Circadian Rhythms: The Day Within

time spiral

credit: gadl

You’ve probably heard that our bodies have an internal clock. That’s why you get tired or hungry at about the same time each day and feel jetlagged when you’ve crossed too many time zones.

Our clocks are set by external cues, such as daylight, alarm clocks, or your roommate grinding coffee beans at an ungodly hour each morning. But our clocks can also function completely independently of all outside cues. This was discovered by a series of studies, dating all the way back to 1938, in which researchers stuck some poor dudes (or occasionally themselves) into dark caves with no light or temperature cycles.2 They found that people continued to function on a near-24 hour schedule.

Our endogenous daily clock, known as our “circadian rhythm”, is not perfect. It tends to run slightly longer than 24 hours and thus needs to be regularly set, or “entrained”, by external cues such as light so that we stay in sync with the real world. But this still means that our brains are capable of telling time and know roughly how long a day is. How is this possible, especially given that the 24 hour day is an arbitrary number determined by the speed of the earth’s rotation?

Molecular timekeeping

Seymour Benzer

Seymour Benzer with a model of his beloved fruit fly (credit: W. A. Harris, PLoS Biology)

It turns out that our brains contain a molecular clock carefully encoded in our DNA. The first genetic component of the clock was identified in fruit flies by the great Seymour Benzer, a total baller who was also the first to show that individual genes can affect ANY kind of behavioral response. In 1971, he and his student Ron Konopka identified three kinds of mutant flies: flies that had a shorter than normal daily rhythm (19 hours), flies that had an extra-long rhythm (28 hours), and flies whose activity was not rhythmic at all.3 These three strains turned out to have different mutations in the exact same gene, which was called period.

By the way guys, this basically never happens. Usually there are so many genes contributing to a given process that every mutant you find has a mutation in a different gene, and the genes you decide to study often end up being the stupid crappy ones and not the exciting one that explains everything. (Yes, I speak from experience.) But Benzer got lucky—three mutants all affecting the same gene meant that that gene must be pretty darn important.

Later studies identified the other core components of the molecular clock and characterized how they function together. The basic idea is that certain proteins are created and destroyed in a feedback loop that takes about 24 hours to complete—so if you can read out the protein levels then you know what time it is.4 This loop consists of several steps:

1) Two proteins called CLOCK and BMAL1 together activate the production of the proteins PER (encoded by period) and CRY.

2) PER and CRY then repress the activity of CLOCK/BMAL1, which prevents production of more PER and CRY.

3) The existing PER and CRY molecules are degraded by yet another set of proteins.

4) Once PER and CRY levels get low enough, the repression of CLOCK and BMAL1 is relieved and they can go back to step 1, cheerfully producing more PER and CRY.

molecular clock

Diagram of the molecular clock. Steps are numbered as described above.

So basically, CLOCK and BMAL1 are like devoted parents popping out a bunch of PER and CRY babies, who turn out to be little brats that terrorize their poor folks into never having any more kids (at least not until the brats are gone). This is the crux of negative feedback: a process produces something that then inhibits the process itself.

When I first learned about the molecular clock, it blew my mind. I mean, this is a complex molecular pathway (which I’ve greatly simplified here) consisting of many different steps whose rates are determined by the biochemistry of DNA transcription and RNA translation and protein binding and degradation… and somehow it just happens to take 24 hours?!

Of course, this isn’t a coincidence. It’s the work of millions of years of evolution. If we lived on another planet with a different day length, evolution would have tweaked the components of the clock to give us a shorter or longer rhythm, as needed.

Early birds and night owls

The molecular clock also explains some of the individual variation in our circadian rhythms. Some of us are night owls who have trouble falling asleep before 1 or 2 am, and want to smash our alarm clock to bits when it blares us into consciousness in the morning (um yes, I may have actually done this). On the other hand, there are you crazies who get up at like 7am for no good reason, even on the weekends. You early birds should consider yourselves lucky that society seems to consider your behavior more virtuous than ours, despite the fact that people’s sleep tendencies are largely dictated by their genetics. Sure, most of us can adapt our schedules to fit our jobs or social life. But we don’t function as well when we’re forced to adjust too far out of the comfort zone that our endogenous clocks have set for us.

The core difference between early birds and night owls seems to be the speed of their circadian cycle. Early birds tend to have a faster cycle, meaning their bodies want to do things earlier than they should. Night owls have a slower cycle, so their rhythms tend to be delayed relative to the real world.

These differences in cycle speed are likely due to genetic variations affecting the molecular clock. The first clock gene implicated in humans, just as in fruitflies, was the period gene, which was found to be mutated in a family of extreme early birds who typically slept from about 7pm to 4am.5 Their mutation makes PER better than usual at repressing its own activation, meaning that step 2 of the loop (see above) occurs more quickly, which means the whole cycle is faster.6 This exceptionally fast cycle leads to the early sleep/wake behaviors observed in this family.

From genes to circuits

Ok, so now you’re an expert on the molecular clock, which enables each cell to keep time on a ~24 hour cycle. But how does that clock actually regulate our behavior—making us wake up, eat, and sleep at certain times of the day? And how do external cues keep the clock synchronized, or allow you to adapt to a new time zone?

Some of these questions can be answered at the molecular level; for example, light-sensitive components of the molecular clock allow it to be reset by daylight. But many of these questions are circuit-level questions. We need to study how the brain senses and translates external cues that entrain the clock (the input pathways) and how the clock activates specific behaviors at specific times (the output pathways). Researchers have identified several of these neural systems—most notably a “master clock” region of the brain called the suprachiasmatic nucleus, where all the input and output pathways likely converge. We still have a long way to go, though, in understanding the inner workings of how our brain keeps time and dictates our daily schedule.

 

Notes:

1. “The day within” is a phrase I heard in a circadian rhythms lecture given by Dr. Norman Ruby at Stanford.

2. Here are just a few examples of the early cave studies:

Kleitman N. Sleep and wakefulness (2nd ed.). University of Chicago Press, Chicago, IL (1963).

This book details some of the earliest cave studies performed in 1938 by the author, Nathaniel Kleitman (often with himself as the subject).

Colin J, Timbal J, Boutelier C, Houdas Y, Siffre M. Rhythm of the rectal temperature during a 6-month free-running experiment. J Appl Physiol 25:170-176 (1968).

This study showed that for a subject in a cave, not only his sleep/wake behavior but also his internal body temperature varied along a roughly 24 hour cycle. (Yes, this means this poor guy was not only alone in a cave for 6 months, but also had a thermometer stuck up his butt most of the time.) The senior author of this paper, Michel Siffre, was another guy who performed many circadian experiments on himself. He published several other papers and books on this subject, though most of them are in French.

Mills JN, Minors DS, Waterhouse JM. The circadian rhythms of human subjects without timepieces or indication of the alternation of day and night. J Physiol 240:567-594 (1974).

As stated in this paper: “We also include some data from a subject D.L. who spent 4 months alone in a deep cave… He had no timepiece and therefore was ‘free-running’, but he had a telephone link to the surface, permanently manned, through which he indicated his times of retiring and rising.” (I feel bad not only for D.L., but also for the technicians who had to sit by the phone 24/7.)

3. Konopka RJ, Benzer S. Clock mutants of Drosophila melanogaster. Proc Natl Acad Sci U S A 68:2112-2116 (1971).

4. Here’s a good review article about the molecular clock, if you want more details:

Partch CL, Green CB, Takahashi JS. Molecular architecture of the mammalian circadian clock. Trends Cell Biol 24:90-99 (2014).

5. Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ, Fu YH. An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome. Science 291:1040-1043 (2001).

6. Xu Y, Toh KL, Jones CR, Shin JY, Fu YH, Ptacek LJ. Modeling of a human circadian mutation yields insights into clock regulation by PER2. Cell 128:59-70 (2007).


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