Experiment
Genetic differences in acute behavioral responses to ethanol contribute to the susceptibility to alcohol use disorder and the reduction of anxiety is a commonly reported motive underlying ethanol consumption among alcoholics. The authors studied the genetic variance in anxiolytic-like responses to ethanol across the BXD recombinant inbred (RI) mouse panel using the light-dark transition model of anxiety.
A total of 597 male mice across 38 strains (see Table S2, Supporting Information) were studied in three phases: provisional mapping, confirmation and fine-mapping. While sex-specific QTL are of significant interest to anxiety and behavioral responses to ethanol, only male mice were used for this initial assessment to reduce phenotypic variation of anxiety-related behaviors and to make the number of animals tractable in terms of manpower and expense.
For the provisional phase, 448 mice from 27 BXD RI strains and their C57BL/6J and DBA/2J progenitor strains were purchased at 8-9 weeks of age from the Jackson Laboratory (Bar Harbor, ME, USA).
Each BXD RI strain consisted of 6-8 mice per treatment and each progenitor strain had 13-16 mice per treatment. For the replication and fine-mapping analyses, the authors acquired, respectively, six and three novel BXD ARI strains (n = 5-10 per strain/treatment) and additional C57BL/6J and DBA/2J progenitor strains (n = 8 per strain/treatment) from Oak Ridge National Laboratory (Oak Ridge, TN, USA) at 6-9 weeks of age.
BXD strain genotypes used for all analyses consisted of approximately 4000 genetic markers obtained from GeneNetwork (www.genenetwork.org/genotypes/BXD.geno). All behavioral measures collected across the BXD strains and their inbred progenitors were evaluated for strain, treatment and strain-by-treatment differences using analysis of variance (ANOVA) with Tukey HSD (honest significant difference) post hoc analysis. Narrow-sense heritability (h2) estimates for all behavioral measures were calculated as previously described by Belknap et al. (1996). Behavioral QTL were identified by strain-mean single locus interval mapping performed with the R/qtl package for R (versions 2.11.1-3.1.1) (Broman et al. 2003; Chesler et al. 2004). The QTL genome-wide adjusted P-values were calculated empirically by permutation analysis with 1000 iterations (Churchill & Doerge 1994).
To examine genotype-by-treatment interactions, a single marker mixed-model QTL analysis was conducted using the lme4 package for R (Bates et al. 2015).
Behavioral phenotypes used for QTL mapping from the 0-5 to 0-10-min intervals are summarized in Table S2.
The authors focus discussion of QTL analysis on the 0- to 5-min interval because of the greater anxiolytic-like responses to ethanol across all strains at this time interval. However, qualitatively identical results were obtained for QTL analysis of the 0- to 5- and 0- to 10-min intervals.
Nearly all BXD RI strains exhibited ethanol-induced anxiolytic-like responses.
Strain-mean single factor interval mapping with permutation-corrected significance levels identified 14 QTL influencing phenotypic variation in anxiety-related behaviors and TLA in response to saline or ethanol injections following mild restraint stress (genome coordinates relative to GRCm38.p1).
Axtldb36 (anxiety-light dark box 36, Saline TLA) maps significantly to Chr 1: 147.39 - 184.42 Mb with a peak LOD score of 3.99 at 159.9 Mb (rs3696645).
Axtldb37 (anxiety-light dark box 37, Saline TLA) maps suggestively to Chr 9: 29.75 - 106.32 Mb with a peak LOD score of 2.39 at 53.77 Mb (rs3689326).
Axtldb38 (anxiety-light dark box 38, Ethanol TLA) maps significantly to Chr 1: 147.39 - 184.42 Mb with a peak LOD score of 3.71 at 157.9 Mb (UT_1_113.684537).
Axtldb39 (anxiety-light dark box 39, Ethanol TLA) maps suggestively to Chr 2: 9.75 - 172.85 Mb with a peak LOD score of 2.48 at 164.2 Mb (rs3675564).
Axtldb40 (anxiety-light dark box 40, Ethanol TLA) maps suggestively to Chr 11: 4.41 - 120.82 Mb with a peak LOD score of 2.16 at 47.93 Mb (rs13481014).
Axtldb41 (anxiety-light dark box 41, Ethanol TLA) maps suggestively to Chr 18: 3.52 - 86.75 Mb with a peak LOD score of 2.68 at 11.08 Mb (rs4231742).
Axtldb42 (anxiety-light dark box 42, Saline TLA) maps suggestively to Chr 1: 29.23 - 184.42 Mb with a peak LOD score of 2.41 at 128.4 Mb (D1Mit139).
Axtldb43 (anxiety-light dark box 43, Saline TLA) maps suggestively to Chr 6: 16.01 - 149.21 Mb with a peak LOD score of 2.74 at 140.1 Mb (gnf06.140.234).
Axtldb44 (anxiety-light dark box 44, Saline TLA) maps suggestively to Chr 11: 8.74 - 121.41 Mb with a peak LOD score of 2.7 at 116.9 Mb (rs13481251).
Axtldb45 (anxiety-light dark box 45, Ethanol TLA) maps suggestively to Chr 1: 13.98 - 197.13 Mb with a peak LOD score of 2.59 at 182.5 Mb (rs13474399).
Axtldb46 (anxiety-light dark box 46, Ethanol TLA) maps significantly to Chr 12: 53.91 - 71.66 Mb with a peak LOD score of 4.32 at 68.67 Mb (rs3716547).
Axtldb47 (anxiety-light dark box 47, Saline TLA) maps suggestively to Chr 1: 96.87 - 169.91 Mb with a peak LOD score of 3.35 at 128.5 Mb (rs13476098).
Axtldb48 (anxiety-light dark box 48, Saline TLA) maps significantly to Chr 11: 114.53 - 120.82 Mb with a peak LOD score of 3.75 at 116.9 Mb (rs13481251).
Axtldb49 (anxiety-light dark box 49, Ethanol TLA) maps suggestively to Chr 1: 14.79 - 190.74 Mb with a peak LOD score of 2.9 at 182.5 Mb (rs13474399).
Axtldb50 (anxiety-light dark box 50, Ethanol TLA) maps significantly to Chr 12: 53.91 - 71.66 Mb with a peak LOD score of 4.37 at 68.67 Mb (rs3716547).
Axtldb46 and Axtldb50 were successfully validated by studies with BXD advanced intercross strains and fine-mapped to a region comprising less than 3.5 Mb. Through integration of genome-wide mRNA expression profiles of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, the authors identified high priority candidate genes within Axtldb46 and Axtldb50, the strongest of which was Ninein (Nin; MGI:105108), a Gsk3-Beta-interacting protein that is highly expressed in the brain.
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