Wednesday, April 20, 2005

Happy Anniversary Tangled Bank

The Tangled Bank

It is a great honor to be the host of the Grand Super-Special First Anniversary Edition of The Tangled Bank. Tangled Bank was the first blog carnival I ever heard of, and is still my favourite (which says a lot as I am a really obsessive blog Carnie).

The Tangled Bank was first announced on April 13th 2004 and the first issue was posted on April 21st 2004. If you check the archives of the Tangled Bank (and newbies should read the year-worth of posts - it's that much fun!) you'll see that the quality of individual posts was always very high, but that the carnival as a whole has grown in size, quality and scop e.

So, of course, I am very excited to announce that this Happy Anniversary Edition is continuing this trend. I enjoyed reading each and every submitted post (an editor's privilege, not "just a job" in this case) and I hope you will love them, too. So, w ithout too much ado, let's get started.

Let's start with the carnival's founder - he deserves the place of honor today. PZ Myers of Pharyngula, as always, edifies us all with some really cool science. This time, PZ chose to teach us how to make a vulva. And no, you have not mistakenly stumbled into the Carnival of Sin - the post is perfectly work-safe.

PZ's post is not a joke, but Joe Dunckley has been writing about scientific April Fools jokes recently and why they don't work (or, rather, why they work too well).

Mike the Mad Biologist uses debunking a creationist explanation of antibiotic resistance to talk about evolution, pleiotropy and other stuff.

If you think the Creationist stickers in textbooks are a new development, think again. Dave, a physicist blogging on Second Order Approximation translates for us the old Osiander Sticker that warns the audience that Copernicus's stuff is "just a theory". No kidding.

Mad House Madman from the Chronicles of a Medical Mad House writes a post about a resident (himself) who recently had a daughter and how he is dealing with the medicine and child at the same time, and how the lines blur (to a hillarious effect).

Chris of Mixing Memory wrote a thought-provoking article on the way scientific community deals with unexpected (paradigm-shifting?) findings.

Kevin P Menard of Technogypsy loves doing science, perhaps even writing papers, but he hates working on patents: its like anti-science: Patent Hell

One cannot go to Carl Zimmer's blog Loom and pick just one post. So, why should I be the one to deprive you of the embarassment of riches? Thus, here are three recent posts: the first about the brain of the "Hobbit" humanoid fossil, the second about the strange sex life of snails, and the third about evolution of the HIV virus.

Syaffolee is a microbiologist so we are used to reading about strange invisible critters like this one about every pathogenic bacteria's nightmare: the bdellovibrio.

This, from my other blog Science And Politics, may look like a science-fiction hypothesis, but it will make you look at viruses in a different light.

These two are my picks - from Keat's Telescope: about bacteria that love salty pretzels but not beer (as far as we know), and how domestication of animals may have been a slower process than we previously thought.

Aydin Orstan of Snailstales writes that Slug Shell offers 'flimsy' evidence for evolution. I love that picture!

Mike of 10000 Birds introduces a magnificient species in this issue: the Black Crowned Night Heron. Gorgeous! My kind of bird....

Bigwig of Silflay Hraka, the genius behind the concept of blog carnivals and founder of the Carnival of Vanities, has a whole series o f beautiful posts about birds (and occasionaly other animals) of Iraq. He sent us his latest entry on Bee-Eaters who may or may not eat (just) bees.

Wolverine Tom, a geologists, sends a post on the Archeocyathids, an extinct invertebrate since the Cambrian.

David of Science and Sensibility, noted a lack of plant related posts in science blogs and set out to write one and, as he writes, "in the process I think I managed to find out why they are so scarce - it was really ha rd!"

Jennifer Forman Orth of the Invasive Species Blog, another regular at Tangled Bank since the very beginning, makes an announcment that you HAVE to read today, April 20th: Death To Smoochy. You'll have to click on the link to see what it is all about.

Ever wonder how long you might have left? Ironman at Political Calculations has put together a tool that models average remaining life expectancy in the U.S.

Orac of Respectful Insolence tells "how bogus alternative medicine doesn't just hurt patients, but siphons off NIH money that could go to more promising research. The anecdote leads into questioning whether the National Center for Complementary and Alternative Medicine is the best use of our limited taxpayer dollars for medical research, given that the NIH budget is going to be flat or even slightly decreasing and the NSF budget actually took a hit last year."

Denni of Liquorice Lovers writes about the many unexpected benefits of liquorice (licorice) root (Glycorrhiza glabra). There are so many listed in that post that I forgot them all...perhaps I should take some licorice to help me remember as one of the proposed benefits is enhancement of memory. Should NIH fund further studies?

Radagast of Rhosgobel wrote this post as a short descriptoin of the evolutionary history of the appendix, aimed primarily at one of his students who had just had an emergency appendectomy.

Nuthatch of Bootstrap Analysis is an ecologist new to blogging. This link is to a "post he wrote following an introduction to his field site, outlining his strong sense of place, as one of the lucky minority of biologists that has spent his whole life a nd career in just one place": Bioregionalism

Revere at Effect Measure wrote about the Trouble in the House of Plastic. "This concerns the just published review of 115 papers on low dose effects of bisphenol A by Fred vom Saal in the current Environmental Health Perspectives. It has already garnered press cove rage but some of the more important points have been ignored. Also a description of why we are concerned with endocrine disruptors."

Pseudonymous UNC Student thought there was something fishy about the new environmenta list student group on campus. After some investigation, the true colors started to appear, described in a three-part post here, here and here.

Students sometimes do not have the perspective on how recent some of the scientific findings in their textbooks are (or how old I am!): Clock Genetics - Short History, right here on Circadiana (and while you're here, look around!)

Dave of Cognitive Daily sent two super-cool posts: Making perceptual categories, or why Nora calls me "Daddy" is about how experts are able to make fine-grained distinctions between items in their field of expertise, and how p eople can be trained to do it; and Why we can't all be divas is a fun little experiment showing that professional musicians can tell when the melody is played in the wrong key, but non-musicians can't (an easi ly observed phenomenon in your local karaoke bar!).

Saint Nate writes about The Conrad Phenomenon. It is a review of a discussed topic in acquired linguistics that actually has very little evidence supporting it.

From Vaughan of Mind Hacks, this post on psychosis and the mundane is a discussion of the mundane and the anomalous in psychosis and how it fits with research into psychosis-like experience in the wider population.

Speaking of hacking brains, here's Carl Zimmer again, on the brave new world of mind-control.

Thank you all for coming. I hope you have enjoyed the Carnival. The next edition of The Tangled Bank will be at Buridan's Ass in two weeks. Send your submissions to: host AT tangledbank DOT net, or pzmyers AT pharyngula DOT com, or to buridans AT buridansass DOT com.

The Tangled Bank.

Sunday, April 17, 2005

Clock Genetics - A Short History

As I have mentioned before, there was quite an angst in the field of chronobiology around 1960s about the lack of undestanding of circadian and other rhythms at cellular and subcellular levels. Experiment involved manipulation of the environment (e.g,. light cycles) and observing outputs (e.g., wheel-running rhythms), while treating the clock, even if its anatomical location was known, as a "black box". Breaking into the black box was one of the most important goals of the field, and, until recently, it was a tough box to break. Is the molecular clock running with 24 hour cycles? Or is it a sum of a series of shorter (ultradian) cycles? Or is it a sum of activities of numerous coupled cells?

Much of the early research on the biochemistry of circadian rhythms was performed in Protists, e.g, Euglena, Paramecium, Acetabularia and Gonyolax polyedra, yet only one clock mutant was discoverd in Chlamidomonas - the protists' genomes proved to tough to crack. When I took my introductory chronobiology course in Spring of 1994., only three clock mutations were known: the tau-mutant in hamsters (Mesocricetus aureatus), the period (per) mutation in fruit-flies (Drosophila melanogaster), and the frequency (frq) mutation in the bread mold (Neurospora crassa).

I have written more about the tau-mutant in hamsters before. While wild-type hamsters have a natural freerunning period in constant darkness almost exactly 24 hours, the tau-mutant has a 20-hour day, while in heterozygotes (crosses between the wild type and mutants) it is somewhere in between: 22 hours. However, it took decades before the real nature of the mutation was cracked. The tau-mutant hamster actually proved to be a good animal model for a familial human circadian disorder (extreme "larkiness"), as it is the mutation in the same gene - Caseine Kinase 1 epsilon - that is responsible for both.

The bread mold (Neurospora crassa) grows mycelia inside glass tubes. Every 24 hours or so it stops growing, grows hyphae and conidiae for a little while, then goes on with mycelial growth. The "freak" (frq) mutation in Neurospora has several different variants with different periods (or lack of rhythm altogether). This mutation has been traced to a gene on the chromosome VII R.

In fruitflies, the period ("per") mutation had three variants. While wild-type Drosophila cycles with about 24 hours period, the perS (short) freeruns with a period of about 19 hours, perL (long) with a period of about 29 hours, and per0 (null) is completely arrhythmic. The mutation was localized to the X chromosome. If a mosaic fruitfly is constructed in which one side of the brain possesses perL and the other side perS, the fly as a whole expresses both rhythms of activity: the 19-hour and the 29-hour rhythm cross over each other on the actograph. If a perS brain is transplanted into the abdomen of the Per0 fly, the previously arrhythmic animal develops a circadian rhythm with perS specific period of 19 hours.

These three mutants were discovered in the 1980s and 1990s, all three quite serendipitously, and not much happened for a while. In the meantime, various models were designed to account for the behaviors of circadian clocks, including in clock mutants. For instance, Ehret came up with a chronon model. In this model, a series of genes induce each other's expression, i.e., protein A induces trasncription of gene B, protein B induces expression of gene C and so on until the last protein in the series, about 24 hours later, induces expression of gene A again. This model is, actually, not that far from the currently understood mechanism of interlocking trasncription/translation feedback loops (see the Dunlap paper I linked to a couple of days ago).

Another interesting model from the past was put forward by Njus. According to his cell-membrane model, daily oscillations in ion concentrations feed back on transport proteins in the membrane. Discarded after the discovery of clock-genes, this model is now getting a second look, as some of the recent data suggest involvement of the membrane (or at least a non-genomic activity of the cell) in circadian rhythm generation in some organisms. I plan to write a whole long post soon about the old and new data that challenge the gene-only model for the circadian clock.

So, everything I wrote above was known in Spring of 1994. The following year, the explosion starts. In 1995, Amita Seghal discovers the second clock gene in the fruitfly: timeless (tim). As the molecular techniques got more and more sophisticated, discovery of new clock genes became a small cottage industry. Discovery of the Clock (clk) gene in the mouse by Joe Takahashi opened up mammalian genome to investigation. Soon, it was seen that the fruitfly and the mouse have very similar molecular players involved in generation of circadian rhythms: there is Period in mammals, Clock in fruitflies, Cycle in fruitflies is the same as Bmal in mammals, both have Cryptochrome (cry) as a component of the system, Caseine kinase 1 epsilon is known in flies as Doubletime.

A couple of dozen genes involved in generation or modulation of circadian rhtyhms in mice and flies have been discovered since then and most have been shown to be working in an almost identical ways in fish, amphibians, reptiles and birds, as well as a number of invertebrates. The other organisms followed suit. White-collar (wc, sometimes refered to as "water closet" in the halls of conferences) in Neurospora, KaiA, B and C in the cyanobacterium Synechococcus, Toc in plants and others soon became known. By about the year 2000 this progress was noted two years in a row by Science Magazine when it gave the field runner-up position in its annual list of most exciting findings of the year. Currently, it is agreed that the circadian clock is the best understood behavioral system at the genetic level. For details, read Dunlap's paper I linked below..

Saturday, April 16, 2005

Downloadable Database of Phase Response Curves

One of the most useful chronobiological databases available online is the PRC Atlas. Compiled by Dr.Carl Johnson of Vanderbilt University, it contains hundreds of published and unpublished Phase-Response Curves. One can sort the Curves by species or by type of stimulus (e.g., light pulses, pulses of varius chemicals, dark pulses on constant-light background, etc.) and one is also able to manipulate (i.e., re-plot) the data to one's own liking.

The page contains links to four important papers/reviews of the utlility of PRC-construction in studies of circadian rhythms as well as a list of further references. The files are available for PC and Mac and one can use them even on very old operating systems as the files were prepared more than a decade ago.

There is also a useful little page of comments on the way PRCs are plotted in the Atlas and are usually plotted (or should be plotted) in the literature.

Unfortunately, the database has not been updated for at least the past 6-7 years, so some more recent PRCs are not included. Still, if one is interested in performing a meta-analysis (e.g., correlating particular circadian properties with ecological niches or phylogenetic histories - I wish someone would actually do this), the data are freely available on this website.ˇ

Tuesday, April 12, 2005

Interpreting The Phase Response Curve

This is the fifth post in a series about mechanism of entrainment. In order to understand the content of this post, you need to read the previous four installments. The first one is here, the second one here, the third is here, and the fourth is here.

A Phase Response Curve (PRC) can be made in three ways:

One can construct a PRC for a single individual. If you have a reasonably long-lived organism, you can apply a number of light pulses over a period of time. The advantage is that you will always know the freerunning period of your organism, and you will know with absolute certainty that the conclusions you draw from the PRC will apply to that individual - information that may be important if the goal is to ensure the maximal health (or reroductive state) of that individual in captivity. The disadvantage is that you have a sample size of one (N=1), so you may need to repeat the experiment with a few more individuals in order to do statistics. Unless, of course, your organism is a blue whale, or platypus, or something endangered, so even data on a single organism are more valuable than none.

Usually one works with a group of animals, each kept isolated from the others and each being exposed to one or more pulses. The resulting PRC is a combined PRC of the individuals in the group. N is greater than one, but the interpretation of results may not apply to each inidividual equally - it is an averaged curve and outliers may behave differently than the group mean suggests. Still, the freerunning period (tau) for each individual is known, so additional analysis for each individual is possible.

Finally, one can construct a group (or population) PRC. For instance, eclosion (hatching out of eggs) is a single developmental event in the life of each individual insect (as well as in some birds, like chickens), yet it is timed by the circadian clock, and the rhythm can be seen if one observes a population. A bunch of eggs ecloses almost simultaneously (e.g., at dawn, or subjective dawn), another bunch about 24 hours later, another bunch another 24 hours later and so on. While individual tau is not known, the population tau is known and can be used for analysis.

It is important to keep in mind, during analysis, if the PRC is individual, combined or population PRC.

We can use the Phase Response Curve to determine if a particular animal will be able to entrain to a particular LD cycle. First, we need to determine what kind of daily shift is needed to entrain the animal. If an individual's freerunning period (tau) is 23 hours, and the period of the entraining cycle (T) is 24 hours, this means that this animal requires a daily phase-shift (delta Phi) to be a delay of 1 hour. The simple formula is tau - T = delta Phi.

Now, we can see at what phase of the circadian rhythm must the onset of light fall in order for stable entrainment to occur. We look at out PRC to find where (on X-axis) can we find a delay (on Y-axis) of 1 hour. We see that this happens at two places: on the negative slope portion in the early subjective night and a couple of hours later on the positive slope of the curve. This is a place to remember: Stable entrainment cannot occur if the light onset falls on the positive-slope portion of the curve. So, we pick the phase at which the 1-hour delay is on the negative slope of the curve. That is the phase at which light has to come on every day for stable entrainment to occur. If errors occur and shift the cycle a little bit in either direction (advance or delay) the nature of the curve ensures that, via a feedback mechanism, the cycle falls back to the correct phase. So, if an error phase-advances the cycle by 10 minutes, the light (next day) will fall on the portion of the curve that delays the rhythm by 1 hour + 10 minutes (70 minutes), bringing the rhythm back to the most stable phase. If an error phase-delays the rhythm by 10 minutes, the light (next day) will fall on the portion of the curve that delays the rhythm by 1 hour - 10 minutes (50 minutes), again bringing the rhythm back to the most stable phase.

If the light falls on the positive-slope portion of the curve, the effect is feed-forward: an error resulting in a slight advance leads to a greater advance which leads to greater advance etc. Eventually the animal will hit the correct phase again and stably entrain, but it may take weeks to get there.

Occasionaly one sees a PRC in which a portion of the negative slope is very steep - greater than 2. Stable entrainment is not possible on this portion either, as very small errors in the phase cause very large shifts of the curve to the left or to the right. The correction (in the next cycle) is then much bigger than the original error leading to unstable shifting back and forth until the light finally hits the correct phase.

Of course, there is another portion of the curve on which entrainment is impossible - the dead zone. Since light has no effect, the rhythm will keep freerunning (ignoring the light cycle) until it encroaches into a phase-delaying or phase-advancing portion of the curve.

Determining the Phase Angle

Let's assume that we are dealing with a nocturnal animal that starts its daily acitivity at CT12. Let's also assume that its freerunning period is 23 hours. From the formula we calculate that it needs a daily delay of 1 hour. From the PRC we find the phase of the rhythm at which 1-hour delay is on a negative slope, e.g., at CT14. From this (14-12=+2h) we calculate that this animal has a positive phase angle of 2 hours. This means that, when stably entrained, it will always start its activity two (circadian) hours before the onset of light-pulse.

Determining the Range of Entrainment

Using the above formula and the PRC one can calculate the lower and upper limits on entrainment of that animal to that type (duration, intensity, quality) of light pulse. Let's assume that the animals' mean tau is 23 hours. From the PRC we see that the greatest phase-delay is 3 hours and the greatest phase-advance is 2 hours. Let's plug in the numbers:
tau - T = delta Phi
23 - T = -3; Tmax = 26h
23 - T = +2; Tmin = 21h.

Thus, this animal cannot entrain to cycles with a period shorter than 21 hours, or longer than 26 hours. If the duration of the pulses used for the construction of the PRC was 3 hours, the lower limit of entrainment is LD3:18 and the upper limit of entrainment is LD3:23. Outside of this range, one is likely to observe relative coordination, scalloping, phase-jumping, or freerunning, but no stable entrainment. And, the coolest thing of all, one can predict from the PRC using very simple math, exactly how the animal will behave in such cycles outside of its range.

Entrainment by Skeleton Photoperiods

Likewise, one can predict the behavior of the animal if exposed to the skeleton LDLD cycles. The first pulse acts as "dawn" and the other one as "dusk". The shift produced by the first pulse determines the phase on which the second pulse will land and the resulting shift. If the sum of the two effects is equal to the daily shifting requirement for stable entrainment, the animal will entrain to the skeleton photocycle. In other words, tau - T = deltaPhi1 + deltaPhi2.

Organisms tend to interpret skeleton photoperiods as shorter of the two possiblities. For instance LDLD 0.25:13.5:0.25:10 can be interpreted as a full photoperiod of LD14:10, or as LD 10.5:13.5. The latter one is shorter, thus prefered by the animal. A phase-jump is likely to occur. However, in some cases, a skeleton photoperiod will be interpreted depending on the phase of the rhythm at which the first light hits at the beginning of the experiment. If it illuminates a phase close to CT0 it will be interpreted as the "dawn" pulse, and if it falls around CT12 as the "dusk" pulse. This ability to entrain both ways to a mid-length (i.e., not too long) skeleton photoperiod is called bistability phenomenon.

I will next plunge into the posts about the use of understanding of entrainment in the study of photoperiodic time measurement (measuring seasonally changing daylengths), but PRC is not done yet. What I have presented so far is the most basic stuff taught in intro college courses. There is a lot more arcane stuff to discuss, but I will come back to it later.

Part 1: Entrainment
Part 2: Phase-Shifting Effects of Light
Part 3: Constructing a Phase-Response Curve
Part 4: Using The Phase Response Curvev

Molecular Basis of Biological Clocks

While, being published in 1999, it is not the most up-to-date paper, this is certainly the most thorough yet most clearly and coherently written review of the molecular basis of circadian rhythms in cyanobacteria (Synechococcus sp.), plants (Arabidopsis thaliana), fungi (Neurospora crassa), insects (Drosophila melanogaster) and mammals (Mus musculus):

Molecular Bases for Circadian Clocks by Jay Dunlapp

Monday, April 11, 2005

Using The Phase Response Curve

This is the fourth post in a series about mechanism of entrainment. In order to understand the content of this post, you need to read the previous three installments. The first one is here, the second one here, and the third is here.

If you look at the Phase Response Curve you made (e.g., this one), you see that, as you follow the curve through the 24-hour cycle, you first encounter a dead zone during the subjective day (VT0 - CT 12) during which light pulses exert no or little effect on the phase of the clock. The line, then, turns down (negative slope) into the delay portion of the curve until it reaches a maximal delay in the early night. It reverses its direction then and goes up (positive slope) until it reaches maximal phase-advances in the late night. Finally, it falls back down again (negative slope) until it jons the X-axis again.

Let's say that, in your experiment, you have used light pulses that were 6 hours long (duration), 100 lux strong (intensity), and containing the full spectrum of visible light (quality). The PRC will tell you how your animals would entrain to a murky, cloudy mid-winter day in high latitudes.

If you used 14-hour pulses of white light of 2000 lux, you would have built a PRC describing entrainment to a nice clear summer day in North Carolina.

These two PRCs would look somewhat similar to each other. All Phase Response Curves are qualitatively the same. There are only quantitative differences.

Keeping duration and intensity constant, but systematically varying quality - using for instance, blue, green, yellow, orange and red light pulses - will result in a series of PRCs of similar shape, yet the sizes of phase-shifts would differ. This series of PRCs can tell us about the spectral sensitivity of the photoreceptive pigment involved in the transduction of light information from the environment to the clock. For instance, strongest responses to orange-red portion of the spectrum suggest rhodopsin or a similar pigment (e.g,. melanopsin). On the other hand, peak response to the blue light suggests a pigment like cryptochrome.

Systematic varying of either duration or intensity of light would also result in construction of a class of PRCs. As intensity (or duration) increases, the sizes of phase-shifts (both advances and delays) also increase (and vice-versa also holds: I have seen nice, low-amplitude PRCs to light-pulses measured in miliseconds). At the same time, you will notice a couple of other things: first, the dead zone is getting progressively narrower, and second, as you look at your raw data, you will notice that fewer and fewer days of transients are needed for the rhythm to achieve the new steady-state after the perturbation by light.

As you keep increasing intensity (or duration) of the light pulse and producing new PRCs, there will be a point - a treshold - at which there will be no more dead zone and no more transients. At this point, each light-pulse elicits an immediate large phase-shift, some as large as 12-hour shifts. It becomes impossible to differentiate between advances and delays (transients used to be a guide - but they are gone now), so the convention is to plot all the data as phase-delays.

This treshold, explained by some elegant yet complex math by Arthur Winfree in a series of books and papers, denotes a switch from a Slow-Resetting PRC (Type I PRC) and Fast-Resetting PRC (Type 0 PRC). The point (of intensity or duration) at which Type I turns into Type 0 PRC is species-specific. Mammals, especially rodents (burrowing nocturnal non-migratory animals) tend to have very small shifts and it requires enermous amounts of light energy to make the switch from Type I to Type 0 PRC. On the other hand, relatively weak stimuli result in Type 0 resetting in a number of plants, fungi and prostists, as well as in some migratory animals, e.g., Japanese quail. Thus the shape and size of PRC can tell us something about the phylogeny and ecology of the organism we are studying.

Here is an example of two human PRCs generated with two intensities of light.

Phase-Response Curves to other stimuli (e.g., dark pulses on a constant light background) tend to have a different shape. Here is a paper that contains some PRCs to various chemicals, including melatonin, serotonin and glutamate. Notice how the curve for glutamate closely matches the curve for light, suggesting that this neurotransmitter may be involved in transmission of light information from the retina to the clock.

Notice also how few data-points were neccessary for the completion of the glutamate PRC. Once the PRC to light has been generated for a particular species, further (more expensive and involved) experiments can be done by applying stimuli (e.g., chemicals) at only three time-points: the phase of greatest advance, the phase of greatest delay, and the dead zone (as control). In experiments performed in this manner, it has been reported that some neurotransmitters and neuromodulators are involved only in phase-delays and others only in phase-advances. This shows how formal analysis aids in the study of underlying physiological mechanisms.

Part 1: Entrainment
Part 2: Phase-Shifting Effects of Light
Part 3: Constructing a Phase-Response Curve

Next: how to use a PRC to study entrainment.

Sunday, April 10, 2005

Constructing the Phase Response Curve

This is the third post in a series about mechanism of entrainment. In order to understand the content of this post, you need to read the first two installments. The first one is here, and the second one here.

After months of applying light pulses to your animals you are ready to analyze and plot your data. You will print out the actographs (see how in the post "On Methodology" in the "Clock Tutorials" category) and you will see many instances of phase-shifts, somewhat like the very last figure in this po st.

For each light pulse you applied to each animal, you measure the direction of the phase-shift (i.e., if it was a delay or an advance) and the size of the shift (e.g., 10 minutes or 10 hours, or whatever may be the case). In order to plot these values, you also need to know the phase of the circadian rhythm at which you have applied the pulse. Usually the onset of the light pulse is used, so if you used a 14-hour long pulse, you mark the time that the light came on, not off. You need to know at which circadian phase did the light come on.

Here comes the hitch. You cannot just start counting hours since the onset of animal's activity. Each individual is going to have a different freerunning period (tau), thus its subjecti ve perception of one hour is going to be different from its neighbor's and likely different from real 60 minutes. You need to know at which point in the motion of the circadian oscillation is the animal at the time of the pulse because, if you just use r eal hours, you will calculate different phases for different individuals. Thus, you have to normalize the phase to reflect this.

If an individual has a freerunning period of, let's say, 22 hours, each of its subjective hours will be shorter tha n the real hour. How much shorter? You calculate 22/24 = 0.92h. Each of its hours is 0.92h long. If you have applied a light pulse 2.76 real hours after the activity onset in a diurnal animal (or 14.76 hours after the activity onset in a nocturnal an imal), you have hit exactly the Circadian Time 3 (CT3).

Another individual has a freerunning period of 26 hours. Each of its subjective hours will be a little longer than the real hour, i.e., it will be 26/24 = 1.08h. Thus, if it is a diurnal an imal, and you find that the pulse started 6.48 hours after the onset of activity, you have hit exactly CT6. If it is a nocturnal animal, you add another 12 hours, i.e., 12 + 6 = CT18.

Now that you have determined the circadian phase of each pulse, direc tion of each phase-shift, and size of each phase-shift, you can start plotting the PRC.

On the X-axis, you use Circadian Time, expressed in circadian hours, thus the axis will go from 0 to 24 and will cover the duration of one circadian cyc le.

On the Y-axis you plot size of phase-shifts (in hours or minutes). Phase-advances are positive numbers and will be plotted above the X-axis. Phase-delays are negative numbers and you will plot them below the X-axis (some of the very earliest PRCs i n the early 1960s have been plotted in reverse - delays positive, advances negative - so be careful when you read the classical literature).

When each phase-shift is represented by a little dot on your graph, draw a best fit line through the data. You h ave just plotted a Phase Response Curve.

What you will see, in most cases, is that there is little or no effect of light pulses administered during the subjective day (CT0 - CT12). This portion of the curve is called the dead zone. As you follow the curve into the early subjective night you will see gradually greater and greater phase-delays (the curve shows a negative slope), followed by a reversal (positive slope): smaller and smaller delays, no effect about mid-night, then greater and greater phase-advances in the late night. Finally, just before "morning", the curve slopes down again and hits zero (joins the X-axis) at about CT24 (=CT0 of the next cycle).

You can see a schematic PRC here.

In the next post, I will try to explain how a Phase Response Curve helps us understand the principles of entrainment.

Part 1:Entrainment,
Part 2:Phase-Shifting Effects of Light..

Phase-Shifting Effects Of Light

In the previous post, I introduced the concept of entrainment of circadian rhythms to environmental cycles. As I stated there, I will focus on non-parametric effects of light (i.e., the timing of onsets and offsets of light) on the phase and perio d of the clock.

Entrainment is a mechanism that forces the internal period (tau) of the biological clock to assume the same period (T) as the environmental cycle, i.e., tau=T. In nature, at least on this planet, this means forcing the biol ogical clock to oscillate with the period of 24h.

Non-parametric properties of light are binary: either the light is on, or it is off. For now, we will ignore parametric properties such as light intensity and light quality (spectral composition). Accor ding to the theory, circadian rhythms are entrained by effects of transitions - lights-on or lights-off - on the phase of the rhythm. Thus, switching on the light in the morning, for instance, induces a phase-shift: either a phase-advance or a phase-delay. This is analogous to re-setting an imprecise wrist-watch every morning. If the watch is running slow, phase-advancing it every morning will reset it to the correct local time. If the watch is running fast, phase-delaying will reset it to the proper time. One can conceptualize biological rhythms to be like slow watches if the endogenous freerunning period (tau) is longer than 24 hours, or like fast watches if the tau is shorter than 24 hours. Experimental evidence in a number of organisms po ints out that effects of lights-on in the morning are what is really important. The evidence for effects of lights-off in the evening is mixed.

If, for example, an animal has a clock with endogenous freerunning period (tau) of 23 hours, it needs to be r eset by phase-delaying it by exactly one hour (23 - 24 = -1h) each day. Another animal may have tau=25 hours and needs to be phase-advanced by exactly one hour (25-24 = +1h) each day. How this requirement is met has been studied by systematic exploration of phase-shifting effects of light and construction of Phase-Response Curves (PRC).

There are eight different experimental protocols that can be used to construct a PRC. They have been shown in many cases to be equivalent to each other (I have published a study demonstrating that two of them are equivalent in my species), yet some questions still remain. Choice of the particular method will depend on the organism studied and the actual question that is asked. I will here describe the simplest method (Aschoff Type I), the protocol that is sure to make your peer-reviewers happy as it is the most straightforward and uncontroversial method and is considered to be "standard".

Individual organisms are kept in isolation in prolonged constant condit ions, including, most importantly, constant darkness (DD), and some type of output of the clock is continuously monitored (e.g., locomotor activity or body temperature in animals, leaf movements in plants, etc.) Every couple of weeks or so, each animal i s exposed to a light pulse of predetermined duration, intensity and spectral composition. What is systematically varied is the phase of the rhythm at which the light-pulse is applied. How is the phase determined?

The animal kept in DD does not experience a real day and a real night. Yet, its behavior indicates that it continues to experience subjective day and subjective night. For instance, for a nocturnal animal, time when it is active is subjective night and time when it is asleep is subjective day. In a diurnal animal it is the reverse: animal is active during its subjective day and asleep during subjective night.

By convention, the time at which the animal initiates its day-specific activity (i.e., waking up for diurnal animals and falling asleep for nocturnal animals) is denoted as Circadian Time Zero (CT0), or sometimes refered to as Internal Time Zero (IT0). That is the time at which dawn would have arrived in the first cycle in DD, i.e., one cycle after the last exp erience of lights-on in LD12:12.

In diurnal animals, time of activity/wakefulness is subjective day and it spans CT0 - CT12. Its subjective night, when it is inactive/asleep, spans CT12-CT24. Likewise, nocturnal animals start their activity (e.g,. whee l-running in rodents) at CT12 and their subjective day also spans CT0-CT12 and subjective night CT12-CT24.

When you look at the records of your experimental animals you will see that light pulses resulted in phase-shifts of the circadian rhythm. In most organisms, light-pulses applied during the subjective day have no effect on the phase, pulses that fell during the early subjective night resulted in phase delays, and pulses targeted at late subjective night produced phase-advances.

You will also notice that it often took several days of gradual phase-advancing before the rhythm assumes the new steady state after the pulse. For phase-delays, fewer days are needed for the assumption of steady-state. These apparent gradual phase-shifts are called tr ansients.

However, remember that circadian systems are multioscillatory in nature. Like the pacemaker (e.g., the SCN in mammals), every other cell in the body also contains a clock that behaves like a slave oscillator. Experiments using two pu lses given just hours apart determined that the pacemaker is phase-shifted by the light-pulse immediately. Activity, temperature, etc. are driven by brain centers other than the pacemaker and thus act as peripheral oscillators. What you see in the records of your animals is gradual resynchronization of these peripheral clocks by the pacemaker.

Part 1: Entrainment
Part 3: Constructing the Phase Response Curve


[Note: This is the first in a series of posts on the analysis of entrainment. I will, for now, supply only the text. Once my computer is fixed I will add figures and edit accordingly.]

The natural, endogenous period of circadian rhythms, as measured in constant conditions, is almost never exactly 24 hours. In the real world, however, the light-dark cycle provided by the Earth's rotation around its axis is exactly 24 hours long. Utility of biological clocks is in retaining a constant phase betwe e n environmental cycles and activities of the organism (so the organism always "does" stuff at the same, most appropriate time of day). Thus, a mechanism must exist to synchronize the internal clock to the environmental cycle, in other words, to force t he biological clock to assume a period of exactly 24 hours. The phenomenon of synchronization of biological rhythms by external cues is called entrainment.

It is important to keep in mind that the environmental cycle does not FORCE the oscillat io n o f biological parameters (e.g,. sleep-wake cycle or body temperature). Instead, it synchronizes the clock which, in turn drives all the other biological phenomena. In other words, it does not turn over an hourglass clock every morning. If that wa s the c ase, failure to turn it over one morning would result in all the sand running out from top to bottom and the clock would stop. But we have seen that turning off the light does not stop the rhythms - they continue to freerun in constant conditions. Entra inment is a process similar to re-setting a wrist-watch. If you have a watch that runs a little slow and accumulates about 5 minutes of delay every day, resetting it 5 minutes forward every morning will keep the watch reasonably well entrained.

Li ght-dark cycles are the most powerful environmental cues for entrainment, although many other cues have been demonstrated to be effective in entrainment of circadian rhythms in particular organisms, including cycles of temperature, atmospheric pressur e, s ound (e.g., conspecific song), feeding schedules, exercise schedules, odors, social cues, etc. While details of physiological pathways that transduce environmental information to the clock may differ between varioius cues in various organisms, the e ssent ial ("formal") properties of entrainment are thought to be the same for all of them. Since light is the strongest synchronizer (Zeitgeber) and most of the studies were performed utilizing light as a cue, most of the discussion in the followi ng ser ies of posts will focus on entrainment by light.

There are two ways of thinking about entrainment by light. One, first suggested by Jurgen Asschoff, focuses on parametric properties of light, i.e., roles of intensity and spectral compositi on ("co lor spe ctrum") of light in entrainment. This is a difficult piece of theory to study and I will not spend too much time on it for now.

I will focus mainly on the non-parametric effects of light, i.e., the roles of timing of onset and o ffset of light on entrainment. Non-parametric analysis of entrainment by light, first thought of and used in elegant experiments by Patricia DeCoursey (in ground squirrels) and Woodie Hastings (in a protist Gonyolax polyedra) and subsequently de veloped by, for instance, Colin Pittendrigh (in Drosophila pseudoobscura) and Jeff Elliott (in hamsters), has been worked out in quite a lot of detail over the years, and is the cornerstone of the field of chronobiology. I will attempt to descr ibe it and explain it as clearly and coherently as I can in a series of posts over the next few days. This is not an easy concept to grasp, so take your time reading, and ask questions in the comments. I will add the figures as soon as I can technically do so.

Part 2:Phase-Shifting Effects Of Light
Part 3: Constructing the Phase Response Curve v

Thursday, April 07, 2005

ClockNews #31: Daylight Savings Etc.

Altering Food and Light Schedules Affects Cancer Genes in Mice

A new study has found that altering food schedules and light/dark exposure in mice modified the expressi on of circadian clock genes and genes involved in carcinogenesis and tumor progression.

The circadian clock regulates the approximate 24-hour cycles of many animals, including mammals. It has been reported that tumors grow faster in animals with a disr u pted circadian clock--which happens, for example, in chronic jet lag--but the molecular mechanism is unclear.

Francis Lévi, M.D., Ph.D., of the French National Institute of Health and Medical Research (INSERM) and University Paris XI at Paul Brousse Hos pital in Villejuif, France, and colleagues compared the expression patterns of circadian clock and cell cycle genes in the livers and tumors of mice synchronized by normal light and dark schedules (normal circadian clock) or with schedules designed to sim ulate chronic jet lag in humans (disrupted circadian clock). They found that meal timing reversed the disrupted circadian clock gene expression patterns and slowed tumor growth in chronic jet lagged mice. The authors conclude that the altered light/da rk o r feeding schedules modified the carcinogenesis and tumor progression.

Mutant gene cause of sleep disorder

A mutant gene behind an "early b ird" sleep disorder has been identified by American researchers. Approximately three-tenths of a percent of the world's population has familial advanced sleep phase syndrome (FASPS), according to the researchers. People with this "time-shift" trai t have b ody clocks that are out of sync with most of the world. Researchers say they have found the genetic culprit for this rare sleep disorder.

People with this sleep disorder do not suffer from sleep deprivation. Rather, they consistently fall asle e p at an early hour and then wake up well before dawn.

Melatonin for jet lag stirs discussion

THE CLAIM: Melatonin can help you conquer jet lag.
T HE FACTS: Some bleary-eyed travelers swear by melatonin as a way to beat jet lag. But experts say research on the hormone's effectiveness is far from clear-cut.

Chronically tired? Or irritable? Maybe it's due to too little sleep

"Many people feel that sleeping is a waste of time, but more are realizing that it's essential to health and well-being," said Sreden.

Daylight Savings Time is a case in point. The day after Americans "spring ahead," a statistical blip occurs in which automobile crashes increase as drivers losing an hour of sleep suffer impaired driving abilities, according to Sreden.

Quiet suffering

Driving while drowsy is a potentially
deadly combination, akin to driving under
the influence of alcohol. Driving
performance after 19 hours of sustained
wakefulness is comparable to driving with
a blood alcohol concentration (BAC) of
0.05 per cent; after 24 hours, the
equivalent to a person with a BAC of 0.10
per cent.

Physicists Find Patterns Within Seemingly Random Events Of Physiological Systems

Finding patterns behind seemingly random events is the signature of a recent trio of research studies coming from the statistical physics group in Boston Universit y's industry for research physicists, findings from this BU group increasingly wed phenomena associated with the inanimate world to those of animate beings -- finding commonalities between stock markets fluctuations, earthquakes, and heart rates, for exam ple, or discovering similarities in mice, men, and other mammals for such fundamental phenomena as wake periods during slumber.

Valdoxan®: A New Approach to The Treatment of Depression

Due to its unique pharmacological profile, Valdoxan is the only antidepressant to have a specific action on circadian rhythms, which are often imbalanced in depressed patients. By improving disturbed wake-sleep patterns, according to Dr Guilleminault, Valdoxan is a bleto relieve sleep complaints of depressed patients with a favourable impact on daytime vigilance.

Respironics Acquires Mini-Mitter Company, Inc., Sleep and Phy siological Monitoring Device Innovator

Mini Mitter develops and markets a range of physiological monitoring products, including devices that measure sleep-wake activity, dermal and core body temperature, heart rate, and energy expenditure.

Oh-oh! I better check if they'll keep making the equipment I use....

Early Bird Sleep Disorder Linked To Mutant Gene

Researchers believe a mutant gene may be responsible for a sleeping disorder that causes people to nod off early, then wake up wide-eyed long before the sun rises.

Why must we spring forward?

Spring forward, fall back.

OK, that memory device helps us recall that we need to set our clocks one hour forward when we go to bed tonight so we'll wake up in sync with daylight-saving time on Sunday. But it also reminds us that if we set th e alarm at our normal time to go to church, we'll be short an hour's sleep.

Worse, we don't get that hour back until the last Sunday in October.

It may not take seven months for us to get used to the new time scheme, but the folks at Circadian, an inter national r esearch and consulting firm, estimate that it does take a couple days for our bodies to adapt to the change.

So, why do we put up with this nuisance? Well, Ben Franklin first proposed daylight-saving time in 1784, but Americans didn't adopt it until Worl d War I to save energy. It worked but proved so unpopular that the country went back to what irritated farmers called "God's time" in 1920. DST returned in World War II, when U.S. clocks were set ahead one hour ye ar-round. After the war, some jurisdiction s continued DST and others abandoned it. Congress later passed the Uniform Time Act of 1966, which doesn't require anyone to observe daylight-saving time, but sets rules for those who do. DST is not observed in Hawaii, American Samoa, Guam, Puerto Rico, t he Virgin I slands, the Eastern Time Zone portion of the Indiana, and by most of Arizona (with the exception of the Navajo Indian Reservation).

The Department of Energy estimates DST reduces electricity use by about 1 percent a day, mainly by reducing th e need for l ighting in evening hours. Some people now argue for year-round DST, but critics say that wastes as much energy on dark winter mornings as it saves in the evening. Russia, further north than the U.S., stays on DST all year and g oes to double-d aylight in the summer.

If you really hate daylight time, you can avoid it by moving to Panama. Equatorial countries don't observe it since their daylight hours don't vary much. If you love it, you can get a double dose by moving to Russi a. For most of u s, the best course is to go to bed a little early Sunday night so our bodies don't show up for work an hour late on Monday.

Scient ists Find Gene that Controls I n somnia

As the saying goes, early to bed, early to rise. But in the world of sleep researchers, that's a disorder as much as the syndrome experienced by people who stay up very late and sleep very late into the morning.

Time Change May Put Sleepier Drivers on Road

Sleep scientists analyzing data from 21 years of car crashes concluded in 2001 that the numbe r of fatal collisions increased slightly on the Sunday and Monday after the switch to daylight saving time. When standard time returns in the fall and clocks are turned back an hour, researchers Jason Varughese of Stanford and Richard P. Allen of Johns Ho pk ins wrote, there also were more accidents, possibly because people were staying out later the Saturday night before.
During daylight saving time, the study showed, the extra hour of sunlight typically occurr ed during peak traffic periods an d c ut the number of fatalities even further. The biggest reduction was in the number of pedestrian deaths, because drivers were better able to see people walking in the street.

Caffeine - are you addicted?

Scientists have claimed that, like methadone users, those who feel they cannot function without a morning coffee may be trying to “stave off withdrawal symptoms”.

Scientists also claim caffeine stimulates the brain in the same way as amphetamines, cocaine and heroin and could be classified as an addictive drug.
Caffeine keeps us awake by blocking the effects of the chemical adenosine, whic h causes us to feel sleepy.

Ad enosine attaches to receptors in the brain and slows down the activity of nerve cells.

Caffeine attaches itself to the same receptors: blocking adenosine, increasing brain cell activity, and producing the buzz obtained from drinking a caffeine-laced b rew.

2:00 A.M. Was Overrated Anyway: Company Offers 12 Reasons to Celebrate Losing an Hour This Weekend

1. Technically speaking, you'll get 13 hours out of your 12-hour cold pill

2. Grogginess is a great excuse for avoiding the in-laws

3. The one person in the house who knows how to re-set the VCR clock gets
a chance to strut his stuff

4. It's one fewer hour of having to lay sl eepless next to yo ur snoring

Scientists use lasers to control flies

Mutant gene s hifts when people a re sleepy

Students lose in sleep cost-benefit ana lysis


Waking up to sleep problems

UCSF study offers insight into human circadian rhythms

Clockwork internalXXÏÏ

Altering Food and Light Schedules Affects Cancer Genes in Mice

Altering Food and Light Schedules Affects Cancer Genes in Mice

A new study has found that altering food schedules and
light/dark exposure in mice modified the expression of
circadian clock genes and genes involved in carcinogenesis
and tumor progression.

The circadian clock regulates the approximate 24-hour
cycles of many animals, including mammals. It has been
reported that tumors grow faster in animals with a
disrupted circadian clock--which happens, for example, in
chronic jet lag--but the molecular mechanism is unclear.

Francis Lévi, M.D., Ph.D., of the French National Institute
of Health and Medical Research (INSERM) and
University Paris XI at Paul Brousse Hospital in Villejuif,
France, and colleagues compared the expression patterns of
circadian clock and cell cycle genes in the livers and tumors
of mice synchronized by normal light and dark schedules
(normal circadian clock) or with schedules designed to
simulate chronic jet lag in humans (disrupted circadian
clock). They found that meal timing reversed the disrupted
circadian clock gene expression patterns and slowed tumor
growth in chronic jet lagged mice. The authors conclude
that the altered light/dark or feeding schedules modified the
expression of circadian clock genes and genes involved in
carcinogenesis and tumor progression.

Monday, April 04, 2005

Genetics Of Super-Larks

Researchers Identify Cause of 'Early Bird' Sleep Disorder


A few rare people who consistently nod off early, then wa ke up wide-eyed much before dawn, can blame a newly-found mutant gene for their sleep troubles, Howard Hughes Medical Institute researchers announced today. This odd "time-shift" trait -- called familial advanced sleep phase syndrome (FASPS) -- was studied in one affected family by neurologist Louis J. Ptacek, a Howard Hughes Medical Institute researcher, and Ying-Hui Fu, at the University of California, San Francisco. Their report appears in the March 31, 2005, issue of the journal Nature.

The sleep-shifting mutation they found is in "a gene that was not previously shown in mammals to be a circadian rhythm gene," Ptacek explained. It's not yet clear how the mutant gene works to shift people's sleep time, their circadian r hythm, he added. But follow-on experiments in fruit flies and mice yielded results that are intriguing. When the mutant gene was inserted into the flies, for example, it did the opposite of what was seen in the human family: it lengthened circadian rhyth m. Yet in genetically engineered mice, the same gene change made the mice early risers -- mimicking what was seen in humans with FASPS.

So, studies of all three organisms -- flies, mice and humans -- "will help us understan d the similarities and differences" in how the gene works in different settings, in different genetic backgrounds, he said. Experiments can be done in mice and flies, with results applying to humans, while the studies of humans can inform what's being see n in the flies and mice.

In addition, "these results show that the gene is a central component of the mammalian circadian clock, and suggest that mammalian and fly clocks may have different regulatory mechanisms, despite th e highly conserved nature of their individual components," the research team wrote in Nature. Such studies may help unravel some of the fundamental mysteries of how circadian rhythms are established and maintained in creatures that have evolved along very different paths.

As for the affected individuals, Ptacek said most are able to live normal lives, and some are proud of being able to arise before dawn and get a lot done while everything is quiet. A few, however, are constantly bothered by living out of sync with everyone else's daily schedule.

"Some of them would never come to a doctor" to find out what's going on with their sleep pattern, Ptacek said, "because they aren't troubled by it. Often, they have adjusted and accommodated their jobs to match their ability. But others are bothered by being out of phase with the rest of the world."

He said the FASPS subjects don't seem to sleep any more or less than o ther people; they just sleep at different times. And there is apparently no connection to the better-known problem called narcolepsy. Ptacek said it was also found -- in the family's six affected individuals -- that "they all have asthma, and they all ha ve migraine headaches, with aura. Now, that could be purely coincidental, but a more important possibility is that these are part of the same syndrome." So far, however, "we haven't even looked at that yet."

He estimated th at a very small number (about .3 percent) of the human population seems to have this "circadian clock" shift. And in earlier research, Ptacek and his colleagues had discovered an entirely different gene that causes a similar clock-shift. Both arise becaus e of so-called point mutations in the genes. This means that altering a single base-pair in the gene's long DNA chain is enough to change a person's sleep behavior. Evidence from tissue culture experiments with the second gene suggests the change causes a protein -- an enzyme called a kinase that is made by the gene - to be less active than normal.

The lead author of the paper in Nature is Ying Xu, a member of the team in San Francisco. Other team members are at the Univers ity of Vermont and the University of Utah.

That "second" gene they mention is Kasein Kinase 1epsilon (also called Doubletime in fruitflies) which, in super-larks, fails to phosphorilate the Period protein. The newly discovered gene is Kasein Kinase 1delta.

Here is another excellent review of the findings.

Sunday, April 03, 2005

Fetal Alcohol Syndrome Affects The Basic Properties Of The Circadian Clock

Alcohol 'binges' in rats during early brain development cause circadian rhythm problems

Rats are nocturnal animals and normally begin their activity slightly after darkness sets in. The rats that had been exposed to alcohol began activities slightly before darkness set in.

When normal rats - or for that matter, humans and other animals - are in situations without environmental cues about day and night, the body's circadian clock generally drives behaviors on a cycle slightly greater than 24 hours. Untreated animals woke up approximately 20 minutes later each day in the absence of a light-dark cycle. The rats that had been exposed to alcohol consistently became active 30 minutes earlier every day.

In situations when the light-dark cycle was shifted six hours earlier, the "jet lag" equivalent for humans having to shift their body clocks when traveling across different time zones, the rats exposed to alcohol in infancy shifted much more quickly, as they did to 15-minute light pulses. While this may sound good to most traveling humans, it reflects permanent changes that have ramifications on how systems in the body function in relation to each other, says Dr. Earnest.

While the paper, quite rightfully, focuses on implications for Fetal Alcohol Syndrome, I find this study quite intriguing from a very basic biological perspective. Circadian clocks are extremelly non-responsive to chemicals.

Apart from heavy water, lithium and a couple of hormones (e.g., melatonin and estrogen), pharmacological agents (and many have been tested) just do not have any effect on the workings of the clock. Alcohol in adults has no effect.

But this study - and David Earnest is one of the top people in the field - shows large alterations in the clockwork due to DEVELOPMENTAL effects of alcohol. And the alterations are in fundamental properties of the clock: period, phase, and response to phase-shifts of the light-dark cycle. It will be very interesting to see what further research on mechanisms reveals: what is the nature of this effect on a cellular and molecular level.e

Science & fun cool stuff
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