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Differential role of CB1 and TRPV1 receptors on anandamide modulation of defensive responses induced by nitric oxide in the dorsolateral periaqueductal gray
Neuropharmacology 62 (2012) 2455e2462
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Differential role of CB1 and TRPV1 receptors on anandamide modulation of defensive responses induced by nitric oxide in the dorsolateral periaqueductal gray S.F. Lisboa*, F.S. Guimarães Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, 14049900 Monte Alegre, Ribeirão Preto, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2011 Received in revised form 6 February 2012 Accepted 10 February 2012
CB1, TRPV1 and NO can regulate glutamate release and modify defensive behaviors in regions related to defensive behavior such as the dorsolateral periaqueductal gray (dlPAG). A possible interaction between the endocannabinoid and nitrergic systems in this area, however, has not been investigated yet. The objective of the present work was to verify if activation of CB1 or TRPV1 receptors could interfere in the flight responses induced in rats by the injection of SIN-1, an NO donor, into the dlPAG. The results showed that local administration of a low dose (5 pmol) of anandamide (AEA) attenuated the flight responses, measured by the total distance moved and maximum speed in an open arena, induced by intra-dlPAG microinjection of SIN-1 (150 nmol). URB597 (0.1 nmol), an inhibitor of anandamide metabolism, produced similar effects. When animals were locally treated with the CB1 receptor antagonist AM251 the effective AEA dose (5 pmol) increased, rather than decreased, the flight reactions induced by SIN1-1. Higher (50e200 nmol) doses of AEA were ineffective and even tended to potentiate the SIN-1 effect. The TRPV1 antagonist capsazepine (CPZ, 30 nmol) prevented SIN-1 effects and attenuated the potentiation of its effect by the higher (200 nmol) AEA dose. The results indicate that AEA can modulate in a dual way the pro-aversive effects of NO in the dlPAG by activating CB1 or TRPV1 receptors. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Cannabinoids CB1 TRPV1 Nitric oxide Periaqueductal gray Defensive behavior Flight reaction
1. Introduction The term cannabinoid refers to compounds present in Cannabis sativa (such as cannabidiol and D9-tetrahydrocannabinol-THC), synthetic agents and endogenous substances, named endocannabinoids (ECs). These ECs, which include anandamide (AEA) and 2arachidonoylglycerol (2-AG), are endogenous lipids synthesized from membranes that behave as natural agonists for the cannabinoid receptors (CB) (Battista et al., 2006; Di Marzo and Petrosino, 2007; Maccarrone et al., 2007). Unlike classical neurotransmitters and neuropeptides, ECs are not stored in vesicles. They are synthesized on-demand in post synaptic neurons after neuronal stimulation (Ligresti et al., 2005) and diffuse to presynaptic terminals, where they can activate CB receptors type 1 (CB1) or 2 (CB2) and decrease the release of neurotransmitters such as glutamate and GABA (De Petrocellis et al., 2004; Wilson and Nicoll, 2002). AEA can also target
* Corresponding author. Tel.: þ55 16 36023209; fax: þ55 16 36332301. E-mail address: [email protected] (S.F. Lisboa). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.02.008
transient receptor potential vanilloid type-1 channel (TRPV1), an ion channel permeable to calcium that, contrary to CB1, could facilitate glutamate release (Xing and Li, 2007). AEA action is limited by its reuptake by an AEA transporter in the post synaptic neuron and subsequently degradation by the enzyme fatty acid amide hydrolase (FAAH) (Beltramo et al., 1997; Di Marzo et al., 1994; Piomelli et al., 1999). CB and TRPV1 receptors, AEA transporter and FAAH are distributed in several regions of the central nervous system related to defensive behaviors (Egertova et al., 2003; Giuffrida et al., 2001; Herkenham et al., 1991; Toth et al., 2005), such as the prefrontal cortex, amygdala, hippocampus, hypothalamus and periaqueductal gray (PAG) (Bandler et al., 2000). This localization suggests that cannabinoids are involved in the modulation of these behaviors (Moreira et al., 2011). The PAG is a mesencephalic brain structure divided into four longitudinal columns: dorsomedial, dorsolateral (dl), lateral and ventrolateral. This structure is proposed to integrate a neural substrate responsible for the coordination of nociceptive, cardiovascular, respiratory and defensive-related responses (Bandler et al., 2000). CB1 and TRPV1 receptors, the AEA transporter and FAAH enzyme are all expressed in the PAG (Casarotto et al., 2011;
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Cavanaugh et al., 2011; Cristino et al., 2006; Egertova et al., 2003; Giuffrida et al., 2001; Herkenham et al., 1991, 1990), suggesting that this structure could mediate some cannabinoid effects (Finn et al., 2003; Lisboa et al., 2008; Martin et al., 1995; Moreira et al., 2007; Resstel et al., 2008b). Accordingly, injections of CB1 receptor agonists into the dorsal PAG induce both anti-nociceptive and antiaversive effects in rats (Finn et al., 2003; Martin et al., 1995) and electrical stimulation of this structure causes release of AEA and anti-nociception via local CB1 receptor activation (Walker et al., 1999). In addition, cannabinoids released in the dlPAG by stressful events may contribute to stress-induced analgesia (Hohmann et al., 2005). Activation of CB1 receptors in the dlPAG of rats can induce anxiolytic-like effects in innate and learned behavioral models (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b). These effects are probably due to reducing glutamate release, since CB1 receptors are coupled to a Gi/o protein (Howlett and Fleming, 1984; Wilson and Nicoll, 2002). Glutamate release in several brain regions is related to stress- and anxiety-related behaviors (Musazzi et al., 2011; Riaza Bermudo-Soriano et al., 2011). In the dlPAG, activation of NMDA receptors induces flight reactions (Aguiar et al., 2006) whereas inhibition of these receptors is anxiolytic (Aguiar and Guimaraes, 2009; Guimaraes et al., 1991; Molchanov and Guimaraes, 2002). Glutamate, by acting on NMDA receptors and increasing Caþþ influx, can activate the neuronal nitric oxide synthase (nNOS) enzyme and increase NO production (Contestabile, 2000). nNOS is highly expressed in the dlPAG (Onstott et al., 1993) and a decrease in NO formation by nNOS inhibitors (Aguiar and Guimaraes, 2009; Guimaraes et al., 1994), NO scavenging (Guimaraes et al., 2005) or antagonism of the main NO cellular target, the enzyme soluble guanylate cyclase (sGC) (de-Oliveira and Guimaraes, 1999), in this region induces decrease in anxiety-like behavior. In an opposite way, flight reactions are induced by administration of nitric oxide donors such as NOC-9 and SIN-1 in the dlPAG (Braga et al., 2009; de Oliveira et al., 2000; Guimaraes et al., 2005). These latter manifestations are similar to those seen to innate threatening stimuli (Bandler and Carrive, 1988; Bittencourt et al., 2004) and have been related to panic attacks (Deakin and Graeff, 1991; Schenberg et al., 2001). Although CB1, TRPV1 and NO can regulate glutamate release and modify defensive behaviors in the dlPAG, no study so far has investigated a possible interaction between the endocannabinoid and nitrergic systems in this area. So, the main objective of the present work was to verify if activation of CB1 or TRPV1 receptors could interfere in the flight responses induced by the injection of SIN-1, an NO donor, into the dlPAG.
1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole-3-carboxa mide (AM251, CB1 antagonist, Tocris) 100 pmol and Capsazepine (CPZ, Tocris) 10 or 30 nmol. SIN-1 was dissolved in saline (0.9% NaCl) and AEA in TocrisolveTM 100 (the formulation is composed of a 1:4 ratio of soya oil/water emulsified with the block co-polymer Pluronic F68). AM251 and URB597 were dissolved in DMSO 10% in saline (0.9% NaCl). Capsazepine was dissolved in DMSO 100%. The solutions were prepared immediately before use and were kept on ice and protected from the light during the experimental session. The doses were chosen based on previous studies that investigated the effects of these drugs after microinjection into the dorsal or dlPAG (de Oliveira et al., 2000; Guimaraes et al., 1991; Lisboa et al., 2007; Moreira et al., 2007; Terzian et al., 2009).
2. Materials and methods
2.6. Histology
2.1. Subjects
After the experiments the rats were anesthetized with urethane (1.25 g/kg, i.p.). Their chests were surgically opened, the descending aortas occluded, the right atrium severed and the brains perfused with 10% formalin through the left ventricle. The brains were post fixed in 10% formalin for 24 h at 4 C, and 40 mm sections were cut with the help of a cryostat (CM 1900, Leica, Germany). Brain sections were stained with 1% neutral red. The placement of the injection needles was identified with the help of the rat brain atlas of (Paxinos and Watson, 1997). The injection sites and a representative photomicrography can be seen in Fig. 1. Rats that received injections outside the aimed area were excluded from analysis.
Male Wistar rats weighing 230e250 g were provided by our local Animal farm facility (Central Animal House facility of University of São Paulo, Ribeirão Preto). The animals were housed in groups of four in a temperature-controlled room (24 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.). Procedures were conducted in conformity with the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals, which are in compliance with international laws and policies. All efforts were made to minimize animal suffering and the experimental protocols were approved by the local Ethical Committee.
2.3. Apparatus The experiments were carried out in a circular open arena (72 cm in diameter with a 50 cm high Plexiglas wall) located in a sound attenuated, temperaturecontrolled (24 C) and 40 lx illuminated room. The rats were videotaped inside the arena and their behavior analyzed with the help of the AnyMaze software (version 4.7, Stoelting). This software detects the position of the animal in the open arena and calculates the distance moved and speed.
2.4. Surgery Rats were anesthetized with 2.5% 2,2,2-tribromoethanol (10 mg/kg, IP) and immobilized in a stereotaxic frame. A stainless steel guide cannula (0.7 mm OD) was implanted unilaterally on the right side aimed at the dlPAG (coordinates: AP ¼ 0 from lambda, L ¼ 1.9 mm at an angle of 16 , D ¼ 4.3 mm). The cannula was attached to the bone with stainless steel screws and acrylic cement. An obturator inside the guide cannula prevented obstruction.
2.5. Procedure 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 200 nL was injected in 30 s using a microsyringe (Hamilton, USA) connected to an infusion pump (Kd Scientific, USA). A polyethylene catheter (PE10) was interposed between the upper end of the dental needle and the microsyringe. In the first set of experiments, animals received a first microinjection into the dlPAG of vehicle or AM251 (100 pmol), followed, 5 min later, by vehicle, AEA (5 pmol) or URB597 (0.01 or 0.1 nmol). 5 min after the second microinjection the animals received a last intra-dlPAG administration of vehicle or SIN-1. In the second set, animals received a first microinjection into the dlPAG of vehicle or AEA (50, 100 or 200 pmol) followed, 5 min later, by vehicle or SIN-1. In the third set, animals received a first microinjection into the dlPAG of vehicle or CPZ (10 or 30 nmol), followed, 5 min later, by vehicle or AEA (200 pmol). 5 min after the second microinjection the animals received a last intra-dlPAG of vehicle or SIN-1. In the last set of experiments all animals received microinjections of saline followed by URB597 (0.01 or 0.1 pmol) or vehicle. Similar to the previous experiment, 5 min later they received a last intra-dlPAG of vehicle or SIN-1.Immediately after the last injection the animals were placed inside the open arena. The distance moved and maximum speed were registered by the AnyMaze software during 10 min.
2.7. Statistical analysis 2.2. Drugs The following drugs were used: 3-morpholinosylnomine hydrochloride (SIN-1, nitric oxide donor; TOCRIS) 150 nmol, Anandamide (AEA, endocannabinoid; Tocris) 5, 50 or 200 pmol, cyclohexyl carbamic acid 30 -carbamoyl-biphenyl-3-yl Ester (URB597, inhibitor of FAAH enzyme; Calbiochem) 0.1 and 0.01 nmol, N-(piperidin-
Results were analyzed by two- or three-way ANOVAs, depending on the treatments being compared (two or three injections). One-way ANOVA followed by the Duncan test were performed for post hoc comparisons. A log transformation was used to achieve homogeneity of variance when necessary. Differences were considered significant at p < 0.05 level.
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Fig. 1. Photomicrograph (A) and histological localization (B) of injection sites located in the dorsolateral periaqueductal gray (black circles) in diagrams based on the atlas of Paxinos and Watson (2006). Due to overlap, the number of points represented is fewer than the real number of rats used in the experiments. The gray circles represent the injections outside the PAG.
3. Results 3.1. Experiment I: a low dose of AEA (5 pmol) attenuated flight reactions induced by SIN-1 administration into the dlPAG via CB1 receptors As previously described (De Oliveira et al., 2001), intra-dlPAG injection of SIN-1 induced flight responses characterized by coordinated running and jumps with escape attempts. This was reflected by an increased total distance traveled (F1,49 ¼ 45.1,
p < 0.001, Fig. 2B) and maximum speed (F1,50 ¼ 13.3, p < 0.005, Fig. 2A). There were significant interactions between treatments (distance traveled, F1,50 ¼ 4.53, p<0.05; maximum speed, F1,49 ¼ 10.11, p<0.005). Post hoc analysis showed that AEA 5 pmol was able to attenuate the effects of SIN-1 in these two variables (Duncan test, p < 0.05, Fig. 2A and B). This effect, however, was prevented by pretreatment with AM251 (p > 0.05, Fig. 2A and B). Moreover, in the presence of AM251, AEA increased rather than decrease the effects of SIN-1 on distance traveled and maximum speed (Fig. 2A and B, Duncan test, p < 0.05).
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Fig. 2. Effects of anandamide (AEA, 5 pmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol) and potentiation of SIN-1 effect by AEA in the presence of AM251 (100 pmol). Data expressed as means SEM. N ¼ 7,5,9,7,7,7,7,9. * indicates significant difference from all other groups that received the same treatment as first injection; þ indicates difference from Veh-Veh and AEA-Veh groups or Veh-Veh-Veh and AM251-AEA-Veh groups (p < 0.05, ANOVA followed by the Duncan test).
3.2. Experiment II: potentiation of endogenous AEA also attenuated flight reactions induced by SIN-1 administration into the dlPAG Confirming the previous results, SIN-1 increased maximum speed (F1,53 ¼ 29.7, p < 0.001, Fig. 3A) and distance traveled (F1,53 ¼ 5.04, p < 0.05, Fig. 3B) in the open arena. The latter effect was prevented by the higher dose (0.1 nmol) of URB597, an inhibitor of AEA metabolism. 3.3. Experiment III: higher doses of AEA did not change the flight reactions induced by SIN-1 In experiment I we showed that pretreatment with the CB1 receptor antagonist before the administration of AEA 5 pmol into the dlPAG enhanced SIN-1 effect. Considering that AEA, usually at higher doses, can also activate TRPV1 receptors and increase glutamate release by dlPAG neurons (Xing and Li, 2007), we performed this experiment to verify the effects of higher doses of AEA on SIN-1 induced flight responses. Results can be seen in Fig. 4A and B. They showed again that intra-dlPAG administration of SIN-1 increased maximum speed (F1,47 ¼ 5.7, p < 0.001) and distance traveled (F1,47 ¼ 58.7, p < 0.001). This effect, however, was not
Fig. 3. Effects of URB597 (URB, 0.01 and 0.1 nmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 11,10,5,15,13,5. * indicates significant difference from all other groups, but not from Veh-URB0.01-SIN-1 group; þ indicates difference from Veh-Veh group (p < 0.05, ANOVA followed by the Duncan test).
prevented by any of the higher doses (50e200 pmol) of AEA. The higher dose of this compound also tended to potentiate SIN-1 effects (Fig. 4A and B), but the differences did not reach statistical significance. 3.4. Experiment IV: TRPV1 receptors are involved in the effects of higher AEA doses on SIN-1 induced flight reactions Although the 10 nmol of the TRPV1 receptor antagonist capsazepine (n ¼ 6e10/group) failed to change the effects of AEA200 þ SIN-1 (data not shown), the higher dose of this antagonist (30 nmol) prevented the effects of SIN-1 on total distance moved (F1,53 ¼ 4.66, p < 0.05, Duncan, p < 0.05, Fig. 5B) and maximum speed (F1,53 ¼ 10.6, p < 0.005, Fig. 5A). This drug was also able to attenuate the potentiation of SIN-1 effects by AEA (200 pmol) observed in total distance traveled (Duncan test, p < 0.05, Fig. 5B). 4. Discussion The present work confirmed previous studies showing that NO donors injected into the dlPAG induce flight responses (Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005; Moreira et al., 2004). The dlPAG, together with the medial hypothalamus and the
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Fig. 4. Lack of effects of higher doses of anandamide (AEA, 50e200 pmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 8,7,5,6,6,7,8,8. * indicates main significant difference between Veh and SIN-1 treated animals (p < 0.05, ANOVA).
amygdala, is part of a system that has been traditionally related to active defensive responses (Graeff, 1994). Stimulation of this brain region in rodents results in responses characterized by galloping, jumping and episodes of wild running similar to those displayed by animals confronting a proximal innate fear stimulus such as a predator (Ambalavanan et al., 1999; Krieger and Graeff, 1985; Schenberg et al., 2001). These responses are called flight reactions and can be induced electrically or by excitatory chemical compounds, such as glutamate agonists, administered into the dlPAG (Aguiar et al., 2006; Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005). Since stimulation of the PAG in neurosurgery patients induces feelings of fear and despair similarly to those described in panic disorder (Nashold et al., 1969) these flight reactions have been related to panic attacks in humans (Deakin and Graeff, 1991; Jenck et al., 1995; Schenberg et al., 2001). NOS inhibitors, NO scavengers and sGC inhibitors injected into the dlPAG cause anxiolytic-like effects in animal models (Aguiar and Guimaraes, 2009; de-Oliveira and Guimaraes, 1999; Guimaraes et al., 2005, 1991, 1994; Molchanov and Guimaraes, 2002; Tonetto et al., 2009) whereas NO donors induce flight reactions (Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005). These latter responses are prevented by local pretreatment with NMDA and non-NMDA glutamate receptor antagonists (Moreira et al., 2004), suggesting, as shown elsewhere (Contestabile, 2000), that the effects of NO donors in the dlPAG are mediated by facilitation of glutamate release. CB1 receptors are largely distributed in the PAG (Herkenham et al., 1990). They inhibit the adenilate cyclase enzyme, decreasing intracellular levels of the second messenger AMPc and
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Fig. 5. Attenuation by capsazepine 30 nmol (CPZ30) of the effects of anandamide (AEA, 200 pmol) on distance traveled (B), but not maximum speed (A), displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 10,8,10,7,5,5,6,6. * indicates significant difference from all other groups (p < 0.05, ANOVA followed by the Duncan test).
consequently decreasing the release of several neurotransmitters, including glutamate (Howlett and Fleming, 1984; Wilson and Nicoll, 2002). Activation of these receptors in the dlPAG induces anxiolytic-like effects (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b) similar to those seen with intra-dlPAG administration of glutamate ionotropic receptor antagonists (Guimaraes et al., 2005, 1991; Molchanov and Guimaraes, 2002). Based on these findings, we hypothesized that the nitrergic and cannabinoid systems could play opposite roles in the regulation of defensive responses in the dlPAG, probably by modulating glutamate release. Accordingly, a low dose of AEA (5 pmol) and the FAAH inhibitor URB597 (0.1 nmol) were able to attenuate the flight responses, measured by the total distance traveled in the open arena and, in the former case, also by the maximum speed, induced by the NO donor SIN-1 administered into the dlPAG. AM251, an antagonist of CB1 receptors, prevented the effects of AEA, indicating that they were being mediated by these receptors. The dose of AM251 used was similar to that able to block the anxiolytic effects of AEA injected into the dlPAG (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b). The antagonist by itself, however, failed to increase the flight responses induced by SIN-1. Although this might indicate that endogenous cannabinoids are not important to modulate these responses, a ceiling effect could also be involved. Actually, the results with URB597, which inhibits AEA metabolism, suggest that enhancement of endogenous cannabinoid levels can, indeed, attenuate flight reactions induced by an NO donor.
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An interaction between the nitrergic and cannabinoid system has also been suggested in other brain areas (Azad et al., 2001; Nasehi et al., 2010; Roohbakhsh et al., 2007). Animals that lack the CB1 gene present an increase in NOS activity in the cerebral cortex, suggesting that CB1 is required to inhibit this activity (Kim et al., 2006). Also, several in vitro studies (Carney et al., 2009; Jones et al., 2008; Maccarrone et al., 2001, 2000a, 2006; Signorello et al., 2010; Waksman et al., 1999) suggest a functional interaction between the cannabinoid and nitrergic systems. The present study provides additional evidence, now in the dlPAG, that these two systems could interact in the central nervous system. AEA can activate both CB1 and TRPV1 receptors, which results in inhibition and facilitation, respectively, of glutamate release (Van Der Stelt and Di Marzo, 2004). These two actions would produce opposite effects on defensive responses. Since the intrinsic efficacy of AEA at TRPV1 is relatively low compared to that observed for this compound at CB1 receptors (Pertwee and Ross, 2002; Ross, 2003), this double mechanism could be involved in the lack of effect observed with the higher doses of AEA in the present work and in the bell-shape dose-response curves commonly observed with cannabinoids, including when administered in the dlPAG (Campos and Guimaraes, 2009; Guimaraes et al., 1990; Moreira et al., 2007). Moreover, in the presence of the CB1 receptor antagonist AM251, the low dose of AEA potentiated, instead of decreasing, the flight responses induced by SIN-1. This agrees with the recent suggestion that in the dlPAG TRPV1 and CB1 receptors might be activated simultaneously at a given synapse to modulate panic-like responses (Casarotto et al., 2011). AEA has shown dual effects on several systems. For example, at low concentration it inhibits, via CB1 receptors, electrically stimulated neuropeptide release from cultured dorsal root ganglia neurons (Ross et al., 2001) while, at higher concentrations, evokes a TRPV1-mediated release (Tognetto et al., 2001). Furthermore, in the presence of a CB1 receptor antagonist, AEA becomes equipotent with capsaicin as a TRPV1 receptor agonist to induce neuropeptide release (Ahluwalia et al., 2003). It was also shown in guinea-pig ileum that AEA increases acetylcholine release in a capsazepinesensitive manner, an effect that was enhanced by the CB1 receptor antagonist, SR141716A (Mang et al., 2001). Similarly, in human neuroblastoma cells, CB1 receptor antagonists potentiate TRPV1-mediated cell death induced by AEA (Maccarrone et al., 2000b). Finally, Kawahara and colleagues (Kawahara et al., 2011) showed that inhibition of the FAAH enzyme unmasks AEA-induced presynaptic inhibition and excitation of glutamatergic synaptic transmission in this region by activating CB1 and TRPV1 receptors, respectively. In the PAG activation of presynaptic TRPV1 receptor has been shown to increase glutamate release (Van Der Stelt and Di Marzo, 2004). Early electrophysiological data showed that capsaicin application to slices of hypothalamus, substantia nigra, locus coeruleus or PAG evokes glutamate release (Liao et al., 2011; Marinelli et al., 2003, 2002; Sasamura et al., 1998) mediated by TRPV1 activation, since the effect was blocked by TRPV1 antagonists. These results suggest that TRPV1 are expressed presynaptically in glutamatergic terminals and that their activation potentiate the release of this neurotransmitter. In agreement with this proposal, it was showed that capsaicin infusion into the dorsal PAG increases glutamatergic synaptic transmission, which was abrogated by TRPV1 antagonist (Xing and Li, 2007). Also, genetic deletion of the TRPV1 gene prevents capsaicin-induced presynaptic excitation in striatum, reinforcing the hypothesis that activation of presynaptic TRPV1 induces glutamate release (Musella et al., 2009). This mechanism could explain the similar anxiolytic effects induced by blockade of NMDA or TRPV1 receptors observed in several animal models (Aguiar and Guimaraes, 2011; Guimaraes
et al., 1991; Molchanov and Guimaraes, 2002; Resstel et al., 2008a; Tonetto et al., 2009). Several pieces of evidence have also suggested the presence of post-synaptic TRPV1 receptors (Chavez et al., 2010; Cristino et al., 2006; Grueter et al., 2010; Micale et al., 2009; Starowicz et al., 2007; Zschenderlein et al., 2011), modulating synaptic inhibition and excitation. For example, a recent study by Zschenderlein and colleagues (Zschenderlein et al., 2011) showed that in the lateral amygdala, after high frequency stimulation, capsaicin activates post-synaptic TRPV1 receptors. At low concentration TRPV1 activation induces the synthesis of AEA, which in turn activates CB1, reduces glutamate release and NOS activation, inhibiting long-term potentiation (LTP). On the other hand, higher levels of AEA, by enhancing intracellular calcium, could activate post-synaptic neuronal NOS, leading to an increase in glutamate release and enhancement of LTP. It remains to be verified if similar mechanisms occur in the PAG. The present data, in addition, indicate the presence of a complex interaction between NO and TRPV1 receptors in this region. Systemic administration of capsaicin has been shown to increase NO synthesis in brain regions related to defensive behavior, such as paraventricular nucleus of hypothalamus, medial amygdala and also the dlPAG (Okere et al., 2000a, 2000b; Okere and Waterhouse, 2006), suggesting that activation of TRPV1 receptors engage a glutamate/NMDA/NO pathway that could be facilitating aversive responses. Our results indicated that blockade of these receptors prevents the effects of exogenous NO, suggesting that this neurotransmitter could be exerting its effect by facilitating a positive feed-back loop involving glutamate and endovanilloids. Further studies are needed to confirm this possibility and investigate its pharmacological mechanisms. In conclusion, the present study showed that a low dose of AEA activates CB1 receptors and attenuate the flight reaction induced by SIN-1, while higher doses of AEA are ineffective, probably by also activating TRPV1 receptors. The antagonism of CB1 receptors in the presence of the low dose of AEA resulted in potentiation of SIN-1 effects, while blockade of TRPV1 receptors previous to the higher dose of AEA unmasked the attenuating effect of AEA. The results indicate, therefore, that AEA can modulate in a dual way the proaversive effects of NO in the dlPAG by activating CB1 or TRPV1 receptors. Acknowledgments The authors thank Eleni T. Gomes and José C. de Aguiar (University of São Paulo) for the technical assistance. The research was supported by a grant from FAPESP (2007/03685-3) SFL and FSG are recipients of FAPESP and CNPq fellowships, respectively. References Aguiar, D.C., Guimaraes, F.S., 2009. Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator. J. Neurosci. Res. 87, 2418e2429. Aguiar, D.C., Guimaraes, F.S., 2011. Blockade of NMDA or NO in the dorsal premammillary nucleus attenuates defensive behaviors. Physiol. Behav. 103, 279e283. Aguiar, D.C., Moreira, F.A., Guimaraes, F.S., 2006. Flight reactions induced by injection of glutamate N-methyl-d-aspartate receptor agonist into the rat dorsolateral periaqueductal gray are not dependent on endogenous nitric oxide. Pharmacol. Biochem. Behav. 83, 296e301. Ahluwalia, J., Urban, L., Bevan, S., Nagy, I., 2003. Anandamide regulates neuropeptide release from capsaicin-sensitive primary sensory neurons by activating both the cannabinoid 1 receptor and the vanilloid receptor 1 in vitro. Eur. J. Neurosci. 17, 2611e2618. Ambalavanan, N., Mariani, G., Bulger, A., Philips, I.J., 1999. Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation. Biol. Neonate 76, 291e300.
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Differential role of CB1 and TRPV1 receptors on anandamide modulation of defensive responses induced by nitric oxide in the dorsolateral periaqueductal gray S.F. Lisboa*, F.S. Guimarães Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, 14049900 Monte Alegre, Ribeirão Preto, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2011 Received in revised form 6 February 2012 Accepted 10 February 2012
CB1, TRPV1 and NO can regulate glutamate release and modify defensive behaviors in regions related to defensive behavior such as the dorsolateral periaqueductal gray (dlPAG). A possible interaction between the endocannabinoid and nitrergic systems in this area, however, has not been investigated yet. The objective of the present work was to verify if activation of CB1 or TRPV1 receptors could interfere in the flight responses induced in rats by the injection of SIN-1, an NO donor, into the dlPAG. The results showed that local administration of a low dose (5 pmol) of anandamide (AEA) attenuated the flight responses, measured by the total distance moved and maximum speed in an open arena, induced by intra-dlPAG microinjection of SIN-1 (150 nmol). URB597 (0.1 nmol), an inhibitor of anandamide metabolism, produced similar effects. When animals were locally treated with the CB1 receptor antagonist AM251 the effective AEA dose (5 pmol) increased, rather than decreased, the flight reactions induced by SIN1-1. Higher (50e200 nmol) doses of AEA were ineffective and even tended to potentiate the SIN-1 effect. The TRPV1 antagonist capsazepine (CPZ, 30 nmol) prevented SIN-1 effects and attenuated the potentiation of its effect by the higher (200 nmol) AEA dose. The results indicate that AEA can modulate in a dual way the pro-aversive effects of NO in the dlPAG by activating CB1 or TRPV1 receptors. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Cannabinoids CB1 TRPV1 Nitric oxide Periaqueductal gray Defensive behavior Flight reaction
1. Introduction The term cannabinoid refers to compounds present in Cannabis sativa (such as cannabidiol and D9-tetrahydrocannabinol-THC), synthetic agents and endogenous substances, named endocannabinoids (ECs). These ECs, which include anandamide (AEA) and 2arachidonoylglycerol (2-AG), are endogenous lipids synthesized from membranes that behave as natural agonists for the cannabinoid receptors (CB) (Battista et al., 2006; Di Marzo and Petrosino, 2007; Maccarrone et al., 2007). Unlike classical neurotransmitters and neuropeptides, ECs are not stored in vesicles. They are synthesized on-demand in post synaptic neurons after neuronal stimulation (Ligresti et al., 2005) and diffuse to presynaptic terminals, where they can activate CB receptors type 1 (CB1) or 2 (CB2) and decrease the release of neurotransmitters such as glutamate and GABA (De Petrocellis et al., 2004; Wilson and Nicoll, 2002). AEA can also target
* Corresponding author. Tel.: þ55 16 36023209; fax: þ55 16 36332301. E-mail address: [email protected] (S.F. Lisboa). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2012.02.008
transient receptor potential vanilloid type-1 channel (TRPV1), an ion channel permeable to calcium that, contrary to CB1, could facilitate glutamate release (Xing and Li, 2007). AEA action is limited by its reuptake by an AEA transporter in the post synaptic neuron and subsequently degradation by the enzyme fatty acid amide hydrolase (FAAH) (Beltramo et al., 1997; Di Marzo et al., 1994; Piomelli et al., 1999). CB and TRPV1 receptors, AEA transporter and FAAH are distributed in several regions of the central nervous system related to defensive behaviors (Egertova et al., 2003; Giuffrida et al., 2001; Herkenham et al., 1991; Toth et al., 2005), such as the prefrontal cortex, amygdala, hippocampus, hypothalamus and periaqueductal gray (PAG) (Bandler et al., 2000). This localization suggests that cannabinoids are involved in the modulation of these behaviors (Moreira et al., 2011). The PAG is a mesencephalic brain structure divided into four longitudinal columns: dorsomedial, dorsolateral (dl), lateral and ventrolateral. This structure is proposed to integrate a neural substrate responsible for the coordination of nociceptive, cardiovascular, respiratory and defensive-related responses (Bandler et al., 2000). CB1 and TRPV1 receptors, the AEA transporter and FAAH enzyme are all expressed in the PAG (Casarotto et al., 2011;
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Cavanaugh et al., 2011; Cristino et al., 2006; Egertova et al., 2003; Giuffrida et al., 2001; Herkenham et al., 1991, 1990), suggesting that this structure could mediate some cannabinoid effects (Finn et al., 2003; Lisboa et al., 2008; Martin et al., 1995; Moreira et al., 2007; Resstel et al., 2008b). Accordingly, injections of CB1 receptor agonists into the dorsal PAG induce both anti-nociceptive and antiaversive effects in rats (Finn et al., 2003; Martin et al., 1995) and electrical stimulation of this structure causes release of AEA and anti-nociception via local CB1 receptor activation (Walker et al., 1999). In addition, cannabinoids released in the dlPAG by stressful events may contribute to stress-induced analgesia (Hohmann et al., 2005). Activation of CB1 receptors in the dlPAG of rats can induce anxiolytic-like effects in innate and learned behavioral models (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b). These effects are probably due to reducing glutamate release, since CB1 receptors are coupled to a Gi/o protein (Howlett and Fleming, 1984; Wilson and Nicoll, 2002). Glutamate release in several brain regions is related to stress- and anxiety-related behaviors (Musazzi et al., 2011; Riaza Bermudo-Soriano et al., 2011). In the dlPAG, activation of NMDA receptors induces flight reactions (Aguiar et al., 2006) whereas inhibition of these receptors is anxiolytic (Aguiar and Guimaraes, 2009; Guimaraes et al., 1991; Molchanov and Guimaraes, 2002). Glutamate, by acting on NMDA receptors and increasing Caþþ influx, can activate the neuronal nitric oxide synthase (nNOS) enzyme and increase NO production (Contestabile, 2000). nNOS is highly expressed in the dlPAG (Onstott et al., 1993) and a decrease in NO formation by nNOS inhibitors (Aguiar and Guimaraes, 2009; Guimaraes et al., 1994), NO scavenging (Guimaraes et al., 2005) or antagonism of the main NO cellular target, the enzyme soluble guanylate cyclase (sGC) (de-Oliveira and Guimaraes, 1999), in this region induces decrease in anxiety-like behavior. In an opposite way, flight reactions are induced by administration of nitric oxide donors such as NOC-9 and SIN-1 in the dlPAG (Braga et al., 2009; de Oliveira et al., 2000; Guimaraes et al., 2005). These latter manifestations are similar to those seen to innate threatening stimuli (Bandler and Carrive, 1988; Bittencourt et al., 2004) and have been related to panic attacks (Deakin and Graeff, 1991; Schenberg et al., 2001). Although CB1, TRPV1 and NO can regulate glutamate release and modify defensive behaviors in the dlPAG, no study so far has investigated a possible interaction between the endocannabinoid and nitrergic systems in this area. So, the main objective of the present work was to verify if activation of CB1 or TRPV1 receptors could interfere in the flight responses induced by the injection of SIN-1, an NO donor, into the dlPAG.
1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole-3-carboxa mide (AM251, CB1 antagonist, Tocris) 100 pmol and Capsazepine (CPZ, Tocris) 10 or 30 nmol. SIN-1 was dissolved in saline (0.9% NaCl) and AEA in TocrisolveTM 100 (the formulation is composed of a 1:4 ratio of soya oil/water emulsified with the block co-polymer Pluronic F68). AM251 and URB597 were dissolved in DMSO 10% in saline (0.9% NaCl). Capsazepine was dissolved in DMSO 100%. The solutions were prepared immediately before use and were kept on ice and protected from the light during the experimental session. The doses were chosen based on previous studies that investigated the effects of these drugs after microinjection into the dorsal or dlPAG (de Oliveira et al., 2000; Guimaraes et al., 1991; Lisboa et al., 2007; Moreira et al., 2007; Terzian et al., 2009).
2. Materials and methods
2.6. Histology
2.1. Subjects
After the experiments the rats were anesthetized with urethane (1.25 g/kg, i.p.). Their chests were surgically opened, the descending aortas occluded, the right atrium severed and the brains perfused with 10% formalin through the left ventricle. The brains were post fixed in 10% formalin for 24 h at 4 C, and 40 mm sections were cut with the help of a cryostat (CM 1900, Leica, Germany). Brain sections were stained with 1% neutral red. The placement of the injection needles was identified with the help of the rat brain atlas of (Paxinos and Watson, 1997). The injection sites and a representative photomicrography can be seen in Fig. 1. Rats that received injections outside the aimed area were excluded from analysis.
Male Wistar rats weighing 230e250 g were provided by our local Animal farm facility (Central Animal House facility of University of São Paulo, Ribeirão Preto). The animals were housed in groups of four in a temperature-controlled room (24 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.). Procedures were conducted in conformity with the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals, which are in compliance with international laws and policies. All efforts were made to minimize animal suffering and the experimental protocols were approved by the local Ethical Committee.
2.3. Apparatus The experiments were carried out in a circular open arena (72 cm in diameter with a 50 cm high Plexiglas wall) located in a sound attenuated, temperaturecontrolled (24 C) and 40 lx illuminated room. The rats were videotaped inside the arena and their behavior analyzed with the help of the AnyMaze software (version 4.7, Stoelting). This software detects the position of the animal in the open arena and calculates the distance moved and speed.
2.4. Surgery Rats were anesthetized with 2.5% 2,2,2-tribromoethanol (10 mg/kg, IP) and immobilized in a stereotaxic frame. A stainless steel guide cannula (0.7 mm OD) was implanted unilaterally on the right side aimed at the dlPAG (coordinates: AP ¼ 0 from lambda, L ¼ 1.9 mm at an angle of 16 , D ¼ 4.3 mm). The cannula was attached to the bone with stainless steel screws and acrylic cement. An obturator inside the guide cannula prevented obstruction.
2.5. Procedure 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 200 nL was injected in 30 s using a microsyringe (Hamilton, USA) connected to an infusion pump (Kd Scientific, USA). A polyethylene catheter (PE10) was interposed between the upper end of the dental needle and the microsyringe. In the first set of experiments, animals received a first microinjection into the dlPAG of vehicle or AM251 (100 pmol), followed, 5 min later, by vehicle, AEA (5 pmol) or URB597 (0.01 or 0.1 nmol). 5 min after the second microinjection the animals received a last intra-dlPAG administration of vehicle or SIN-1. In the second set, animals received a first microinjection into the dlPAG of vehicle or AEA (50, 100 or 200 pmol) followed, 5 min later, by vehicle or SIN-1. In the third set, animals received a first microinjection into the dlPAG of vehicle or CPZ (10 or 30 nmol), followed, 5 min later, by vehicle or AEA (200 pmol). 5 min after the second microinjection the animals received a last intra-dlPAG of vehicle or SIN-1. In the last set of experiments all animals received microinjections of saline followed by URB597 (0.01 or 0.1 pmol) or vehicle. Similar to the previous experiment, 5 min later they received a last intra-dlPAG of vehicle or SIN-1.Immediately after the last injection the animals were placed inside the open arena. The distance moved and maximum speed were registered by the AnyMaze software during 10 min.
2.7. Statistical analysis 2.2. Drugs The following drugs were used: 3-morpholinosylnomine hydrochloride (SIN-1, nitric oxide donor; TOCRIS) 150 nmol, Anandamide (AEA, endocannabinoid; Tocris) 5, 50 or 200 pmol, cyclohexyl carbamic acid 30 -carbamoyl-biphenyl-3-yl Ester (URB597, inhibitor of FAAH enzyme; Calbiochem) 0.1 and 0.01 nmol, N-(piperidin-
Results were analyzed by two- or three-way ANOVAs, depending on the treatments being compared (two or three injections). One-way ANOVA followed by the Duncan test were performed for post hoc comparisons. A log transformation was used to achieve homogeneity of variance when necessary. Differences were considered significant at p < 0.05 level.
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Fig. 1. Photomicrograph (A) and histological localization (B) of injection sites located in the dorsolateral periaqueductal gray (black circles) in diagrams based on the atlas of Paxinos and Watson (2006). Due to overlap, the number of points represented is fewer than the real number of rats used in the experiments. The gray circles represent the injections outside the PAG.
3. Results 3.1. Experiment I: a low dose of AEA (5 pmol) attenuated flight reactions induced by SIN-1 administration into the dlPAG via CB1 receptors As previously described (De Oliveira et al., 2001), intra-dlPAG injection of SIN-1 induced flight responses characterized by coordinated running and jumps with escape attempts. This was reflected by an increased total distance traveled (F1,49 ¼ 45.1,
p < 0.001, Fig. 2B) and maximum speed (F1,50 ¼ 13.3, p < 0.005, Fig. 2A). There were significant interactions between treatments (distance traveled, F1,50 ¼ 4.53, p<0.05; maximum speed, F1,49 ¼ 10.11, p<0.005). Post hoc analysis showed that AEA 5 pmol was able to attenuate the effects of SIN-1 in these two variables (Duncan test, p < 0.05, Fig. 2A and B). This effect, however, was prevented by pretreatment with AM251 (p > 0.05, Fig. 2A and B). Moreover, in the presence of AM251, AEA increased rather than decrease the effects of SIN-1 on distance traveled and maximum speed (Fig. 2A and B, Duncan test, p < 0.05).
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Fig. 2. Effects of anandamide (AEA, 5 pmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol) and potentiation of SIN-1 effect by AEA in the presence of AM251 (100 pmol). Data expressed as means SEM. N ¼ 7,5,9,7,7,7,7,9. * indicates significant difference from all other groups that received the same treatment as first injection; þ indicates difference from Veh-Veh and AEA-Veh groups or Veh-Veh-Veh and AM251-AEA-Veh groups (p < 0.05, ANOVA followed by the Duncan test).
3.2. Experiment II: potentiation of endogenous AEA also attenuated flight reactions induced by SIN-1 administration into the dlPAG Confirming the previous results, SIN-1 increased maximum speed (F1,53 ¼ 29.7, p < 0.001, Fig. 3A) and distance traveled (F1,53 ¼ 5.04, p < 0.05, Fig. 3B) in the open arena. The latter effect was prevented by the higher dose (0.1 nmol) of URB597, an inhibitor of AEA metabolism. 3.3. Experiment III: higher doses of AEA did not change the flight reactions induced by SIN-1 In experiment I we showed that pretreatment with the CB1 receptor antagonist before the administration of AEA 5 pmol into the dlPAG enhanced SIN-1 effect. Considering that AEA, usually at higher doses, can also activate TRPV1 receptors and increase glutamate release by dlPAG neurons (Xing and Li, 2007), we performed this experiment to verify the effects of higher doses of AEA on SIN-1 induced flight responses. Results can be seen in Fig. 4A and B. They showed again that intra-dlPAG administration of SIN-1 increased maximum speed (F1,47 ¼ 5.7, p < 0.001) and distance traveled (F1,47 ¼ 58.7, p < 0.001). This effect, however, was not
Fig. 3. Effects of URB597 (URB, 0.01 and 0.1 nmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 11,10,5,15,13,5. * indicates significant difference from all other groups, but not from Veh-URB0.01-SIN-1 group; þ indicates difference from Veh-Veh group (p < 0.05, ANOVA followed by the Duncan test).
prevented by any of the higher doses (50e200 pmol) of AEA. The higher dose of this compound also tended to potentiate SIN-1 effects (Fig. 4A and B), but the differences did not reach statistical significance. 3.4. Experiment IV: TRPV1 receptors are involved in the effects of higher AEA doses on SIN-1 induced flight reactions Although the 10 nmol of the TRPV1 receptor antagonist capsazepine (n ¼ 6e10/group) failed to change the effects of AEA200 þ SIN-1 (data not shown), the higher dose of this antagonist (30 nmol) prevented the effects of SIN-1 on total distance moved (F1,53 ¼ 4.66, p < 0.05, Duncan, p < 0.05, Fig. 5B) and maximum speed (F1,53 ¼ 10.6, p < 0.005, Fig. 5A). This drug was also able to attenuate the potentiation of SIN-1 effects by AEA (200 pmol) observed in total distance traveled (Duncan test, p < 0.05, Fig. 5B). 4. Discussion The present work confirmed previous studies showing that NO donors injected into the dlPAG induce flight responses (Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005; Moreira et al., 2004). The dlPAG, together with the medial hypothalamus and the
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Fig. 4. Lack of effects of higher doses of anandamide (AEA, 50e200 pmol) on maximum speed (A) and distance traveled (B) displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 8,7,5,6,6,7,8,8. * indicates main significant difference between Veh and SIN-1 treated animals (p < 0.05, ANOVA).
amygdala, is part of a system that has been traditionally related to active defensive responses (Graeff, 1994). Stimulation of this brain region in rodents results in responses characterized by galloping, jumping and episodes of wild running similar to those displayed by animals confronting a proximal innate fear stimulus such as a predator (Ambalavanan et al., 1999; Krieger and Graeff, 1985; Schenberg et al., 2001). These responses are called flight reactions and can be induced electrically or by excitatory chemical compounds, such as glutamate agonists, administered into the dlPAG (Aguiar et al., 2006; Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005). Since stimulation of the PAG in neurosurgery patients induces feelings of fear and despair similarly to those described in panic disorder (Nashold et al., 1969) these flight reactions have been related to panic attacks in humans (Deakin and Graeff, 1991; Jenck et al., 1995; Schenberg et al., 2001). NOS inhibitors, NO scavengers and sGC inhibitors injected into the dlPAG cause anxiolytic-like effects in animal models (Aguiar and Guimaraes, 2009; de-Oliveira and Guimaraes, 1999; Guimaraes et al., 2005, 1991, 1994; Molchanov and Guimaraes, 2002; Tonetto et al., 2009) whereas NO donors induce flight reactions (Braga et al., 2009; De Oliveira et al., 2001; Guimaraes et al., 2005). These latter responses are prevented by local pretreatment with NMDA and non-NMDA glutamate receptor antagonists (Moreira et al., 2004), suggesting, as shown elsewhere (Contestabile, 2000), that the effects of NO donors in the dlPAG are mediated by facilitation of glutamate release. CB1 receptors are largely distributed in the PAG (Herkenham et al., 1990). They inhibit the adenilate cyclase enzyme, decreasing intracellular levels of the second messenger AMPc and
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Fig. 5. Attenuation by capsazepine 30 nmol (CPZ30) of the effects of anandamide (AEA, 200 pmol) on distance traveled (B), but not maximum speed (A), displayed by rats in an open arena after intra-dlPAG injection of vehicle (Veh) or SIN-1 (150 nmol). Data expressed as means SEM. N ¼ 10,8,10,7,5,5,6,6. * indicates significant difference from all other groups (p < 0.05, ANOVA followed by the Duncan test).
consequently decreasing the release of several neurotransmitters, including glutamate (Howlett and Fleming, 1984; Wilson and Nicoll, 2002). Activation of these receptors in the dlPAG induces anxiolytic-like effects (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b) similar to those seen with intra-dlPAG administration of glutamate ionotropic receptor antagonists (Guimaraes et al., 2005, 1991; Molchanov and Guimaraes, 2002). Based on these findings, we hypothesized that the nitrergic and cannabinoid systems could play opposite roles in the regulation of defensive responses in the dlPAG, probably by modulating glutamate release. Accordingly, a low dose of AEA (5 pmol) and the FAAH inhibitor URB597 (0.1 nmol) were able to attenuate the flight responses, measured by the total distance traveled in the open arena and, in the former case, also by the maximum speed, induced by the NO donor SIN-1 administered into the dlPAG. AM251, an antagonist of CB1 receptors, prevented the effects of AEA, indicating that they were being mediated by these receptors. The dose of AM251 used was similar to that able to block the anxiolytic effects of AEA injected into the dlPAG (Lisboa et al., 2008; Moreira et al., 2007; Resstel et al., 2008b). The antagonist by itself, however, failed to increase the flight responses induced by SIN-1. Although this might indicate that endogenous cannabinoids are not important to modulate these responses, a ceiling effect could also be involved. Actually, the results with URB597, which inhibits AEA metabolism, suggest that enhancement of endogenous cannabinoid levels can, indeed, attenuate flight reactions induced by an NO donor.
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An interaction between the nitrergic and cannabinoid system has also been suggested in other brain areas (Azad et al., 2001; Nasehi et al., 2010; Roohbakhsh et al., 2007). Animals that lack the CB1 gene present an increase in NOS activity in the cerebral cortex, suggesting that CB1 is required to inhibit this activity (Kim et al., 2006). Also, several in vitro studies (Carney et al., 2009; Jones et al., 2008; Maccarrone et al., 2001, 2000a, 2006; Signorello et al., 2010; Waksman et al., 1999) suggest a functional interaction between the cannabinoid and nitrergic systems. The present study provides additional evidence, now in the dlPAG, that these two systems could interact in the central nervous system. AEA can activate both CB1 and TRPV1 receptors, which results in inhibition and facilitation, respectively, of glutamate release (Van Der Stelt and Di Marzo, 2004). These two actions would produce opposite effects on defensive responses. Since the intrinsic efficacy of AEA at TRPV1 is relatively low compared to that observed for this compound at CB1 receptors (Pertwee and Ross, 2002; Ross, 2003), this double mechanism could be involved in the lack of effect observed with the higher doses of AEA in the present work and in the bell-shape dose-response curves commonly observed with cannabinoids, including when administered in the dlPAG (Campos and Guimaraes, 2009; Guimaraes et al., 1990; Moreira et al., 2007). Moreover, in the presence of the CB1 receptor antagonist AM251, the low dose of AEA potentiated, instead of decreasing, the flight responses induced by SIN-1. This agrees with the recent suggestion that in the dlPAG TRPV1 and CB1 receptors might be activated simultaneously at a given synapse to modulate panic-like responses (Casarotto et al., 2011). AEA has shown dual effects on several systems. For example, at low concentration it inhibits, via CB1 receptors, electrically stimulated neuropeptide release from cultured dorsal root ganglia neurons (Ross et al., 2001) while, at higher concentrations, evokes a TRPV1-mediated release (Tognetto et al., 2001). Furthermore, in the presence of a CB1 receptor antagonist, AEA becomes equipotent with capsaicin as a TRPV1 receptor agonist to induce neuropeptide release (Ahluwalia et al., 2003). It was also shown in guinea-pig ileum that AEA increases acetylcholine release in a capsazepinesensitive manner, an effect that was enhanced by the CB1 receptor antagonist, SR141716A (Mang et al., 2001). Similarly, in human neuroblastoma cells, CB1 receptor antagonists potentiate TRPV1-mediated cell death induced by AEA (Maccarrone et al., 2000b). Finally, Kawahara and colleagues (Kawahara et al., 2011) showed that inhibition of the FAAH enzyme unmasks AEA-induced presynaptic inhibition and excitation of glutamatergic synaptic transmission in this region by activating CB1 and TRPV1 receptors, respectively. In the PAG activation of presynaptic TRPV1 receptor has been shown to increase glutamate release (Van Der Stelt and Di Marzo, 2004). Early electrophysiological data showed that capsaicin application to slices of hypothalamus, substantia nigra, locus coeruleus or PAG evokes glutamate release (Liao et al., 2011; Marinelli et al., 2003, 2002; Sasamura et al., 1998) mediated by TRPV1 activation, since the effect was blocked by TRPV1 antagonists. These results suggest that TRPV1 are expressed presynaptically in glutamatergic terminals and that their activation potentiate the release of this neurotransmitter. In agreement with this proposal, it was showed that capsaicin infusion into the dorsal PAG increases glutamatergic synaptic transmission, which was abrogated by TRPV1 antagonist (Xing and Li, 2007). Also, genetic deletion of the TRPV1 gene prevents capsaicin-induced presynaptic excitation in striatum, reinforcing the hypothesis that activation of presynaptic TRPV1 induces glutamate release (Musella et al., 2009). This mechanism could explain the similar anxiolytic effects induced by blockade of NMDA or TRPV1 receptors observed in several animal models (Aguiar and Guimaraes, 2011; Guimaraes
et al., 1991; Molchanov and Guimaraes, 2002; Resstel et al., 2008a; Tonetto et al., 2009). Several pieces of evidence have also suggested the presence of post-synaptic TRPV1 receptors (Chavez et al., 2010; Cristino et al., 2006; Grueter et al., 2010; Micale et al., 2009; Starowicz et al., 2007; Zschenderlein et al., 2011), modulating synaptic inhibition and excitation. For example, a recent study by Zschenderlein and colleagues (Zschenderlein et al., 2011) showed that in the lateral amygdala, after high frequency stimulation, capsaicin activates post-synaptic TRPV1 receptors. At low concentration TRPV1 activation induces the synthesis of AEA, which in turn activates CB1, reduces glutamate release and NOS activation, inhibiting long-term potentiation (LTP). On the other hand, higher levels of AEA, by enhancing intracellular calcium, could activate post-synaptic neuronal NOS, leading to an increase in glutamate release and enhancement of LTP. It remains to be verified if similar mechanisms occur in the PAG. The present data, in addition, indicate the presence of a complex interaction between NO and TRPV1 receptors in this region. Systemic administration of capsaicin has been shown to increase NO synthesis in brain regions related to defensive behavior, such as paraventricular nucleus of hypothalamus, medial amygdala and also the dlPAG (Okere et al., 2000a, 2000b; Okere and Waterhouse, 2006), suggesting that activation of TRPV1 receptors engage a glutamate/NMDA/NO pathway that could be facilitating aversive responses. Our results indicated that blockade of these receptors prevents the effects of exogenous NO, suggesting that this neurotransmitter could be exerting its effect by facilitating a positive feed-back loop involving glutamate and endovanilloids. Further studies are needed to confirm this possibility and investigate its pharmacological mechanisms. In conclusion, the present study showed that a low dose of AEA activates CB1 receptors and attenuate the flight reaction induced by SIN-1, while higher doses of AEA are ineffective, probably by also activating TRPV1 receptors. The antagonism of CB1 receptors in the presence of the low dose of AEA resulted in potentiation of SIN-1 effects, while blockade of TRPV1 receptors previous to the higher dose of AEA unmasked the attenuating effect of AEA. The results indicate, therefore, that AEA can modulate in a dual way the proaversive effects of NO in the dlPAG by activating CB1 or TRPV1 receptors. Acknowledgments The authors thank Eleni T. Gomes and José C. de Aguiar (University of São Paulo) for the technical assistance. The research was supported by a grant from FAPESP (2007/03685-3) SFL and FSG are recipients of FAPESP and CNPq fellowships, respectively. References Aguiar, D.C., Guimaraes, F.S., 2009. Blockade of NMDA receptors and nitric oxide synthesis in the dorsolateral periaqueductal gray attenuates behavioral and cellular responses of rats exposed to a live predator. J. Neurosci. Res. 87, 2418e2429. Aguiar, D.C., Guimaraes, F.S., 2011. Blockade of NMDA or NO in the dorsal premammillary nucleus attenuates defensive behaviors. Physiol. Behav. 103, 279e283. Aguiar, D.C., Moreira, F.A., Guimaraes, F.S., 2006. Flight reactions induced by injection of glutamate N-methyl-d-aspartate receptor agonist into the rat dorsolateral periaqueductal gray are not dependent on endogenous nitric oxide. Pharmacol. Biochem. Behav. 83, 296e301. Ahluwalia, J., Urban, L., Bevan, S., Nagy, I., 2003. Anandamide regulates neuropeptide release from capsaicin-sensitive primary sensory neurons by activating both the cannabinoid 1 receptor and the vanilloid receptor 1 in vitro. Eur. J. Neurosci. 17, 2611e2618. Ambalavanan, N., Mariani, G., Bulger, A., Philips, I.J., 1999. Role of nitric oxide in regulating neonatal porcine pulmonary artery smooth muscle cell proliferation. Biol. Neonate 76, 291e300.
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