Regulation of Siesta by the Central Circadian Clock in the Brain and its Physiological Role in Memory Consolidation

概要

Siesta has been regarded as an evolutionally conserved, brain circadian clock-regulated fundamental physiology. A number of putative physiological benefits of siesta have been reported in humans, which include increased performance, improved alertness, and enhanced memory formation. Epidemiological studies even suggest that taking a proper siesta reduces the incidence of Alzheimer’s disease. However, these potential benefits and their underling molecular and/or neuronal mechanisms are currently not experimentally demonstrated.

　The master clock in the hypothalamic suprachiasmatic nucleus (SCN) plays a critical role in regulating the sleep/wake cycle. It has been reported that SCN neurons expressing vasoactive intestinal polypeptide are involved in the suppression of locomotor activity during siesta. However, a direct mechanism regulating sleep in siesta is not known. Our laboratory previously identified an SCN- enriched siesta-associated gene (SSAG) that mediates regulation of body temperature during siesta. Because the regulation of sleep and circadian changes in body temperature are intimately linked to each other, I assumed that SSAG in the SCN might play a role in inducing sleep for siesta.

　In Chapter 1, using electroencephalogram (EEG), I found that SSAG deletion in the SCN causes a deficit in siesta sleep. In Chapter 2, using infrared thermography, I found that SSAG in the SCN regulates simultaneously sleep and body temperature during siesta. In Chapter 3, I employed synaptic inhibitor tetanus toxin and revealed that SSAG-positive SCN neurons mediate regulation of sleep and body temperature during siesta. In Chapter 4, using step-down passive avoidance test and novel object recognition test, I showed that siesta sleep promotes episodic memory formation in mice.

Chapter 1: SSAG in the SCN is required for proper siesta sleep.
I generated conditional SSAG SCN knockout mice (SSAG SCN-KO mice) and examined circadian sleep/wake profile using EEG. I found that SSAG SCN-KO mice were normal in spectral distribution of EEG power, total time, episode number and duration for wake, non-rapid eye movement (NREM), and rapid eye movement sleep in the entire 24-hour day. I also observed that locomotor activity of SSAG SCN-KO mice was comparable to that of control wildtype mice. However, a significant deficit in siesta was observed for SSAG SCN-KO mice: SSAG SCN-KO mice stayed awake in the middle of night/active phase of mice. These results demonstrate that SSAG in the SCN is essential for proper siesta regulation.

Chapter 2: Correlated dysregulation of siesta sleep and body temperature in SSAG SCN-KO mice. To elucidate the temporal relationship between behaviour and body temperature during siesta, I employed infrared thermography. I found that SSAG SCN-KO mice often stayed in a “sphinx-like” posture, with their head up, suggesting arousal; and their body temperatures, either interscapular skin surface or core body temperature, were not adequately decreased as compared to control mice.

Chapter 3: SSAG-positive SCN neurons mediate regulation of sleep and body temperature in siesta.
Next, instead of using SSAG SCN-KO mice, I analysed SSAG-Cre knock-in mice injected with adeno-associated virus encoding Cre-dependent tetanus toxin transgene into the SCN. I found that expression of tetanus toxin in SSAG-positive SCN neurons resulted in a lack or very low degree of siesta sleep and body temperature drop. These results indicate that SSAG-positive neurons in the SCN mediate sleep and body temperature during siesta.

Chapter 4: Siesta sleep is necessary for memory formation.
Epidemiologic studies suggest that siesta may help enhance learning and memory formation in humans. I took advantage of our mouse model to investigate the effects of siesta sleep on memory formation. To this end, I used novel object recognition test and step-down passive avoidance test. I found that SSAG SCN-KO mice trained at Zeitgeber time (ZT) 14 (i.e., before siesta time) and tested at ZT22 (i.e., after siesta time) showed reduced memory performance, as compared with control SSAG floxed mice (ZT0 denotes lights-on and ZT12 lights-off). In contrast, memory formation of SSAG SCN-KO mice trained at ZT22 and tested at ZT6 (i.e., memory formation during extra-siesta time) was not impaired. Sleep profiles during ZT22–6 were comparable between SSAG SCN-KO and SSAG floxed mice. These results demonstrate that SSAG-mediated siesta sleep is required for memory formation during the dark/active phase in mice.

Based on the results from Chapters 1–4, I have, for the first time, experimentally demonstrated the physiological contribution of siesta to memory formation by identifying and studying the function of the siesta-regulating gene in the central circadian clock structure in the brain.