Friday, January 27, 2006

Plant Photoperiodism

TroutGrrrl on the Science And Sarcasm blog wrote a nice post on plant photoperiodism. As an animal kind of person, it's unlikely I would have ever tackled this topic so it is nice to see someone else doing it so I can link. For background information, you may want to check my earlier posts on Seasonality and Photoperiodism.

Wednesday, January 25, 2006

Persistence In Perfusion

Mammals have only one circadian pacemaker - the suprachiasmatic nucleus (SCN). Apparently all the other cells in the body contain circadian clocks, too, but only the SCN drives all the overt rhythms. Without the SCN, there are no rhythms - the peripheral clocks either get out of phase with each other, or their clocks stop ticking altogether.

If you place various tissues in a dish, the SCN cycles indefinitely. All other tissues are capable of only a few oscillations in the absence of a daily signal from the SCN. The mammalian pineal secretes melatonin rhythmically, but only in a animal with an intact SCN. The retina also synthesizes melatonin rhythmically, but only if the SCN is present. The melatonin never exits the eye anyway and the clock (and melatonin) in the retina is thought to regulate only local events, e.g., daily rhythms of rod-cone dominance and disk-shedding.

In non-mammalian vertebrates, SCN (although not as precisely located so far) can also be a pacemaker but it is not the only one. In some species, the pineal gland is the pacemaker (e.g., house sparrow, some lizards). In other species it may be the retina. In some, like the pigeon, both the pineal and the eye are pacemakers - only complete removal of both eyes and the pineal renders the whole animal arrhythmic.

Capability to study a pacemaker in a dish is quite important, as it eliminates from the study all the effects of interactions between multiple clocks or feedback from target organs. The SCN of mammals is easy to culture and much progress has been made in understanding the mammalian pacemaker in vitro.

The pineal of a number of species belonging to all vertebrate classes is also relatively easy to culture. The chicken pineal is so easy to keep in a flow-through setup, that there is a whole industry devoted to the study of cell biology of this organ. People who study the chicken pineal even have their own journal - Journal of Pineal Reearch - and their own Gordon Research Conference (it just ended - I wish I could have gone there).

The retina of the eye is a bit more difficult organ to culture. It is very metabolically active and requires high oxygen levels in the culture media. The eyes of lampreys, fish and frogs have been successfully cultured quite a while ago. Eyes of lizards were cultured first about ten years ago or so. Mammalian eye was a little more difficult to do, but nevertheless, Dr.Gianluca Tosini managed to do it in mice and hamsters a few years back, but he is regarded as Grand Master of in vitro chronobiology. While subsequent studies of these tissues were productive in understanding the vertebrate circadian system, the problem with all of these cultured retinae is that none of them is the main pacemaker in its species. In each case, the pacemaker of the animal was either the SCN or the pineal, and what was cultured was a 'slave oscillator'.

What was needed was a culture of an ocular clock from an animal in which the eye is the main or sole circadian pacemaker. That meant a bird, preferably the Japanese quail. Several of the top people in the field tried to culture an avian retina, but to no avail. One could not detect any melatonin secreted by the cultured tissue. Another one measured whopping levels of melatonin but no rhythm at all. Nothing ever got published on those failed attempts, but such information gets exchanged over beer at the conferences.

Perhaps there is really a need for a Journal of Negative, Inconclusive and Unpublishable Results, to deter people from trying, again and again, experiments that have failed before. On the other hand, past failures are not deterrents to everyone. Some people thrive on challenge. One such person is my lab-mate and friend, Christopher Steele, freshly a PhD, happy in his new job up in New England.

His Dissertation was focused on the eye as a pacemaker in the circadian system of Japanese quail. He was particularly interested in the way the two eyes communicate with each other in order to remain synchronized, and in the neural and hormonal signals going from the eyes to the rest of the system, and from the rest of the system to the eyes.

It would have been great for his work if he could isolate the retina and examine how it responds to various hormones and neurotransmitters, or to light pulses and light-cycles. But it was supposed to be impossible to do! But Chris is a veteran of two wars, not to mention endless melatonin hormone assays - nothing is impossible for him. He got the funding, he got the green light from the PI, he got Dr.Tosini to start a collaboration, and he got to work.

Speak of frustration! There is a reason why all those people before gave up on this problem - it's hard! Over and over and over again, Chris tried and tried, tweaking one little thing at a time, to no avail. A time came when I had a feeling that our advisor was ready to call it quits and was just trying to find a way to suggest that to Chris politely.

But Chris wanted to have another go at it. This time, he decided that all the technical stuff was perfected and doing fine - the method of removing retinae from the eyes, the syringe pumps, the media, the fraction collector, the sterility of the whole setup. Instead, he used his knowledge of biology for a change and decided to supplement the media with precursors of melatonin, either tryptophan or serotonin:
The wells containing retinas perfused with tryptophan-rich media were almost as bad as anything he did before - one cycle, perhaps two, then everything crashes and there is no more melatonin to detect. But with added serotonin, he got this:Ha! It worked. And then it worked again. And again. Of course, this poses all source of new questions. Why did quail retina require supplementation with serotonin while that was not needed for culturing eyes from lampreys, African claw-frogs, green iguanas or hamsters? Would addition of serotonin to flow-through culture also make the quail pineal exhibit melatonin rhythms?

Well, Chris was called up again and spent another year in yet another war. In the meantime, the data just sat there in the corner of the lab, waiting for him to come back. Before he went away, Chris taught me how to use the culture system and I did one run, which did not work, but I knew two days into the experiment why it was not working. Anyway, now I know how to do it and, if anyone ever lets me into a lab again, I intend to use this technique in the future. Once Chris came back, he analyzed the data and the paper is now, finally, out in print.

Now you also understand that the title of this post is a double-entendre. The circadian pacemakers of the quail retina persist in perfusion, but also Chris persisted in trying the perfusion and the persistence paid off.

Monday, January 23, 2006

ClockNews #33

Getting enough sleep

Contrary to the idea that more sleep is better, an editorial in the journal Sleep by Daniel Kripke, a professor of psychiatry at UC San Diego, cites data from Japan and the United States showing that the ideal sleep duration is between 61⁄2 and 71⁄2 hours. Across large numbers of subjects, longevity rates actually decrease as sleep duration shifts beyond this range. Those who slept less than four hours or more than nine or 10 hours were found to have mortality rates greater than those in the middle range.

I wanna be sedated

The insomnia epidemic is now affecting kids
Morning Exercise Provides Top Benefits

When is the best time to exercise? Most experts would say that any time you can find to exercise is the best time. For some people, it’s nearly impossible to find the time to exercise at all, and for others there’s no way they can break a sweat until the evening. But if a.m. exercise may be an option, we’ve got a few reasons why working out in the morning is best…
Darkness unveils vital metabolic fuel switch 5-AMP

Constant darkness throws a molecular switch in mammals that shifts the body's fuel consumption from glucose to fat and induces a state of torpor in mice, a research team led by scientists at The University of Texas Medical School at Houston reports in the Jan. 19 edition of Nature.
Don't be SAD...get out there!

Getting outdoors and then getting active is one of the most effective ways to lessen the symptoms of the winter blues.
The month of no-sun days

It's not the rain that disrupts the body's circadian rhythms, potentially leading to depression; it's the latitude and the amount of daylight someone is exposed to, he said.

Sunday, January 22, 2006

Serotonin, Melatonin, Immunity and Cancer

I love Miss Frizzle from the cartoon "The Magic School Bus". She always says "Make connections, kids, make connections!" Here I'll try to make some tentative connections between two recent papers, both concerning health issues in humans.

The first paper, brought to my attention by Corpus Callosum, a blog I belatedly placed on my blogroll here only today, is titled Commonly Used Antidepressants May Also Affect Human Immune System, which is a high-hype way of presenting a finding that some types of immune cells appear to communicate using serotonin as a signal. To be precise, the dendritic cells, a type of antigen-presenting cell in the immune system, can uptake and relase serotonin, which in turn excites T-cells in a rapid fashion. Thus, manipulation of serotonin by pharmacological agents (as in treatment of depression) may have - as yet unknown - effects on the immune system.

Well, I have a hammer so I see nails everywhere. Whenever I see "serotonin" I wonder if melatonin is also involved. Why? Because the two appear to be connected almost in everything.

First, serotonin is a biochemical precursor of melatonin (you can see the pathway here). Thus, if there is more serotonin now, there can potentially be more melatonin later. If serotonin is lacking now, there will be a lack of melatonin later.

Second, the two substances appear to be ubiquitous throughut the body and their receptors are found apparently everywhere (what are Melatonin receptors doing in the quail ovaries and eggs?).

Third, the two substances almost always have opposite effects. While one stimulates intestinal movement, the other one inhibits it. There are many examples of such antagonism.

And, it has been known for a while now that melatonin enhances immune response. My friend and colleague Chris Moore published several papers on this topic. For instance, in this paper, he showed that exposure to constant light, which supresses melatonin synthesis and release, inhibits both the cellular and the humoral immunity, and that addition of melatonin to drinking water boosts immunity in a dose-dependent manner in quail kept in constant light. In another paper he showed that supression of melatonin by light is not neccessary for additional melatonin to boost the immune response beyond that seen normally. Also, it appears that melatonin enhances immunity via an opiate route. Finally, removing one source of melatonin, the pineal, reduced the immune response, while, strangely, removal of the other source of melatonin, the eyes, did not have an effect.

So, if melatonin boosts immunity, we can expect serotonin to do the opposite, i.e., to supress the immune response. Does that mean that taking Prozac is bad for your immune response? Who knows - it is too early to tell.

The second paper comes by the way of Tara Smith from Aetiology, one of the dozen or so science bloggers who recently joined the Seed group's ScienceBlogs (also added to my blogroll today). She explains it very well in her post, so head over there for more.

In the paper, a series of experiments in nude rats with hepatomas and in rats with transplants of human breast cancer tissue, shows that a) exposure to constant light decreases melatonin, b) both the rat and the human cancer cells express melatonin receptors, c) perfusion with melatonin-rich blood slows down cancer and d) perfusion with melatonin-depleted blood speeds up cancer. Out of several different treatments, one of the types of melatonin-depleted blood came from women working in bright light during night shift.

The effect of melatonin on cancer tissues was direct, via a linoleic acid pathway. But, considering the paper I discussed above, isn't it reasonable to expect that effects of bright light at night will also have an effect on immunity, i.e., the supression of melatonin release would supress immune response and also allow the cancers to form and spread? It is well known, after all, thet night-shift nurses tend to suffer much more, from a variety of diseases including breats cancer, than nurses that always work day-shifts.

Finally, melatonin does not work only directly, as a hormone. It is also a signal of time. A person working on a rotating shift is constantly 'jet-lagged', i.e., all the little clocks in various tissues are out of sync with each other. This in itself, melatonin or no melatonin, should be pretty bad for one's health. After all, in a spectacular series of experiments in 1950s, Dr.Janet Harker showed that cockroaches containing two pacemakers entrained several hours out of phase with each other (e.g., NYC time in the left lobe and New Zealand time in the right lobe) developed intestinal cancer - something rarely seen in insects.

Friday, January 20, 2006

Diversity of insect circadian clocks - the story of the Monarch butterfly

There are pros and cons to the prevalent use of just a dozen or so species as standard laboratory models. On one hand, when a large chunk of the scientific community focuses its energies on a single animal, techniques get standardized, suppliers produce affordable equipment and reagents, experiments are more likely to get replicated by other labs, it is much easier to get funding, and the result is speedy increase in knowledge.

On the other hand, there are drawbacks. One is narrow focus which can breed arrogance. The worst offenders are people who work with rats. They rarely put the word "rat" in the title of the paper, and often it is not even found in the abstract, introduction and discussion of the paper. One has to dig through the materials and methods to find out, although if you know about this, the very fact that the species is not noted in the title is a dead giveaway that it is a paper about rats. Some of the papers dealing with humans also make the same mistake of not pointing out the species in the title.

One of the most important animal laboratory models for the study of genetics and molecular biology is the fruitfly Drosophila melanogaster. For a century now, almost all advances in knowledge in these areas came from fruitfly research first, then this knowledge got applied to other species, e.g., mice and humans.

Last month, a paper came out that highlights both the pros and the cons of the "model" approach. On one hand, all the techniques used in the work were developed by fruitfly researchers and are now standard methods, easily replicable between labs.

On the other hand, it shows how important it is to sometimes move away from the models and take a reality check: is the mechanism described in the model animal generalizable to other animals or is it idiosyncratic to the model. The papers dealing with models, including fruitflies (and of course rats!), often make the implicit claim for generalizibility (helps funding!) without data to support this claim.

The model of the molecular mechanism of the circadian clock has been initially developed in the fruitfly and massive research is still going on in this animal. It is regarded as a reference model in a way - models developed later in mice, bread-mold, Arabidopsis plant, Synechococcus bacterium, etc, are always compared to the fruitfly model to look for similarities and differences. In a sense, it is the 'deafult' model in chronobiology.

This paper took a look at a non-model animal and found out that the fruitfly mechanism does not appear to be even typical of other insects. Steven Reppert and colleages at the University of Massachusets Medical School are studying circadian system in Monarch butterflies (mainly in order to better understand migratory orientation).
In this paper they discover that the Monarch, unlike the fruitfly, has two copies of a clock gene called Cryptochrome (CRY). One copy (CRY1) is very similar to that of Drosophila. The other copy (CRY2), however, is much more similar to the mouse version of the gene.

In the brain pacemakers of fruitflies, CRY is not the core component of the clock but is a blue-light photoreceptor. In the peripheral tissues, the same gene may be a component of the clock (it represses expression of some other clock genes).

In mammals, CRY is not directly photosensitive, but is a core clock gene and a strong repressor of expression of other clock genes.

In Monarchs, as they show in this paper, CRY1 is responsive to light, just like the CRY of fruitflies. The CRY2, though, does not respond to light, but represses expression of other genes, just like the mouse CRY.

The best thing about this paper, though, is that the authors then went on and looked into genebanks of several other insect species and, lo and behold, discovered CRY2 in a few more insects, including moths, honeybees, mosquitoes and flour beetles. Actually, the honeybees and flour beetles appear to have ONLY the mammalian-like version of the gene.

They also plotted the phylogeny of the CRY gene, showing the genealogical relationship between the fruitfly-like and mouse-like versions of CRY, both versions presumably resulting from a gene duplication some time in the past (the apparent precursor, bacterial photolyase, appears as only one copy in E.coli and its function is in DNA repair).
The PERIOD protein does not enter the nucleus in the Chinese silkmoth and the Monarch butterfly. Thus, at least in these two insects, the molecular mechanism of the circadian clock must be different from that of the fruitfly. Presence of the mammalian-like version of the CRY gene, a potent gene repressor, suggests that it may be fulfilling the function of PER in these species. Thus, there appears to be more than one way to run a clock in an insect and the fruitfly mechanism is not as 'standard' at previously thought.

And working with Monarch butterflies must be great fun!

Haisun Zhu,1 Quan Yuan,1 Oren Froy, Amy Casselman, and Steven M. Reppert, 2005, The two CRYs of the butterfly.Current Biology, Vol 15, R953-R954, 6 December 2005.

Circadian Rhythms, or Not, in Arctic Reindeer

There are two main hypotheses - not mutually exclusive - for the adaptive value of having a circadian clock. One is the Internal Synchronization hypothesis, stating that the circadian clock serves to synchronize biochemical and physiological processes within the body. The second is the External Synchronization hypothesis, stating that the circadian clock serves to syncronize the physiology and behavior to the natural environment.

The prediction from the Internal Hypothesis is that circadian rhythms in various physiological parameters - for instance body temperature, release of various hormones, cell division - will persist in organisms that live in non-rhythmic environments, e.g., in caves, underground or in the depths of the ocean. Measuring such parameters, especially in the wild, is difficult and expensive, though, so not much work has been done. Work in the laboratory, mostly in fruitflies and hamsters, brings indirect support for this hypothesis.

The predicition from the External Hypothesis is that circadian rhythms of behavior will not persist in organisms that live in arrhythmic environments. This has been shown in a number of fossorial animals in the field.

A research group from Norway has recently published a short paper in Nature (I saw these data on a poster at a meeting in 1999 - just goes to show how long it takes to publish in Nature!), looking at an animal that lives in an environment that is sometimes rhythmic, sometimes not - the Arctic. They have monitored, for a whole year, the gross locomotor activity of 12 reindeer. Six of those were in Svaldbard (78 degrees North) and the other six were of another subspecies in northern Norway (70 degrees North).
In the Arctic, for several months during the winter there is only darkness - it is the long polar night. Likewise, it is one continuous day through the months of summer. However, for a few weeks in spring and again in fall, there is a clear light-dark cycle in the environment.

What the group discovered was that, in both subspecies, there was no detectable rhythm during the one long day of summer. This is consistent with the data in a number of laboratory animals - constant bright light tends to supress circadian rhythmicity. Reindeer, like most ruminants, have ultradian rhythms of activity, i.e., tend to roam and forage in bursts.

During spring and fall, both subspecies entrained to the external light-dark cycle, although the rhythm in Norway deer appeared much more robust. It is unclear if the apparent rhythms in the more Northern population were entrained circadian rhythms or masking effects (i.e., direct effects of light on activity) of the LD cycle.

During the winter, though, the Norway reindeer exhibited a freerunning rhythm of activity, but the Svaldbard deer were again not showing any rhythms. An absence of a freerunning rhythm in constant darkness is a rare finding in animal chronobiology, and the data strongly support the External Synchronization hypothesis.
On the left - Norway deer, on the right - Svaldbard deer. Every black dot is a bout of activity and white dot a bout of inactivity. X-axis is a double-plotted 24-hour day. Y-axis (going from top to bottom) shows one day of the year in each row.

However, people from the same group (mostly Karl-Arne Stokkan) have shown before that Svaldbard reindeer (as well as Svaldbard ptarmigan, a gallinaceous bird, which is also behaviorally arrhythmic in a similar experiment) have no rhythm of melatonin secretion during the long polar night in winter either, something that contradicts the Internal Synchronization hypothesis to some extent. On the other hand, people and seals living at the same latitudes have circadian rhythms in melatonin release.

Circadian pacemakers in the suprachiasmatic nuclei signal time of day using various neural and hormonal mechanisms. One of the mechanisms involves rhythmic stimuulation of melatonin release from the pineal gland. Other hormones may be Arginine Vasopressing, Cortisol, brain peptides, etc. It is possible that melatonin signal acts on brain centers that drive various behaviors, while other neural and humoral signals drive rhythms in biochemistry and physiology of various internal organs. It is, thus, possible that animals in arrhythmic environments have no rhythms in melatonn release or behavior but still retain rhythms in physiology - something that can be tested by, for instance, continuous monitoring of body temperature in the field - hard and expensive, but not impossible to do. If this is the case, that both Internal and External Synchronization hypotheses will be supported.

Circadian clocks are so ubiqutous, it is difficult to come up with a conclusive evidence for their adaptive function. Finding animals, like reindeer, that are not rhythmic in constant natural environments but exhibit rhythms when the environment is rhythmic, add some strength to the notion that the biological clocks are evolved adaptations.

And of course, not being sleepy on the night of December 25th certainly helps Rudolf, Prancer and friends work more efficiently in delivering presents.

van Oort BE, Tyler NJ, Gerkema MP, Folkow L, Blix AS, Stokkan KA. Circadian organization in reindeer. Nature. 2005 Dec 22;438(7071):1095-6.

Thursday, January 19, 2006

Sleep And Loneliness

Over at Cognitive Daily (at its new digs on Seed Magazine's new stable of Science Blogs), either Dave or Greta (can't tell which one) comment on an interesting study that suggests that lonely people have less efficient sleep. The study invollved college students without depression and incorporated measurements of sleep quality both in the lab and at home.

They conclude:
Whether loneliness causes poor sleep is not answered by this study, which can only show a correlation between loneliness and inefficient sleep. Perhaps poor sleep causes loneliness, or perhaps some other condition causes both loneliness and poor sleep (though individuals suffering from depression, possibly the most likely culprit, were excluded from this study).
If you have any crazy college sleep/sleep deprivation stories, share them in their comment thread. I'll be reading them over there with great interest.

Perhaps being gregarious exposes one to more germs. On the other hand, it appears that having sex boosts the immune system (from Countess and Blondesense). Perhaps having sex also makes one sleep better, too?

Cacioppo, J.T., Hawkley, L.C., Berntson, G.G., Ernst, J.M., Gibbs, A.C., Stickgold, R., & Hobson, J.A. (2002). Do lonely days invade the nights? Potential social modulation of sleep efficiency. Psychological Science, 13(4), 384-387.

Wednesday, January 18, 2006

How Period and Timeless Interact in Fruitflies

A brand new paper is making a splash in the field these days - so much that you can find the press release in three places: here, here and here, this last one being the coolest as it contains a movie and three podcasts!

One of the biggest problems in circadian biology is to account for such a long time - 24 hours - it takes for the whole transcription-translation feedback loop to run its course through a single cycle. Biochemical reactions tend to happen at much shorter time scales. Some mathematical models tried to invent possible mechanisms (with no basis in experimental findings), while molecular biologists, in general, tended to ignore the problem altogether.

Still, I have seen in a few talks over the years some evidence that some circadian clock gene mutations affect only a portion of the cycle. In other words, if a mutation makes the endogenous period in a mutant shorter than that in a wild type, it is not because the whole cycle runs faster, but because one phase of the cycle runs faster. If I remember correctly from various talks, it was almost always the late-afternoon/early-evening phase that got affected.

Now, Young, Saez and Meyer published a paper in Science that sheds some light on the problem. In the process, they also significantly alter the model of the Drosophila circadian clock. You can see the old model in a movie here (see if you can play the movie directly by clicking on this), actually a series of movies produced a couple of years ago. You can see the new model here:
The big difference (let's completely ignore a dozen other molecular players for now) is in the behavior of two core clock proteins: PERIOD and TIMELESS. It was believed, until now, that it takes about six hours for the two proteins to find each other in the cytoplasm and that once they do, they bind to each other (heterodimerize), which is neccessary for their entry into the nucleus where they act to activate expression of some genes and supress expression of some other genes. No need for complicated details now.

However, some very recent studies indicate that the two proteins break off from each other immediatelly before re-entering the nucleus, and they enter the nucleus alone, not as parts of a dimer.

What this new study does is demonstrate that the two proteins, as soon as they are formed, easily find each other and bind to each other, then sit idly in the cell for six hours before breaking off from each other and entering the nucleus. Also, mutations of Tim have no effect, while mutations of Per alter the duration the two proteins sit together as a dimer.

From the press release:
"They discovered that, rather than randomly colliding, the two proteins bind together in the cytoplasm almost immediately and create what Young and Meyer refer to as an "interval timer." Then, six hours after coming together, the complexes rapidly break apart and the proteins move into the nucleus singly, all of them within minutes of each other. "Some switch is thrown at six hours that lets the complex explode. The proteins pop apart and roll into the nucleus," Young says. "Somehow, implanted within the system is a timer, formed by Period and Timeless, that counts off six hours. You have a clock within a clock."
Corpus Callosum comments:
What they found was that the interaction between the two proteins -- somewhat like the resonant frequency of a crystal, used as a timekeeper in an electronic circuit -- acts as a fundamental, but figurative, egg timer within a cell.
It is possible that this is how it works.

One caveat though. The ability of the dimer to stay together for six hours, and the ability of the Per mutations to alter this timing, are not neccessarily the inherent property of the dimer. In other words, it may not have anything to do with resembling properties of a crystal.

They may just as well be the result of interactions between the dimer and other proteins in the cell, some of which may be influenced by the signalling coming from other cells (e.g., from neighboring pacemaker neurons).

In other words, the ability of the dimer to stay put for six hours may be a property of the dimer, or it may be a property of the multicellular system as whole.

Friday, January 13, 2006

New sleep medicine blog

Sleep Disorders is a blog written by an MD who is a sleep specialist.

Wednesday, January 11, 2006

HHMI Website

Howard Hughes Medical Institute which funds a number of researchers in the field, has made, a couple of years ago, an excellent website about circadian rhythms. There, you can find news, scientists' profiles, teacher's manuals, some really cool animations and the Biological Clock Virtual Museum. Most definitely a site worth your visit.

Science & fun cool stuff
Circle of Science Assessment
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