Difficulties in fine-mapping quantitative trait loci (QTLs) are a major impediment to progress in the molecular dissection of complex traits in mice. Here we show that genome-wide high-resolution mapping of multiple phenotypes can be achieved using a stock of genetically heterogeneous mice. We developed a conservative and robust bootstrap analysis to map 843 QTLs with an average 95% confidence interval of 2.8 Mb. The QTLs contribute to variation in 97 traits, including models of human disease (asthma, type 2 diabetes mellitus, obesity and anxiety) as well as immunological, biochemical and hematological phenotypes. The genetic architecture of almost all phenotypes was complex, with many loci each contributing a small proportion to the total variance. Our data set, freely available at http://gscan.well.ox.ac.uk, provides an entry point to the functional characterization of genes involved in many complex traits.

The interaction between genotype and environment is recognized as an important source of experimental variation when complex traits are measured in the mouse, but the magnitude of that interaction has not often been measured. From a study of 2448 genetically heterogeneous mice, we report the heritability of 88 complex traits that include models of human disease (asthma, type 2 diabetes mellitus, obesity, and anxiety) as well as immunological, biochemical, and hematological phenotypes. We show that environmental and physiological covariates are involved in an unexpectedly large number of significant interactions with genetic background. The 15 covariates we examined have a significant effect on behavioral and physiological tests, although they rarely explain .10% of the variation. We found that interaction effects are more frequent and larger than the main effects: half of the interactions explained .20% of the variance and in nine cases exceeded 50%. Our results indicate that assays of gene function using mouse models should take into account interactions between gene and environment.

The Wistar-Kyoto (WKY) rat exhibits physiological and behavioral similarities to endophenotypes of human depression. In the forced swim test (FST), a well-characterized antidepressant-reversible test for behavioral despair in rodents, WKYs express characteristics of behavioral despair; increased immobility, and decreased climbing. To map genetic loci linked to behavior in the FST, we conducted a quantitative trait loci (QTL) analysis of the segregating F2 generation of a WKY x Fisher 344 (F344) reciprocal intercross. Using linear-model-based genome scans to include covariate (sex or lineage)-by-QTL interaction effects, four significant QTL influencing climbing behavior were identified. In addition, we identified three, seven, and two suggestive QTL for climbing, immobility, and swimming, respectively. One of these loci was pleiotropic, affecting both immobility and climbing. As found in human linkage studies, several of these QTL showed sex- and/or lineage-dependent effects. A simultaneous search strategy identified three epistatic locus pairs for climbing. Multiple regression analysis was employed to characterize the joint contributions of these QTL and to clarify the sex- and lineage-dependent effects. As expected for complex traits, FST behavior is influenced by multiple QTL of small effect, each contributing 5%-10%, accounting for a total 10%-30% of the phenotypic variance. A number of loci mapped in this study share overlapping candidate regions with previously identified emotionality QTL in mice as well as with susceptibility loci recognized by linkage or genome scan analyses for major depression or bipolar disorder in humans. The presence of these loci across species suggests that these QTL may represent universal genetic factors contributing to mood disorders.

The Wistar Kyoto (WKY) rat is hyperreactive to stress and exhibits depressive-like behavior in several standard behavioral tests. Because patients with depressive disorders often exhibit disruptions in the circadian rhythm of activity, as well as altered secretory patterns of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid hormones, we tested the hypothesis that these phenomena occur in the WKY rat. Plasma ACTH and corticosterone levels remained significantly higher after the diurnal peak for several hours in WKY rats relative to Wistar rats. Also, plasma levels of thyroid-stimulating hormone were significantly higher in WKY relative to Wistar rats across the 24-h period, despite normal or slightly higher levels of 3,5,3'-triiodothyronine. In addition, under constant darkness conditions, WKY rats exhibited a shorter free running period and a decreased response to a phase-delaying light pulse compared with Wistar rats. In several ways these results are similar to those seen in other animal models of depression as well as in depressed humans, suggesting that the WKY rat could be used to investigate the genetic basis for these abnormalities.

There is a clear link between altered circadian rhythms and depressive disorders, although the nature of this relationship is unknown. In addition, exercise affects both mood and alters clock function. To investigate the relationship between circadian rhythms, depression, and exercise, 3-wk-old mice housed on a 12:12-h light-dark cycle were exposed to chronic stress (CS) for 6 wk before being placed into constant darkness (DD). One-half of both the control and stressed mice were given access to a running wheel. Stressed mice consumed significantly less of a 2% sucrose solution during CS and exhibited a significant increase in immobility in the forced swim test 3 wk after the termination of stress relative to control mice. These effects were more pronounced in mice without running wheels. Stressed mice also exhibited altered percent distribution of total activity and increased fragmentation of daily activity rhythms during CS relative to control mice. Alterations in percent distribution were more pronounced in animals without running wheels. No activity rhythm changes were seen in DD, and there were no differences in light-induced phase shifts between stressed and control mice. These results suggest that CS causes long-term depressive-like symptoms but does not have long-lasting effects on activity rhythms. These changes were more pronounced in mice without running wheels, suggesting that exercise may protect against the harmful effects of stress.

Phase-shifting effects of timed calorie restriction were investigated in mice during exposure to a 12:12-h light-dark cycle. Food-anticipatory activity (FAA), the output of a food-entrainable pacemaker, was expressed before the time of feeding whether mice received daily hypocaloric food (3.3 g of chow/day) or normocaloric food (5 g of chow/day) at zeitgeber time (ZT) 2 (ZT12 = lights off). Subsequently, mice were placed in constant darkness and fed ad libitum. The onset of the nocturnal period of locomotor activity was phase advanced by 1 h in calorie-restricted mice compared with normocalorie-fed controls. The phase advance still occurred when FAA was prevented by restraining calorie-restricted mice. Giving hypocaloric food at ZT2, ZT10, ZT14, or ZT22 phase advanced the nocturnal pattern of activity by 1, 3, 1, and 1 h, respectively. After transfer to constant darkness, FAA free ran in parallel with the normal nocturnal period of locomotor activity. A light pulse during the early subjective night phase delayed both components. These results indicate that 1) timed calorie restriction under a light-dark cycle can phase advance the light-entrainable pacemaker with a phase-dependent magnitude, 2) FAA feedback is not crucial for the observed phase advance, and 3) the light-entrainable pacemaker may control the period of the food-entrainable pacemaker in mice fed ad libitum.

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.

The Wistar-Kyoto (WKY) rat exhibits several behavioral and hormonal abnormalities often associated with depression. One of the hallmarks of depression consists of alterations in the sleep-wake cycle, particularly in rapid eye movement (REM) sleep. If the WKY rat is indeed an animal model for depression, we hypothesized that it should also show sleep abnormalities relative to the control strain, the Wistar (WIS) rat Under baseline conditions, WKY rats showed a 50% increase in total REM sleep time during the 12 h light phase and an increase in sleep fragmentation during both the light and dark phase. The WKY rats also exhibited lower EEG power densities over the entire frequency range (0.2-25.0 Hz) during REM sleep. After a 6 h sleep deprivation, the REM sleep rebound was more pronounced during the dark but not the light phase in the WKY rats. Since the WKY rat represents a genetic model for depression with altered EEG sleep patterns, this strain may be particularly useful for investigating the relationship between depression and sleep abnormalities.