Psychopharmacology (2008) 199:223230 DOI 10.

1007/s00213-008-1168-x

ORIGINAL INVESTIGATION

Involvement of 5HT1A receptors in the anxiolytic-like effects of cannabidiol injected into the dorsolateral periaqueductal gray of rats
Alline Cristina Campos & Francisco Silveira Guimares

Received: 28 January 2008 / Accepted: 8 April 2008 / Published online: 1 May 2008 # Springer-Verlag 2008

Abstract Rationale Cannabidiol (CBD) is a non-psychotomimetic constituent of Cannabis sativa plant that induces anxiolytic effects. However, the brain sites and mechanisms of these effects remain poorly understood. The dorsolateral periaqueductal gray (dlPAG) is a midbrain structure related to anxiety that contains receptors proposed to interact with CBD such as 5HT1A. In addition, since CBD has been shown to inhibit anandamide metabolism, CB1 receptors could also be involved in the effects of this cannabinoid. Objectives To investigate if the dlPAG could be a possible site of the anxiolytic effects induced by CBD and if these effects depend on CB1 or 5HT1A receptors. Materials and methods Male Wistar rats with cannulae aimed at the dlPAG were tested in the elevated plus maze (EPM) and the Vogel conflict test (VCT). Results CBD injected into the dlPAG produced anxiolyticlike effects in the EPM with a bell-shaped doseresponse curve. The anxiolytic effect of CBD was confirmed in the VCT. These effects were prevented by WAY100635, a 5HT1A receptor antagonist, but not by AM251, an antagonist of CB1 receptors. Conclusion These results suggest the CBD interacts with 5HT1A receptors to produce anxiolytic effects in the dlPAG. Keywords Cannabinoids . Anxiety . Serotonin . Animal model
A. C. Campos : F. S. Guimares (*) Department of Pharmacology, School of Medicine of Ribeiro Preto, Campus USP, Av. Bandeirantes 3900, 14049-900 Monte Alegre, Ribeiro Preto, SP, Brazil e-mail: fsguimar@fmrp.usp.br

Introduction The term cannabinoids refers to a heterogeneous group of molecules that act on cannabinoid receptors. They can be divided into three groups: endogenous (endocannabinoids), synthetic, and phytocannabinoids (for review, see Russo and Guy 2006). The latter group is constituted by terpenophenolic substances extracted from Cannabis sativa. This plant contains several phytocannabinoids, including two major compounds: 9-tetrahydrocannabinol (9THC) and cannabidiol (CBD; Mechoulam and Gaoni 1965). 9-THC acts as a cannabinoid receptor (CB) partial agonist and induces psychotomimetic effects, hypothermia, and analgesia (Devane et al. 1988). CBD is a nonpsychotomimetic constituent of cannabis that shows low affinity for CB1 receptors in vitro (Bisogno et al. 2001). CBD systemic administration produces several pharmacological effects, including antiinflammatory, immunomodulatory (Malfait et al. 2000), anticonvulsive, neuroprotective, antipsychotic, and anxiolytic (Carlini et al. 1973; Guimares et al. 1990; Zuardi et al. 1995; Moreira and Guimares 2005). However, the molecular mechanisms underlying these effects remain poorly understood. CBD could facilitate the endocannabinoid signaling by inhibiting the cellular uptake and enzymatic hydrolysis of endocannabinoids (Bisogno et al. 2001). It can bind to CB1, 5HT1A, and TRPV1 receptors at micromolar concentration range (Bisogno et al. 2001, Russo et al. 2005, Thomas et al. 2007) and has been proposed to activate vanilloid (TRPV1) and serotonergic (5HT1A) receptors and inhibit adenosine uptake (Bisogno et al. 2001; Russo et al. 2005; Carrier et al. 2006). Earlier work demonstrated that CBD is able to antagonize the anxiogenic effect of high doses of 9-THC (Zuardi et al. 1981). Subsequent studies showed that

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systemically injected CBD induces anxiolytic effects in animal models such as the elevated plus maze (EPM), Vogel conflict test, and contextual fear conditioning (Guimares et al. 1990; Moreira et al. 2006; Resstel et al. 2006). The brain sites of these effects, however, are still unknown. Several brain structures have been related to the coordination of anxiety-like behavior. Among them, the periaqueductal gray (PAG) has received considerable attention. The PAG is a mesencephalic structure divided along its rostro-caudal axis into dorsomedial, dorsolateral (dlPAG), lateral, and ventrolateral columns. It is proposed to be part of a neural substrate responsible for the coordination of both nociceptive responses and anxiety-related behaviors (Beijamini and Guimares 2006; Bandler et al. 2000, for review). PAG expresses a significant number of receptors that could potentially interact with CBD, such as CB1 (Herkenham et al. 1991), TRPV1 (Cristino et al. 2006), and 5HT1A (De Paula Soares and Zangrossi 2004; Nogueira and Graeff 1995). Moreover, the PAG could mediate the antinociceptive, anxiolytic-like, and antiaversive effects of other cannabinoids (Hohmann et al. 2005; Moreira et al. 2007). Thus, the aim of this study was to investigate if the dlPAG could be a brain site related to the anxiolytic effects of systemically administered CBD and if these effects involve activation of CB1 or 5HT1A receptors.

Materials and methods Animals Male Wistar rats weighing 220240 g at the beginning of the experiments were housed in pairs in a temperaturecontrolled room (241C) under standard laboratory conditions with free access to food and water and a 12-h light/ 12-h dark cycle (lights on at 06:30 A.M.). Procedures were conducted in conformity with the Brazilian Society of Neuroscience and Behavior guidelines for the care and use of laboratory animals, which are in compliance with international laws and policies. The experiment protocols were approved by the local Ethical Committee. All efforts were made to minimize animal suffering. Apparatus Two behavioral tests aimed at detecting anxiolytic drug effects were employed, the EPM and the VCT. The former test consisted of two opposite open arms (5010 cm) crossed at a right angle by two arms of the same dimensions enclosed by 40-cm-high walls with no roof. The maze was located 50 cm above the floor, and a 1-cmhigh edge made of Plexiglas surrounded the open arms to prevent falls. The experiment took place in a sound-

attenuated, temperature-controlled (251C) room, illuminated by three 40-W fluorescent bulbs placed 4 m above the apparatus. Rodents naturally avoid the open arms of the EPM, and anxiolytic compounds typically increase the exploration of these arms without changing the number of enclosed arm entries (File 1992; Carobrez and Bertoglio 2005). The Ethovision software (V. 1.9, Noldus, Netherlands) was employed for behavioral analysis in the EPM. It detects the position of the animal in the maze and calculates the number of entries and time spent in open and enclosed arms. For these calculations, a 6-cm-large exclusion zone was added between the center of the maze and each arm so that most of the animals body should be in the open or enclosed arm for an entry to be registered. The VCT was performed in a Plexiglas box (length: 42 cm, width: 25 cm, height: 20 cm) with a stainless grid floor. A metallic spout of a drinking bottle containing water was projected into the box. The contact of the animal with the spout and the grid floor closed an electrical circuit controlled by a sensor (AnxioMeter model 102, Columbus, OH, USA) that recorded the number of licks on the metallic spout. Every 20th lick produced a 0.5-mA shock for 2 s. The apparatus registered the total number of licks and shocks delivered during the test period. The whole apparatus was located inside a soundattenuated cage. To control for possible antinociceptive drug effects in the dlPAG that could interfere in the Vogel test, the animals were also submitted to the tail-flick test (DAmour and Smith 1941). The apparatus consisted of an acrylic platform with a niquelchrome wire coil (EFF 300, Insight Instruments, Ribeiro Preto, Brazil) maintained at room temperature (2426C). The coil temperature can be raised at 9C/s by the passage of electric current. The system had a cut-off time of 6 s to prevent tissue damage when the coil temperature approached 80C. Drugs Cannabidiol (kindly supplied by Dr. Raphael Mechoulam, Hebrew University, Jerusalem, Israel) was dissolved in grape seed oil. The CB1 cannabinoid receptor antagonist N(piperidin-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4methyl-1Hpyrazole-3 carboxamide (AM251, TOCRIS) was dissolved in DMSO 10% in saline (0.9% NaCl). The 5HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamide maleate (WAY-100635, Sigma, USA) was dissolved in saline. The solutions were prepared immediately before the tests and administered in a volume of 0.2 L into the dlPAG. Surgery One hundred and seventy-nine rats were anesthetized with 2.5% 2,2,2-tribromoethanol (10 mg/kg, i.p.) and fixed in a

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stereotaxic frame. A stainless steel guide cannula (0.6 mm OD) was implanted unilaterally on the right side aimed at the dlPAG (coordinates: AP=0 from lambda, L=1.9 mm, D=4.0 mm; Paxinos and Watson 1997). The cannula was inserted at an angle of 16 to prevent damage of the venous sinus. It was attached to the bones with stainless steel screws and acrylic cement. An obturator inside the guide cannula prevented obstruction. Procedure The experiments took place 7 days after surgery. Independent groups of animals were used in all experiments. Intracerebral injections were performed with a thin dental needle (0.3 mm OD) introduced through the guide cannula until its tip was 1.0 mm below the cannula end. A volume of 0.2 L was injected in 20 s using a microsyringe (Hamilton, USA) connected to an infusion pump (Kd Scientific, USA). A polyethylene catheter (PE 10) was interposed between the upper end of the dental needle and the microsyringe. Experimental design Experiment 1 The animals received intra-dlPAG injections of vehicle or CBD (1560 nmol) and were placed 10 min later in the center of the EPM facing an enclosed arm. The number of entries and time spent in the open and enclosed arms were recorded for 5 min. Experiment 2 The animals were water-deprived for 48 h before the test. After the first 24 h of deprivation, they were allowed to drink freely for 3 min in the test cage in order to find the drinking bottle spout. Some animals did not find the spout and were not included in the experiment. Twenty-four hours later, they received intra-dlPAG injections of vehicle or CBD 30 nmol and 10 min later were placed in the apparatus. The test period lasted for 3 min, and the animals received a 0.5-mA shock through the bottle spout every 20 licks. The number of punished licks was registered. To control for possible drug influence on water consummatory drive, independent groups of animals were submitted to the same protocol without receiving any electrical shock. The procedure was similar to the one already used and validated in the laboratory (Moreira et al. 2006). Experiment 3 Independent groups of animals were gently handled, and their tails were laid across the coil. The heat was applied to a portion of the ventral surface of the tail between 4 and 6 cm from its end. The time to withdraw the tail was recorded as tail-flick latency. The electric current was calibrated to provoke this reflex within 2.53.5 s in non-treated animals (Molchanov and Guimares 2002). The

tail-flick latency was measured at 5-min intervals until a stable baseline (BL) was obtained over three consecutive trials. The tail withdrawal latency was measured at 10-min intervals after drug administration for up to 30 min (Molchanov and Guimares 2002). The tail-flick latency were normalized using an antinociception index (AI; Pedigo et al. 1975) according to the formula AI=(TL BL)/(6BL). Experiment 4 The animals received intra-dlPAG injections of vehicle or AM251 100 pmol (dose based on Moreira et al. 2006) followed 5 min later by vehicle or CBD (30 nmol). Ten minutes after the second injection, the animals were submitted to the EPM test. Experiment 5 Similar to experiment 4, except that the animals received vehicle or WAY100635 0.37 nmol (dose based on De Paula Soares and Zangrossi 2004) before being submitted to the EPM test.

Histology After the behavioral tests, the rats were sacrificed under deep urethane anesthesia and perfused through the left ventricle of the heart with isotonic saline followed by 10% formalin solution. The brains were removed, and after a minimum period of 5 days immersed in a 10% formalin solution, 50-m sections were obtained in a Cryostat (Cryocut 1800). The injection sites were identified in diagrams from the Paxinos and Watsons (1997) atlas and are illustrated in Fig. 1. Rats receiving injections of the active dose of CBD outside the dlPAG were included in an additional group (out). Statistical analysis The percentages of entries and time spent in the open arms (100open/open+enclosed) during the 5-min sessions in the EPM were calculated for each animal. These results plus the number of enclosed arm entries and the number of licks in the Vogel test were also analyzed by one-way ANOVA. Data from the tail-flick experiment were analyzed by a repeated measure ANOVA followed by one-way ANOVAs at each time. The Duncan test was employed for multiple comparisons. Differences were considered significant at p<0.05 level.

Results The injection sites in the dlPAG can be seen in Fig. 1. In the first experiment, CBD produced a bell-shaped dose

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Fig. 2 Anxiolytic-like effect of CBD (1560 nmol) microinjected into the dlPAG of rats submitted to the EPM. Columns represent means SEM. The open columns represent the percent of entries in the open arms, while the hatched columns represent the percent of the time spent in the open arms. Animals receiving CBD (30 nmol) injections outside the dlPAG were joined in an out group. Asterisks indicate significant difference from vehicle (Veh, p<0.05, ANOVA followed by the Duncan test; n=7, 6, 9, 5, 11, respectively)

received the active dose (30 nmol) of CBD injections outside the dlPAG (out) were not different from the control group. No effects were found in the number of enclosed arms entries. The active dose of CBD in the EPM (30 nmol) also increased the number of punished licks (F(2, 20) =7.35, p= 0.004; p<0.01) in the VCT (Fig. 3) without changing the number of unpunished licks (Table 1). The animals that received CBD outside the dlPAG (out) were also not different from controls. Results from the tail-flick test can be seen in Fig. 4. The repeated measures ANOVA revealed a significant effect of treatment (F(2, 14) =108.4, p<0.001), time (F(6, 84) =19.21, p<0.001), and treatmenttime (F(12, 84) =25.07, p<0.001). ANOVAs performed at each time showed that morphine (5 mg/kg) produced antinociceptive effects 10, 15, 20, and 25 min after injection when compared to CBD and vehicle (F(2, 14) =34.0 to 147.9, p<0.001). Fifteen minutes after injection, CBD also produced a small but significant

Fig. 1 Left panel Histological localization of site injections (0.2 L) in the dlPAG (full circles) and deep layers of superior colliculus (empty circles) in diagrams based on Paxinos and Watsons (1997) rat brain atlas. Right panel Representative microphotography of an injection site in the dlPAG in a coronal brain slice. Bar=500 m

response curve (Fig. 2), with the dose of 30 nmol increasing the percentage of entries (F(4, 33) =3.5; p=0.01; Duncan test, p<0.05) and time spent (F(4, 33) =2.6; p=0.045, Duncan test, p<0.05) in the open arms of the EPM as compared to animals that received vehicle. Animals that

Fig. 3 Effects of CBD (30 nmol) or vehicle injected into the dlPAG of rats submitted to the Vogel conflict test. Bars represent the mean (SEM) total number of punished licks in the 3-min session. Animals receiving CBD injections outside the dlPAG were joined in an out group. Asterisk indicates significant difference from vehicle (p<0.05, ANOVA followed by the Duncan test; n=7, 8, 8, respectively)

Psychopharmacology (2008) 199:223230 Table 1 Effects of intra-dlPAG administration of CBD (30 nmol) on the number of unpunished licks of rats that had been water-deprived for 48 h Treatment Vehicle CBD Unpunished licks 685.1154.6 612.992.9

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Data are expressed as mean (SEM, n=57/group). No significant effect was found

antinociceptive effect (p < 0.05) compared to vehicle. However, contrary to morphine, the AI values after CBD injection were not different from baseline. In the third experiment, we investigated if a previous injection of AM251, an antagonist of CB1 receptors, could antagonize the anxiolytic-like effects of CBD observed in the dose of 30 nmol (Fig. 5). Similar to the first experiment, CBD increased the percentage of time spent in the open arms (F(4, 28) =3.02, p=0.03, Duncan test, p<0.05) without changing the number of enclosed arms entries. It also tended to increase the percentage of open arm entries, but the results did not reach significance (F(4, 28) =1.28). Previous injection of AM251 into the dlPAG failed to prevent the increase in the percentage of time spent in the open arms induced by CBD. Animals that received CBD outside the dlPAG (out) were not different from the control group. Results of the fourth experiment can be seen in Fig. 6. Previous injection of WAY100635, an antagonist of 5HT1A receptors, prevented the anxiolytic-like effects of CBD in the dlPAG. CBD increased the percentage of open arms

Fig. 5 Effects of the CB1 receptor antagonist AM251 (100 pmol) followed by CBD or vehicle (Veh) injected into the dlPAG. Animals receiving vehicle + CBD injections outside the dlPAG were joined in an out group. Further specifications as in Fig. 2. Asterisks represent statistical differences from vehicle (p<0.05, ANOVA followed by Duncan test; n=7, 9, 5, 7, 6, respectively)

entries (F(4, 24) =3.37, p=0.043; Duncan test, p<0.05) when compared to animals that received vehicle. No effects in enclosed arms entries were found.

Discussion The present study showed that CBD injected into the dlPAG increased exploration of the open arms without changing the number of entries into the enclosed arms of the EPM, an animal model based on the rodent natural aversion to elevated and open places. This result is usually interpreted as indicating an anxiolytic effect (File 1992).

Fig. 4 Time course of the effects of vehicle, CBD (30 nmol) or morphine (5 mg/kg) in the tail-flick test. Each point represents the mean (SEM) latency of tail withdrawal. Asterisks indicate difference from all other groups, number sign indicates difference from vehicle (p<0.001; n=5, 6, 6, respectively)

Fig. 6 Effects of the 5HT1A receptor antagonist WAY100635 (0.37 nmol) followed by CBD or vehicle (Veh) injected into the dlPAG. Animals receiving vehicle + CBD injections outside the dlPAG were joined in an out group. Further specifications as in Fig. 2. Asterisk represents statistical differences from vehicle (p<0.05, ANOVA followed by Duncan test; n=6, 3, 6, 6, 8, respectively)

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The effect seems to be mediated by the dlPAG since injections of the active dose of CBD outside this region were not effective. The anxiolytic-like effect of CBD in the dlPAG was confirmed in the Vogel conflict test. This model is based on the classical GellerSeifter paradigm (Geller and Seifer 1962). Similar to our results with CBD, anxiolytic drugs typically increase the number of punished licks without affecting the number of unpunished licks (Vogel et al. 1971). CBD also produced an antinociceptive effect in the tail-flick test after intra-dlAPG injection. However, this effect is unlikely to be responsible for the observed anticonflict effect since it was small, not differing from baseline values, and occurred only 15 min after the injection, whereas the VCT was performed 10 min after drug administration. Moreover, systemic administration of morphine, at similar doses used in the present study, does not produce antipunishment effects in a suppressed schedule-induced drinking paradigm (Prez-Padilla and Pelln 2007). CBD can facilitate endocannabinoid-mediated neurotransmission by decreasing anandamide hydrolysis or reuptake (Bisogno et al. 2001). This effect could be related to its anxiolytic effects in the dlPAG since direct injection of anandamide into this region induced anxiolytic-like effects in the EPM that are prevented by local prior administration of AM251 (Moreira et al. 2007). Although usually described as a CB1 receptor antagonist, AM251 can also produce inverse cannabimimetic effects. However, it shows greater potency in opposing effects induced by CB1 agonists than producing inverse effects at CB1 receptors, suggesting that at low concentrations, this drug could act as a neutral CB1 receptor antagonist (Pertwee 2005). In our study, the same dose of AM251 (100 pmol) that was able to antagonize the effects of intra-dlPAG administered anandamide failed to prevent the anxiolytic effects of CBD. It is improbable, therefore, that these effects are being mediated by facilitation of CB1 receptormediated neurotransmission. There is also evidence that CBD could act, at micromolar concentration range, as an agonist of 5HT1A receptors in vitro (Russo et al. 2005) and in vivo (Mishima et al. 2005). The 5HT1A receptor is a Gi-coupled-receptor that, when activated, enhances K+ currents and inhibits adenylyl cyclase activity (Raymond et al. 1999). These receptors act as inhibitory auto-receptors in serotonergic neurons in the raphe nuclei but are also localized postsynaptically in several brain regions, including the PAG, amygdala, hippocampus, and frontal cortex. The PAG receives serotonergic projections from the dorsal raphe nuclei (see Millan 2003, for review), and activation of 5HT1A receptors promotes the control of anxiety states and the hypothalamuspituitaryadrenal axis during stress

responses (Carrasco and Van de Kar 2003). Corroborating the proposal that CBD could facilitate 5HT1A-mediated neurotransmission, its anxiolytic effect in the dlPAG was prevented by previous treatment with WAY100635, a highaffinity (Ki ranging from 0.1 to 2.2 nM, Hamon et al. 1990, Corradetti et al. 2005) 5HT1A receptor antagonist. The dose of WAY100635 was the same that was able to block the anxiolytic effects of 8-OH-DPAT, a 5HT1A agonist, in the dlPAG (Zanoveli et al. 2003; De Paula Soares and Zangrossi 2004). Despite the present results, however, CBD has other proposed mechanisms of action, such as blockade of adenosine uptake (Carrier et al. 2006) and antagonism of the putative cannabinoid receptor GPR55 (Ryberg et al. 2007). The involvement of these mechanisms in the anxiolytic effects of CBD in the dlPAG remains to be tested. The higher dose of CBD (60 nmol) was ineffective in the dlPAG. Systemic administration of CBD also produced a bell-shaped doseresponse curve in rats submitted to the EPM (Guimares et al. 1990), an effect also observed with other cannabinoids (Moreira et al. 2006). CBD can activate TRPV1 receptors at micromolar concentration range (Bisogno et al. 2001). These receptors are expressed in the PAG (Cristino et al. 2006) where they can facilitate glutamate release (Palazzo et al. 2002). It was recently showed that capsazepine, a TRPV1 antagonist, blocks the anxiogenic effects of high doses of anandamide in the prefrontal cortex (Rubino et al. 2007), raising the possibility that a similar mechanism could be involved in CBD effects in the PAG. Preliminary data from our laboratory support this possibility (Campos and Guimares, unpublished data), but further experiments are needed to address this question. In conclusion, the present results suggest that activation of 5HT1A receptors located in the dlPAG could be one of the mechanisms of the anxiolytic effect observed with this compound after systemic administration.
Acknowledgments We thank Dr. Eleni T. Gomes and Jos Carlos de Aguiar for technical support. This research was supported by grants from FAPESP and CNPq. ACC was a recipient of a FAPESP fellowship.

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