The endocannabinoid (eCB) system, named from the plant Cannabis sativa, comprises cannabinoid receptors (CB), endogenous lipid ligands such as 2-arachidonoyl glycerol (2-AG) and n-arachidonoyl ethanolamine (AEA or anandamide), and enzymes involved in synthesis and degradation of these ligands. Whereas CB receptors are unique to Chordates, enzymes for eCB synthesis and degradation occur throughout the animal kingdom and several eCB-like lipids have been identified in plants. CB1 receptors are the most abundant eCB receptors in Vertebrates and are widely expressed in the brain. CB1-mediated signaling regulates various aspects of adult plasticity and brain development, including wiring of neuronal connections. In the zebrafish embryo, previous data showed that CB1 knockdown causes defects in anterior commissure formation. Since this axonal system contains Gonadotropin Releasing Hormone (GnRH) fibers, we investigated whether pharmacologic modulation of the CB1 receptor could modify GnRH axonal pathfinding and fasciculation in zebrafish embryos. We treated transgenic GnRH3::GFP zebrafish embryos with various CB1 antagonists from 0 to 72 hours post-fertilization (hpf) and analyzed several parameters such as survival, hatching time and morphology. We also performed morpholino-mediated CB1 knockdown and monitored the expression levels of key genes potentially involved in CB1-mediated effects by Real-Time RT-PCR. CB1 antagonist treatment produced 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 placode 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. Taken together these results indicate that during early zebrafish development, CB1 acts as a regulator of axonal pathfinding of GnRH neurons. Future experiments will elucidate if the CB1 miss-regulation also affects GnRH neuron migration from the olfactory placode to the hypothalamus and whether other neuronal systems are affected.

INTERFERENCE WITH THE CANNABINOID RECEPTOR CB1 INDUCES MISWIRING OF GNRH AXONS IN ZEBRAFISH (DANIO RERIO) EMBRYOS

COTTONE, Erika;D'ATRI, ILARIA;POMATTO, VALENTINA;MERLO, Giorgio Roberto;BOVOLIN, Patrizia
2017-01-01

Abstract

The endocannabinoid (eCB) system, named from the plant Cannabis sativa, comprises cannabinoid receptors (CB), endogenous lipid ligands such as 2-arachidonoyl glycerol (2-AG) and n-arachidonoyl ethanolamine (AEA or anandamide), and enzymes involved in synthesis and degradation of these ligands. Whereas CB receptors are unique to Chordates, enzymes for eCB synthesis and degradation occur throughout the animal kingdom and several eCB-like lipids have been identified in plants. CB1 receptors are the most abundant eCB receptors in Vertebrates and are widely expressed in the brain. CB1-mediated signaling regulates various aspects of adult plasticity and brain development, including wiring of neuronal connections. In the zebrafish embryo, previous data showed that CB1 knockdown causes defects in anterior commissure formation. Since this axonal system contains Gonadotropin Releasing Hormone (GnRH) fibers, we investigated whether pharmacologic modulation of the CB1 receptor could modify GnRH axonal pathfinding and fasciculation in zebrafish embryos. We treated transgenic GnRH3::GFP zebrafish embryos with various CB1 antagonists from 0 to 72 hours post-fertilization (hpf) and analyzed several parameters such as survival, hatching time and morphology. We also performed morpholino-mediated CB1 knockdown and monitored the expression levels of key genes potentially involved in CB1-mediated effects by Real-Time RT-PCR. CB1 antagonist treatment produced 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 placode 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. Taken together these results indicate that during early zebrafish development, CB1 acts as a regulator of axonal pathfinding of GnRH neurons. Future experiments will elucidate if the CB1 miss-regulation also affects GnRH neuron migration from the olfactory placode to the hypothalamus and whether other neuronal systems are affected.
2017
Second Joint Meeting of Société Zoologique de France and Unione Zoologica Italiana
Torino
18-23 settembre 2017
The evolution of animal diversity: a comparative approach
Donna D., Moietta M., Palestrini C., Peretto P., Roggero A.
46
46
978-88-905691-7-3
Cottone, Erika; D’Atri, Ilaria; Zuccarini, Giulia; Pomatto, Valentina; Gothilf, Yoav; Merlo, Giorgio; Bovolin, Patrizia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1648111
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