Experiment
Excessive and/or inappropriate fear is one of the most prominent symptoms of anxiety disorders, in particular disorders of fear regulation such as posttraumatic stress disorder. Knowledge of the specific genes that confer susceptibility will aid in both diagnosis and the development of more efficacious treatments.
Conditioned fear (CF) is a form of Pavlovian learning in which an aversive unconditioned stimulus (US) is paired with a previously neutral cue (conditional stimulus, CS). CF is heritable in both mice and humans.
Previously a set of chromosome substitution strains (CSS), in which a single chromosome from the A/J (AJ) strain was introgressed onto a C57BL/6J (B6) background, was used to identify a number of alleles that influenced CF [J:121951]. One of the strongest findings was that C57BL/6J-Chr 10A/J/NaJ (CSS-10) had increased freezing to cue and freezing to context compared to B6. A large QTL, Frct3 (fear conditioning traits 3), was identified on mouse Chromosome 10.
In the current study C57BL/6J-Chr 10A/J/NaJ (CSS-10) male mice were backcrossed to C57BL/6J(B6) female mice to create F1 progeny. F1 offspring were subsequently intercrossed generating 83 F2 (45 male, 38 female) progeny to replicate the mapping of the previously identified QTL Frct3 on Chr 10. Using the F2 population significant, overlapping QTL were identified on Chr 10 for increased freezing at multiple traits related to CF.
Table 1:
QTL Frct3a (fear conditioning traits 3a) mapped between 121262479-127659097 Mb (B37) on Chr 10 with a LOD score of 3.99 for behavior linked to 'tone 1, day 1' where baseline freezing was measured. Mice were exposed to two 30-second tones (85dB, 3kHz) that coterminated with a 2-second, 0.5mA foot shock. Baseline freezing was defined as the average percent of time beginning 30 seconds after the mice were placed in the test chambers and ending 150 seconds later.
QTL Frct3b 9 (fear conditioning traits 3b) mapped between 121262479-127659097 Mb (B37) on Chr 10 with a LOD score of 13.49 for behavior linked to 'average tone day 1' where the average time spent freezing to both tones on day 1 was measured.
QTL Frct3c (fear conditioning traits 3c) mapped between 117560107-127659097 Mb (B37) on Chr 10 with a LOD score of 3.29 for behavior linked to 'context' when freezing to context was measured on day 2 where the testing environment was identical to day 1 but neither tones nor shocks were presented.
QTL Frct3d (fear conditioning traits 3d) mapped between 110233294-127659097 Mb (B37) on Chr 10 with a LOD score of 4.73 for behavior linked to 'average tone day 3' where the average percent time spent freezing during the 2 30-second tones was measured in an altered context.
The A/J allele was associated with increased freezing for all four traits. The overlapping regions of the QTL spanned a 10.1 Mb region between 117560107-127659097 Mb
Next two congenic lines were created by backcrossing C57BL/6J-Chr10 10A/J/NaJ F2 mice with B6 mice to generate heterozygous congenic founders which were then used to create congenic breeder pairs. DNA was extracted from congenic mice and genotyped to define the boundaries of the congenic segments. The first congenic line (Line 1), had a congenic region that spanned from 122.387-129.068 Mb (Build 37; rs29362176, rs45982283). The second congenic line (Line 2), which was derived from Line 1, had a congenic region spanning 127.277-129.068 Mb (Build 37; rs3723970, rs45982283).
Genotyping was done with a combination of custom assays and sequencing of SNPs known to be polymorphic between B6 and AJ. For Line 1 the congenic region began between rs29380524 (122.384 Mb) and rs29362176 (122.387 Mb). For Line 2 the congenic region began between rs29382217 (127.264) and rs3723970 (127.277 Mb). Both congenic regions ended between rs13480830 (128.964 Mb) and rs45982283 (129.068 Mb). Thus, these congenic strains allowed the assessment of a ~ 6.7Mb region (Line 1) and a ~1.8 Mb region (Line 2) using a method similar to the sequential analysis. In total 59 mice from 23 litters were tested.
Figure 1:
Congenic Line 1 reduced the QTL interval for Frct3 (containing QTL Frct3a (1c), Frct3b (1d), Frct3c (1e), and Frct3d (1f)) from a 10.1 Mb to an app 6.7 Mb region mapping between 122.387 Mb (rs29362176) and 129.068 Mb (rs45982283).
Subcongenic Line 2 revealed significant differences in freezing to both tones (QTL Frct3b, Fig 1d) and freezing to cue (QTL Frct3d, Fig 1f) compared to B6 littermates refining the locations to a 1.8 Mb region mapping between 127.277 Mb (rs3723970) and 129.068 Mb (rs45982283)
Haplotype mapping followed by polymerase chain reaction was used to identify one gene, Rnf41, that was differentially expressed in both congenic lines relative to B6 and one, Shmt2, which was differentially expressed between Line 1 and B6.
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