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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology


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The daily rhythm of life is maintained by a circadian clock which allows an organism to coordinate a self-sustained circadian rhythm. The time kept by a circadian clock enables the organism to respond physiologically to daily environmental changes. This rhythm can be maintained endogenously, even after long periods of time in total darkness. Light entrainment is the resetting mechanism by which light exposure can alter the phase of the circadian cycle. Prokaryotes, fungi, plants and animals have been reported to be entrained by light triggering of the circadian pacemaker, which generates the oscillation and regulates various output rhythms. In such manner, an organism's physiology and behavior can be temporally regulated.

Light entrainment is a more complex mechanism than a simple resetting due to light exposure. When an organism in constant darkness is exposed to a pulse of light during its putative "day," there is no resetting of the clock. Phase shift is affected only by exposure during putative night. Further, exposure at different times during the night has a different effect. A pulse early in the putative night induces a phase delay, while a pulse late in the night induces an advance. Thus it is context, as well as stimulus, that triggers light entrainment.


Three major classes of photoreceptors are present in plants: phototropins, cryptochromes and phytochromes. With these three types of receptors, plants are able to detect a wide range of the spectrum, from ultraviolet to far red. Based on this information, plants are able to maintain an endogenous clock that regulates a diverse array of activities including leaf opening, fragrance emission, carbon metabolism and leaf motion.

In contrast to mammals, cryptochromes are not a vital part of the entrainment mechanism in plants. Indeed, it has been shown that no single photoreceptor is entirely responsible for entrainment in plants. Another difference between the clock systems of plants and those of animals is that plants can actively regulate expression of photoreceptors as part of the feedback governing timekeeping. Animals cannot and therefore must use only downstream regulators. Initial work also indicates that the entrainment and regulation systems of plants have much more cross talk and more plasticity than their counterpart systems in animals.

Figure 1. Feedback mechanism governing the circadian rhythm of Arabidosis Thaliana demonstrating regulation of every step of the cascade, including phototransduction


How is circadian rhythm regulated in Drosophila?

Drosophila serves as a good model for circadian clock research since it is a well studied organism and many of the mechanisms governing its clock are conserved in other species. The Drosophila circadian oscillator is composed of two intracellular feedback loops in gene expression: a PER/TIM loop and a CLK-CYC loop. CLK–CYC heterodimers bind to PER and TIM promoters and activate transcription of both PER and TIM. The DBT and Shaggy/GSK3 protein kinases regulate PER/TIM accumulation and nuclear transportation. TIM binds to phosphorylated PER-DBT and stabilizes the complex. PER is also stabilized by PP2a, which removes phosphates that were added to PER. The TIM–PER–DBT complexes are phosphorylated by Shaggy which, promotes their transport into the nucleus. TIM–PER–DBT complexes then bind to CLK–CYC, thereby removing CLK–CYC from the PER and TIM promoters and inhibiting PER and TIM transcription. In the nucleus, PER and TIM eventually dissociate, and PER is phosphorylated and degraded by DBT. Two more cycling transcription factors, Vrille and PDP1, form a second feedback loop by regulating transcription CLK and attributing the oscillation of PER/TIM loop.

Figure 2. Drosophila circadian clock (Image taken from S. Panda, 2002)

Entrainment Mechanisms

  • Light exposure shifts the phase of the circadian oscillator. In Drosophila, mRNAs for TIM and PER are highest at the beginning of the night; TIM and PER proteins start to accumulate in the cytoplasm. In the middle of the night, as accumulation of PER-TIM-DBT complexes in the nucleus, CLK–CYC induced transcription was inhibited leading mRNAs of PER/TIM drop. Several photoreceptors are activated by light, and these photoreceptors then trigger the degradation of TIM via tyrosine phosphorylation. The light dependent degradation of TIM in the late night is accompanied by stable phase advances in the temporal regulation of the PER and TIM biochemical rhythms.
Figure 3. The immunocytochemistry assays on flies’ sections of heads with an anti-TIM antibody. (A)–(F) at the following times: ZT16 (A), ZT18 (B), ZT20 (C), ZT22 (D), ZT23.9 (E), and ZT1 (F). TIM expression is detectable in nuclei from ZT18 to ZT23.9. The western blot is Tim oscillation. (Image taken from M. Hunter-Ensor, 1996)
  • A different link between light and central clock of Drosophila is cryptochrome (CRY) which is a blue-light sensitive pigment protein and plays a different role in the light entrainment. CRY transcription is influenced by other clock genes such as PER, TIM, CLK, and CYC. CRY protein levels are significantly affected by light exposure. Importantly, circadian photosensitivity is increased in a CRY-overexpressing strain. (Emery, 1998) It has been shown CRY binds to TIM in a light dependent way and causes fast degradation of TIM upon such binding, relieving its action as a transcriptional inhibitor. These physiological and genetic data link a specific photoreceptor molecule to circadian rhythm. CRY serves a major photoreceptor dedicated to the resetting of circadian rhythms in Drosophila.
Figure 4. Cry mutant was found abolishing the phase response curve to a brief pulse of light (Image taken from R. Stanewsky, 1998)


How similar are fly and mammel circadian rhythm regulation?

The light-responsive properties of the circadian pacemaker are conserved between flies and rodents. Are these timekeeping mechanisms conserved? Suprachiasmatic nucleus (SCN) have been shown the target of direct retinal projections and required for entrainment by light. The rhythm was eliminated when SCN was ablated. In mammals, a heterodimer, CLOCK and BMAL1, binds to the transcription promoters of PER and CRY, and activates their transcription. CKIε, homologue of Drosophia DBT, phosphorylates cytoplasmic PER and triggers its degradation. There are three different forms of PER (mPER1, mPER2, mPER3) and two different forms CRY proteins (mCRY1, mCRY2) in mammalians. The PER binds to CRY and CKIε to form a repression complex that translocates back into the nucleus. The complex interacts directly with CLOCK and BMAL1, and results in loss of their activation activity.

Figure 5. Mammalian circadian clock (Image taken from S. Panda, 2002)

Are different light entrainment mechanisms used in mammals?

Since mouse TIM does not show function similarly as in Drosophila to control rhythm and mouse cryptochromes (mCRY1 and mCRY2) have no distinct role in being photoreceptors for entrainment, mammals likely use different mechanisms of light entrainment. Eye loss in mammals abolishes light entrainment, indicating that the eyes provide the major source of light information to the clock pacemakers. Mice without both rods and cones photoreceptors are blind, but they still retain many eye-mediated responses to light. Retinal ganglion cells express melanopsin, a photo pigment that confers this photosensitivity. However, the melanopsin deficient mutants only show attenuation of circadian rhythm. Recently, the study done by S. Panda et al. showed that outer-retinal degeneration with a deficiency in melanopsin mutant exhibits complete loss of light entrainment of the circadian oscillator. We can conclude that both the unique characteristics of melanopsin and photoreceptor cell type provide integrated inputs to SCN for light entrainment

Figure 6 Photoinhibition levels by extension of light into the anticipated dark phase. It shows either the melanopsin-containing or the classical outer-retinal photoreceptors are sufficient for transducing photic information to critical brain areas. (Image taken from S. Panda, 2006)

Mechanism of Entrainment In Mammals

Phase change in the clock cycle can be effected by both photic and non-photic (usually endogenous) stimuli. By definition, only photic input defines light entrainment. Photic shifts take place through the direct retinohypothalimic tract (RHT). Entrainment depends on release of glutamate from the RHT nerve terminals in the SCN. This release induces a calcium influx which, in turn, induces phosphorylation of a CRE-binding (CREB) protein. Phosporylated CREB then travels to the nucleus, where it initiates transcription of clock-mediating genes. Recent work has indicated that histamine, rather than glutamate, is the final neurotransmitter in the entraining sequence, however this is still under debate. The same studies also indicate that histamine may be both the final neurotransmitter in the photic and non-photic pathways.

Figure 7. Entrainment mechanism within the SCN in mammals (Image taken from E Jacobs, 2000)


1.S. Panda, J.B. Hogenesch and S.A. Kay, Nature, 2002, 417,329-335

2.Emery, P., So, W. V., Kaneko, M., Hall, Cell, 1998, 95(5), 669-79

3.R. Stanewsky, M. Kaneko, P. Emery, B. Beretta, K. Wager-Smith, S. A. Kay, M. Rosbash, J. C. Hall , Cell, 1998, 95(5), 681-79

4.M. Hunter-Ensor, A. Ousley, and A. Sehgal, Cell, 1996, 84, 677–685

5.R. G. Foster, Neuron, 1998, 20, 829

6.M.S. Freedman, R.J. Lucas,B. Soni, M. von Schantz, M. Muñoz, Z. David-Gray, R. Foster, Science, 1999, 284, 5413, 502 – 504

7.S. Panda, I. Provencio, D. C. Tu, S.S. Pires, M.D. Rollag, A.M. Castrucci, M.T. Pletcher, T.K. Sato, T. Wiltshire, M. Andahazy, S.A. Kay, R.N. Van Gelder, J.B. Hogenesch, Science, 2006, 301, 525-527

8. Fankhauser, C; Staiger, D. Planta, 2002, 216, 1-16

9. Jacobs, EH; Yamatodani, A; Timmerman, H. Trends in Pharmacological Sciences, 2000, 21, 293-298.

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