5-HT1A receptors are involved in the anxiolytic effect of Δ9-tetrahydrocannabinol and AM 404, the anandamide transport inhibitor, in Sprague–Dawley rats

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Abstract

The mechanism mediating the effects of cannabinoids on anxiety-related responses appear to involve cannabinoid CB1 and non-CB1 receptors. However, other neurotransmitters may play a role in such effect. This study shows evidence of an interaction between endocannabinoid system and serotonin (5-HT), 1A receptor subtype on anxiety-like behavior in Sprague–Dawley rats. The exogenous cannabinoid agonist, Δ9-tetrahydrocannabinol (THC), and N-(4-hydroxyphenyl)-arachidonylamide, the anandamide transporter inhibitor (AM 404) were evaluated in the elevated plus maze test. THC (0.075–0.75 mg/kg i.p.), given 30 min and AM 404 (0.75–1.25 mg/kg i. p.), given 60 min before the test, exhibited a dose–response anxiolytic effect evaluated in terms of increase in the percentage of total entries and time spent in the open and decrease of total entries and time spent in the closed arms. The anxiolytic effect obtained with the maximal active dose of both THC (0.75 mg/kg) and AM 404 (1.25 mg/kg) was blocked by the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide dihydro chloride (WAY-100635 (300 μg/kg, s.c.), given 30 min before THC or 15 min before AM 404. The combination of an ineffective dose of THC (0.015 mg/kg) or AM 404 (0.015 mg/kg) on anxiety-related responses with an ineffective dose of the 5HT1A receptor agonist, 8-Hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) (7.5 μg/kg, i.p.), led to a synergistic effect. No interference with spontaneous motor activity, evaluated in an activity cage for 5 min, in rats given the drugs alone or in combination, was found. These data suggest that the anxiolytic effect produced by endo- and eso-cannabinoids is modulated by 5-HT1A receptors.

Introduction

Recent findings suggest a role for endocannabinoid signalling in the modulation of anxiety-related behaviors (Piomelli et al., 1999, Piomelli, 2003, Viveros et al., 2005). Pharmacological blockade of the enzyme fatty acid amide hydrolase (FAAH), which is responsible for intracellular anandamide degradation, produces anxiolytic effects in adult rats tested in the elevated zero maze (Gaetani et al., 2003) and in the isolation-induced ultrasonic vocalization paradigm in rat pups (Kathuria et al., 2003). In a recent paper (Bortolato et al., 2006, Rutkowska et al., 2006) clear evidence that anandamide has a role in the regulation of anxiety states, has been provided. Specifically the peripheral injection of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonylamide (AM404) or cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597), the active inhibitor of fatty acid amide hydrolase, exhibited anxiolytic-like effects in different rat models of anxiety. These effects were accompanied by an increased brain level of anandamide and were prevented by cannabinoid CB1 receptor blockade.

AM 404, significantly decreased restraint-induced serum corticosterone release in mice suggesting a role of endocannabinoid system in the environmental stress (Patel et al., 2004; Patel and Hillard, 2006). An inhibition of anandamide transporter by AM 404 in rat brain neurons and slices (Beltramo et al., 1997, Beltramo and Piomelli, 2000) and human astrocytoma cells (Piomelli et al., 1999) in vitro was shown. The compound, given i.v., induced vasodilatation (Calignano et al., 1997) in guinea-pigs, while it enhanced and prolonged anandamide-induced analgesia (Beltramo et al., 1997) in rats, in vivo. On the other hand, AM404 did not elicit catalepsy or analgesia in rats (Beltramo and Piomelli, 2000) while it was found to potentiate the effects of anandamide exogenously administered (Rodriguez de Fonseca et al., 2005). An hypokinetic effect in Wistar rats was reported after treatment with AM 404 at a dose of 10 mg/kg, which was reversed by rimonabant (Giuffrida et al., 2000). AM 404 (5 mg/kg) acted as a psychodysleptic drug, altering prepulse inhibition in mice through a cannabinoid CB1 receptor (Fernandez-Espejo and Galan-Rodriguez, 2004) and elicited antidepressant-like effects in the forced swimming test in rats (Hill and Gorzalka, 2005).

On the other hand, exogenous cannabinoids like Δ9-tetrahydrocannabinol (THC) as well as genetic manipulation participate in regulating a variety of emotional responses (Witkin et al., 2005). Thus, several studies showed that cannabinoid agonists can induce anxiogenic and anxiolytic-like responses in rodents, depending on the dose and the environmental conditions (Rodriguez de Fonseca et al., 1996). Low doses of cannabinoids usually induce an anxiolytic-like effect, whereas high doses cause the opposite response (Marın et al., 2003, Viveros et al., 2005). More recent findings have shown that both (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl) cyclohexanol (CP 55,940) or R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl]-pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-(1-aphthalenyl)-methanone mesylate (WIN 55,212-2) produced anxiolytic effects in mice, tested in the elevated plus maze, at low doses only (Patel and Hillard, 2006). On the other hand, THC also produced the same effect in the light–dark test, in mice (Berrendero and Maldonado, 2002). Furthermore, endocannabinoid system, through the activation of cannabinoid CB1 receptors, seem to be involved in the regulation of emotional behavior, as revealed by the anxiogenic-like response induced in rats by the acute treatment of the cannabinoid CB1 receptor antagonist, rimonabant (Navarro et al., 1997). An increase in the basal level of anxiety during the light–dark test or in the plus maze test in cannabinoid CB1 knockout mice (Haller et al., 2002, Haller et al., 2004, Martin et al., 2002), was observed. Whereas, cannabinoid CB1 receptor mutant mice show an anxiolytic-like phenotype in the shock-probe burying test (Degroot and Nomikos, 2006). The mechanism by which cannabinoids would control anxiety-related behavior is not still elucidated.

Pharmacological (Griebel, 1995, Griebel et al., 1997, Griebel et al., 2005) and genetic (Holmes, 2001) studies have well documented the role of serotonergic system in the regulation of anxiety. Until now, scarce attention has been paid to the interaction of cannabinoids with serotonin. A cross-talk between cannabinoid CB1 and 5-HT receptors in rat (Devlin and Christopoulos, 2002) cerebellar and bovine (Kimura et al., 1998) synaptic membranes, using radioligand binding assay, was reported. In addition, co-expression of cannabinoid CB1 receptors and different 5-HT receptor subtypes in neuronal subpopulations of the cerebellum (Devlin and Christopoulos, 2002) and forebrain (Hermann et al., 2002), was found. In vivo, a study by Molina-Holgado et al. (1997), reported changes in 5-HT content and uptake sites in different brain regions following acute and chronic perinatal maternal exposure of THC. An involvement of 5-HT neurons in mediating cannabinoid effects such as antiemesis (Fan, 1995), hypothermia (Malone and Taylor, 1998), some types of analgesia (Fan, 1995), sleep (Mendelson and Basile, 2001) and feeding (Rowland et al., 2001), has been already found.

Among different subtypes, 5-HT1A receptors, projecting from mid-brain raphe nuclei to limbic areas, have received particular attention. In the elevated plus maze, 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT), dose-dependently induced an anxiolytic like effect in Sprague–Dawley rats, which was reversed by the pre-treatment with the 5-HT1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide dihydrochloride (WAY-100635) (Collinson and Dawson, 1997). Similar effect was obtained using Wistar rats in the ultrasonic vocalization test (De Vry et al., 2004). On the other hand, WAY-100635, per se, was found to produce a biphasic anxiolytic effect in the plus maze test (Griebel et al., 1999).

Very few data exist about the interaction between endocannabinoid and 5-HT1A system. Pre-treatment with the 5-HT1A receptor subtype antagonist, WAY-100635, attenuated the anxiogenic, but not the anxiolytic effect of CP 55,940 (Marco et al., 2004).

On the basis of the above evidence, the present study was designed to test in vivo the involvement of 5-HT1A subtype receptors on emotional-like behavior induced by acute injection of AM 404 and THC in Sprague–Dawley rats, using the elevated plus maze. In order to test this hypothesis, we studied the possible interaction between the anandamide transport inhibitor, AM 404, or THC and the 5-HT1A receptor antagonist WAY-100635 or agonist, 8-OH-DPAT in the modulation of elevated plus maze. This test is presently the most widely used model of anxiety-like behavior for drug discovery in experimental research (Dawson and Tricklebank, 1995) and it is based on the naturalistic conflict between the tendency of rats to explore a novel environment and the aversive properties of a brightly lit open area (Pellow et al., 1985).

Section snippets

Subjects

At the beginning of the experiment, maze-naïve non-handled male Sprague–Dawley rats (Charles-River, Calco, Italy) weighing 150–175 g were housed in cages (10 per cage), in a climatically controlled colony room under a 12-h light–dark cycle (lights on at 8:00 a.m.). Food and water were continuously available and each animal was handled daily during the seven days preceding the experiment. The day before the experiment animals were individually housed and randomly assigned to each experimental

Results

The effect of THC and AM 404 on elevated plus maze behavior, is shown in Table 1. Systemic administration of both drugs produced a significant effect on emotional behavior. The percentage of entries made in the open arms (χ2 = 15.60, P < 0.001 for THC; χ2 = 16.64, P < 0.0001 for AM 404) increased. Furthermore, the percentage of time spent in the open (χ2 = 13.25, P < 0.01 for THC; χ2 = 12.38, P < 0.01 for AM 404) increased and that in the closed arms (χ2 = 11.54, P < 0.01 for THC; χ2 = 15.55 P < 0.01 for AM 404)

Discussion

The present findings provide for the first time a dose-dependent anxiolytic effect, in Sprague–Dawley rats of THC and a further evidence for AM 404, as shown by the increase in the number of entries and time spent in the open arms of the elevated plus maze. The maximal effective dose of AM 404 (1.25 mg/kg) produced an increase of open arm time of about 300% similar to that (400%) obtained for the classical anxiolytic compound, diazepam (Bortolato et al., 2006). However, THC at the maximal

Acknowledgement

This work was supported by a grant of Italian Ministry of Research and Technology (PRIN, COFIN 2004) to M.S.

References (59)

  • H. Hermann et al.

    Coexpression of the cannabinoid receptor type 1 with dopamine and serotonin receptors in distinct neuronal subpopulations of the adult mouse forebrain

    Neuroscience

    (2002)
  • M.N. Hill et al.

    Pharmacological enhancement of cannabinoid CB(1) receptor activity elicits an antidepressant-like response in the rat forced swim test

    Eur. Neuropsychopharmacol.

    (2005)
  • S. Hogg

    A review of the validity and variability of the elevated plus-maze as an animal model of anxiety

    Pharmacol. Biochem. Behav.

    (1996)
  • A. Holmes

    Targeted gene mutation approaches to the study of anxiety-like behavior in mice

    Neurosci. Biobehav. Rev.

    (2001)
  • B.G. Kelley et al.

    Delta 9-tetrahydrocannabinol antagonizes endocannabinoid modulation of synaptic transmission between hippocampal neurons in culture

    Neuropharmacology

    (2004)
  • S. Marın et al.

    Involvement of the kappa-opioid receptor in the anxiogenic-like effect of CP 55,940 in male rats

    Pharmacol. Biochem. Behav.

    (2003)
  • W.B. Mendelson et al.

    The hypnotic actions of the fatty acid amide, oleamide

    Neuropsychopharmacology

    (2001)
  • F. Molina-Holgado et al.

    Maternal exposure to delta 9-tetrahydrocannabinol (delta 9-THC) alters indolamine levels and turnover in adult male and female rat brain regions

    Brain Res. Bull.

    (1997)
  • S. Pellow et al.

    Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat

    J. Neurosci. Methods

    (1985)
  • M.P. Viveros et al.

    Endocannabinoid system and stress and anxiety responses

    Pharmacol. Biochem. Behav.

    (2005)
  • P.M. Zygmunt et al.

    The anandamide transport inhibitor AM404 activates vanilloid receptors

    Eur. J. Pharmacol.

    (2000)
  • M. Beltramo et al.

    Carrier-mediated transport and enzymatic hydrolysis of the endogenous cannabinoid 2-arachidonylglycerol

    Neuroreport

    (2000)
  • M. Beltramo et al.

    Functional role of high-affinity anandamide transport, as revealed by selective inhibition

    Science

    (1997)
  • M. Beltramo et al.

    Reversal of dopamine D(2) receptor responses by an anandamide transport inhibitor

    J. Neurosci.

    (2000)
  • F. Berrendero et al.

    Involvement of the opioid system in the anxiolytic-like effects induced by Δ9-tetrahydrocannabinol

    Psychopharmacology

    (2002)
  • D.L. Boger et al.

    Structural requirements for 5-HT2A and 5-HT1A serotonin receptor potentiation by the biologically active lipid oleamide

    Proc. Natl. Acad. Sci.

    (1998)
  • M.M. Bortolato et al.

    Anxiolytic-Like properties of the anandamide transport inhibitor AM404

    Neuropsychopharmacology

    (2006)
  • D. Braida et al.

    Cannabinoid-induced working memory impairment is reversed by a second generation cholinesterase inhibitor in rats

    Neuroreport

    (2000)
  • J.P. Chhatwal et al.

    Enhancing cannabinoid neurotransmission augments the extinction of conditioned fear

    Neuropsychopharmacology

    (2005)
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