Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2.

Background and Objectives: The cannabinoid receptor 2 (CB2) was previously implicated in brain functions, including complex behaviors. Here, we assessed the role of CB2 in selected swimming behaviors in zebrafish larvae and developed an in vivo upscalable whole-organism approach for CB2 ligand screening. Experimental Approach: Using CRISPR-Cas9 technology, we generated a novel null allele (cnr2upr1 ) and a stable homozygote-viable loss-of-function (CB2-KO) line. We measured in untreated wild-type and cnr2upr1/upr1 larvae, photo-dependent (swimming) responses (PDR) and center occupancy (CO) to establish quantifiable anxiety-like parameters. Next, we measured PDR alteration and CO variation while exposing wild-type and mutant animals to an anxiolytic drug (valproic acid [VPA]) or to an anxiogenic drug (pentylenetetrazol [PTZ]). Finally, we treated wild-type and mutant larvae with two CB2-specific agonists (JWH-133 and HU-308) and two CB2-specific antagonists, inverse agonists (AM-630 and SR-144528). Results: Untreated CB2-KO showed a different PDR than wild-type larvae as well as a decreased CO. VPA treatments diminished swimming activity in all animals but to a lesser extend in mutants. CO was strongly diminished and even more in mutants. PTZ-induced inverted PDR was significantly stronger in light and weaker in dark periods and the CO lower in PTZ-treated mutants. Finally, two of four tested CB2 ligands had a detectable activity in the assay. Conclusions: We showed that larvae lacking CB2 behave differently in complex behaviors that can be assimilated to anxiety-like behaviors. Mutant larvae responded differently to VPA and PTZ treatments, providing in vivo evidence of CB2 modulating complex behaviors. We also established an upscalable combined genetic/behavioral approach in a whole organism that could be further developed for high-throughput drug discovery platforms.