Modulating Swimming Behaviors in Wildtype and Cannabinoid Receptors (CB1 & CB2) Mutant Zebrafish Larvae
Authors: Colon‐Cruz Luis , Varshney Gaurav , Acevedo Agnes , Bonano Keisy , Burgess Shawn , Yudowski Guillermo , Behra Martine 
Published: June 2021

Discovery of new psychiatric drugs has been painfully slow, while diagnosed psychiatric disorders have risen, creating an urgent need for novel approaches bolstering this domain. A major drawback is that promising drugs, found by classic approaches like in vitro/in silico systems or neuronal cells, will have to be tested for safety and efficacy in a complex organism. Rodents are usually the preferred model because of an advanced understanding of the central nervous system (CNS) function and the availability of well‐accepted behavioral tests. However, ethical considerations and prohibitive costs do not allow rapid multiple dosages testing of a high number of animals to account for the inherent variability of candidate drugs that get overseen. We propose to use zebrafish larvae as an intermediate model. This lower vertebrate offers the possibility to perform up‐scalable behavioral screens in a genetic tractable model and has been successfully used for drug safety and discovery. It is particularly suited to further assess the genetic contribution to a pharmacological response which can give insight into the drug's mode of action, but also assert its safety in genetics variants. As prove of principle, we focused on a well‐known neuronal signaling pathway: the endocannabinoid system (eCBs), which is mediated via two cannabinoid receptors 1 and 2 (CB1 and CB2, encoded by cnr1 and cnr2 genes respectively). The classical view of CB1 acting in the CNS and CB2 in the peripheral immune system has been recently challenged by mounting evidence revealing that CB2 is also expressed in the CNS and some CB2‐dependent neuromodulation. To investigate further their role as neuromodulators and how the receptors work alone or in combination, we generated CB1 and CB2 loss‐of‐function mutations using CRISPR/Cas9. Wild type and mutant larvae, lacking one or both CBs, were recorded in a photo‐dependent swimming response assay (PDR). We focused on well‐accepted anxiety‐like parameters: hypo/hyper‐activity, center avoidance, and photo‐stimulated startle response. Next, we evaluated larvae’ PDR performance in presence of different neurotropic compounds. We chose a well‐described anxiogenic drug Pentylenetetrazole, Diphenhydramine (main component of Benadryl®) which has a narcotic effect, and copper, a commonly found water contaminant known to induce anxiety‐like behaviors. We found that PDR alterations were clearly genotyped and compound specific, therefore demonstrating the eCBs signaling role in those anxiety‐like behaviors. Furthermore, it is also proving that our composite approach is a quantifiable read‐out for drug efficacy. This work lays the foundations for biomedical platforms to screen neurotropic molecules in variable genetic context to reinforce drug discovery. In addition, it can be extended to dissect neuronal signaling pathway(s) by systematic use of loss‐of‐function mutations in genes relevant for understanding altered‐behaviors lined to neuropsychiatric disorders.Support or Funding InformationR25 GM061838, MBRS‐Research Initiative for Scientific Enhancement (RISE) Program/NSF‐CREST (#HRD‐1137725)‐Puerto Rico Center of Environmental Neuroscience (PRCEN)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.