The effects of repeated anandamide treatment on orexin, weight loss, and physical activity in zebrafish (Danio rerio) during caloric restriction

Cannabis is widely used as a therapeutic to increase appetite in chemotherapy patients and to reduce anxiety in patients with stress disorders. While Cannabis use increases appetite and reduces anxiety, chronic use is paradoxically linked to lower rates of obesity. The effects of acute exposure to endocannabinoids have been studied, but the effects of repeated exposure has not. As endocannabinoids play a pivotal role in food consumption, overstimulation of the endocannabinoid system over time may cause a tolerance to cannabinoids, leading to lower basal endocannabinoid signaling and food consumption. Endocannabinoids regulate many neuronal pathways including reward, sleep, and hunger. The endocannabinoid 2-arachidonoylethanolamine (Anandamide or AEA) modulates physical activity during periods of moderate-intensity exercise and is implicated in the experience of a ‘runners high.’  Understanding how this system responds to repeated stimulation could provide insights into the link between endocannabinoids, weight loss, and physical activity. This study investigated how the repeated treatment with anandamide impacts physical activity and weight loss in zebrafish (Danio rerio) during a 21-day caloric restriction. Anandamide treatment significantly increased physical activity, but this increase gradually reduced over the course of the experiment. Caloric restriction alone showed reduced physical activity, as measured by the average velocity over a 10-minute observation period (0.15 cm/sec) compared to the negative control group (0.173 cm/sec), however the group treated with both AEA and caloric restriction had the highest average movement (0.200 cm/sec). Caloric restriction was verified via qPCR for orexin, a neuropeptide involved in regulating hunger. Groups subjected to caloric restriction had a greater expression of orexin RNA compared to the non-calorically restricted groups. It was hypothesized that anandamide treatment would increase orexin signaling when food is limited, as an increase in physical activity would cause an increase in hunger. Interestingly, there were no differences in orexin expression due to anandamide treatment, and no change in cannabinoid receptor (CB1) expression was found. The results of this study indicate that anandamide treatment stimulates physical activity during periods of caloric restriction, however it does not stimulate orexin signaling. The groups treated with anandamide both showed weight loss similar to caloric restriction alone, indicating that repeated treatment of anandamide increases weight loss. These reductions in weight were likely independent of stress, as there were no differences in the rates of thigmotaxis, a common marker of stress in zebrafish, between the treatments during the course of the experiment.