The circadian clock stops ticking during deep hibernation in the European hamster

Abstract

Hibernation is a fascinating, yet enigmatic, physiological phenomenon during which body temperature and metabolism are reduced to save energy. During the harsh season, this strategy allows substantial energy saving by reducing body temperature and metabolism. Accordingly, biological processes are considerably slowed down and reduced to a minimum. However, the persistence of a temperature-compensated, functional biological clock in hibernating mammals has long been debated. Here, we show that the master circadian clock no longer displays 24-h molecular oscillations in hibernating European hamsters. The clock genes Per1, Per2, and Bmal1 and the clock-controlled gene arginine vasopressin were constantly expressed in the suprachiasmatic nucleus during deep torpor, as assessed by radioactive in situ hybridization. Finally, the melatonin rhythm-generating enzyme, arylalkylamine N-acetyltransferase, whose rhythmic expression in the pineal gland is controlled by the master circadian clock, no longer exhibits day/night changes of expression but constantly elevated mRNA levels over 24 h. Overall, these data provide strong evidence that in the European hamster the molecular circadian clock is arrested during hibernation and stops delivering rhythmic output signals.

Fig. 1.

European hamster's hibernation pattern and experimental paradigm. (A) Typical Tb recording (animal B17) illustrating the major phases of the hibernation cycle of the European hamster. Three weeks of recording (November 25 to December 14, 2005) show five hibernation cycles. The ambient temperature (Ta) of the climatic room was set at 6 ± 2°C (dashed line). The horizontal black bar represents the period detailed in B. (B) Focus on a single hibernation cycle (December 6 to December 10, 2005). Entrance into hibernation (Tb < 34°C) occurred during nighttime. (C) Experimental paradigm used for the experiment. For the euthermic group, the hamsters had a Tb close to 37°C and were aroused from hibernation for >48 h. These animals were killed at mid-day (D, 1300; n = 6), mid-night (N, 0100; n = 6), or subjective mid-day (SD, 1300; darkness from the previous day; n = 5). For the hibernating group, only the animals entering torpor (Tb <34°C) between 0000 and 0200 (mid-night) were considered. Their killing occurred 36 h (day; n = 4), 48 h (night; n = 5), or 60 h (subjective day; no lights on for the last day; n = 7) later. Light and dark bars indicate day and night, respectively; dark gray bar, subjective day.

Fig. 2.

The circadian expression of clock genes in the SCN is abolished under deep hibernation. In situ hybridization was used to examine gene expression in euthermic and hibernating hamsters during daytime, nighttime, or during the subjective day. For all genes, the upper panel displays representative autoradiograms for each condition, and the lower panel shows the relative mRNA levels. (A–C) The expression of the clock genes Per1 (A), Per2 (B), and Bmal1 (C) exhibits circadian fluctuations in euthermic hamsters but not in hibernating animals. During hibernation, Per1 expression is elevated (A), Per2 expression remains low (B), and Bmal1 expression is intermediate between daytime and nighttime levels in euthermic hamsters (C). (D) Similarly, the expression of the clock-controlled gene Avp is rhythmic in euthermic but not in hibernating hamsters, in which its expression remains low. (E) The expression of the immediate early gene c-fos is dramatically increased in hibernating relative to euthermic hamsters, without significant day/night fluctuations. Data are shown as a percentage of the maximum and represent the means ± SEM (n = 4–7). Bars labeled with different characters (a, b, c) are significantly different from each other (P < 0.05).

Fig. 3.

Aa-nat is constitutively expressed in the pineal gland of hibernating hamsters. (A) Representative autoradiograms and quantification of Aa-nat mRNA demonstrate marked day/night changes of expression in euthermic animals, in contrast to hibernating hamsters, which display persistently high expression level. Data are shown as percentage of the maximum and represent the means ± SEM (n = 4–7). (B) Plasma melatonin concentration displays the expected large day/night difference in euthermic animals but not in hibernating hamsters, which exhibit constantly low levels. Data are means ± SEM (n = 4–7). ***, P < 0.001.