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The endocannabinoid (eCB) system is a complex network of proteins and ligands primarily found in the central nervous system. Though the eCB system is most notorious for producing psychotropic effects following consumption of the Cannabis sativa plant, it also modulates a wide range of physiological processes including neuronal development, neuroinflammation, anxiety, memory, appetite, lipid homeostasis, pain, and immunity. Harnessing the eCB system has been a successful therapeutic strategy for treating diseases, as in the cases of cannabidiol (CBD) for epilepsy treatment, or Cannabis as an anti-emetic for patients undergoing chemotherapy. Though there have been great strides in our understanding of the eCB system, unanswered questions remain in regards to its potential for additional therapeutic uses, toxicological considerations for Cannabis consumers, and more fundamentally, the mechanistic workings of eCB system signaling in the context of neural circuitry.One approach to gain insights on the eCB system is through utilization of zebrafish (Danio rerio), a powerful model organism used in biological research. Proteins of interest can be easily targeted by both pharmacological agents and genetic alterations within zebrafish, allowing for examination of complex phenotypes resulting from desired perturbations. A diverse panel of zebrafish behavioral assays are available, allowing for examination of memory, addiction, sociability, aggression, and anxiety-like behaviors. Unlike mammalian models, development is relatively fast and breeding produces a large amount of progeny, allowing for quick generation progression and large sample sizes. Also, unlike mammals, development occurs externally and the brain is transparent, allowing for accessible and clear visualization of neurodevelopment, neuronal activity, and fluorescent markers of interest throughout the whole brain. These attributes make zebrafish an excellent model for studying the eCB system in vivo. In this dissertation, we explore the roles of several eCB proteins through utilization of genetic and pharmacological manipulation of the zebrafish model.We start off in Chapter 1 with an in-depth examination of techniques in zebrafish that have paved the way for gaining insights on neurobiological mechanisms. The techniques we cover include forward and reverse genetic screens, and chemical screens, which have shed light on mechanisms of neuronal subtype differentiation and maintenance, habituation, candidate genes implicated in autism spectrum disorders, genes involved in electrical synapse formation, and regulators of sleep/wake states, to name a few examples.Next, in Chapter 2, we introduce the known roles of the eCB system in vertebrates, with an emphasis on the zebrafish model. We describe each component of the eCB system, how each eCB protein and ligand work together to facilitate signaling cascades, and the involvement of the eCB system in addiction, development, anxiety, lipid homeostasis, appetite, immunity, and neuroinflammation. We examine spatial expression of two eCB genes, cb1 and cnrip1a, in developing zebrafish larvae, and quantitative expression of 18 eCB genes across 10 developmental stages and 11 organs in adult fish.In the next two chapters, we use techniques described in Chapter 1 to target eCB genes described in Chapter 2. In Chapter 3, we utilize CRISPR-Cas9 gene editing to produce knockout zebrafish lines of cb1, dagla, daglb, abhd4, mgll, and faah. Following homology analysis, we determined that all 6 of these genes are conserved in zebrafish with amino acid sequence identity ranging from 45-70%. After sequencing each eCB knockout line, we found that all mutant alleles contained a frameshift mutation, suggesting a deleterious effect. We proceeded to phenotype dagla knockout fish and observed in increase in locomotor activity, as well as alterations in mRNA transcript of dagla, gpr55a, and fas. We next examine the effects of pharmacological agents that target CB1 (WIN55212-2, 2-arachadonoylglycerol [2-AG], anandamide [AEA]), MGLL (MJN110, JZL-184) and FAAH (PF-3845) in Chapter 4. Through the light-dark preference assay, we observed changes in dark avoidance (an anxiety-like behavior) and locomotor activity following eCB protein-targeting drug treatment. These genetic and pharmacological studies provide an excellent opportunity for gaining insight on individual gene involvement in behavior and physiological processes.In Chapter 5, we focus on one gene, cb1, and further examine its role in anxiety; in particular, we aim to gain a greater understanding of the neuronal populations that are involved in facilitating eCB-mediated changes in anxiety behavior. Corroborating human and rodent studies, we determine that zebrafish have increased anxiety-like behavior following either pharmacological or genetic inhibition of CB1. By leveraging high resolution in situ techniques, we discovered region-specific colocalization of cb1 mRNA with pallial and hypothalamic glutamatergic and subpallial GABAergic neuronal markers. We produced a transgenic CB1 zebrafish line, allowing access to CB1-expressing cells and visualization of anatomical connectivity throughout the entire brain.In conclusion, this dissertation reveals the behavioral effects of perturbing eCB genes, furthering our understanding of the roles of each eCB protein, and provides insight on the mechanism by which CB1 modulates anxiety-like behavior. From this work, 7 new eCB zebrafish lines were produced, allowing for further studies to uncover mechanistic roles of cb1, dagla, daglb, abhd4, mgll, and faah. Uncovering the roles of each eCB gene on biological processes not only demystifies this broadly expressed and highly conserved pathway, but also paves the way for insight on potential therapeutic uses and toxicological effects of eCB-targeting pharmacological agents.

Obesity and related metabolic disorders have reached epidemic levels with dire public health consequences. Efforts to stem the tide focus on behavioral and pharmacological interventions. Several hypolipidemic pharmaceutical agents target endogenous lipid receptors, including the peroxisomal proliferator activated receptor alpha (PPAR alpha) and cannabinoid receptor 1 (CB1). To further the understanding of these clinically relevant receptors, we elucidated the biochemical basis of PPAR alpha activation by lipoprotein lipolysis products and the metabolic and transcriptional responses to elevated endocannabinoid signaling. PPAR alpha is activated by fatty acids and their derivatives in vitro. While several specific pathways have been implicated in the generation of PPAR alpha ligands, we focused on lipoprotein lipase mediated lipolysis of triglyceride rich lipoproteins. Fatty acids activated PPAR alpha similarly to VLDL lipolytic products. Unbound fatty acid concentration determined the extent of PPAR alpha activation. Lipolysis of VLDL, but not physiological unbound fatty acid concentrations, created the fatty acid uptake necessary to stimulate PPAR alpha. Consistent with a role for vascular lipases in the activation of PPAR alpha, administration of a lipase inhibitor (p-407) prevented PPAR alpha dependent induction of target genes in fasted mice. Apolipoprotein CIII, an endogenous inhibitor of lipoprotein lipase, regulated access to the lipoprotein pool of PPAR alpha ligands. Our results support a role for the local generation of PPAR alpha ligands by lipase activity. The endocannabinoid system regulates diverse physiological functions, including energy balance. While loss of CB1 signaling has more pronounced effects on lipid parameters than expected based on weight loss alone, direct demonstration of endocannabinoid regulation of lipid metabolism is lacking. To test the effects of endogenously produced cannabinoids on lipid metabolism, independent of alterations in food intake, we analyzed tissues from mice treated with IDFP, an organophosphorus inhibitor of endocannabinoid breakdown. IDFP administration inhibited hepatic monoacylglycerol lipase leading to elevated levels of 2-arachidonylglycerol. We found that IDFP administration caused accumulation of apoE depleted VLDL. HDL particles accumulated apoE and failed to transfer it to VLDL in vitro. Importantly, these effects were prevented by pharmacological inhibition of CB1 and absent in plasma from CB1 mice. IDFP also caused a CB1-dependent increase in hepatic triglycerides. Thus, endocannabinoids inhibit the transfer of apoE from HDL to VLDL leading to apoE depletion of triglyceride rich lipoproteins. Microarray analysis allowed us to determine the effects of IDFP on expression of genes involved in lipid metabolism and to discover novel cannabinoid responsive genes. IDFP increased expression of lipogenic and SREBP2 target genes in a CB1-dependent fashion. On a global scale, pre-administration of a CB1 antagonist prevented many of the IDFP induced alterations in gene expression. IDFP decreased expression of genes involved in amino acid metabolism and inflammation. PCR analysis of selected mRNAs confirmed several of the key array findings. Our work indicates that endocannabinoids exert a large influence on hepatic lipid metabolism independent of food intake and suggest that peripherally restricted CB1 antagonists may be of therapeutic value. Overall, these findings shed light on the endogenous mechanisms of PPAR alpha activation and the hepatic responses to Cb1 activation. This information may help guide the continuing effort to develop treatments for metabolic disease.