Genetic Modulation of Initial Sensitivity to Δ9-Tetrahydrocannabinol (THC) Among the BXD Family of Mice

Abstract

Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, Δ9-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, we demonstrated significant sex and strain differences in acute responses to THC following administration of a single dose (10 mg/kg, i.p.) in C57BL/6J (B6) and DBA/2J (D2) inbred mice. To determine the extent to which these differences are heritable, we quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 members of the BXD family of inbred strains derived by crossing and inbreeding B6 and D2 mice. Acute THC responses (initial sensitivity) were quantified as changes from baseline for: 1. spontaneous activity in the open field (mobility), 2. body temperature (hypothermia), and 3. tail withdrawal latency to a thermal stimulus (antinociception). Initial sensitivity to the immobilizing, hypothermic, and antinociceptive effects of THC varied substantially across the BXD family. Heritability was highest for mobility and hypothermia traits, indicating that segregating genetic variants modulate initial sensitivity to THC. We identified genomic loci and candidate genes, including Ndufs2, Scp2, Rps6kb1 or P70S6K, Pde4d, and Pten, that may control variation in THC initial sensitivity. We also detected strong correlations between initial responses to THC and legacy phenotypes related to intake or response to other drugs of abuse (cocaine, ethanol, and morphine). Our study demonstrates the feasibility of mapping genes and variants modulating THC responses in the BXDs to systematically define biological processes and liabilities associated with drug use and abuse.

Keywords: BXD family; C57BL/6; DBA/2; QTL; THC; addiction; cannabis; drug response.

FIGURE 1

Experimental Overview. (A) Overview of the generation of the BXD RI population. This family of strains has been derived at several points in time from both F2 and advanced intercrosses and there are now ∼150 strains available. (B) Description of daily treatment regime. Initial sensitivity is measured by subtracting Day 1 trait values from those of Day 0 for each individual. (C) Timing of daily trait measurements for body temperature (Temp), time mobile in the open field (OF), and latency to withdrawal the tail in response to a thermal stimulus (TF) are shown as minutes post-injection.

FIGURE 2

Variation in Initial Response to THC Among BXDs. A significant effect of THC treatment relative to baseline (p < 0.001) was observed for all traits. Initial response to THC is shown as the difference between baseline and initial THC treatment. Strains are shown on the x-axis and initial response (difference between baseline response on day 0 and response to THC on day 1) is shown on the y-axis. Negative values indicate a decrease in response on day 1 compared to day 0 and positive values indicate an increase in response. Parental strains are indicated in blue (B6) and red (D2) and BXD strains are shown in black. (A) There is a significant effect of strain (p < 0.001) on initial response to THC for time spent mobile in the open field 30 min post-injection. Male and female responses were combined as there were no significant interaction effects involving sex. Responses to THC varied ∼90-fold among strains for the mobility trait. (B) The antinociceptive effects of THC were quantified using tail withdrawal latency in response to a thermal stimulus at 60 min post-injection. Male and female responses were combined as there were no significant interaction effects involving sex. Response to THC varied nearly 8-fold across strains, however, no significant effects of strain were observed for the antinociception trait. (C,D) There is a significant main effect of strain (p < 0.05) on initial hypothermic response 60 min post-injection of THC for both females (C) and males (D). Hypothermic response to THC in females varies 13-fold across strains compared to 24-fold variation in response in males. Summarized B6 and D2 responses shown for comparison (Parks et al., 2020).

FIGURE 3

THC Initial Response Trait Correlations and Genetic Co-Regulation. (A) Trait correlation network constructed after calculating Pearson’s correlation on ranked trait values to generate Spearman’s rank order correlation coefficient (Rho or P). The hypothermia trait was split by sex due to a condition-by-sex interaction effect on body temperature change in response to THC. Changes in time mobile in the open field and body temperature in males following a single THC (10 mg/kg) exposure are significantly (p < 0.01) correlated. Weak (not significant) correlations are also observed between mobility and hypothermia in females following a single THC exposure and between changes in male and female hypothermia in response to THC. (B) Genetic regulation of THC initial response traits is observed, albeit at a suggestive level with no traits passing the threshold for genome-wide significance (p < 0.05). Interval maps for each trait shown as a different line color corresponding to the correlation network in (A). The strength of association (LOD) on the y-axis is plotted for each trait across the genome (megabase position on each chromosome or Chr) on the x-axis. Change in mobility and male body temperature are regulated by the same suggestive locus on Chr 11 (zoomed region in boxed area). (C) Use of an alternative linear mixed-model QTL mapping method (GEMMA, see section “Materials and Methods”) resulted in replication of genetic co-regulation of initial motor and hypothermia THC response traits from the same Chr 11 interval. Strength of association or –log(P) values (y-axis) shown for each marker (blue dots) within the Chr 11 QTL interval for mobility and temperature (males) initial THC response traits. Mapping with linear mixed-models can account for population family structure or kinship within the BXD population that is not addressed using traditional interval mapping. SNP density plotted in orange on the x-axis. (D) Scatterplot describing the relationship between average change in time mobile in the open field in response to THC (X-axis) and average change in male body temperature in response to THC (Y-axis) for each BXD strain based on Spearman’s rank order correlation.