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.
Highlights
The endocannabinoid system is a key modulator of excitatory and inhibitory neuronal activity[1] and its dysregulation has been linked to several psychiatric disorders.[2,3,4,5] The two cannabinoid receptors (CB1 and cannabinoid receptor 2 (CB2)) belong to the G protein-coupled receptor family.They are both activated by endogenous ligands[6,7] and exogenous compounds such as D9-THC, the main psychoactive component in cannabis.[8]
When adding a classical anxiogenic drug, pentylenetetrazol (PTZ), a well-characterized GABAA inhibitor in many animal models,[32,33,34,35,36] we found that larvae lacking CB2 presented an increase in swimming activity and a decrease in center occupancy (CO) when compared with wild type
We identified a two-nucleotide deletion (D2: CT) just 3 base pair upstream of the protospacer adjacent motif site introducing a translation frameshift, which would predictably create an early stop codon in aa position 159
Summary
The endocannabinoid (eCB) system is a key modulator of excitatory and inhibitory neuronal activity[1] and its dysregulation has been linked to several psychiatric disorders.[2,3,4,5] The two cannabinoid receptors (CB1 and CB2) belong to the G protein-coupled receptor family They are both activated by endogenous ligands (eCBs)[6,7] and exogenous compounds such as D9-THC, the main psychoactive component in cannabis.[8] CB1 is highly expressed in the CNS and implicated in numerous neurological diseases (for review, see Marco et al.,[3] Kendall and Yudowski,[5] Bilkei-Gorzo,[9] Di Marzo et al.,[10] and Pavlopoulos et al.[11]). Mutant larvae responded differently to VPA and PTZ treatments, providing in vivo evidence of CB2 modulating complex behaviors. We established an upscalable combined genetic/behavioral approach in a whole organism that could be further developed for high-throughput drug discovery platforms
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