group showed that the local administration of AEA into the dorsolateral PAG (dlPAG) induces anxiolytic-like effect in several animal models of anxiety, such as the elevated plus maze (EPM), the Vogel conflict and in fear conditioned tests [12,22,23]. In all cases, the effects of AEA were mediated by CB1 receptors. Contrary to AEA, little is known about the role of 2-AG on the modulation of anxiety-related behaviors. Previous studies showed an increase in 2-AG in the PAG after foot shock stress, where this endocannabinoid mediates stress-induced analgesia [24]. In line with the notion that this endocannabinoid could also modulate aversive reactions [13,25], the administration of the MGL inhibitor JZL-184 reduced the anxiety measured in the marble-burying, elevated zero maze and elevated plus maze (EPM) tests [26–28]. While AEA promote anti-aversive effects through CB1-R mechanisms, the anxiolytic-like responses observed after augmentation of 2-AG signaling were both CB1- and CB2-dependent [26,28]. Thus, the aim of this study was to test the hypothesis that the dlPAG could be one of the brain regions involved in the anxiolyticlike effects of 2-AG. We also investigated whether 2-AG effects are mediated by CB1 or CB2 receptors.
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with no roof [32]. The arms were located 50 cm above the floor. The maze was in a sound-attenuated, temperature-controlled (23 ◦ C) room, illuminated by one 40-W fluorescent light placed 1.5 m away from the apparatus. The Any-Maze software (V. 4.5, Stoelting, IL, USA) was employed for behavioral analysis. Each session lasted for 5 min. After each trial, the maze was cleaned with an alcohol solution and properly dried [12]. 2.5. Procedure 2.5.1. Intra-dlPAG injection Seven days after surgery the animals were randomly assigned to one of the treatment groups. The injection was performed by gently holding the animal to remove the obturator and introduce the guide cannula (12 mm). A volume of 0.2 L (1 L/min) was injected in 30 s using a microsyringe (Hamilton, USA) connected to an infusion pump (Insight, Brazil) through a polyethylene catheter (PE10) that was interposed between the upper end of the dental needle and the syringe. In order to prevent reflux, the needle was left in place for 30 s after the end of each injection. 2.5.2. Experiment 1 The animals received intra-dlPAG injections of vehicle (0.2 L) or 2-AG (5, 50 or 500 pmol/0.2 L) and ten minutes later were exposed to the EPM during 5 min. 2.5.3. Experiment 2 In order to verify if the effects of 2-AG were dependent on CB1 activation, the animals were pre-treated in the dlPAG with vehicle (0.2 L) or AM251 (100 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or 2-AG (50 pmol/0.2 L). 10 min after the last injection they were exposed to the EPM test (5 min). 2.5.4. Experiment 3 We also verified if the effects of 2-AG were mediated by CB2 receptors. Thus, in order to achieve the dose that will be employed in the next experiment, the animals received intra-dlPAG injections of vehicle or AM630 (10, 100 or 1000 pmol/0.2 L), and were exposed to the EPM test 10 min later. 2.5.5. Experiment 4 The animals received intra-dlPAG injection of vehicle (0.2 L) or AM630 (1000 pmol/0.2 L) followed, 5 minutes later, by vehicle (0.2 L) or 2-AG (50 pmol/0.2 L). Ten minutes after the last injection they were exposed to the EPM test.
2. Materials and methods 2.1. Animals Male wistar rats weighing 220–240 g were obtained from the Biotery Center of the Institute of Biological Sciences, UFMG, after approval by the Ethics Committee for Research on Animal Experimentation (CETEA/UFMG no. 66/2010). The rats were kept in groups of five animals per cage, with free access to food and water under light–dark cycle of 12 h (beginning at 06:30 a.m.) and controlled temperature (24 ± 1 ◦ C). All the experiments were conducted between 8 a.m. and 12 a.m. 2.2. Drugs The drugs utilized in the present study were 2-AG (Tocris® ): 5, 50 and 500 pmol diluted in saline sterile, [1,1 -biphenyl]-3-yl-carbamic acid cyclohexyl ester, URB602 (MGL inhibitor, Cayman Chemical® ): 30, 100 and 300 pmol diluted in 50% DMSO in phosphate buffer solution; N(piperidine-1yl)-5-(4-iodophenyl)-1-(2,4dichlorophenyl)-4-methyl 1 pyrazolcarboxamide, AM251 (CB1 receptor antagonist, Sigma® ): 100 pmol diluted in 10% DMSO in sterile saline; 6-iodo-2-methyl-1(2-morpholinoethyl)-1H-indol-3-yl)(4-methoxyphenyl) methanone, AM630 (CB2 receptor antagonist, Tocris® ): 10, 100 and 1000 pmol diluted in saline sterile. The dose ranges were selected based on previous works injecting these substances into brain regions and/or on comparative affinity (Ki) for the respective receptor target [12,22,29]. 2.3. Surgery The animals were anesthetized with ketamine (60 mg/kg) and xylazine (8 mg/kg) i.p. and fixed to a stereotaxic apparatus. A stainless steel guide cannula (0.6 mm 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). The cannula was attached to the bones with stainless steel screws and acrylic cement. An obturator inside the guide cannula to prevented obstruction was utilized [30,31]. Animals were injected with pentabiotic and the analgesic, antipyretic and anti-inflammatory Banamine® (flunixin meglumine, 1 ml/kg). 2.4. Apparatus The EPM consisted of two opposite wood open arms (50 cm × 10 cm) crossed at right angle by two arms of the same dimensions enclosed by 40-cm high walls
2.5.6. Experiment 5 In order to verify if the inhibitor of MGL would induce anxiolytic-like effect, a dose–response curve for URB 602 was performed. Thus, the animals received vehicle or URB602 (30, 100 or 300 pmol/0.2 L) 10 min before being submitted to the EPM test. 2.5.7. Experiment 6 To test if the effects of URB602 were related to CB1 activation, the animals received intra-dlPAG injection of vehicle (0.2 L) or AM251 (100 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or URB602 (100 pmol/0.2 L). 10 min after the last injection they were exposed to the EPM. 2.5.8. Experiment 7 This last experiment was conducted to verify if the effects of URB602 were dependent on CB2 receptors. For this, the animals received intra-dlPAG injection of vehicle (0.2 L) or AM630 (1000 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or URB602 (100 pmol/0.2 L). Ten minutes after the last injection they were exposed to the EPM. 2.6. Histology After the behavioral tests, the rats were deeply anesthetized with Urethane (25%, 5 mL/kg) and underwent intracardiac perfusion with saline 0.9% followed by 10% formalin solution. After that, a dental needle was inserted through the guide cannula and 0.2 L of methylene blue was injected. The brains were removed and stored in 10% formalin for three days. Then, 50 m sections were obtained in a cryostat (Microm HM 505 N). The injection sites were identified in diagrams from the Paxinos and Watson’s atlas [33]. The injection sites can be seen in Fig. 1. Rats that received injections outside the aimed area were excluded from analysis. 2.7. Statistical analysis The percentages of entries and time spent in the open arms [100 × open/(open + enclosed)] during the 5-min sessions in the EPM were calculated for each animal. These results and the number of enclosed arm entries were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan test for multiple comparisons. Differences were considered significant at p < 0.05 level.
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Fig. 1. Diagrams containing histological localization of injection sites based on the atlas of Paxinos and Watson [33]. The circles represent the injection sites in dlPAG.
3. Results Experiment 1: The intra-dlPAG injection of 2-AG (50 pmol/0.2 L) significantly increased the percentage of entries in the open arms as compared to control group (F(3,26) = 4.22, p = 0.012, Duncan test p < 0.05; Fig. 2). There was no difference between groups in the percentage of time spent in the open arms. Moreover, there was no effect in the number of enclosed arms entries (F(3,26) = 0.36, p = 0.775), suggesting that the 2-AG did not affect basal motor activity. Experiment 2: In this experiment, the injection of 2-AG (50 pmol/0.2 L) in the dlPAG increased the percentage of time spent in the open arms in the dlPAG (F(3,27) = 2.967, p = 0.05; Duncan p < 0.05; Fig. 3). The pre-treatment with AM251 (100 pmol/0.2 L) prevented this effect. No effect was found in the number of enclosed arms entries (F(3,27) = 0.156, p = 0.925). Experiment 3: Intra-dlPAG injection of AM630 (10, 100 and 1000 pmol/0.2 L) did not induce any alteration in the percentage of time spent (F(3,17) = 0.715, p = 0.552) and in the number of entries (F(3,17) = 1.31, p = 0.292) in the open arms. Moreover, no significant difference was observed in the number of entries in enclosed arms
(F(3,17) = 0.061, p = 0.979). Since AM630 did not modify the behavioral responses in the EPM, the dose of 1000 pmol was chosen for the subsequent experiment. Experiment 4: The intra-dlPAG injection of AM630 (1000 pmol/0.2 L) attenuated the increase in the percentage of time spent into the open arms caused by 2-AG (50 pmol/0.2 L) (F(3,27) = 2.267, p = 0.103; Duncan test, p < 0.05; Fig. 4). Moreover, no effects were observed in the number of entries in the enclosed arms (F(3,27) = 2.035, p = 0.131). Experiment 5: The MGL (enzyme responsible for the hydrolysis of 2-AG) inhibitor, URB602 (100 pmol/0.2 L), injected intradlPAG significantly increased the percentage of time (F(3,26) = 2.015, p = 0.136, Duncan test p < 0.05) spent in the open arms as compared to controls (Fig. 5). No effect was found in the number of enclosed arms entries (F(3,26) = 0.217, p = 0.883), suggesting that the drugs did not affect basal motor activity. Experiment 6: AM251 (100 pmol/0.2 L) was able to prevent the increase in the percentage of time (F(3,26) = 4.283, p = 0.013; Duncan test, p < 0.05) spent into the open arms promoted by URB602 (100 pmol/0.2 L). The URB 602 significantly increased the number of entries into the open arms (F(3,26) = 5.98, p = 0.003; Duncan
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Fig. 2. Effects of 2-AG (5, 50 or 500 pmol/0.2 L) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage of time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle group (p < 0.05, ANOVA followed by the Duncan test; n = 7–14/group).
test, p < 0.05), and this effect was partially prevented by AM251. No effect was found in the number of enclosed arms entries (F(3,26) = 0.09, p = 0.964; Fig. 6). Experiment 7: AM630 (1000 pmol/0.2 L) blocked the increase in the number of entries (F(3,31) = 4.525, p = 0.009; Duncan test, p < 0.05) into the open arms promoted by URB602 (100 pmol/0.2 L). No effect was observed in the number of enclosed arms entries (F(3,31) = 0.763, p = 0.523; Fig. 7). 4. Discussion In the present study we verified the possible role of 2-AG in the dlPAG in the modulation of anxiety-like behavior. Our results
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Fig. 3. Effects of pre-treatment with AM251 (100 pmol) followed by 2-AG (50 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group (p < 0.05, ANOVA followed by the Duncan test; n = 6–10/group).
showed that intra-dlPAG administration of this endocannabinoid increased the exploration of the open arms in the EPM, indicating an anxiolytic-like effect. These effects were also observed after the inhibition of endogenous 2-AG hydrolysis with the compound URB602. In investigating the mechanisms involved in these responses, we observed that both CB1 and CB2 receptors contribute to the anxiolytic-like effects. The dlPAG is proposed to be part of a neural substrate responsible for the coordination of both nociceptive responses and
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Fig. 4. Effects of pre-treatment with AM630 (1000 pmol) followed by 2-AG (50 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group and # indicates significant difference from vehicle + 2-AG group (50 pmol; p < 0.05, ANOVA followed by the Duncan test; n = 6–9/group).
Fig. 5. Effects of URB602 (30, 100 or 300 pmol/0.2 L) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle (p < 0.05, ANOVA followed by the Duncan test; n = 6–8/group).
anxiety-related behaviors [34–36]. Several studies have shown that AEA and synthetic cannabinoid compounds injected into this region modulate aversion-related responses in animal models [12,22,23,37]. Finn and colleagues showed that local administration of HU210, a synthetic cannabinoid, attenuated the escape responses induced by intra-dorsal PAG injections of the excitatory amino acid d,l-homocysteic acid [37]. Anxiolytic-like effects were observed after local administration of AEA in several models [12,22,23]. Similarly, in the present study we observed that 2-AG induces antiaversive effects in the dlPAG in animals exposed to EPM test. The dose–response curve of 2-AG effects in the EPM was bellshaped, with higher doses being ineffective. This effect resembles the yield complex responses observed with other cannabinoids
in experimental models of anxiety. In line with this, systemic injections of cannabinoid agonists induce anxiolytic-like effect in the EPM test at lower doses, becoming ineffective at higher doses [38,39]. Moreover, local injections of 9 -THC, the main psychoactive substance present in Cannabis sativa, into brain regions responsible for the control of anxiety related behaviors such as amygdala, hippocampus and pre-frontal cortex also exhibited this action profile [40]. Although it is not known the exact mechanisms responsible for these effects, it could be related to the ability of cannabinoids to modulate the release of neurotransmitters such as glutamate and GABA that have opposite actions on anxiety states [1,41].
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Fig. 6. Effects of AM251 (100 pmol) followed by URB602 (100 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group, # indicates significant difference from vehicle + URB602 (100 pmol; p < 0.05, ANOVA followed by the Duncan test; n = 7–8/group).
Recently, Gregg and colleagues [36] showed that the enzyme diacilglicerol lipase-␣ (DGL), responsible for 2-AG synthesis, is expressed in the dlPAG. They also observed that footshock stress elicits the rapid formation of 2-AG in this region, and this effect was blockade by local injection of a drug that selectivity inhibits the DGL [36]. These results suggest that during stressful situations the 2-AG signaling could be recruited in order to inhibit aversive responses. Our study is in agreement with this possibility, since we observed that intra-dlPAG injection of a drug that inhibits the hydrolysis of this endocannabinoid induces anxiolytic-like. In addition, these data corroborate studies demonstrating that systemic injections of other MGL inhibitor, JZL-184, promoted anxiolytic-like
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Fig. 7. Effects of AM630 (1000 pmol) followed by URB602 (100 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group and # indicates significant difference from vehicle + URB602 (p < 0.05, ANOVA followed by the Duncan test; n = 7–11/group).
effect in EPM and elevated zero maze paradigms [26,42]. Similar effects were reported in the marble-burying test [27]. In order to verify which cannabinoid receptor is involved in the effects of 2-AG, we locally pre-treated the animals with CB1 or CB2 antagonists before injections of 2-AG and URB602 into the dlPAG. Neither antagonist was effective by themselves at the doses employed in this study. Yet, they blocked the anxiolytic-like effects observed by intra-dlPAG injection of 2-AG as well as the hydrolysis inhibitor, suggesting that both CB1 and CB2 receptors are involved in these responses. Several studies have shown that CB1R mediates the behavioral responses induced by cannabinoids agonists [12,21,39,43,44]. Moreover, other studies also described CB1 dependent mechanism for the anxiolytic-like effects induced by systemic injection of 2-AG and a MGL inhibitor in the marble burying test in mice and EPM test in rats [27,28]. Regarding the observation that 2-AG effects in the dlPAG were also dependent on CB2, this result could be related to the fact that
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this endocannabinoid binds both receptors [3,6,45]. Although it has been initially proposed that CB2 receptors are mainly expressed in immune cells [46], recent studies indicate that they are also expressed in the brain and could modulate behavioral responses [47–50]. Accordingly, García-Gutiérrez and Manzanares showed that over-expression of CB2 receptors decreased vulnerability to anxiety [47]. Moreover, the anxiolytic-like effects induced by systemic inhibition of 2-AG hydrolysis in mice were mediated by CB2 but not CB1 receptor activation [26]. In conclusion, the present work showed that facilitation of 2-AG signaling in the dlPAG induces anxiolytic-like effects in rats tested in the EPM test. We also showed that both CB1 and CB2 receptors are involved in these responses. These results, therefore, support the proposal that modulation of the endocannabinoid system in this region is important for aversive reactions, and suggest that drugs that interfere with 2-AG signaling constitute a potential therapeutic strategy for anxiety related disorders.
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This research was supported by grants from CAPES, CNPq, FAPESP (2012/17626-7) and FAPEMIG (APQ-01038-11, PRONEM: APQ-04625-10)
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Research report
Modulation of anxiety-like behavior by the endocannabinoid 2-arachidonoylglycerol (2-AG) in the dorsolateral periaqueductal gray A.F. Almeida-Santos a,1 , P.H. Gobira a,1 , L.C. Rosa a,b , F.S. Guimaraes b,c,2 , F.A. Moreira a , D.C. Aguiar a,∗ a Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Pres. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil b Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049900, Brazil c Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, Brazil
h i g h l i g h t s • • • •
We verified the role of the endocannabinoid 2AG on anxiety behaviors mediated by dlPAG. The augmentation of 2AG signaling induces anxiolytic-like effects in the E.P.M. test. These effects were also observed with the MGL inhibitor. This anxiolytic-like effect in the EPM was dependent on activation of CB1 and CB2 receptors.
a r t i c l e
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Article history: Received 24 April 2013 Received in revised form 16 May 2013 Accepted 18 May 2013 Available online 25 May 2013 Keywords: 2-Arachidonoylglycerol (2-AG) Endocannabinoids Anxiety Elevated plus maze Dorsolateral periaqueductal gray
a b s t r a c t Anandamide and 2-arachidonoylglycerol (2-AG) are the two main endocannabinoids, exerting their effects by activating type 1 (CB1r) and type 2 (CB2r) cannabinoid receptors. Anandamide inhibits anxiety-like responses through the activation of CB1r in certain brain regions, including the dorsolateral periaqueductal gray (dlPAG). 2-AG also attenuates anxiety-like responses, although the neuroanatomical sites for these effects remained unclear. Here, we tested the hypothesis that enhancing 2-AG signaling in the dlPAG would induce anxiolytic-like effects. The mechanisms involved were also investigated. Male Wistar rats received intra-dlPAG injections of 2-AG, URB602 (inhibitor of the 2-AG hydrolyzing enzyme, mono-acylglycerol lipase - MGL), AM251 (CB1r antagonist) and AM630 (CB2r antagonist). The behavior was analyzed in the elevated plus maze after the following treatments. Exp. 1: vehicle (veh) or 2-AG (5 pmol, 50 pmol, and 500 pmol). Exp. 2: veh or URB602 (30 pmol, 100 pmol or 300 pmol). Exp. 3: veh or AM251 (100 pmol) followed by veh or 2-AG (50 pmol). Exp. 4: veh or AM630 (1000 pmol) followed by veh or 2-AG. Exp. 5: veh or AM251 followed by veh or URB602 (100 pmol). Exp. 6: veh or AM630 followed by veh or URB602. 2-AG (50 pmol) and URB602 (100 pmol) significantly increased the exploration of the open arms of the apparatus, indicating an anxiolytic-like effect. These behavioral responses were prevented by CB1r (AM251) or CB2r (AM630) antagonists. Our results showed that the augmentation of 2-AG levels in the dlPAG induces anxiolytic-like effects. The mechanism seems to involve both CB1r and CB2r receptors. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Endocannabinoids are a class of bioactive lipids synthesized on demand from membrane phospholipids in postsynaptic neurons, acting as retrograde messengers (for review, see [1]).
∗ Corresponding author. Tel.: +55 31 3409 2718. E-mail addresses: [email protected], [email protected] (D.C. Aguiar). 1 These authors contributed equally to this work. 2 Tel.: +55 16 36023209; fax: +55 16 36332301. 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.05.027
The main representatives are the arachidonic acid derivates arachidonoyl ethanolamide, (AEA, also termed anandamide), and 2-arachidonoylglicerol (2-AG [2–4]). These substances exert their effects mainly through two Gi protein-coupled receptors, named by their order of discovery as cannabinoid receptors type 1 (CB1) and type 2 (CB2) [5]. Both AEA and 2-AG have slightly less affinity for CB2 as compared to CB1 receptor [2,3,6]. The mechanism of inactivation of these compounds involves a transport into cells followed by enzymatic hydrolysis through specific enzymes. AEA is preferentially degraded by fatty acid amide hydrolase (FAAH), whereas the mono-acylglycerol lipase (MGL) is the main responsible for 2-AG [7–11].
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Emerging evidence implicates endocannabinoids in a wide range of physiological and pathological processes, including the control of emotional states [9,12–15]. In line with this is the extensively expression of CB1 receptors in brain regions responsible for defensive reactions such as the hypothalamus, pre-frontal cortex (PFC), amygdala and periaqueductal gray (PAG, [16–19]). Moreover, systemic administration of the endocannabinoid transport inhibitor AM404 induced CB1-mediated anxiolytic-like effect associated with increased levels of AEA [20]. Further, genetic or pharmacological inhibition of FAAH, which increases the levels of AEA, also induces anxiolytic-like effects via CB1 mechanisms [9,21]. In order to verify the brain sites responsible for AEA effects, previous work from our group showed that the local administration of AEA into the dorsolateral PAG (dlPAG) induces anxiolytic-like effect in several animal models of anxiety, such as the elevated plus maze (EPM), the Vogel conflict and in fear conditioned tests [12,22,23]. In all cases, the effects of AEA were mediated by CB1 receptors. Contrary to AEA, little is known about the role of 2-AG on the modulation of anxiety-related behaviors. Previous studies showed an increase in 2-AG in the PAG after foot shock stress, where this endocannabinoid mediates stress-induced analgesia [24]. In line with the notion that this endocannabinoid could also modulate aversive reactions [13,25], the administration of the MGL inhibitor JZL-184 reduced the anxiety measured in the marble-burying, elevated zero maze and elevated plus maze (EPM) tests [26–28]. While AEA promote anti-aversive effects through CB1-R mechanisms, the anxiolytic-like responses observed after augmentation of 2-AG signaling were both CB1- and CB2-dependent [26,28]. Thus, the aim of this study was to test the hypothesis that the dlPAG could be one of the brain regions involved in the anxiolyticlike effects of 2-AG. We also investigated whether 2-AG effects are mediated by CB1 or CB2 receptors.
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with no roof [32]. The arms were located 50 cm above the floor. The maze was in a sound-attenuated, temperature-controlled (23 ◦ C) room, illuminated by one 40-W fluorescent light placed 1.5 m away from the apparatus. The Any-Maze software (V. 4.5, Stoelting, IL, USA) was employed for behavioral analysis. Each session lasted for 5 min. After each trial, the maze was cleaned with an alcohol solution and properly dried [12]. 2.5. Procedure 2.5.1. Intra-dlPAG injection Seven days after surgery the animals were randomly assigned to one of the treatment groups. The injection was performed by gently holding the animal to remove the obturator and introduce the guide cannula (12 mm). A volume of 0.2 L (1 L/min) was injected in 30 s using a microsyringe (Hamilton, USA) connected to an infusion pump (Insight, Brazil) through a polyethylene catheter (PE10) that was interposed between the upper end of the dental needle and the syringe. In order to prevent reflux, the needle was left in place for 30 s after the end of each injection. 2.5.2. Experiment 1 The animals received intra-dlPAG injections of vehicle (0.2 L) or 2-AG (5, 50 or 500 pmol/0.2 L) and ten minutes later were exposed to the EPM during 5 min. 2.5.3. Experiment 2 In order to verify if the effects of 2-AG were dependent on CB1 activation, the animals were pre-treated in the dlPAG with vehicle (0.2 L) or AM251 (100 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or 2-AG (50 pmol/0.2 L). 10 min after the last injection they were exposed to the EPM test (5 min). 2.5.4. Experiment 3 We also verified if the effects of 2-AG were mediated by CB2 receptors. Thus, in order to achieve the dose that will be employed in the next experiment, the animals received intra-dlPAG injections of vehicle or AM630 (10, 100 or 1000 pmol/0.2 L), and were exposed to the EPM test 10 min later. 2.5.5. Experiment 4 The animals received intra-dlPAG injection of vehicle (0.2 L) or AM630 (1000 pmol/0.2 L) followed, 5 minutes later, by vehicle (0.2 L) or 2-AG (50 pmol/0.2 L). Ten minutes after the last injection they were exposed to the EPM test.
2. Materials and methods 2.1. Animals Male wistar rats weighing 220–240 g were obtained from the Biotery Center of the Institute of Biological Sciences, UFMG, after approval by the Ethics Committee for Research on Animal Experimentation (CETEA/UFMG no. 66/2010). The rats were kept in groups of five animals per cage, with free access to food and water under light–dark cycle of 12 h (beginning at 06:30 a.m.) and controlled temperature (24 ± 1 ◦ C). All the experiments were conducted between 8 a.m. and 12 a.m. 2.2. Drugs The drugs utilized in the present study were 2-AG (Tocris® ): 5, 50 and 500 pmol diluted in saline sterile, [1,1 -biphenyl]-3-yl-carbamic acid cyclohexyl ester, URB602 (MGL inhibitor, Cayman Chemical® ): 30, 100 and 300 pmol diluted in 50% DMSO in phosphate buffer solution; N(piperidine-1yl)-5-(4-iodophenyl)-1-(2,4dichlorophenyl)-4-methyl 1 pyrazolcarboxamide, AM251 (CB1 receptor antagonist, Sigma® ): 100 pmol diluted in 10% DMSO in sterile saline; 6-iodo-2-methyl-1(2-morpholinoethyl)-1H-indol-3-yl)(4-methoxyphenyl) methanone, AM630 (CB2 receptor antagonist, Tocris® ): 10, 100 and 1000 pmol diluted in saline sterile. The dose ranges were selected based on previous works injecting these substances into brain regions and/or on comparative affinity (Ki) for the respective receptor target [12,22,29]. 2.3. Surgery The animals were anesthetized with ketamine (60 mg/kg) and xylazine (8 mg/kg) i.p. and fixed to a stereotaxic apparatus. A stainless steel guide cannula (0.6 mm 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). The cannula was attached to the bones with stainless steel screws and acrylic cement. An obturator inside the guide cannula to prevented obstruction was utilized [30,31]. Animals were injected with pentabiotic and the analgesic, antipyretic and anti-inflammatory Banamine® (flunixin meglumine, 1 ml/kg). 2.4. Apparatus The EPM consisted of two opposite wood open arms (50 cm × 10 cm) crossed at right angle by two arms of the same dimensions enclosed by 40-cm high walls
2.5.6. Experiment 5 In order to verify if the inhibitor of MGL would induce anxiolytic-like effect, a dose–response curve for URB 602 was performed. Thus, the animals received vehicle or URB602 (30, 100 or 300 pmol/0.2 L) 10 min before being submitted to the EPM test. 2.5.7. Experiment 6 To test if the effects of URB602 were related to CB1 activation, the animals received intra-dlPAG injection of vehicle (0.2 L) or AM251 (100 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or URB602 (100 pmol/0.2 L). 10 min after the last injection they were exposed to the EPM. 2.5.8. Experiment 7 This last experiment was conducted to verify if the effects of URB602 were dependent on CB2 receptors. For this, the animals received intra-dlPAG injection of vehicle (0.2 L) or AM630 (1000 pmol/0.2 L) followed, 5 min later, by vehicle (0.2 L) or URB602 (100 pmol/0.2 L). Ten minutes after the last injection they were exposed to the EPM. 2.6. Histology After the behavioral tests, the rats were deeply anesthetized with Urethane (25%, 5 mL/kg) and underwent intracardiac perfusion with saline 0.9% followed by 10% formalin solution. After that, a dental needle was inserted through the guide cannula and 0.2 L of methylene blue was injected. The brains were removed and stored in 10% formalin for three days. Then, 50 m sections were obtained in a cryostat (Microm HM 505 N). The injection sites were identified in diagrams from the Paxinos and Watson’s atlas [33]. The injection sites can be seen in Fig. 1. Rats that received injections outside the aimed area were excluded from analysis. 2.7. Statistical analysis The percentages of entries and time spent in the open arms [100 × open/(open + enclosed)] during the 5-min sessions in the EPM were calculated for each animal. These results and the number of enclosed arm entries were analyzed by one-way analysis of variance (ANOVA) followed by the Duncan test for multiple comparisons. Differences were considered significant at p < 0.05 level.
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Fig. 1. Diagrams containing histological localization of injection sites based on the atlas of Paxinos and Watson [33]. The circles represent the injection sites in dlPAG.
3. Results Experiment 1: The intra-dlPAG injection of 2-AG (50 pmol/0.2 L) significantly increased the percentage of entries in the open arms as compared to control group (F(3,26) = 4.22, p = 0.012, Duncan test p < 0.05; Fig. 2). There was no difference between groups in the percentage of time spent in the open arms. Moreover, there was no effect in the number of enclosed arms entries (F(3,26) = 0.36, p = 0.775), suggesting that the 2-AG did not affect basal motor activity. Experiment 2: In this experiment, the injection of 2-AG (50 pmol/0.2 L) in the dlPAG increased the percentage of time spent in the open arms in the dlPAG (F(3,27) = 2.967, p = 0.05; Duncan p < 0.05; Fig. 3). The pre-treatment with AM251 (100 pmol/0.2 L) prevented this effect. No effect was found in the number of enclosed arms entries (F(3,27) = 0.156, p = 0.925). Experiment 3: Intra-dlPAG injection of AM630 (10, 100 and 1000 pmol/0.2 L) did not induce any alteration in the percentage of time spent (F(3,17) = 0.715, p = 0.552) and in the number of entries (F(3,17) = 1.31, p = 0.292) in the open arms. Moreover, no significant difference was observed in the number of entries in enclosed arms
(F(3,17) = 0.061, p = 0.979). Since AM630 did not modify the behavioral responses in the EPM, the dose of 1000 pmol was chosen for the subsequent experiment. Experiment 4: The intra-dlPAG injection of AM630 (1000 pmol/0.2 L) attenuated the increase in the percentage of time spent into the open arms caused by 2-AG (50 pmol/0.2 L) (F(3,27) = 2.267, p = 0.103; Duncan test, p < 0.05; Fig. 4). Moreover, no effects were observed in the number of entries in the enclosed arms (F(3,27) = 2.035, p = 0.131). Experiment 5: The MGL (enzyme responsible for the hydrolysis of 2-AG) inhibitor, URB602 (100 pmol/0.2 L), injected intradlPAG significantly increased the percentage of time (F(3,26) = 2.015, p = 0.136, Duncan test p < 0.05) spent in the open arms as compared to controls (Fig. 5). No effect was found in the number of enclosed arms entries (F(3,26) = 0.217, p = 0.883), suggesting that the drugs did not affect basal motor activity. Experiment 6: AM251 (100 pmol/0.2 L) was able to prevent the increase in the percentage of time (F(3,26) = 4.283, p = 0.013; Duncan test, p < 0.05) spent into the open arms promoted by URB602 (100 pmol/0.2 L). The URB 602 significantly increased the number of entries into the open arms (F(3,26) = 5.98, p = 0.003; Duncan
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Fig. 2. Effects of 2-AG (5, 50 or 500 pmol/0.2 L) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage of time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle group (p < 0.05, ANOVA followed by the Duncan test; n = 7–14/group).
test, p < 0.05), and this effect was partially prevented by AM251. No effect was found in the number of enclosed arms entries (F(3,26) = 0.09, p = 0.964; Fig. 6). Experiment 7: AM630 (1000 pmol/0.2 L) blocked the increase in the number of entries (F(3,31) = 4.525, p = 0.009; Duncan test, p < 0.05) into the open arms promoted by URB602 (100 pmol/0.2 L). No effect was observed in the number of enclosed arms entries (F(3,31) = 0.763, p = 0.523; Fig. 7). 4. Discussion In the present study we verified the possible role of 2-AG in the dlPAG in the modulation of anxiety-like behavior. Our results
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Fig. 3. Effects of pre-treatment with AM251 (100 pmol) followed by 2-AG (50 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group (p < 0.05, ANOVA followed by the Duncan test; n = 6–10/group).
showed that intra-dlPAG administration of this endocannabinoid increased the exploration of the open arms in the EPM, indicating an anxiolytic-like effect. These effects were also observed after the inhibition of endogenous 2-AG hydrolysis with the compound URB602. In investigating the mechanisms involved in these responses, we observed that both CB1 and CB2 receptors contribute to the anxiolytic-like effects. The dlPAG is proposed to be part of a neural substrate responsible for the coordination of both nociceptive responses and
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Fig. 4. Effects of pre-treatment with AM630 (1000 pmol) followed by 2-AG (50 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group and # indicates significant difference from vehicle + 2-AG group (50 pmol; p < 0.05, ANOVA followed by the Duncan test; n = 6–9/group).
Fig. 5. Effects of URB602 (30, 100 or 300 pmol/0.2 L) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle (p < 0.05, ANOVA followed by the Duncan test; n = 6–8/group).
anxiety-related behaviors [34–36]. Several studies have shown that AEA and synthetic cannabinoid compounds injected into this region modulate aversion-related responses in animal models [12,22,23,37]. Finn and colleagues showed that local administration of HU210, a synthetic cannabinoid, attenuated the escape responses induced by intra-dorsal PAG injections of the excitatory amino acid d,l-homocysteic acid [37]. Anxiolytic-like effects were observed after local administration of AEA in several models [12,22,23]. Similarly, in the present study we observed that 2-AG induces antiaversive effects in the dlPAG in animals exposed to EPM test. The dose–response curve of 2-AG effects in the EPM was bellshaped, with higher doses being ineffective. This effect resembles the yield complex responses observed with other cannabinoids
in experimental models of anxiety. In line with this, systemic injections of cannabinoid agonists induce anxiolytic-like effect in the EPM test at lower doses, becoming ineffective at higher doses [38,39]. Moreover, local injections of 9 -THC, the main psychoactive substance present in Cannabis sativa, into brain regions responsible for the control of anxiety related behaviors such as amygdala, hippocampus and pre-frontal cortex also exhibited this action profile [40]. Although it is not known the exact mechanisms responsible for these effects, it could be related to the ability of cannabinoids to modulate the release of neurotransmitters such as glutamate and GABA that have opposite actions on anxiety states [1,41].
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Fig. 6. Effects of AM251 (100 pmol) followed by URB602 (100 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group, # indicates significant difference from vehicle + URB602 (100 pmol; p < 0.05, ANOVA followed by the Duncan test; n = 7–8/group).
Recently, Gregg and colleagues [36] showed that the enzyme diacilglicerol lipase-␣ (DGL), responsible for 2-AG synthesis, is expressed in the dlPAG. They also observed that footshock stress elicits the rapid formation of 2-AG in this region, and this effect was blockade by local injection of a drug that selectivity inhibits the DGL [36]. These results suggest that during stressful situations the 2-AG signaling could be recruited in order to inhibit aversive responses. Our study is in agreement with this possibility, since we observed that intra-dlPAG injection of a drug that inhibits the hydrolysis of this endocannabinoid induces anxiolytic-like. In addition, these data corroborate studies demonstrating that systemic injections of other MGL inhibitor, JZL-184, promoted anxiolytic-like
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Fig. 7. Effects of AM630 (1000 pmol) followed by URB602 (100 pmol) administered intra-dlPAG of rats tested in the EPM. The bars represent mean ± S.E.M. of the percentage time spent (upper panel) and entries in the open arms (middle panel) and the number of entries in the closed arms (lower panel). * indicates significant difference from vehicle + vehicle group and # indicates significant difference from vehicle + URB602 (p < 0.05, ANOVA followed by the Duncan test; n = 7–11/group).
effect in EPM and elevated zero maze paradigms [26,42]. Similar effects were reported in the marble-burying test [27]. In order to verify which cannabinoid receptor is involved in the effects of 2-AG, we locally pre-treated the animals with CB1 or CB2 antagonists before injections of 2-AG and URB602 into the dlPAG. Neither antagonist was effective by themselves at the doses employed in this study. Yet, they blocked the anxiolytic-like effects observed by intra-dlPAG injection of 2-AG as well as the hydrolysis inhibitor, suggesting that both CB1 and CB2 receptors are involved in these responses. Several studies have shown that CB1R mediates the behavioral responses induced by cannabinoids agonists [12,21,39,43,44]. Moreover, other studies also described CB1 dependent mechanism for the anxiolytic-like effects induced by systemic injection of 2-AG and a MGL inhibitor in the marble burying test in mice and EPM test in rats [27,28]. Regarding the observation that 2-AG effects in the dlPAG were also dependent on CB2, this result could be related to the fact that
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this endocannabinoid binds both receptors [3,6,45]. Although it has been initially proposed that CB2 receptors are mainly expressed in immune cells [46], recent studies indicate that they are also expressed in the brain and could modulate behavioral responses [47–50]. Accordingly, García-Gutiérrez and Manzanares showed that over-expression of CB2 receptors decreased vulnerability to anxiety [47]. Moreover, the anxiolytic-like effects induced by systemic inhibition of 2-AG hydrolysis in mice were mediated by CB2 but not CB1 receptor activation [26]. In conclusion, the present work showed that facilitation of 2-AG signaling in the dlPAG induces anxiolytic-like effects in rats tested in the EPM test. We also showed that both CB1 and CB2 receptors are involved in these responses. These results, therefore, support the proposal that modulation of the endocannabinoid system in this region is important for aversive reactions, and suggest that drugs that interfere with 2-AG signaling constitute a potential therapeutic strategy for anxiety related disorders.
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Acknowledgments [25]
This research was supported by grants from CAPES, CNPq, FAPESP (2012/17626-7) and FAPEMIG (APQ-01038-11, PRONEM: APQ-04625-10)
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