Cry1-/- circadian rhythmicity depends on SCN intercellular coupling

Abstract

In mammals, the suprachiasmatic nucleus (SCN) is the central pacemaker organizing circadian rhythms of behavior and physiology. At the cellular level, the mammalian clock consists of autoregulatory feedback loops involving a set of "clock genes," including the Cryptochrome (Cry) genes, Cry1 and Cry2. Experimental evidence suggests that Cry1 and Cry2 play distinct roles in circadian clock function. In mice, Cry1 is required for sustained circadian rhythms in dissociated SCN neurons or fibroblasts but not in organotypic SCN slices or at the behavioral level, whereas Cry2 is not required at any of these levels. It has been argued that coupling among SCN cellular oscillators compensates for clock gene defects to preserve oscillatory function. Here we test this hypothesis in Cry1(-/-) mice by first disrupting intercellular coupling in vivo using constant light (resulting in behavioral arrhythmicity) and then examining circadian clock gene expression in SCN slices at the single cell level. In this manner, we were able to test the role of intercellular coupling without drugs and while preserving tissue organization, avoiding the confounding influences of more invasive manipulations. Cry1(-/-) mice (as well as control Cry2(-/-) mice) bearing the PER2::LUC knock-in reporter were transferred from a standard light:dark cycle to constant bright light (~650 lux) to induce arrhythmic locomotor patterns. In SCN slices from these animals, we used bioluminescence imaging to monitor PER2::LUC expression in single cells. We show that SCN slices from rhythmic Cry1(-/-) and Cry2(-/-) mice had similarly high percentages of functional single-cell oscillators. In contrast, SCN slices from arrhythmic Cry1(-/-) mice had significantly fewer rhythmic cells than SCN slices from arrhythmic Cry2(-/-) mice. Thus, constant light in vivo disrupted intercellular SCN coupling to reveal a cell-autonomous circadian defect in Cry1(-/-) cells that is normally compensated by intercellular coupling in vivo.

Figure 1

Effects of LL on locomotor behavior of Cry-deficient mice. A. Representative double plotted wheel-running actograms of Cry1−/− and Cry2−/− mice that were transferred to LL on the first day of the record. The majority of Cry-deficient mice quickly became arrhythmic after transfer to LL, but a minority of animals retained circadian rhythms for the first few weeks under LL. B. Proportion of Cry1−/− and Cry2−/− animals that developed arrhythmic activity patterns within seven weeks under LL (left) and latency to develop arrhythmia (right). Numbers within columns represent total sample sizes for animals held under LL for at least seven weeks and used for behavioral analyses. R: rhythmic mice, AR: arrhythmic mice.

Figure 2

Effects of genotype and LL-induced behavioral arrhythmicity on PER2::LUC rhythms of SCN slices in vitro illustrated by baseline-subtracted time series. A–B. PER2::LUC rhythms of SCN slices from rhythmic Cry1−/− (n = 5) and Cry2−/− (n = 3) mice were significantly clustered according to circadian time (A) but not dissection time (B). Rayleigh plots depict the phase distribution of peak PER2::LUC on the first cycle in vitro, plotted in angular degrees. Lines represent mean angular vectors and those extending outside the inner circle indicate significant clustering for that group as determined by the Rayleigh test. C. PER2::LUC rhythms of SCN slices from arrhythmic Cry1−/− (n = 7) and Cry2−/− (n = 5) mice were clustered according to dissection time and genotype.

Figure 3

Reduced PER2::LUC rhythmicity of single cells in SCN slices from behaviorally arrhythmic Cry1−/− mice. A. Representative background-subtracted time series of PER2::LUC rhythms displayed by SCN neurons from rhythmic and arrhythmic Cry1−/− and Cry2 −/− mice (five neurons per panel illustrated with different colors). More examples are shown in Figures S1–S4. B. SCN slices from arrhythmic Cry1−/ − mice had fewer rhythmic neurons relative to slices from rhythmic Cry1−/− mice, arrhythmic Cry2−/− mice, or rhythmic Cry2−/− mice. C. The goodness-of-fit (percentage of total variance in the PER2::LUC trace accounted for by a best fit sine wave) was reduced in SCN cells from Cry1−/− mice, and was lowest in Cry1−/− cells from arrhythmic mice. Average number of cells extracted per slice: 40 cells. * p < 0.05. * Tukey’s HSD, p < 0.05.

Figure 4

Viability of Cry1−/− SCN slices from arrhythmic mice. A. SCN slices from Cry1−/− animals were at least as bright as Cry2−/− slices (right). * Different from all other groups, Tukey’s HSD, p < 0.05. B. Example of a SCN slice from an arrhythmic Cry1−/ − mouse that initially displayed very low amplitude rhythms, but then high amplitude and sustained rhythms after two medium changes.