Fear conditioning provides an opportunity to survey a range of clinically relevant processes including short and long-term memory, context generalization, and memory extinction, making it an efficient tool with which to probe the genetics of fear dependent behavior. To map fear related QTLs, we subjected a population of inbred mouse strains to a standard fear conditioning procedure and follow-up memory tests. We then combined behavioral phenotype data with SNP genotypes and tissue specific gene expression to search for candidate genes and related networks associated with fear phenotypes. Across 48 behavioral endpoints, we mapped a total of 27 QTLs, highlighting the complexity of behavioral regulation and showcasing the value of HMDP for mapping fear loci.
The inbred strains of the HMDP were not randomly selected, but were, in fact, carefully chosen to avoid, insofar as possible, high correlation of non-linked genome segments. Nevertheless, there are some shared segments across the genome due to bottlenecks in the breeding and the history of the strains. EMMA endeavors to correct for these artifacts in the association analysis. However, some caution should be applied to the interpretation of the mapping results, since bias may remain which cannot be overcome by the analysis of the data.
The strongest behavioral QTL in our investigation was for the phenotype cue immobility and had two peak markers on chromosome 7. These markers were located in the adjacent genes Tyr
and had identical P
values of 4.4 × 10-9
, yet there were recombination breakpoints between them. Many HMDP strains have mutations in Tyr
and are albino, resulting in possibly learning and memory deficits due to decreased visual acuity. However, a study that examined this allele specifically showed that it plays only a minor role in cue immobility and that additional loci are likely to influence fear conditioning [16
is an attractive candidate gene for this locus, since it has previously been shown to be involved in hippocampal LTP.
We surveyed the architecture of transcriptional regulation across two brain regions. We found a smaller number of cis and trans eQTLs in the hippocampus than in the striatum. This diminution may be caused by signal dilution due to the heterogeneous cellular nature of the hippocampus. However we found that the cis and trans eQTLs in the two tissues overlapped significantly, indicating that DNA polymorphism has a robust effect in modulating gene expression across tissues.
By simplifying the gene expression data into modules, we identified groups of genes that are related to fear related behavior. Two such modules in the hippocampus (CF1 and CF2) showed strong correlations with context-dependent fear measures, allowing identification of networks of genes whose co-expression co-varied with fear phenotypes across the HMDP. We assigned priorities to genes within each module based on their level of intramodular connectivity and mapped loci responsible for regulating MEs in both hippocampus and striatum. Cued and context immobility were phenotypically similar as they clustered together in the behavioral dendrogram. However, the two identified modules did not show strong correlations with cued fear, confirming suggesting that the two different types of fear are expressed through different neural and/or molecular pathways.
A hub gene in CF1 (Psmd6) and two of the most highly connected genes in CF2 (Ube2a
) have been shown to play roles in ubiquitination. Interestingly, others have shown that ubiquitin-mediated proteolysis is involved in initiating long-term stable memory, as both specific removal of specific inhibitory proteins and gene induction are likely to be critical players in fear conditioning [45
]. Other components in these modules may be implicated by association in these genetic pathways and provide attractive targets for further investigation.
Structural equation modeling allowed us to identify single markers that influenced the expression of single genes which in turn influence fear related phenotypes. We identified five genes with causal relationships for fear-related phenotypes in the hippocampus and striatum including 6330503K22RIK, Rps15a, Kif3a, Stard7, and Plvap.