Exposure to cannabinoid receptor 1 ligands induces miswiring of GnRH3 and AgRP1 axons in zebrafish embryos

Cannabinoid receptors 1 (CB1) are G-protein coupled receptors, they are widely expressed in the central nervous system, and are part of an important neuro-modulatory system: the endocannabinoid system. This system is composed of neuro-modulatory lipids, the endocannabinoids, of which anandamide (AEA) and 2-arachydonoylglycerol (2-AG) are the best characterized, their synthetic and degradative enzymes and their receptors. Among the latter, CB1 receptors have been recognized as regulators of brain development, including wiring of neuronal connections: the endocannabinoids have been proposed to activate intracellular pathways that involve the control of neuronal actomyosin cytoskeleton, participating in the regulation of axonal guidance and pathfinding. CB1 receptors are also targets of Phyto-cannabinoids (e.g. Cannabis-derived 9-THC), and it has been reported that children exposed in utero to cannabis present neurobehavioral and cognitive impairments, suggesting that the exposure to exogenous ligands during embryonic development could affect the endocannabinoid system and the cellular mechanisms regulated by it. In the zebrafish embryo, previous data showed that CB1 receptor knockdown causes abnormal axonal growth in forebrain areas, characterized by failure in fasciculation of axonal tracts, in particular at the level of the anterior commissure. Since this area is rich in Gonadotropin Releasing Hormone (GnRH) and Agouti-related peptide (AgRP) expressing fibers, the aim of this study was to assess whether pharmacological modulation of CB1 could modify GnRH and AgRP axonal pathfinding during zebrafish early neurodevelopment. Fluorescence immunocytochemical analysis suggested that, during early developmental stages, CB1 is expressed in forebrain areas where both GnRH3 and AgRP1 fibers are present and co-localize with GnRH3-expressing axons. Transgenic GnRH3:EGFP and agrp1:mCherry embryos treated with increasing concentrations of different CB1 ligands (agonist, antagonist and reverse agonist) from 0 to 72-96hpf, showed axon misrouting and abnormal pathfinding of both GnRH3 and AgRP1 fibers in the anterior commissure. These data were confirmed by morpholino-mediated CB1 knockdown, providing very similar phenotypes. Following exposure with either CB1 agonist, antagonist or reverse agonist, CB1 gene expression was significantly up-regulated, as well as GnRH3 and AgRP1 expression levels. Moreover, CB1 pharmacological modulation influenced the expression of some genes involved in axonal growth and cell migration, such as Stmn2a/b, Negr1 and Sez6a/b, highlighting the possible involvement of novel molecular mechanisms. These results indicate for the first time that, during development, CB1 acts as regulator of axonal pathfinding on GnRH and AgRP neurons. Future experiments will elucidate whether the CB1 miss-regulation in these very early stages will have effects on sexual maturation and reproduction, as well as feeding behavior. Moving from the animal model to humans, the implications of these results are important in the contest of pre-natal exposure to cannabis.