Effects of environmental and physiological covariates on sex differences in unconditioned and conditioned anxiety and fear in a large sample of genetically heterogeneous (N/Nih-HS) rats.

Physiological and environmental variables, or covariates, can account for an important portion of the variability observed in behavioural/physiological results from different laboratories even when using the same type of animals and phenotyping procedures. We present the results of a behavioural study with a sample of 1456 genetically heterogeneous N/Nih-HS rats, including males and females, which are part of a larger genome-wide fine-mapping QTL (Quantitative Trait Loci) study. N/Nih-HS rats have been derived from 8 inbred strains and provide very small distance between genetic recombinations, which makes them a unique tool for fine-mapping QTL studies. The behavioural test battery comprised the elevated zero-maze test for anxiety, novel-cage (open-field like) activity, two-way active avoidance acquisition (related to conditioned anxiety) and context-conditioned freezing (i.e. classically conditioned fear). Using factorial analyses of variance (ANOVAs) we aimed to analyse sex differences in anxiety and fear in this N/Nih-HS rat sample, as well as to assess the effects of (and interactions with) other independent factors, such as batch, season, coat colour and experimenter. Body weight was taken as a quantitative covariate and analysed by covariance analysis (ANCOVA). Obliquely-rotated factor analyses were also performed separately for each sex, in order to evaluate associations among the most relevant variables from each behavioural test and the common dimensions (i.e. factors) underlying the different behavioural responses. ANOVA analyses showed a consistent pattern of sex effects, with females showing less signs of anxiety and fear than males across all tests. There were also significant main effects of batch, season, colour and experimenter on almost all behavioural variables, as well as "sex × batch", "sex × season" and "sex × experimenter" interactions. Body weight showed significant effects in the ANCOVAs of most behavioural measures, but sex effects were still present in spite of (and after controlling for) these "body weight" effects. Factor analyses of relevant variables from each test showed a two-fold factor structure in both sexes, with the first factor mainly representing anxiety and conditioned fear in males, while in females the first factor was dominated by loadings of activity measures. Thus, besides showing consistent sex differences in anxiety-, fear- and activity-related responses in N/Nih-HS rats, the present study shows that females' behaviour is predominantly influenced by activity while males are more influenced by anxiety. Moreover, the results point out that, besides "sex" effects, physiological variables such as colour and body weight, and environmental factors as batch/season or "experimenter", have to be taken into account in both behavioural and quantitative genetic studies because of their demonstrated influences on phenotypic outcomes.

Fine mapping of bone structure and strength QTLs in heterogeneous stock rat.

We previously demonstrated that skeletal structure and strength phenotypes vary considerably in heterogeneous stock (HS) rats. These phenotypes were found to be strongly heritable, suggesting that the HS rat model represents a unique genetic resource for dissecting the complex genetic etiology underlying bone fragility. The purpose of this study was to identify and localize genes associated with bone structure and strength phenotypes using 1524 adult male and female HS rats between 17 to 20 weeks of age. Structure measures included femur length, neck width, head width; femur and lumbar spine (L3-5) areas obtained by DXA; and cross-sectional areas (CSA) at the midshaft, distal femur and femoral neck, and the 5th lumbar vertebra measured by CT. In addition, measures of strength of the whole femur and femoral neck were obtained. Approximately 70,000 polymorphic SNPs distributed throughout the rat genome were selected for genotyping, with a mean linkage disequilibrium coefficient between neighboring SNPs of 0.95. Haplotypes were estimated across the entire genome for each rat using a multipoint haplotype reconstruction method, which calculates the probability of descent at each locus from each of the 8 HS founder strains. The haplotypes were then tested for association with each structure and strength phenotype via a mixed model with covariate adjustment. We identified quantitative trait loci (QTLs) for structure phenotypes on chromosomes 3, 8, 10, 12, 17 and 20, and QTLs for strength phenotypes on chromosomes 5, 10 and 11 that met a conservative genome-wide empiric significance threshold (FDR=5%; P<3×10(-6)). Importantly, most QTLs were localized to very narrow genomic regions (as small as 0.3 Mb and up to 3 Mb), each harboring a small set of candidate genes, both novel and previously shown to have roles in skeletal development and homeostasis.

Gene expression in hippocampus as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in the development/expression of anxiety/fear, we analyzed the gene expression profile in the hippocampus of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock is a unique genetic resource for the fine mapping of quantitative trait loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety, fearfulness or other complex traits. We selected high- and low-anxious NIH-HS rats according to the number of avoidance responses they performed in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety/fearfulness tests, i.e. the elevated zero-maze and a "novel-cage activity" test. Three weeks after behavioral testing, the hippocampus was dissected and prepared for the microarray study. There appeared 29 down-regulated and 37 up-regulated SNC-related genes (fold-change>|2.19|, FDR<0.05) in the "Low-anxious" vs. the "High-anxious" group. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, nine relevant genes (Avpr1b, Accn3, Cd74, Ltb, Nrg2, Oprdl1, Slc10a4, Slc5a7 and RT1-EC12), tested for validation through qRT-PCR, have either neuroendocrinological or neuroinmunological/inflammation-related functions, or have been related with the hippocampal cholinergic system, while some of them have also been involved in the modulation of anxiety or stress-related (neurobiological and behavioral) responses (i.e. Avpr1b, Oprdl1). The present work confirms the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis or mechanisms involved in anxiety and/or fear, and suggest that some MHC-(neuroinmunological/inflammation)-related pathways, as well as the cholinergic system within the hippocampus, may play a role in shaping individual differences in trait anxiety.

Gene expression in amygdala as a function of differential trait anxiety levels in genetically heterogeneous NIH-HS rats.

To identify genes involved in anxiety/fear traits, we analyzed the gene expression profile in the amygdala of genetically heterogeneous NIH-HS rats. The NIH-HS rat stock has revealed to be a unique genetic resource for the fine mapping of Quantitative Trait Loci (QTLs) to very small genomic regions, due to the high amount of genetic recombinants accumulated along more than 50 breeding generations, and for the same reason it can be expected that those genetically heterogeneous rats should be especially useful for studying differential gene expression as a function of anxiety-(or other)-related traits. We selected high- and low-anxious NIH-HS rats differing in their number of avoidances in a single 50-trial session of the two-way active avoidance task. Rats were also tested in unconditioned anxiety tests (e.g., elevated zero-maze). Three weeks after behavioural testing, the amygdala was dissected and prepared for the microarray study. There appeared 6 significantly down-regulated and 28 up-regulated genes (fold-change >|2|, FDR<0.05) between the low- and high-anxious groups, with central nervous system-related functions. Regression analyses (stepwise) revealed that differential expression of some genes could be predictive of anxiety/fear responses. Among those genes for which the present results suggest a link with individual differences in trait anxiety, six relevant genes were examined with qRT-PCR, four of which (Ucn3, Tacr3, H2-M9 and Arr3) were validated. Remarkably, some of them are characterized by sharing known functions related with hormonal HPA-axis responses to (and/or modulation of) stress, anxiety or fear, and putative involvement in related neurobehavioural functions. The results confirm the usefulness of NIH-HS rats as a good animal model for research on the neurogenetic basis of anxiety and fear, while suggesting the involvement of some neuropeptide/neuroendocrine pathways on the development of differential anxiety profiles.

Combined sequence-based and genetic mapping analysis of complex traits in outbred rats.

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Coping style and stress hormone responses in genetically heterogeneous rats: comparison with the Roman rat strains.

The purpose of the present study was to evaluate for the first time the stress-induced hypothalamus-pituitary-adrenal (HPA), adrenocorticotropic hormone (ACTH), corticosterone and prolactin responses of the National Institutes of Health genetically heterogeneous rat stock (N/Nih-HS rats) in comparison with responses of the relatively high and low stress-prone Roman Low- (RLA-I) and High-Avoidance (RHA-I) rat strains. The same rats were also compared (experiment 1) with respect to their levels of unconditioned anxiety (elevated zero-maze test), novelty-induced exploratory behavior, conditioned fear and two-way active avoidance acquisition. In experiment 2, naive rats from these three strains/stocks were evaluated for "depressive-like" behavior in the forced swimming test. N/Nih-HS and RLA-I rats showed significantly higher post-stress ACTH, corticosterone and prolactin levels than RHA-I rats. N/Nih-HS rats also presented the highest context-conditioned freezing responses, extremely poor two-way avoidance acquisition and very low novelty-induced exploratory behavior. Experiment 2 showed that, compared to RHA-I rats, N/Nih-HS and RLA-I rats displayed significantly less struggling (escape-directed) and increased immobility responses in the forced swimming test. Factor analysis of data from experiment 1 showed associations among behavioral and hormonal responses, with a first factor comprising high loadings of elevated zero-maze variables and lower loadings of conditioned fear, two-way avoidance acquisition and hormonal measures, while a second factor mainly grouped conditioned fear and two-way avoidance acquisition with novelty-induced exploration and post-stress prolactin. Thus, regarding their anxiety/fearfulness, passive coping style, "depressive-like" and stress-induced hormonal responses the N/Nih-HS rats resemble the phenotype profiles of the relatively high-anxious and stress-prone RLA-I rat strain.