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Inducible clocks: Living in an unpredictable world

Clifford B. Saper and Patrick Fuller
Department of Neurology, Harvard Medical School , Beth Israel Deaconess Medical Center , USA

The suprachiasmatic nucleus (SCN) is the master light-entrainable circadian pacemaker in the mammalian circadian timing system (CTS). However, the mechanisms by which the SCN signals are translated into a range of circadian behaviors are only now being elucidated. Previous work from our laboratory demonstrated that the SCN control of circadian rhythms of sleep-wakefulness, locomotor activity, feeding, and corticosteroid levels, all depend on a multi-synaptic relay from the SCN to the ventral subparaventricular region and from the latter to the dorsomedial nucleus of the hypothalamus (DMH).

The CTS is also capable of entraining to food availability. If food availability is restricted to the normal sleep portion of the circadian cycle, animals will begin to show an preprandial increases in wakefulness, locomotor activity, body temperature, and corticosteroid cycles a few hours before the anticipated food presentation, essentially inverting their normal circadian patterns of behavior and physiology. Not only does food entrainment override the temporal information provided by the light-dark cycle, but these anticipatory phenomenon persist in SCN lesioned animals and in the absence of the periodic forcing stimulus, i.e., food availability. Recent work by our laboratory has demonstrated the critical role for the DMH in food and light entrainment, suggesting that it is a key center for regulating circadian rhythms.

The location of the oscillator that is set in motion by food entrainment (i.e., food-entrainable oscillators) remains unresolved. In surveys of clock gene expression in mouse brains during food entrainment, Mieda and Yanagisawa showed that the circadian clock genes per1 and per2 are induced in the DMH and the nucleus of the solitary tract/area postrema by restricted feeding. We wanted to know if these clock genes in the DMH continue to cycle after food entrainment, so in collaboration with Drs. Chuck Weitz and Darko Knutti, we cultured DMH explants from Per1-luc rats, either on ad lib feeding or on restricted feeding. Remarkably, Per1 expression in the DMH explants following food entrainment demonstrated self-sustaining and high amplitude oscillations in vitro. By contrast, we did not observe self-sustaining Per1 oscillations in DMH explants from animals fed ad lib.

We next studied the ability of BMAL1 mice, who lack detectable circadian function at the molecular and behavioral levels, to entrain to restricted feeding. In our study, BMAL1 (-/-) mice were arrhythmic in constant dark (DD) and did not entrain to our light-dark schedule (LD 12:12 ), instead demonstrating an ultradian pattern of body temperature (T b) and locomotor activity distributed throughout the biological day and night. BMAL1 (-/-) mice also failed to entrain to a 4-hour window of restricted feeding. We subsequently injected an adeno-associated viral vector containing the BMAL1 gene into the SCN of BMAL1 (-/-) mice. Injections into the SCN restored the circadian rhythm of T b and light entrainment, but not food entrainment. Experiments with injections into the DMH are currently under way.

These studies provide support for the concept that the brain contains specialized circadian circuitry that can induce clock gene expression and oscillations in response to food deprivation followed by refeeding, to permit animals the optimal chance of being awake and ready when food is available.