5-HT1A receptors are involved in the anxiolytic effect of Δ9-tetrahydrocannabinol and AM 404, the anandamide transport inhibitor, in Sprague–Dawley rats
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.
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2021, Progress in Brain ResearchCitation Excerpt :However, this role is complex and animal studies suggest a biphasic U-shape effect of cannabinoids on anxiety- and depressive-like behaviors. Cannabinoid agonists such as WIN55,212-2, THC or HU-210 at low doses; and anandamide, or FAAH or endocannabinoid uptake inhibitors, can improve depressive-like behaviors and induce anxiolytic-like effects in animals (Bambico et al., 2007, 2012, 2016; Berrendero and Maldonado, 2002; Bortolato et al., 2007; Braida et al., 2007; Gobbi et al., 2005; Hill and Gorzalka, 2005a; Kathuria et al., 2003; McLaughlin et al., 2007, 2012; Rubino et al., 2008). On the other hand, anxiogenic responses have also been reported with anandamide, WIN55,212-2 or CP55,940 at higher doses in rodents (Borcel et al., 2004; Franklin et al., 2013a; Rubino et al., 2008).