UCSF

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.