The mu opioid receptor (MOR) is a diversely regulated target for the alleviation of pain in the clinical setting. However, untoward side effects such as tolerance, dependence, respiratory suppression, constipation, and abuse liability detract from the general activation of these receptors. Studies in genetically modified rodent models suggest that activating G protein signaling pathways while avoiding phosphorylation of the receptor or recruitment of b-arrestin scaffolding proteins could preserve the analgesic properties of MOR agonists while avoiding certain side effects. With the development of novel MOR "biased" agonists, which lead to preferential activation of G protein pathways over receptor phosphorylation, internalization, or interaction with other effectors, this hypothesis can be tested in a native, physiological setting. Overall, it is clear that the MOR is not a simple on-off switch and that the diverse means by which the receptor can be regulated may present an opportunity to refine therapeutics for the treatment of pain.
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 (
cnr2
upr1
) and a stable homozygote-viable loss-of-function (CB2-KO) line. We measured in untreated wild-type and
cnr2
upr1/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.
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