Female but not male rats show biphasic effects of low doses of Δ9-tetrahydrocannabinol on anxiety: can cannabidiol interfere with these effects?
Graphical abstract
Introduction
Cannabis is still considered an illicit drug, even though its medicinal use has increased over the last few years. Among cannabis active compounds, cannabidiol (CBD) is the most studied due to its safety profile and therapeutic potential for several diseases (Bitencourt et al., 2021; Melas et al., 2021; Peters et al., 2021; Stern et al., 2018). On the other hand, Δ9-tetrahydrocannabinol (THC) is the primary psychoactive component responsible for cannabis recreational and undesirable clinical effects. However, THC is present in several medicinal preparations alone or combined with CBD (Freeman et al., 2019). Synthetic THC (dronabinol) is approved for nausea and vomiting in patients undergoing chemotherapy (Serafimovska et al., 2020). Sativex®, which contains a ratio of ~1:1 of THC and CBD, is approved for multiple sclerosis spasticity at doses varying from 1.0 to 20 mg (MacCallum and Russo, 2018). Considering that high doses of THC potentially produce undesirable effects, some authors have addressed the importance of using low to ultra-low doses of THC. In this line, low doses of THC reverse cognitive impairment in old mice (Bilkei-Gorzo et al., 2017), also, ultra-low doses of THC improved cognitive performance in a spatial memory test in old female mice (Sarne et al., 2018) and did not affect anxiety (Fishbein-Kaminietsky et al., 2014), an undesirable effect of THC.
Anxiety disorders have a higher prevalence in women (Marvaldi et al., 2021). Sexual dimorphism is a relevant factor that interferes with mood, including anxiety-like behaviors, and increases susceptibility to a stressful stimulus (Altemus et al., 2014; Shansky et al., 2004). Besides, evidence suggests that females are more sensitive to Cannabis effects than males (Cooper and Craft, 2018). In line with this, developmental exposure to CBD increased anxiety in female offspring, but not males (Wanner et al., 2021). Treatment with 5.0 mg/kg of THC increased only the female rats’ avoidance in the open-field test and c-Fos expression in stress-related brain areas (Ruiz et al., 2021). However, others have shown that the anxiogenic effect of high doses of THC is observed in both male and female mice (Bruijnzeel et al., 2019; Kasten et al., 2019). Thus, the available preclinical data vary widely in dosage and evaluate the effects of conventional and high THC doses on anxiety. Identifying the acute effects of lower doses of THC and THC combined with CBD on anxiety in females is necessary for developing appropriate cannabis-based treatments.
The serotoninergic and dopaminergic transmissions in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) are essential to regulate anxiety and motivated behaviors in rodents and humans (Everett et al., 2021; Liu et al., 2019; Marcus et al., 2020; Stern et al., 2010). CBD and THC interact with multiple targets, including serotonin 5-HT1A receptors (Campos et al., 2012; Raymundi et al., 2020a; O'Sullivan and Kendall et al., 2010). Chronic treatment with THC 1.0 mg/kg in adolescent rats reduced the dorsal raphe nucleus's neural activity, and knockout mice for the 5-HT2A receptor were insensitive to the anxiolytic effects of a low THC dose (De Gregorio et al., 2020; Viñals et al., 2015). Regarding the dopaminergic neurotransmission, enhancing endocannabinoid transmission in the ventral tegmental area (VTA) abolished the avoidance behavior induced by reducing dopamine release. Systemic administration of the CB1/CB2 agonist WINN55,212–2 increased neuronal activity in the VTA and dopamine efflux in the NAc (Cheer et al., 2004; Wenzel and cheer, 2018).
Considering the increasing use of medicinal cannabis preparations containing THC alone or THC and CBD combined in different proportions, and the lack of studies investigating the effects of THC and THC plus CBD in female rats, this study aimed to investigate the effects of low doses of THC alone and combined with CBD on anxiety-like behavior in male and female rats exposed to the elevated plus-maze (EPM). In part of these animals, we also analyzed the serotoninergic and dopaminergic contents in the mPFC and NAc. The results showed that females are more sensitive to the effects of low THC doses on anxiety than males. CBD potentiated the anxiolytic and abolished the anxiogenic effect of THC. The anxiogenic effect of THC was associated with increased mPFC and NAc dopamine levels, whereas increased mPFC serotonin accompanied the THC anxiolytic effect.
Section snippets
Animals
Three months-old female and male Wistar rats weighing 230–300 g were used in the present work. The animals were obtained from the local vivarium and housed in groups of 4–5 per cage (50 × 30 × 15 cm), under light: dark cycle of 12 h (beginning of the light phase at 7:00 h), in a temperature-controlled room (22 ± 1 °C), with access to food and water ad libitum for at least seven days before testing. The experimental protocols were approved by the Ethics Committee for the use of animals in the
Experiment 1: effects of THC in female rats tested in the EPM
Fifty animals were randomly allocated to four groups (n = 12–13/group) based on the treatment (vehicle, 0.075, 0.1, or 1.0 mg/kg of THC) administered 20 min before the EPM testing.
One-way ANOVA showed significant treatment effects for %OAT [F(3,46) = 23.4; p = 0.000001; η2 = 0.60] and %OAE [F(3,46) = 13.0; p = 0.000001; η2 = 0.46], but not EAE [F(3,46) = 0.80; p = 0.50; η2 = 0.05]. Newman-Keuls post-hoc tests showed that animals treated with THC 1.0 mg/kg presented lower %OAT (p = 0.0001; Fig. 2
Discussion
Female rats systemically treated with THC 0.075 or 0.1 mg/kg showed enhanced open arms exploration compared to controls, suggesting an anxiolytic-like effect. This result agrees with that reported in male rats and mice tested in the EPM (Onaivi et al., 1990; Braida et al., 2007; Rubino et al., 2007), and other anxiety tests (e.g., Berrendero and Maldonado, 2002), although the most effective dose was usually higher in those studies. In contrast, a higher dose of THC (1.0 mg/kg) decreased open
Conclusions
The present work reports differences in the sensitivity to THC effects on anxiety between male and female rats, suggesting that females would benefit from lower doses of THC. CBD potentiated the anxiolytic-like effect of a low THC dose, suggesting an additive/synergic action. It also prevented the anxiogenic effects of a higher THC dose, supporting the proposal that it could counteract the potential unwanted effects of THC. THC and CBD effects could be associated with changes in dopamine and
Author contribution
CAJS: Conceptualization, designed and performed the experiments, Formal analysis and Data curation, Project administration and Resources, Writing – review & editing, Writing – original draft and final revision and editing of the manuscript. AMR: Conceptualization, designed and performed the experiments, Formal analysis and Data curation, Writing – review & editing, Writing – original draft and final revision and editing of the manuscript. BZS: Conceptualization, designed and performed the
Role of funding source
Our study was supported by Brazilian grants from Fundação Araucária (convênio 043/2020), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2017/24,304–0), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; 409,255/2018–1) and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. Fundação Araucária, CNPq and CAPES had no further role in study design; in the collection, analysis and interpretation of data; in the
Declaration of competing interest
FSG is a co-inventor (Mechoulam R, JC, Guimaraes FS, AZ, JH, Breuer A) of the patent “Fluorinated CBD compounds, compositions and uses thereof. Pub. No.: WO/2014/108,899. International Application No.: PCT/IL 2014/050,023″ Def. US no. Reg. 62193296; July 29, 2015; INPI on August 19, 2015 (BR1120150164927). The University of São Paulo has licensed the patent to Phytecs Pharm (USP Resolution No. 15.1.130002.1.1). The University of São Paulo has an agreement with Prati-Donaduzzi (Toledo, Brazil)
Acknowledgements
We thank Gisele Oliveira Guaita, from the Department of Pharmacology, UFPR, for her technical support on the neurochemical analysis. We thank HempMeds® Brazil for kindly donating the CBD and Aura Pharma, Uruguay, for kindly donating the THC.
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