Experiment
Cannabinoid receptor 1 activation by the major psychoactive component in cannabis, 19-tetrahydrocannabinol (THC), produces motor impairments, hypothermia, and analgesia upon acute exposure. In previous work, the authors 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, the authors quantified acute responses to a single dose of THC (10 mg/kg, i.p.) in males and females from 20 strains 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.
Genetic mapping is performed using strain means and not individual level data. Here the authors applied two different methods to compute QTL probability given strain genotypes and initial responses to THC using GeneNetwork (Mulligan et al., 2017). The first method applied was traditional simple regression (Haley-Knott or HK) and the second was genome-wide efficient mixed model association [GEMMA; (Zhou and Stephens, 2012)] with the "leave one chromosome out" (LOCO) option [rationale for using both methods described in Mulligan et al. (2018)]. Prior to performing genome scans, the strain-averaged distribution for each trait was checked for normality using the probability plot function in GeneNetwork. All traits were approximately normally distributed and used for QTL mapping.
For both HK and GEMMA, a dense panel of 7,321 markers was used for mapping. QTL were considered noteworthy if: (1) a genome-wide suggestive level (p < 0.63, equivalent to a 63% probability of a false positive or one false positive per genome scan) was reached following permutation (1000 tests) using the HK method, and (2) the peak marker association [-log(p) > 3] detected using GEMMA overlapped the QTL mapped by HK.
Genome-wide efficient mixed model association permutation was performed for each trait as described previously (Mulligan et al., 2018). For trait 21481, the 95th percentile (significant) threshold and 67th percentile (suggestive) threshold were 4.8 and 3.8 LOD, respectively. For trait 21483, the 95th percentile (significant) threshold and 67th percentile (suggestive) threshold were 4.5 and 3.6 LOD, respectively. For trait 21485, the 95th percentile (significant) threshold and 67th percentile (suggestive) threshold were 4.7 and 3.7 LOD, respectively. For trait 21487, the 95th percentile (significant) threshold and 67th percentile (suggestive) threshold were 4.7 and 3.7 LOD, respectively.
A 1.5-LOD drop from the peak marker (HK mapping) was used to define an approximate ~95% confidence interval (CI) for a QTL of interest (Alonso-Blanco et al., 2006). For each QTL CI, a complete list of mouse reference genes was generated using the UCSC Genome Browser (RRID:SCR_005780) Table Browser Tool (Group: Genes and Gene Predictions; Track: NCBI RefSeq; Genome: GRCm38/mm10). Genes in each QTL CI were then prioritized based on several criteria.
No genome-wide significant QTL (LOD > ~3.5) were detected for any trait (Figure 3B) using traditional interval HK mapping methods. However, seven suggestive QTL (LOD > 2.5) were detected for all THC initial response traits (genome coordinates relative to GRCm38/mm10):
Thcir1 (THC initial response 1, time mobile) maps to Chr 11: 80,036,498 - 90,077,597 bp with a peak LOD score of 2.8 (GEMMA -log(p) value of 3.2) at 89,612,498 bp.
Thcir2 (THC initial response 2, hypothermia, males only) maps to Chr 11: 80,036,498 - 89,612,498 bp with a peak LOD score of 3.2 (GEMMA -log(p) value of 3.9) at 82,187,018 bp.
Thcir3 (THC initial response 3, hypothermia, males only) maps to Chr 19: 27,531,578 - 36,395,869 bp with a peak LOD score of 2.5 (GEMMA -log(p) value of 3.7) at 32,887,054 bp.
Thcir4 (THC initial response 4, hypothermia, females only) maps to Chr 13: 107,382,731 - 115,929,261 bp with a peak LOD score of 2.6 (GEMMA -log(p) value of 3.7) at 110,449,36 bp.
Thcir5 (THC initial response 5, antinociception) maps to Chr 1: 168,144,357 - 172,235,364 bp with a peak LOD score of 2.9 (GEMMA -log(p) value of 3.9) at 170,949,421 bp.
Thcir6 (THC initial response 6, antinociception) maps to Chr 4: 102,851,020 - 109,169,766 bp with a peak LOD score of 2.5 (GEMMA -log(p) value of 3.3) at 107,548,653 bp.
One suggestive QTL was detected by GEMMA analysis only:
Thcir7 (THC initial response 7, antinociception) maps to Chr 5: 53,611,846 bp with a -log(p) value of 4.3.
Top positional candidate genes include: Ndufs2 (Thcir5); Scp2 (Thcir6); Rps6kb1/p70s6K (Thcir2); Pde4d (Thcir4); and Pten (Thcir3). These genes are likely to play a role in modulating initial response to THC based on location within modulatory QTL, putative functional sequence variants between B and D haplotypes that segregate among BXD progeny, evidence of genetic control of expression in brain tissue, and previously reported involvement in endocannabinoid/cannabinoid receptor signaling pathways.
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