Prolonged Cannabidiol Treatment Lacks on Detrimental Effects on Memory, Motor Performance and Anxiety in C57BL/6J Mice

Eva M. Schleicher, Frederik W. Ott, Melanie Müller, Barbara Silcher, Marius E. Sichler,
Maximilian J. Löw, Jannek M. Wagner and Yvonne Bouter

Frontiers in Behavioral Neuroscience, 2019, Volume 13, Article 94

doi : 10.3389/fnbeh.2019.00094

 

The Cannabis plant contains more than 100 currently known phytocannabinoids. Regarding the rising consumption of the non-psychotropic phytocannabinoid cannabidiol (CBD) in people’s everyday life (e.g., beauty products, food and beverages), the importance of studies on the influence of CBD on healthy humans and rodents is evident. Therefore, the behavioral profile of CBD was investigated with a battery of behavioral tests, including motor, anxiety, and memory tests after prolonged CBD treatment. Adult C57Bl/6J wildtype (WT) mice were daily intraperitoneally injected with 20 mg/kg CBD for 6 weeks starting at two different points of ages (3 months and 5 months) to compare the influence of prolonged CBD treatment with a washout period (former group) to the effects of long term CBD treatment (current group). Our results show that CBD treatment does not influence motor performance on an accelerating Rotarod test, while it also results in a lower locomotor activity in the open field (OF).

No influence of CBD on spatial learning and long term memory in the Morris Water Maze (MWM) was observed. Memory in the Novel Object Recognition test (NORT) was unaffected by CBD treatment. Two different anxiety tests revealed that CBD does not affect anxiety behavior in the Dark-Light Box (DLB) and OF test. Although, anxiety is altered by current CBD treatment in the Elevated Plus Maze (EPM). Moreover, CBDtreated C57Bl/6J mice showed an unaltered acoustic startle response (ASR) compared to vehicle-treated mice. However, current CBD treatment impairs prepulse inhibition (PPI), a test to analyze sensorimotor gating. Furthermore, prolonged CBD treatment did not affect the hippocampal neuron number. Our results demonstrate that prolonged CBD treatment has no negative effect on the behavior of adult C57Bl/6J mice.

Keywords : cannabidiol, behavior, morris water maze, anxiety, cannabinoid system, cannabis

INTRODUCTION

Over the last years, there has been growing interest in the therapeutic potential of the phytocannabinoid cannabidiol (CBD) occurring naturally in the plant Cannabis sativa/indica, commonly known as marijuana. Several studies showed that CBD is involved, among others, in immunomodulatory, anti inflammatory, antiemetic, anticonvulsant, anxiolytic, antipsychotic, muscle relaxant and neuroprotective processes (Atakan, 2012; Burstein, 2015; Watt and Karl, 2017). Interestingly, the interest in studying CBD initially came through its interaction with the probably most commonly recognized constituent of the cannabis plant, 9-Tetrahydrocannabinol (THC). CBD was first isolated by Adams et al. (1940) in 1940 and its structure was elucidated 23 years later (Cunha et al., 1980). The pharmacological mechanisms of THC are the most well understood among the more than 100 other currently known phytocannabinoids (Mechoulam et al., 2014). Whereas THC is dependent from CB1-, and CB2-receptor binding, the mechanism of action for CBD is still not fully understood (De Petrocellis and Di Marzo, 2010). There are several other receptors that appear to be involved in the therapeutic effect of CBD, such as TRPV1-, PPARg-, 5-HT1A-, and GPR55-Receptors (Zygmunt et al., 1999; Bouaboula et al., 2005; Russo et al., 2005; O’Sullivan, 2007; Ryberg et al., 2007).

Beneficial impacts of CDB on multiple diseases, such as multiple sclerosis (Mecha et al., 2013), brain ischemia (Schiavon et al., 2014; Mori et al., 2017) and epilepsy (Patra et al., 2019) have been shown in animal models. Several studies support the beneficial effects of CBD for treating neuropsychiatric disorders, particularly affective disturbances as anxiety, depression and schizophrenia (Micale et al., 2013; Kucerova et al., 2014; Blessing et al., 2015). An anxiolytic effect has also been observed in healthy humans (Cunha et al., 1980). Recently, Stark et al. (2019) showed that early treatment with CBD can even prevent the appearance of schizophrenia-like deficits. Surprisingly, few studies have examined the possible effects of prolonged CBD treatment on healthy mice. Studies of CBD effects have been mostly restricted to its acute effect and less is known about the efficiency after chronic CBD treatment. Therefore, the purpose of this study was to investigate the consequences of prolonged CBD treatment on the behavior of healthy C57BL/6J animals. In addition, we analyzed the effects of CBD on behavior after a washout period.

MATERIALS AND METHODS

Animals and Drug Treatment C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in this study with an equal distribution of male and female mice. All animals were handled according to
the guidelines of the Federation of European Laboratory Animal Science Association (FELASA) and approved by the ‘‘Lower Saxony State Office for Consumer Protection and Food Safety’’ (LAVES). Mice were kept in individually ventilated cages (IVC, 32  16  14 cm; Tecniplast, Hohenpeißenberg, Germany) in groups up to five. Water and food were available ad libitum. Powdered CBD (THC Pharm GmbH, Frankfurt/Main, Germany) was dissolved in equal amounts of 2.5 ml Tween 80 (Carl Roth GmbH, Karlsruhe, Germany) and 2.5 ml 100% ethanol and diluted in 45 ml of 0.9% NaCl solution. A vehicle
control treatment group was set up in the exact same way, with the exception of CBD. Mice were assigned to either CBD or vehicle-treated groups and treated daily with an intraperitoneal injection containing the injection volume of 10 ml/kg body weight for 6 weeks starting at the age of 3 months (in the following called ‘‘former’’) or 5 months (in the following called ‘‘current’’; Figure 1). Mice were treated with 20 mg/kg body weight of CBD. Mice were weighed weekly and the injection volume was adjusted accordingly. In the current group, treatment continued during behavioral testing and lasted until the day of sacrifice. Behavioral testing started for all mice at the age of 6 months and mice were sacrificed with 26 weeks.

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