ABSTRACT
Genetic heterogeneity underlies many phenotypic variations observed in circadian rhythmicity. Continuous distributions in measures of circadian behavior observed among multiple inbred strains of mice suggest that the inherent contributions to variability are polygenic in nature. To identify genetic loci that underlie this complex behavior, we have carried out a genome-wide complex trait analysis in 196 (C57BL/6J X BALB/cJ)F(2) hybrid mice. We have characterized variation in this panel of F(2) mice among five circadian phenotypes: free-running circadian period, phase angle of entrainment, amplitude of the circadian rhythm, circadian activity level, and dissociation of rhythmicity. Our genetic analyses of these phenotypes have led to the identification of 14 loci having significant effects on this behavior, including significant main effect loci that contribute to three of these phenotypic measures: period, phase, and amplitude. We describe an additional locus detection method, genome-wide genetic interaction analysis, developed to identify locus pairs that may interact epistatically to significantly affect phenotype. Using this analysis, we identified two additional pairs of loci that have significant effects on dissociation and activity level; we also detected interaction effects in loci contributing to differences of period, phase, and amplitude. Although single gene mutations can affect circadian rhythms, the analysis of interstrain variants demonstrates that significant genetic complexity underlies this behavior. Importantly, most of the loci that we have detected by these methods map to locations that differ from the nine known clock genes, indicating the presence of additional clock-relevant genes in the mammalian circadian system. These data demonstrate the analytical value of both genome-wide complex trait and epistatic interaction analyses in further understanding complex phenotypes, and point to promising approaches for genetic analysis of such phenotypes in other mammals, including humans.
Subject(s)
Behavior, Animal , Circadian Rhythm/genetics , Drosophila Proteins , Epistasis, Genetic , Genome , Mice, Inbred BALB C/genetics , Mice, Inbred C57BL/genetics , Photoreceptor Cells, Invertebrate , Animals , Behavior, Animal/physiology , Cell Cycle Proteins , Chromosome Mapping , Crosses, Genetic , Cryptochromes , Eye Proteins/genetics , Female , Flavoproteins/genetics , Fourier Analysis , Genetic Linkage , Genetic Markers , Male , Mice , Nuclear Proteins/genetics , Period Circadian Proteins , Proteins/genetics , Receptors, G-Protein-Coupled , Running , Symbiosis/genetics , Transcription FactorsABSTRACT
The Clock mutation lengthens periodicity and reduces amplitude of circadian rhythms in mice. The effects of Clock are cell intrinsic and can be observed at the level of single neurons in the suprachiasmatic nucleus. To address how cells of contrasting genotype functionally interact in vivo to control circadian behavior, we have analyzed a series of Clock mutant mouse aggregation chimeras. Circadian behavior in Clock/Clock <--> wild-type chimeric individuals was determined by the proportion of mutant versus normal cells. Significantly, a number of intermediate phenotypes, including Clock/+ phenocopies and novel combinations of the parental behavioral characteristics, were seen in balanced chimeras. Multivariate statistical techniques were used to quantitatively analyze relationships among circadian period, amplitude, and suprachiasmatic nucleus composition. Together, our results demonstrate that complex integration of cellular phenotypes determines the generation and expression of coherent circadian rhythms at the organismal level.
Subject(s)
Chimera/genetics , Chimera/physiology , Circadian Rhythm/genetics , Trans-Activators/genetics , Animals , Behavior, Animal/physiology , CLOCK Proteins , Cluster Analysis , Fourier Analysis , Genetic Linkage , Genotype , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Mutant Strains , Multigene Family , Multivariate Analysis , Mutation , Penetrance , Periodicity , Phenotype , Suprachiasmatic Nucleus/metabolism , Trans-Activators/metabolismABSTRACT
The most commonly measured mouse behavior in fear conditioning tests is freezing. A technical limitation, particularly for genetic studies, is the method of direct observation used for quantifying this response, with the potential for bias or inconsistencies. We report the use of a computerized method based on latency between photobeam interruption measures as a reliable scoring criterion in mice. The different computer measures obtained during contextual fear conditioning tests showed high correlations with hand-scored freezing; r values ranged from 0.87 to 0.94. Previously reported strain differences between C57BL/6J and DBA/2J in context-dependent fear conditioning were also detected by the computer-based system. In addition, the use of computer-scored freezing of 199 (BALB/cJ x C57BL/6J)F2 mice enabled us to detect a suggestive gender-dependent chromosomal locus for contextual fear conditioning on distal chromosome 8 by QTL analysis. Automation of freeze scoring would significantly increase efficiency and reliability of this learning and memory test.
