Hibernation with rhythmicity: the circadian clock and hormonal adaptations of the hibernating Asiatic toads (Bufo gargarizans)

Abstract

Hibernation is one of the fundamental strategies in response to cold environmental temperatures. During hibernation, the endocrine and circadian systems ensure minimal expenditure of energy for survival. The circadian rhythms of key hormones, melatonin (MT), corticosterone (CORT), triiodothyronine (T₃ ), and thyroxine (T₄ ), and the underlying molecular regulatory mechanisms of hibernation have been well determined in mammals but not in ectotherms. Here, a terrestrial hibernating species, Asiatic toad (Bufo gargarizans), was employed to investigate the plasma CORT, MT, T₃ , and T₄ ; and the retina, brain, and liver mRNA expression of the core clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle ARNT-like 1 (Bmal1), cryptochrome (Cry) 1 and 2, and period (Per) 1 and 2, at 7-time points over a 24-h period under acute cold (1 day at 4°C), and hibernation (45 days at 4°C). Our results showed that the circadian rhythms of the core clock genes were rather unaffected by acute cold exposure in the retina, unlike the brain and liver. In contrast, during hibernation, the liver clock genes displayed significant circadian oscillations, while those in the retina and brain stopped ticking. Furthermore, plasma CORT expressed circadian oscillations in both groups, and T₃ in acute cold exposure group, whereas T₄ and MT did not. Our results reveal that the plasma CORT and the liver sustain rhythmicity when the brain was not, indicating that the liver clock along with the adrenal clock synergistically maintains the metabolic requirements to ensure basic survival in hibernating Asiatic toads.

Keywords: Asiatic toad; circadian rhythm; clock genes; cold exposure; hibernation; metabolic hormones.