ABSTRACT This proposal will address the critical need to define for cannabinoid exposure: 1) the most sensitive developmental exposure windows; 2) the dose- and sex-dependence of adverse outcomes; 3) developmentally relevant molecular mechanisms of persistent adverse effects; and 4) the relative developmental toxicity of other cannabinoids available to consumers. Cannabis/∆9-tetrahydro- cannabinol (THC) is the most commonly used illicit drug by pregnant women, and cannabidiol (CBD) is readily available over the counter with suggested benefits in pregnancy for morning sickness, stress, and sleeplessness. Similarly, other minor cannabinoids are marketed directly to consumers with numerous health claims. Because of maternal use, pre- and post-natal cannabinoid exposures occur during critical stages of children’s brain development despite our lack of understanding of the acute and long-term consequences. In addition, cannabinoid exposure (e.g. via vaping) frequently occurs during adolescence, another sensitive time of brain development and neuronal pruning. In our zebrafish model system, we have observed persistently altered adult behavior after embryos were exposed to THC and CBD during early development. Our central hypothesis is that exposure to cannabinoids causes alterations in inflammatory mediators in the developing brain leading to the persistent alterations in behavior throughout development and into adulthood. Our research framework, depicted as an adverse outcome pathway (AOP), will specifically investigate three aims to measure: 1) morphological and persistent behavioral alterations in anxiety/locomotion; 2) time-of-exposure susceptibilities; and 3) neuroinflammation resulting from THC and CBD developmental exposure. We will use the highly relevant, predictive, and high throughput zebrafish model, including three transgenic lines, to assess the spatial and temporal relationships between microglial response, gene expression, and persistent behavioral adverse outcomes. Cannabinoid-mediated changes in neuroinflammation gene/protein expression and altered cellular trajectories will be identified using single nucleiRNAseq and LC-MS/MS protein validation in larval and adult brains. The dependence of the specific cannabinoid receptors in observed toxicities will be determined by using cannabinoid receptor 1 and 2 null lines. Our ongoing and productive collaborations, including work with three NIH-COBRE core facilities, will be leveraged to inform the developmental origins of health and disease caused by cannabinoid exposure. The proposed research is significant because it will provide new, relevant information to guide cannabinoid policy and healthcare decisions made by pediatricians, obstetricians, and policy- makers needed to ensure public health and safety.

PROJECT NARRATIVE Pre-, peri- and postnatal exposures to ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are increasing with expanded use of marijuana, hemp-derived supplements, and minor cannabinoid- containing products available and marketed directly to consumers for an array of health claims. This project will address knowledge gaps associated with developmental consequences of exposure to THC, CBD, and other minor cannabinoids, with a particular emphasis on determining how early life exposures contribute to acute and latent effects into adulthood. Our long-term goal is to define critical periods of susceptibility, the dose dependence of adverse outcomes, and molecular mechanisms of action of cannabinoids that can be used to guide cannabis/CBD policy decisions and ensure public health safety.