Antiaversive Effects of Cannabinoids:  Is the Periaqueductal Gray Involved?

doi:10.1155/2009/625469

Journal List > Neural Plast > v.2009; 2009

Neural Plast. 2009; 2009: 625469.

Published online 2008 December 2. doi: 10.1155/2009/625469.

PMCID: PMC2593468

Antiaversive Effects of Cannabinoids: Is the Periaqueductal Gray Involved?

F. A. Moreira, D. C. Aguiar, A. C. Campos, S. F. Lisboa, A. L. Terzian, L. B. Resstel, and F. S. Guimarães^*

Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3900, 14049900 Ribeirão Preto, SP, Brazil

*F. S. Guimarães: Email: fsguimar/at/fmrp.usp.br

Recommended by Robert Adamec

Received May 14, 2008; Revised August 12, 2008; Accepted September 9, 2008.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Cannabinoids play an important role in activity-dependent changes in synaptic activity and can interfere in several brain functions, including responses to aversive stimuli. The regions responsible for their effects, however, are still unclear. Cannabinoid type 1 (CB1) receptors are widely distributed in the central nervous system and are present in the periaqueductal gray (PAG), a midbrain structure closely involved in responses related to aversive states. Accordingly, exposure to stressful stimuli increases endocannabinoid (eCB) levels in the PAG, and local administration of CB1 agonists or drugs that facilitate eCB-mediated neurotransmission produces antinociceptive and antiaversive effects. To investigate if these drugs would also interfere in animal models that are sensitive to anxiolytic drugs, we verified the responses to intra-PAG injection of CB1 agonists in rats submitted to the elevated plus-maze, the Vogel punished licking test, or contextual aversive conditioning model. The drugs induced anxiolytic-like effects in all tests. The same was observed with the transient receptor potential vanilloid type 1 (TRPV1) antagonist capsazepine and with cannabidiol, a nonpsychotomimetic phytocannabinoid that produces anxiolytic-like effects after systemic administration in humans and laboratory animals. These results, therefore, suggest that the PAG could be an important site for the antiaversive effects of cannabinoids.

1. Introduction

Cannabis sativa plant has been used for various purposes since the dawn of civilizations [1, 2], although only in the middle of twentieth century were its chemical constituents identified. Among its major components, there are the phytocannabinoids cannabinol, cannabidiol (CBD), and Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the latter accounting for most of cannabis effects [3–5]. The mechanisms of Δ⁹-THC effects started to be unveiled in the late 80s, with the discovery of CB1 receptors [6, 7]. Soon afterwards, the first endogenous agonist (arachidonoyl ethanolamide, AEA) was isolated and named anandamide, after the Sanskrit word “ananda” for “bliss” [8]. A second endocannabinoid, 2-arachidonoyl glycerol [9], and another cannabinoid receptor, called CB2 [10], soon followed. Selective antagonists were developed, such as rimonabant and AM251, supporting the notion that the CB1 receptor is the major responsible for the behavioral effects of cannabinoids [11, 12]. The expression of this receptor is considerably high in several brain regions such as the basal ganglia, cerebral cortex, hippocampus, amygdale, hypothalamus, and periaqueductal gray (PAG) [13, 14].

CB1 receptors are believed to be located in presynaptic terminals [15]. They activate Gi proteins that inhibit adenylate cyclase and calcium channels and enhance potassium currents, thereby reducing neural firing and neurotransmitter release [16]. This complements the fact that endocannabinoids are synthesized on a stimulus-dependent manner in postsynaptic neurons and immediately diffuse to the synaptic cleft [16]. Thus, contrary to classical neurotransmitters, endocannabinoids act “on demand” as retrograde messengers, inhibiting neural activity. Their effects cease by internalization followed by hydrolysis in neurons. It is still controversial whether endocannabinoids move through the cell membrane passively or are internalized by a putative transporter. Although the latter remains to be identified [17, 18], pharmacological tools were developed, such as AM404, which are able to inhibit it and, thereby, increase CB1 receptor activation by AEA [18]. Inside neurons, AEA and 2-AG are catabolized by fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MGL), respectively [19]. Possibly, FAAH is located in postsynaptic neurons, whereas MGL is expressed in the presynapse [17]. Selective inhibitors of either FAAH (URB597) or MGL (URB602) have been developed, which provide the possibility of enhancing CB1 receptor activation by increasing the brain levels of endocannabinoids. Studies with these drugs as well as with genetically modified mice have related endocannabinoids to several functions of the central nervous system (for review, see [20]).

Other putative components of this system are the transient receptor potential vanilloid type 1 (TRPV1), the peroxisome-proliferator activated receptor, and the G protein-coupled receptor GPR55. Although anandamide binds to all these receptors, their functions remain uncertain [21]. In addition, an allosteric site in the CB1 receptor has been identified [22] and there is the possibility that, contrary to the initial thoughts, CB2 receptors may indeed be relevant for behavioral responses [23, 24]. Finally, more substances have been proposed as endocannabinoids, such as arachidonoyl dopamine, virodhamine, and noladin ether [20].

2. Cannabinoids and Anxiety

Natural or synthetic cannabinoids or CB1 receptor antagonists often yield complex responses in experimental models of anxiety. As summarized in Table 1, several authors have noticed bell-shaped dose-response curves in animal models predictive of anxiogenic- or anxiolytic-like activity, namely, the elevated plus maze (EPM), the elevated zero maze (EZM), the light dark test (DLT), and the Vogel conflict test (VCT). CB1 receptor agonists tend to be anxiolytic in lower doses, whereas higher doses may be anxiogenic [25]. However, compounds that enhance endocannabinoid effects, such as inhibitors of AEA uptake or hydrolysis, appear to produce only anxiolytic effects without bell-shaped dose-response curves (Table 1).

Table 1

Effects of cannabinoids and drugs that interfere with the endocannabinoid system in animal models predictive of anxiolytic- or anxiogenic-like activity. (AEA: AEA; Δ⁹-THC: Δ⁹-tetrahydrocannabinol; CBD: cannabidiol; EPM: elevated plus-maze; EXM: (more ...)

The reasons for these complex effects remain unknown. One possibility could be that these drugs would interfere with diverse brain regions which have different roles in the modulation of anxiety-like responses. However, the sites responsible for the effects of cannabinoids remain poorly investigated. CB1 receptors, as well as the putative protein responsible for internalization of AEA and the enzyme FAAH, are expressed in several regions of CNS related to anxiety, aversion, and defensive behaviors, including the prefrontal cortex, amygdala, hippocampus, hypothalamus, and PAG [13, 14]. These structures are proposed to be part of a system responsible for the elaboration of behavioral and autonomic responses to aversive stimuli. They are possible neural sites whose malfunction would lead to psychiatric pathologies such as generalized anxiety and panic disorders [26]. In this context, anxiolytic drugs would act by normalizing the functions of these structures [27, 28]. Moreover, this brain aversive system would be responsible for behavioral suppression in animal models predictive of anxiolytic-like activity. Generally, models of experimental anxiety rely on exposing animals to situation that generates conflicts between approach and avoidance, which can be generated by the drive of exploring a new, though, aversive environment, or by a source of reward that is associated with punishment. Anxiolytic-like drugs injected either systemically or into these structures shift the conflict toward approach responses [27, 28]. Thus, these models provide invaluable insights into the neurobiology of anxiety and the pharmacology of anxiolytic compounds. As discussed below, we have used direct drug administration in animals submitted to these models for studying the possible role of the PAG in the antiaversive actions of cannabinoids.

3. Anxiolytic Effects of Cannabinoids in the Periaqueductal Gray

The PAG is a mesencephalic structure that surrounds the cerebral aqueduct and can be divided along its rostrocaudal axis into dorsomedial, dorsolateral (dlPAG), lateral, and ventrolateral columns [29]. It is an important site in ascending pain transmission and a major component of a descending pain inhibitory system. Moreover, this structure receives glutamatergic projection from forebrain regions and sends descendent pathways to motor outputs and to autonomic centres that control blood pressure and heart rate [26]. The dorsal columns (dPAG) are possibly responsible for the elaboration of active defensive behaviors (see [26], for review). Lesions of the dPAG inhibit fear and anxiety produced by stimulation of the amygdala whereas stimulation of this region induces threat display associated with vocalization and strong flight responses [26]. In the caudal ventrolateral PAG, however, immobility has been described as the main outcome of local stimulation [30].

CB1 receptors are distributed along the various columns of this structure [13]. Moreover, administration of CB1 agonists increases Fos expression [31] and brain metabolic activity in the PAG of rats [32], suggesting that this structure could be involved in the effects of systemically administered cannabinoids. In agreement with this proposal, injection of CB1 receptor agonists into the dlPAG of rats has been shown to induce antinociceptive responses [33] and electric stimulation of the dorsal and lateral columns induces antinociception via activation of CB1 receptors accompanied by local AEA release [34]. Furthermore, subcutaneous formalin injection, a painful stimulus, substantially increased the release of AEA in the PAG [34, 35].

Concerning the possible involvement of PAG-endocannabinoid system on modulation of anxiety-like behaviors, an initial study showed that local administration of HU210, a potent CB1 agonist, attenuated the flight responses induced by dPAG injections of the excitatory amino acid D,L-homocysteic acid (see [36, Table 2]). In a subsequent study, where the injections were restricted to the dorsomedial PAG, HU210 decreased hyperlocomotion induced by aversive ultrasound stimulation, but failed to change freezing responses. Moreover, HU210 effects were not entirely blocked by previous local injection of a CB1 receptor antagonist [70].

Considering these initial results, we decided to further investigate a possible influence of the PAG-endocannabinoid system on anxiety-like behaviors in rats submitted to different animal models of anxiety (Table 2). First, we showed that AEA injected into the dlPAG increased the exploration of the open arms of the elevated plus maze (EPM) [71], a model based on a natural conflict between exploratory behavior and innate fear of open spaces. The effects of AEA were similar to those observed with classical anxiolytic benzodiazepines [72] and were blocked by previous treatment with AM251, a CB1 receptor antagonist. These effects were also potentiated by previous treatment with AM404, an inhibitor of AEA uptake/metabolism. AM404 by itself, however, was without effect in this model. AEA produced an inverted U-shaped dose-response curve, with higher doses being ineffective [71].

Table 2

Effects of Cannabinoid-related drugs injected into the PAG of rats submitted to animal models of anxiety-related behaviors. (AEA: anandamide; ACEA: arachidonyl-2-chloro-ethylamide; CBD: cannabidiol; EPM: elevated plus-maze; VCT: Vogel conflict test; CFC: (more ...)

To confirm a possible anticonflict effect of AEA in the dlPAG, we used the Vogel conflict test (VCT) [73], an animal model of anxiety not based on innate fear but instead on suppression of punished responses learned during the test. In this model, water-deprived rodents are exposed to a conflict between licking the spout of a bottle containing water and receiving a mild shock on the tong [74]. Anxiolytics that potentiate the action of γ-aminobutyric acid such as the benzodiazepines typically increase the number of punished licks [75]. AEA also induced anxiolytic-like effects in the VCT at the same dose range observed in the EPM (Table 2). Different from the results obtained in the latter model, AM404 was also able to increase the number of punished licks (Table 2). Although the causes of these contradictory results are not clear, they could involve the distinct animal models of anxiety employed. Brain endocannabinoids have been proposed to act as a “stress buffer system” [76], recruited by highly demanding situations. It was possible that the VCT, by involving pain and water deprivation, engages the endocannabinoid system in the dlPAG to a greater extent than the EPM. Actually, as discussed above, painful stimuli such as those used in the VCT have already been showed to increase AEA in this region [77].

We have further investigated this effect by intra-dlPAG administration of AEA and AM404 in rats submitted to a contextual fear conditioning paradigm, an animal model that also involves pain exposure [78]. Animals re-exposed to an environment where they had being previously submitted to an aversive stimulation, such as electrical footshocks, show behavioral and cardiovascular changes characterized by immobility (freezing) and mean arterial pressure (MAP) and heart rate (HR) increases [79, 80]. Although electrical or chemical stimulation of the dorsal portion of PAG is usually related with flight reactions, it can also produce freezing responses and increased cardiovascular activity [26]. Re-exposure to an aversively conditioned context increases neuronal activity in the PAG [81, 82], and PAG lesions block freezing to aversively conditioned stimulus [83, 84]. dlPAG microinjection of AEA or AM404 blocked the expression of the conditioned aversive responses [78]. This effect was inhibited by local pretreatment with AM251, reinforcing the involvement of CB1 receptors.

Altogether, these results suggest that the endocannabinoid system in the dlPAG can modulate responses to aversive stimuli. The mechanisms of these effects are still unclear. Using brain slices of the rat PAG, Vaughan et al. [85] showed that cannabinoids act via CB1 receptors to inhibit GABAergic and glutamatergic synaptic transmission. The efficacy of endogenous cannabinoids was limited by uptake and breakdown since AEA was only able to inhibit evoked inhibitory postsynaptic currents in the presence of the AT inhibitor, AM404. Several studies indicate that GABA- and glutamate-mediated neurotransmissions in the dPAG play opposite roles. While the former tonically inhibits defensive responses, the latter facilitates them [26]. Thus, CB1-mediated inhibitory effects on these two neurotransmitter systems could be one of the explanations for the observed bell-shaped dose-response curve induced by AEA in this region as well as the contradictory results regarding the effects of cannabinoids on anxiety (see Table 1 and text bellow for a discussion on the possible involvement of TRPV1 receptors).

These mechanisms may explain the effects in the PAG, yet they do not necessarily apply to other brain regions. In some areas, the levels of CB1 receptor expression can be higher in GABAergic (particularly in cholecystokinin-containing basket cells) as compared to glutamatergic neurons, with cannabinoid effects favoring impairment of inhibitory mechanisms mediated by the former neuronal population [16]. However, it remains to be further investigated how these neural subpopulations contribute to specific behavioral effects of cannabinoids. In addition, GABAergic and glutamatergic neurons may have different sensitivity to CB1 agonists or antagonists depending on the species under investigation. For instance, Haller et al. [52] observed opposite effects in mice and rats tested with the same doses of a cannabinoid in models of anxiety-like behavior (see Table 1). Inhibitory and excitatory currents were differentially affected in the hippocampi of these species, providing a possible basis for the discrepancies in the behavioral responses. Since we have employed rats as subjects in all our experiments, studies in other species could further consolidate our hypothesis that glutamatergic and GABAergic inhibitions would mediate anxiolytic- and anxiogenic-like effects of cannabinoids, respectively. For a more extensive discussion on the relevance of diverse neural subpopulations for the effects of cannabinoids, see [89].

4. Cannabidiol

Cannabidiol (CBD) is a major nonpsychotomimetic constituent of Cannabis sativa that is able to antagonize the anxiogenic and psychotomimetic effects of high doses of Δ⁹-THC [90, 91]. It also promotes anxiolytic-like effects in several animal models (see [44–47], Table 1). In addition, CBD induces anxiolytic effects in healthy volunteers in the simulated public speaking test, a model of clinical anxiety, and in subjects submitted to a functional imaging analysis study [92, 93]. However, as commonly seem with other cannabinoids in animal models of anxiety, experiments with CBD yield bell-shaped dose-response curves, low doses being anxiolytic, and higher doses being ineffective [44]. The mechanisms for these actions remain poorly understood. CBD has low affinity for CB1 or CB2 receptors and could facilitate the endocannabinoid signalling by inhibition of AEA uptake or its enzymatic hydrolysis. It can also act as an agonist of TRPV1 or 5HT1A receptors [94, 95].

Considering that the PAG, in addition to CB1 [14], also expresses a significant number of TRPV1 and 5HT1A receptors [96, 97], we decided to verify if this region could be related to the effects of CBD. We found that CBD microinjections into the dlPAG produced anxiolytic-like effects in rats submitted to the EPM or the VCT [86] (Table 2). The effects in the EPM, however, also showed a bell-shaped dose-response curve, but were not blocked by previous local administration of AM251 [86], employed at the same dose that was able to antagonize the anxiolytic-like effects of AEA and AM404 (Table 2). The anxiolytic-like effects of CBD, however, were prevented by WAY100635, an antagonist of 5HT1A receptors. Activation of these Gi-coupled-receptors enhances K+ currents and inhibits adenylyl cyclase activity [98]. They act as inhibitory autoreceptors in serotonergic neurons in the raphe nuclei but are also localized postsynaptically in several brain regions, including the PAG, amygdala, hippocampus, and frontal cortex. Actually, the PAG receives serotonergic projections from the dorsal raphe nuclei, and local activation of 5HT1A receptors promotes the control of anxiety states and the hypothalamus-pituitary-adrenal axis during stress responses [99]. Thus, 5HT1A receptors located in the PAG are possibly involved in the anxiolytic-like effects of CBD, a hypothesis corroborated by several studies showing that agonists of these receptors produce anxiolytic effects in the PAG [100, 101].

5. TRPV1 Receptors Methods

TRPV1 receptors belong to a large family of calcium-permeable cation channels [102]. They can be activated by elevation in temperature, pH decrease, or by exogenous ligands such as capsaicin, the pungent ingredient of red hot chilli peppers [103, 104]. They have been related to pain transmission and inflammatory responses in the peripheral nervous system. In addition to environmental stimuli, endocannabinoids such as AEA and N-arachidonyldopamine can also activate TRPV1 receptors. As a consequence, they can also be denominated endovanilloids [104, 105].

TRPV1 receptors are expressed in various brain regions related to anxiety, including the PAG [106, 107], where they can regulate glutamate release. Corroborating this proposal, local infusion of capsaicin produces antinociception by increasing glutamate release in this region [108]. In addition, activation of presynaptic TRPV1 receptors produced an excitatory effect in the firing activity of dlPAG neurons [109]. Glutamate is the main excitatory neurotransmitter in the central nervous system, and the injection of NMDA antagonist receptors into the dlPAG promotes anxiolytic effects in the EPM and VCT [110].

Few studies, however, have investigated the role of TRPV1 in anxiety. Systemic administration of capsazepine, a TRPV1 antagonist, induced anxiolytic-like effects in rats submitted to the EPM (Table 1) [69]. More recently, Marsch et al. [111] demonstrated that TRPV1-deficient mice show decreased anxiety in the EPM and light-dark test. Accordingly, the dual FAAH/TRPV1 blocker N-arachidonoyl-serotonin is able to induce CB1-mediated anxiolytic-like effects more potently than selective blockers of FAAH or TRPV1, further suggesting opposite roles for CB1 and TRPV1 receptors [112].

To further investigate the role of TRPV1 on anxiety modulation, we verified the effects of intra-dlPAG injection of capsazepine in rats submitted to the EPM and VCT. This drug decreased anxiety-like behaviors in both models (Table 2), suggesting that TRPV1 receptors facilitate anxiety responses in the PAG. The fact that AEA and CBD can also activate TRPV1 receptors [94, 104, 105] could help to explain the bell-shaped dose-response curves usually found with these compounds regarding their anxiolytic effects (Tables 1 and 2). In agreement with this proposal, it was recently showed that capsazepine blocks the anxiogenic effects of high doses of AEA in the prefrontal cortex [113]. It remained to be tested if similar effects could occur in the dlPAG. In an initial study, we confirmed this possibility, showing that intra-dlPAG pretreatment with an ineffective dose of capsazepine was able to turn the higher, ineffective dose of the CBD into an anxiolytic one (Table 2).

6. Conclusions

The pieces of evidence revised above suggest that the PAG, particularly its dorsolateral column, is involved in the modulatory effects of cannabinoids on defensive responses. This does not mean that the PAG is the only or the most relevant structure accounting for the antiaversive properties of cannabinoids. Other authors have also identified brain sites where CB1 receptor activation induces anxiolytic-like effects. Injection of low doses of Δ⁹-THC either in the ventral hippocampus (5 μg) or in the prefrontal cortex (10 μg) resulted in anxiolytic-like effects; whereas in the amygdala (1 μg), opposite results were reported [114]. An early work has also shown anxiogenic-like effect of Δ⁹-THC in this brain region [115] Moreover, intraprefrontal cortex injection of low or high doses of methanandamide induces CB1-mediated anxiolytic- or TRPV1-mediated anxiogenic-like effects, respectively [113]. Other authors have also investigated brain sites mediating nociceptive responses, antidepressive-like activity, and rewarding effects of cannabinoids [89].

In conclusion, local administration of CB1 agonists into the dlPAG produces anxiolytic-like effects in several animal models. These effects are prevented by AM251, indicating that they are being mediated by activation of CB1 receptors, possibly by presynaptic inhibition of glutamate release (see Figure 1). Results with AM404, an AEA metabolism/uptake inhibitor, also suggest that local synthesis of endocannabinoids in the dlPAG can modulate defensive responses, at least under high-aversive conditions. The results also showed that the dlPAG could be involved in the reported anxiolytic effects of CBD, a nonpsychotomimetic phytocannabinoid. This compound, however, appears to act by activating 5HT1A receptors (Figure 1). Finally, activation of vanilloid TRPV1 receptors in the dlPAG seems to facilitate defensive responses (Figure 1) and may be, in part, responsible for the bell-shaped dose-response curves of the anxiolytic effects of AEA and CBD. A balance between CB1- and TRPV1-activations is a possible mechanism through which endogenous AEA could control aversive responses.

Figure 1

Possible effects of cannabinoids in the dlPAG. Glutamatergic inputs from forebrain structures such as the dorsomedial part of the ventromedial hypothalamic nucleus (dmVMH) and dorsal premammilary hypothalamic nucleus (PmD) activate a local neural substrate (more ...)

Acknowledgments

The authors would like to thank the excellent technical support provided by J. C. Aguiar and E. T. Gomes. This research is supported by grants from FAPESP and CNPq. D. C. Aguiar, A. C. Campos, S. F. Lisboa, and L. B. Resstel are recipients of FAPESP fellowships. S. F. Lisboa and A. L. Terzian received a CNPq fellowship.

References

Booth M. Cannabis: A History. London, UK: Banton Books; 2003.

Courtwright DT. Forces of Habit: Drugs and the Making of the Modern World. Cambridge, Mass, USA: Harvard University Press; 2001.

Zuardi AW. History of cannabis as a medicine: a review. Revista Brasileira de Psiquiatria. 2006;28(2):153–157. [PubMed]

Mechoulam R. Marijuana chemistry. Science. 1970;168(936):1159–1166. [PubMed]

Paton WDM. Pharmacology of marijuana. Annual Review of Pharmacology. 1975;15:191–220.

Devane WA, Dysarz FA, III, Johnson MR, Melvin LS, Howlett AC. Determination and characterization of a cannabinoid receptor in rat brain. Molecular Pharmacology. 1988;34(5):605–613. [PubMed]

Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature. 1990;346(6284):561–564. [PubMed]

Devane WA, Hanus L, Breuer A, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science. 1992;258(5090):1946–1949. [PubMed]

Mechoulam R, Ben-Shabat S, Hanus L, et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochemical Pharmacology. 1995;50(1):83–90. [PubMed]

10.

Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature. 1993;365(6441):61–65. [PubMed]

11.

Rinaldi-Carmona M, Barth F, Héaulme M, et al. SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS Letters. 1994;350(2-3):240–244. [PubMed]

12.

Gatley SJ, Gifford AN, Volkow ND, Lan R, Makriyannis A. ¹²³I-labeled AM251: a radioiodinated ligand which binds in vivo to mouse brain cannabinoid CB₁ receptors. European Journal of Pharmacology. 1996;307(3):331–338. [PubMed]

13.

Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. The Journal of Neuroscience. 1991;11(2):563–583. [PubMed]

14.

Tsou K, Brown S, Sañudo-Peña MC, Mackie K, Walker JM. Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience. 1998;83(2):393–411. [PubMed]

15.

Egertov M, Giang DK, Cravatt BF, Elphick MR. A new perspective on cannabinoid signalling: complementary localization of fatty acid amide hydrolase and the CB1 receptor in rat brain. Proceedings of the Royal Society B. 1998;265(1410):2081–2085. [PubMed]

16.

Wilson RI, Nicoll RA. Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature. 2001;410(6828):588–592. [PubMed]

17.

Howlett AC, Barth F, Bonner TI, et al. International Union of Pharmacology. XXVII. Classification of cannabinoid receptors. Pharmacological Reviews. 2002;54(2):161–202. [PubMed]

18.

Giuffrida A, Beltramo M, Piomelli D. Mechanisms of endocannabinoid inactivation: biochemistry and pharmacology. Journal of Pharmacology and Experimental Therapeutics. 2001;298(1):7–14. [PubMed]

19.

McKinney MK, Cravatt BE. Structure and function of fatty acid amide hydrolase. Annual Review of Biochemistry. 2005;74:411–432.

20.

Pacher P, Bátkai S, Kunos G. The endocannabinoid system as an emerging target of pharmacotherapy. Pharmacological Reviews. 2006;58(3):389–462. [PubMed]

21.

Brown AJ. Novel cannabinoid receptors. British Journal of Pharmacology. 2007;152(5):567–575. [PubMed]

22.

Price MR, Baillie GL, Thomas A, et al. Allosteric modulation of the cannabinoid CB₁ receptor. Molecular Pharmacology. 2005;68(5):1484–1495. [PubMed]

23.

Van Sickle MD, Duncan M, Kingsley PJ, et al. Identification and functional characterization of brainstem cannabinoid CB₂ receptors. Science. 2005;310(5746):329–332. [PubMed]

24.

Gong J-P, Onaivi ES, Ishiguro H, et al. Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain Research. 2006;1071(1):10–23. [PubMed]

25.

Viveros MP, Marco EM, File SE. Endocannabinoid system and stress and anxiety responses. Pharmacology Biochemistry and Behavior. 2005;81(2):331–342.

26.

Graeff FG. Neuroanatomy and neurotransmitter regulation of defensive behaviours and related emotions in mammals. Brazilian Journal of Medical and Biological Research. 1994;27(4):811–829. [PubMed]

27.

McNaughton N, Gray JA. Anxiolytic action on the behavioural inhibition system implies multiple types of arousal contribute to anxiety. Journal of Affective Disorders. 2000;61(3):161–176. [PubMed]

28.

McNaughton N, Corr PJ. A two-dimensional neuropsychology of defense: fear/anxiety and defensive distance. Neuroscience & Biobehavioral Reviews. 2004;28(3):285–305. [PubMed]

29.

Bandler R, Keay KA, Floyd N, Price J. Central circuits mediating patterned autonomic activity during active vs. passive emotional coping. Brain Research Bulletin. 2000;53(1):95–104. [PubMed]

30.

Bandler R, Depaulis A. Elicitation of intraspecific defence reactions in the rat from midbrain periaqueductal grey by microinjection of kainic acid, without neurotoxic effects. Neuroscience Letters. 1988;88(3):291–296. [PubMed]

31.

Patel NA, Moldow RL, Patel JA, Wu G-D, Chang SL. Arachidonylethanolamide (AEA) activation of FOS proto-oncogene protein immunoreactivity in the rat brain. Brain Research. 1998;797(2):225–233. [PubMed]

32.

Chin C-L, Tovcimak AE, Hradil VP, et al. Differential effects of cannabinoid receptor agonists on regional brain activity using pharmacological MRI. British Journal of Pharmacology. 2008;153(2):367–379. [PubMed]

33.

Martin WJ, Patrick SL, Coffin PO, Tsou K, Walker JM. An examination of the central sites of action of cannabinoid-induced antinociception in the rat. Life Sciences. 1995;56(23-24):2103–2109. [PubMed]

34.

Walker JM, Huang SM, Strangman NM, Tsou K, Sañudo-Peña MC. Pain modulation by release of the endogenous cannabinoid anandamide. Proceedings of the National Academy of Sciences of the United States of America. 1999;96(21):12198–12203. [PubMed]

35.

Hohmann AG, Suplita RL., II Endocannabinoid mechanisms of pain modulation. The AAPS Journal. 2006;8(4):E693–E708. [PubMed]

36.

Finn DP, Jhaveri MD, Beckett SRG, et al. Effects of direct periaqueductal grey administration of a cannabinoid receptor agonist on nociceptive and aversive responses in rats. Neuropharmacology. 2003;45(5):594–604. [PubMed]

37.

Onaivi ES, Green MR, Martin BR. Pharmacological characterization of cannabinoids in the elevated plus maze. Journal of Pharmacology and Experimental Therapeutics. 1990;253(3):1002–1009. [PubMed]

38.

Valjent E, Mitchell JM, Besson M-J, Caboche J, Maldonado R. Behavioural and biochemical evidence for interactions between Δ⁹-tetrahydrocannabinol and nicotine. British Journal of Pharmacology. 2002;135(2):564–578. [PubMed]

39.

Berrendero F, Maldonado R. Involvement of the opioid system in the anxiolytic-like effects induced by Δ⁹-tetrahydrocannabinol. Psychopharmacology. 2002;163(1):111–117. [PubMed]

40.

Patel S, Hillard CJ. Pharmacological evaluation of cannabinoid receptor ligands in a mouse model of anxiety: further evidence for an anxiolytic role for endogenous cannabinoid signalling. Journal of Pharmacology and Experimental Therapeutics. 2006;318(1):304–311. [PubMed]

41.

Braida D, Limonta V, Malabarba L, Zani A, Sala M. 5-HT_1A receptors are involved in the anxiolytic effect of Δ⁹-tetrahydrocannabinol and AM 404, the anandamide transport inhibitor, in Sprague-Dawley rats. European Journal of Pharmacology. 2007;555(2-3):156–163. [PubMed]

42.

Schramm-Sapyta NL, Cha YM, Chaudhry S, Wilson WA, Swartzwelder HS, Kuhn CM. Differential anxiogenic, aversive, and locomotor effects of THC in adolescent and adult rats. Psychopharmacology. 2007;191(4):867–877. [PubMed]

43.

Rubino T, Sala M, Viganò D, et al. Cellular mechanisms underlying the anxiolytic effect of low doses of peripheral Δ⁹-tetrahydrocannabinol in rats. Neuropsychopharmacology. 2007;32(9):2036–2045. [PubMed]

44.

Guimarães FS, Chiaretti TM, Graeff FG, Zuardi AW. Antianxiety effect of cannabidiol in the elevated plus-maze. Psychopharmacology. 1990;100(4):558–559. [PubMed]

45.

Guimarães FS, de Aguiar JC, Mechoulam R, Breuer A. Anxiolytic effect of cannabidiol derivatives in the elevated plus-maze. General Pharmacology. 1994;25(1):161–164. [PubMed]

46.

Moreira FA, Aguiar DC, Guimarães FS. Anxiolytic-like effect of cannabidiol in the rat Vogel conflict test. Progress in Neuro-Psychopharmacology and Biological Psychiatry. 2006;30(8):1466–1471. [PubMed]

47.

Resstel LB, Joca SRL, Moreira FA, Corrêaa FMA, Guimarães FS. Effects of cannabidiol and diazepam on behavioural and cardiovascular responses induced by contextual conditioned fear in rats. Behavioural Brain Research. 2006;172(2):294–298. [PubMed]

48.

Giuliani D, Ferrari F, Ottani A. The cannabinoid agonist HU 210 modifies rat behavioural responses to novelty and stress. Pharmacological Research. 2000;41(1):45–51. [PubMed]

49.

Jiang W, Zhang Y, Xiao L, et al. Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects. The Journal of Clinical Investigation. 2005;115(11):3104–3116. [PubMed]

50.

Hill MN, Gorzalka BB. Enhancement of anxiety-like responsiveness to the cannabinoid CB₁ receptor agonist HU-210 following chronic stress. European Journal of Pharmacology. 2004;499(3):291–295. [PubMed]

51.

Haller J, Varga B, Ledent C, Freund TF. CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1-specific agents. Behavioural Pharmacology. 2004;15(4):299–304. [PubMed]

52.

Haller J, Mátyás F, Soproni K, et al. Correlated species differences in the effects of cannabinoid ligands on anxiety and on GABAergic and glutamatergic synaptic transmission. European Journal of Neuroscience. 2007;25(8):2445–2456. [PubMed]

53.

Arévalo C, de Miguel R, Hernández-Tristán R. Cannabinoid effects on anxiety-related behaviours and hypothalamic neurotransmitters. Pharmacology Biochemistry and Behavior. 2001;70(1):123–131.

54.

Marín S, Marco E, Biscaia M, et al. Involvement of the κ-opioid receptor in the anxiogenic-like effect of CP 55,940 in male rats. Pharmacology Biochemistry and Behavior. 2003;74(3):649–656.

55.

Marco EM, Pérez-Alvarez L, Borcel E, et al. Involvement of 5-HT1A receptors in behavioural effects of the cannabinoid receptor agonist CP 55,940 in male rats. Behavioural Pharmacology. 2004;15(1):21–27. [PubMed]

56.

Genn RF, Tucci S, Marco E, Viveros M-P, File SE. Anxiolytic and anxiogenic effects of the cannabinoid agonist CP 55,940 in animal tests of anxiety. Journal of Psychopharmacology. 2003;17:p. A27.

57.

Genn RF, Tucci S, Marco EM, Viveros MP, File SE. Unconditioned and conditioned anxiogenic effects of the cannabinoid receptor agonist CP 55,940 in the social interaction test. Pharmacology Biochemistry and Behavior. 2004;77(3):567–573.

58.

Chakrabarti A, Ekuta JE, Onaivi ES. Neurobehavioral effects of anandamide and cannabinoid receptor gene expression in mice. Brain Research Bulletin. 1998;45(1):67–74. [PubMed]

59.

Scherma M, Medalie J, Fratta W, et al. The endogenous cannabinoid anandamide has effects on motivation and anxiety that are revealed by fatty acid amide hydrolase (FAAH) inhibition. Neuropharmacology. 2008;54(1):129–140. [PubMed]

60.

Bortolato M, Campolongo P, Mangieri RA, et al. Anxiolytic-like properties of the anandamide transport inhibitor AM404. Neuropsychopharmacology. 2006;31(12):2652–2659. [PubMed]

61.

Kathuria S, Gaetani S, Fegley D, et al. Modulation of anxiety through blockade of anandamide hydrolysis. Nature Medicine. 2003;9(1):76–81. [PubMed]

62.

Naidu PS, Varvel SA, Ahn K, Cravatt BF, Martin BR, Lichtman AH. Evaluation of fatty acid amide hydrolase inhibition in murine models of emotionality. Psychopharmacology. 2007;192(1):61–70. [PubMed]

63.

Moreira FA, Kaiser N, Monory K, Lutz B. Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors. Neuropharmacology. 2008;54(1):141–150. [PubMed]

64.

Rutkowska M, Jamontt J, Gliniak H. Effects of cannabinoids on the anxiety-like response in mice. Pharmacological Reports. 2006;58(2):200–206. [PubMed]

65.

Navarro M, Hernández E, Muñoz RM, et al. Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxiety-like responses in the rat. NeuroReport. 1997;8(2):491–496. [PubMed]

66.

Haller J, Bakos N, Szirmay M, Ledent C, Freund TF. The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. European Journal of Neuroscience. 2002;16(7):1395–1398. [PubMed]

67.

Griebel G, Stemmelin J, Scatton B. Effects of the cannabinoid CB1 receptor antagonist rimonabant in models of emotional reactivity in rodents. Biological Psychiatry. 2005;57(3):261–267. [PubMed]

68.

Rodgers RJ, Evans PM, Murphy A. Anxiogenic profile of AM-251, a selective cannabinoid CB1 receptor antagonist, in plus-maze-naïve and plus-maze-experienced mice. Behavioural Pharmacology. 2005;16(5-6):405–413. [PubMed]

69.

Kasckow JW, Mulchahey JJ, Geracioti TD., Jr. Effects of the vanilloid agonist olvanil and antagonist capsazepine on rat behaviours. Progress in Neuropsychopharmacology and Biological Psychiatry. 2004;28(2):291–295.

70.

Finn DP, Jhaveri MD, Beckett SRG, Kendall DA, Marsden CA, Chapman V. Cannabinoids modulate ultrasound-induced aversive responses in rats. Psychopharmacology. 2004;172(1):41–51. [PubMed]

71.

Moreira FA, Aguiar DC, Guimarães FS. Anxiolytic-like effect of cannabinoids injected into the rat dorsolateral periaqueductal gray. Neuropharmacology. 2007;52(3):958–965. [PubMed]

72.

Pellow S, File SE. Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacology Biochemistry and Behavior. 1986;24(3):525–529.

73.

Lisboa SF, Resstel LB, Aguiar DC, Guimarães FS. Activation of cannabinoid CB₁ receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test. European Journal of Pharmacology. 2008;593(1–3):73–78. [PubMed]

74.

Vogel JR, Beer B, Clody DE. A simple and reliable conflict procedure for testing anti-anxiety agents. Psychopharmacologia. 1971;21(1):1–7. [PubMed]

75.

Millan MJ. The neurobiology and control of anxious states. Progress in Neurobiology. 2003;70(2):83–244. [PubMed]

76.

Viveros M-P, Marco E-M, Llorente R, López-Gallardo M. Endocannabinoid system and synaptic plasticity: implications for emotional responses. Neural Plasticity. 2007;2007:12 pages. Article ID 52908.

77.

Hohmann AG, Suplita RL, Bolton NM, et al. An endocannabinoid mechanism for stress-induced analgesia. Nature. 2005;435(7045):1108–1112. [PubMed]

78.

Resstel LB, Lisboa SF, Aguiar DC, Corrêa FMA, Guimarães FS. Activation of CB1 cannabinoid receptors in the dorsolateral periaqueductal gray reduces the expression of contextual fear conditioning in rats. Psychopharmacology. 2008;198(3):405–411. [PubMed]

79.

Fanselow MS. Conditional and unconditional components of post-shock freezing. The Pavlovian Journal of Biological Science. 1980;15(4):177–182. [PubMed]

80.

Resstel LB, Joca SR, Guimarães FG, Corrêa FMA. Involvement of medial prefrontal cortex neurons in behavioural and cardiovascular responses to contextual fear conditioning. Neuroscience. 2006;143(2):377–385. [PubMed]

81.

Carrive P, Lee J, Su A. Lidocaine blockade of amygdala output in fear-conditioned rats reduces Fos expression in the ventrolateral periaqueductal gray. Neuroscience. 1999;95(4):1071–1080. [PubMed]

82.

Carrive P, Leung P, Harris J, Paxinos G. Conditioned fear to context is associated with increased Fos expression in the caudal ventrolateral region of the midbrain periaqueductal gray. Neuroscience. 1997;78(1):165–177. [PubMed]

83.

Amorapanth P, Nader K, LeDoux JE. Lesions of periaqueductal gray dissociate-conditioned freezing from conditioned suppression behaviour in rats. Learning & Memory. 1999;6(5):491–499. [PubMed]

84.

LeDoux JE, Iwata J, Cicchetti P, Reis DJ. Different projections of the central amygdaloid nucleus mediate autonomic and behavioural correlates of conditioned fear. The Journal of Neuroscience. 1988;8(7):2517–2529. [PubMed]

85.

Vaughan CW, Connor M, Bagley EE, Christie MJ. Actions of cannabinoids on membrane properties and synaptic transmission in rat periaqueductal gray neurons in vitro. Molecular Pharmacology. 2000;57(2):288–295. [PubMed]

86.

Campos AC, Guimarães FS. Involvement of 5HT1A receptors in the anxiolytic-like effects of cannabidiol injected into the dorsolateral periaqueductal gray of rats. Psychopharmacology. 2008;199(2):223–230. [PubMed]

87.

Terzian AL, Aguiar DC, Guimarães FS, Moreira FA. Modulation of anxiety-like behaviour by Transient Receptor Potential Vanilloid Type 1 Channel (TRPV1) located in the dorsolateral periaqueductal gray. European Neuropsychopharmacology. In press.

88.

Canteras NS. The medial hypothalamic defensive system: hodological organization and functional implications. Pharmacology Biochemistry and Behavior. 2002;71(3):481–491.

89.

Moreira FA, Lutz B. The endocannabinoid system: emotion, learning and addiction. Addiction Biology. 2008;13(2):196–212. [PubMed]

90.

Zuardi AW, Shirakawa I, Finkelfarb E, Karniol IG. Action of cannabidiol on the anxiety and other effects produced by Δ⁹-THC in normal subjects. Psychopharmacology. 1982;76(3):245–250. [PubMed]

91.

Zuardi AW, Crippa JAS, Hallak JEC, Moreira FA, Guimarães FS. Cannabidiol, a Cannabis sativa constituent, as an antipsychotic drug. Brazilian Journal of Medical and Biological Research. 2006;39(4):421–429. [PubMed]

92.

Zuardi AW, Cosme RA, Graeff FG, Guimarães FS. Effects of ipsapirone and cannabidiol on human experimental anxiety. Journal of Psychopharmacology. 1993;7(1):82–88.

93.

de Souza Crippa JA, Zuardi AW, Garrido GEJ, et al. Effects of cannabidiol (CBD) on regional cerebral blood flow. Neuropsychopharmacology. 2004;29(2):417–426. [PubMed]

94.

Bisogno T, Hanuš L, De Petrocellis L, et al. Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide. British Journal of Pharmacology. 2001;134(4):845–852. [PubMed]

95.

Russo EB, Burnett A, Hall B, Parker KK. Agonistic properties of cannabidiol at 5-HT1a receptors. Neurochemical Research. 2005;30(8):1037–1043. [PubMed]

96.

Cristino L, de Petrocellis L, Pryce G, Baker D, Guglielmotti V, Di Marzo V. Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience. 2006;139(4):1405–1415. [PubMed]

97.

Nogueira RL, Graeff FG. Role of 5-HT receptor subtypes in the modulation of dorsal periaqueductal gray generated aversion. Pharmacology Biochemistry and Behavior. 1995;52(1):1–6.

98.

Raymond JR, Mukhin YV, Gettys TW, Garnovskaya MN. The recombinant 5-HT_1A receptor: G protein coupling and signalling pathways. British Journal of Pharmacology. 1999;127(8):1751–1764. [PubMed]

99.

Carrasco GA, Van de Kar LD. Neuroendocrine pharmacology of stress. European Journal of Pharmacology. 2003;463(1–3):235–272. [PubMed]

100.

Zanoveli JM, Nogueira RL, Zangrossi H., Jr. Serotonin in the dorsal periaqueductal gray modulates inhibitory avoidance and one-way escape behaviours in the elevated T-maze. European Journal of Pharmacology. 2003;473(2-3):153–161. [PubMed]

101.

de Paula Soares V, Zangrossi H., Jr. Involvement of 5-HT_1A and 5-HT₂ receptors of the dorsal periaqueductal gray in the regulation of the defensive behaviours generated by the elevated T-maze. Brain Research Bulletin. 2004;64(2):181–188. [PubMed]

102.

Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997;389(6653):816–824. [PubMed]

103.

Szallasi A, Blumberg PM. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacology Reviews. 1999;51(2):159–212.

104.

van der Stelt M, Di Marzo V. Endovanilloids: putative endogenous ligands of transient receptor potential vanilloid 1 channels. European Journal of Biochemistry. 2004;271(10):1827–1834. [PubMed]

105.

Marinelli S, Di Marzo V, Florenzano F, et al. N-arachidonoyl-dopamine tunes synaptic transmission onto dopaminergic neurons by activating both cannabinoid and vanilloid receptors. Neuropsychopharmacology. 2007;32(2):298–308. [PubMed]

106.

Mezey É, Tóth ZE, Cortright DN, et al. Distribution of mRNA for vanilloid receptor subtype 1 (VR1), and VR1-like immunoreactivity, in the central nervous system of the rat and human. Proceedings of the National Academy of Sciences of the United States of America. 2000;97(7):3655–3660. [PubMed]

107.

McGaraughty S, Chu KL, Bitner RS, et al. Capsaicin infused into the PAG affects rat tail flick responses to noxious heat and alters neuronal firing in the RVM. Journal of Neurophysiology. 2003;90(4):2702–2710. [PubMed]

108.

Palazzo E, de Novellis V, Marabese I, et al. Interaction between vanilloid and glutamate receptors in the central modulation of nociception. European Journal of Pharmacology. 2002;439(1–3):69–75. [PubMed]

109.

Xing J, Li J. TRPV1 receptor mediates glutamatergic synaptic input to dorsolateral periaqueductal gray (dl-PAG) neurons. Journal of Neurophysiology. 2007;97(1):503–511. [PubMed]

110.

Molchanov ML, Guimarães FS. Anxiolytic-like effects of AP7 injected into the dorsolateral or ventrolateral columns of the periaqueductal gray of rats. Psychopharmacology. 2002;160(1):30–38. [PubMed]

111.

Marsch R, Foeller E, Rammes G, et al. Reduced anxiety, conditioned fear, and hippocampal long-term potentiation in transient receptor potential vanilloid type 1 receptor-deficient mice. The Journal of Neuroscience. 2007;27(4):832–839. [PubMed]

112.

Micale V, Cristino L, Tamburella A, et al. Anxiolytic effects in mice of a dual blocker of fatty acid amide hydrolase and transient receptor potential vanilloid type-1 channels. Neuropsychopharmacology. In press.

113.

Rubino T, Realini N, Castiglioni C, et al. Role in anxiety behavior of the endocannabinoid system in the prefrontal cortex. Cerebral Cortex. 2008;18(6):292–301.

114.

Rubino T, Guidali C, Vigano D, et al. CB1 receptor stimulation in specific brain areas differently modulate anxiety-related behaviour. Neuropharmacology. 2008;54(1):151–160. [PubMed]

115.

Onaivi ES, Chakrabarti A, Gwebu ET, Chaudhuri G. Neurobehavioral effects of Δ⁹-THC and cannabinoid (CB1) receptor gene expression in mice. Behavioural Brain Research. 1996;72(1-2):115–125. [PubMed]