Experiment
Genome-wide mapping of QTL was performed affecting locomotor activity in a novel environment, as well as QTL associated with methamphetamine (MA)-induced locomotor activity in an (C57BL/6J x DBA/2J)F2 and an Aap:B6,D2-G8 (F8 AIL) advanced intercross line population of mice (NCBI Build 37; mm9).
Testing was conducted over three consecutive days during the light phase. On the first and second days of testing, mice received an intraperitoneal (i.p.) injection of physiological saline and were then immediately placed in individual activity chambers where locomotor activity was recorded for 30 min. On the third day of testing, mice received an i.p. injection of 2 mg/kg MA and were then immediately placed in the activity chambers to measure locomotor activity for 30 min. Methamphetamine response was defined as the total distance traveled on day three during the 30-min test beginning immediately after drug administration. Locomotor activity was measured using automated Versamax activity chambers. DNA was extracted and genotyped from both the F2 and F8 populations.
Linkage association analysis was performed in the combined population (676-F2, 552-F8) using the R package QTLRel with a mixed model. For each analysis, P<0.05 significance thresholds were estimated using 1000 permutations. Sex was included as an interactive covariate.
Fig 2:
Six significant QTL associated with locomotor activity in a novel environment were identifed in the integrated results in both the 0-15 minute (p<0.05, LOD>3.93) and 0-30 minute (p<0.05, LOD>4.03), day 1, saline test, intervals:
QTL Bllact1 (base line locomotor activity 1) mapped to Chromosome 1;
QTL Bllact2 (base line locomotor activity 2) mapped to Chromosome 4;
QTL Bllact3 (base line locomotor activity 3) mapped to Chromosome 6;
QTL Bllact4 (base line locomotor activity 4) mapped to Chromosome 9;
QTL Bllact5 (base line locomotor activity 5) mapped to Chromosome 11;
QTL Bllact6 (base line locomotor activity 6 ) mapped to Chromosome X.
Seven significant QTL associated with methamphetamine induced locomotor activation were identified in the integrated results:
QTL Milac1 (methamphetamine induced locomotor activation 1) mapped to Chromosome 1 in the MA, day 3, 0-15 minute test interval (p<0.05, LOD>3.98) and in the 0-30 minute test interval (p<0.05, LOD>3.84).
QTL Milac2 (methamphetamine induced locomotor activation 2) mapped to Chromosome 8 in the MA, day 3, 0-15 minute test interval (p<0.05, LOD>3.98) and in the 0-30 minute test interval (p<0.05, LOD>3.84).
QTL Milac3 (methamphetamine induced locomotor activation 3) mapped to Chromosome 9 with significance in the MA, day 3 tests, in all 3 timed test intervals.
QTL Milac4 (methamphetamine induced locomotor activation 4) mapped to Chromosome 11 with significance in the MA, day 3 tests, in all 3 timed test intervals.
QTL Milac5 (methamphetamine induced locomotor activation 5) mapped to Chromosome 12 in the MA, day 3, 15-30 minute test interval (p<0.05, LOD>4.01) and in the 0-30 minute test interval (p<0.05, LOD>3.84).
QTL Milac6 (methamphetamine induced locomotor activation 6) mapped to Chromosome 15 in the MA, day 3, 0-15 minute test interval (p<0.05, LOD>3.98).
QTL Milac7 (methamphetamine induced locomotor activation 7) mapped to Chromosome 16 with significance in the MA, day 3 tests, in all 3 timed test intervals.
Because the use of F2 and F8 AIL mice is most interpretable when there is concurrence between the QTL locations in both generations, the analysis was further constrained to QTL that were evident in both the F2 and the F8 cohorts. This left three QTL for activity on day 1 (in a novel environment: Bllact1, Bllact4 and Bllact6 ) and three QTL for activity on day 3 (MA response: Milac3, Milac6 and Milac7).
The 30 minute testing period was divided into 6 consecutive five-minute bins in order to determine if a particular time point was driving each QTL. In both Bllact1 (Figure 3a, 3b) and Bllact2 (Figure 3e, 3f), the 0-5 minute time bin was largely responsible for the QTL. In contrast, Bllact4 (Figure 3c, 3d) reached its peak during the 5-10 minute bin. For methamphetamine response, Milac6 (Figure 4c, 4d) peaked during the 0-5 minute bin and Milac3 (Figure 4a, 4b) and Mila7 (Figure 4e, 4f) both showed the strongest effect at the 10-15 minute bin. The 1.8-LOD intervals for these six QTLs ranged from 1.5 to 50.0 Mb, with a median width of 15.6 Mb. Table 1:
QTL Bllact1 mapping to Chr 1 peaked at marker rs8245216 (173.174 Mb) with a LOD score of 15.4 in the 5-10 minute interval. The confidence interval spanned from 172.357 to 173.832 Mb and included 20 genes with non-synonymous coding SNPs (Table S2).
QTL Bllact2 mapping to Chr 4 peaked at marker rs13478002 (136.412 Mb) with a LOD score of 7.1 in the 0-5 minute interval. The confidence interval spanned from 133.375 to 141.023 Mb and included 64 genes with non-synonymous coding SNPs (Table S2).
QTL Bllact6 mapping to Chr X peaked at marker gnfX.086.039 (99.159 Mb) with a LOD score of 6.2 in the 0-5 minute interval. The confidence interval spanned from 81.842 to 131.953 Mb and included 12 genes with non-synonymous coding SNPs (Table S2).
QTL Milac3 mapping to Chr 9 peaked at marker rs3655717 (68.205 Mb) with a LOD score of 10.7 in the 10-15 minute interval. The confidence interval spanned from 57.888 to 72.491 Mb and included 24 genes with non-synonymous coding SNPs (Table S2).
QTL Milac6 mapping to Chr 15 peaked at marker rs13482642 (75.523 Mb) with a LOD score of 9.3 in the 0-5 minute interval. The confidence interval spanned from 68.959 to 85.617 Mb and included 52 genes with non-synonymous coding SNPs (Table S2).
QTL Milac7 mapping to Chr 16 peaked at marker rs4186744 (54.759 Mb) with a LOD score of 7.6 in the 10-15 minute interval. The confidence interval spanned from 42.090 to 70.632 Mb and included 42 genes with non-synonymous coding SNPs (Table S2).
Table S1 lists eQTL co-mapping with Bllact1, Bllact2, Bllact6, Milac3, Milac6, and Milac7. Table includes gene symbol and name, as well as Mb location for each gene, mean expression, max LOD, and eQTL chromosome and Mb location.
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