Effects of aging on central and peripheral mammalian clocks

Abstract

Circadian organization changes with age, but we do not know the extent to which age-related changes are the result of alterations in the central pacemakers, the peripheral oscillators, or the coupling mechanisms that hold the system together. By using transgenic rats with a luciferase (luc) reporter, we assessed the effects of aging on the rhythm of expression of the Period 1 (Per1) gene in the suprachiasmatic nucleus (SCN) and in peripheral tissues. Young (2 months) and aged (24-26 months) Per1-luc transgenic rats, entrained to light-dark cycles, were killed, and tissues were removed and cultured. Per1-luc expression was measured from 10 tissues. In the SCN, the central mammalian pacemaker, Per1-luc expression was robustly rhythmic for more than 7 weeks in culture. The only difference between SCN rhythmicity in young and old rats was a small but significant age-related shortening of the free-running period. Circadian rhythmicity in some peripheral tissues was unaffected by aging, whereas rhythmicity in other tissues was either phase advanced relative to the light cycle or absent. Those tissues that were arrhythmic could be induced to oscillate by application of forskolin, suggesting that they retained the capacity to oscillate but were not being appropriately driven in vivo. Overall, the results provide new insights into the effects of aging on the mammalian circadian system. Aging seems to affect rhythms in some but not in all tissues and may act primarily on interactions among circadian oscillators, perhaps attenuating the ability of the SCN to drive damped oscillators in the periphery.

Fig 1.

SCN from aged rats display robust circadian oscillations in vitro. Representative circadian rhythms of Per1-luc luminescence from cultured SCN explanted from young (A) and aged (B) rats. The tissue was explanted just before lights-off (arrows). Light output (in counts/min) is plotted against previous light onset (hour 0). Four SCN from aged rats were maintained in vitro for 40 days before recording (two examples are shown in C and D). Circadian rhythms persisted in all four explants, and as expected, the explants were out of phase with one another after 40 days in culture.

Fig 2.

Circadian rhythms of cultured cornea, pituitary, kidney, pineal, and PVN. Data were plotted as in Fig. 1 (A and B). Aging did not alter the robustness of circadian oscillations in cornea and pituitary. Shown are representative recordings from cornea of young (A) and aged (B) rats and pituitary from young (C) and aged (D) rats. Aging did alter the phase of kidney, pineal, and PVN. Shown are examples of circadian rhythms from kidney of young (E) and old (F), pineal from young (G) and old (H), and PVN from young (I) and old (J) animals. There was no difference in the robustness of these oscillations between young and old animals. Peak Per-luc activity in kidney and pineal cultures from aged rats were ≈4-h phase advanced compared with tissue from young rats. A sharp peak at the beginning of subjective day (J) was observed in most PVN from aged rats but not from young rats (see Results and Fig. 5 for details).

Fig 3.

Aging disrupted the circadian rhythms in RCA and lung. Luminescence of cultured RCA from young (A) and old (B) and from lung of young (D) and old (E) animals are plotted against previous light onset (hour 0). RCA and lung from young rats showed clear circadian oscillations, but RCA and lung from old rats showed weak or no circadian oscillation. Circadian oscillation in old tissue could be induced by forskolin stimulation (arrows) in previously arrhythmic RCA (C) and lung (F).

Fig 4.

Aging shortened the circadian free-running period of the SCN but not of the pituitary or cornea. Mean period (± SEM) of SCN, pituitary, and cornea are shown. *, Statistically significant (P < 0.001, t test). These periods were based on the first 6–7 days of culture. The sample size is shown in Fig. 5.

Fig 5.

Phase map for central and peripheral circadian oscillators. The peak of the circadian oscillation was determined during the interval between 12 h and 36 h in culture. The average times (± SEM) of peaks were plotted against the time of last lights-on. •, data from young rats; ○, data from aged rats. The light–dark cycle to which the animals were exposed before killing (black and white bar) is shown at Top. (Right) The sample size is shown (number of rhythmic tissues/number of tissues tested). *, Statistically significant (P < 0.01, t test); **, the pituitary in two aged rats had tumors and consequently were excluded from the analysis. Arc, arcuate nucleus.