Cannabinoid modulation of predator fear: involvement of the dorsolateral periaqueductal gray

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1 International Journal of Neuropsychopharmacology (1), 17, CINP 1 doi:1.117/s Cannabinoid modulation of predator fear: involvement of the dorsolateral periaqueductal gray ARTICLE S. F. Lisboa, L. H. A. Camargo, A. C. Magesto, L. B. M. Resstel and F. S. Guimarães Department of Pharmacology, School of Medicine of Ribeirão Preto, Ribeirão Preto, SP, and Centre for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, Brazil Abstract The present study investigated the effects of systemic or intra-dorsolateral periaqueductal gray (dlpag) administration of CB1 agonists on behavioural changes induced in rats by predator (a live ) exposure, a model of panic responses. Since nitric oxide (NO) and cannabinoid neurotransmission are proposed to interact in the dlpag to modulate defensive responses, we also investigated if NO is involved in the biphasic effects of anandamide (AEA) injected into the dlpag. The results showed that systemic administration of WIN,1- or intra-dlpag AEA attenuated the defensive behaviours caused by exposure. Both compounds produced biphasic curves. The cannabinoid receptor type 1 (CB1) antagonist AM1 prevented the panicolytic effect of AEA whereas a neuronal NOS inhibitor turned the ineffective high dose of AEA into an effective one. These results suggest that modulation of the cannabinoid system could be a target in the treatment of panic disorders. However, the biphasic effects of these compounds could limit their therapeutic potential. Received August 13; Reviewed 3 September 13; Revised 1 November 13; Accepted 19 December 13; First published online 17 January 1 Key words: Cannabinoids, dorsolateral periaqueductal gray, nitric oxide, panic, predator exposure. Introduction Defensive behaviours elicited by real or potential threats vary depending on factors such as stimuli nature, intensity and distance (Graeff, 199; Gray and McNaughton, ). While the actual presence of a predator induces behavioural changes such as flight and freezing, signs of its potential presence (e.g. predator odour) produce changes such as risk assessment. Behavioural and pharmacological analyses suggest that these distinct behavioural reactions are associated with panic attacks and anxiety in humans, respectively (Blanchard et al., 1997). Several pieces of evidence indie that these responses are mediated by parallel brain circuits engaged by distinct stimuli such as pain, aggressive conspecifics and predators (Gross and Canteras, 1). The circuit responsible for responses to predators is proposed to involve the lateral amygdala, the posterior part of the basomedial amygdala, the dorsomedial part of the ventromedial hypothalamus, the ventrolateral part of the premammilary nucleus and the midbrain dorsolateral periaqueductal gray (dlpag) (Gross and Canteras, 1). Address for correspondence: S. F. Lisboa, Department of Pharmacology, School of Medicine of Ribeirão Preto, Av. Bandeirantes, 39, 19-9, Monte Alegre. Ribeirão Preto, SP, Brazil. Tel.: Fax: sabrinalisboa@usp.br Recurrent episodes of panic attacks, described as intense feelings of distress and fear accompanied by increased autonomic activity, is a key feature of panic disorder (Katon, ; Roy-Byrne et al., ). The dorsal PAG (dpag) is part of a neural defensive system that has been closely associated with panic attacks (Gray and McNaughton, ; Schenberg et al., 1; Del-Ben and Graeff, 9). There is convincing clinical and experimental evidence indiing that these attacks are related to behavioural responses to proximal threats such as predator exposure, which are organized by this same region (Graeff, ). Electrical stimulation of the dpag in humans during neurosurgical procedures induces feelings of intense fear and imminent death along with autonomic alterations (Nashold et al., 199) that closely resemble a panic attack (Schenberg et al., 1). More recently, neuroimaging studies demonstrated an increase in dpag activity in patients in panic (Del-Ben and Graeff, 9) or in healthy volunteers exposed to a proximal threatening stimulus (Mobbs et al., 7). Pre-clinical experiments with rodents have also shown that dpag electrical or chemical stimulation causes intense flight responses accompanied by autonomic changes (Krieger and Graeff, 198; Beckett and Marsden, 199; Schenberg et al., 1), which are attenuated by systemic or local administration of panicolytic drugs such as serotonin selective reuptake inhibitors (Schutz et al., 198; Jenck et al., 199; Schenberg et al., 1; Hogg et al., ). on 7 July 18

2 119 S. F. Lisboa et al. In addition to classical neurotransmitters such as glutamate, GABA and serotonin, atypical neurotransmitters such as nitric oxide (NO) have also been proposed to modulate defensive responses in the dpag (Guimaraes et al., ; Fogaca et al., 1). Different from classical transmitter molecules, NO and other atypical transmitters are not stored in vesicles, but are formed on demand in post-synaptic neurons from where they can diffuse and interfere in pre-synaptic function (Esplugues, ; Garthwaite, 8). The neuronal isoform of the NO synthase (nnos) enzyme is widely expressed in the dlpag (Onstott et al., 1993) and its inhibition induces anti-aversive effects in several animal models, including predator exposure (Guimaraes et al., 199; Aguiar and Guimaraes, 9; Tonetto et al., 9). Endocannabinoids (ECBs) are another important class of atypical neurotransmitters. The cannabinoid receptor type 1 (CB1), the most expressed G-protein coupled receptor in the central nervous system, is present in the dlpag, acting pre-synaptically to decrease the release of neurotransmitters such as glutamate and GABA (Herkenham et al., 199, 1991; Tsou et al., 1998; Mezey et al., ; Wilson and Nicoll, ; De Petrocellis et al., ; Toth et al., ; Cavanaugh et al., 11). Anandamide (AEA), one of the main ECBs, is also a common ligand for the inotropic transient receptor potential vanilloid 1 (TRPV1), an ion channel permeable to calcium that, contrary to CB1, could facilitate glutamate release (Xing and Li, 7). Both CB1 and TRPV1 are expressed in the dpag (Cristino et al., ). TRPV1 antagonists or CB1 activation by AEA or synthetic agonists in the dlpag induce anxiolytic effects and attenuate the flight responses caused by direct PAG electrical or chemical stimulation (Moreira et al., 7; Lisboa et al., 8; Campos and Guimaraes, 9; Terzian et al., 9; Casarotto et al., 1; Fogaca et al., 1; Lisboa and Guimaraes, 1), suggesting that these receptors play an opposite modulatory role on defensive behaviours. Cat odour, which induces anxiogenic-like effects, modified the expression of several ECBs components in the PAG, such as NAPE-PLD and FAAH, enzymes responsible for the synthesis and degradation of AEA, respectively. It also decreased the expression of enzymes involved in the synthesis of the other main ECB, -arachidonyl-glycerol (Sutt et al., 8). The effects of cannabinoids on defensive behaviours induced by a live predator, however, have not yet been investigated. Considering that this procedure is proposed as a model useful to detect panicolytic effects (Blanchard et al., 1997; Campos et al., 13), in the present work we investigated if systemic or intra-dlpag administration of the cannabinoid agonists WIN,1- and AEA, respectively, is able to decrease defensive behaviours in rats exposed to a live predator (a ). We also verified if AEA effects are actually being mediated by CB1 receptors. Finally, since nitrergic and ECB neurotransmissions are proposed to interact in the dlpag to modulate defensive responses (Fogaca et al., 1; Lisboa et al., 13), we investigated if NO is responsible for the biphasic effects of AEA injected into the dlpag. Materials and method Subjects Adult male Wistar rats ( 7 g) were obtained from the colony of pathogen-free rats maintained by the Pharmacy School of Ribeirão Preto, University of São Paulo. They were housed in groups of four with free access to food and water in a temperature-controlled room ( C), 1 h light/dark cycle. An adult male (3 kg), kept at the animal farm of our University Campus with free access to food and water, was used throughout the study. A white dummy, of approximately the same size as the live, was used as control. The experiments were carried out according to the Brazilian Society of Neuroscience and Behaviour guidelines for care and use of laboratory animals, and all efforts were made to minimize animal suffering. The experiment protocol was approved by the local ethical committee (-). Drugs The CB1/CB agonist (R)-(+)-[,3-Dihydro--methyl-3- (-morpholinylmethyl)pyrrolo[1,,3-de]-1,-benzoxazin-- yl]-1-naphthalenylmethanone mesylate or WIN,1- mesylate (WIN, SIGMA;.1,.3,., 1.,., mg/kg) was dissolved in 1% DMSO in NaCl.9% and administered in a volume of 1 ml/kg. The endocannabinoid anandamide (AEA;.,,, 1, pmol/. μl; TOCRIS) was dissolved in TocrisolveTM 1 (provided with the drug; 1: ratio of soya oil/water emulsified with the block co-polymer Pluronic F8). The CB1 antagonist N-(piperidin-1yl)--(-iodophenyl)-1-(,-dichlorophenyl)- -methyl-1hpyrazole-3-carboxamide (AM1; 1 pmol/. μl; TOCRIS) was dissolved in 1% DMSO in NaCl.9%. The nnos inhibitor N -[Imino(propylamino) methyl]-l-ornithine hydrochloride (NPA; 1 nmol/. μl; TOCRIS) was dissolved in sterile isotonic saline (NaCl.9%). Dose-response curves were obtained for WIN and AEA. AM1 and NPA doses were based on previous studies (Pamplona et al., ; Moreira et al., 7; Aguiar and Guimaraes, 9; Lisboa and Guimaraes, 1). Apparatus The exposure apparatus consisted of a rectangular box (8 cm) with Plexiglas walls and a metal grid floor. The apparatus was loed in a sound-attenuated and illuminated room. It was divided into two compartments by a metal grid wall. In the experimental session, each rat was placed in the middle of the compartment opposite to the one containing the live or dummy, on 7 July 18

3 Cannabinoid modulation of predator fear 119 facing the other compartment. The box was designed to comfortably contain the and to provide enough space for measuring the rat presence proximal or distal to the compartment. The rat compartment was divided into two equal parts (near and distant to the compartment) by a virtual line. Previous studies using this apparatus show that when the live was not present the rats have no preference for any part of the box, whereas they escape to the -distant part and tend to remain there when the predator is present. Cat exposure also increases immobility time (Beijamini and Guimaraes, ; Moreira and Guimaraes, 8; Aguiar and Guimaraes, 9). The motor behaviour evaluation was carried out in a circular open arena (7 cm in diameter with a cm high Plexiglas wall) loed in a sound attenuated, temperature-controlled ( C) room, illuminated by three W fluorescent bulbs placed m above the apparatus, as previously described (Lisboa and Guimaraes, 1). The rats were videotaped during the 1 min inside the arena and their behaviour was analysed with the help of the AnyMaze software (Stoelting, USA). This software detects the position of the animal in the open arena and calculates the distance moved and maximum speed. Experimental procedure Experiment I The first set of experiments verified if systemic administration of a cannabinoid agonist could attenuate the fear-behaviour elicited by predator exposure. Animals received an i.p. injection of icle or WIN,1- (.1. mg/kg) to establish a dose-response curve and were exposed 3 min later to a or live for 1 min. Additional group of animals receiving the higher dose of WIN were submitted to an open field in order to evaluate possible locomotor effects. The next experiments were aimed to verify if the dlpag could be a brain site for cannabinoid action and which mechanisms are involved in their effects. Seven days after surgery, an independent group of animals were randomly assigned to one of the treatment groups. Intra-dlPAG injections were performed with a thin dental needle (.3 mm OD) introduced through the guide cannula until its tip was 1. mm below the cannula end. A volume of. μl was injected in 3 s using a microsyringe (USA) connected to an infusion pump (Kd Scientific, USA). A polyethylene heter (PE1) was interposed between the upper end of the dental needle and the microsyringe. Surgery and intra-dlpag injection Rats were anaesthetized with.%,,-tribromoethanol (Sigma-Aldrich, USA, 1 mg/kg, IP) and immobilized in a stereotaxic frame (David Kopf). A stainless steel guide cannula (.7 mm OD) was implanted unilaterally on the right side aimed at the dlpag (coordinates: AP= from lambda, L =1.9 mm at an angle of 1, D=.3 mm) (Paxinos and Watson, 7). The cannula was attached to the skull with stainless steel screws and acrylic cement. An obturator inside the guide cannula prevented obstruction. Histology At the end of experiments, rats were anaesthetized with chloral hydrate (%, 1 ml/kg, i.p.), their chests were surgically opened, the descending aortas occluded, the right atrium severed and the brains perfused with 1% formalin through the left ventricle. The brains were post fixed in 1% formalin for h at C and μm sections were cut with the help of a cryostat (CM 19, Leica, Germany). The placement of the injection needles was identified with the help of the rat brain atlas of Paxinos and Watson (7). A representative photomicrography and the injection sites can be seen in Fig. 1. Rats that received injections outside the aimed area were excluded from analysis. Confirming previous findings (Moreira et al., 7), no effect was observed when the CB1 agonists were injected outside the dlpag (data not shown). Experiment II In order to verify if the dlpag could be a brain site for cannabinoid action, we performed a dose-response curve for AEA in the dlpag. A group of animals received micro-injection of icle or AEA (. pmol/. μl) and 1 min later were exposed to the or to the live for 1 min. Experiment III To verify if CB1 receptors could be involved in the observed effects of AEA, an independent group of animals received a first micro-injection into the dlpag of icle or AM1 (1 pmol/. μl) followed, min later, by icle or AEA ( or pmol/. μl). Ten minutes after the last micro-injection the animals were exposed to the or live for 1 min. Experiment IV Considering that the lack of effect of higher AEA doses could involve nitrergic mechanisms (Lisboa and Guimaraes, 1; Lisboa et al., 13), a group of animals received a first intra-dlpag micro-injection of icle or NPA (1 nmol/. μl) followed, min later, by icle or AEA ( pmol/. μl). Ten minutes later they were exposed to the or live for 1 min. The animal behaviour was recorded by a video camera and analysed by the AnyMaze software. The program detected the animal position in the observation box on 7 July 18

4 119 S. F. Lisboa et al. (a) (b) Bregma- 7. mm Bregma- 7.3 mm Bregma- 7. mm Fig. 1. (a) Representative photomicrography of a injection site in the dlpag. The black arrowhead indies the cannula end while the white arrowhead indies the injection site. dmpag: dorsomedial periaqueductal gray, lpag: ateral periaqueductal gray, aq: aqueduct. Fig. 1. (b) Histological localisation of injection sites in the dlpag (black circles) in diagrams based on the atlas of Paxinos and Watson (7). Due to overlap, the number of points represented is smaller than the real number of rats used in the experiments. The grey circles represent the injections outside the PAG. and calculated the time spent and the distance moved in the area close to the compartment. Immobility time (freezing), considered as the cessation of the movements except those associated with breathing, was analysed manually by an observer blind to the animal s treatment. Statistical analysis The data were analysed by Student t-test or one-way analysis of variance (ANOVA) followed by the Dunnett or Student-Newman-Keuls (S-K-N) post-hoc tests. Data from experiment I and III did not reach homogeneity of variance even after a log transformation and, therefore, were analysed by Kruskal-Wallis followed by the Mann- Whiney test. Differences were considered significant at p. level. Results Experiment I: systemic administration of low doses of WIN,1- decreased fear in rats exposed to a live predator The exposure to the live increased immobility time (t =1., p<.1, n=1 1/group; Fig. a) and decreased total distance moved (t = 3., p=.7; Fig. d), time spent in the area close to the compartment (t =3.8, p=.1; Fig. b) and the distance moved in this compartment (t =3.9, p<.1; Fig. e) and in the area far from the compartment (t =., p <.; Fig. f). In addition, it increased time spent in the compartment far from the (t =3., p <.; Fig. c). Some animals also showed flight attempts characterized by jumps toward the roof of the box. WIN mg/kg decreased distance moved (F,7 =3., p =., n =7 1/group, Dunnett p =.1; Fig. d) and on 7 July 18

5 Cannabinoid modulation of predator fear 1197 (a) (d) Immobility time (s) Total distance moved (m) (b) Time spent near the compartment (s) (e) 9 Distance moved near the compartment (m) (c) (f ) Time spent far from compartment (s) 3 1 Distance moved far from the compartment (m) Fig.. Effect of systemic administration of the cannabinoid compound WIN,1- on behavioural responses evoked by predator exposure. Cat exposure increased immobility time (a) decrease time spent near (b) and far (c) from the compartment, decreased total distance moved (d) distance moved near (e) and far from the compartment (f). WIN decreased and increased immobility time at the doses of. and mg/kg, respectively (a). WIN.1,.3,. and mg/kg increased time spent near the (b). The. mg/kg dose also increased the distance moved near the (e) and the mg/kg dose also decreased time spent (c) and distance moved far from the (e). Data are expressed as mean±e.p.m. p<. (Dunnett s or Mann-Whitney test) indies difference from icle group. n=7 1 animals/group. time spent (F,7 =.9, p <.1, Dunnett p<.1; Fig. c) in the area far from the compartment. WIN mg/kg increased immobility time whereas WIN. mg/kg decreased this behaviour (χ = 1.1, D.F. =, p=., Mann-Whitney, p=.; Fig. a). WIN.1,.3,. and mg/kg increased the time close to the compartment (χ =18.8, D.F. =, p=., Mann-Whitney, p <.1; Fig. b). Considering that high doses of cannabinoid compounds can reduce locomotion (Patel and Hillard, ), we performed an additional experiment to investigate if the higher dose ( mg/kg) of WIN was affecting locomotor behaviour. The drug failed to change the total distance moved (p >., Fig. 3a) and average speed (p >., Fig. 3b) but decreased the maximum locomotor speed (t 1 =.9, p<.; Fig. 3c). on 7 July 18

6 1198 S. F. Lisboa et al. (a) (b) (c) 1 Veh WIN Veh WIN Veh WIN Total distance (m) Average speed (m/s) Fig. 3. Effect of systemic administration of the cannabinoid compound WIN,1- on locomotor behaviour in rats evaluated in an open-field. WIN did not altered total distance moved (a) and average speed (b), but decreased the maximum locomotor speed (c). Data are expressed as mean±e.p.m. p<. (Student t-test). n = animals/group. Maximum speed (m/s) Experiment II: AEA administration into the dlpag attenuated fear-behaviour elicited by predator exposure Cat exposure increased immobility time (t 17 =1.1, p <.1, n =9 1/group; Fig. a) and decreased the total distance moved (t 17 =3.98, p=.1; Fig. d), as well as the distance moved in the compartments near (t 17 =.9, p=.1; Fig. e) and far (t 17 =.7, p=.1; Fig. f) from the compared to the animals exposed to the dummy. AEA, at the doses of., and pmol, attenuated immobility time (ANOVA F,8 =., p=.1; Dunnett s p <., n =9 1/group; Fig. a). AEA pmol was also able to increase the total distance moved (F,8 =.8, p<.1; Dunnett s p<.1; Fig. d) and distance moved in the compartment close to (F,8 =.; Dunnett s p <.; Fig. e) and far from the (F,8 = 3.1, p <.; Fig. f). AEA. tended to increase the distance moved in the compartment near the (Dunnett s p=.; Fig. e). Experiment III: involvement of dlpag CB1 receptors in AEA-attenuated fear response evoked by predator exposure Pre-treatment with the CB1 antagonist AM1 prevented the decrease in the immobility time induced by AEA pmol (F 3,9 =8., p=.1, n= 11/group, S-N-K, p<.; Fig. a). It also prevented AEA-induced increase in total distance moved (F 3,9 =9., p<.1, S-N-K, p <.; Fig. d) and distance moved in the compartments close to (F 3,9 =3.8, p<., S-N-K, p<.; Fig. e) and far from the (F 3,9 =.3; p=., S-N-K, p<.; Fig. f). Pre-treatment with AM1 also attenuated the effect of AEA pmol on immobility time (F 3,9 =.1, p=., n = 11/group, S-N-K, p <.; Fig. a). Although pmol did not alter the other parameters, it decreased the total distance (F 3,9 =3.3, p<., S-N-K, p<.; Fig. d) and time spent (logarithm data; F 3,9 =8., p <.1, S-N-K, p <.; Fig. b) in the compartment close to the and increased the time spent far from the (χ =11.9, D.F.=3, p=.8, Mann-Whitney, p=., different from AE1+, p =.1 AM1 + different from icle+icle; Fig. c) in combination with AM1 pre-treatment. Experiment IV: involvement of nnos in the lack of effects of a high dose of AEA ( pmol) in the fear response induced by predator exposure Considering that the absence of effects of higher doses of cannabinoids in the dlpag could be due to increased production of NO (Lisboa et al., 13), we administered NPA before AEA pmol to observe if this combination could attenuate fear behaviour elicited by predator exposure. AEA did not induce any effect per se. In combination with NPA, however, AEA decreased immobility time (F 3, =.9, p<., n = 7/group, S-N-K, p <.; Fig. 7a) and increased the total distance moved (F 3, =., p <., S-N-K, p <.; Fig. 7d) and distance moved close to (F 3, =3.9, p<., S-N-K, p<.; Fig. 7e) and far from the compartment (F 3, =., p<., S-N-K, p<.; Fig. 7f). Discussion Cat exposure induced defensive behaviours such as freezing and flight attempts. It also decreased general exploratory activity, particularly in the compartment close to the compartment. These results agree with those from several studies showing a significant increase in immobility time and reduction in locomotor activity and non-defensive behaviours, such as auto-cleaning, after predator exposure (Blanchard and Blanchard, 1989; McGregor et al., ; Beijamini and Guimaraes, ; Aguiar and Guimaraes, 9). Systemic administration of a cannabinoid compound, WIN,1-, modified these behaviours, either decreasing or increasing them depending on the dose used. dlpag injection of the endocannabinoid AEA also produced a biphasic effect, attenuating fear behaviour at low doses but having no effect at higher doses. The former effect depends on CB1 receptor activation whereas the lack of effects by the higher dose involve interference with the nitrergic system. Biphasic effects of cannabinoids on defensive responses are frequently observed. Systemic administration of the non-selective cannabinoid agonist WIN,1- was previously showed to induce an anxiolytic-like effect (Viveros et al., ; Patel and Hillard, ; on 7 July 18

7 Cannabinoid modulation of predator fear 1199 (a) (d) Immobility time (s) 3 1 Total distance moved (m) (b) (e) 8 Time spent near the compartment (s) Distance moved near the compartment (m) (c) (f ) 8 Time spent far from compartment (s) 3 1 Distance moved far from the compartment (m) Fig.. Effect of anandamide (AEA) administration into the dlpag on behavioural responses evoked by predator exposure. Cat exposure increased immobility time (a), decreased total distance moved (d), distance moved near to (e) and far from the compartment (f). AEA., and pmol (. μl) decreased immobility time (a). AEA pmol also increased total distance moved (d), the distance moved near to (e) and far from the (f). Data are expressed as mean±e.p.m. p<. (Dunnett s) indies difference from. n=9 11 animals/group. Gobira et al., 13) and to impair acquisition (Pamplona and Takahashi, ) and facilitate extinction (Pamplona et al., ) of hippocampus-dependent aversive memories. In addition, it inhibited stress-induced alterations in corticosterone levels and in hypothalamic pituitary adrenal (HPA) negative feedback (Ganon- Elazar and Akirav, 9, 1). These effects seem to be mediated by CB1 receptors (Viveros et al., ), probably by decreasing glutamate release (Rey et al., 1). On the other hand, higher doses of cannabinoid compounds induce anxiogenic-like effects in different animal models (Viveros et al.,, 7), including odour exposure (Arnold et al., 1). The attenuation of defensive responses induced by low doses of WIN disagree with a recent study showing that this drug did not induce panicolytic-like effect in the elevated-t-maze model (Gobira et al., 13), a model also based on innate fear responses. This discrepancy could be related to the different animal model and doses employed. Gobira et al. (13) measured the latency on 7 July 18

8 1 S. F. Lisboa et al. (a) Immobility time (s) 3 1 (d) Total distance moved (m) 1 1 AM1 AM1 AM1 AM1 (b) Time spent near the compartment (s) (e) Distance moved near the compartment (m) AM1 AM1 AM1 AM1 (c) Time spent near the compartment (s) 3 1 (f) Distance moved from the compartment (m) 8 AM1 AM1 AM1 AM1 Fig.. Participation of CB1 receptors in the effect of AEA pmol. Intra-dlPAG AEA decreased immobility time (a), increased total distance moved (d), and distances moved near to (e) and far from the compartment (f). Pre-treatment with AM1 (1 pmol/. μl) attenuated these effects. Data are expressed as mean±e.p.m. p<. (S-N-K) indies difference from all other groups and p<. (S-N-K) compared to - group. n= 11 animals/group. to escape from the open arm of the elevated T-maze, a single and quick response, whereas the present study evaluated behavioural changes induced by a proximal presence of a predator over a much longer time period (1 min). Several mechanisms have been proposed to explain the biphasic effects induced by cannabinoids on defensive responses. They could depend on changes in a GABA/ glutamate neurotransmission balance, with anxiolytic and anxiogenic effects favouring the former and latter, respectively (Rey et al., 1). Corroborating this proposition, it was recently showed that the anxiolyticlike effect of a low dose of a synthetic cannabinoid and the anxiogenic-like effect of a higher dose of the same compound disappear in mice lacking CB1 receptors in forebrain glutamatergic and GABAergic neurons, respectively (Rey et al., 1). Another possible mechanism, at least for some cannabinoids such as WIN and AEA, is activation of TRPV1 receptors, calcium-selective ion channels that facilitate glutamate release (Xing and Li, 7; Ho et al., 1; Moreira et al., 1a). In this case, low doses of these compounds would decrease glutamate on 7 July 18

9 Cannabinoid modulation of predator fear 11 (a) (d) 1 Immobility time (s) 3 1 Total distance moved (m) 1 AM1 AM1 AM1 AM1 (b) (e) Time spent near the compartment (s) 3 1 Distance moved near the compartment (m) 3 1 $ AM1 AM1 AM1 AM1 (c) (f ) 8 Time spent far from compartment (s) $ Distance moved far from the compartment (m) 3 1 AM1 AM1 AM1 AM1 Fig.. Participation of CB1 receptors in the effect of intra-dlpag AEA pmol. AEA decreased immobility time (a). Although AEA did not altered other parameters, pre-treatment with AM1 (1 pmol/. μl) decreased time spent near to the compartment (b), increased the time spent far from (c) and decreased distance moved near to the compartment (e). Data are expressed as mean±e.p.m p<. (S-N-K) indie difference from all other groups, p<. (S-N-K or Mann-Whitney test) compared to - group and $p<. compared to - group (S-N-K or Mann-Whitney test). n= 11 animals/group. release through a CB1-mediated mechanism, whereas higher dose would increase it by activation TRPV1 receptors (Moreira et al., 1a). In addition to the effects on anxiety, higher doses of cannabinoid compounds can decrease locomotor behaviour (Patel and Hillard, ). In the present study the higher dose of WIN ( mg/kg) failed to change total distance moved and average speed in animals tested in the open field. This dose, however, did decrease maximum locomotor speed. This result, therefore, suggests that the increased in immobility time induced by this dose of WIN could involve, at least in part, interference in locomotor activity. The observation that electrical stimulation of the dorsal and dorsolateral portions of PAG induces defensive reactions in animals and fear and anxiety in humans (Nashold et al., 199; Schenberg et al., 1; Mobbs et al., 7) indies that this brain region could be part of the encephalic circuit that processes these on 7 July 18

10 1 S. F. Lisboa et al. (a) 3 (d) 1 Immobility time (s) Total distance moved (m) NPA1 NPA1 NPA1 NPA1 AEA AEA AEA AEA (b) (e) 8 Time spent near the compartment (s) 3 1 Distance moved near the compartment (m) $ NPA1 NPA1 NPA1 NPA1 AEA AEA AEA AEA (c) (f) Time spent far from compartment (s) 3 1 Distance moved far from the compartment (m) 3 1 NPA1 NPA1 NPA1 NPA1 AEA AEA AEA AEA Fig. 7. Participation of NO formation in the absence of effect of higher dose of intra-dlpag AEA ( pmol). After pre-treatment with NPA (1 nmol/. μl), AEA decreased immobility time (a), increase the total distance moved (d), distance moved near to (e) and far from the compartment (f). Data are expressed as mean±e.p.m. p<. (S-N-K) indies difference from all other groups and $p<. (S-N-K) from NPA1- group. n = 7 animals/group. behaviours. Defensive reactions that follow electrical or chemical stimulation of dlpag share many similarities with those induced by predator exposure (Graeff, 199; Blanchard et al., 1997; Campos et al., 13). For example, rats exposed to a present freezing behaviour and analgesia as well as fear conditioning to the place where exposition had occurred (Lester and Fanselow, 198; Blanchard et al., 1998). These changes are reduced by lesions of the amygdale (Blanchard and Blanchard, 197) or dpag (Fanselow, 1991). Considering the involvement of dlpag in defensive behaviour and its role as a possible brain site for cannabinoid anxiolytic effects (Fogaca et al., 1; Moreira et al., 1b), we investigated if AEA administration into this structure could also interfere in the defensive responses observed in rats exposed to a live. Corroborating this possibility, intra-dlpag administered AEA, at the doses of., and pmol, significantly attenuated the defensive responses induced by exposure. Together, these results suggest that cannabinoids on 7 July 18

11 Cannabinoid modulation of predator fear 13 can produce, in addition to anxiolytic-like effects (Moreira et al., 7; Lisboa et al., 8; Resstel et al., 8), panicolytic-like effects (Lisboa and Guimaraes, 1). The panicolytic effects of AEA in the dlpag were attenuated by AM1, indiing that they are mediated by CB1 receptors. This result agree with others studies employing different animal models (Finn et al., 3, ; Moreira et al., 7; Lisboa et al., 8; Resstel et al., 8; Lisboa and Guimaraes, 1) and suggest that activation of local CB1 receptors could lead to a reduction in glutamate release and NO formation, promoting anti-aversive effects (Lisboa et al., 8, 13; Resstel et al., 8; Lisboa and Guimaraes, 1). Predator exposure increases NO production (Chiavegatto et al., 1998) and induces activation of NADPH-d containing neurons, an indirect marker for NOS enzyme, in the dlpag, an effect prevented by a NMDA receptor antagonist (Beijamini and Guimaraes, ). In addition, inhibition of NO formation or antagonism of NMDA receptors in this region produces effects similar to AEA, attenuating defensive behaviour changes evoked by predator exposure (Aguiar and Guimaraes, 9). These treatments also caused anxiolytic-like effects in rats exposed to the elevatedplus-maze (Guimaraes et al., 1991, 199). Together, these results indie that glutamate- and NO-mediated neurotransmission in the dlpag modulate defensive responses induced by different threat stimuli, including predator exposure. Conceivably, cannabinoids could, by activating CB1 receptors, decrease glutamate release and consequent NO formation, exerting an anti-aversive effect. Biphasic effects of cannabinoids on defensive behaviours can also be observed after intra-cerebral administration of these compounds. For example, intra-dlpag administration of AEA in rats induced an anxiolytic-like effect in the EPM at low, but not at high doses (Moreira et al., 7; Lisboa et al., 13). In agreement with this observation, we showed that the higher doses of AEA (1 and pmol) were ineffective. Moreover, in the presence of AM1, the dose of pmol of AEA, which was also ineffective in some parameters, decreased the time and distance moved closer to the compartment and increased the time spent far from the compartment, suggesting increased fear. Previous results showed that, in the presence of CB1 blockade, an effective dose of AEA that attenuated flight reaction induced by a NO donor in the dlpag, induced an opposite (pro-aversive) effect (Lisboa and Guimaraes, 1). On the other hand, higher doses of AEA potentiated flight reactions induced by NO donor administration into the dlpag (Lisboa and Guimaraes, 1). In addition, intra-dlpag pre-treatment with ineffective doses of a TRPV1 or NMDA antagonists (Fogaca et al., 13) or inhibitors of the NO pathway (Lisboa et al., 13) caused anxiolytic-like effects in the EPM when combined with a high (ineffective) dose of AEA. These results suggest that the lack of effect of higher doses of AEA could depend on NO formation and/or increased NMDA activation. To investigate if similar mechanisms are also involved in the lack of behavioural effect of high doses of AEA in the predator exposure model, we pre-treated animals with the selective nnos inhibitor NPA before injection of the higher dose of AEA ( pmol). In the presence of NPA, AEA pmol attenuated the behavioural response induced by exposure, indiing that the lack of effect of this higher dose of AEA when given alone depends on NO formation. The mechanism of this interaction is not completely clear, but in a recent study Zschenderlein and co-workers (Zschenderlein et al., 11) suggested that in the lateral amygdala activation of post-synaptic TRPV1 receptors could facilitate presynaptic release of glutamate by increasing intracellular calcium with a consequent stimulation of nnos and NO formation. NO could then act pre-synaptically as a retrograde messenger and enhance the release of glutamate (Zschenderlein et al., 11). Although it remains to be tested if a similar mechanism occurs in the dlpag, the TRPV1 agonist capsaicin was shown to increase NO formation in defensive-related brain regions such as the medial amygdala, paraventricular nucleus of the hypothalamus and dlpag (Okere et al., a, b; Okere and Waterhouse, ). In conclusion, the present study indied that CB1 agonists injected either systemically or into the dlpag, attenuate defensive responses evoked by predator exposure. Higher doses of these compounds produced no or even pro-aversive effects, possible by increasing NO formation. The results suggest that modulation of the cannabinoid system could be a target in the treatment of panic disorders. However, the biphasic effects of these compounds could limit their therapeutic potential and should be carefully considered when choosing cannabinoid doses. Conflict of Interest The authors declare no conflict of interest. Acknowledgments The authors thank José C. de Aguiar and Eleni T. Gomes (University of São Paulo) for the excellent technical assistance. The research was supported by grants from FAPESP (7/999-9, 1/17-7). SFL, ACM and LBMR are recipients of FAPESP fellowship. FSG and LHAC are recipients of CNPq fellowship. References Aguiar DC, Guimaraes FS (9) 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 87:18 9. on 7 July 18

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