Experiment
To explore the genetic networks governing the biology of conditional fear, the authors used a systems genetics approach to analyze a hybrid mouse diversity panel (HMDP) with high mapping resolution. The HMDP combines inbred and RI lines to create a panel of 100 strains with great resolution and statistical power. The HMDP consists of 29 classical inbred strains supplemented with 71 RI strains derived from C57BL/6J crossed with either DBA/2J, A/J or C3H/HeJ.
To identify regions of the genome associated with fear-related behavior, 700 male mice from the HMDP were subjected to a fear conditioning procedure and characterized on 48 unique behavioral phenotypes drawn from different test phases. Mice were tested for cued and contextual fear acquired through a Pavlovian conditioning procedure. Immobility (freezing) is a classical measure of fear triggered by an environmental threat. The authors also monitored other measures of fear including velocity, thigmotaxis (wall-preference), path shape, and habituation. On day one, a mouse is placed in a cage where an auditory conditional stimulus (CS) tone is played for fifteen seconds followed by a brief foot shock. Training consisted of three tone-shock pairings. The next day, the mouse returned to the same chamber and contextual fear is indexed through a collection of behavioral endpoints including immobility. On the third day, the mouse is placed in a novel chamber and given a series of CS presentations with no foot shock. Cued fear is quantified across the same behavioral endpoints used to assess contextual fear.
The classical inbred and RI strains were genotyped previously by the Broad Institute (classical) and the Wellcome Trust Center for Human Genetics (RI). The genotypes of the RI lines at the Broad SNPs were imputed from the Wellcome Trust genotypes. Only SNPs with a minor allele frequency greater than or equal to 10% were used in the analysis to minimize false positives due to small sample size. All genome coordinates are based on NCBI build 35 (mm7) of the mouse genome.
Using the above phenotypes as quantitative traits, the authors performed a genome-wide association study (GWAS) to identify loci associated with each of the behavioral traits. The authors corrected for the confounding effects of relatedness and population structure between strains using efficient mixed model association (EMMA). The authors mapped loci for the behavioral phenotypes using EMMA and 101,629 SNPs.
A total of 30 behavioral QTL were mapped with FDR < 0.05:
Ptfc1 (pre-training fear conditioning 1, thigmotaxis mean distance to point) maps to Chr 9 with a peak p-value of 1.14 x 10^-6 at 61,060,175 bp.
Ptfc2 (post-training fear conditioning 2, velocity mean) maps to Chr 15 with a peak p-value of 2.92 x 10^-6 at 5,887,595 bp.
Ptfc3 (pre-training fear conditioning 3, immobility mean) maps to Chr 2 with a peak p-value of 1.77 x 10^-6 at 6,186,281 bp.
Ptfc4 (pre-training fear conditioning 4, immobility mean) maps to Chr 7 with a peak p-value of 1.31 x 10^-6 at 126,370,751 bp.
Ptfc5 (post-training fear conditioning 5, immobility mean) maps to Chr 8 with a peak p-value of 4.41 x 10^-6 at 68,297,006 bp.
Pcfc1 (precue fear conditioning 1, immobility mean) maps to Chr 7 with a peak p-value of 5.58 x 10^-9 at 94,641,553 bp.
Pcfc2 (precue fear conditioning 2, immobility mean) maps to Chr 7 with a peak p-value of 5.58 x 10^-9 at 94,744,373 bp.
Pcfc3 (precue fear conditioning 3, immobility mean) maps to Chr 7 with a peak p-value of 5.14 x 10^-8 at 107,177,259 bp.
Cufcd1 (cue fear conditioning 1, immobility mean) maps to Chr 3 with a peak p-value of 1.56 x 10^-6 at 103,364,188 bp.
Cufcd2 (cue fear conditioning 2, immobility mean) maps to Chr 3 with a peak p-value of 3.44 x 10^-6 at 130,123,970 bp.
Cufcd3 (cue fear conditioning 3, immobility mean) maps to Chr 4 with a peak p-value of 2.58 x 10^-6 at 6,678,672 bp.
Cufcd4 (cue fear conditioning 4, immobility mean) maps to Chr 7 with a peak p-value of 4.40 x 10^-9 at 94,641,553 bp.
Cufcd5 (cue fear conditioning 5, immobility mean) maps to Chr 7 with a peak p-value of 4.40 x 10^-9 at 94,744,373 bp.
Cufcd6 (cue fear conditioning 6, immobility mean) maps to Chr 7 with a peak p-value of 7.06 x 10^-9 at 104,540,350 bp.
Cufcd7 (cue fear conditioning 7, immobility mean) maps to Chr 15 with a peak p-value of 1.76 x 10^-6 at 37,521,578 bp.
Cufcd8 (cue fear conditioning 8, immobility mean) maps to Chr 19 with a peak p-value of 3.80 x 10^-6 at 26,658,546 bp.
Pcfc4 (precue fear conditioning 4, mobility mean) maps to Chr 7 with a peak p-value of 1.37 x 10^-6 at 94,641,553 bp.
Pcfc5 (precue fear conditioning 5, mobility mean) maps to Chr 7 with a peak p-value of 1.37 x 10^-6 at 94,744,373 bp.
Pcfc6 (precue fear conditioning 6, thigmotaxis mean distance to point) maps to Chr 1 with a peak p-value of 3.17 x 10^-6 at 163,397,742 bp.
Cufcd9 (cue fear conditioning 9, thigmotaxis mean distance to point) maps to Chr 11 with a peak p-value of 1.24 x 10^-8 at 48,065,799 bp.
Pcfc7 (precue fear conditioning 7, thigmotaxis mean) maps to Chr 2 with a peak p-value of 3.36 x 10^-6 at 151,612,920 bp.
Pcfc8 (precue fear conditioning 8, thigmotaxis mean) maps to Chr 11 with a peak p-value of 2.20 x 10^-6 at 52,523,068 bp.
Pcfc9 (precue fear conditioning 9, thigmotaxis mean) maps to Chr 13 with a peak p-value of 3.73 x 10^-6 at 72,750,827 bp.
Cfcd2 (contextual fear conditioning 2, thigmotaxis mean distance to point) maps to Chr 1 with a peak p-value of 1.22 x 10^-6 at 172,955,973 bp.
Cfcd3 (contextual fear conditioning 3, thigmotaxis mean distance to point) maps to Chr 8 with a peak p-value of 3.62 x 10^-6 at 53,062,087 bp.
Cfcd4 (contextual fear conditioning 4, thigmotaxis mean distance to point) maps to Chr 9 with a peak p-value of 2.16 x 10^-6 at 61,070,635 bp.
Cfcd5 (contextual fear conditioning 5, meander mean) maps to Chr 2 with a peak p-value of 3.65 x 10^-6 at 129,472,283 bp.
Cfcd6 (contextual fear conditioning 6, immobility mean) maps to Chr 2 with a peak p-value of 3.32 x 10^-6 at 128,198,673 bp.
Cfcd7 (contextual fear conditioning 7, immobility mean) maps to Chr 6 with a peak p-value of 5.22 x 10^-8 at 71,209,634 bp.
Cfcd8 (contextual fear conditioning 8, mobility extinction) maps to Chr 11 with a peak p-value of 4.27 x 10^-6 at 70,800,475 bp.
Pcfc1 is located within the Tyrosinase (Tyr) gene. Since the HMDP is composed of inbred mouse strains, a number are homozygous for a recessive mutation in Tyr leading to an albino coat color (26 of 94 strains phenotyped). Pcfc2 has the same p-value as Pcfc1 and lies in the glutamate receptor gene metabotropic 5 (Grm5), which is involved in glutamatergic neurotransmission. Homozygous null mice for Grm5 have been shown to have reduced hippocampal long term potentiation (LTP) and impaired spatial learning. These mice also have a behavioral phenotype associated with a rodent model of schizophrenia. Polymorphism at this locus may contribute to a variance in motor activity as a conditioned response to a tone.
By integrating fear phenotypes, transcript profiling data from hippocampus and striatum and also genotype information, two gene co-expression networks correlated with context-dependent immobility were identified. The authors prioritized the key markers and genes in these pathways using intramodular connectivity measures and structural equation modeling. Highly connected genes in the context fear modules included Psmd6, Ube2a and Usp33, suggesting an important role for ubiquitination in learning and memory.
In addition, the authors surveyed the architecture of brain transcript regulation and demonstrated preservation of gene co-expression modules in hippocampus and striatum, while also highlighting important differences. Rps15a, Kif3a, Stard7, 6330503K22RIK, and Plvap were among the individual genes whose transcript abundance were strongly associated with fear phenotypes.
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