Experiment
The expanded panel of BXD RI mice was used to identify quantitative trait loci (QTLs) associated with sensitivity to locomotor stimulatory and motor incoordinating effects of alcohol. The goals of the current study were to determine whether previously reported QTLs were replicated and refined and to determine whether novel QTLs would be identified.
The BXD RI set is generated from inbred C57BL/6J and DBA/2J parental strains. Until recently, only 30 strains of BXD mice were available, but the panel has been expanded with the addition of 46 new strains of BXD RI mice (Peirce et al., 2004).
Following an i.p. dose of 2.25 g/kg of ethanol (EtOH) or saline control, locomotor activation was assessed using an activity chamber and motor incoordination was assessed using the accelerating rotarod. Male and female BXD mice from over 55 strains from both the original and the expanded RI sets were tested. Two treatment paradigms were utilized to evaluate the effects of EtOH versus saline treatment-order.
Each mouse was given the same tests in the same order with a 3-day testing regimen utilized. Half of the mice received saline first and half of the mice received EtOH first. On the first day, all mice were given training trials on the accelerating rotarod. On the second day, mice were given an injection of the appropriate solution. Testing was begun 10 minutes after the injection to allow time for the EtOH to be absorbed into the system prior to evaluation in the elevated plus maze. After the 10 minute wait, mice were given a 5-minute test in the elevated plus maze to measure anxiety, followed immediately by a 20-minute test in the activity chamber.
After the activity chamber, mice were given 3 trials on the accelerating rotarod with blood collected at 60 minutes after injection to measure blood ethanol concentrations (BECs). After completion of testing, mice were returned to the housing room. On the third day, the testing was identical to that of the second day except that the elevated plus maze was no longer a novel environment and thus was not repeated, and the solution injected was different between the 2 days. Two groups were tested: group 1 (SE) was given saline on the first day followed by EtOH on the second day and group 2 (ES) was given EtOH on the first day and saline on the second day.
QTLs were identified using WebQTL, which can be found at GeneNetwork (www.genenetwork.org; NCBI Build 37). The likelihood ratio statistic was used to determine genome-wide significant and suggestive QTLs. Significant (p<0.05) levels for each QTL were determined following 1,000 permutation tests.
Table 3:
Activity chamber - Significant QTL:
Saline Exposed:
QTL Acq1 (activity chamber QTL 1) mapped to Chromosome 4 between 47.1-50.2 cM, LRS=23.3, for timepoint saline10 for both sexes. There were no significant effects for sex or treatment-order so all animals were grouped together. The DBA/2J allele increased the trait value at this locus.
A significant QTL, Actres1 (activity response to ethanol/saline 1), was also identified in the ES group on Chromosome 4 for timepoint saline20 that was treatment-order specific but had no significant sex effect so males and females were grouped together within each treatment group. The DBA/2J allele increased the trait value at this locus, which mapped between 47.6-50.4 cM.
A significant QTL, Actrse1 (activity response to saline/ethanol 1), was also identified in female mice in the SE group on Chromosome 4 mapping between 53.3 and 57.0 cM. The DBA/2J allele increased the trait value at this locus.
A fourth significant QTL, Actrse2 (activity response to saline/ethanol 2), also mapped to Chromosome 4 between 53.0 and 54.6 cM for total saline in female mice in the SE group. The DBA/2J allele increased the trait value at this locus also.
EtOH-exposed:
In the Ethanol5 group QTL Acq2 (activity chamber QTL 2) was mapped to Chromosome 2 between 60.8-64.6 cM in male mice. There were no significant treatment-order effects, male mice were combined across treatment groups. The DBA/2J allele increased the trait value at this locus.
In the Ethanol10 group QTL Actres2 (activity response to ethanol/saline 2) was mapped to Chromosome 2 between 60.7 and 74.1 cM in male mice in the ES group. The DBA/2J allele increased the trait value at this locus.
In the Ethanol15 group QTL Actres3 (activity response to ethanol/saline 3) was mapped to Chromosome 2 between 60.8 and 63.1 cM in male mice in the ES group. The DBA/2J allele increased the trait value at this locus.
In the Ethanol15 group QTL Actrse3 (activity response to saline/ethanol 3) was mapped to Chromosome 18 between 12.4 and 18.6 cM in female mice in the SE group. The C57BL/6J allele increased the trait value at this locus.
In the Ethanol20 group QTL Actres4 (activity response to ethanol/saline 4) was mapped to Chromosome 2 between 60.8 and 62.9 cM, LRS=23.4, in male mice in the ES group. The DBA/2J allele increased the trait value at this locus.
In the Ethanol20 group QTL Actrse4 (activity response to saline/ethanol 4) was mapped to Chromosome 9 between 43.6 and 48.5 cM in female mice in the SE group. The DBA/2J allele increased the trait value at this locus.
In the Ethanol20 group QTL Actrse5 (activity response to saline/ethanol 5) was mapped to Chromosome 18 between 12.2 and 17.6 cM in female mice in the SE group. The C57BL/6J allele increased the trait value at this locus.
In the total ethanol score of the ES group QTL Actres5 (activity response to ethanol/saline 5) mapped to Chromosome 2 between 59.9 and 69.8 cM. The DBA/2J allele increased the trait value at this locus.
Activity Chamber Difference QTL:
A measure of activity difference scores revealed significant QTL in the 15 to 20 minute differences. QTL Acdes1 (activity chamber difference ethanol/saline 1) mapped to Chromosome 2 between 59.4 and 62.9 cM in the male mice of the ES group. The DBA/2J allele increased the trait value at this locus.
QTL for the total difference in activity scores were identified on Chrs 2 and 5:
QTL Actdes1 (activity chamber total difference ethanol/saline 1) mapped to Chromosome 2 between 59.4 and 69.3 cM in male mice of the ES group.The DBA/2J allele increased the trait value at this locus.
QTL Actdse1 (activity chamber total difference saline/ethanol 1)) mapped to Chromosome 5 between 17.5 and 22.4 cM in the male mice of the SE group. The DBA/2J allele increased the trait value at this locus.
Rotarod QTL:
A significant QTL for Rotarod training, QTL Rotse1 (rotarod training saline/ethanol 1), mapped to Chromosome 3 between 26.5 and 28.2 cM in combined sexes of the SE group. The C57BL/6J allele increased the trait value at this locus.
Another, QTL Rotse2 (rotarod training saline/ethanol 2), mapped to Chromosome 15 between 34.1 and 35.5 cM, LRS=17.0, in combined sexes of the SE group. The C57BL/6J allele increased the trait value at this locus.
Another, QTL Rotse3 (rotarod training saline/ethanol 3), mapped to Chromosome 16 between 10.1 and 10.8 cM, LRS=18.4, in combined sexes of the SE group. The DBA/2J allele increased the trait value at this locus.
A significant QTL for saline time and rotarod training, QTL Rotst1 (rotarod training saline time 1), mapped to Chromosome 15 between 33.9 and 34.7 cM in combined sexes of the SE group. The C57BL/6J allele increased the trait value at this locus.
A significant QTL for ethanol time and rotarod training, QTL Rotet1 (rotarod training ethanol time 1), mapped to Chromosome 7 between 76.8 and 80.4 cM in female mice of both treatment groups. The C57BL/6J allele increased the trait value at this locus.
A significant locus, Rosd1 (rotarod saline difference 1), for saline difference in rotarod difference score mapped to Chromosome 7 between 66.8 and 76.5 cM, LRS= 22.0, in female mice in the ES group. The C57BL/6J allele increased the trait value at this locus.
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