Clocks in Bacteria IV: Clocks in other bacteria

For decades, it was thought that prokaryotes did not have circadian clocks. Then, a clock was discovered in a unicellular cyanobacterium, Synechococcus (later also in Synechocystis [1] and Trichodesmium [2]) which quickly became an important model in the study of circadian rhythms in general. Still, it was thought, for ten years or so, that no other prokaryotes had a circadian clock. Recently, the clock genes were found in filamentous (chain-forming) cyanobacteria, as well as a whole host of other bacteria and archaea. However, having clock genes does not neccessarily translate into having a functioning clock - the genes may have other functions (e.g., photoreception, or DNA repair) in bacteria other than Synechococcus.

So, two recent papers tried to address this question - do photosynthetic bacteria exhibit circadian rhythms? And the results of the two studies, in two different species of bacteria, have some interesting similarities to each other, so let's look at them in parallel.

Van Praag et al.[3], used Rhodospirillum rubrum, a gram-negative purple non-sulfur bacteria. Min et al.[4], also chose a purple photosynthetic bacterium Rhodobacter sphaeroides. In the former, the measured output was hydrogenase uptake, while in the latter a battery of luciferase reporter genes was inserted in the genome - strains exhibiting fluoresecence (presumably those in which the construct got inserted behind a promoter) were used in the study.

In the first study (click on images to enlarge), hydrogenase uptake was measured in unoxic (anaerobic) conditions in constant light (LL) at 32oC, and in constant darkness at 32oC and 16oC. In each of the three conditions, a rhythm was observed. The period of the freerunning rhythms was markedly different between the three conditions. In LL-32oC, period was ultradian: 12.1 hours. In DD at 32oC, the period was also ultradian: 14.8 hours. Only in DD at 16oC was the rhythm within a circadian range: 23.4 hours.



In the second study, light output was measured in three experiments. In all three, bacteria were assayed in constant darkness at 23oC. In the first and second groups, bacteria were pre-treated and their putative clocks entrained by a warm-cold-warm cycle prior to release into constant conditions. In the third group, the pre-treatments was exposure to a light-dark cycle prior to release into constant conditions. The first group was tested under aerobic conditions, while the second and the thir group were tested under anaerobic conditions.

Again, rhythms were observed in all three groups. What was observed was a difference in phase at which the rhythm begins dependent on the type of entraining cycle preceding the testing. The most important difference, however, was the difference in the freerunning period between the aerobic and anaerobic treatments. In the aerobic group, period was circadian: 20.5 hours. In the anaerobic conditions, the period was ultradian: 10.6 and 12.7 in groups II and III respectively.


What does this all mean? Temperature, light and oxygenation all appeared to have an effect on period. These experiments are difficult to do - if one was working with rodents or insects, the natural thing would be to test a large number of animals at several different temperatures to test for the possible lack of temperature compensation of the circadian rhythm, as well as at several different light intensities to test for the Aschoff's Rule. It is possible that this is a circadian clock that is not well temperature compensated, that is extremely sensitive to light, and that is based on the red-ox environment.

The way the studies have been reported, it is not clear that the rhythms are actually circadian, or if it just happened that some of the rhythms fell into the circadian range by accident. What is clear is that these bacteria generate endogenous rhythms. Are these rhythms circadian or not, and if so, are they driven by core-clock genes kaiA, kaiB and kaiC remains to be elucidated in the future.

[1] Aoki S, Kondo T, Wada H, and Ishiura M (1997) Circadian rhythm of the cyanobacterium Synechocystis sp. strain PCC 6803 in the dark. J Bacteriology 179:5751-5755.

[2] Chen YB, Domonic B, Mellon MT, and Zehr JP (1998) Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous nonheterocystous cyanobacterium Trichodesmium sp. strain IMS 101. J Bacteriology 180:3598-3605.

[3] Esther Van Praag, Robert Degli Agosti and Reinhard Bachofen, Rhythmic Activity of Uptake Hydrogenase in the Prokaryote Rhodospirillum rubrum, JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 15 No. 3, June 2000 218-224

[4] Hongtao Min, Haitao Guo, Jin Xiong, Rhythmic gene expression in a purple photosynthetic bacterium, Rhodobacter sphaeroides, FEBS Letters 579 (2005) 808–812

Previously in this series:
Circadian Clocks in Microorganisms
Clocks in Bacteria I: Synechococcus elongatus
Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria
Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria

Politics of Periodicity

How I wish I could see this seminar at The Johns Hopkins University:

Thurs., April 27, 4 p.m. "Circadian Oscillators in the Brain: Politics of Periodicity," a Biology seminar with Eric Herzog, Washington University; 100 Mudd. HW

Eric is a great speaker - I wonder what is he going to talk about! I am assuming that "politics" refers to the "negotiation" between different types of clock cells in the mammalian SCN, each with a different endogenous period, as to what period will the final output have. This will entail signalling mechanisms between cells, I assume, as well as phase-shifting properties of the cells.

Anyone there at John Hopkins who can go, watch and blog this?

Drinking mothers - perpetually jet-lagged offspring

Prenatal alcohol exposure can alter circadian rhythms in offspring

Children with fetal alcohol spectrum disorders (FASD) suffer from a variety of behavioral alterations. For example, they may exhibit alterations in sleeping and eating patterns, which may indicate that their circadian systems which control biological rhythms have been affected by alcohol exposure during development. A rodent study in the May issue of Alcoholism: Clinical & Experimental Research confirms that alcohol exposure during a period equivalent to the third human trimester influences the ability to synchronize circadian rhythms to light cues.

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For this study, researchers exposed male Sprague-Dawley rats to 6.0 g/kg of alcohol per day (n = 8), using an artificial rearing procedure, from postnatal days four through nine. The alcohol level represented heavy binge drinking. An artificially reared control group (n = 8) and a normally reared control group (n = 8) were also included in the study design. At 10 to 12 weeks of age, wheel-running behavior was continuously measured for eight days under a 12-hour light/12-hour dark (LD) cycle. Then the cycle was delayed by six hours and the rats were exposed to a new LD cycle for an additional six days. Their adjustment to the new cycle was evaluated.

------------snip---------------------

"This is the equivalent to a person undergoing exposure to 'jet lag,'" noted Earnest. "Basically, if you take a human and go across a number of time zones from east to west, similar to the light/dark cycle of these animals, some people will shift quickly, and some will not, and may even experience some physical problems or illness because of effects on their immune system. The responses of the alcohol-treated animals indicated that they resynchronized to the shifted light/dark cycle more slowly than the control animals."

The implications of these results for humans, added Earnest, are much broader than the term "jet lag" might indicate. "These individuals are going to have difficulties, in terms of their ability to function, while traveling across time zones and also during shift work," he said. "There are a couple of prominent examples in history regarding this: the Exxon Valdez and Chernobyl. The captain of the Exxon Valdez was not only working shift work, but he was drinking too, and unable to maintain a normal, necessary performance. With Chernobyl, the shift-work schedules were inappropriate and, at the time that the accident happened, poor mental and physical performances contributed to the disaster."

The underlying message, said Thomas, is that drinking alcohol during pregnancy can have long-lasting damaging effects to the offspring. "There is currently no known safe amount of alcohol that can be consumed during pregnancy, so it is best to abstain from alcohol drinking during pregnancy. We need to better understand the mechanisms of this dysfunction to determine if there are ways to mitigate the circadian dysfunction and behavioral dysregulation associated with developmental alcohol exposure."

Sleepwalking with Ambien

Evil Monkey of the Neurotopia blog has a good rundown on the recent finding that patients on Ambien walk and eat in their sleep.

Sleep Photoblogging

I've seen this picture on a gazillion Lefty blogs this morning and was toying with the idea of posting it here with a snarky remark. Now that Sleepdoctor has it up, I cannot be left behind, so here it is:
Vice President, dreaming of quail...

The end of Polyphasic Sleep

Michael Breus PHD, ABSM, of Sleep Disorders Blog looks at Steve Pavlina's end of the Polyphasic Sleep Experiment. From what I've seen, everyone who tried it quit in the end. Nobody lasted long enough for any negative physical consequences to kick-in (phase-shifting the clock several times a day every day for months is most definitely not good for your health in the long term).

They all quit for social reasons - you cannot live out of sync with the family and the rest of the civilization. Read whar Dr.Breus has to say in: Sleep Hacker Backs Off.

Clocks in Bacteria III: Evolution of Clocks in Cyanobacteria

As you probably know, my specialty are birds, so writing this series on clocks in microorganisms was quite an eye-opener for me and I have learned a lot. The previous two posts cover the clocks in the cyanobacterium Synechococcus elongatus, the first bacterium in which circadian rhythms were discovered and, thus, the species most studied to date.

The work in Synechococcus has uncovered a cluster of three genes - kaiA, kaiB and kaiC - that are essential for circadian rhytmicity in this species. kaiA positively regulates the kaiBC promoter and overexpression of kaiC represses the kaiBC promoter. Deletion of any one of the three genes leads to the complete loss of rhythmicity.

Synechococcus is a unicellular cyanobacterium. It was thought that circadian clock evolved in it due to incompatibility between nitrogen fixation and photosynthesis. Thus, temporal separation of these two processes was needed, phosynthesis occuring only during the day, while nitrogen fixation was relagated to the night time. It is known that filamentous cyanobacteria, those that build chains of cell, utilize a different strategy, that of spatial separation, some cells being involved in nitrogen fixation and others in photosynthesis. The two cell types exchange the end-results of those processes. Thus, it was thought that filamentous cyanobacteria have no need for a circadian clock.

However, it appears that Synechococcus is not the only bacterium to have a clock. Laboratory of Eviatar Nevo in Israel has taken a look at another cyanobacterium, this time a filamentous, chain-forming species, Nostoc linckia, and the work that ensued suggests that a number of other bacteria may possess a circadian clock as well [1,2,3].
Cyanobacteria are some of the oldest organisms on Earth, at least 3.5 billion years old, appearing in the fossil record relatively soon after the split between Eubacteria and Archaea (3.8 billion years ago). For most of the evolutionary history of cyanobacteria, the environment was very harsh, and UV radiation was one of the major factors influencing the evolution of prokaryotes. For most of that evolutionary history, the environment has undergone large changes, not just in oxygen levels, but also in the levels of UV radiation.

Volodymir Dvornik, Eviatar Nevo and collaborators hypothesized that a circadian clock, involved in temporal processing of light (including UV light) may be an important adaptation in all cyanobacteria and have detected the kaiABC cluster in Nostoc. Moreover, they hypothesized that Nostoc living in harsh, exposed environments (on sun-bathed slopes of so-called Evolution Canyons in Israel) would show greater mutation rate and higher nuclotide polymorphisam in the kai genes than Nostoc living on less harsh slopes of the Canyons. This is exactly what they found [1].

Some of the data from that study was intiguing - suggesting gene duplications and horizontal gene transfer of kai genes. So, they followed this up with a study of kai genes in a number of species of cyanobacteria [2] and later in a number of species of Eubacteria and Archea [3]. Here is the tree of kaiC (right) compared to the tree of 16S rRNA genes (left) - with quite amazing overlap:
Their analysis suggests that kaiC is the oldest element of the complex, while the kaiA is the youngest. kaiA occurs only in cyanobacteria, while kaiB, kaiC and the kaiBC complex occur in other types of bacteria and Archaea. There are also two types of kaiC: short and long. The long, double-domain kaiC (dd-kaiC) is found only in photosynthetic bacteria. Likewise, kaiBC cluster is found only in photosynthetic bacteria. Here is the tree of the kaiBC cluster:
Non-photosynthetis bacteria tend to have the short version of kaiC (sd-kaiC), as well as independent kaiB elsewhere in the genome (i.e., not in a cluster with kaiC). Analysis of the trees of kai gene evolution sugests many duplication events, as well as many occurences of gene loss and horizontal tranfer. Curiously, all the horizontal tranfers occured from cyanobacteria, as donors, to other types of bacteria and Archaea as recipients. Here is the proposed evolutionary history of the kai genes:
Thus, a number of bacteria and Archaea posses one, two or three kai genes, sometimes in multiple copies. Does that mean they have functioning circadian clocks?

Bacteria other than cyanobacteria do not have kaiA. Deletion of kaiA in Synechococcus abolishes rhythms. It is not inconceivable that a different gene (and several additional transcription factors besides kaiA are involved in the Synechococcus clock, so there is no lack of potential candidates) may fulfill that role in other microorganisms. Still, Synechococcus is the only prokaryote in which circadian rhythms have been measured and studied (OK, there is a recent exception - but you will have to wait for the next post to hear about it). Is it possible that kai genes in other bacteria have other functions and only in cynobacteria they got exapted for the circadian role? Time and new research will tell.

Previously in this series:
Circadian Clocks in Microorganisms
Clocks in Bacteria I: Synechococcus elongatus
Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria

References:

[1] Volodymyr Dvornyk, Oxana Vinogradova, and Eviatar Nevo, Long-term microclimatic stress causes rapid adaptive radiation of kaiABC clock gene family in a cyanobacterium, Nostoc linckia, from “Evolution Canyons” I and II, Israel, PNAS, February 19, 2002, vol. 99, no. 4, 2082–2087

[2] Volodymyr Dvornyk, Eviatar Nevo, Evidence for Multiple Lateral Transfers of the Circadian Clock Cluster in Filamentous Heterocystic Cyanobacteria Nostocaceae, JMol Evol (2004) 58:341–347

[3] Volodymyr Dvornyk, Oxana Vinogradova, and Eviatar Nevo, Origin and evolution of circadian clock genes in prokaryotes, PNAS, March 4, 2003, vol. 100, no. 5, 2495–2500

ClockNews #38

Asleep or awake we retain memory

Sleeping helps to reinforce what we've learned. And brain scans have revealed that cerebral activity associated with learning new information is replayed during sleep. But, in a study published in the open access journal PLoS Biology, Philippe Peigneux and colleagues at the University of Liege demonstrate for the first time that the brain doesn't wait until night to structure information. Day and night, the brain doesn't stop (re)working what we learn.

The science of lost sleep in teens

"Some of our kids are literally sleep-walking through life, with some potentially serious consequences," Millman said. "As clinicians and researchers, we know more now than ever about the biological and behavioral issues that prevent kids from getting enough sleep. But the National Sleep Foundation did something powerful: They asked teens themselves about their sleep. The results are startling and should be a wake-up call to any parent or pediatrician."

Children who sleep less are three times more likely to be overweight

The less a child sleeps, the more likely he or she is to become overweight, according to researchers from Universit Laval's Faculty of Medicine in an article published in the latest edition of the International Journal of Obesity. The risk of becoming overweight is 3.5 times higher in children who get less sleep than in those who sleep a lot, according to researchers Jean-Philippe Chaput, Marc Brunet, and Angelo Tremblay. These results come from data collected among 422 grade school students aged 5 to 10. The scientists measured the weight, height, and waist size of each participant. Information on the children's lifestyle and socioeconomic status was obtained through phone interviews with their parents.

Sleep apnea treatment benefits the heart

Patients with obstructive sleep apnea have enlarged and thickened hearts that pump less effectively, but the heart abnormalities improve with use of a device that helps patients breathe better during sleep, according to a new study in the April 4, 2006, issue of the Journal of the American College of Cardiology.

Low Stairway to Heaven

SAD arises via fluctuation in melatonin, a chemical produced in response to darkness. Those with SAD suffer from depression during the winter, as the ratio of dark to light hours increases. Melatonin produces a certain drowsiness that causes your circadian rhythms to fall out of sync with the day-night cycles of the environment. Further, some research shows that darkness decreases serotonin levels, which negatively affects mood. Certain SAD sufferers find remedy in “light box treatment”—they expose themselves to artificial light for a few hours a day and become less depressed. I like to think of the steps as my own light box treatment—give me a few hours in front of Low and my mood dramatically improves.

Getting enough Zzzzzs? Kids often don’t

You can force a kid to bed, but you can’t make him fall asleep. Especially if he — or she — is a teenager. It’s not adolescent rebellion. It’s adolescent metabolism. They physically cannot fall asleep because their bodies’ internal clocks are on sort of a chronic daylight-saving time overdrive — which worsens through their teen years, ultimately tapering off in their 20s, according to Leigh Heithoff, clinical specialist with BryanLGH Medical Center West’s Department of Sleep Medicine.

What's holding up the sandman for so many of us?

Unfortunately, with the ease of writing and filling a prescription and the mostly good press these new drugs have gotten to date, millions of people are now taking them without first exploring the reasons for their sleep problems and possible nondrug routes to cure them.

Eye cells that don't see, but regulate

As any good high school biology student can tell you, the human eye sees light with special cells called rods and cones. But when George C. Brainard experimented with shining various colors of light into people's eyes, something odd happened: A specific shade of blue light was most effective at shutting down the body's production of melatonin - the "hormone of darkness" that helps regulate sleep and the body's internal clock. Yet that shade of blue is not one of the colors best detected by rods and cones. His conclusion, shared by others conducting studies on blind people and animals: There must be some unknown cells in the eye - some that responded to light but had nothing to do with seeing. Since that experiment at Thomas Jefferson University, reported in 2001, other scientists have indeed found the new cells, as well as the gene that controls them. Only in the last year has a consensus emerged about how the new cells work.

Sleep has become the new obsession

First it was looks, then weight. Now, the new Western obsession is sleep - or a lack of it. But even experts don't agree on how much people really need

Integrating Transcriptomics and Proteomics

An example of time-shifted discord would be that of the mammalian 24-hour circadian clock, in which regulatory proteins such as Period (mPER) exhibit a four- to eight-hour delay between protein and transcript expression.

Aging-Related Sex Dependent Loss of the Circulating Leptin 24-Hour Rhythm in the Rhesus Monkey

The adipocyte-derived hormone leptin plays a pivotal role in the regulation of body weight and energy homeostasis. Many studies have indicated that the circulating levels of leptin show a 24-h rhythm, but the exact cause and nature of this rhythm is still unclear. In the present study we remotely collected blood samples every h from young and old, male and female rhesus monkeys, and examined their 24-h plasma leptin profiles. In both the young males (10-11 yrs) and young females (7-13 yrs) a clear 24-h plasma leptin rhythm was evident, with a peak occurring ~4 h into the night and a nadir occurring ~1 h into the day (lights on from 0700-1900 h). A 24-h plasma leptin rhythm was also observed in the old males (23-30 yrs), even when they were maintained under constant lighting conditions (continuous dim illumination of ~100 lux). In marked contrast, plasma leptin concentrations were relatively constant across the day and night in old perimenopausal and postmenopausal females (17-24 yrs), regardless of the lighting schedule. These data establish that rhesus monkeys, like humans, show a daily nocturnal rise in plasma leptin, and show that the magnitude of this rhythm undergoes a sex-specific aging-dependent attenuation. Furthermore, they suggest that the underlying endocrine mechanism may be driven in part by a circadian clock mechanism.

Wake-Up Call for Sleep Tech

A bad night's sleep is reason for a very big business. Sleeping pills, led by Ambien, rack up more than $2 billion a year in the United States. Then there is the revenue from overnight stays at sleep clinics, over-the-counter pills, a parade of gimmicks and a thriving business for sleep specialists.
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"Sleep is the new sex." So says psychologist Arthur J. Spielman, associate director of the Center for Sleep Disorders Medicine & Research at New York Methodist Hospital in Brooklyn, New York. "People want it, need it, can't get enough of it." The same could be said of profits. Spielman is co-author of The Insomnia Answer (Perigee Books, 2006). He is also developing light-delivering goggles that are supposed to help people reset the circadian rhythms that govern when they nod off and wake up, so they fall asleep faster and stay asleep longer. Sleep is also the new snake oil -- the promise of a good snooze from a book or a bed or a bottle. It's easy pickings.

I'm so tired of being tired

"I'm so tired," said my friend the last time we met for lunch. "The doctor said I have chronic fatigue."
She was not sleeping more than six hours a night, didn't find time to eat well or exercise, and was always achy, so it was not surprising that she felt exhausted. But now there was a label attached to her symptoms that made her feel depressed and alarmed.

CNN Presents Classroom: Sleep: A Dr. Sanjay Gupta Special

Set your VCR to record the CNN Special Classroom Edition: Sleep: A Dr. Sanjay Gupta Special when it airs commercial-free on Monday, April 17, 2006, from approximately 4:10 -- 5:00 a.m. ET on CNN. (A short feature begins at 4:00 a.m. and precedes the program.)

Lighting for the Aging Eye

In the bath, avoid fluorescents, Gilbertson advises. Instead, opt for 100-percent color rendering light bulbs, positioned on either side of your bathroom mirror. Consider installing a dimmer on bathroom lights. Research shows that very low-level regular light, or light in the red spectrum, maximizes night vision while minimizing the disruption of our circadian rhythm, Blitzer says.

Top tips to better sleep

One in three people get less than five hours of sleep a night, according to new research. But Dr John Shneerson, director of the Sleep Centre, Papworth Hospital, Cambridge, says just being aware of some simple tricks can help sleep sufferers achieve a good night's rest. "Sticking to regular bedtimes, helping the body to unwind and avoiding certain foods and drinks in the evening can induce drowsiness and enhance sleep," he says.

Ramelteon Showed Significant Reduction in Time to Fall Asleep With No Evidence of Rebound Insomnia or Withdrawal Effects

Results of a sub-analysis from a phase 3 clinical study showed that Rozerem™ (ramelteon) significantly reduced time to fall asleep in adults with chronic insomnia and showed no evidence of rebound insomnia or withdrawal effects.

Night shift: Late-night work can be bad for your health

According to studies done in the past five years by Harvard University, the National Cancer Institute and the Fred Hutchinson Cancer Research Center in Seattle, women who work the night shift have an increased risk of breast and colorectal cancer. These women have a 60 percent higher risk of breast cancer than women who have never worked nights, the research says. Because of the circadian rhythm -- the 24-hour cycle of sleep and wakefulness -- the body will never be fooled into thinking it's daytime when it's dark out. However, Attarian says there are people who over time can get used to the off-hours. Because of their genetic make-up, Attarian says, some people are just night owls.

Remodeling of astrocytes, a prerequisite for synapse turnover in the adult brain?

A stitch in time, it’s all in the mind

Time slows down in some Kung Fu movie sequences. Jet Li’s foot takes forever to land. Michelle Yeoh’s riposte is glacially slow. This showcases a state-of-the-mind technique called entering the zone: Tai chi masters say this enables aspirants to ‘go faster by going slower.’ While sceptics scoff at such paradoxes, scientists are discovering that, like biofeedback, humans may have more conscious control over their measurement and perception of time than previously thought. Some say this could even lead to chemical cues to shrinking eternity and stretching fleeting moments for those who want to throw away their watch and rock around the clock.

Bed Rest May Not Be Helpful for Threatened Miscarriage

An opinion piece in the March 24 issue of The New York Times highlights a controversial issue in obstetrics: the value of bed rest for threatened miscarriage. Although this intervention is widely prescribed, evidence of its efficacy is limited or absent, and some experts suggest that there may be deleterious effects.

Smart strategies that help you become a beautiful dreamer

Naps can be lifesavers, but if you overdo them, it may be tough to nod off during normal sleeping hours. And you don't want to nap for more than an hour or so at a stretch. "The problem is, when you start getting into two- or three-hour naps, you start resetting your circadian cycle," Dr. Hartse says. (That's your body's internal clock.)

REM sleep and paranormal phenomena

Lindsay links to an interesting article in the Washington Post - Near-Death Experiences Linked to Sleep Cycles:

As many as 10 percent of survivors of heart attacks report having a near-death experience -- such as feelings of transcendence, being surrounded by light or floating outside their bodies.

New research announced yesterday suggests a biological explanation for such phenomena: People with near-death experiences are more likely to have different sleep-wake mechanisms in their brains.

In a study comparing 55 people with near-death experiences with 55 people who had no such experiences, neurologist Kevin Nelson of the University of Kentucky found that people who reported such experiences were also more likely to report a phenomenon known as "REM intrusion," where things normally experienced during sleep carry over into wakefulness. REM is an acronym for rapid eye movement, one of the phases of sleep.

Such people wake up but still feel paralyzed or hear sounds that others do not -- as the vestiges of sleep fall away, those experiences disappear. It is not considered a disorder, but merely a variant of the brain's sleep-wake cycle.

Nelson, who published his findings in the journal Neurology, said the extreme fear or feeling of danger brought on by imminent death might trigger the brain mechanism that governs the transition between sleep and wakefulness, leading people to experience various dreamlike phenomena.

The neurologist added that religious and cultural beliefs clearly influenced near-death experiences, and stressed that his findings only spoke to how such a brain mechanism might work, and not why it would work that way.

She also links to the abstract of the paper, a Nature magazine coverage of the connection between near-death experiences and REM sleep and a related article on the connection between sleep paralysis and alien abductions. A year ago, Chris Mooney published a good article on that connection as well:

Our bodies are paralyzed while we undergo REM sleep, and for good reason (lest we act out our dreams and injure ourselves). But in some small number of cases we can actually start to wake up before paralysis wears off, and yet still remain in a dreaming state. What results is hallucination, often of some extremely scary stuff. It appears that humans have always experienced sleep paralysis and sought to explain it, resulting in well known stories of incubi and succubi--demons thought to sexually attack people in their sleep--as well as related tales from other eras and cultures.

Sleepy Americans

Study Shows that Americans are Besieged by Sleeplessness

More and more Americans work and walk around like sleep-deprived zombies, in part due to growing work hours and poor choices made in an environment that is potentially always "on" due to television and the Internet. That's according to a study published last week by the National Academy of Sciences' Institute of Medicine.

The study found that chronic sleep disorders now affect a whopping 50 million to 70 million Americans. Millions more are deprived of sleep on a semi-regular basis. In addition to environment, factors owing to physiology play a role, too. Among other things, more and more Americans are obese, which can interfere with slumber.

The study noted that drug companies are rushing sleep medicines into the sleepless void, targeting the trend as an emerging market. Some 43 million prescriptions for sleep aids were filled last year, and four new drugs will be released over the next year and a half. New sleep centers have sprung up unaccredited throughout the land, tempting Americans. "You can get a sleep study done in a strip mall now without ever meeting with a [qualified] specialist," James Wyatt of the Sleep Disorders Center at Rush University Medical Center in Chicago told the Chicago Tribune.

As noted in the sleep study, the growth in disorders seems to be the product of a failure to deal effectively with environment. That's part of a larger societal trend, say other psychologists. The New York Times ran a piece last week noting that current research shows some 9 percent of Americans suffer from problems related to "high impulsiveness."

Heritability of snoring

Interesting, in today's New York Times:

In the Genes: When the Littlest Family Member Snores, Too

Snoring may be genetic. Children who snore are almost three times as likely as others to have parents who snore. And snoring and sleep-disordered breathing are twice as common in children who test positive for allergies.

Sleep-disordered breathing — snoring is one symptom of it — is associated with poor school performance, cardiovascular troubles and daytime behavioral problems like attention-deficit hyperactivity disorder.

Researchers studied 1-year-old children participating in the Cincinnati Childhood Allergy and Air Pollution Study. Among the 681 children, 105 were habitual snorers — that is, they snored more than three nights a week. The children were also examined for allergies to various foods and other substances using a skin-prick test.

Having a positive allergy test almost doubled the risk for snoring, and having one parent who snored almost tripled the risk. Being African-American more than tripled the risk that a child would be a habitual snorer. The results were published yesterday in the journal Chest.

"If you have a child who snores frequently or loudly," said Dr. Maninder Kalra, an author of the study, "we recommend evaluation by a sleep specialist." Treatment for sleep-disordered breathing in children may involve surgery to remove enlarged tonsils or adenoids, or the use of a breathing device during sleep. Dr. Kalra is an assistant professor of medicine at Cincinnati Children's Hospital Medical Center.

Sleep-disordered breathing can be definitively diagnosed only in a sleep laboratory, and the authors point out that one limitation of their study is that they did not perform this definitive test on each child. Further, the research relied on the reporting of parents, which may not have always been accurate.

Chossat's Effect in humans and other animals

If you know what Chossat's Effect is, I guess you are a) a physiologist, b) expert in thermoregulation, and c) old. This is term that got expunged from the scientific lexicon a few decades ago, in an effort - correct me if I am wrong on this - spearheaded by the U.S. textbook companies, to replace scientific terminology named after the discoverers (and sometimes even Latin and Greek terms) with bland English neologisms.

But I love Schwann's Cells, Fallopian Tubes (or Mullerian Ducts), Purkinje Fibers, Broca's Area and the amazing Bundle of His! Those terms are memorable, make it easy to sneak in some historical context into teaching science, and have an emotional effect of bringing forth images of ancient scientists working under candlelight, sacrificing their eyesight and health, their social standing and sometimes even their lives, in the feverish hunger for knowledge.

So, what is Chossat's Effect? It comes from a certain 19th century French scientist who was studying the physiology of starvation [1]. The 'modern' term for this effect is "fasting-induced nocturnal hypothernia" (doesn't that sound like something that would prompt the students in the classroom to immediatelly stop paying attention to the teacher and instead pick-up their cell-phones and start text-messaging their friends?).

Actually, this is a very interesting area of research that is very tightly connected to circadian biology. This post is likely to be long, so feel free to skim and just focus on the first part if you are into birds, second part if you are interested in mammals, and the last part if you are into humans.

Birds

All warm-blooded animals (and yes, that includes at least some reptiles, not to mention a few heat-producing plants like stink-cabbage) exhibit a daily rhythm of body temperature. If an animal is active during the day (diurnal) and sleeps during the night, reducing the metabolic rate during the night is a good way to save energy.

Some of the smallest birds, like swifts and hummingbirds, need to feed continuously in order to stay alive. At night, when they are not able to forage (flowers are closed, it's hard to see, and owls are hunting at the time), they drop their metabolic rate, and thus body temperature, quite dramatically. The body temperature gets down as low as the environmental temperature, sometimes daringly close to the freezing point. The total drop can be as large as 40 degrees Celsius in some instances! This is called daily torpor (yup, click on that link - it is an excellent blog post) and the metabolic rate drops as much as 95% [2, 3]. This is like full-scale winter hibernation EVERY DAY!

Chossat's effect does not refer to daily torpor, though. It describes a drop in temperature during the night that is larger than the usual circadian fluctuation, in animals undergoing fasting, e.g., during spells of very bad weather (e.g., hurricanes).

Normal amplitude (daily maximum minus nightly minimum) of body temperature in birds with normal access to food ranges between about 1 and 2 degrees Celsius. For instance, a daily maximum may be 41 degrees and the nightly minimum may be 39 degrees (yes, the birds are much warmer than mammals, which makes them inhospitable to microbes that cause many mammalian diseases), which calculates to 2 degrees of amplitude.

During fasting (or food deprivation in the laboratory), the nightly minima drop down to lower levels than in fed birds. The minimum gets lower and lower with each additional night. Importantly, the daily maxima do not change at all. It is thought that it is advantageous for birds to retain their normal metabolic rates during the day so they can immediately resume foraging once the bad weather subsides. Also, if the bad weather persists for too long, the birds need the daytime metabolic rates in order to fly away [4].

According to John Wingfield's "Emergency Life-History Stage" hypothesis [5], an individual's perception of inclement weather directly affect the levels of stress hormones (e.g., corticosterone). An individual who does not perceive the bad weather to be "too bad", will reduce daytime activity and reduce night-time temperature in order to save energy - this individual has made a decision to sit it out.

On the other hand, an individual who perceives bad weather to be "really bad" (or if it lasts too long) will have higher levels of stress hormones and will attempt to fly away during the day. This is not the same mechanism as the seasonal migration, which is usually a nocturnal flight, i.e., they do not experience Zugunruhe, just stress. Stressed birds do not attempt to escape at night, at which time they have allowed their body temperature to drop by several degrees.

Nocturnal hypothermia has been studied in a large number of species of birds (see, for examples, references # 6-12), but most of the work was performed on pigeons [13-15] and quail [16]. Not all avian species exhibit this response. Laurilla at al. [18] write:

"On the other hand, many large birds that are adapted to long fasting periods as a part of their life histories, e.g. penguins and geese (Cherel et al., 1988; Castellini andRea, 1992), owls (Hohtola et al., 1994) and some raptors (McKechnie andLovegrove, 1999) do not show marked hypothermia during fasting. Some species enter hypothermia upon food restriction only when isolated from conspecifics in a laboratory environment, while in the field they remain normothermic by huddling. These observations have even led some authors to question the usefulness of the concept of hypothermia (Lovegrove andSmith, 2003)."[8]

Here is a graphic example of a fasting-induced nocturnal hypothermia in quail (from[17]). The period between the two triangles is the time (3 days) during which the birds had water but no food. Before and after, birds were fed ad libitum. Below is a graph that shows the difference between the temperature minima during the first, second and third day (left) and night (right) of food deprivation in comparison to the last three days and nights of normal feeding prior to the fasting treatment:
Much of the more recent research is looking at other environmental cues that can modify the Chossat's effect, as well as the involvement of the circadian clock in this time-specific form of thermoreguluation. For instance, some of the ambient cues that affect the response include ambient temperature [16, 20], ambient light [17], photoperiod [18, 19], single vs. repeated fasting [18, 19], caloric food restriction vs. complete food deprivation [13], social situation, e.g., opportunity for huddling [8] and presence of stationary vs. flying predators [19, 20]. Here is an example of an effect of ambient temperature on nocturnal hypothermia in fasted pigeons (from [20]). Lower the ambient temperature, deepeer the Chossat's effect:
Here is the effect of the presence of a predator (from [2]). In the presence of a perched hawk (P), nocturnal hypothermia reached normally low levels. In the presence of the flying hawk (F), temperature did not drop as much. Presumably, the pigeons kept the metabolic rate high enough to be able to fly fast if needed:
As stated above, hypothermia occurs only during the night while the temperature during the days remains normal. However, all the studies are performed either in natural conditions of day and night or in light-dark cycles in the laboratory. In constant darkness, the circadian rhythm of temperature persists and hypothermia is apparent. Moreover, the temperature drops both at the minima during the 'subjective night' and at the maxima during the 'subjective day' (from [17]):
This suggests that light has a direct (or "masking") effect on body temperature during the light-phase of the cycle. But is this effect acting directly on the thermoregulatory centers in the hypothalamus or is it mediated by the circadian clock that drives the rhythm of body temperature? In Japanese quail, the circadian pacemakers are located in the eyes. When the eyes are removed [17], both the daily maxima in the light-phase and the nightly minima during the dark phase drop, suggesting that the effect is mediated via the circadian clock, as the light perceived by the photoreceptors in the pineal gland and in the deep brain is incapable of keeping the daily maxima from dropping:
Mammals

Some small mammals, such as smallest rodents and shrews, exhibit a full-blown daily torpor either normally [21] or in response to fasting [22]. Here is an example of a daily torpor of a mouse-opposum:
In nocturnal animals, which many mammals are, body temperature is high at night when the animals are active and it drops during the day when the animals are sleeping. In rats, fasting induces diurnal hypothermia, i.e., drop of the daily minimum during the day (black circles, compared to pre- and post- treatment values in white symbols) while the nightly maxima remain unaffected [23]:
Chronic caloric food restriction leads to the drop in both the daily minima and nightly maxima of temperature [24].

All the studies until recently have studied responses in relatively small animals (both birds and mammals) with high metabolic rates and high energy needs. But do larger animals, like humans, also exhibit Chossat's effect? After all, the first documented case, that by Chossat himself, was in a dog. This was repeated recently [25]. But even dogs are pretty small compared to humans.

Recently, researchers have addressed this question in a number of species of large mammals, including sheep, goats, horses and yaks [26-29]. Some additional environmental cues were also studied, including the effects of shearing on the circadian temperature rhythm in sheep [30]. Here is a record from a goat:
Notice that, unlike in birds, both the maxima and minima gradually go down.

But, as far as I could find by digging through the literature, nobody has ever performed a similar study in humans. I am assuming that it has been noticed if body temperature drops in fasted humans, but I am not aware of a study systematically addressing this question.

Humans

A few years ago I was teaching one of many sections of an Animal Anatomy and Physiology Course. This course requires students to perform a research project. One group of students studied the effects of fasting on body temperature and blood pressure in humans.

They found 8 subjects, all healthy, athletic, non-drinking, non-smoking students ages 19-23. They were instructed to eat normally during the Day1 of the experiment. They subequently spent 36 hours in a house drinking only water and eating nothing. Every four hours, temperature and pressure were measured. By using kids' digital ear thermometers and manual sphigmomanometers they managed, for the most part, not to awaken the subjects during the night. Here are examples of body temperature of three of the subjects - Night1, followed by Day2 and Night 2:























Here are the pooled data for all eight subjects, starting with Day2 and followed by Night1 and Night2 plotted on top of each other for comparison:
Obviously, body temperature of Night2, after a day of fasting, was lower than that of Night1, after the day of normal feeding. I do not have their raw data any more, but if I remember correctly, the data for blood pressure looked very similar. I heard they had a huge breakfast, courtesy of the young researchers, at the end of the experiment.

So, Chossat's Effect appears to be operating in humans as well. Now, this is cool in itself, and I sure hope that someone with access to good clinical lab repeats this study, but there is something else about these data that really excites me. This finding can be used as a tool for studying something entirely different!

The Hypothesis

One of the first demonstrations that humans have daily rhythms involved the time-of-day dependence of time perception. In other words, our subjective "feel" of the speed of passage of time changes systematically with the time of day. At the same time, it has been known for a couple of centuries now that the subjective time perception is also altered during fever. And we know that circadian clock governs daily rhtyhms of body temperature. So, what affects the time perception: time of day or body temperature? If the time passes faster in the evening than at dawn, is it because of the circadian clock acting on the time-perception brain-centers directly, or because we are warmer at the time (which is also driven by the circadian clock)?

This question has haunted circadian researchers for decades and they have devised ever more elaborate experiments to tease the two hypotheses apart, with no avail - we still do not know. But, if by depriving the subjects of food, we can dissociate clock-time from temperature, perhaps we can address this question after all. If the subjective perception of 1 minute (do not use 1 second or 1 hour - those are durations unsuited for this experiment) is similar between the night after a fed day and the night after the fasting day, then the perception is directly driven by the circadian clock. If, on the other hand, perception of a minute changes systematically between the two nights, then we conclude that it is body temperature that affects subjective time perception. Please, someone do this! And if you do, or even if you just want to replicate the Chossat's Effect in humans, I would appreciate it if you would properly cite this post:

Bora Zivkovic, Chossat's Effect in humans and other animals (April 9, 2006), blog Circadiana, http://circadiana.blogspot.com/2006/04/chossats-effect-in-humans-and-other.html

REFERENCES:

[1] M. Chossat, Sur l'inanition, Paris, 1843

[2] Hiebert, S.M. 1990. Energy costs and temporal organization of torpor in the rufous hummingbird (Selasphorus rufus). Physiological Zoology . 63:1082-1097.

[3] Hiebert, S.M. 1991. Seasonal differences in the response of rufous hummingbirds to food restriction: body mass and the use of torpor. Condor 93:526-537.

[4] Tobias Wang, Carrie C.Y. Hung, David J. Randall, THE COMPARATIVE PHYSIOLOGY OF FOOD DEPRIVATION: From Feast to Famine, Annual Review of Physiology, January 2006, Vol. 68, Pages 223-251

[5] Wingfield, JC; Maney, DL; Breuner, CW; Jacobs, JD; Lynn, S; Ramenofsky, M; Richardson, RD, Ecological bases of hormone-behavior interactions: The "emergency life history stage", American Zoologist [Am. Zool.]. Vol. 38, no. 1, pp. 191-206. 1998.

[6] Tracy A. Maddocks, Fritz Geiser, Energetics, Thermoregulation and Nocturnal Hypothermia in Australian Silvereyes, Condor, Vol. 99, No. 1 (Feb., 1997) , pp. 104-112

[7] Randi Eidsmo Reinertsen and Svein Haftorn, The effect of short-time fasting on metabolism and nocturnal hypothermia in the Willow Tit Parus montanus, Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology, Volume 154, Number 1 (January 1984): 23 - 28

[8] Barry G. Lovegrove and Gary A. Smith, Is 'nocturnal hypothermia' a valid physiological concept in small birds?: a study on Bronze Mannikins Spermestes cucullatus, Ibis, Volume 145, Issue 4, Page 547 - October 2003

[9] MacMillen RE, Trost CH., Nocturnal hypothermia in the Inca dove, Scardafella inca, Comp Biochem Physiol. 1967 Oct;23(1):243-53.

[10] Colleen T. Downs, Mark Brown, NOCTURNAL HETEROTHERMY AND TORPOR IN THE MALACHITE SUNBIRD (NECTARINIA FAMOSA).

[11] Waite, TA, Nocturnal hypothermia in gray jays Perisoreus canadensis wintering in interior Alaska, ORNIS SCAND. Vol. 22, no. 2, pp. 107-110. 1991.

[12] Cécile Thouzeau, Claude Duchamp, and Yves Handrich, Energy Metabolism and Body Temperature of Barn Owls Fasting in the Cold, Physiological and Biochemical Zoology, volume 72 (1999), pages 170–178

[13] Rashotte ME, Henderson D., Coping with rising food costs in a closed economy: feeding behavior and nocturnal hypothermia in pigeons, J Exp Anal Behav. 1988 Nov;50(3):441-56.

[14] R. Graf, S. Krishna and H. C. Heller, Regulated nocturnal hypothermia induced in pigeons by food deprivation, Am J Physiol Regul Integr Comp Physiol 256: R733-R738, 1989

[15] Michael E. Rashotte, Iuri F. Pastukhov, Eugene L. Poliakov, and Ross P. Henderson, Vigilance states and body temperature during the circadian cycle in fed and fasted pigeons (Columba livia), Am J Physiol Regul Integr Comp Physiol 275: R1690-R1702, 1998

[16] Hohtola E, Hissa R, Pyornila A, Rintamaki H, Saarela S., Nocturnal hypothermia in fasting Japanese quail: the effect of ambient temperature, Physiol Behav. 1991 Mar;49(3):563-7.

[17] Herbert Underwood, Christopher T. Steele and Bora Zivkovic, Effects of Fasting on the Circadian Body Temperature Rhythm of Japanese Quail, Physiology & Behavior, Vol. 66, No. 1, pp. 137–143, 1999

[18] Mirja Laurila, Tiina Pilto, Esa Hohtola, Testing the flexibility of fasting-induced hypometabolism in birds: effect of photoperiod and repeated food deprivations, Journal of Thermal Biology 30 (2005) 131–138

[19] MIRJA LAURILA, THERMOREGULATORY CONSEQUENCES OF STARVATION AND DIGESTION IN BIRDS, PhD Dissertation, Faculty of Science, Department of Biology, University of Oulu, 2005 (http://herkules.oulu.fi/isbn9514277147/isbn9514277147.pdf)

[20] Mirja Laurila, Esa Hohtola, The effect of ambient temperature and simulated predation risk on fasting-induced nocturnal hypothermia of pigeons in outdoor conditions, Journal of Thermal Biology 30 (2005) 392–399

[21] Francisco Bozinovic, Gricelda RuÍz, Arturo CortÉs & Mario Rosenmann, Energetics, thermoregulation and torpor in the Chilean mouse-opossum Thylamys elegans (Didelphidae), Revista Chilena de Historia Natural 78: 199-206, 2005

[22] Lovegrove BG, Raman J, Perrin MR., Daily torpor in elephant shrews (Macroscelidea: Elephantulus spp.) in response to food deprivation, J Comp Physiol [B]. 2001 Feb;171(1):11-21.

[23] Kei Nagashima, Sadamu Nakai, Kenta Matsue, Masahiro Konishi, Mutsumi Tanaka, and Kazuyuki Kanosue, Effects of fasting on thermoregulatory processes and the daily oscillations in rats, Am J Physiol Regul Integr Comp Physiol 284: R1486–R1493, 2003.

[24] Yoda T, Crawshaw LI, Yoshida K, Su L, Hosono T, Shido O, Sakudara S, Fukuda Y & Kanosue K (2000) Effects of food deprivation on daily changes in body temperature and behavioural thermoregulation in rats. Am J Physiol 278: R134-R139.

[25] G. Piccione, G. Caola and R. Refinetti, Daily Rhythms of Blood Pressure, Heart Rate, and Body Temperature in Fed and Fasted Male Dogs, J. Vet. Med. A 52, 377–381 (2005)

[26] Giuseppe Piccione, Giovanni Caola, Roberto Refinetti, Circadian rhythms of body temperature and liver function in fed and food-deprived goats, Comparative Biochemistry and Physiology Part A 134 (2003) 563–572

[27] Piccione, G., Caola, G., Refinetti, R., 2002a. Circadian modulation of starvation-induced hypothermia in sheep and goats. Chronobiol. Int. 19, 531–541.

[28] Piccione, G., Caola, G., Refinetti, R., 2002b. The circadian rhythm of body temperature of the horse. Biol. Rhythm Res. 33, 113–119.

[29] Xing-Tai Han, Ao-Yun Xie, Xi-Chao Bi, Shu-Jie Liu and Ling-Hao Hu, Effects of high altitude and season on fasting heat production in the yak Bos grunniens or Poephagus grunniens, British Journal of Nutrition (2002), 88, 189–197

[30] Giuseppe Piccione, Giovanni Caola, and Roberto Refinetti, Effect of shearing on the core body temperature of three breeds of Mediterranean sheep, Small Ruminant Research 46 (2002) 211–215

Influence of Light Cycle on Dominance Status and Aggression in Crayfish

Understanding the role of serotonin in depression has led to development of anti-depressant drugs, like Prozac. Much of the research in this area has been performed in Crustaceans: lobsters and crayfish. The opposite behavioral state of depression, something considered a normal state, could possibly best be described as self-confidence.

Self –confidence is expressed differently in different species, but seems to always be tied to high status in a social hierarchy. In crayfish, self-confidence is expressed as aggression. When two individuals meet, they engage in aggressive behavior (Fig. 1, see below).

Outcome of this “fight” determines the social status and leads to long-term changes in the brain, particularly the serotonergic system, associated with either winning (self-confidence) or losing (depression). Thus, experiencing a victory alters the behavior in a way that makes subsequent victories more likely and vice versa (Goessmann et al., 1999; Huber et al., 1997; Issa et al., 1999). Experimental manipulations of the serotonergic system by, for instance, injections of serotonin or Prozac, result in changes in aggressive behavior and social status.

Two standard experimental practices are used in the study of aggression in crustaceans. In one, two or more individuals are placed together in an aquarium and left there for a long period of time (days to weeks). After the initial aggressive encounters, the social status of an individual can be deduced from its control of resources, like food, shelter and mates.

In the other paradigm, two individuals are allowed to fight for a brief period of time (less than an hour), after which they are isolated again and re-tested the next day at the same time of day. Neither of the two approaches is capable of addressing two questions: how fast do the changes in the brain start to occur, and, what if any effect can time of day have on the behavior. Because of this, the existing literature, sometimes implicitly sometimes explicitly, suggests that the rule "once a winner, always a winner" is true, and that the neural changes are fast and induced by a single agonistic encounter.

To address these two questions, Amy Hughes, then an undergraduate student in our department, now an epidemiology graduate student at the University of Minnesota, and I have designed a different experimental procedure. This study began as a project in the Animal Anatomy and Physiology laboratory class in which Amy was my student. However, the initial data were interesting and we easily persuaded Dr.Robert Grossfeld to help support further study as Amy's honors research project. Apart from securing funding, animals, equipment and space, Dr.Grossfeld proved as a valuable resource on crustacean neurobiology and on keeping the research focused and 'on target'.

We kept individual crayfish in social isolation for a month prior to the experiment. They were kept in a light-dark cycle consisting of 12 hours of light and 12 hours of darkness. Then, we matched the individuals by size and tested their aggressive behavior and social status by behavioral observations for about 15 minutes every 3 hours over a period of 24 hours. One set of tests was initiated at “dawn” or lights-on time, which was 8am and ended at 8am on the next day. The other set of tests started at dusk, or lights-off time, which was 8pm and ended at 8pm the following day. We scored the behaviors using the scoring systems modified from standard literature (Karavanich and Attema 1998, see Table 1).

It has been known for quite a long time now that crayfish are nocturnal animals and that the levels of general locomotor activity are much greater during the night than during the day (Page and Larimer 1972). However, no specific behaviors were monitored in such studies. Analysis of our data shows that aggressive behavior is much more intense and frequent during first three hours of night than at other times during the 24-hour cycle (Fig.2). This is somewhat different from the general locomotor activity which tends to be high throughout the night, not concentrated to the early portion of the night.

Expectation that brain changes associated with winning or losing are initiated very fast were met by about half of the tested pairs of animals in each data-set. In these animals, the social status was determined during the first or second time-point and remained stable for the rest of the experiment (Figs 3 and 4).

However, in about a half of the pairs in each data-set, the stable social hierarchy was not achieved during the 24-hour period of the experiment. Levels of aggression and the final outcome varied during this period, suggesting that the brain changes in these individuals did not occur as fast, and that there is considerable plasticity in behavioral states during the first 24 hours of social interactions (Fig. 5).

Interestingly, more switches in social status occurred during the day than during the night. This can be explained in several different ways. For instance, a very high level of aggression may be necessary to trigger the changes in the brain, and such highly aggressive encounters are most likely to occur in the early night. Thus, encounters during the previous day were not intense enough to result in long-term changes in the brain. Further research needs to be done to study the rate at which an experience of victory or loss triggers permanent changes in the brain.

Also, there is no strong evidence in the literature that the two individuals can actually recognize each other. However, they can recognize the social status of their opponents. During the fights, crayfish squirt urine at each other (Zulandt Schneider et al. 2001, also see figure on the right where urine is colored with a fluorescent dye). Chemical signature of the urine was not studied at all so far, but it apparently carries information about the brain-state of the animal.

Since socially dominant animals have higher levels of serotonin in their nervous system, it is likely that the chemical message in the urine is related to serotonin. The most likely candidate, a direct metabolite of serotonin, is melatonin. A recent study has shown that levels of melatonin in these animals are very high in the early night, drop off somewhat during the rest of the night and are practically undetectable during the day (Tilden et al. 2003). Our results are consistent with the notion that urinary melatonin (or its metabolite) may be the signal carrying information about social status, as the most intense fights and most definitive outcomes occurred during the early night. This hypothesis needs to be tested in the future.

This research has been displayed on the poster session of the Undergraduate Research Sympsium at North Carolina State University. It is unlikely that any of the participants in this study will follow up on this work in the near future. Though there is not sufficient data to publish a 'real' paper, we feel that the information should be available online for those who study aggression, social dominance, serotonin, melatonin and circadian rhythms in crustaceans.

The project, as presented by Amy on the poster, is reproduced below. We will appreciate proper attribution in case you follow up on this study. The correct reference is:

Amy Hughes, Bora Zivkovic and Robert Grossfeld, Influence of Light Cycle on Dominance Status and Aggression in Crayfish (April 6, 2006), Circadiana blog; http://circadiana.blogspot.com/2006/04/influence-of-light-cycle-on-dominance.html

Influence of Light Cycle on Dominance Status and Aggression in Crayfish

Amy Hughes, Bora Zivkovic and Robert Grossfeld

INTRODUCTION

Conflicts arise within a population of social animals over the allocation of important resources. Interactions between crayfish determine a social hierarchy both when resources are in dispute and when they are not. Even when no resource except space is contested, crayfish will fight. Several factors determine the outcomes of these contests, including physical attributes of an organism and past experience (Goessmann et al., 1999; Huber et al., 1997; Issa et al., 1999; Zulandt Schneider et al., 2000).

In most previous studies of crustacean aggressive behavior, animals were tested at the same time for consecutive days. In those experiments, winning enhanced further success by heightening aggression, whereas losing decreased subsequent chances for dominance by lowering aggression.

Recognition of the opponent’s attributes plays a role in changes in social status, e.g. detection of the opponent’s relative dominance status and recognition of a prior opponent (Goessmann et al., 1999; Karavanich & Attema, 1998; Zulandt Schneider et al., 2000).

The purpose of this study was to test the hypothesis that the establishment and stability of a dominance hierarchy may be plastic over sequential pairings during the first day, and influenced by time of day.

MATERIALS AND METHODS

• Forty-four adult male crayfish (Procambarus clarkii) were kept in separate containers with no visual or physical contact for one month on a LD12:12 light cycle (lights on automatically at 8:00 AM and lights off at 8:00 PM). They were fed fish food pellets every 7 days.
• Pairs of 20-40 g inter-molt males were matched closely by weight (within 1 g).
• For testing, each pair was placed in a small container [30 cm (L) x 20 cm (W) x 15 cm (D)] filled approximately two-thirds full with fresh water.
• The same pairs encountered each other for approximately 15 min every 3 h over a 24-h period.
• One set (11 pairs) was tested first at the time of lights-off (i.e. 8:00 PM).
• The other set (11 pairs) was tested first at the time of lights-on (i.e. 8:00 AM).
• The time of initiation of a fight, duration of the fight, number and type of agonistic behaviors, and final “winner” of the encounter were recorded based on visual observation. The total agonistic behavior scores were compared using a one-tailed Student t- test.

Figure 1. Two crayfish in a fight













Table 1. The scoring table of behavior. Modified from Karavanich and Attema 1998. Insert: an example of a meral spread.

Retreat – moving away, turning away: -1

Strike and Rip – using claws unrestrained: +4

Claw Lock – using claws to grasp: +3

Meral Spread – threatening display of claws: +2

Approach – walking towards, turning towards other animal: +1



Figure 2: Agonistic interactions escalate during the early evening following lights off (i.e. 8:00 PM). n=44; p<0.0001.

Figure 3: Behavior scores of crayfish tested first at 8:00 PM
In 7 of 11 pairs, the animal that established dominance at the first or second time point (8:00 PM or 11:00 PM, respectively) remained the more aggressive animal the entire 24 h.
Figure 4: Behavior scores of crayfish tested first at 8:00 AM
In 5 of 11 pairs, the animal that established dominance at the first or second time point (8:00 AM or 11:00 AM, respectively) remained the more aggressive animal the entire 24 h.
Figure 5: In 10 pairs, a stable dominance relationship was not established during the first two time points.
The number of switches in social status was greater during the light phase than during the dark phase (p<0.01). In 4 of 11 pairs (8:00 PM set), dominance switched over the 24 hours:
In 6 of 11 pairs (8:00 AM set), dominance switched over the 24 hours:
CONCLUSIONS

Crayfish agonistic behaviors are most frequent during early evening following lights-off as compared to early morning following lights-on.

In about half of the pairs, the relative levels of aggression (“dominance status”) remained stable after the first or second encounter, whereas in the other pairs it changed at one or more times during the 24 hour testing period, especially during the daytime. The latter results suggest plasticity in social status during the first day that crayfish are paired.

REFERENCES

Balzer I.; Espinola I.R.; Fuentes-Pardo B., Daily variations of immunoreactive melatonin in the visual system of crayfish, Biology of the Cell, Volume 89, Number 8, November 1997, pp. 539-543(5)

Goessmann, C., Hemelrijk, C., and Huber, R. (2000). The formation and maintenance of crayfish hierarchies: Behavioral and self-structuring properties. Behav Ecol Sociobiol. 48:418-428.

Huber, R., Smith, K., Delago, A., Isaksson, K., and Kravitz, E. A. (1997). Serotonin and aggressive motivation in crustaceans: Altering the decision to retreat. Proc. Natl. Acad. Sci. USA. 94: 5939-5942.

Issa, F.A., Adamson, D.J. and Edwards, D.H. (1999). Dominance hierarchy formation in juvenile crayfish Procambarus clarkii. J. Exp. Biol. 202:3497-3506.

Karavanich, C. and Attema, J. (1998). Individual recognition and memory in lobster dominance. An. Behav.. 56:1553-1560.

Page,Terry L. and Larimer, James L. (1972), Entrainment of the circadian locomotor activity rhythm in crayfish: The role of the eyes and caudal photoreceptor, Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, Volume 78, Number 2: 107 - 120

Tilden, Andrea R. , Brauch Rebecca, Ball Ryan, Janze Aura M. ,Ghaffari Ali H. , Sweeney Catherine T., Yurek Jamie C. and Cooper Robin L. (2003), Modulatory effects of melatonin on behavior, hemolymph metabolites, and neurotransmitter release in crayfish, Brain Research 992: 252–262

Zulandt Schneider, R. A., Huber, R. and Moore, P.A. (2001), Individual and status recognition in the crayfish, Orconectes rusticus: The effects of urine release on fight dynamics. Behav. 138:137-153.

Clocks in Bacteria II: Adaptive Function of Clocks in Cyanobacteria

In the previous two posts, here and here, I have mentioned how the discovery of circadian clocks in Cyanobacteria changed the way we think about the origin and evolution of circadian clocks. Quite soon after the initial discovery, the team from Carl Johnson's laboratory published two papers [1,2] describing a more direct test of adaptive function of circadian clocks in the Synechococcus elongatus.
Wild-type and various clock-mutants in Synechoccocus, when raised in isolation in light-dark cycles, have comparable reproductive rates. When raised in constant light, they fare even a little better, i.e., multiply faster. Thus, in isolation, clock does not appear to confer adaptive advantage.
However, when the strains are cultured together, two strains grown in the same petri-dish, and exposed to light-dark cycle, the strain whose endogenous period is closer to the period of the environmental cycle "wins" the contest. This suggests that circadian clock confers fitness in rhythmic environments. In constant light, arrhythmic mutants outperform rhythmic strains.
Here is how Johnson describes the experiments (from a book chapter not available online):

"The authors’ laboratory tested the adaptive significance of circadian programs by using competition experiments between different strains of the cyanobacterium Synechococcus elongatus (Ouyang et al., 1998; Woelfle et al., 2004). For asexual microbes such as S. elongatus, differential growth of one strain under competition with other strains is a good measure of reproductive fitness. In pure culture, because the strains grew at about the same rate in constant light and in LD cycles, there did not appear to be a significant advantage or disadvantage in having different circadian periods when the strains were grown individually. The fitness test was to mix different strains together and to grow them in competition to determine whether the composition of the population changes as a function of time. The cultures were diluted at intervals to allow growth to continue. Different period mutants were used to answer the question, ‘‘Does having a period that is similar to the period of the environmental cycle enhance fitness?’’ The circadian phenotypes of the strains used had freerunning periods of about 22 h (B22a, C22a) and 30 h (A30a, C28a). These strains were determined by point mutations in three different clock genes: kaiA (A30a), kaiB (B22a), and kaiC (C22a, C28a). Wild type has a period of about 25 h under these conditions. When each of the strains was mixed with another strain and grown together in competition, a pattern emerged that depended on the frequency of the LD cycle and the circadian period. When grown on a 22-h cycle (LD 11:11), the 22 h-period mutants could overtake wild type in the mixed cultures. On a 30-h cycle (LD 15:15), the 30 h-period mutants could defeat either wild type or the 22 h-period mutants. On a ‘‘normal’’ 24-h cycle (LD 12:12), the wild-type strain could overgrow either mutant (Ouyang et al., 1998). Note that over many cycles, each of these LD conditions have equal amounts of light and dark (which is important, as photosynthetic cyanobacteria derive their energy from light); it is only the frequency of light versus dark that differs among the LD cycles. Figure 1 shows results from the competition between wild type and the mutant strains (Ouyang et al., 1998).

Clearly, the strain whose period most closely matched that of the LD cycle eliminated the competitor. Under a nonselective condition (in this case, constant light), each strain was able to maintain itself in the mixed cultures. Because the mutant strains could defeat the wild-type strain in LD cycles in which the periods are similar to their endogenous periods, the differential effects that were observed are likely to result from the differences in the circadian clock. A genetic test was also performed to demonstrate that the clock gene mutation was specifically responsible for the differential effects in the competition experiment (Ouyang et al., 1998). Because the growth rate of the various cyanobacterial strains in pure culture is not detectably different, these results are most likely an example of ‘‘soft selection’’ where the reduced fitness of one genotype is seen only under competition (Futuyma, 1998).

In a test of the extrinsic versus intrinsic value of the clock system of cyanobacteria, wild type was competed with an apparently arrhythmic strain (CLAb). As shown in Fig. 2, the arrhythmic strain was defeated rapidly by wild type in LD 12:12, but under competition in constant light, the arrhythmic strain grew slightly better than wild type (Woelfle et al., 2004). Taken together, results show that an intact clock system whose freerunning period is consonant with the environment significantly enhances the reproductive fitness of cyanobacteria in rhythmic environments; however, this same clock system provides no adaptive advantage in constant environments and may even be slightly detrimental to this organism. Therefore, the clock system does not appear to confer an intrinsic value for cyanobacteria in constant conditions."

It is telling how many control experiments they had to do in order to eliminate various alternative explanations. They had to show that mutations in clock genes do not have additional effects on the ability of the cell to grow and reproduce. Check. They had to show that clock mutations do not affect the ability of the cells to utilize the food and light energy. Check. They had to show that clock mutations do not affect any conceivable way by which one strain can, perhaps by secreting chemicals, actively disrupt the health of the other strain. Check. And in the end, although they demonstrated that "resonance", i.e., similarity between environmental cycle and the intrinsic period confers some advantage, they still could not state with certainty that this "proves" that the circadian clock has an adaptive function.
Here is Johnson [3] again:

"The original adaptation of circadian clocks was presumably to enhance reproductive fitness in natural environments, which are cyclic (24h) conditions. We can refer to this situation as an adaptation to extrinsic conditions. However, some researchers have proposed that circadian clocks may additionally provide an "intrinsic" adaptive value (Klarsfeld 1998; Paranjpe 2003 and Pittendrigh 1993). That is, circadian pacemakers may have evolved to become an intrinsic part of internal temporal organization and, as such, may have become intertwined with other traits that influence reproductive fitness in addition to their original role for adaptation to environmental cycles. Note that a rigorous evolutionary biologist would no longer consider an intrinsic value for clocks to be an adaptation if their original extrinsic value has been lost. However, if clocks retain extrinsic value and additionally accrue intrinsic value, then they would still be considered an adaptation."

Testing if a trait is an adaptation is a very difficult task. Testing if something as ubiquitous as a circadian clock is an adaptation is even harder. Can you imagine testing if using ATP for energy storage, or using DNA for information storage are adaptations? Are there organisms that do not use these, so we can use them in comparative or competitive studies?

In his book Adaptation and Environment (1990), Robert Brandon came up with five criteria that need to be satisfied in order to determine if a trait is an adaptation (thanks to Robert Skipper for a reminder of this):

"One must have

1. evidence that selection has occurred;
2. an ecological explanation of the fact that some types are better adapted than others;
3. evidence that the trait in question is heritable;
4. information about the structure of the population, including both demic structure and the structure of the selective environment;
5. phylogenetic information concerning what has evolved from what."

The early cyanobacterial studies have shown criterion #3 to be correct. The competitive assay studies started cracking the criterion #2. In the next post on this topic, I will describe some studies that started investigating the criterion #5, with some additional evidence for criteria Nos. 1, 2 and 4. Apparently, we still have a long way to go. Johnson again:

An example from the circadian literature of a ‘‘just-so’’ story that the author has personally promulgated is that of ‘‘temporal separation’’ of photosynthesis and nitrogen fixation in cyanobacteria. In nitrogen-fixing unicellular bacteria, nitrogen fixation is often phased to occur at night. Nitrogen (N2) fixation is inhibited by low levels of oxygen, which poses a dilemma for photosynthetic bacteria because photosynthesis generates oxygen. Mitsui et al. (1986) proposed that the nocturnal phasing of nitrogen fixation was an adaptation to permit N2 fixation to occur when photosynthesis was not evolving oxygen, and the author has repeated this hypothesis in several publications (Johnson et al., 1996, 1998). This hypothesis would predict that cyanobacterial growth in constant light would be slower than in a light/dark (LD) cycle because nitrogen fixation would be inhibited under these conditions and therefore the growing cells might rapidly become starved for metabolically available nitrogen. The problem is that cyanobacteria grow perfectly well in constant light—in fact, they grow faster in constant light than in LD cycles, presumably because of the extra energy they derive from the additional photosynthesis. This result is inconsistent with the ‘‘temporal separation’’ hypothesis. It does not mean that the ‘‘temporal separation’’ hypothesis is incorrect—in fact, the author believes that under appropriate (but as yet untested) conditions of medium, light, and carbon dioxide, the ‘‘temporal separation’’ hypothesis will emerge triumphant. Nevertheless, the point here is that ‘‘temporal separation in cyanobacteria’’ is an example of a ‘‘just-so’’ circadian story that we like to tell without its being rigorously supported by appropriate data. This was the conclusion of Gould and Lewontin (1979) for many investigations in the field of population biology, and this criticism is on target."

[1] YAN OUYANG, CAROL R. ANDERSSON, TAKAO KONDO, SUSAN S. GOLDEN, AND CARL HIRSCHIE JOHNSON, Resonating circadian clocks enhance fitness in cyanobacteria, Proc. Natl. Acad. Sci. USA, Vol. 95, pp. 8660–8664, July 1998

[2] Mark A. Woelfle, Yan Ouyang, Kittiporn Phanvijhitsiri and Carl Hirschie Johnson, The Adaptive Value of Circadian Clocks: An Experimental Assessment in Cyanobacteria, Current Biology, Vol. 14, 1481–1486, August 24, 2004,

[3] Carl Hirschie Johnson, Testing the Adaptive Value of Circadian Systems, Methods in Enzymology, Volume 393 , 2005, Pages 818-837