ClockNews#20: Zebrafish, Math-Modelling and SAD

For Zebrafish, That Certain Glow
http://www.nytimes.com/2005/02/01/science/01obse.html

With its relatively small genome and its ability to produce mutations that are analogous to those in humans, the humble zebrafish is already a valuable tool in genetics research. But it just got better: researchers at the University of Houston have developed a transgenic zebrafish that glows according to its biological clock.
The fish, which incorporates a gene responsible for the flash of fireflies, should help scientists better understand the genetic basis of circadian rhythms, the metabolic processes linked to cycles of light and dark. Already, the Houston researchers have used it to study how the circadian clock is kick-started in the developing zebrafish.
"What we've done is inserted a recombinant gene into the germ line of these fish," said Dr. Gregory M. Cahill, an associate professor and the co-author of a paper describing the work with Dr. Maki Kaneko in the open-access journal Public Library of Science Biology.
In their work, the firefly gene is put under the control of a zebrafish gene that is a "biological clock promoter": it controls the expression of the firefly gene according to circadian rhythms. So the firefly gene produces more of an enzyme called luciferase during the morning than the evening.
When zebrafish larvae are put in a special solution, the luciferase causes the larvae to luminesce. This glow, while faint, can be detected by a machine. When measured over time, it reflects the functioning of the fish's internal clock.
With this approach, Dr. Cahill said, large numbers of larvae can be measured at once. "You want to identify mutants where the clock runs too fast or too slow," he said. But mutations are rare, so a lot of zebrafish are needed. "In theory, you can test up to 2,000 animals at a time with this," he said.
Once clock-affecting mutations are discovered, they can be compared with the human genome to better understand the genetic factors behind jet lag or other conditions that affect the body's rhythms. Similar comparative work is being done with fruit flies and mice. But Dr. Cahill said the new technique had some advantages. Unlike fruit flies (and like humans), zebrafish are vertebrates. And they reproduce much more abundantly than mice. "The nice thing about fish is that we can do lots of these, relatively inexpensively," he said.

[Update, added 2-2-2005]:

A much better review:

Glow-in-the-dark zebrafish at UH hold keys to biological clocks
http://www.innovations-report.de/html/berichte/biowissenschaften_chemie/bericht-39656.html

Professor Gregory M. Cahill’s research illuminates a ’first’ in this species Using genetically altered zebrafish that glow in the dark, University of Houston researchers have found new tools that shed light upon biological clock cycles. Gregory M. Cahill, associate professor of biology and biochemistry at UH, and Maki Kaneko, a fellow UH researcher who is now at the University of California-San Diego, presented their findings in a paper titled "Light-dependent Development of Circadian Gene Expression in Transgenic Zebrafish," appearing Feb. 1 in the Public Library of Science’s PLoS Biology, an online journal that, along with PLoS Medical, is committed to making scientific and medical literature a public resource. "By injecting the luc gene that makes fireflies glow into our zebrafish, our bottom-line finding goes back to nature versus nurture," Cahill said. "We found that these per3-luc zebrafish contain something in their genetic makeup that gets their clocks ticking without parental influence, however, we determined that it does take some sort of environmental input for the clock to start. In this case it was exposure to light/dark cycles after the fourth day of development, about the age when the fish start to swim and feed." The researchers used zebrafish (danio rerio) because they yield such a high output of spawn, with hundreds of eggs being laid by each female per week. This gives the scientists a better chance of identifying mutant fish whose biological clocks run fast or slow, providing the ability to trace the specific genes that create the anomaly. Putting UH a bit ahead of other institutions engaged in this type of research, Cahill and his team will be able to analyze more than 2,000 zebrafish per week. The per3-luc zebrafish is the first vertebrate system available for this level of high-throughput measurement. "Because we can test so many zebrafish at a time, the one in a thousand odds of finding a mutant are more easily and efficiently attainable," Cahill said. "Ultimately, this type of research can help with tracing why humans develop such things as sleep disorders or mental illnesses like depression." Per3 is the naturally occurring clock-regulated gene. The protein that it encodes is produced at highest levels near dawn, and when the luc gene is inserted into it, the luciferase protein is produced in a similar way. The result is that these fish glow rhythmically, emitting more light during the day than during the night. The amount of light is below the level of detection by the human eye, but it is easily measured with an instrument called a luminometer. "This has given us the tool we need to find other parts of systems that influence biological clocks," Cahill said. "We are optimistic that this will shed light upon such things as reproduction in other light-dependent animals." These findings have laid the groundwork for further study along these lines. With a team now built, UH graduate students who assisted with this project are now trained to work with Cahill to implement the next steps of this research. Prior to coming to UH in 1994, Cahill was a research assistant professor in the Department of Anatomy and Cell biology at the University of Kansas Medical Center in Kansas City and received his postdoctoral training at Emory University. He received his doctorate in biology and neuroscience from the University of Oregon in Eugene, where he studied the mechanisms of circadian responses to light. He graduated with his bachelor of science from the College of Biological Sciences at the University of Minnesota in Minneapolis/St. Paul. His research interests include molecular, cellular and physiological mechanisms of vertebrate circadian rhythmicity, photoreceptor cell and molecular biology, and neurobiology. He is a member of the Society for Research on Biological Rhythms and the Society for Neuroscience and is currently funded under a $1.2 million National Institutes of Health grant through 2007 as the principal investigator on "Genetic analysis of zebrafish circadian rhythmicity," under which this latest study falls.

I just downloaded the paper and printed it out for slow reading at home. Here is the abstract:

Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish
Maki Kaneko1¤ , Gregory M. Cahill1*
http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371/journal.pbio.0030034

1 Department of Biology and Biochemistry, University of Houston, Texas, United States of America

The roles of environmental stimuli in initiation and synchronization of circadian oscillation during development appear to vary among different rhythmic processes. In zebrafish, a variety of rhythms emerge in larvae only after exposure to light-dark (LD) cycles, whereas zebrafish period3 (per3) mRNA has been reported to be rhythmic from day 1 of development in constant conditions. We generated transgenic zebrafish in which expression of the firefly luciferase (luc) gene is driven by the zebrafish per3 promoter. Live larvae from these lines are rhythmically bioluminescent, providing the first vertebrate system for high-throughput measurement of circadian gene expression in vivo. Circadian rhythmicity in constant conditions was observed only after 5–6 d of development, and only if the fish were exposed to LD signals after day 4. Regardless of light exposure, a novel developmental profile was observed, with low expression during the first few days and a rapid increase when active swimming begins. Ambient temperature affected the developmental profile and overall levels of per3 and luc mRNA, as well as the critical days in which LD cycles were needed for robust bioluminescence rhythms. In summary, per3-luc zebrafish has revealed complex interactions among developmental events, light, and temperature in the expression of a clock gene.

Received September 23, 2004; Accepted November 19, 2004; Published February 1, 2005
DOI: 10.1371/journal.pbio.0030034
Copyright: © 2005 Kaneko and Cahill. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abbreviations: BAC, bacterial artificial chromosome; cps, counts per second; DD, constant darkness; Kmr, kanamycin resistance gene; LD, light-dark; luc, luciferase gene; MESA, maximum entropy spectral analysis; per, period gene; qPCR, quantitative PCR; SEM, standard error of the mean
Academic Editor: Ueli Schibler, University of Geneva, Switzerland
*To whom correspondence should be addressed. E-mail: gcahill@uh.edu
¤ Current address: Division of Biology, University of California-San Diego, La Jolla, California, United States of America
Citation: Kaneko M, Cahill GM (2005) Light-Dependent Development of Circadian Gene Expression in Transgenic Zebrafish. PLoS Biol 3(2): e34.]



NYU profs finding out what makes the biological clock tick
http://www.nyunews.com/news/campus/8742.html

A pair of NYU researchers have developed a model to chart and explain the biological clock that exists in every cell of the human body. Daniel Forger, an NYU mathematician and biologist, and Charles Peskin, a professor at NYU's Courant Institute of Mathematical Sciences and Center for Neural Science, have developed a mathematical model that explains circadian rhythm, the remarkably prcecise 24-hour biological clock found in cells in the body. If this precision is affected, it may have drastic consequences, Forger said. Jet lag, the habitual urge to sleep at certain times and drug symptoms are all directly affected by the circadian clock's timekeeping. Mutations or pharmaceutical drugs could affect the circadian clock, changing its rhythm, he said. "The circadian clock has also been linked to Alzheimer's and cancer," Forger said. "This model can begin to answer some of these questions." The scientists aim to trump previous models in terms of accuracy, tracking and recording large amounts of molecular data that would otherwise be too much for researchers, Forger said. "It's hard to keep track of the numbers of molecules in a cell over time," he said. "That's what makes this model so great. We can manipulate numbers of molecules." But problems can arise with these rhythms. "Clocks wind down over time," Forger said. "That basically happens in cells, too - the clock within." NYU Assistant Professor of Biology and Neural Science Justin Blau has researched circadian rhythms and recognizes the far-reaching effects of the new model. "As for human implications, it would be interesting to use the model to see how the clock can be reset by light most efficiently - which could help travelers adapt to a new time zone as quickly as possible," Blau said. The researchers validated their results with experimental data regarding concentrations of protein molecules within the cells of mice. "What is great about this model is that it makes clear predictions that can be tested experimentally," Blau said. "So this could be a new dawn for math-biology, where work in math leads to experiments in biology." Forger agreed that the research has great potential for the math and science world. "It's a first step," Forger said. •


Beat The Blues This Winter
http://www.ediets.com/news/article.cfm/cmi_919113

"Fall back" will have an incredible impact for millions that are plagued by Seasonal Affective Disorder (SAD), commonly referred to as the "Winter Blues." Research in Ontario suggests that between 2 and 3 percent of the general population may have SAD. Another 15 percent have a less severe experience described as the "winter blues."
Recent studies suggest that SAD is more common in northern countries, where the winter day is shorter and it is usually characterized by feelings of sadness, anxiety, and lethargy caused by the overproduction of melatonin, a sleep hormone produced by the brain. Symptoms may also include irritability, cravings for sweet or starchy foods, and significant weight gains.
Weather often affects your mood. Some people, however, are vulnerable to a type of depression that follows a seasonal pattern. For them, the shortening days of late autumn are the beginning of a type of clinical depression that can last until spring. This condition is called "Seasonal Affective Disorder," or SAD.

What Causes SAD?

Research into the causes of SAD is ongoing. However, SAD is thought to be related to seasonal variations in light. A "biological internal clock" in the brain regulates our circadian (daily) rhythms and the production of neurotransmitters that regulate sleep, mood, and appetite.
For many thousands of years, the cycle of human life revolved around the daily cycle of light and dark. We were alert when the sun was up; we slept when our world was in darkness. The introduction of electricity has relieved us of the need to be active mostly in the daylight hours, but our biological clocks may still be telling our bodies to sleep as the days shorten. This puts us out of step with our daily schedules.

What are the Symptoms?

SAD can be difficult to diagnose, since many of the symptoms are similar to those of other types of depression or bipolar disorder. Generally, symptoms that recur during the winter may indicate the presence of SAD. They may include:
Change in appetiteCraving for sweet or starchy foodsWeight gainDecreased energyFatigueTendency to oversleepDifficulty concentratingIrritabilityFeelings of anxiety and despair
The symptoms of SAD generally disappear when spring arrives. For some people, this happens suddenly with a short time of heightened activity. For others, the effects of SAD gradually dissipate.

How is SAD Treated?

If you feel depressed for long periods during autumn and winter, if your sleep and appetite patterns change dramatically and you find yourself thinking about suicide, you should seek professional help. There is effective treatment for SAD. Even people with severe symptoms can get rapid relief once they begin treatment.
People with mild symptoms can benefit from spending more time outdoors during the day and by arranging their environments so that they receive maximum sunlight. Keep curtains open during the day. Move furniture so that you sit near a window. Installing skylights and adding lamps can also help.
Exercise relieves stress, builds energy and increases your mental and physical well-being. Build physical activity into your lifestyle before SAD symptoms take hold. Make a habit of taking a daily noon-hour walk. The activity and increased exposure to natural light can raise your spirits.
Many people with SAD respond well to exposure to bright, artificial light. "Phototherapy," or light therapy, involves sitting under a special fluorescent light box once or twice a day. A health care professional should be consulted before beginning this kind of treatment.
Exposure to bright light stimulates the pineal gland, which suppresses the secretion of melatonin, the sleep hormone commonly overproduced by SAD sufferers. A high fidelity light source of 10,000 LUX, such as the Verilux HappyLite Sunshine Simulator works by providing daylight balanced, soothing, glare-free light in a concentrated "dose."
Increasing your exposure to light, monitoring your diet, sleep patterns and exercise levels are important first steps in maintaining your health and regulating your Circadian Rhythms. For those who are severely affected by SAD, devising a treatment plan with a health care professional consisting of light therapy, medication and/or cognitive-behavioral therapy may help to relieve these depressive symptoms.

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ClockNews #19: It's slower than you thought

Body clock is slow to adjust
http://www.nashuatelegraph.com/apps/pbcs.dll/article?AID=/20050130/ENTERTAINMENT/101300055

Resetting the body’s biological clock to adjust to
dramatic time changes may take more tinkering than once thought, researchers
have found.Scientists at the University of Pittsburgh found that the common
practice of gradually moving bedtime up or back a bit at a time before an
overseas trip to help reduce the impact of jet lag does work, but only to a
certain point.The study is part of an ongoing project to come up with the best
way to help astronauts prepare their sleep habits for space travel, but the
findings may help anyone who has to deal with time or schedule changes."When we
have to change our sleep schedule, we often wonder if we should make the change
all at once or more gradually over several days or weeks. This research has the
eventual aim of helping us make that decision in the best way possible," said
Dr. Timothy Monk, a professor of psychiatry at Pitt’s medical school.Keeping
sleep-wake cycles straight is particularly hard for people living in space for
long periods, cut off from the natural cues on Earth. Like most animals, humans
have a biological clock in our head that’s able to keep time, getting us ready
to sleep at night and wakefulness during the day using rhythms with a period of
about 24 hours, called the circadian rhythms.But in orbit, the sunrise-sunset
cycle runs for just 90 minutes, and after being away from the natural 24-hour
cycle for several months, the biological clock starts to be thrown off balance,
with sleep and wakeful alertness suffering.In addition, astronauts often have to
be awake and alert at off-times to carry out experiments or vehicle maneuvers.
NASA has tried to meet those needs with a long-standing set of guidelines that
specify how much an astronaut’s bedtime can change from one day to the next,
generally favoring a "trickling in" approach of delaying sleep by up to two
hours when possible."The thought was that mission schedulers could trickle in a
series of two-hour phase delays without incurring any negative consequences as
far as sleep quality and alertness," Monk said. "However, based on the findings
from our experiment, that assumption might be quite wrong."In their experiment,
which was described in the December issue of the journal Aviation, Space and
Environmental Medicine, the researchers followedvolunteers who spent 16 days on
a "mission" in a time isolation facility at Pitt. They went through a series of
nine repeated two-hour delays in bedtime.The effect on circadian rhythms was
measured through tests for alertness, mood and core body temperature. At the
same time, sleep was monitored to assess duration and quality.Over the course of
the study, Monk’s team found that the circadian clock did adjust itself, but
only by about one hour a night, rather than the two hours expected under NASA’s
guidelines.As a result, the subjects eventually went through a massive
flattening of their natural sleep-wake rhythm, which led to significant
disruptions in sleep and lowered alertness when they were awake.The team is now
moving ahead with tests of other sleep cycle adjustments to see how subjects
respond. "There is always some cost to performing tasks when we expect to be
asleep, but by the end of our experiments, we should be able to tell which
approach - gradual delays, gradual advances or changes all at once - will lead
to the least disruption of an astronaut’s sleep and alertness," Monk
said.

Clocks Online

Here is a good website on Circadian Clocks:
Biological Clock Web Site

Forty-Five Years of Pittendrigh's Empirical Generalizations

It appears that every scientific discipline has its own defining moment, an event that is touted later as the moment of "birth" of the field. This can be a publication of a paper (think of Watson and Crick) or a book ("Origin of Species" anyone?). In the case of Chronobiology, it was the 1960 meeting at Cold Spring Harbor. The book of Proceedings from the Meeting (Symposia on Quantitative Biology, Vol.XXV) is a founding document of the field: I have two copies, my advisor has three (all heavily used and annotated).

The 1960 meeting was not the first one. There were a few others before, e.g., one in Stockholm, Sweden, another in Feldafing, Germany. But the Cold Spring Harbor meeting was special. Why? I don't know - I wasn't even born yet. I have a hunch that there were several aspects of this symposium that made it different from the preceding meetings. First, the sheer number of participants was larger thus, perhaps, reaching a critical mass, or crossing a treshold needed for the group to feel as if they are not just congregating individuals but a part of something bigger. Additionally, being a part of a venerable tradition of powerful meetings at Cold Spring Harbour may have signalled to the group that they were finally taken seriously by a broader scientific community.

Second, it appears that this was the first time all the participants really understood that the diverse phenomena they were studying were unified by more than just appearance of oscillations, but that they were different aspects of just one basic biological phenomenon for which, for the first time, they had a name: circadian rhythms and clocks (as well as other circa- rhythms), the term coined by Franz Halberg in 1959.

It is fun to read the Proceedings every now and then and compare the state of science, as well as way of thinking, between 1960 and 2005. One obvious and expected difference is in genetics. Today almost every paper has a picture of a gel. In 1960. Watson and Crick were still busy. The lack of understanding of molecular (the often-used term then was "subcellular") mechanism was a frequent lament in the papers comprising the Proceedings, and was subsituted with an unusual (to our eyes) amount of complex mathematical modelling (and that was before the personal computers!).

Another thing that immediatelly catches one's attention is the enormous number of species studied compared to current reliance on about a dozen model organisms (e.g., human, rat, mouse, hamster, chicken pineal in vitro, Xenopus frog, zebrafish, fruitfly, Neurospora bread mold, Arabidopsis plant, Synechococcus cyanobacteria). Particularly, the number of plants and protists is amazing, as the current chronobiology is so animal-centered, mainly due to the need to use models for human disease for the purpose of funding.

If you ask researchers today who the pioneers of the field are, the most likely list of "Fathers of Chronobiology" will emerge, including Colin Pittendrigh, Jurgen Aschoff, Gustav Kramer, Erwin Bunning, Karl von Frisch, August Forell, Curt Richter, Frank Brown, Max Renner, Rutger Weaver, Woodie Hastings, Eberhard Gwinner, John Palmer, Franz Halberg, Michael Menaker and Serge Daan, among others (the last three are still active, the rest are retired or dead). If you asked the 1960 participants, including all of the mentioned men, they would have probably trotted out a list that looks like this: Ingeborg Beling, Anthonia Kleinhoonte, Rose Stoppel, Beatrice Sweeney, Patricia DeCoursey, Janet Harker, Miriam Bennett, Dorothea Minis and Ursula von StPaul. Huh? "Mothers of Chronobiology"? What happened?

The proportion of the women in chronobiology is today, as it apparently always was, very high, even for biology which is rating the best among natural sciences. I am not going to risk a "Summers" mistake and suggest anything remotely like a genetic explanation (e.g., cyclicity of woman's physiology attracting women to study cycles). Yet today, most of the Big Names are men, while in the past at least half of the Big Names were women. There are many women inhabiting the labs and doing marvelous research today, but rarely as heads of Big Labs. Patricia DeCoursey is still active and, a living legend as she is, she can do whatever she wants. Amita Seghal and Carla Green also come to mind as current big female stars with their own Big Labs. But otherwise it is men, men, men (hey, I am a man and I want to have a Big Lab and become a Big Name, too). Why has the situation changed over the decades?

Big Names today are people with Big Labs. Male-dominated culture results in more men heading Big Labs. At the same time, expense of research ensures that much of the work is neccessarily done in Big Labs. It is difficult nowadays to get exciting work funded, done, published and revered if your lab is a little dark room in the basement. Half a century ago, the picture was different. Science did not require exorbitant amounts of money, huge lab space, dozens of technicians and students, and rapid rate of publishing. One could spend years in a dark basement room and finally emerge with such exciting and novel discoveries as to immediatelly become a Big Name.

The male-centered culture ensured that best and brightest male students got into the biggest, most popular labs, leaving the female candidates with remnants - working with some semi-crazy professor in the basement who is doing some weird semi-scientific stuff. It is a big risk, but if that crazy professor is onto something revolutionary, the final payback can be huge. Until the 1960s, chronobiology was regarded as weird stuff. Gustav Kramer was partically funded by Duke University Department of Parapsychology! It was as far in the left field as you could get in science. It was the daring loners who did the best of the earliest stuff, not the thousands of mainstream scientists involved in doing regular science of the day in big laboratories.

Today, it is difficult being a weird loner. Only mainstream, bandwaggon science gets funded at all. Chronobiology is now as mainstream as can be. There is almost no issue of Science Magazine without at least one paper on the topic. Does it mean that it has lost some of its creativity? It is interesting to see, reading the Proceedings, what did the last 45 years bring to the field. While nothing was known about clock genes and the molecular mechanism, it was strongly anticipated and some fairly plausible models were put forward, not too different from what it turned out to be in the end.

On the other hand, it comes as a shock for the current researcher in the field to read the Proceedings and realize that practically everything we know now was already known in 1960. We added detail, lots of detail, but no new concepts, no new rules, no new creative experimental protocols. For instance, look at the list of Empirical Generalizations about biological clocks from the beginning of the paper by Colin Pittendrigh (probably the most cited paper in the field ever):

I. CRs (circadian rhtyhms) are defined as those rhythms whoe tauFR (freerunning period) is an approximation to the period of the earth's rotation. [This is a definition that is still used]

II. CRs are ubiquitous in living systems (both in terms of all organisms on Earth and in terms of all functions within an organism) [ We now know that many bacteria, as well as deep-oceanic organisms do not have clocks, while in some subterranean animals the clock has degraded somewhat. Every function in every body is under circadian control]

III. CRs are endogenous [Yes, they are generated within the organism, not by organisms responding to environmental cycles - this was a big controversy in the 1950s]

IV. CRs are self-sustaining oscillations [Yes, they still are]

V. CRs are innate [Yes. We have identified a whole suite of genes and understand how they work]

VI. CRs occur autonomously at all levels of organization [Yes, though we tend to concentrate on molecules today]

VII. The system displays remerkable precision [True]

VIII. Period is open to spontaneous or induced shifts within a range of values [Correct]

IX. Species differ in the range of relizable values of period [True]

X. Period may show aftereffects of the regime immediately preceding the steady-state freerun being studied [True, I will write more about this later]

XI. CRs are temprature-independent [True]

XII. Period is light-intensity dependent (Aschoff's Rule) [This is generally true, exceptions have been found, nobody is studying this phenomenon any more, I will write more about it later]

XIII. CRs are entrainable by a restricted class of environmental periodicities [True]

XIV. The phase of a freerunning CR can be shifted by single perturbations [True]

XV. Transients always precede attainment of a new steady-state [Exceptions have been found since then, but nobody is studying the phenomenon, I will write more about it later]

XVI. CRs have so far proved surprisingly intractable to chemical perturbation [True. Heavy water, lithium, melatonin and a couple of other hormones are the only substances that affect the clock].

As you can see, everything important about clocks has been figured out by 1960 and has stood the test of time since then.

[Colin S. Pittendrigh]


If anything, today's crop of students is less sophisticated - some of the papers from 1960 induce a headache, that is how conceptually difficult it is to understand them. Yet they are not bogus or useless. Those are very important papers in the field, resulting from years of very creative experimenting, and should be a part of training of every new chronobiologist. But, they are not taught in courses. They are not read at lab meetings. Much of the lore is now lost.

Those who forget history are liable to repeat it. Those who are unaware of the early research in the field can make big mistakes. I have seen a young researcher present his paper at a meeting and he tried to interpret his data with an explanation that was disproven about 50 years ago. Prompted by a person in the audience (who actually did that research 50 years ago), the young guy admitted he had never read or even heard of that series of papers. And this was not some obscure series of papers, this was doctoral research of one of the founding leaders of the field, something that should be everyone's required reading.

Reading the old literature (from late 1920s till late 1980s) can be an excerise in frustration. There are creative ideas and plausible hypotheses that nobody has tested yet. There are lines of research that have been abandoned, yet modern techniques should make the continuation easy and fun, but nobody is doing it. There are species that promise to be great models for modern research, but nobody is looking at them any more. Finally, there is a long list of papers with data that directly contradict the current understanding of the way the circadian clock works. Nobody is revisiting those data to repeat the experiments or explain them in light of current knowledge.

When one reads those papers, one starts to doubt if the current model is correct. The recent data are compatible with the present consensus model, but some of the old data are not. If all you know is the current model and the current data, and that is the only way you think about it, you will never realize that there may be alternative models that are compatible with ALL data, both recent and old. The recent research assumes that the model is correct and tries to work out the fine details. Knowledge of the old data should force people to directly test if the model is correct or not. But who is going to get funded to repeat a 1928 experiment?

I plan to devote a whole future post to the whole gammut of data contradiciting the current molecular model. But I first have to make my way to describing the model itself, and that will take several posts over the next few weeks.


Related:WWDD1: Darwinian Method
WWDD2: Darwin on Time
WWDD3: Whence Clocks?
WWDD4: Power of Darwinian Method
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ClockNews #18: Orexin in Huntington's Disease

BIOMEDICINE: A Good Night's Sleep?

One of the distressing symptoms of progressive neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases is the severe disruption of sleep patterns, which leads to anxiety and distress in both patients and their caregivers. Two recent papers now shed light on the cellular and molecular mechanisms of sleep disruption in Huntington's disease (HD).

Petersen et al. find that HD patients and R6/2 mice (which mimic many of the features of human HD) exhibit progressive loss of brain neurons in the hypothalamus-- a key regulator of many different processes, including sleep. The hypothalamic neurons that die in HD patients and mice
produce the neuropeptide orexin, loss of which has been implicated in narcolepsy. Decreasing amounts of orexin in the cerebrospinal fluid of HD patients could thus be used as a marker of HD progression.

Orexin neurons in the hypothalamus also innervate the suprachiasmatic nucleus, which
drives circadian sleep/ wake cycles by regulating the transcription of several
key "clock" genes. Morton et al. report that progressive disruption of circadian
behavior in R6/2 mice is accompanied by marked alterations in the expression of
the mPer2 and mBmal1 clock genes. These findings help to explain why HD patients
suffer from markedly increased daytime sleepiness and night wakefulness, and
hopefully will contribute to better management of these distressing symptoms.

Hum. Mol. Genet. 14, 39 (2005); J. Neurosci. 25, 157 (2005).

http://www.sciencemag.org/content/vol307/issue5709/twil.shtml

ClockNews #17: Melanopsin

Melanopsin plays a key role in the inner workings of the eye

http://www.innovations-report.com/html/reports/life_sciences/report-39491.html

A Brown University team has found that a protein called
melanopsin plays a key role in the inner workings of mysterious, spidery cells
in the eye called intrinsically photosensitive retinal ganglion cells, or
ipRGCs. Melanopsin, they found, absorbs light and triggers a biochemical cascade
that allows the cells to signal the brain about brightness. Through these
signals, ipRGCs synchronize the body’s daily rhythms to the rising and setting
of the sun. This circadian rhythm controls alertness, sleep, hormone production,
body temperature and organ function. Brown researchers, led by neuroscientist
David Berson, announced the discovery of ipRGCs in 2002. Their work was
astonishing: Rods and cones aren’t the only light-sensitive eye cells. Like rods
and cones, ipRGCs turn light energy into electrical signals. But while rods and
cones aid sight by detecting objects, colors and movement, ipRGCs gauge overall
light intensity. Numbering only about 1,000 to 2,000 out of millions of eyes
cells, ipRGCs are different in another way: They have a direct link to brain,
sending a message to the tiny region that controls the body clock about how
light or dark the environment is. The cells are also responsible for narrowing
the pupil of the eye. "It’s a general brightness detection system in the eye,"
said Berson, the Sidney A. Fox and Dorothea Doctors Fox Professor of
Ophthalmology and Visual Sciences. "What we’ve done now is provide more details
about how this system works." The research, published in the current issue of
Nature, provides the first evidence that melanopsin is a functional sensory
photopigment. In other words, this protein absorbs light and sets off a chain of
chemical reactions in a cell that triggers an electrical response. The study
also showed that melanopsin plays this role in ganglion-cell photoreceptors,
helping them send a powerful signal to the brain that it is day or night. The
team made the discovery by inserting melanopsin into cells taken from the
kidneys and grown in culture. These cells, which are not normally sensitive to
light, were transformed into photoreceptors when flooded with melanopsin. In
fact, the kidney cells responded to light almost exactly the way ipRGCs do,
confirming that melanopsin is the photopigment for ganglion-cell photoreceptors.
"This resolves a key question about the function of these cells," Berson said.
"And so little is known about them, anything we learn is important." Berson and
his team made another intriguing finding: The biochemical cascade sparked by
melanopsin is closer to that of eye cells in invertebrates like fruit flies and
squid than in spined animals such as mice, monkeys or humans. "The results may
well tell us that this is an extremely ancient system in terms of evolution,"
Berson said. "We may have a bit of the invertebrate in our eyes." The research
team from Brown included lead author and post-doctoral research associate Xudong
Qiu and post-doctoral research associate Kwoon Wong, both in the Department of
Neuroscience, as well as graduate students Stephanie Carlson and Vanitha Krishna
in the Neuroscience Graduate Program. Tida Kumbalasiri and Ignacio Provencio
from the Uniformed Services University of the Health Sciences also contributed
to the research.

Ha, strike one for melanopsin! This is one of the ongoing big battles in chronobiology. On one side is the Melanopsin "mafia", led by Foster, Provencio and Berson. Their strategy is to show that melanopsin is the only photopigment involved in circadian photoentrainment and completely ignore the existence of other pigments.

On the other hand, there is the opposing Cryptochrome "mafia" (Sankar, Ruby, van Gelder) whose tactic is not just to show that cryptochrome is the pigment, but also that melanopsin is not. Both sides have some very strong and some very fishy data to show.

Of course, there is the third group, the sophisticates led by Mrosovsky who realize that a complex, and somewhat redundant system is what should be expected from evolutionary theory in the first place. They, just to make a point, produce great data demonstrating that yes, classical rods and cones (wit htheir pigments rhodopsin and color-opsins) also play a role in entrainment.

With each pigment tuned to a different wavelength of light, and the spectral composition of natural light changing over the course of the day, isn't it reasonable to expect that a finely tuned photodetection system would be able to track such changes in the environment?

Anyway, what is exciting about this study is the ancient origin of the signaling cascade, something that has been semi-expected by the field, as circadain photoreception is an older function for photoreception than vision.

The next big question: are deep-brain extraretinal photoreceptors in birds, reptiles, amphibians and fish also ancient? How about their pineal and parapineal (frontal organ) photoreceptors? What photopigments and transduction signalling cascades are involved?

...and there's more:

Cells see the light with melanopsin
http://www.nature.com/news/2005/050124/full/050124-13.html

Thanks to the rod and cone cells in our eyes, our
brains can use light to build images. Recent studies identified a third type of
cell that responds to light and dark. Three research groups have now confirmed
that melanopsin is the pigment that this cell-type uses, opening possible
avenues for treating blind people.
In the classic model, mammals have two
types of light-detecting cells, called photoreceptors, in the retina at the back
of their eye. Rod cells use the rhodopsin pigment to pick up dim light, and cone
cells use related pigments to discriminate colour.
But three years ago,
scientists found a third type of light-sensitive cell. In such cells, a pigment
called melanopsin is used to tell night from day. But apparently the visual
parts of the brain do not use this information. Instead, these cells communicate
with the neurons at the base of the brain that set the daily body cycle.
For
example, mice without working rods or cones cannot see images. But researchers
showed that they can still use a small set of melanopsin-containing cells in the
retina to adjust their biological clocks. Exactly how melanopsin worked,
however, remained a mystery.
Convincing conversion
Now researchers have
proved that melanopsin is a light-sensitive pigment, by activating the gene for
it inside non-vision cells, and converting them into photoreceptors. The results
of their work appear this week in the journals Science1 and Nature2,3.
Previous studies had shown that a small number of ganglion cells need
melanopsin to respond to light. "But that doesn't prove it's a photopigment, it
just shows that it's crucial," says neuroscientist Mark Hankins of Imperial
College in London.
By making embryonic mouse neurons produce melanopsin, he
and his team made them sensitive to light. "This shows that it's melanopsin that
functions as a photopigment," says Hankins.
Likewise, another group
demonstrated that frog eggs also became light-sensitive when injected with the
genes for melanopsin. The third team converted human embryonic kidney cells
using this pigment.
"It was fantastic," says Satchidananda Panda, a
biologist at the Salk Institute for Biological Studies in La Jolla, California,
who investigated the frog eggs. He explains that very few light-sensitive
proteins still work in the cells of different species.
Illuminations
Melanopsin resembles pigments in invertebrates' eyes, in that light makes
the cells containing it more active. Pigments in vertebrates' rods and cones
have the opposite effect, inhibiting their cells. This may help biologists
understand the evolution of the circadian rhythm system in humans, says Panda.
The results also underline the possibility of conferring visual powers on
unlikely cells. "If you could put the melanopsin gene into cells then you could
make the normally non-sensitive ones become light-sensitive," says Ron Douglas,
a vision researcher at City University in London.
"It's quite important,
because there are some forms of blindness where the rods and cones are lost,"
says Hankins. In the future, converting other cells in these people's eyes with
melanopsin could help them to form images.

References

1. Panda,
S. et al. Science 307, 600-604 doi:10.1126/science.1105121 (2005).
2.
Melyan, Z. et al. Nature advanced online publication doi:10.1038/nature03344
(2005).
3. Qiu, X. et al. Nature advanced online publication
doi:10.1038/nature03345 (2005).

Update:

Another excellent review of the work:
Opsin mediates circadian clock
http://www.biomedcentral.com/news/20050128/02/

...and one that is designed to catch the eye and to appeal to the non-scientists and ends up being naively over-optimistic:
Genetic discovery could help restore sight to the blind
http://news.ft.com/cms/s/a4cc14da-70d1-11d9-b572-00000e2511c8.html

Tag:circadian

ClockNews #16: Circadian Rhythm in Visual Sensitivity

NYU biologists find new function for pacemaker neurons
http://www.eurekalert.org/pub_releases/2005-01/nyu-nbf012505.php

A study by New York University researchers reveals a
new function for the nerve cells that regulate circadian rhythms of behavior in
fruit flies.

The nerve cells, called pacemaker neurons, contain a molecular
clock that controls a 24-hour circadian rhythm in activity similar to the
rhythms in sleep/wake cycles found in humans and many other organisms. It was
previously known that pacemaker neurons receive visual signals to reset their
molecular clocks, but scientists did not have any evidence that they transmitted
information to their target cells, as most other neurons do.

The current study shows that pacemaker neurons do in fact transmit signals and are required
for a rapid behavior, according to the paper, published in the January 20th
issue of Neuron. The study was conducted by Esteban O. Mazzoni, a graduate
student in NYU's Biology Department, Biology Professor Claude Desplan, and
Assistant Biology Professor Justin Blau. The finding suggests it may be possible
to identify genes that can be used to treat problems such as sleep disorders and
jet lag.

The researchers examined the role that pacemaker neurons play in
helping Drosophila larvae avoid light. Drosophila is a species of fruit fly
commonly used in biological research. Fruit fly larvae foraging for food avoid
light, presumably to keep away from predators. Unlike adult Drosophila, the
larvae only have one structure for gathering visual cues, called Bolwig's Organ.
This organ senses the amount of light in the environment and transmits that
information to the pacemaker neurons to reset their molecular clocks.

In the experiments described by Mazzoni, Desplan, and Blau, fly larvae were placed in
the center of a Petri dish with one side dark and the other illuminated. Normal
larvae exhibited the natural behavior and clustered on the dark side. However,
when the larvae had their pacemaker neurons disabled, they were as blind as
larvae that had their light-sensing organs removed and distributed themselves
evenly between the light and dark halves of the Petri dish.

Further experiments showed that, in addition to transmitting the light information, the
pacemaker neurons also modulate the sensitivity of larvae to light, generating a
circadian rhythm in visual sensitivity. The experiments revealed that fruit fly
larvae are most sensitive to light at dawn and least sensitive toward dusk.
The study demonstrates that pacemaker neurons are doing much more than
scientists had suspected. They not only relay visual signals to target cells,
but are also act as filters, using their molecular clocks to adjust the
intensity of the transmitted signal depending on the time of day.

Almost all of the genes that make up Drosophila's molecular clock have counterparts with
similar functions in mammals. Because of this similarity, it may be possible to
identify genes in fruit flies that can be used to treat problems in people, such
as sleep disorders and jet lag.

Hmmmm, this is one messily written press release. I have not seen the paper yet, but from what I can glean from the release, the pacemakers control a circadian rhythm in photosensitivity. When the pacemaker is deleted, the rhythm is abolished. This may or may not be sufficient to explain the data, i.e., the larvae are not blind, but just do not "care" any more about light intensity. I will update once I read the actual paper.


Categories:Clock News

Tags:circadian

ClockNews #15: Dreams

Study Disputes the Randomness of Dreams
http://www.rednova.com/news/display/?id=112054

Your wildest dreams probably start soon after your head hits
the pillow.
A new study finds that more aggressive, emotionally charged
dreams tend to occur in the early, rapid-eye-movement (REM) period of sleep,
whereas deeper, non-REM slumber encourages gentler, kinder dreaming.
The finding that the brain compartmentalizes dreams into two separate sleep periods
may also put to rest the theory that dreams are no more than a meaningless
rehash of random images, according to the researchers.

Sleep Blogging

Flickr Photos of sleeping people (and animals) - cute!


Tags:circadian,sleep

Clock Tutorial #4: On Methodology

So, are you ready to do chronobiological research? If so, here are some of the tips - the thought process that goes into starting one's research in chronobiology.

First, you need to pick a question. Are you interested in doing science out of sheer curiosity to discover stuff that nobody knew before (a very noble, but hard-to-fund pursuit)? Or would you prefer your work to be applicable to human medicine or health policy, veterinary medicine, conservation biology, or agriculture?

Do you want to figure out some nitty-gritty details about the molecular basis of circadian rhythms, or neural connections between pacemakers in a mammalian brain? Perhaps you want to know if a cave animal still has a functioning clock, or if a microorganism has a clock at all. Or perhaps, you are interested in tests of adaptive function of biological rhythms and want to do your research out in the field. You may be looking for an organism to evaluate if it can become a new model, or you already know which of the current model organisms you are going to use.

Are you a kind of person who revels in a competitive area, rushing to publish as many papers as quickly as possible, each paper certain to add just a little bit to the current knowledge, all the papers accumulating reputation for you in the long run? Or are you a kind of person who prefers to tackle a difficult and risky project with no guarantee of success but a high return if it does succeed in the end, a project that may take years to accomplish while you live in obscurity, but can potentially result in a minor revolution in the thinking within the field once it is done? Think hard about the kind of personality you are and how thick is your skin.

No matter what your project is going to be, if you are in chronobiology, you have to be able to continuously monitor biological rhythms in your organisms for at least several cycles. Rare are the experiments in which you can make do without it. For instance, if you are looking at circadian rhythms of gene expression, you still need to know the phase of the cycle at which you are taking your samples. You can only know the phase if you are monitoring some kind of output of the circadian system.

The most widely used overt rhythms in laboratory research are behavioral rhythms, e.g., gross locomotor activity, wheel-running activity in rodents (and cockroaches), perch-hopping rhythms in passerine birds (songbirds), tube-running behavior in fruitflies, feeding or drinking rhythms in some other animals, etc. The advantages of behavioral rhythms are the ease and low cost of monitoring them (a LED diode, or a simple switch will do the trick). The disadvantages are the sensitivity of behaviors to various environmental events. For instance light (or darkness) may directly stimulate (or inhibit) behavior regardless of the phase of the clock. Mice are much more light-shy than rats, for instance, though both are nocturnal rodents. Darkness may inhibit feeding in some diurnal animals. The ability of an environmental cue to directly induce changes in the measured output is called masking, and one needs to be aware of this in one's model animal, either from published literature, or by running a set of experiments to determine the appearance and/or intensity of masking. If masking effects make the project impossible, one should lookk at other possible outputs, e.g., body temperature, heart-rate, blood-pressure, oxygen-consumption, blood levels of a hormone (e.g., melatonin). The best thing to do, if technically feasible, is to simultaneously monitor two or more overt rhythms in each animal.

Here is an example of a laboratory setup for studying circadian rhythms in rodents. A hamster, in this case, is housed in a cage that contains a running wheel. The running wheel has a switch that registers every revolution of the wheel and sends that information to a computer. The computer puts a time stamp on each data point, and collects the data over long periods of time.



For visual analysis of the data, computer software was developed that presents the data in a graphical format called an actograph. As you can see in the figure above, an actograph has 24 hours of the day plotted on the X axis. The data from the first day are plotted on the top, the second day is plotted immediatelly below the first day, the third day data below the second, etc. Each time point (e.g., in 10-minute bins) is depicted either as white or black. White denotes times when wheel was not moving. Black denotes times when the hamster was running in the wheel.

Once the whole actograph is printed out, one can see long-term patterns. In this example, the hamster was kept in a light-dark cycle (LD) with 12 hours of light (from 7am till 7pm) and 12 hours of darkness (LD 12:12). From the actograph, we can see that the circadian clock driving wheel-running in this hamster has entrained (synchronized) to the LD cycle - the hamster was running in the wheel at the beginning of each night. However, at his point, we are still not certain that what we see is the real output of the circadian clock, as the same pattern would emerge if light exerted a masking effect on behavior by inhibiting wheel-running. Thus, at this point in our research project, the rhythm is properly called a diurnal rhythm, not a circadian rhythm (the insect folks like to call this diel rhythm).

How do we know if the observed rhythm is really circadian? By releasing the animal into constant conditions, usually constant darkness (DD). In some animals, DD inhibits feeding, thus we have to use very dim constant light (dLL), often of a single wavelength (e.g., green). In photosynthetizing organisms, like plants, we usually use constant light (LL). Here is an example of a diurnal animal (a gallinaceous bird) kept initially in LD cycles, then released into DD. The measured overt rhythm here is core body temperature (measured by radiotransmitters implanted into its abdominal cavity, with a receiver and a computer translating transmitted radiofrequqncy into degrees Celsius). White represents body temperature below the mean temperature of that particular day, while black represents times (in 10-minute bins) when the temprature was above the daily mean.



Notice how the temperature rhythm entrains to the LD cycle but, after release into DD (after about two weeks), continues to cycle indefinitely. This is called a freerunning rhythm. The morning rise of body temperature occurs a little bit earlier every day, thus the inherent, endogenous, genetically determined period of the freerunning rhythm of this bird is shorter than 24 hours. Actually, in this example, it is about 22.5 hours.

Another way to plot data is a strip chart. This method allows one to plot only a rather small number of days/cycles, but has an advantage of showing the amplitude and the exact shape of the rhythm. For instance, this is a strip chart of human body temperature over just a single cycle.




Actually it is more than a simple strip chart, as it is a composite of four groups of humans. Each data point is an average of measurements taken at that particular time point from the whole group. Further, each group has been held at a different ambient temperature. Comparison of the four strip charts all plotted together tells us the amount of masking that environmetal temperature can exert on the clock-controlled rhythm of core body temperature.

More on entrained and freerunning rhythms in the next installment of Clock Tutorials

Tags:
circadian
sleep

ClockNews#14: Student SAD, Napping at Work, and Lunesta

Dealing with students’ seasonal affective disorder
http://www.nashuatelegraph.com/apps/pbcs.dll/article?AID=/20050123/YOUTH/101230071

SAD seems to be more predominant in January and February and
affects more women and young people than men. Symptoms may include:n Weight
gain.n Excessive sleeping.n An overall feeling of sadness that seems to leave in
the spring and summer.n Carbohydrate cravings.

Sleeping duty and wake-up calls
http://timesofindia.indiatimes.com/articleshow/998742.cms

Admittedly, at 11.40 in the a.m., the Nakate naptime
was out of line by any shift paradigm, but this should not give employers carte
blanche to declare all naps prejudicial to company interests, ultra vires of the
Industrial Relations Act, and tantamount to culpable workicide. On the contrary,
the enlightened annadata would make a post-lunch snooze compulsory so as to
cleanse the brain of the morning's rigours. Any HR ministry will tell you of the
need for detoxification. Long before it got anointed by business gurus as a
power nap, I've been a practising believer. It's the equivalent of pressing the
Refresh button; net-net, I, the tasks at hand, and my bosses benefit; deprived,
my page expires. The world's most vibrant peoples all swear by the virtues of
the afternoon siesta; the more libidinous recharge their batteries with the
equally time-honoured — and energising — custom, of the baporiyu, or afternoon
dalliance with a clandestine dream-girl. However, as everyone, from armies to
Anil Ambani, knows, catching a nap is quite different from being caught napping.
Also, as everyone from Rip van Winkle to Kumbahakaran knew, you have to observe
the cardinal rule of all therapy: you have to be mindful of the fine line that
separates the benefits of sleep from an over-doze.

Makers of new sleep aid plan ads during late TV
http://www.fortwayne.com/mld/journalgazette/living/10714830.htm

On late-night television, the companion of insomniacs
everywhere, that’ll buy a lot of advertising, and the drug, named Lunesta, is
expected to be one of this year’s most heavily marketed medications.
Lunesta breaks new ground on at least two counts. When taken at bedtime, the drug not
only puts insomnia sufferers to sleep for a full six or seven hours; it also
carries a low risk of grogginess the next day. This double-barreled approach
offers a balm for those who tend to awaken frequently during the night or too
early in the morning.

ClockNews#13: Lunesta

About a new sleeping pill, Lunesta:

Possible Help for Troubled Sleepers
http://www.14wfie.com/Global/story.asp?S=2841997&nav=3w6rVTZE

Now, there's a drug on the market that can help you get some
shut-eye and not just for a while, but indefinitely.

Wow! I can immediatelly think of many drugs that can put you to sleep indefinitely. How about cyanide? Botulinum? You fall asleep and never wake up for sure! Sorry, just joking at inept writing. Lunesta is supposed to be safe for life-long use. Be cautious about any and every drug, though....


A personal story of an insomniac:
Abnormal sleeping patterns formed at birth http://www.pahrumpvalleytimes.com/2005/01/21/opinion/bree.html

People are supposed to get older and grow out of wanting to
stay up until all hours of the night, and stop worrying that something
spectacular will happen during the wee hours of the night. I must have missed
that part of growing up.

ClockNews #12: Zebrafish Research at BU

MED researcher probes sleep at new zebrafish facility
http://www.bu.edu/bridge/archive/2005/01-21/zebra.html

"The gentle bubbling sound from hundreds of fish tanks in
Irina Zhdanova’s laboratory could lull a person to sleep. The silvery striped
zebrafish inside the shoe-box-size aquariums, however, are nodding off for other
reasons: Zhdanova is investigating how the hormone melatonin regulates sleep in
the fish, research that may someday help insomniacs get a good night’s rest.
The humble zebrafish, common in pet stores, has achieved biology stardom in
recent years. It is now the organism of choice for studying human development,
genetics, and a wide range of diseases. Zhdanova, a MED associate professor of
anatomy and neurobiology, studies the biology of sleep and the role of melatonin
in circadian rhythms, the daily cycles in physiological processes such as
wakefulness and sleep. She recently discovered that zebrafish and humans have a
lot in common when it comes to regulating their internal clocks: the diurnal
fish have a pineal gland in their brains that secretes melatonin, which lulls
them into a sleep-like stupor and affects the timing of the sleep-wake cycle.

In a recently renovated laboratory on the Medical Campus,
Zhdanova and her colleagues are putting the finishing touches on a zebrafish
facility containing several hundred tanks. It eventually will house tens of
thousands of zebrafish, with adjacent rooms for microscopes and other equipment
for studying the fish. The zebrafish lab is the first to be built at BU and for
now is dedicated exclusively to Zhdanova’s research, but she’s eager to help
other BU researchers build their own facilities.

A fish earns its stripes

Zebrafish research still is a relatively new field, but
Zhdanova says its popularity "in the United States and abroad is exploding."
Zebrafish are in vogue for many reasons: they’re easier to keep than frogs,
mice, and monkeys, and researchers can maintain large numbers of the
one-inch-long fish within close quarters. They are prolific, laying about 200
eggs a week, and their clear embryos develop quickly and are ideal for observing
developing organs. Researchers have sequenced nearly the entire zebrafish genome
and are already studying human diseases in the fish. "Of course, nonhuman
primates are the best models for sleep research, because they are our closest
relatives," Zhdanova says, "but maintaining them is expensive and
labor-intensive, and a lot of ethical issues are rightfully involved in using
them." Because of this, Zhdanova’s research team focuses primarily on zebrafish,
but also extends its most promising work at a separate facility with parallel
studies in rhesus monkeys.

Specifically, Zhdanova wants to better understand how
melatonin works at the molecular level to affect sleep, circadian rhythms, and
cognitive function. Researchers have known about melatonin for nearly 50 years,
but it’s still unclear how it interacts with certain brain structures and,
perhaps, with other tissues in the body. The hormone can shift a person’s
circadian clock forward or backward, tricking the body into thinking the
previous night has been extended or the coming night has arrived early. "It’s
nontoxic and its effect is very subtle," she says. "It does not work like
typical hypnotics that completely knock you out. We know a lot about the effects
of melatonin, but we still don’t know how it works to promote sleep and maintain
it."

Zebrafish may give researchers a window on how and where
melatonin works. "The beauty of these fish, in addition to many other things, is
that they are transparent during development," Zhdanova says. "Through the egg
you can see the entire embryo. Within 48 hours after fertilization it is already
swimming, and the larvae are also transparent. Under the microscope you can see
all the structures of the body and the brain, especially if some are highlighted
by fluorescent proteins or dyes." Melatonin appears to interact differently with
different cells, and Zhdanova wants to identify the different proteins that bind
melatonin in the brain and in other tissues. To do this, she inserts a gene in
the fish’s DNA that produces a fluorescent green protein when a nearby gene is
activated. In this way, she’ll be able to see where the glowing melatonin goes
in the fish, and which genes in the zebrafish genome encode the melatonin
receptors.
More important, though, she can conduct these studies while the
fish are alive and swimming. With a video monitoring system, she can monitor
their behavior over time as the levels of melatonin rise and fall in their
bodies.

The video-monitoring system was invented by Dr.Greg Cahill (at U. of Texas at Houston, I believe). The tiny fish larvae are placed in a 96-well plate and a camera is positioned above. A special piece of software was developed to analyze daily rhythms of activity (random swimming around the well) for each individual fish. Behavioral output such as gross motor activity has its pros and cons, but works perfectly well for the kind of research that Dr.Zhdanova is doing.

Better sleep

Before Zhdanova began working with zebrafish four years ago,
she was interested primarily in sleep in higher vertebrates. Born in Kiev,
Ukraine, she trained as a medical doctor, earned her Ph.D. in behavioral
physiology, and studied psychiatric diseases such as manic depression in St.
Petersburg. She was impressed that "99 percent of these diseases are correlated
with altered sleep patterns that might reflect their biological roots" and
became interested in the complicated biochemistry involved in regulating sleep.
She came to Boston for a postdoc at an MIT sleep lab and investigated the role
of melatonin in sleep.

Zhdanova at first worked mainly with elderly patients who
had age-related insomnia, and she showed that low doses of melatonin helped them
fall asleep and sleep through the night. But as a "hobby project" in 2000, she
and an undergraduate student developed an automated system for recording the
behavior of zebrafish. Video cameras traced the fish’s movements, providing a
comprehensive record of their activity day and night. She wanted to know if the
fish respond to melatonin the way people do. "These larvae can actually breathe
through their skin," she says, "and can absorb a lot of things from the water,
including melatonin. The interesting thing we saw was that melatonin had a very
similar effect in the zebrafish as it does in monkeys and humans. It would slow
them down, but they were not anesthetized. If you disturbed them even a little,
they would wake up. Since then, I’ve loved zebrafish."

In subsequent studies, Zhdanova proved that melatonin does
in fact promote sleep in zebrafish and that it somehow affects several tissues
at once, slowing down the heart and lowering body temperature. Her other
research was showing that low doses of melatonin administered at night to
children with insomnia stemming from severe neurological diseases also helped
them get to sleep.

The same property of permeability of the zebrafish skin to melatonin was used by another researcher, Dr.Keith Barrett (then at The Howard Hughes Center for Biological Timing at Northwestern University in Chicago, IL), to develop a method for monitoring physiological circadian rhythms in zebrafish. He also placed fish larvae in a 96-well plate, but has modified the plate somewhat, allowing the water/medium to flow through each well at a steady rate (via tiny input and output tubes). The medium coming out of each well was collected by a fraction collector and each hour the test tubes were automatically moved and the medium would start flowing into the next set of test tubes. This way, each tube contained the medium collected from a single well (fish) over a period of one hour.

As the zebrafish produce melatonin, and the melatonin permeates the skin, it naturally leaks out into the medium. Thus, Keith only had to assay the samples for melatonin concentrations in order to monitor daily rhythms of melatonin synthesis and release in zebrafish. While a great system, overcoming the problems of a gross behavioral output, it is unsuited for Dr.Zhdanova's work as she is supplementing melatonin into the medium in the first place.

The goal now is to better understand how melatonin works, in
the hope of someday finding safe and effective medicines for treating insomnia
in people. "The combination of the two model organisms in our labs is in many
respects ideal for doing this," Zhdanova says. "We have two diurnal species that
are at very different evolutionary stages. Molecular biology, genetics, drug
discovery, and drug testing are excellent things to do in zebrafish, for
example, because you can have excellent statistics. We can record simultaneously
the behavior of 80 or 100 or 160 different fish. That’s impossible to do in
humans, and it’s very difficult to do in monkeys." But when a promising drug
pans out in zebrafish, Zhdanova can then test it in monkeys.
The hope, she says, is that this powerful new model organism will shed light on a poorly understood behavior. "We still don’t really know what sleep is," she says. "Its
physiological function is still an enigma." "

ClockNews#11: The Myth of Sleepless People

You have probably seen one of those news-wires before:

UKRAINIAN MALE WIDE AWAKE FOR 20 YEARS
http://en.rian.ru/rian/index.cfm?prd_id=160&msg_id=5309602&startrow=1&date=2005-01-14&do_alert=0

KIEV, January 14 (RIA Novosti) - Fyodor Nesterchuk,
63, of the town of Kamen-Kashirsky, Ukraine's Volynsky region, has not been
asleep for the past 20 years. No soporific can do the job and no doctor has
managed to put him to sleep even for half an hour, reports the Ukrainian private
ICTV channel.
Having survived such a long period of ailment, the Volynsky
resident has already reconciled himself to it: the pensioner works as an
insurance agent in the daytime and reads at night. "I start with periodicals,
then pass to fiction. When the eyes get tired, I switch off the light and keep
dozing in the vain attempt to fall asleep," says Nesterchuk who has already
re-read the whole home library for several times.
Nesterchuk's insomnia is
treated as an exception from the rules and explained by physicians as a
side-effect of the past diseases. "If one feels comfortable, this is no
pathology," said Fyodor Koshel, chief of the Lutsk city health department.
"Nesterchuk does not look exhausted because of insomnia."

Whenever these people are brought into the lab, hooked up to EEG and monitored overnight, they are always found to sleep just fine, thank you, except that they do not remember having slept afterwards.



It's not enough just to reduce residents' hours
http://www.ama-assn.org/amednews/2005/01/17/prsf0117.htm
A new literature review reveals mixed results for patient safety when work hours are cut.

"There's been a general perception that by decreasing
work hours, by definition we'll be improving patient safety," she said. "This
review shows that can happen, but it doesn't always happen. You must do it in a
manner that still provides excellent patient care."
Dr. Fletcher said the way
scheduling changes are implemented makes a big difference in whether patient
safety improves as a result. The program in the Harvard study was able to add an
intern to make the new schedule work.
"They demonstrated that by decreasing
hours and adding an extra person, there were fewer errors," she said. "Our
review shows that you don't always reduce errors by just reducing hours."

How about paying attention to the residents' circadian rhythms?

"Systematic Review: Effects of Resident Work Hours on Patient Safety," Annals of Internal Medicine, Dec. 7, 2004, in pdf (www.annals.org/cgi/reprint/141/11/851.pdf)

...and another one of those over-precise oversimplifications:

Timing Is Everything
http://www.cbsnews.com/stories/2005/01/18/earlyshow/health/main667522.shtml

By understanding and adapting to your body's daily
rhythms, you can do everything more effectively, from exercise to intercourse,
researchers say. "Organic Style" magazine editor Janie Pyun gave The Early Show
co-anchor Harry Smith an ides of when your internal clock dictates is the best
time to take on activities ranging from exercising to eating sweets to - having
sex.

Time Perception

Chris of the "Mixing Memory" blog (http://mixingmemory.blogspot.com/) is a cognitive scientist and he occasionally writes about time perception (his other stuff is very cool, too). Here are short snippets from three of his best, longest and most detailed posts on this topic:

Temporal Attitudes
http://mixingmemory.blogspot.com/2004/10/temporal-attitudes.html

In English, we have two primary ways of speaking about time
in terms of space. Either we are moving forward through time (e.g., "We're
coming up on midterms") or time is moving past us (e.g., "The deadline is fast
approaching"). These to ways of speaking about time in spatial terms imply
different directions. In the first, things are moving forward from us; in the
second, things are moving from our front to our back. The ambiguous sentence
Boroditsky uses in her studies is, "Next Wednesday's meeting has been moved
forward two days." Ordinarily, about half of the people who read this interpret
it as meaning that the meeting has been moved to Monday, while the other have
read it as meaning the meeting has been moved to Friday.
-------------snip----------------------
Another interesting finding people whose native language conceptualizes time with a different directionality (e.g., vertical instead of horizontal) interpret temporal statements differently. When primed with pictures depicting a directionality consistent with their native language's description of time, people are faster to verify
the truth of certain statements about temporal relations (e.g., April comes after March). This implies that our time concepts are heavily influenced by the way our languages relate time and space conceptually.

Time Perception I (neurology)
http://mixingmemory.blogspot.com/2004/12/by-request-time-perception-i.html

Like reasoning, there is a whole hell of a lot of research
on time perception, and I've tossed around several ideas about how to approach
the topic in a blog post. There are so many issues, and almost all of them are
very interesting, that I am still not exactly sure what I want to do. More than
likely, it's going to take a series of posts, but I've got to start somewhere,
so I'll start with the neuroscience. In a subsequent post, I'll talk about
different factors that affect the cognitive perception of time. God only knows
what comes after that.The neuroscience of time perception has recently become a
hot area of study. So far, several brain regions have been found to be involved
in different aspects of time perception. The most widely studied are the
cerebellum and the basal ganglia, but other non-cortical regions, such as the
inferior parietal lobes, and cortical regions such as the inferior prefontal
cortex, dorsolateral prefrontal cortex, anterior cingulate gyrus, and the
supplementary motor area also play roles. I'll take each of these regions in
order, and in no particular order.

Time Perception II: Cognitive Factors
http://mixingmemory.blogspot.com/2004/12/time-perception-ii-cognitive-factors.html

There are five main types of cognitive and affective
factors that influence time perception: attention, modality, arousal, affective
valence, and linguistic factors. I'm going to go through each factor and
describe the ways in which they affect time perception, and some of the research
demonstrating these effects, starting with
modality.
-----------snip-----------------------
Humans are very accurate
measurers of time at relatively short intervals (from milliseconds to minutes),
with both the mean perceived time and the standard(the function of this
relationship has a slope near 1 deviation of duration judgements varying
linearly with elapsed time. This second property (the linear relationship
between duration and the standard deviation of duration judgements) indicates
that time perception obeys Weber's Law, such that the absolute sensitivity of
time judgements is independent of the length of the actual duration. Factors (in
addition to those discussed below) that affect duration judgements include: the
order in which stimuli are perceived (time-order errors), whether the interval
is filled or empty (filled intervals are perceived as longer than empty ones),
and the length of time between the event and the duration judgement (durations
are remembered as having been longer if there is a delay in
recall).
-----snip---------------
We've all had the
experience of time flying when we are doing something interesting, or time
dragging on when we are bored. For some time, psychologists have theorized that
this is due to the sharing of attentional resources between the processing of
the stimuli and the processing of temporal information. When a stimulus attracts
a significant amount of attention, there is less attention available to process
time. This results in a shortening of perceived duration judgements. When more
attention can be given to temporal information, time judgements tend to be more
accurate. For instance, when participants were presented with interesting
stories in a prospective judement task, they judged them to be shorter than
stories of the same length that were less interesting.

Categories: Clock Tutorials
Tags:circadian,sleep

ClockQuote #2 (Poe)

They who dream by day are cognizant of many things which escape those who
dream only by night. (Edgar Allan Poe)

ClockNews #10: SAD and Night-time Driving Accidents

Sunshine alternatives for those with seasonal affective disorder
http://www.hannibal.net/stories/011905/hap_0119050022.shtml

As winter approaches and the days get shorter; millions of
people in the U.S. once again develop the sadness and loss of energy that is
characteristic of seasonal affective disorder (SAD). SAD is a mood disorder
related to the seasonal variations of light. As the seasons change, there is a
shift in our circadian rhythm, or our biological internal clock.

Driving Drowsy
http://www.southbendtribune.com/stories/2005/01/19/living.20050119-sbt-MICH-D1-By_PATTY_LEVINE.sto

Almost every segment of our society is now working round the
clock. A report in the most recent issue of the New England Journal of Medicine
found that interns were twice as likely to have a motor vehicle accident after
working an extended shift of more than 24 hours, and more than five times as
likely to have a near-miss collision. The interns who participated in this study
also reported falling asleep while driving or while stopped in traffic.
As people get less and less sleep, their body clock, or circadian rhythm, becomes
more disrupted, and drowsy driving is the result. But the problem is more than
just getting enough sleep; when we sleep is also an important factor.
Most sleep-related accidents occur between 2 and 6 a.m., those hours when humans are biologically programmed to sleep.

Clocks and Bipolar Disorder

This is an interesting short review about the link between the circadian clock and the manic/depressive disorder:

Circadian Rhythms Factor in Rapid-Cycling Bipolar Disorder
http://www.angelfire.com/home/bphoenix1/circ.html

....even if circadian abnormalities are neither the sole nor
the primary cause of bipolar illness, it is possible that circadian
interventions can have therapeutic utility. Compared to psychotropic
medications, circadian interventions are relatively flexible therapeutic
modalities; they have a rapid onset and offset of action, and their clinical
effects may be altered by changing the time that they are administered. This
flexibility may be particularly useful in rapid-cycling bipolar patients, whose
frequent mood cycles may require rapid alterations in their therapeutic regimen.
Further research will indicate what, if any, role circadian dysfunction plays in
the pathogenesis of rapid-cycling bipolar disorder, and whether circadian
interventions can be helpful to these often treatment-resistant patients.

One thing they do not mention is that Lithium is one of the most effective drugs in the treatment of bipolar disorder. Lithium is also one of the very rare substances that is capable of altering properties of circadian rhythms (lengthening the period). If I remember correctly, a couple of years ago Eric Herzog and colleagues published a paper demonstrating that Lithium lengthens the period of the circadian rhythms of individual dispersed cells of the suprachisamatic nucleus in a dish. The work until then was all on effects of Lithium on rhythmicity of whole animals, thus various feedback loops between the clock and other brain areas could not be discounted.


Categories: Clock Tutorials
Tags:circadian,sleep

Pushing Sleeping Pills - Beware!

From two excellent blogs come these comments:

Sleep for Sale: Is insomnia the next big disease needing drug therapy?
http://oracknows.blogspot.com/2005/01/sleep-for-sale-is-insomnia-next-big.html

...an interesting article(http://www.motherjones.com/news/outfront/2005/01/12_402.html) on the marketing of sleeping pills for the weary and whether it's going to be an example of overblown pharmaceutical company advertising in the near future. The main problem, as I see it, is that the benefits of these drugs are surprisingly modest and will no doubt be exaggerated in the marketing campaigns. Also, two out of the three new drugs are related to Ambien (http://www.ambien.com/), which has some problems of its own, mainly if the patient takes it too soon before wanting to go to sleep, as it can cause confusion and amnesia(http://sleepdisorders.about.com/od/medication/a/ambieneffects.htm) if you take it and stay awake.

Feeding the vicious cycle
http://majikthise.typepad.com/majikthise_/2005/01/feeding_the_vic.html

Each generation of sleeping pills is relatively homogenous and faces common clinical and regulatory barriers. So, the makers of zolpidem, zopiclone, and zaleplon face similar challenges even though their competing fiercely with each other for market share.
The following set of quasi-incommensurable messages provides a veritable smorgasbord of rationales for prescribing the maximum number of sleeping pills to the broadest segment of the population for the longest period of time.

Housekeeping and Some Caveats

This is not the final "look" of this site. I am working on making links to a number of useful websites, archives, "recent posts", and generally sprucing this place up. I will also form "categories" for easier searches, and have finally figured out how to put images into my posts...

In a couple of weeks, once the site looks the way I want it to, I will send an e-mail to every chronobiologist in the world, asking them to come and check it out, post comments, send me suggestions, perhaps do some guest-blogging. Hopefully, some of them will use it as an educational tool when they teach courses about biological clocks.

Which leads me to a warning to students. If you copy even a smallest snippet of content from this blog and paste it into your term paper, you are very likely to get in trouble for plagiarism. This blog shows up in every search engine I could think of checking (from Google, Yahoo and MSN, to specialized blog-searches like Technorati etc.). Almost all colleges, and many high schools, now have special software for detection of plagiarism. Even the oldest and seemingly out-of-touch instructors use the Web to keep up with news, search for images to include in their lectures, etc. If they teach about clocks, either whole specialized courses in chronobiology, or just single lectures within broader courses in Biology, Physiology or Behavior, they are highly likely to find themselves reading this site. So, beware. Use this blog as a source of information, not as a source of whole sentences. OK? OK!?

What This Blog Is NOT About: Biorhythms

When I first took a class on Biological Clocks (eleven years ago), the instructor explained why biorhythms are not science. This was done with such fun, and as aside, I did not take it seriously. I did not realize I was supposed to study this exercise in Baloney Detection. I was surprised when I saw the question on the first mid-term exam, asking us to debunk biorhythms point-by-point. I lost several points there. I have learned since then to pay attention to everything a professor says, even if it seems to be a funny story. Since then, I also retain the personal distaste for the whole Fleiss/Freud story, which you can read here (so I don't have to waste my valuable time writing it myself):

http://skepdic.com/biorhyth.html (also here: http://www.canonball.bigpondhosting.com/biorhythms.html and here: http://www.rathinker.co.kr/skeptic/biorhyth.html)

The theory of biorhythms is a pseudoscientific theory that
claims our daily lives are significantly affected by rhythmic cycles overlooked
by scientists who study biological rhythms. Biochronometry is the scientific
study of rhythmicity and biological cycles or "clocks," such as the circadian
(from the Latin circa and dia; literally, "about a day"). Circadian rhythms are
based upon such things as our sensitivity to light and darkness, which is
related to our sleep/wakefulness patterns. Biorhythms is not based upon the
scientific study of biological organisms. The cycles of biorhythm theory did not
originate in scientific study, nor have they been supported by anything
resembling a scientific study. The theory has been around for over one hundred
years and there has yet to be a scientific journal that has published a single
article supporting the theory. There have been some three dozen studies
supporting biorhythm theory but all of them have suffered from methodological
and statistical errors (Hines, 1998). An examination of some 134 biorhythm
studies found that the theory is not valid (Hines, 1998). It is empirically
testable and has been shown to be false. Terence Hines believes that this fact
implies that biorhythm theory "can not properly be termed a pseudoscientific
theory." However, when the advocates of an empirically testable theory refuse to
give up the theory in the face of overwhelming evidence against it, it seems
reasonable to call the theory pseudoscientific. For, in fact, the adherents to
such a theory have declared by their behavior that there is nothing that could
falsify it, yet they continue to claim the theory is scientific....

...now go and read the whole thing, including the details of studies that tested the biorhytm "hypothesis". Try to understand the logic behind the biorhythms and why that kind of logic makes it (and similar ideas) pseudoscientific. Then follow the links and references for more good stuff. And, by the way, the term "biochronometry" used in the passage above was an early suggestion (by Mike Menaker) for the name of the discipline which is now established under the name of "chronobiology" - the topic of this blog.

Belief in biorhythms is one of the "benign" superstitions. Its believers are not very numerous. They do not, generally, try to "evangelize" their belief and their belief usually does not harm anyone else. A couple of shady businesses peddle "biorhythm calculators" but those are cheap and one needs to purchase only one in a lifetime, so this is not one of those foibles that can easily lead to personal financial devastation. Thus, this is not something that magazines like "Skeptic" and "Skeptical Inquierer" urgently feel needs constant hammering in every issue.

Martin Gardner wrote a beautiful chapter in one of his books telling the whole strange history of biorhythms, including the possible Platonic amorous relationship (followed by a falling out) between Wilhelm Fliess and Sigmund Freud, the fight over primacy with Herman Swoboda, the mystery of stolen files during the WWI, the subsequent addition of the third cycle by Teltcher, and, again, mysterious loss of files during the WWII. John Palmer wrote an excellent article debunking the whole construct from a scientific point of view (I'll try to find my copy somewhere in this mess so I can add the reference later).

As stated in the beginning of the article linked above, this "theory" is testable and has been tested a number of times, thus proven to be wrong. So why do some people still believe it?

Imagine taking a sample of 100 biorhythm believers and giving them a questionnaire or an interview. How many of them are aware that this was tested and proven wrong? One? More likely none? These are not the kind of people who would gravitate towards reading "Skeptic". They do not know the whole thing is bogus. Of course they do not know or use Sagan's Baloney Detection Kit. Of course, their understanding of science is that it is a boring subject they were forced to take in high school and somehow managed to get a D. They are likely to be really nice, sweet people, and probably quite innately smart. So why didn't they figure it out by themselves? Because it never occured to them to ask the question "Is this true or not?" When they first heard of it, they thought it was fun, they started charting their biorhythms and never once stopped to think about what they were doing. The influences of biorhythms are supposed to be "subtle" so no matter what kind of day you really had you can shoehorn it into agreement with your biorhythm state for that day...kinda like a horoscope, you can always imagine a "good fit".

When you are a kid, you believe everything you see, hear or read. When I was a kid I read all the books by Erich von Daniken and believed every word in them. But I grew out of it. Most people do not. They keep believing stuff just because a person who looks like a person with authority said so. Additionally, most people are too focused on their everyday lives, their relationships, job, and leisure, to ever stop and think about big questions or to question their deeply held beliefs. They just live from one day to another in sweet oblivion about life's persistent questions. Can't blame them, really. Except when they use their under-exercised brains to make decisions that affect all of us, like participation in elections. It is easy to swallow Frank Luntz's linguistic constructs if one never doubted anything in one's life. I guess the Social Security must be in crisis - after all, no less an authority than the President himself said so.

Biorhythms are quite benign, they tend not to invoke a terribly emotional response in believers, they are testable and tested negative, so perhaps they may be an ideal test-case for teaching the schoolkids about skepticism, about the difference between science, pseudoscience and nonsense, about historically silly ways some of these hoaxes originated, and about proper application of the Baloney Detection Kit. I don't see any powerful lobby fighting against the inclusion of such a lesson into the curriculum, yet this may provide the spark that triggers a lifelong habit of critical thinking in a substantial proportion of the population - an experiment that may be worth doing for the sake of the country.


Categories: Clock Tutorials
Tags:circadian,sleep