STONED
Studies To Observe Novel in vivo Endocannabinoid-related Drugs (STONED) is a research project within SWIRL which is investigating the effects of the human endocannabinoids, 2-arachidonylglycerol and anandamide, and phytocannabinoids such as cannabidiol on Lumbriculus variegatus. While conventional invertebrate models, such as C. elegans and Drosophila, lack an endocannabinoid system the annelid worms are known to express cannabinoid receptor homologs (Salzet & Stefano, 2002), and so L. variegatus is a valuable tool in unravelling the underlying physiology and pharmacology of the endocannabinoid system.
STONED aims to examine the behavioural effects of endo- and phytocannabinoids in L. variegatus and investigate the presence or absence of cannabinoid receptor homologs in this species.
Concentration-dependent response of the novel in vivo research animal Lumbriculus variegatus to cannabidiol
Julanta Carriere, Claire Price, Lisa J Wallace, Aidan Seeley
Swansea Worm Integrative Research Laboratory
Swansea University Medical School
Swansea University
Introduction
Cannabidiol (CBD) is an investigative non-psychoactive cannabinoid from Cannabis sativa, and the endocannabinoid system is highly conserved in animals and signalling can be excitatory, inhibitory, or modulatory [1]. Lumbriculus variegatus is a species of aquatic worm which has been shown as a novel in vivo pharmacology research animal [2]. Here we demonstrate the lethal dose of 50% of the population (LD50) and the behavioural effects of CBD on L. variegatus stereotypical movements and free, unstimulated, locomotor activity.
Methods
The LD50 was determined following 24-hour exposure to CBD (0-250μM) and measuring the numbers of worms which survived post-exposure. Stereotypical movements were recorded following tactile stimulation of anterior and posterior regions before CBD exposure, after 10-minute exposure to CBD (0-20μM), and 10 minutes and 24 hours after removal of drug compounds and subsequent incubation in pondwater using methods previously described [2]. Unstimulated free locomotion was measured by rapid image collection of L. variegatus under the same conditions. Statistical significance was determined by paired t-tests or a two-way ANOVA.
Results
Concentrations ≥30µM resulting in a 100% fatality rate with an LD50 of 23.00µM (21.74 - 24.26µM 95% CI). Exposure to ≥5μM CBD significantly inhibited body reversal (p<.05, n=8) and helical swimming (p<.05, n=8). Free, unstimulated movement increased by 26.6±8.68% (p=.0212, n=8) 10 minutes after removal of 5µM. Long term effects were observed 24 hours post-exposure to 10µM and 20µM with movement decreased by 29.58±11.64% (p=.0188, n=8) and 39.8±13.52% (p=.0120, n=8), respectively.
Conclusion
We demonstrate CBD has toxicity and concentration-dependent effects in the micromolar range, in contrast to C. elegans which lack toxicity even in the millimolar range [3] . This demonstrates L. variegatus have a unique physiology compared to conventional invertebrates such as C. elegans and Drosophila, which proposed to lack a full endocannabinoid system [4]. Further study of cannabinoids in L. variegatus may provide meaningful and impactful research within the field, offering a wealth of research opportunities.
1. Silver RJ. The Endocannabinoid System of Animals. Animals. 2019;9(9):686. doi:10.3390/ani9090686
2. Seeley A, Bellamy C, Davies NA, Wallace MJ. Lumbriculus variegatus: A novel organism for in vivo pharmacology education. Pharmacol Res Perspect. 2021;9(5):e00853. doi:10.1002/prp2.853
3. Land MH, Toth ML, MacNair L, et al. Effect of Cannabidiol on the Long-Term Toxicity and Lifespan in the Preclinical Model Caenorhabditis elegans. Cannabis Cannabinoid Res. 2021;6(6):522-527. doi:10.1089/can.2020.0103
4. Salzet M, Stefano GB. The endocannabinoid system in invertebrates. Prostaglandins Leukot Essent Fatty Acids. 2002;66(2):353-361. doi:10.1054/plef.2001.0347
