Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2

Abstract

<b>Background and Objectives:</b> 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 <i>in vivo</i> upscalable whole-organism approach for CB2 ligand screening. <b>Experimental Approach:</b> Using CRISPR-Cas9 technology, we generated a novel null allele (<i>cnr2^upr1</i> ) and a stable homozygote-viable loss-of-function (CB2-KO) line. We measured in untreated wild-type and <i>cnr2^upr1/upr1</i> 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). <b>Results:</b> 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. <b>Conclusions:</b> 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 <i>in vivo</i> 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.