Abstract The cannabis plants marijuana and hashish are the most commonly used substances of abuse by women of reproductive age. But, concerningly, frequent use of cannabis by pregnant women may result in lasting cognitive and neuro-behavioral issues in children that were exposed in utero. The mechanisms by which cannabinoids in cannabis plants influence the structure and function of the developing brain are not well understood. Previous studies in animal models indicate that the main cannabinoid receptor, CB1R, affects formation of neuronal circuits by signaling though various molecular factors such as the Netrin receptor DCC, PKA and RhoA. However, we lack comprehensive knowledge of essential mechanisms by which CB1R influences neuronal circuit formation during brain development. For many years, our laboratory determined signaling mechanisms for Wnt and Cadherin factor, -catenin, in development of the visual projection in tadpoles of the vertebrate frog model Xenopus laevis. This is an ideal system for studying neuronal circuit formation because of its strong genetic similarity to humans, amenability to molecular and genetic manipulation in single retinal ganglion cells and imaging of individual retinal ganglion cells with altered molecular signaling directly in their native environment. We now propose to determine whether CB1R inhibits Wnt/Cadherin/- catenin signaling to regulate growth cone filopodia and axon pathfinding features in situ. This proposal stems from work from others showing that CB1R inhibits Wnt and destabilizes -catenin in cancer cells, and Wnt mediated destabilization of -catenin downregulates Cadherin cell-cell adhesion, and our recently published paper showing that CB1R and -catenin oppositely modulate growth cone filopodia in retinal ganglion cells. We will test two aims: 1) Quantify axon pathfinding and growth cone defects following manipulation of CB1R in situ. Our previously published data showed that pharmacological manipulation of CB1R perturbs growth cone filopodia and axon fasciculation in situ. We will now assess whether cell- autonomous (morpholino based) loss-of-function of CB1R in individual retinal ganglion cells alters multiple growth cone and axon pathfinding parameters. 2) Establish functional interactions between CB1R and Wnt/Cadherin signaling in retinal ganglion cells. We will determine whether phenotypic effects of CB1R loss- of-function on growth cone filopodia and axon pathfinding features are rescued by expression of factors in the canonical Wnt signaling pathway (Axin, APC) and mutants of key players in Cadherin cell-cell adhesive complex (-catenin, -catenin). This data will determine a novel and essential signaling mechanism for cannabinoids in neuronal circuit development in a vertebrate model. Given the conservation of these signaling pathways, these results may also establish a fundamental mechanism for endocannabinoids in formation of neuronal circuits in human fetuses, and for how increased prenatal cannabis exposure could disrupt establishment of neuronal circuits, and lead to persistent cognitive and neurobehavioral deficits in children.

Project Narrative The cannabis plants marijuana and hashish are the most commonly used substances of abuse by women of reproductive age. Concerningly, frequent exposure to cannabis during fetal stages can lead to defects in brain development and neurobehavioral and cognitive deficits in children. The work proposed here will help explain how increased prenatal cannabis exposure affects the developing brain by defining key cellular processes that are impacted.