Age-Related Changes in the Circadian System Unmasked by Constant Conditions

Abstract

Circadian timing systems, like most physiological processes, cannot escape the effects of aging. With age, humans experience decreased duration and quality of sleep. Aged mice exhibit decreased amplitude and increased fragmentation of the activity rhythm, and lengthened circadian free-running period in both light-dark (LD) and constant dark (DD) conditions. Several studies have shown that aging impacts neural activity rhythms in the central circadian clock in the suprachiasmatic nucleus (SCN). However, evidence for age-related disruption of circadian oscillations of clock genes in the SCN has been equivocal. We hypothesized that daily exposure to LD cycles masks the full impact of aging on molecular rhythms in the SCN. We performed ex vivo bioluminescent imaging of cultured SCN slices of young and aged PER2::luciferase knock-in (PER2::LUC) mice housed under LD or prolonged DD conditions. Under LD conditions, the amplitude of PER2::LUC rhythms differed only slightly between SCN explants from young and aged animals; under DD conditions, the PER2::LUC rhythms of aged animals showed markedly lower amplitudes and longer circadian periods than those of young animals. Recordings of PER2::LUC rhythms in individual SCN cells using an electron multiplying charge-coupled device camera revealed that aged SCN cells showed longer circadian periods and that the rhythms of individual cells rapidly became desynchronized. These data suggest that aging degrades the SCN circadian ensemble, but that recurrent LD cycles mask these effects. We propose that these changes reflect a decline in pacemaker robustness that could increase vulnerability to environmental challenges, and partly explain age-related sleep and circadian disturbances.

Keywords: PER2::luciferase; aging; imaging; suprachiasmatic nucleus.

Figure 1.

Effects of aging on PER2::LUC rhythms in SCN explants from mice maintained under LD cycles. A, Representative double-plotted actograms of wheel-running activity in young and aged PER2::LUC mice maintained under LD cycles. Lighting conditions are indicated at the top of the figure; open bars indicate light phases, and closed bars indicate dark phases. Asterisks indicate the timing of the killing of the animals. B, Typical examples of the PER2::LUC rhythm of SCN explants from mice maintained under LD cycles, as measured by PMT. C, Phase map of peak PER2::LUC rhythms in the SCN. Each point represents the average peak of PER2::LUC rhythms in each cycle, plotted relative to the LD cycle prior to the killing of the animals. D, Amplitude of PER2::LUC rhythms in the SCN. The amplitude of each oscillation was determined as the sum of the absolute values of the lowest and highest points (counts). Data are shown as the mean ± SD. n = 5 per group.

Figure 2.

Effects of aging on PER2::LUC rhythms in SCN explants from mice maintained in DD. A, Representative double-plotted actograms of wheel-running activity in young and aged PER2::LUC mice maintained in DD. Asterisks indicate the timing of the killing of the animals. B, Typical examples of PER2::LUC rhythms of SCN explants from mice maintained in DD for 10 d, as measured by PMT. C, Phase map of peak PER2::LUC rhythms in the SCN. Each point represents the average peak of PER2::LUC rhythms in each cycle, plotted relative to the circadian time prior to the killing of the animals. D, Amplitude of PER2::LUC rhythms in the SCN. The amplitude of each oscillation was determined as the sum of the absolute value of the lowest and highest points (counts). Data are shown as the mean ± SD. n = 5 per group. *p < 0.05, **p < 0.01 for young vs. aged animals (Bonferroni post hoc comparison).

Figure 3.

Effects of aging on PER2::LUC rhythms in SCN explants from mice maintained in DD. A, Representative images of PER2::LUC in SCN explants collected from young and aged mice maintained in DD for 10 d, recorded by an EM-CCD camera at 30, 42, 108, and 120 h after the start of recording. B, Representative graphical (top) and raster (below) plots of PER2::LUC rhythms of individual cells in SCN explants of young and aged SCNs. C, D, Phase spreads (C) and normalized amplitudes (D) of PER2::LUC rhythms averaged over young (red) and aged (green) SCNs are shown over five cycles. E, Ratios of free-running circadian period change from the first to the fifth cycle of PER2::LUC rhythms in 50 individual SCN cells are shown. Data are shown as the mean ± SD. n = 6 per group. *p < 0.05 (t test). Scale bar, 100 μm.