Identification of Common and Separate Mechanisms Governing Circadian Locomotor Activity and Body Temperature

概要

Almost all organisms, including humans, display daily rhythms of behaviour and physiology generated by an endogenous mechanism called circadian clock. In mammals, these rhythms are established by transcription/translation-based autoregulatory feedback loops, in which non-coding cis-regulatory elements on the promoter of the clock genes are believed to play a key role in generating circadian transcription. Genetic evidence supporting this model, however, is still exclusively based on the effects of mutations in the protein-coding sequences of the clock genes. Therefore, while non-coding circadian cis-elements are assumed to be important for daily maintenance of behaviour and physiology, there is currently no direct evidence to corroborate this notion.

Because locomotor activity rhythms (LAR) and body temperature rhythms (BTR) are both robust and easy to be measured, they have been used to detect circadian rhythms in living animals. Interestingly, BTR is separately regulated from LAR, and BTR has essential roles in maintaining circadian energy homeostasis and entrainment of peripheral tissue clocks. A previous study in which subsets of neurons in the brain of rats were ablated suggests that LAR and BTR are controlled by different output pathways that originate from the suprachiasmatic nucleus (SCN). However, molecules involved in regulating BTR have not been identified so far.

Methodologically, there are several critical problems in measurements of BTR. In many studies, BTR measurements were performed by implanting a thermal sensor into abdominal cavity of animals. This requires surgery. Moreover, a sensor device is often large for small laboratory animals such as mice, making these experiments highly invasive. In addition, long-term measurement of BTR with high time-resolution over several days is difficult due to limited data storage performance of thermal sensor devices. LAR is usually detected by infrared sensor, but this conventional method also has several limitations. Firstly, it is difficult to accurately quantify locomotor activities because the sensitivity differs between infrared sensor devices, and the sensitivity changes gradually over time due to mechanical degradation. Secondly, mice often show non-locomotor activities such as feeding, grooming, and postural adjustments, but these subtle changes in activity cannot be distinguished via a conventional infrared sensor. These limitations have hampered elucidation of whether and how BTR and LAR are temporally related to each other.

In Chapter 1, using mice with a point mutation of the cis-regulatory element E′-box in the promotor of Period2 (Per2), I showed that circadian transcription of Per2 and other clock genes was drastically attenuated in cells extracted from the mutant mice, indicating that circadian core clock cycling is achieved through the Per2 E′-box. Furthermore, these mutant mice cannot maintain proper LAR and BTR. In Chapter 2, I showed that the calcitonin receptor, a G-protein coupled receptor (GPCR) that is abundantly expressed in the SCN, is involved in BTR control without affecting LAR. In Chapter 3, I established a new method for simultaneous measurement of body movements and body surface temperature (BST) using an infrared camera to elucidate their detailed temporal relationship.

Chapter 1: Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity.
To determine whether cis-element-mediated transcription/translation-based feedback loops is required for the formation of the circadian clock, I focused on the E'-box located near the transcriptional start site of Per2. I found that the mutation of the Per2 E'-box attenuated the circadian rhythms of cultured cells and tissues, and prevented mice from maintaining proper circadian rhythms in locomotor activity and body temperature. These results provide the first genetic evidence that non-coding element-based Per2 transcription is essential for generating cell-autonomous clock and for maintaining organismal circadian behaviour and body temperature rhythmicity.

Chapter 2: Calcitonin receptor modulates body temperature rhythms in mammals.
The neurons in the hypothalamic SCN, the center of the body circadian clock, are known to projects to various brain regions and involved in shaping circadian rhythms of many behavioral and physiological states. However, their molecular mechanisms are largely unknown. The calcitonin receptor (Calcr) is a GPCR that is abundantly expressed in the SCN, but its contribution to BTR has been completely unknown. To elucidate the role of Calcr, I simultaneously measured BTR and LAR of Calcr-deficient mice. LAR of Calcr-deficient mice were comparable to that of wild-type mice. However, BTR of Calcr-deficient mice are significantly different from that of wild-type mice. BTR of wild-type animal normally displays bimodal two peaks, one in the early night and at dawn, with a deep trough at midnight, but BTR of Calcr-deficient mice lost this characteristic dip at midnight and remained relatively unchanged throughout the night. Thus, I have identified for the first time a molecule involved in mid-night BTR control without affecting LAR.

Chapter 3: Temporal relationships between body temperature and behaviour revealed by thermographic imaging.
To elucidate the temporal relationship between behaviour and body temperature in mice, I developed a method able to trace simultaneously body movements and body surface temperature (BST). To this end, I used infrared video camera. It is known that locomotor activity and body temperature are highly correlated; basically, body temperature is high when animals are locomotorily active. As a result, locomotion is considered to elevate body temperature. However, I found that changes in BST are not always associated with locomotor activity changes. Interestingly, video analysis revealed that mice exhibit non-locomotor activities just before start of locomotion and that BST is increased in association with these non-locomotor activities. I also found that significant BST variations occur even when animals are at rest. Thus, my thermographic video imaging identified thermoregulation which is independent of locomotor activities.

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