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.

Detection and toxicity assessment of waterborne contaminants are crucial for protecting human health and the environment. Development of easy-to-implement, rapid and cost-effective tools to measure anthropogenic effects on watersheds are critical for responsible management, particularly in times of increasing development and urbanization. Traditionally, environmental toxicology has focused on limited endpoints, such as lethality and fertility, which are directly affecting population levels. However, more sensitive readings are needed to assess sub-lethal effects. Monitoring of contaminant-induced behavior alterations was proposed before, but is difficult to implement in the wild and performing it in aquatic laboratory models seem more suited. For this purpose, we adapted a photo-dependent swimming response (PDR) that was previously described in zebrafish larva. We first asked if PDR was present in other aquatic animals. We measured PDR in larvae from two freshwater prawn species (Macrobrachium rosenbergii, MR, and Macrobrachium carcinus, MC) and from another fish the fathead minnow (FHM, Pimephales promelas). In all, we found a strong and reproducible species-specific PDR, which is arguing that this behavior is important, therefore an environmental relevant endpoint. Next, we measured PDR in fish larvae after acute exposure to copper, a common waterborne contaminant. FHM larvae were hyperactive at all tested concentrations in contrast to ZF larvae, which exhibited a concentration-dependent hyperactivity. In addition to this well-accepted anxiety-like behavior, we examined two more: photo-stimulated startle response (PSSR) and center avoidance (CA). Both were significantly increased. Therefore, PDR measures after acute exposure to this waterborne contaminant provided as sensitive readout for its detection and toxicity assessment. This approach represents an opportunity to diagnostically examine any substance, even when present in complex mixtures like ambient surface waters. Mechanistic studies of toxicity using the extensive molecular tool kit of ZF could be a direct extension of such approaches.

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

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