Elsevier

Neuropharmacology

Volume 62, Issue 1, January 2012, Pages 373-384
Neuropharmacology

Effect of cannabidiol on sleep disruption induced by the repeated combination tests consisting of open field and elevated plus-maze in rats

https://doi.org/10.1016/j.neuropharm.2011.08.013Get rights and content

Abstract

Patients with post-traumatic stress disorder (PTSD) frequently complain of having sleep disturbances, such as insomnia and rapid eye movement (REM) sleep abnormality. Cannabidiol (CBD), a psycho-inactive constituent of marijuana, reduces physiological non-REM (NREM) sleep and REM sleep in normal rats, in addition to generating its anxiolytic effect. However, the effects of CBD on anxiety-induced sleep disturbances remain unclear. Because anxiety progression is caused by persistent stress for a period of time, we employed the repeated combination tests (RCT) consisting of a 50-min open field (OF) and a subsequent 10-min elevated plus-maze (EPM) for four consecutive days to simulate the development of anxiety. Time spent in the centre arena of OF and during open arms of the EPM was substantially decreased in latter days of RCT, suggesting the habituation, which potentially lessens anxiety-mediated behavioural responses, was not observed in current tests. CBD microinjected into the central nucleus of amygdala (CeA) significantly enhanced time spent in centre arena of OF, increased time during the open arms and decreased frequency of entry to the enclosed arms of EPM, further confirming its anxiolytic effect. The decrease of NREM sleep during the first hour and the suppression of REM sleep during hours 4–10 after the RCT represent the similar clinical observations (e.g. insomnia and REM sleep interruption) in PTSD patients. CBD efficiently blocked anxiety-induced REM sleep suppression, but had little effect on the alteration of NREM sleep. Conclusively, CBD may block anxiety-induced REM sleep alteration via its anxiolytic effect, rather than via sleep regulation per se.

This article is part of a Special Issue entitled ‘Anxiety and Depression’.

Graphical abstract

Highlights

► Repeated combination test (RCT) simulates post-traumatic stress disorder (PTSD)-induced sleep disruptions. ► Anxiety behaviours are persistent and REM sleep decreases after RCT. ► CBD reduces anxiety and blocks RCT-induced REM sleep suppression. ► CBD reduces NREM sleep and REM sleep in normal rats. ► Effect of CBD on RCT-induced REM sleep suppression is via its anxiolytic action.

Introduction

Anxiety disorders are the most common psychiatric disorders associated with sleep disruptions. In fact, sleep disturbances are included in the diagnostic criteria in two categories of anxiety disorders: general anxiety disorder (GAD) and post-traumatic stress disorder (PTSD) (American Psychiatry Association, 1994). Patients with GAD frequently complain to have difficulties falling asleep, difficulties staying asleep, restless sleep, and unrefreshing sleep (American Psychiatry Association, 1994, Anderson et al., 1984), and persistent symptoms of increased arousal, insomnia, nightmares and disrupted rapid eye movement (REM) sleep are common sleep disorders in patients with PTSD (American Psychiatry Association, 1994, Horowitz et al., 1980, Mellman et al., 2002, Mellman and Davis, 1985, Neylan et al., 1998). Although anxiousness and worries are usually the causes of sleep disturbances in anxiety patients, sleep-specific pathologies and the other factor, such as stress per se, may develop and exacerbate sleep disruptions. The causal connection between sleep problems and anxiety are difficult to determine in the clinical aspect, and therefore the employment of an animal model of anxiety to study this relationship becomes adequate. The open field (OF) has been widely used to assess the behavioural effects of anxiety in rodents (Hall, 1936). Confrontation of the novel environment provided by the OF produces conflicting motivations between fear and exploration (Prut and Belzung, 2003, Welker, 1957). More thigmotaxis and less locomotion activity indicate greater anxiety, whereas greater exploration and more ambulation to the centre arena of the OF reflect less emotion (Choleris et al., 2001). The elevated plus-maze (EPM), which is another rodent model for anxiety and used as a screening test for putative anxiolytics, consists of two open and two enclosed arms elevated 60 cm from the floor. This model is based on rodent’s aversion of height and openness (Pellow et al., 1985, Treit et al., 1993). Because the progression of anxiety may be caused by the persistence of stress for a long period of time, we would like to mimic this anxiety development by exposing the animals to repeated stressors. However, a habituation behaviour may occur after repeated exposure to the OF. This anxiety-like behaviour is greater expressed during initial exposure to the OF, and exploration behaviour is increased as exposure time in the OF increases or with repeated exposure to the OF, indicating the lessened anxiety (Ivinskis, 1970, Makino et al., 1991). In contrast, anxiety behaviours expressed in the EPM have demonstrated a lack of habituation of exploration in the open arms because of the innate aversiveness of openness and height (Graeff et al., 1998, Treit et al., 1993). We herein employed the repeated combination tests (RCT), consisting of a 50-min of OF and a subsequent 10-min of EPM, for four consecutive days to simulate the anxiety development and anxiety-induced sleep disruptions.

Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) are two major constituents of the Cannabis sativa (ElSohly, 2002, Mechoulam, 1970). Two cannabinoid receptors, CB1 and CB2, are coupled to the pertussis toxin-sensitive G-protein, Gi,o, which inhibits adenylate cyclase and subsequently reduces the conversion of ATP to cyclic AMP (cAMP) (Chevaleyre et al., 2006). Δ9-THC has a strong binding affinity for central CB1 receptors and produces psychoactive properties, including the anxiogenic effect (Grotenhermen, 2006), whereas CBD exhibits a low binding affinity for CB1 receptors and has a non-psychoactive property (Grotenhermen, 2006, Mechoulam et al., 2002). The pharmacological property of the anxiolytic effect of CBD has been demonstrated in both animal researches (Guimaraes et al., 1990, Mechoulam et al., 2002, Pickens, 1981) and human studies (Crippa et al., 2004, Fusar-Poli et al., 2010), and becomes significant for therapeutic purposes. CBD also possesses sleep modulation effects; intracerebroventricular (ICV) administration of CBD during the light period increases wakefulness and decreases REM sleep in rats (Murillo-Rodríguez et al., 2006). We previously demonstrated that microinjection of CBD into the central nucleus of amygdala (CeA) decreases NREM sleep with limited effects on REM sleep suppression in normal rats (Yi et al., 2008). However, effects of CBD on anxiety-induced sleep disturbances have not been elucidated yet. CeA plays a key role in emotional processing, anxiety-related physiological and behavioural responses (Graeff et al., 1998), and its related behavioural responses, i.e. sleep–wake activity (Sanford et al., 1995). Here we determined the sleep disturbances induced after exposure of rats to RCT. Both effects of CBD, microinjected into the CeA, on sleep disturbances and the anxiolytic were evaluated. Comparing to the effects of CBD on spontaneous sleep regulation, present results would further elucidate whether the action of CBD on anxiety-induced sleep disturbance is mediated by its sleep regulation per se or is the consequence of anxiolytic action.

Section snippets

Substances

Stock solution of CBD (Tocris, Bristol, UK) was dissolved in 2% dimethyl sulfoxide (DMSO). These stock solutions were stored at −20 °C until administration. The doses of CBD used in these experiments were 0.5 and 1.0 μg. The total volume for each injection was 1 μl.

Animals

Male Wistar rats (250–300 g; National Laboratory Animal Breeding and Research Center, Taiwan) were used in these experiments. These animals were anesthetized (ketamine/xylazine; 87/13 mg/kg), and injected with an analgesic (morphine)

Behavioral measures of the repeated combination tests (RCT)

The values represented in Fig. 2 were the time differences for rats spending in the centre arena of the OF between second-day and first-day exposures of combination tests (Δ-day2), between third-day and first-day exposures (Δ-day3), and between fourth-day and first-day exposures (Δ-day4). The measures during the OF exposure were also divided into two 25-min blocks. The time for rats spending in the centre arenas of the OF during the total 50 min of exposure was gradually decreased as rats were

Discussion

Persistent sleep disturbances, such as increased arousal, reduced sleep efficiency caused by increased wakefulness from sleep, insomnia, nightmares, disrupted REM sleep, and periodic limb movements, are highly prevalent in PTSD patients (American Psychiatry Association, 1994, Harvey and Bryant, 1998, Horowitz et al., 1980, Koren et al., 2002, Mellman et al., 2001, Mellman et al., 2002, Mellman and Davis, 1985, Neylan et al., 1998). Growing evidence indicates that sleep disturbances are the core

Statement of interest

None. Authors have indicated no financial conflicts of interest.

Acknowledgements

This work was supported by National Science Council grant NSC96-2628-B-002-029-MY3. We thank Mr. Brian Chang for his help with English revision and Mr. Yi-Fong Tsai’s technical assistance in this project.

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