A novel quantitative trait locus on mouse chromosome 18, "era1," modifies the entrainment of circadian rhythms

Abstract

Study objective: The mammalian circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus conveys 24-h rhythmicity to sleep-wake cycles, locomotor activity, and other behavioral and physiological processes. The timing of rhythms relative to the light/dark (LD12:12) cycle is influenced in part by the endogenous circadian period and the time of day specific sensitivity of the clock to light. We now describe a novel circadian rhythm phenotype, and a locus influencing that phenotype, in a segregating population of mice.

Methods: By crossbreeding 2 genetically distinct nocturnal strains of mice (Cast/Ei and C57BL/6J) and backcrossing the resulting progeny to Cast/Ei, we have produced a novel circadian phenotype, called early runner mice.

Results: Early runner mice entrain to a light/dark cycle at an advanced phase, up to 9 hours before dark onset. This phenotype is not significantly correlated with circadian period in constant darkness and is not associated with disruption of molecular circadian rhythms in the SCN, as assessed by analysis of period gene expression. We have identified a genomic region that regulates this phenotype-a major quantitative trait locus on chromosome 18 (near D18Mit184) that we have named era1 for Early Runner Activity locus one. Phase delays caused by light exposure early in the subjective night were of smaller magnitude in backcross offspring that were homozygous Cast/Ei at D18Mit184 than in those that were heterozygous at this locus.

Conclusion: Genetic variability in the circadian response to light may, in part, explain the variance in phase angle of entrainment in this segregating mouse population.

Figure 1

Daily rhythms of wheel running in individual mice representing CE (A), B6 (B), F1 (C) and BX (D-E) mouse cohorts. Mice were kept in LD12:12 followed by DD. Time spent in darkness is indicated by a gray background. The timing of the LD12:12 cycle is further indicated by the white and black bars at the top of panel A.

Figure 2

Variability in the timing of wheel running activity onset in BX mice under LD12:12. Daily wheel running profiles from CE × (CE × B6) backcross mice and inbred parental (CE and B6) strains. A-I: Seven-day wheel running records from individual BX offspring (A-G) that varied with respect to the timing of wheel onset relative to the LD12:12 cycle (indicated by white and black bars at top) and from CE (H) and B6 (I) mice. A'-I': Wheel running profiles in hourly bins (group mean ± SEM) from backcross offspring exhibiting wheel running more than 5 (A'), 5 (B'), 4 (C'), 3 (D'), 2 (E'), 1 (F') h before lights-off, or at lights-off (G'), and from CE (H') and B6 (I') mice. Sample sizes are shown in each panel. J: A histogram showing the distribution of wheel running onset times relative to lights-off in the BX population.

Figure 3

Per1 and 2 gene expression in the SCN and cerebral cortex of BX mice. Time of day dependence of relative per1 (A) and per2 (B) gene expression in anteroventral hypothalamus tissue punches from early runner BX (black bars) and control (gray bars) mice. Inset, Per1 gene expression relative to β-actin expression was significantly higher in anteroventral hypothalamus tissue punches from B6 mice exposed to light in the subjective night (ZT13-14; labeled ‘L’) than in time of day controls (labeled ‘D’; *P < 0.001 vs. control, Student's T).

Figure 4

A graph of the genome-wide scan for a QTL. The dashed lines represent the scores for a Haley-Knott regression and the solid lines represent scores based on maximum likelihood (EM algorithm). Note the high LOD score (3.9) for chromosome 18.

Figure 5

Chromosome 18 QTL: effects on phase angle of entrainment and circadian period in DD. A, phase angle of entrainment, as assessed by extrapolation of DD wheel running data. B, circadian period in DD. Data are from mice that were homozygous CE (n=56; black bars) or heterozygous CE/B6 (n=33; gray bars) at D18Mit184.

Figure 6

Chromosome 18 QTL: representative phase shifts caused by 3-hr light exposure in constant dark. Mice were subjected to 3-h light pulses (50 lux) at CT4, CT12 and CT18. Data are plotted in modulo-tau format, such that each horizontal bin contains the data from two double-plotted circadian cycles. Pulses occurred during the tenth circadian cycle shown. Mice on the left were homozygous CE at D18Mit184, while those on the right were heterozygous at D18Mit184.

Figure 7

Chromosome 18 QTL: effects on the phase shifting effect of light exposure in DD. Upper panel, entire data set of individual phase shifts caused by 3-h light exposure in constant dark in mice genotyped as homozygous CE (black circles) or heterozygous CE/B6 (gray circles) at D18Mit184. Lower panel, group mean and SEM phase shifts caused by 3-h light exposure in constant dark in mice genotyped as homozygous CE (black bars) or heterozygous CE/B6 (gray bars) at D18Mit184. Data points were merged into 3-h circadian bins for statistical analysis. * P < 0.05 vs. CE/B6, same time point.