ORIGINAL INVESTIGATION

Involvement of 5HT1A receptors in the anxiolytic-like effectsof cannabidiol injected into the dorsolateral periaqueductalgray of rats

Alline Cristina Campos & Francisco Silveira Guimarães

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

AbstractRationale Cannabidiol (CBD) is a non-psychotomimeticconstituent of Cannabis sativa plant that induces anxiolyticeffects. However, the brain sites and mechanisms of theseeffects remain poorly understood. The dorsolateral peri-aqueductal gray (dlPAG) is a midbrain structure related toanxiety that contains receptors proposed to interact withCBD such as 5HT1A. In addition, since CBD has beenshown to inhibit anandamide metabolism, CB1 receptorscould also be involved in the effects of this cannabinoid.Objectives To investigate if the dlPAG could be a possiblesite of the anxiolytic effects induced by CBD and if theseeffects depend on CB1 or 5HT1A receptors.Materials and methods Male Wistar rats with cannulaeaimed at the dlPAG were tested in the elevated plus maze(EPM) and the Vogel conflict test (VCT).Results CBD injected into the dlPAG produced anxiolytic-like effects in the EPM with a bell-shaped dose–responsecurve. The anxiolytic effect of CBD was confirmed in theVCT. These effects were prevented by WAY100635, a5HT1A receptor antagonist, but not by AM251, anantagonist of CB1 receptors.Conclusion These results suggest the CBD interacts with5HT1A receptors to produce anxiolytic effects in thedlPAG.

Keywords Cannabinoids . Anxiety . Serotonin .

Animal model

Introduction

The term cannabinoids refers to a heterogeneous group ofmolecules that act on cannabinoid receptors. They can bedivided into three groups: endogenous (endocannabinoids),synthetic, and phytocannabinoids (for review, see Russoand Guy 2006). The latter group is constituted byterpenophenolic substances extracted from Cannabis sativa.This plant contains several phytocannabinoids, includingtwo major compounds: Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD; Mechoulam and Gaoni1965). Δ9-THC acts as a cannabinoid receptor (CB) partialagonist and induces psychotomimetic effects, hypothermia,and analgesia (Devane et al. 1988). CBD is a non-psychotomimetic constituent of cannabis that shows lowaffinity for CB1 receptors in vitro (Bisogno et al. 2001).

CBD systemic administration produces several pharma-cological effects, including antiinflammatory, immunomod-ulatory (Malfait et al. 2000), anticonvulsive, neuroprotective,antipsychotic, and anxiolytic (Carlini et al. 1973; Guimarãeset al. 1990; Zuardi et al. 1995; Moreira and Guimarães2005). However, the molecular mechanisms underlyingthese effects remain poorly understood. CBD could facilitatethe endocannabinoid signaling by inhibiting the cellularuptake and enzymatic hydrolysis of endocannabinoids(Bisogno et al. 2001). It can bind to CB1, 5HT1A, andTRPV1 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 adenosineuptake (Bisogno et al. 2001; Russo et al. 2005; Carrier et al.2006).

Earlier work demonstrated that CBD is able to antago-nize the anxiogenic effect of high doses of Δ9-THC(Zuardi et al. 1981). Subsequent studies showed that

Psychopharmacology (2008) 199:223–230DOI 10.1007/s00213-008-1168-x

A. C. Campos : F. S. Guimarães (*)Department of Pharmacology,School of Medicine of Ribeirão Preto, Campus USP,Av. Bandeirantes 3900,14049-900 Monte Alegre, Ribeirão Preto, SP, Brazile-mail: fsguimar@fmrp.usp.br

systemically injected CBD induces anxiolytic effects inanimal models such as the elevated plus maze (EPM),Vogel conflict test, and contextual fear conditioning(Guimarães et al. 1990; Moreira et al. 2006; Resstel et al.2006). The brain sites of these effects, however, are stillunknown. Several brain structures have been related to thecoordination of anxiety-like behavior. Among them, theperiaqueductal gray (PAG) has received considerableattention. The PAG is a mesencephalic structure dividedalong its rostro-caudal axis into dorsomedial, dorsolateral(dlPAG), lateral, and ventrolateral columns. It is proposed tobe part of a neural substrate responsible for the coordinationof both nociceptive responses and anxiety-related behaviors(Beijamini and Guimarães 2006; Bandler et al. 2000, forreview). PAG expresses a significant number of receptorsthat could potentially interact with CBD, such as CB1(Herkenham et al. 1991), TRPV1 (Cristino et al. 2006), and5HT1A (De Paula Soares and Zangrossi 2004; Nogueiraand Graeff 1995). Moreover, the PAG could mediate theantinociceptive, anxiolytic-like, and antiaversive effects ofother cannabinoids (Hohmann et al. 2005; Moreira et al.2007). Thus, the aim of this study was to investigate if thedlPAG could be a brain site related to the anxiolytic effectsof systemically administered CBD and if these effectsinvolve activation of CB1 or 5HT1A receptors.

Materials and methods

Animals

Male Wistar rats weighing 220–240 g at the beginning ofthe experiments were housed in pairs in a temperature-controlled room (24±1°C) under standard laboratory con-ditions with free access to food and water and a 12-h light/12-h dark cycle (lights on at 06:30 A.M.). Procedures wereconducted in conformity with the Brazilian Society ofNeuroscience and Behavior guidelines for the care and useof laboratory animals, which are in compliance withinternational laws and policies. The experiment protocolswere approved by the local Ethical Committee. All effortswere made to minimize animal suffering.

Apparatus

Two behavioral tests aimed at detecting anxiolytic drugeffects were employed, the EPM and the VCT. The formertest consisted of two opposite open arms (50×10 cm)crossed at a right angle by two arms of the samedimensions enclosed by 40-cm-high walls with no roof.The maze was located 50 cm above the floor, and a 1-cm-high edge made of Plexiglas surrounded the open arms toprevent falls. The experiment took place in a sound-

attenuated, temperature-controlled (25±1°C) room, illumi-nated by three 40-W fluorescent bulbs placed 4 m abovethe apparatus. Rodents naturally avoid the open arms of theEPM, and anxiolytic compounds typically increase theexploration of these arms without changing the number ofenclosed arm entries (File 1992; Carobrez and Bertoglio2005). The Ethovision software (V. 1.9, Noldus, Nether-lands) was employed for behavioral analysis in the EPM. Itdetects the position of the animal in the maze and calculatesthe number of entries and time spent in open and enclosedarms. For these calculations, a 6-cm-large exclusion zonewas added between the center of the maze and each arm sothat most of the animal’s body should be in the open orenclosed arm for an entry to be registered. The VCT wasperformed in a Plexiglas box (length: 42 cm, width: 25 cm,height: 20 cm) with a stainless grid floor. A metallic spout ofa drinking bottle containing water was projected into the box.The contact of the animal with the spout and the grid floorclosed an electrical circuit controlled by a sensor (Anxio-Meter model 102, Columbus, OH, USA) that recorded thenumber of licks on the metallic spout. Every 20th lickproduced a 0.5-mA shock for 2 s. The apparatus registeredthe total number of licks and shocks delivered during the testperiod. The whole apparatus was located inside a sound-attenuated cage. To control for possible antinociceptive drugeffects in the dlPAG that could interfere in the Vogel test, theanimals were also submitted to the tail-flick test (D’Amourand Smith 1941). The apparatus consisted of an acrylicplatform with a niquelchrome wire coil (EFF 300, InsightInstruments, Ribeirão Preto, Brazil) maintained at roomtemperature (24–26°C). The coil temperature can be raisedat 9°C/s by the passage of electric current. The system hada cut-off time of 6 s to prevent tissue damage when the coiltemperature approached 80°C.

Drugs

Cannabidiol (kindly supplied by Dr. Raphael Mechoulam,Hebrew University, Jerusalem, Israel) was dissolved ingrape seed oil. The CB1 cannabinoid receptor antagonist N-(piperidin-1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1Hpyrazole-3 carboxamide (AM251, TOCRIS) wasdissolved in DMSO 10% in saline (0.9% NaCl). The5HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-cyclohexanecarboxamidemaleate (WAY-100635, Sigma, USA) was dissolved insaline. The solutions were prepared immediately before thetests and administered in a volume of 0.2 μL into the dlPAG.

Surgery

One hundred and seventy-nine rats were anesthetized with2.5% 2,2,2-tribromoethanol (10 mg/kg, i.p.) and fixed in a

224 Psychopharmacology (2008) 199:223–230

stereotaxic frame. A stainless steel guide cannula (0.6 mmOD) was implanted unilaterally on the right side aimed atthe dlPAG (coordinates: AP=0 from lambda, L=1.9 mm,D=4.0 mm; Paxinos and Watson 1997). The cannula wasinserted at an angle of 16° to prevent damage of the venoussinus. It was attached to the bones with stainless steelscrews and acrylic cement. An obturator inside the guidecannula prevented obstruction.

Procedure

The experiments took place 7 days after surgery. Indepen-dent groups of animals were used in all experiments.Intracerebral injections were performed with a thin dentalneedle (0.3 mm OD) introduced through the guide cannulauntil its tip was 1.0 mm below the cannula end. A volumeof 0.2 μL was injected in 20 s using a microsyringe(Hamilton, USA) connected to an infusion pump (KdScientific, USA). A polyethylene catheter (PE 10) wasinterposed between the upper end of the dental needle andthe microsyringe.

Experimental design

Experiment 1 The animals received intra-dlPAG injectionsof vehicle or CBD (15–60 nmol) and were placed 10 minlater in the center of the EPM facing an enclosed arm. Thenumber of entries and time spent in the open and enclosedarms were recorded for 5 min.

Experiment 2 The animals were water-deprived for48 h before the test. After the first 24 h of deprivation,they were allowed to drink freely for 3 min in the test cage inorder to find the drinking bottle spout. Some animals did notfind the spout and were not included in the experiment.Twenty-four hours later, they received intra-dlPAG injectionsof vehicle or CBD 30 nmol and 10min later were placed in theapparatus. The test period lasted for 3 min, and the animalsreceived a 0.5-mA shock through the bottle spout every 20licks. The number of punished licks was registered. To controlfor possible drug influence on water consummatory drive,independent groups of animals were submitted to the sameprotocol without receiving any electrical shock. The proce-dure was similar to the one already used and validated in thelaboratory (Moreira et al. 2006).

Experiment 3 Independent groups of animals were gentlyhandled, and their tails were laid across the coil. The heatwas applied to a portion of the ventral surface of the tailbetween 4 and 6 cm from its end. The time to withdraw thetail was recorded as tail-flick latency. The electric currentwas calibrated to provoke this reflex within 2.5–3.5 s innon-treated animals (Molchanov and Guimarães 2002). The

tail-flick latency was measured at 5-min intervals until astable baseline (BL) was obtained over three consecutivetrials. The tail withdrawal latency was measured at 10-minintervals after drug administration for up to 30 min(Molchanov and Guimarães 2002). The tail-flick latencywere normalized using an antinociception index (AI;Pedigo et al. 1975) according to the formula AI=(TL−BL)/(6−BL).

Experiment 4 The animals received intra-dlPAG injectionsof vehicle or AM251 100 pmol (dose based on Moreira etal. 2006) followed 5 min later by vehicle or CBD (30nmol). Ten minutes after the second injection, the animalswere submitted to the EPM test.

Experiment 5 Similar to experiment 4, except that theanimals received vehicle or WAY100635 0.37 nmol (dosebased on De Paula Soares and Zangrossi 2004) beforebeing submitted to the EPM test.

Histology

After the behavioral tests, the rats were sacrificed underdeep urethane anesthesia and perfused through the leftventricle of the heart with isotonic saline followed by 10%formalin solution. The brains were removed, and after aminimum period of 5 days immersed in a 10% formalinsolution, 50-μm sections were obtained in a Cryostat(Cryocut 1800). The injection sites were identified indiagrams from the Paxinos and Watson’s (1997) atlas andare illustrated in Fig. 1. Rats receiving injections of theactive dose of CBD outside the dlPAG were included in anadditional group (out).

Statistical analysis

The percentages of entries and time spent in the open arms(100×open/open+enclosed) during the 5-min sessions inthe EPM were calculated for each animal. These resultsplus the number of enclosed arm entries and the number oflicks in the Vogel test were also analyzed by one-wayANOVA. Data from the tail-flick experiment were analyzedby a repeated measure ANOVA followed by one-wayANOVAs at each time. The Duncan test was employed formultiple comparisons. Differences were considered signif-icant at p<0.05 level.

Results

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

Psychopharmacology (2008) 199:223–230 225

response curve (Fig. 2), with the dose of 30 nmol increasingthe percentage of entries (F(4, 33)=3.5; p=0.01; Duncantest, 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 ascompared to animals that received vehicle. Animals that

received the active dose (30 nmol) of CBD injectionsoutside the dlPAG (out) were not different from the controlgroup. No effects were found in the number of enclosedarms entries.

The active dose of CBD in the EPM (30 nmol) alsoincreased the number of punished licks (F(2, 20)=7.35, p=0.004; p<0.01) in the VCT (Fig. 3) without changing thenumber of unpunished licks (Table 1). The animals thatreceived CBD outside the dlPAG (out) were also notdifferent from controls.

Results from the tail-flick test can be seen in Fig. 4. Therepeated measures ANOVA revealed a significant effect oftreatment (F(2, 14)=108.4, p<0.001), time (F(6, 84)=19.21,p<0.001), and treatment×time (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, and25 min after injection when compared to CBD and vehicle(F(2, 14)=34.0 to 147.9, p<0.001). Fifteen minutes afterinjection, CBD also produced a small but significant

Fig. 2 Anxiolytic-like effect of CBD (15–60 nmol) microinjected intothe dlPAG of rats submitted to the EPM. Columns represent means ±SEM. The open columns represent the percent of entries in the openarms, while the hatched columns represent the percent of the timespent in the open arms. Animals receiving CBD (30 nmol) injectionsoutside the dlPAG were joined in an out group. Asterisks indicatesignificant difference from vehicle (Veh, p<0.05, ANOVA followedby the Duncan test; n=7, 6, 9, 5, 11, respectively)

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

Fig. 1 Left panel Histological localization of site injections (0.2 μL)in the dlPAG (full circles) and deep layers of superior colliculus(empty circles) in diagrams based on Paxinos and Watson’s (1997) ratbrain atlas. Right panel Representative microphotography of aninjection site in the dlPAG in a coronal brain slice. Bar=500 μm

226 Psychopharmacology (2008) 199:223–230

antinociceptive effect (p<0.05) compared to vehicle.However, contrary to morphine, the AI values after CBDinjection were not different from baseline.

In the third experiment, we investigated if a previousinjection of AM251, an antagonist of CB1 receptors, couldantagonize the anxiolytic-like effects of CBD observed inthe dose of 30 nmol (Fig. 5). Similar to the first experiment,CBD increased the percentage of time spent in the openarms (F(4, 28)=3.02, p=0.03, Duncan test, p<0.05) withoutchanging the number of enclosed arms entries. It alsotended to increase the percentage of open arm entries, butthe results did not reach significance (F(4, 28)=1.28).Previous injection of AM251 into the dlPAG failed toprevent the increase in the percentage of time spent in theopen arms induced by CBD. Animals that received CBDoutside the dlPAG (out) were not different from the controlgroup.

Results of the fourth experiment can be seen in Fig. 6.Previous injection of WAY100635, an antagonist of 5HT1Areceptors, prevented the anxiolytic-like effects of CBD inthe dlPAG. CBD increased the percentage of open arms

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

Discussion

The present study showed that CBD injected into thedlPAG increased exploration of the open arms withoutchanging the number of entries into the enclosed arms ofthe EPM, an animal model based on the rodent naturalaversion to elevated and open places. This result is usuallyinterpreted as indicating an anxiolytic effect (File 1992).

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

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

Table 1 Effects of intra-dlPAG administration of CBD (30 nmol) onthe number of unpunished licks of rats that had been water-deprivedfor 48 h

Treatment Unpunished licks

Vehicle 685.1±154.6CBD 612.9±92.9

Data are expressed as mean (±SEM, n=5–7/group). No significanteffect was found

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

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The effect seems to be mediated by the dlPAG sinceinjections of the active dose of CBD outside this regionwere not effective. The anxiolytic-like effect of CBD in thedlPAG was confirmed in the Vogel conflict test. This modelis based on the classical Geller–Seifter paradigm (Gellerand Seifer 1962). Similar to our results with CBD,anxiolytic drugs typically increase the number of punishedlicks without affecting the number of unpunished licks(Vogel et al. 1971). CBD also produced an antinociceptiveeffect in the tail-flick test after intra-dlAPG injection.However, this effect is unlikely to be responsible for theobserved anticonflict effect since it was small, not differingfrom baseline values, and occurred only 15 min after theinjection, whereas the VCT was performed 10 min afterdrug administration. Moreover, systemic administration ofmorphine, at similar doses used in the present study, doesnot produce antipunishment effects in a suppressed sched-ule-induced drinking paradigm (Pérez-Padilla and Pellón2007).

CBD can facilitate endocannabinoid-mediated neuro-transmission by decreasing anandamide hydrolysis or re-uptake (Bisogno et al. 2001). This effect could be related toits anxiolytic effects in the dlPAG since direct injection ofanandamide into this region induced anxiolytic-like effectsin the EPM that are prevented by local prior administrationof AM251 (Moreira et al. 2007). Although usuallydescribed as a CB1 receptor antagonist, AM251 can alsoproduce inverse cannabimimetic effects. However, itshows greater potency in opposing effects induced byCB1 agonists than producing inverse effects at CB1receptors, suggesting that at low concentrations, this drugcould act as a neutral CB1 receptor antagonist (Pertwee2005). In our study, the same dose of AM251 (100 pmol)that was able to antagonize the effects of intra-dlPAGadministered anandamide failed to prevent the anxiolyticeffects of CBD. It is improbable, therefore, that theseeffects are being mediated by facilitation of CB1 receptor-mediated neurotransmission.

There is also evidence that CBD could act, at micromo-lar concentration range, as an agonist of 5HT1A receptorsin 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 adenylylcyclase activity (Raymond et al. 1999). These receptors actas inhibitory auto-receptors in serotonergic neurons in theraphe nuclei but are also localized postsynaptically inseveral brain regions, including the PAG, amygdala,hippocampus, and frontal cortex. The PAG receivesserotonergic projections from the dorsal raphe nuclei (seeMillan 2003, for review), and activation of 5HT1Areceptors promotes the control of anxiety states andthe hypothalamus–pituitary–adrenal axis during stress

responses (Carrasco and Van de Kar 2003). Corroboratingthe proposal that CBD could facilitate 5HT1A-mediatedneurotransmission, its anxiolytic effect in the dlPAG wasprevented by previous treatment with WAY100635, a high-affinity (Ki ranging from 0.1 to 2.2 nM, Hamon et al. 1990,Corradetti et al. 2005) 5HT1A receptor antagonist. Thedose of WAY100635 was the same that was able to blockthe anxiolytic effects of 8-OH-DPAT, a 5HT1A agonist, inthe dlPAG (Zanoveli et al. 2003; De Paula Soares andZangrossi 2004).

Despite the present results, however, CBD has otherproposed mechanisms of action, such as blockade ofadenosine uptake (Carrier et al. 2006) and antagonism ofthe putative cannabinoid receptor GPR55 (Ryberg et al.2007). The involvement of these mechanisms in theanxiolytic effects of CBD in the dlPAG remains to betested.

The higher dose of CBD (60 nmol) was ineffective in thedlPAG. Systemic administration of CBD also produced abell-shaped dose–response curve in rats submitted to theEPM (Guimarães et al. 1990), an effect also observed withother cannabinoids (Moreira et al. 2006). CBD can activateTRPV1 receptors at micromolar concentration range(Bisogno et al. 2001). These receptors are expressed inthe PAG (Cristino et al. 2006) where they can facilitateglutamate release (Palazzo et al. 2002). It was recentlyshowed that capsazepine, a TRPV1 antagonist, blocks theanxiogenic effects of high doses of anandamide in theprefrontal cortex (Rubino et al. 2007), raising the possibil-ity that a similar mechanism could be involved in CBDeffects in the PAG. Preliminary data from our laboratorysupport this possibility (Campos and Guimarães, unpub-lished data), but further experiments are needed to addressthis question.

In conclusion, the present results suggest that activationof 5HT1A receptors located in the dlPAG could be one ofthe mechanisms of the anxiolytic effect observed with thiscompound after systemic administration.

Acknowledgments We thank Dr. Eleni T. Gomes and José Carlos deAguiar for technical support. This research was supported by grantsfrom FAPESP and CNPq. ACC was a recipient of a FAPESPfellowship.

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