Role of endocannabinoid signaling in a preference/aversion circuitry

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PROJECT SUMMARY The ability to seek reward and avoid potential threats is fundamental to the fitness and survival of all animals from early life stages. Our research aims to address circuit-wide mechanisms with cellular and molecular clarity employing larval zebrafish. As a vertebrate genetic model organism, zebrafish shares considerable similarity with mammals. In both mammals and larval zebrafish (See Preliminary data section), the lipid neurotransmitters endocannabinoids (eCB) and the neuropeptide hypothalamic corticotropin releasing factor (Hy CRF) are known to regulate motivated behaviors. However, an understanding of their roles circuit-wide at cellular resolution is currently lacking. Larval zebrafish with a relatively simple and transparent brain of ~100K neurons (compared to ~75 million in the mouse, and ~100 billion in the human brain) is well suited to address this question. New regulatory principles uncovered in simpler systems will lay foundation for studying more complex systems. Free-living with the need to hunt for food and avoid predators, larval zebrafish display readily observable approach and avoidance behaviors in response to environmental stimuli, drugs, or social cues. Here I propose to elucidate the role of eCB and Hy CRF, brain-wide at cellular resolution, in the context of light/dark preference, a fundamental motivated behavior conserved across species. Larval zebrafish avoid dark, which can be enhanced by stressors and alleviated by anxiolytics. Our preliminary data show that ablation of Hy CRF neurons ameliorates, whereas inhibition of the cannabinoid receptor CB1 enhances, dark avoidance. We have genetically disrupted major genes in the eCB signaling pathway, including CB1 (primarily neural) and CB2 (primarily immune) receptors, receptor-interacting proteins (CNRIP1a and CNRIP1b), eCB synthesis enzymes (e.g. DAGLa, DAGLb, ABHD4), and eCB degradation enzymes (MGLL, FAAH). These knockout animals are valuable resources for understanding signaling specificity by uncovering which receptors and ligands and associated regulatory proteins are involved in specific behavioral regulation. Furthermore, we have established brain-wide calcium imaging and computational platforms for examining the activity and plasticity of distributed neural circuits at cellular resolution. In this application, built on these preliminary data and our expertise in studying brain development and function employing zebrafish, we will test the hypothesis that eCB signaling regulates dark avoidance circuitry that involves Hy CRF neurons. We will gain circuit-wide understanding and uncover new cell types/molecules for future studies of circuit assembly and plasticity under stress or drug treatment in a highly accessible brain. Impact and Outcomes: If successful, this project will achieve, for the first time to our knowledge, a cellular resolution circuit-wide understanding of eCB signaling in relation to Hy CRF in a fundamental motivated behavior. Such improved understanding at the whole circuitry level shall lay foundation for informing future marijuana policy and for tackling disease states associated with perturbed CRF or eCB signaling.