Welcome to the first edition of our new column, #NCintroduces, which aims to give students, early career researchers and those interested in the field an introduction to an exciting area or idea in neuroscience. In this installment, Tom Naughton, a UCL (London, UK) Biomedical Sciences graduate and now-lab technician at the UCL Cancer Institute, introduces the complex world of circadian rhythms.
How a genetic feedback loop tunes biology
As the Earth basks in the light of the sun, its rotation produces a 24-hour cycle of daylight and darkness. Much of life on Earth has adapted to this relentless rhythm. Single-celled and complex multicellular organisms synchronize their behavior with the daily solar cycles, causing both behavioral and molecular processes to lock into phase with a natural rhythm. Whilst biological patterns may differ from species to species, the rule is usually the same: they oscillate with a period of close to 24 hours, otherwise known as a circadian rhythm. 
I say “close to”, because the oscillation rarely matches a day exactly. It is often slightly longer, or slightly shorter than the day’s length. The existence of an external stimulus (known as a zeitgeber, or entrainment signal), like the steady rhythm of daylight, resets the internal clock every day. In fact, the activity patterns of organisms tend to drift, or ‘free-run’, without this input, becoming progressively earlier or later each day, according to the period of the internal cycle. As soon as the light/dark cycle returns, they snap back into the correct rhythm, resulting in a ‘phase-shift’. 
“Therefore the entrainment signal is not the cause of the rhythmicity of the organism, as the rhythm still exists in its absence…”
Therefore the entrainment signal is not the cause of the rhythmicity of the organism, as the rhythm still exists in its absence. Instead, circadian cycles are intrinsic, and can be influenced by an external signal. The most prevalent and powerful entrainment signal is daylight, but it is not the only one. For example, strict feeding regimes have also been shown to maintain the rhythmicity of internal clocks. 
The study of biological rhythms has been awarded its own title: chronobiology. From chronobiological investigations, we have learned that animal behavior is governed by an oscillator, which cycles with a circadian rhythm and can respond to one or more external stimuli.
In the multitude of different organisms that exist on Earth, many different oscillators have evolved. Model organisms, whose clock systems have been studied in detail, range from fruit flies to bacteria. In mammals, which this article focuses on, an area of the brain known as the suprachiasmatic nucleus of the hypothalamus (SCN), located just above the region where the two optic nerves intersect, is thought to be the home of this oscillator.
The mammalian oscillator in action
The mammalian SCN is made up of a mixed population of neurons , which are intrinsically rhythmic. In other words, they show impulse patterns that oscillate with a circadian rhythm.  Destroying the SCN tissue in a live animal disrupts rhythmic behavior, and by transplanting SCN tissue from healthy donor animals into those afflicted, it is possible to restore it. 
“Even when SCN neurons are dissociated from their host and kept alive in a dish, this basic rhythmicity remains…”
Even when SCN neurons are dissociated from their host and kept alive in a dish, this basic rhythmicity remains , meaning that the process is robust and continues without external input. These neurons, as they keep time, communicate with other areas of the brain, driving rhythmic secretion of hormones, influencing the body’s homeostasis and driving the machinery that controls sleep and wakefulness. 
But what causes the neurons of the SCN to exhibit these circadian patterns of activity? In other words, if we think of organic behavior as the clock’s face, what are the gears that turn it? It turns out that these cells run on a kind of molecular clockwork – a network of genes that are activated and deactivated with an approximately 24-hour rhythm. At the core of this network are four genetic families: Clock, Bmal1, Period (per) and cryptochrome (cry) genes. 
Running like clockwork
Within each cell, CLOCK and BMAL1 proteins are synthesized, and then bind to one another, forming a complex structure that can induce transcription of per and cry genes. As PER and CRY proteins accumulate in the cell, they interrupt the activity of the CLOCK/BMAL1 complex, thus switching off their own production. Eventually, due to consistent protein degradation by nuclear enzymes, their levels drop below a critical point. The inhibition of CLOCK/BMAL1 is released, and the cycle begins again. This feedback loop is tweaked by kinase and phosphatase enzymes, which modulate the activity and stability of the proteins, giving the oscillation its period of approximately 24 hours. As well as activating per and cry genes, CLOCK/BMAL1 facilitates transcription of other genes that operate outside of the function of the clock, meaning that the complex can influence and regulate other processes. 
This is just a basic snapshot of the molecular oscillator in action; a number of other genes, including those that regulate Clock and Bmal1, help to keep the clock in-phase, with a steady recurrence.
Modern genetic techniques allow us to visualize the rhythmic expression of clock-associated proteins, like PER, in real-time. As a result, we can correlate the rhythmic expression of these proteins and the pattern of firing in SCN neurons.  The same study allows us to conclude that the neurons are intrinsically responsive to light as an entrainment signal.
Resetting the clock
In order for light to influence SCN neurons in situ, another component must bridge the gap between them. Light information is conveyed to the SCN from unique cells in the retina, known as photosensitive retinal ganglion cells (pRGCs). These cells feed information directly connected to the SCN.  Mutant mice that are visually blind, but with intact pRGCs, are still able to reset their clock in the same way as healthy mice.  They are not blind to the entrainment signal.
The bundles of connections that pass from pRGCs in the retina to the SCN form what is known as the retinohypothalamic tract (RHT). Destroying the relay between the environment and the oscillator by surgically cutting the wires that connect them causes animals to free-run in normal conditions. These animals are blind to the zeitgeber, and lose the ability to entrain to the light/dark cycle. 
“…we can conclude with some certainty that blue light interferes with circadian systems.”
Photoreceptors in the retina use light-sensitive pigments to convert light information into an electrical signal, and pRGCs are no exception. They express a unique pigment, known as melanopsin.  pRGCs have been shown to be most sensitive to light with a wavelength of 484 nanometers , which lies within the blue region of the visible light spectrum. As such, we can conclude with some certainty that blue light interferes with circadian systems. How much of an impact this has remains to be determined empirically, but those apps that stop your laptop screen emitting blue light in the evening may be doing more than you realize.
Clocks in the periphery
The neurons of the SCN are not the only mammalian cells to run on molecular clockwork. In fact, various tissues rhythmically express clock genes , although interestingly, the rhythm is less stable than in the cells of the SCN. This has led to the proposal of a circadian hierarchy, where the SCN acts as a master timekeeper, synchronizing the clocks in peripheral tissues.
“…untangling how circadian processes work has profound implications for our physical and mental health.”
So the mystery of chronobiology is becoming clearer as more pieces of the genetic and functional puzzle are discovered and understood. More is yet to be uncovered, especially with regard to how the interplay between master and peripheral clocks works. This is important because untangling how circadian processes work has profound implications for our physical and mental health.
Protection from damage
For example, enzymatic repair of DNA damage appears to be regulated with a circadian rhythm, with high levels of enzyme activity during the day (when the sun is at its peak intensity), and low levels at night.  Considering that DNA damage can act as a key contributor to cancer-causing genetic mutation, disruption of our circadian rhythms could have a detrimental impact on our cancer defense mechanisms. In fact, when mice are subjected to lab conditions that simulate consistent jet lag, not only is the rhythm of regular clock genes diminished, but the expression of key anticancer genes is altered as well . The same study has indicated that these conditions correspond to an increase in growth of transplanted tumor cells over time, and this effect can be overcome somewhat by exposing the mice to another time-dependent stimulus, such as a rhythmic feeding schedule.
In addition, shift-work disorder, which occurs when shift-workers experience circadian disruption, has been linked to higher scores on standardized depression scales in nurses. 
A lot more work is needed if we are to understand the epidemiology of circadian disruption. And mammalian chronobiology is just one aspect of the whole field – a wide range of organisms are being studied, which have diverged over the course of history, but operate around the central theme of cycling networks of gene transcription/translation.
“With insightful research and lateral thinking, our species could be the first to master our own biological time, rather than being slaves to it.”
The full impact of biological time, both from a sociological and a biological standpoint, is yet to be uncovered. But the evidence suggests that it could be enormous. The astonishing thing is that something so fundamental to our existence is still poorly understood. With insightful research and lateral thinking, our species could be the first to master our own biological time, rather than being slaves to it.
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