Exposure to Cannabinoid Receptor 1 ligands induces miswiring of GnRH axons in the brain of zebrafish embryos

INTRODUCTION: CB1 cannabinoid receptors (CB1) are the targets of marijuana (Cannabis spp)-derived phytocannabinoids (e.g. Δ9-THC) and endocannabinoids (AEA and 2-AG). Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments [1]. CB1 cannabinoid receptors are widely expressed in the brain and they have been recently recognized as regulators of brain development, including wiring of neuronal connections. In the zebrafish (zf) embryo, previous data showed that CB1 knockdown causes defects in axonal fasciculation in the anterior commissure [2]. Since this area is particularly rich in Gonadotropin Releasing Hormone (GnRH) fibers, we assessed whether pharmacologic modulation of the CB1 receptor could modify GnRH axonal pathfinding and fasciculation in zebrafish embryos. METHODS: We treated transgenic GnRH3::GFP zf embryos with increasing concentrations of various CB1 antagonists from 0 to 72 hpf. We also performed morpholino-mediated CB1 knockdown and analyzed several parameters such as survival, hatching time and morphology. Expression levels of key genes potentially involved in CB1-mediated effects were monitored by Real-Time RT-PCR. RESULTS: Following CB1 antagonist treatment, we found a reduction in GnRH neuropil extension and axon misrouting in the anterior commissure. Morpholino-mediated downregulation of CB1 expression reduced the number of GnRH3::GFP positive cells in the olfactory epithelium while not changing their position. Finally, we observed that CB1 knockdown downregulates the expression of two genes involved in axonal growth and cell migration, namely Stmn2a/b and Sez6a/b. CONCLUSIONS: Taken together these results indicate that during early zebrafish development, CB1 acts as a regulator of axonal pathfinding on GnRH cells. Future experiments will elucidate if the CB1 miss-regulation also affects GnRH neuron migration from the olfactory placode to the hypothalamus, with consequent effects on sexual maturation and reproduction.