Identification of gene variants mediating the behavioral and physiological response to THC

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Cannabis is one of the most widely used psychotropic drugs in the United States and changes in public policy are expected to further increase use. Current cultivars of cannabis have been selected for high Δ9- tetrahydrocannabinol (THC) levels and THC levels have tripled over the past few decades. There is increasing evidence that use of high potency cannabis containing high levels of THC increases the risk of adverse health and behavioral consequences. Many of the specific effectors (e.g., receptors, receptor signaling pathways) mediating response to THC have not yet been fully identified and virtually nothing is known about how individual genetic differences influence response. This lack of knowledge represents a critical barrier in understanding health risks and behaviors associated with high potency cannabis use. To address these issues we have developed a powerful pharmacogenomic screen in the BXD recombinant inbred mouse population to identify gene variants in effector proteins that regulate cannabinoid 1 receptor (CB1) levels, signal transduction, termination of signaling, and/or trafficking following acute exposure to high-dose (10 mg/kg, i.p.) THC. Previously, we demonstrated strain differences in the level of the major effector of THC responses, CB1, between BXD parental strains and demonstrated heritable variation in acute responses to THC in parental strains and BXDs. We have already identified suggestive quantitative trait loci (QTLs) and candidate causal QTL genes (QTGs) in our pharmacogenomic screen using only a subset (i.e., 20) of BXDs. Here, we expand our work to identify genetic modulators of CB1 protein levels (Aim 1) and to identify and validate genetic modulators of acute responses to high-dose THC (Aim 2). In Aim 1A we use a targeted proteomics approach to quantify striatal CB1 levels in membrane-enriched protein fractions in parents, reciprocal F1, and BXD strains. Differential expression in parental strains, allele-specific expression in the F1 and linkage mapping in the BXDs will identify and validate QTLs containing trans-acting regulators of striatal CB1. In Aim 1B we extend the analysis to two other regions and in Aim 1C we include exposure to high-dose THC. In Aim 2A we screen a large panel (i.e., 80) of BXDs for differential CB1 activation quantified as hypolocomotion, hypothermia, and antinociception following VEH or THC treatment and identify QTLs accounting for at least 30% of the variation in each trait at a target precision of ±2 Mb. In Aim 2B we generate matched proteomes and transcriptomes for parental strains and F1 and generate transcriptome data for expression QTL (eQTL) mapping in BXDs with the goal of facilitating the identification of causal QTGs. In Aim 2C we validate the biological role of QTGs in cannabinoid receptor signaling pathways and responses to THC. This is the first pharmacogenomic screen to identify variation in effectors of CB1 signaling and will reveal new targets underlying responses to high-dose THC and behaviors associated with cannabis use in rodents and humans.