Our results highlight a specific genomic region on chromosome 3 as a major contributor to the variation of CORT response derived from the HAB/LAB mouse model. The strong contribution of genomic factors of this region seems to be essential for regulating stress-related reactivity of the HPA axis.
Here, we described first of all a difference in CORT secretion in HAB vs. LAB mice upon a 15-min restraint stressor, with LAB mice secreting more CORT compared to HAB mice. Thus, HAB mice appear to have a rather blunted stress response. Although, in general, high levels of CORT response are described to be associated with high anxiety states
[34, 35], the opposite is true for HAB vs. LAB mice. This finding was replicated in CD-1 mice bred for differences in CORT responses to a stressor, with lower HPA axis reactivity mice showing more passive exploratory behaviour
. In addition, a stronger increase in CORT has been demonstrated after social defeat in LAB than in HAB rats, probably due to the more active coping style of the former in response to any kind of stressor
We could not detect any major difference in locomotion of HAB vs. LAB mice in the EPM, indicating that the animals’ anxiety-related phenotype is mainly independent of effects based on differing locomotion. Though, we cannot completely reject a confounding influence of locomotion on behaviour, especially as HAB vs. LAB male mice exhibited nominally significant differences in locomotion in the OF. On the other hand, we could also detect nominally significant differences in exploratory behaviour on the EPF for HAB vs. LAB males, a test much less affected by differences in locomotion.
Linkage studies were applied in rats and mice to a wide variety of complex phenotypes, including alcohol preference, anxiety-related, depression-like and exploratory behaviours or HPA axis function
[37–40]. Based on the study by Williams et al., where a comparable breeding strategy led to the identification of about 300 genotypic markers useful for complex trait analysis
, our mouse population seems to fulfil the requirements for linkage analysis. In addition, loading of the phenotypes of different behavioural tests in F2 on independent PCs underlines the good segregation of characteristic traits from the original HAB and LAB mouse lines.
Anxiety phenotypes of EPM and OF tests mainly loaded on two separate PCs showing limited comparability and predictability of measurements from different anxiety tests in freely segregating F2 animals. This is consistent with findings in other studies
[42, 43] supporting the idea that different paradigms reflect – at least partially – different facets of trait anxiety. Similarly, in the F2 generation, phenotypes representing anxiety, depression and endocrine stress response loaded on separate PCs, reflecting strongly reduced comorbidity of these traits compared to F0 HAB and LAB mice. This resembles findings of another F2 study, where a connection between depression-like behaviour and stress reactivity was rejected
. However, as solely based on low loadings of other behavioural phenotypes in the stress-related phenotypic cluster, a connection between these phenotypes cannot be completely denied, being in line with the idea of anxiety and stress reactivity representing multifactorial and polygenic traits with minor contributions of many genes. It is, therefore, likely that associated phenotypes and analogous comorbidities in the clinical situation such as those between anxiety, depression-like behaviours and HPA axis/stress reactivity share contributing genetic factors that are hardly detectable. This is also underlined by a recent study, proposing a network approach to analyse overlapping symptoms in the concept of comorbidity
As stress CORT and increase in CORT are highly correlated (due to the low levels of basal CORT, resulting in almost near-to-identical values for the two parameters) and placed on the independent PC6 representing the only prominent loadings, both might be considered equally representative of the neuroendocrine system response. Stress CORT is exemplarily shown (Figure
4) for genetic analysis. Identified by sequential linkage analysis of trait cosegregation in a full three-generation pedigree and F2 intercross mapping, a CORT-related QTL was detected on mouse chromosome 3 between 41.3 cM (closest marker: rs13477268 at 40.5 cM) and 43.3 cM (closest marker: rs4138887 at 45.3 cM) with a linkage peak at 42.3 cM. Two-QTL scans showed an evidence for epistasis between the region of the peak location at 42.3 cM and the locus at 35.3 cM (closest marker: rs6376008 at 37.9 cM). The association analysis of freely segregating F2 mice derived from a HAB x LAB cross identified a prominent trait co-occurrence with rs13477268 and two highly correlated nearby markers, rs4138887 and rs6376008. The high correlation of nearby SNPs (rs13477268, rs4138887 and rs6376008) points to the presence of a broad linkage disequilibrium (LD) block, indicating that the identified group of significant markers represents the effect of the true locus underlying the neuroendocrine stress response.
Taken together, our findings highlight the region from 37.9 cM to 45.2 cM of mouse chromosome 3 as an endocrine stress response-specific locus. The additionally identified regions on the X chromosome, only showing nominally significant association with the traits stress CORT and increase in CORT, provide an impetus for the involvement of sex-specific mechanisms in influencing the respective trait, which is supported by other studies
Comparative genome analyses of mouse chromosome 3 showed rat and human homologues on chromosomes 2 (rat) and 1 and 4 (human), respectively
. The markers that were linked to stress CORT and increase in CORT are not located near to any neurotransmitter receptor gene or other candidate genes for HPA axis regulation mapped to date in mice (according to NCBI, Ensembl and UCSC databases).
A particularly interesting candidate gene for the trait of neuroendocrine stress response is Pde4dip, a locus characterised by the maximum likelihood estimate of QTL location and involved in epistasis. The encoded protein, myomegalin, is responsible for cardiac contractility during adrenergic stimulation
. The rat homologues of our identified QTL (Figure
4A) are highly overlapping with previously identified QTLs for cardiovascular disease-related phenotypes. These overlaps include rat QTLs for blood pressure and heart rate
 and bradycardia
. As these rat QTLs also include the genomic locus of Pde5a, which is capable of modulating acute and chronic cardiac stress responses
[50, 51], represented by rs13477379 in our study, it makes this gene an interesting additional candidate, although not included in the region of strongest linkage. Particularly as acute CORT responses show a pronounced effect on the cardiovascular system
, the previously described cardiovascular QTLs in this genomic region are in line with our findings.
The identified QTL is located in a region that also overlaps with a QTL for ethanol-induced CORT responses in mice
 and is homologous to a QTL for fasting cortisol levels in humans
. Both ethanol administration and fasting have been shown to provoke an increase in CORT secretion and glucocorticoid production, similar to acute stress responses
[55, 56]. Finally, the 7-cM region, identified to be linked to stress CORT in the present study, is completely homologous with one of the QTLs (Srcrt-1) Solberg et al. described to influence stress CORT in their rat model
, with the difference that the linkage in our study can be limited to a single, smaller region, which is strongly linked to the stress CORT phenotype. In case of Solberg et al., Srcrt-1 shows the second strongest effect on stress CORT, behind a marker on rat chromosome 6 (homologous to mouse chromosome 12) of five linked genomic loci altogether
The strongly linked 7-cM genomic region on chromosome 3 harbours at least five genes involved in steroid, most prominently glucocorticoid, synthesis (Table
, emphasising a strong effect on the stress CORT phenotype. Although these genes are primarily focussed on androgen, oestrogen and progesterone synthesis (Table
4), their enzyme products are known to play a vital role in the synthesis of all glucocorticoids, among them CORT (KEGG pathway mmu00140;
http://www.genome.jp/kegg/). This strongly supports a conserved genomic and, thereby, heritable effect on stress reactivity. Interestingly, a recent study indicates that corticosteroids can be synthesised endogenously in the brain
. Therefore, the genomic region identified in our study might also affect corticosteroids directly in the brain. The second functionally enriched group (Table
4), albeit showing only nominal significance, is related to cholesterol biosynthesis, which is the first metabolite required for glucocorticoid synthesis
. Another genomic site containing genes coding for glutamate receptors and other transmembrane proteins (NCBI) is related to the locus at 35.3 cM, which is in epistatic interaction with the identified locus at 42.3 cM.
While many mechanisms of genomic and non-genomic regulation remain largely unclear in terms of activation vs. inhibition of the HPA axis
, our results strongly point to a genetic influence of the 7-cM region. The additionally identified regions on the X chromosome, only showing nominally significant linkage to the stress CORT trait, provide an impetus for the involvement of sex-specific mechanisms in influencing the respective trait, which is supported by other studies