Altered Swimming Behaviors in Zebrafish Larvae Lacking Cannabinoid Receptor 2

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 ( 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.

Cnr2 is important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors

“…To dissociate auditory from visual responses, animals were either first exposed to 90-minute of constant light followed by 90-minute of constant darkness (Fig.8A to E), or vice versa (Fig.8F to J). For animals first exposed to light, as we had previously demonstrated the baseline swimming activity (SA) for mutant animals was lower than for wild type (Fig.8A, yellow box) [8]. In response to sound stimulation (green dashed lines in 8A yellow box, B and D) the SA was only slightly increased in wild type by ~14% (black, SAwt-1 = 6.80 vs. SAwt-0 = 7.65 cm/min, p < 0.0001), but by ~63% in mutant larvae (magenta in D, SAcnr2-1 = 2.87 vs. SAcnr2-0 = 4.67cm/min, p < 0.0001).…”

“…To elucidate the putative role of cnr2 in those sensory epithelia, we used the stable lossof-function mutant line (cnr2 upr1 ) that we had previously generated [8], to monitor HC development and regeneration in the LL in the absence of cnr2 (Figure1-supplement 1). We visualized functional HCs using FM 1-43 [48].…”

“…Based on the perturbation that we observed in vesicle distribution at the ribbons in mutant retina, we hypothesized that mutant animals challenged by even slight variations in light intensity would not be able to fully readjust during gradual changes. To test this, we first reproduced our previously published experimental setup in which we recorded wild type and mutant larvae SA after an initial 30min adaptation to dark, followed by 4 successive 10-minute light/10minute dark periods (Figure 8-supplement 1A [8]). As expected, in mutant larvae (magenta squares) the SA was significantly reduced in light and increased in dark periods.…”

“…By contrast, expression, and function of the second well characterized cannabinoid receptor (CNR2) remained more elusive until recently, in part due to interspecies differences at the genomic and tissue/organ expression levels [5]. The previously accepted notion that CNR2 was mostly restricted to the immune system has been repeatedly challenged [6], and expression in the mammalian [7] and fish brain [8] as well as associated behavioral changes were demonstrated. In sensory organs, Cnr2 expression was described in several cell types like photoreceptors of the adult retina in various mammals [9] as well as in fish [10].…”

“…We found that the cnr2 gene was expressed in all HCs of the inner ear and the LL, starting at 3day post-fertilization (dpf) onward. In cnr2 loss of function animals (cnr2 upr1 ) that we had previously generated [8], notably HCs in NMs of the LL were not strongly affected in their development, regeneration, or innervation. However, we found profound perturbation in the size and distribution of synaptic ribbons which was more evident in mature HCs.…”