Daniele C. Aguiar

Anxiolytic-like effect of cannabidiol in the rat Vogel conflict test.

Cannabidiol (CBD) is a major constituent of the Cannabis sativa plant. It inhibits the anxiogenic activity of high doses of Delta9-tetrahydrocannabinol and induces anxiolytic-like effects. However, the mechanisms underlying the actions of CBD are unknown. Therefore, the aim of the present study was to test the effects of CBD in the Vogel test, a widely used animal model of anxiety. In addition, it was verified if these effects would depend on benzodiazepine-receptor activation. After 24 h of water deprivation, male Wistar rats were subjected to an initial 3-min non-punished (pre-test) drinking session. This was followed by an additional 24-h period of water deprivation followed by a 3-min punished-licking session (test). Diazepam (3 mg/kg) or CBD (2.5, 5 or 10 mg/kg) were intraperitoneally injected 30 min before the test session. CBD (10 mg/kg) and diazepam had similar anticonflict effects, increasing the number of punished licks. The effect of diazepam, but not of CBD, was prevented by the benzodiazepine-receptor antagonist flumazenil (10 mg/kg). To exclude that the anticonflict effects were reflecting non-specific drug effects, we checked the effects of CBD on water consumption and nociceptive response. The drug did not interfere on the former variable in a non-punished test session. Moreover, contrary to morphine (5 mg/kg), CBD was ineffective in the tail-flick test. In conclusion, CBD induced an anticonflict effect not mediated by benzodiazepine receptors or by non-specific drug interference on nociceptive threshold or water consumption. These results reinforce the hypothesis that this cannabinoid has anxiolytic properties.

Anxiolytic-like effect of cannabinoids injected into the rat dorsolateral periaqueductal gray

Contradictory results exist concerning the effects of systemic injections of CB(1) cannabinoid receptor agonists on anxiety-related behaviors. Direct drug administration into brain structures related to aversive responses can potentially help to clarify the role of cannabinoids on anxiety. One such structure is the midbrain dorsolateral periaqueductal gray (dlPAG). Therefore, the aim of this study was to test the hypothesis that the activation of the CB(1) receptor in the dlPAG would attenuate anxiety-related behaviors. Male Wistar rats with cannula aimed at the dlPAG received injections of the endogenous cannabinoid anandamide, the anandamide transport inhibitor AM404, the anandamide analogue ACEA or the CB(1) receptor antagonist AM251, and were submitted to the elevated plus maze (EPM), an animal model of anxiety. Anandamide (0.5-50pmol) and ACEA (0.05-5pmol) induced anxiolytic-like effects with bell-shaped dose-response curves, the higher doses being ineffective. The anandamide anxiolytic effect was potentiated by AM404 (50pmol) and prevented by AM251 (100pmol). Neither AM404 (0.5-50pmol) nor AM251 (1-100pmol) alone modified the animal behavior in the EPM. The present study suggests that the dlPAG is a possible neuroanatomical site for anxiolytic-like effects mediated by CB(1) agonists. Furthermore, this work supports the importance of neuronal uptake as a mechanism that limits the in vivo actions of anandamide.

Role of nitric oxide in brain regions related to defensive reactions.

Nitric oxide synthase (NOS) positive neurons are located in most brain areas related to defensive reactions, including the dorsolateral periaqueductal grey (dlPAG). NOS inhibitors injected into this structure induce anxiolytic-like responses whereas NO donors promote flight reactions. Intra-dlPAG administration of carboxy-PTIO, a NO scavenger, or ODQ, a soluble guanylate cyclase inhibitor, produced anxiolytic-like effects on rats exposed to the elevated plus-maze (EPM). A double-staining experiment using NADPHd histochemistry and c-Fos immunohistochemistry in rats exposed to a cat or to the EPM showed increased activation of NO producing neurons in the dlPAG, paraventricular and lateral nuclei of hypothalamus and dorsal raphe nucleus. Cat exposure also increased activation of NOS neurons in the medial amygdala, dorsal pre-mammillary nucleus and bed nucleus of stria terminalis. Local infusion into the dlPAG of a glutamate NMDA-receptor antagonist (AP7) or a benzodiazepine agonist (midazolam) completely prevented the flight reactions induced by intra-dlPAG administration of SIN-1, a NO donor. The responses were also inhibited by the 5-HT2A/C agonist DOI but not by a 5-HT1A agonist. These results suggest a modulatory role for NO on brain areas related to defensive reactions, probably by interacting with glutamate, serotonin and/or GABA-mediated neurotransmission.

Modulation of anxiety-like behaviour by Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels located in the dorsolateral periaqueductal gray.

The endocannabinoid anandamide is a possible agonist at the Transient Receptor Potential Vanilloid Type 1 (TRPV1) channel, in addition to its agonist activity at cannabinoid type 1 (CB1) receptor. In the midbrain dorsolateral periaqueductal gray (dlPAG) our previous data showed that CB1 activation induces anxiolytic-like effects. However, the role of TRPV1 has remained unclear. Thus, in the present study we tested the hypothesis that this channel would contribute to the modulation of anxiety-like behaviour in the dlPAG. Male Wistar rats received local injections of the TRPV1 antagonist capsazepine (10-60 nmol) and were submitted to the elevated plus-maze (EPM) and to the Vogel test. In addition, animals received local injections of capsaicin (0.01-1 nmol), a TRPV1 agonist, and were tested in the same models. In accordance with our hypothesis, capsazepine produced anxiolytic-like effects both in the EPM and in the Vogel test. Capsaicin mimicked these results, which might be attributed to its ability to quickly desensitize the channel. Altogether, our data suggest that, while CB1 receptors seem to inhibit aversive responses in the dlPAG, TRPV1 could facilitate them. Thus, CB1 and TRPV1 may have opposite functions in modulating anxiety-like behaviour in this region.

Opposing Roles for Cannabinoid Receptor Type-1 (CB(1)) and Transient Receptor Potential Vanilloid Type-1 Channel (TRPV1) on the Modulation of Panic-Like Responses in Rats

The midbrain dorsal periaqueductal gray (dPAG) has an important role in orchestrating anxiety- and panic-related responses. Given the cellular and behavioral evidence suggesting opposite functions for cannabinoid type 1 receptor (CB1) and transient receptor potential vanilloid type-1 channel (TRPV1), we hypothesized that they could differentially influence panic-like reactions induced by electrical stimulation of the dPAG. Drugs were injected locally and the expression of CB1 and TRPV1 in this structure was assessed by immunofluorescence and confocal microscopy. The CB1-selective agonist, ACEA (0.01, 0.05 and 0.5 pmol) increased the threshold for the induction of panic-like responses solely at the intermediary dose, an effect prevented by the CB1-selective antagonist, AM251 (75 pmol). Panicolytic-like effects of ACEA at the higher dose were unmasked by pre-treatment with the TRPV1 antagonist capsazepine (0.1 nmol). Similarly to ACEA, capsazepine (1 and 10 nmol) raised the threshold for triggering panic-like reactions, an effect mimicked by another TRPV1 antagonist, SB366791 (1 nmol). Remarkably, the effects of both capsazepine and SB366791 were prevented by AM251 (75 pmol). These pharmacological data suggest that a common endogenous agonist may have opposite functions at a given synapse. Supporting this view, we observed that several neurons in the dPAG co-expressed CB1 and TRPV1. Thus, the present work provides evidence that an endogenous substance, possibly anandamide, may exert both panicolytic and panicogenic effects via its actions at CB1 receptors and TRPV1 channels, respectively. This tripartite set-point system might be exploited for the pharmacotherapy of panic attacks and anxiety-related disorders.

Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 channels in fear and anxiety-two sides of one coin?

The transient receptor potential vanilloid type 1 channel (TRPV1; originally vanilloid receptor VR1) is activated in peripheral terminals of nociceptive fibers by noxious heat, low pH, and natural products such as capsaicin, the pungent ingredient of red-hot chilli peppers. Evidence has been accumulating that TRPV1 is expressed also in the brain, where it seems to be involved in antinociception, locomotor control, and regulation of affective behaviors. This ion channel might be activated by arachidonoyl ethanolamide (anandamide), the endogenous agonist of the cannabinoid type 1 (CB(1)) receptor. However, while CB(1) activation leads to a decrease in intracellular calcium and attenuation of synaptic transmission, anandamide binding to TRPV1 results in elevated calcium levels and potentiated synaptic transmission. This suggests a tripartite regulatory system with antagonistic effects of CB(1) and TRPV1, which are tied together by the same endogenous ligand. Such a system may have important implication for the modulation of behavioral responses. The present commentary elaborates on this interplay between CB(1) receptors and TRPV1 channels in the context of fear- and anxiety-related behaviors.

Activation of cannabinoid CB1 receptors in the dorsolateral periaqueductal gray induces anxiolytic effects in rats submitted to the Vogel conflict test.

There are contradictory results concerning the effects of systemic injections of cannabinoid agonists in anxiety-induced behavioral changes. Direct drug administration into brain structures related to defensive responses could help to clarify the role of cannabinoids in these changes. Activation of cannabinoid CB(1) receptors in the dorsolateral periaqueductal gray induces anxiolytic-like effects in the elevated plus maze. The aim of this work was to verify if facilitation of endocannabinoid-mediated neurotransmission in this region would also produce anxiolytic-like effects in another model of anxiety, the Vogel conflict test. Male Wistar rats (n=5-9/group) with cannulae aimed at the dorsolateral periaqueductal gray were water deprived for 24 h and pre-exposed to the apparatus where they were allowed to drink for 3 min. After another 24 h-period of water deprivation, they received the microinjections and, 10 min later, were placed into the experimental box. In this box an electrical shock (0.5 mA, 2 s) was delivered in the spout of a drinking bottle at every twenty licks. The animals received a first microinjection of vehicle (0.2 microl) or AM251 (a cannabinoid CB(1) receptor antagonist; 100 pmol) followed, 5 min later, by a second microinjection of vehicle, anandamide (an endocannabinoid, 5 pmol), AM404 (an inhibitor of anandamide uptake, 50 pmol) or URB597 (an inhibitor of Fatty Acid Amide Hydrolase, 0.01 or 0.1 nmol). Anandamide, AM404 and URB597 (0.01 nmol) increased the total number of punished licks. These effects were prevented by AM251. The results give further support to the proposal that facilitation of CB(1) receptor-mediated endocannabinoid neurotransmission in the dorsolateral periaqueductal gray modulates defensive responses.

The endocannabinoid and endovanilloid systems interact in the rat prelimbic medial prefrontal cortex to control anxiety-like behavior.

Cannabinoid receptor 1 (CB(1)) agonists usually induce dose-dependent biphasic effects on anxiety-related responses. Low doses induce anxiolytic-like effects, whereas high doses are ineffective or anxiogenic, probably due to activation of Transient Receptor Potential Vanilloid Type 1 (TRPV(1)) channels. In this study we have investigated this hypothesis by verifying the effects of the CB(1)/TRPV(1) agonist ACEA injected into the prelimbic medial prefrontal cortex (PL) and the participation of endocannabinoids in the anxiolytic-like responses induced by TRPV(1) antagonism, using the elevated plus-maze (EPM) and the Vogel conflict test (VCT). Moreover, we verified the expression of these receptors in the PL by double labeling immunofluorescence. ACEA induced anxiolytic-like effect in the intermediate dose, which was attenuated by previous injection of AM251, a CB(1) receptor antagonist. The higher and ineffective ACEA dose caused anxiogenic- and anxiolytic-like effects, when injected after AM251 or the TRPV(1) antagonist 6-iodonordihydrocapsaicin (6-I-CPS), respectively. Higher dose of 6-I-CPS induced anxiolytic-like effects both in the EPM and the VCT, which were prevented by previous administration of AM251. In addition, immunofluorescence showed that CB(1) and TRPV(1) receptors are closely located in the PL. These results indicate that the endocannabinoid and endovanilloid systems interact in the PL to control anxiety-like behavior.

Antiaversive Effects of Cannabinoids: Is the Periaqueductal Gray Involved?

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.

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

Innate fear stimulus induces activation of neurons containing the neuronal nitric oxide synthase enzyme (nNOS) in defensive‐related brain regions such as the dorsolateral periaqueductal gray (dlPAG). Intra‐dlPAG administration of nitric oxide synthase (NOS) inhibitors and glutamate antagonists induce anxiolytic‐like responses. We investigated the involvement of nitric oxide (NO) and glutamate neurotransmission in defensive reactions modulated by dlPAG. We tested if intra‐dlPAG injections of the selective nNOS inhibitor, N‐propyl‐L‐arginine (NP), or the glutamate antagonist, AP7 (2‐amino‐7‐phosphonoheptanoic acid), would attenuate behavioral responses and cellular activation induced by predator exposure (cat). Fos‐like immunoreactivity (FLI) was used as a marker of neuronal functional activation, whereas nNOS immunohistochemistry was used to identify NOS neurons. Cat exposure induced fear responses and an increase of FLI in the dlPAG and dorsal premammillary nucleus (PMd). NP and AP7 attenuated the cat‐induced behavioral responses. Whereas NP tended to attenuate FLI in the dlPAG, AP7 induced a significant reduction in cellular activation of this region. The latter drug, however, increased FLI and double‐labeled cells in the PMd. Cellular activation of this region was significantly correlated with time spent near the cat (r = 0.7597 and 0.6057 for FLI and double‐labeled cells). These results suggest that glutamate/NO‐mediated neurotransmission in the dlPAG plays an important role in responses elicit by predator exposure. Blocking these neurotransmitter systems in this brain area impairs defensive responses. The longer time spent near the predator that follows AP7 effect could lead to an increased cellular activation of the PMd, a more rostral brain area that has also been related to defensive responses.