Category Archives: Medicine

Whenever I read a paper from Karl-Arne Stokkan’s lab, and I have read every one of them, no matter how dense the scientese language I always start imagining them running around the cold, dark Arctic, wielding enormous butterfly nets, looking for and catching reindeer (or ptarmigans, whichever animal the paper is about) to do their research.

If I was not so averse to cold, I’d think this would be the best career in science ever!
It is no surprise that their latest paper – A Circadian Clock Is Not Required in an Arctic Mammal (press release) – was widely covered by the media, both traditional and blogs, See, for example, The Scientist, BBC, Scientific American podcast and Wired Science.
Relevant, or just cool?
It is hard to find a science story that is more obviously in the “that’s cool” category, as opposed to the “that’s relevant” category. For the background on this debate, please read Ed Yong, David Dobbs, DeLene Beeland, Colin Schultz, and the series of Colin’s interviews with Carl Zimmer, Nicola Jones, David Dobbs, Jay Ingram, Ferris Jabr, Ed Yong and Ed Yong again.
I agree, it is a cool story. It is an attention-grabbing, nifty story about charismatic megafauna living in a strange wilderness. I first saw the work from the lab in a poster session at a conference many years ago, and of all the posters I saw that day, it is the reindeer one that I still remember after all these years.
Yet, the coolness of the story should not hide the fact that this research is also very relevant – both to the understanding of evolution and to human medicine. Let me try to explain what they did and why that is much more important than what a quick glance at the headlines may suggest. I did it only part-way a few years ago when I blogged about one of their earlier papers. But let me start with that earlier paper as background, for context.
Rhythms of Behavior
In their 2005 Nature paper (which was really just a tiny subset of a much longer, detailed paper they published elsewhere a couple of years later), Stokkan and colleagues used radiotelemetry to continuously monitor activity of reindeer – when they sleep and when they roam around foraging.
You should remember that up in the Arctic the summer is essentially one single day that lasts several months, while the winter is a continuous night that lasts several months. During these long periods of constant illumination, reindeer did not show rhythms in activity – they moved around and rested in bouts and bursts, at almost unpredictable times of “day”. Their circadian rhythms of behavior were gone.
But, during brief periods of spring and fall, during which there are 24-hour light-dark cycles of day and night, the reindeer (on the northern end of the mainland Norway, but not the population living even further north on Svaldbard which remained arrhythmic throughout), showed daily rhythms of activity, suggesting that this species may possess a circadian clock.
Rhythms of Physiology
In a couple of studies, including the latest one, the lab also looked into a physiological rhythm – that of melatonin synthesis and secretion by the pineal gland. Just as in activity rhythms, melatonin concentrations in the blood showed a daily (24-hour) rhythm only during the brief periods of spring and fall. Furthermore, in the latest paper, they kept three reindeer indoors for a couple of days, in light-tight stalls, and exposed them to 2.5-hour-long periods of darkness during the normal light phase of the day. Each such ‘dark pulse’ resulted in a sharp rise of blood melatonin, followed by just as abrupt elimination of melatonin as soon as the lights went back on.

Rhythms of gene expression
Finally, in this latest paper, they also looked at the expression of two of the core clock genes in fibroblasts kept in vitro (in a dish). Fibroblasts are connective tissue cells found all around the body, probably taken out of reindeer by biopsy. In other mammals, e.g., in rodents, clock genes continue to cycle with a circadian period for a very long time in a dish. Yet, the reindeer fibroblasts, after a couple of very weak oscillations that were roughly in the circadian range, decayed into complete arrhytmicity – the cells were healthy, but their clocks were not ticking any more.

What do these results suggest?
There is something fishy about the reindeer clock. It is not working the same way it does in other mammals studied to date. For example, seals and humans living in the Arctic have normal circadian rhythms of melatonin. Some other animals show daily rhythms in behavior. But in reindeer, rhythms in behavior and melatonin can be seen only if the environment is rhythmic as well. In constant light conditions, it appears that the clock is not working. But, is it? How do we know?
During the long winter night and the long summer day, the behavior of reindeer is not completely random. It is in bouts which show some regularity – these are ultradian rhythms with the period much shorter than 24 hours. If the clock is not working in reindeer, i.e., if there is no clock in this species, then the ultradian rhythms would persist during spring and fall as well. Yet we see circadian rhythms during these seasons – there is an underlying clock there which can be entrained to a 24-hour light-dark cycle.
This argues for the notion that the deer’s circadian clock, unless forced into synchrony by a 24 external cycle, undergoes something called frequency demultiplication. The idea is that the underlying cellular clock runs with a 24-hour period but that is sends signals downstream of the clock, triggering phenotypic (observable) events, several times during each cycle. The events happen always at the same phases of the cycle, and are usually happening every 12 or 8 or 6 or 4 or 3 or 2 or 1 hours – the divisors of 24. Likewise, the clock can trigger the event only every other cycle, resulting in a 48-hour period of the observable behavior.
If we forget for a moment the metaphor of the clock and think instead of a Player Piano, it is like the contraption plays the note G several times per cycle, always at the same moments during each cycle, but there is no need to limit each note to appear only once per cycle.
On the other hand, both the activity and melatonin rhythms appear to be driven directly by light and dark – like a stop-watch. In circadian parlance this is called an “hourglass clock” – an environmental trigger is needed to turn it over so it can start measuring time all over again. Dawn and dusk appear to directly stop and start the behavioral activity, and onset of dark stimulates while onset of light inhibits secretion of melatonin. An “hourglass clock” is an extreme example of a circadian clock with a very low amplitude.
While we mostly pay attention to period and phase, we should not forget that amplitude is important. Yes, amplitude is important. It determines how easy it is for the environmental cue to reset the clock to a new phase – lower the amplitude of the clock, easier it is to shift. In a very low-amplitude oscillator, onset of light (or dark) can instantly reset the clock to Phase Zero and start timing all over again – an “hourglass” behavior.
The molecular study of the reindeer fibroblasts also suggests a low-amplitude clock – there are a couple of weak oscillations to be seen before the rhythm goes away completely.
There may be other explanations for the observed data, e.g., masking (direct effect of light on behavior bypassing the clock) or relative coordination (weak and transient entrainment) but let’s not get too bogged down in arcane circadiana right now. For now, let’s say that the reindeer clock exists, that it is a very low-amplitude clock which entrains readily and immediately to light-dark cycles, while it fragments or demultiplies in long periods of constant conditions.
Why is this important to the reindeer?
During long night of the winter and the long day of the summer it does not make sense for the reindeer to behave in 24-hour cycles. Their internal drive to do so, driven by the clock, should be overpowered by the need to be flexible – in such a harsh environment, behavior needs to be opportunistic – if there’s a predator in sight: move away. If there is food in sight – go get it. If you are full and there is no danger, this is a good time to take a nap. One way to accomplish this is to de-couple the behavior from the clock. The other strategy is to have a clock that is very permissive to such opportunistic behavior – a very low-amplitude clock.
But why have clock at all?
Stokkan and colleagues stress that the day-night cycles in spring help reindeer time seasonal events, most importantly breeding. The calves/fawns should be born when the weather is the nicest and the food most plentiful. The reindeer use those few weeks of spring (and fall) to measure daylength (photoperiod) and thus time their seasonality – or in other words, to reset their internal calendar: the circannual clock.
But, what does it all mean?
All of the above deals only with one of the two hypotheses for the adaptive function (and thus evolution) of the circadian clock. This is the External Synchronization hypothesis. This means that it is adaptive for an organism to be synchronized (in its biochemistry, physiology and behavior) with the external environment – to sleep when it is safe to do so, to eat at times when it will be undisturbed, etc. In the case of reindeer, since there are no daily cycles in the environment for the most of the year, there is no adaptive value in keeping a 24-hour rhythm in behavior, so none is observed. But since Arctic is highly seasonal, and since the circadian clock, through daylength measurement (photoperiodism) times seasonal events, the clock is retained as an adaptive structure.
This is not so new – such things have been observed in cave animals, as well as in social insects.
What the paper does not address is the other hypothesis – the Internal Synchronization hypothesis for the existence of the circadian clock – to synchronize internal events. So a target cell does not need to keep producing (and wasting energy) to produce a hormone receptor except at the time when the endocrine gland is secreting the hormone. It is a way for the body to temporally divide potentially conflicting physiological functions so those that need to coincide do so, and those that conflict with each other are separated in time – do not occur simultaneously. In this hypothesis, the clock is the Coordination Center of all the physiological processes. Even if there is no cycle in the environment to adapt to, the clock is a necessity and will be retained no matter what for this internal function, though the period now need not be close to 24 hours any more.
What can be done next?
Unfortunately, reindeer are not fruitflies or mice or rats. They are not endangered (as far as I know), but they are not easy to keep in the laboratory in large numbers in ideal, controlled conditions, for long periods of time.
Out in the field, one is limited as to what one can do. The only output of the clock that can be monitored long-term in the field is gross locomotor activity. Yet, while easiest to do, this is probably the least reliable indicator of the workings of the clock. Behavior is too flexible and malleable, too susceptible to “masking” by direct effects of the environment (e.g., weather, predators, etc,). And measurement of just gross locomotor activity does not tell us which specific behaviors the animals are engaged in.
It would be so nice if a bunch of reindeer could be brought into a lab and placed under controlled lighting conditions for a year at a time. One could, first, monitor several different specific behaviors. For example, if feeding, drinking and defecation are rhythmic, that would suggest that the entire digestive system is under circadian control: the stomach, liver, pancreas, intestine and all of their enzymes. Likewise with drinking and urination – they can be indirect indicators of the rhytmicity of the kidneys and the rest of the excretory system.
In a lab, one could also continuously monitor some physiological parameters with simple, non-invasive techniques. One could, for example monitor body temperature, blood pressure and heart-rate, much more reliable markers of circadian output. One could also take more frequent blood samples (these are large animals, they can take it) and measure a whole plethora of hormones along with melatonin, e.g., cortisol, thyroid hormones, progesterone, estrogen, testosterone, etc (also useful for measuring seasonal responses). One could measure metabolites in urine and feces and also gain some insight into rhythms of the internal biochemistry and physiology. All of that with no surgery and no discomfort to the animals.
Then one can place reindeer in constant darkness and see if all these rhythms persist or decay over time. Then one can make a Phase–Response Curve and thus test the amplitude of the underlying oscillator (or do that with entrainment to T-cycles, if you have been clicking on links all along, you’ll know what I’m talking about). One can test their reproductive response to photoperiod this way as well.
Finally, fibroblasts are peripheral cells. One cannot expect the group to dissect suprachiasmatic nuclei out of reindeer to check the state of the master pacemaker itself. And in a case of such a damped circadian system, testing a peripheral clock may not be very informative. Better fibroblasts than nothing, but there are big caveats about using them.
Remember that the circadian system is distributed all around the body, with each cell containing a molecular clock, but only the pacemaker cells in the suprachiasmatic nucleus are acting as a network. In a circadian system like the one in reindeer, where the system is low-amplitude to begin with, it is almost expected that peripheral clocks taken out of the body and isolated in a dish will not be able to sustain rhythms for very long. Yet those same cells, while inside of the body, may be perfectly rhythmic as a part of the ensemble of all the body cells, each sending entraining signals to the others every day, thus the entire system as a whole working quite well as a body-wide circadian clock. This can be monitored in real-time in transgenic mice, but the technology to do that in reindeer is still some years away.
Finally, one could test a hypothesis that the reindeer clock undergoes seasonal changes in its organization at the molecular level by comparing the performance of fibroblasts (and perhaps some other peripheral cells) taken out of animals at different times of year.
What’s up with this being medically relevant?
But why is all this important? Why is work on mice not sufficient and one needs to pay attention to a strange laboratory animal model like reindeer?
First, unlike rodents, reindeer is a large, mostly diurnal animal. Just like us.

Second, reindeer normally live in conditions that make people sick, yet they remain just fine, thank you. How do they do that?
Even humans who don’t live above the Arctic Circle (or in the Antarctica), tend to live in a 24-hour society with both light and social cues messing up with our internal rhythms.
We have complex circadian systems that are easy to get out of whack. We work night-shifts and rotating shifts and fly around the globe getting jet-lagged. Jet-lag is not desynchronization between the clock and the environment, it is internal desynchronization between all the cellular clocks in our bodies.
In the state of almost permanent jet-lag that many of us live in, a lot of things go wrong. We get sleeping disorders, eating disorders, obesity, compromised immunity leading to cancer, problems with reproduction, increase in psychiatric problems, the Seasonal Affective Disorder, prevalence of stomach ulcers and breast cancer in night-shift nurses, etc.
Why do we get all that and reindeer don’t? What is the trick they evolved to stay healthy in conditions that drive us insane and sick? Can we learn their trick, adopt it for our own medical practice, and use it? Those are kinds of things that a mouse and a rat cannot provide answers to, but reindeer can. I can’t think of another animal species that can do that for us. Which is why I am glad that Stokkan and friends are chasing reindeer with enormous butterfly nets across Arctic wasteland in the darkness of winter 😉
Lu, W., Meng, Q., Tyler, N., Stokkan, K., & Loudon, A. (2010). A Circadian Clock Is Not Required in an Arctic Mammal Current Biology, 20 (6), 533-537 DOI: 10.1016/j.cub.2010.01.042