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Cannabis Cannabinoid Res. 2019 Jun 14;4(2):88-101. doi: 10.1089/can.2018.0025. eCollection 2019.

Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2.

Author information

1
Department of Anatomy and Neurobiology, Medical Sciences Campus, University of Puerto Rico (MSC-UPR), San Juan, Puerto Rico.
2
Department of Anatomy and Neurobiology, Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico.
3
Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma.
4
National Human Genome Research Institute, NHGRI/NIH, Bethesda, Maryland.
5
Puerto Rico Clinical and Translational Research Consortium, Medical Sciences Campus, University of Puerto Rico (MSC-UPR), San Juan, Puerto Rico.

Abstract

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.

KEYWORDS:

AM-360; JWH-133; PTZ; VPA; cannabinoid receptor 2 knockout; zebrafish larva

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