Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Master of Science




Laviolette, Steven


Emerging evidence has elucidated the anxiolytic properties of the cannabis-derived phytochemicals cannabidiol (CBD) and d-limonene and the ‘Entourage Effect’ wherein, multiple components of cannabis synergize to produce stronger effects than their pure counterparts. However, no studies have yet explored this effect in combinations of CBD and limonene. Thus, the present thesis investigated the anxiolytic and synergistic potential of concurrently administered intra-nucleus accumbens shell CBD (1 ng/0.5 μl or 5 ng/0.5 μl) and inhaled limonene (200 μl or 2000 μl). Additionally, the role of the 5-HT1A receptor in mediating these effects was examined by co-application of the antagonist NAD299 and by the assessment of downstream molecular biomarkers. Findings from this study demonstrated for the first time that relative to their isolated counterparts, combinations of limonene and CBD more effectively reduces symptoms of anxiety, with the observed reversal of these effects with NAD299 elucidating a role at the 5-HT1A receptor.

Summary for Lay Audience

Current medications for anxiety disorders are fraught with adverse side-effects such as ongoing drug dependence, withdrawal, and memory loss. Thus, there is a critical need for the development of novel pharmacotherapies with safer and more tolerable profiles. While cannabis use has been associated with an increased risk of psychosis, these effects are associated with the cannabinoid tetrahydrocannabinol (THC). In contrast, extensive evidence has demonstrated that the cannabis-derived phytochemicals, cannabidiol (CBD) and d-limonene, possess anxiolytic properties, with several studies associating the anti-anxiety effects of CBD with specific brain regions associated with reward and mood dysfunction, namely the nucleus accumbens shell (NASh).

The citrus-scented monoterpene, d-limonene, has been shown to reduce anxiety in pre-clinical and clinical assays in a similar biochemical manner to CBD. In addition to their analogous properties, emerging evidence has alluded to a phenomenon known as the ‘Entourage Effect’ (EE). The Entourage Effect posits that multiple components in the cannabis plant interact to produce a stronger influence than each component in isolation, i.e., a synergistic effect. This effect is documented mainly for THC and other cannabis-derived phytochemicals and there is currently no knowledge of how combinations of CBD and limonene may produce clinically synergistic effects. Thus, this research project examined dose combinations of co-applied intra-NASh CBD and inhaled d-limonene that may provide the greatest anxiolytic efficacy, as well as assess associated changes in protein expression in the brain by pre-clinical modeling of anxiety-related behavioural and molecular assays in rodents.

The results of this study demonstrate for the first time the EE-potentiated anxiolytic effects of concurrently administered CBD and d-limonene. Additionally, by utilizing a specific molecular antagonist (NAD299) and by assessing changes in anxiety-related biomarkers, this thesis elucidated the potential 5-HT1A receptor-mediated signalling brain pathways targeted by these formulations. Ultimately, these findings combined with the evidence that CBD and limonene exhibit remarkably safe pharmacological profiles, highlight the therapeutic potential of cannabis-derived compounds and their prospective use as a natural alternative or adjunct to current anti-anxiety medications.