Category Archives: Chronobiology

A new paper just came out today on PLoS-Biology: Glucocorticoids Play a Key Role in Circadian Cell Cycle Rhythms. The paper is long and complicated, with many control experiments, etc, so I will just give you a very brief summary of the main finding.
One of the three major hypotheses for the origin of circadian clocks is the need to shield sensitive cellular processes – including cell division – from the effects of UV radiation by the sun, thus relegating it to night-time only:

The cyclic nature of energetic availability and cycles of potentially degrading effects of the sun’s ultraviolet rays on particular pigmented enzymes, provided the selective environment. A cell with a timer can predict the changes and adjust its metabolic activities to minimize energetic and material loss. This cell will outcompete the other cells in the Archeozoic sea (Pittendrigh 1967).

Biological clocks in various organisms regulate timing of many different biochemical, physiological and behavioral events, but the circadian control of cell-cycle is really ubiqutous – it has been found in everything from bacteria to humans.
In many large organisms, the distinction between pacemakers and peripheral clocks is mainly in the ability of the pacemaker to synchronize itself to the outside environment and to send daily signals that act to synchronize all the peripheral clocks in all the cells in the body. The local clocks, then, regulate timing of local events.
In some organisms, peripheral clocks are also capable of direct sampling of the environment. Zebrafish is one such animal – its peripheral clocks (in every cell of the body) are photosensitive and entrainable directly by light-dark cycles. Only in constant light conditions does the brain pacemaker assume the role of the “conductor of the orchestra”, synchronizing all the clocks in the body.
In vertebrates, it has been thought for a long time now that the central pacemaker (the SCN in the hypothalamus of the brain) uses, among else, cortisol as a signal for synchronizing the peripheral clocks. It times the release of corticotropins from the pituitary which in turn releases cortisol from the adrenal gland into the circulation at particular time of day. Various tissues are sensitive to cortisol and will use its surge as a timing/entraining signal.
In this paper, circadian rhythms of cell-division were shown to get attenuated in mutants that do not produce corticotropins (and thus do not produce cortisol). However, the clock genes still cycle normally in the periphery. Placing the fish in continous bath of cortisol agonist reinstates the circadian rhythms of cell division.
This suggests that cortisol is not a timing signal from the center to the periphery as the peripheral clocks keep cycling in its absence (and entrain directly – no need for any input from the eyes).
This also suggests that cortisol is neccessary for the coupling of the peripheral clock mechanism and its own output – the cell cycle. The presence of cortisol need not be rhythmic – it just needs to be there if the clock is to time the daily rhythms of cell division.

There are several journals dedicated to biological rhythms or sleep. Of those I regularly check only two or three of the best, so I often miss interesting papers that occur in lower-tier journals. Here is one from December 2006 that caught my eye the other day:
Mammalian activity – rest rhythms in Arctic continuous daylight:

Activity – rest (circadian) rhythms were studied in two species of Arctic mammals living in Arctic continuous daylight with all human-induced regular environmental cues (zeitgebers) removed. The two Arctic species (porcupine and ground squirrel) lived outdoors in large enclosures while the Arctic summer sun circled overhead for 82 days. Would local animals maintained under natural continuous daylight demonstrate the Aschoff effect described in previously published laboratory experiments using continuous light, in which rats’ circadian activity patterns changed systematically to a longer period, expressing a 26-hour day of activity and rest? The outdoor experiments reported here, however, showed that under natural continuous daylight, both species (porcupine and ground squirrel) had specific times of activity and rest on a nearly 24-hour scale, and their activity peaks did not come later each day. The daily rhythms of the two species were recorded using implanted physiological radio capsules, and from direct observation.

You may recall that I wrote about a similar study in a much larger Arctic mammal – the reindeer, which loses the overt behavioral rhythmicity during the long summer. Apparently, these two small mammals, the porcupine and ground squirrel are different.
In the press release, they explain:

It seemed that although the scientists were very careful not to provide time cues of any sort, the animals had managed to latch onto something that gave them regularity.
“I have written for years that experimental animals seem to be hungry for cues, or time signals, to keep on a regular cycle,” Folk said. “So we tried to figure out what cue the wild animals were using, and we could find only one thing that kept a 24 hour periodicity. At Barrow, the sun travels in a circle overhead for 82 days, but at midnight the circle is tipped to the north.
“We postulate that the animals are conscious of where the sun is in the sky and that the nearness of the sun to the horizon could be a clue to animals, and even plants, to keep on a 24-hour schedule.”

This is an interesting hypothesis: not just using the clock to orient by Sun, but also using the Sun posiiton to entrain the clock. I hope this gets tested and that this was not just a case of investigators missing an alternative environmental cue. Changes in the Earth’s magnetic field show daily oscillations and are potentially one of such alternative cues that animals could use. Just like Dr.Folk states in the article, I’d also like to see this study replicated in Arctic birds, as they are known to be sensitive to the magnetic field which they can use for migratory orientation.

No other aspect of behavioral biology is as well understood at the molecular level as the mechanism that generates and sustains circadian rhythms. If you are following science in general, or this blog in particular, you are probably familiar with the names of circadian clock genes like per, tim, clk, frq, wc, cry, Bmal, kai, toc, doubletime, rev-erb etc.
The deep and detailed knowledge of the genes involved in circadian clock function has one unintended side-effect, especially for people outside the field. If one does not stop and think for a second, it is easy to fall under the impression that various aspects of the circadian oscillations, e.g., period, phase and amplitude, are determined by the clock genes. After all, most of these genes were discovered by the study of serendipitously occuring mutations, usually period-mutations.
If the circadian properties are really deteremined by clock genes, then the predictions from this hypothesis are that: 1) every cell in the body shows the same period (phase, amplitude, etc.), 2) every cell in the body has the same period throughout its life, 3) every cell removed from the body and placed in the dish continues to oscillate with the same period as it had inside the body, and 4) the properties of the circadian rhythms are not alterable by environmental influences.
Stated this bluntly, one has to recoil in horror: of course it is not determined by genes! But without such an exercise in thinking, much work and writing (especially by the press) tacitly assumes the strict genetic determination. However, the experimental data show this not to be true. Period (and other properties) of the whole organism’s rhythms are readily modified by environmental influences, e.g., light intensity (Aschoff Rule), heavy water, lithium, sex steroids and melatonin. They change with age and reproductive state. There is individual variation even in clonal species (or highly-inbred laboratory strains). The period of the rhythms measured in cells or cell-types in a dish is not always the same as exhibited by the same cells inside the organism. Finally, the occurrence of splitting (of one unified circadian output into two semi-independent components differing in period)suggests that two or more groups of circadian pacemakers simultaneously exhibit quite different periods within the same animal.
Several years ago, Dr. Eric Herzog (disclosure – a good friend) has shown that even the individual pacemaker cells within the same SCN (suprachiasmatic nucleus – the site of the main mammalian circadian clock in the brain) exhibit different periods. When dispersed in culture, pacemaker neurons (originally taken from a single animal) tend to show a broad variation in periods (amplitudes, etc…).
As they grow cellular processes, two neurons in a dish may touch and form connections. As soon as they do, their periods change and from then on the two cells show the SAME period, i.e., they are synchronized. As more and more cells connect, they build an entire network of neurons, all cycling in sync with each other – same period, same phase, same amplitude. This is assumed to happen inside the whole animal as well – the unconnected SCN neurons of the fetus start making connections just before (or immediately after, depending on the species) birth and as a result, an overt, whole-organism rhythm emerges out of arrhytmic background.
But, what molecules are involved in cell-cell communication that allows the pacemaker cells to synchronize their rhythms? For several years, the most likely candidate was thought to be GABA, an inhibitory neurotransmitter produced by all SCN cells. Sara Aton, a graduate student in Eric Herzog’s lab (now postdoc at UPenn), set out to test this hypothesis. Over a few years and several papers, a different story emerged, culminating in this months paper in PNAS:
GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons. (by Aton SJ, Huettner JE, Straume M and Herzog ED).
What the paper shows – and there is a lot of detail there, so you can read the paper for yourself if interested, or at least the media coverage (here, here and here) – is that various perturbations of the GABA system, either at the synthesis end or the reception end, have, at best, some mild effects on amplitude and phase. There was no effect of GABA on period of individual pacemaker neurons. Yet, effect on period is neccessary for mutual synchronization of cells into a network. Instead, VIP (vasoactive intestinal polypeptide) was shown to be the agent that, by modulating period, allowed spatially coupled cells to also temporally couple – to synchronize their circadian oscillations.
This is a much more important finding than you may think at the first glance. The naive idea of a single clock driving all the overt rhythms has been abandoned for more than half a century. Every important problem in chronobiology – coherence of rhythms, temperature compensation, communication between pacemakers and peripheral clocks, entrainment to environmental cycles, etc. – hinges on the properties of the multi-clock networks. Understanding the biochemical mechanism by which pacemaker cells syncronize with each other is thus a key finding that will allow us to study those phenomena at a cellular and molecular level. Right now, and due to Sara Aton’s work, VIP is the “handle” we will use to revisit those old problems and test our pet hypotheses about the coupling of circadian systems in various animals (the reasonable assumption being that mouse is not unique in using VIP and that this molecule is probably used for the same function in all vertebrates). We can now study exactly how two cells communicate by VIP to synchronize their clocks – is the pattern of VIP release, for instance, used as a kind of temporal “code”?
Probably the most important such phenomenon to study is splitting. Different kinds of spliting have been observed in lizards, starlings, tree-shrews (Tupaia), mice, rats, hamsters, marmosets and many other animals under various experimental conditions, e.g., constant light, constant darkness, removal of the pineal, infusion with testosterone, or exposure to skeleton photocycles. Splitting can be induced by highly artificial experimental protocols, e.g., alternate eye-patching, or they may appear spontaneusly in animals out in the wild, something that can be replicated in the laboratory.
In some cases, it has been shown that the splitting is lateral, i.e., left SCN drives one component and the right SCN drives the other. In the case of alternate eye-patching, it is reasonable to hypothesize that the same thing is happening. But in other cases, it is more likely that each SCN splits into two subsets of neurons, synchronized within but not between the two groups. Is VIP the synchronizer in both groups? In all cases? In all animals?
If, for instance, rhythms split into two componenets under the influence of testosterone, is VIP used for coupling within each of those two semi-independent “clocks”? Does one group of neurons, insensitive to testosterone, use VIP to synchronize its output, while the other group of neurons, under the influence of testosterone, changes its period and also uses VIP to synchronize its output?
Now that we know to use VIP and not GABA as an entry into the system, all of these questions will be much more amenable to future research – an exciting prospect for me and many others in the field.

Randy Nelson is a wonderful person, an engaging speaker and the author of the best textbook on Behavioral Endocrinology. I heard that he is also a great teacher which does not surprise me and he has a talent for attracting some of the best students and postdocs to work in his lab. Oh, by the way, he also does some great research.
For decades, the study of seasonality and photoperiodism was a hustling bustling field, until everyone jumped on the clock-gene bandwagon. Randy Nelson is one of the rare birds to remain in the photoperiodism field, coming out every year with more and more exciting papers. Here is just the latest one – supercool! [excerpt under the fold]

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