Experiment
Fear conditioning (FC) may provide a useful model for some components of post-traumatic stress disorder (PTSD).
In the current study, an F2 intercross between (C57BL/6J x DBA/2J) mice generated 620 F2 mice and 567 mice from an Aap:B6,D2-G8 advanced intercross line. B6 and D2 inbred mice were chosen as the progenitor strains in order to take advantage of the vast amount of bioinformatics resources associated with these strains and because these strains display robust differences in fear conditioning.
Fear conditioning testing consisted of a 5 min test that occurred three times over consecutive days during the light phase, between 0800 and 1,700h.
On test day 1, mice were placed into the fear conditioning (FC) chamber. 180 s later, mice were exposed twice to the conditioned stimulus (CS), which consisted of an 85 dB, 3 kHz tone that persisted for 30 s and co-terminated with the unconditioned stimulus (US), which was a 2s, 0.5 mA foot shock delivered through the stainless steel floor.
Two measures were calculated for QTL mapping: (1) baseline freezing, defined as average percent time freezing beginning 30s after the mice were placed into the test chambers, and ending 150s later (30180s; pre-training freezing), and (2) time spent freezing to
each CS presentation, calculated by averaging the percent time spent freezing to the tone presentations (180210s, 240270s; freezing to toneday 1).
Test day 2 began exactly 24 h after the start of test day 1. On test day 2, the testing environment was identical to day 1; however, neither tones (CS) nor shocks (US) were presented. On day 2, freezing to context was used for QTL mapping; this was defined as average percentage of time freezing in response to the test chamber during the same period of time as pre-training freezing (30180s; freezing to context).
Test day 3 began exactly 24 h after the start of test day 2. On test day 3, the context was altered in several ways: (1) a different experimenter conducted the testing and wore a different style of gloves, (2) the transfer cages had no bedding, (3) the metal shock grid, chamber door and one wall were covered with hard white plastic, (4) yellow film was placed over the chamber lights, (5) chambers and plastic surfaces were cleaned with 0.1% acetic acid solution, and (6) the vent fan was partially obstructed to alter the background noise.
On day 3, the tone (CS) was presented at the same times as on day one, but no foot shocks (US) were paired with it. On day 3, freezing to cue was defined as the average percent time spent freezing during the two 30s CS presentations (180210s, 240270s; freezing to cue) and used for QTL mapping.
Genome wide analysis was performed on the integrated data from both the F2 and G8 populations for pre-taining freezing, freezing to tone/shock day 1, freezing to context, and freezing to cue. Using 1,000 permutations, significance thresholds for these traits weredetermined to range from 3.92 to 4.03 LOD. No QTLs reached genome-wide significance
for pre-training freezing.
Two QTLs associated with freezing to tone day 1 (on chromosomes 1 and 13) were identified, five QTLs associated with freezing to context (on chromosomes 1, 2, 5, 10, and 13) and four QTLs associated with freezing to cue (on chromosomes 1, 2, 5 and 13), Table 1:
Condition fear QTL: freezing to tone, day 1:
QTL Cfq13 (conditioned fear QTL 13) mapped to Chromosome 1 with a LOD score of 14.0 at 109.515 Mb, p<0.05. The 1.5 LOD support interval mapped between 104.264 and 127.206 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
QTL Cfq14 (conditioned fear QTL 14) mapped to Chromosome 13 with a LOD score of 6.4 at 55.240 Mb, p<0.05. The 1.5 LOD support interval mapped between 46.459 and 73.083 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
Condition fear QTL: freezing to context, day 2:
QTL Cfq15 (conditioned fear QTL 15) mapped to Chromosome 1 with a LOD score of 6.9 at 132.415 Mb, p<0.05. The 1.5 LOD support interval mapped between 129.953 and 136.107 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
QTL Cfq16 (conditioned fear QTL 16) mapped to Chromosome 2 with a LOD score of 4.6 at 91.483 Mb, p<0.05. The 1.5 LOD support interval mapped between 73.315 and 107.506 Mb. The C57BL/6J SNP at this locus was attributed to increased freezing.
QTL Cfq17 (conditioned fear QTL 17) mapped to Chromosome 5 with a LOD score of 5.3 at 76.761 Mb, p<0.05. The 1.5 LOD support interval mapped between 73.058 and 112.437 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
QTL Cfq18 (conditioned fear QTL 18) mapped to Chromosome 10 with a LOD score of 4.8 at 12.931 Mb, p<0.05. The 1.5 LOD support interval mapped between 3.470 and 27.855 Mb. The C57BL/6J SNP at this locus was attributed to increased freezing.
QTL Cfq19 (conditioned fear QTL 19) mapped to Chromosome 13 with a LOD score of 5.2 at 67.311 Mb, p<0.05. The 1.5 LOD support interval mapped between 40.134 and 73.083 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
Conditioned fear: freezing to cue, day 3:
QTL Cfq20 (conditioned fear QTL 20) mapped to Chromosome 1 with a LOD score of 5.5 at 111.832 Mb, p<0.05. The 1.5 LOD support interval mapped between 93.792 and 129.953 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
QTL Cfq21 (conditioned fear QTL 21) mapped to Chromosome 2 with a LOD score of 5.4 at 84.707 Mb, p<0.05. The 1.5 LOD support interval mapped between 80.456 and 108.521 Mb. The C57BL/6J SNP at this locus was attributed to increased freezing.
QTL Cfq22 (conditioned fear QTL 22) mapped to Chromosome 5 with a LOD score of 5.4 at 76.761 Mb, p<0.05. The 1.5 LOD support interval mapped between 64.258 and 91.490 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
QTL Cfq23 (conditioned fear QTL 23) mapped to Chromosome 13 with a LOD score of 6.9 at 55.240 Mb, p<0.05. The 1.5 LOD support interval mapped between 47.380 and 58.826 Mb. The DBA/2J SNP at this locus was attributed to increased freezing.
Using a variety of bioinformatic approaches including eQTL that co-mapped with the QTL and
SNP analysis within the 1.5 LOD intervals, numerous candidate genes were identified that had previously been implicated in fear learning in animal models, including Bcl2, Btg2, Dbi, Gabr1b, Lypd1, Pam and Rgs14. See Table 2 for supporting references.
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