The popularity of CBD is rapidly escalating (Leas et al., 2019). Epidolex, a CBD-rich cannabis extract, has been approved by the United States for the treatment of a narrow classification of pediatric epilepsies (Franco et al., 2021). However, its unapproved uses for the treatment of pain, anti-emesis, anxiety, sleep, and stress reduction has led to an increase in CBD exposure during infancy, childhood, and adolescence (Aran et al., 2018; Bertrand et al., 2018; Sarrafpour et al., 2020; Moss et al., 2021). Although many early studies point to benefits in children and adolescents across numerous indications such as epilepsy, anxiety, hyperactivity, and autism spectrum disorder (Shannon and Opila-Lehman, 2016; Aran et al., 2018; Bar-Lev Schleider et al., 2019; Barchel et al., 2019; Masataka, 2019; Poleg et al., 2019; Franco et al., 2021), CBD’s therapeutic potential could be thwarted if adverse developmental consequences are observed.

The impact of CBD on the developing brain is not well-characterized, and our understanding of its consequences has been limited, in part, by the relative recency of its popularity and the lack of data collection at long-term longitudinal time points. Here, we used a well-characterized normally developing healthy mouse model and exposed them to repeated suprathreshold anxiolytic doses of CBD for a prolonged 3-week time course during the mouse adolescent period (Brust et al., 2015). This is the first study to assess the impact of multiple daily CBD doses in mice during this important developmental stage. Although we detected less weight gain during the duration of our study in CBD-treated female mice, we failed to detect any detrimental effects on general locomotor activity, anxiety, or learning and spatial memory. Surprisingly, we revealed an improvement in the learning rate of a spatial memory task in CBD-treated mice compared to those administered the vehicle.

CBD is currently being used off-label or under investigation for treating numerous conditions in children and adolescents including autism-spectrum disorder (Poleg et al., 2019), anxiety (Masataka, 2019), insomnia (Shannon and Opila-Lehman, 2016), depression, and substance abuse (Laczkovics et al., 2021). Weight management is also a common reason for cannabinoid consumption (Bersani et al., 2016; Davis, 2016; Rossi et al., 2018). We observed a sex-specific effect on weight gain; female mice treated with CBD gained less weight over the 3-week exposure period than vehicle-treated females or male mice. Since food intake was not assessed, we cannot resolve whether these effects resulted from reduced food intake, changes in metabolic function, or a combination. However, CBD has been shown to reduce food intake in numerous rodent and human studies (Ignatowska-Jankowska et al., 2011; Farrimond et al., 2012; Devinsky et al., 2016; Linge et al., 2016). Notably, those effects were not shown to be sex specific, but stronger effects of CBD and other cannabinoids have been observed in females compared to males in both rodents and humans (Cooper and Craft, 2018; Spindle et al., 2020). Therefore, sex differences need to be accounted for in further research involving CBD and the translation to the clinic.

Our exposure period starting at PND 25 and ending at PND 45 represents the mouse brain development period similar to human adolescence (Brust et al., 2015). It’s possible that adverse effects on the developing brain and behavior could result from repeated CBD exposure earlier in development. 20 mg/kg CBD administration to pregnant dams once daily (i.e., same dose but half the exposure frequency as the current study) during gestation and lactation in rats led to heightened anxiety assessed in the marble burying task in female offspring (Wanner et al., 2021). Interestingly, the female offspring also showed improved spatial memory in the Y-maze, which is consistent with our finding of a faster spatial learning rate in the CBD-exposed cohort. Notably, we did not assess measures of cognitive flexibility or working memory that rely heavily on prefrontal cortical circuitry (Hamilton and Brigman, 2015) and undergo maturation during this adolescent time period (Klune et al., 2021), which makes them vulnerable to pharmacological perturbation.

We assessed spatial learning and memory using the Barnes Maze procedure. The hippocampus plays a critical role in spatial memory formation (Bird and Burgess, 2008) and is functionally coupled to the prefrontal cortex during navigational search strategies in the Barnes Maze (Negrón-Oyarzo et al., 2018). Hippocampal neurogenesis is important for the acquisition of spatial memory across a variety of behavioral paradigms (Lieberwirth et al., 2016), and reducing neurogenesis impairs Barnes Maze performance (Raber et al., 2004). C57BL/6J mice raised in standard laboratory housing experience sub-maximum hippocampal neurogenesis, and CBD can increase adult neurogenesis through modulating activity of cannabinoid type I receptors (CB1) (Wolf et al., 2010). This modulation may occur through inhibition of FAAH-mediated degradation of hippocampal anandamide signaling by CBD (Bisogno et al., 2001; Ibeas Bih et al., 2015). Further, this increase in neurogenesis protects against an impairment to neurogenesis and resulting anxiety resulting from a chronic stress paradigm (Campos et al., 2013). Since multiple daily i.p. injections can be stress-inducing, one potential explanation for the faster acquisition rate in the Barnes Maze by CBD-treated mice is that CBD either enhanced neurogenesis or protected against the stress-induced reduction from repeated i.p. injections. Further studies are needed to elucidate this mechanism.

CBD has over 65 known targets in the brain and body that are activated at varying doses (Ibeas Bih et al., 2015). It’s still not clear which of these targets promote CBD’s many proposed therapeutic actions, although some have been better characterized. Of particular relevance in adolescent use, some involve, though not limited to, GPR55 in epilepsies resulting from heterozygous mutation to the Scn1a gene (Kaplan et al., 2017), 5-HT1a receptors in anxiety (Gomes et al., 2011), depression (Linge et al., 2016), and protection against acute stressors (Resstel et al., 2009), and PPARs and TRPV receptors on digestive and gut health (Couch et al., 2019). Further, CBD inhibition of fatty acid amino hydrolase (FAAH), the primary degradation enzyme for the endocannabinoid, anandamide, is implicated or proposed to reduce compulsive behavior (Casarotto et al., 2010), anxiety (Moreira et al., 2008), borderline personality disorder (Kolla et al., 2020), and autism spectrum disorder (Aran et al., 2019). It’s reasonable to hypothesize that modulating these systems during critical stages of development could impact the brain in ways that go undetected in our broad mouse behavioral assays but may present in humans through more subtle investigative techniques or in more complex assessments. These human studies are largely lacking, but in one study of epileptic patients, subtle changes in regional white matter density were detected following 12 weeks of CBD use (Thompson et al., 2020). However, alterations were not associated with changes in cognitive function or adaptive behavioral skills. Follow-up safety studies in a human clinical population will need to be conducted in several years when currently using children and adolescents become adults.

Effective CBD dosing differs as a function of therapeutic indication. In pre-clinical acute-exposure studies, CBD reduced anxiety in the elevated plus maze at doses between 2.5 and 10 mg/kg, but this effect was lost at a higher 20 mg/kg dose in rats (Guimarães et al., 1990). We confirmed that an acute CBD dose of 5 mg/kg, but not 10 mg/kg, reduced anxiety in the elevated plus maze in mice. Our decision to test the effect of a higher, 20 mg/kg dose, was based on the presumption that a suprathreshold dose for anxiety-related behaviors, which is one of the most common reasons for off-label CBD use (Moltke and Hindocha, 2021), would impact more neural mechanisms and have more pronounced effects, should they occur. We also chose to use a suprathreshold dose for anxiety-related behavior, albeit in healthy mice, to account for dose escalation that may occur with tolerance (Long et al., 2012). This 20 mg/kg dose has been shown to improve social deficits in a mouse model expressing autism spectrum disorder behaviors (Kaplan et al., 2017). Further, clinical and pre-clinical mouse studies demonstrate CBD’s efficacy in reducing epileptic seizures at doses up to around 20 mg/kg (Devinsky et al., 2017) and 100 mg/kg (Kaplan et al., 2017), respectively. It remains possible that a higher dose than the one tested here would have more lasting effects on brain function and behavior. It is similarly possible that a lower CBD dose may contribute to more lasting effects on brain development if a high dose, by impacting different targets or modulating them at different levels, counteracted the impairing effect of a lower dose. While the evidence seems to suggest that high doses lead to more acute adverse effects than lower doses (Devinsky et al., 2018), there has been no long-term investigation of CBD’s long-term dose-response effects on brain development, thereby highlighting the need for a dose-response assessment at different developmental stages.

We report that multiple daily doses of a moderate CBD dose throughout the adolescent developmental period does not negatively impact locomotor behavior, anxiety, and spatial learning in healthy C57BL/6J mice. Further, the faster acquisition rate of a spatial learning task may highlight CBD’s potential protective benefits against stressors. While sex-specific impacts of CBD on weight gain during this period need to be considered and monitored, these findings do not detract from the progression of clinical studies investigating CBD’s therapeutic potential in an adolescent population.