The Circadian Rhythm Story: Past, Present and Future
This is a modal window.
The media could not be loaded, either because the server or network failed or because the format is not supported.
Formal Metadata
| Title |
| |
| Title of Series | ||
| Number of Parts | 340 | |
| Author | ||
| License | CC Attribution - NonCommercial - NoDerivatives 4.0 International: You are free to use, copy, distribute and transmit the work or content in unchanged form for any legal and non-commercial purpose as long as the work is attributed to the author in the manner specified by the author or licensor. | |
| Identifiers | 10.5446/45075 (DOI) | |
| Publisher | ||
| Release Date | ||
| Language |
Content Metadata
| Subject Area | ||
| Genre | ||
| Abstract |
|
00:00
Lecture/Conference
00:37
Lecture/Conference
01:02
Lecture/ConferenceComputer animation
01:48
Lecture/ConferenceComputer animation
02:58
Lecture/Conference
03:39
Computer animationLecture/Conference
04:22
Lecture/Conference
04:47
Computer animation
05:47
Lecture/Conference
06:25
Lecture/Conference
06:51
Lecture/Conference
07:15
Computer animation
07:46
Lecture/Conference
08:11
Lecture/ConferenceComputer animation
08:43
Lecture/Conference
09:12
Computer animation
09:55
Lecture/Conference
10:34
Computer animationMeeting/Interview
12:48
Computer animation
19:31
Lecture/Conference
20:08
Computer animation
25:07
Lecture/Conference
25:32
Lecture/Conference
26:30
Lecture/Conference
26:56
Computer animation
Transcript: English(auto-generated)
00:14
Good morning, everyone. We are the new kids on the block, as you just heard.
00:22
And in the interest of time, I'm not going to go through the things I've memorized. Guten morgen, bonjour, buenas dias, bokeh tov, saber kachir, et cetera. So it's a great pleasure to be here, and I'm looking forward to a fantastic week.
00:42
So this is where we begin this story on October 2 when we were telephoned about this prize. And the point of this slide is to say that there's really a fourth winner here, and it's Drosophila melanogaster,
01:04
our companion in arms. And this is the fifth Nobel Prize that the fruit fly has won. Beginning in 1933 with the great pioneer T.H. Morgan, who actually won the first American Nobel Prize
01:21
in physiology or medicine. I learned that fact at the Swedish embassy in November, having not known it myself. And so these five Nobel Prizes for the fruit fly turned out to be a source of amusement because one of the perks I received from this prize
01:44
was to be on the American radio show Wait, Wait, Don't Tell Me. And Paula Poundstone, this fantastic comedienne, said on the show, she said, five, five Nobel Prizes? How do those little animals fly with five metals, those heavy metals around their neck?
02:02
So let me begin where this story begins and tell you that circadian rhythms arose because this inexorable rotation of the Earth, the light-dark cycle and in most of the Earth, daily changes in temperature, is the environmental cue to which life has adapted
02:23
well before the atmosphere achieved its current gaseous constitution, well before nutrition was anything like what is available today for organisms. And so these are the properties that circadian organisms share.
02:42
These clocks continue in constant darkness, so they're self-sustained. And as a consequence, the periods, the endogenous period of organisms are not exactly 24 hours. They're a little shorter or a little longer, as I'll show you. The organisms entrain the light in temperature
03:00
and that's why we are exactly 24 hours because of the daily light cycle. The purpose of these rhythms is to anticipate what is going to happen in the outside world. So the early bird gets the worm and the early worm avoids being eaten by the bird.
03:22
And lastly, there are internal events which are governed by the clock. Many of them, it's more efficient, it's better to do A than B than C than D than do A, B, C, D all at the same time. That's the rationale. So here's an example of two different strains
03:41
of fruit flies in constant darkness. And you can see that the locomotor activity rhythm of these flies is tilted either to the left in a short period variant or to the right in a long period variant, to some extent illustrating the fact that animals, we are about 24 hours and 15 minutes human beings.
04:04
So not completely on 24 hours. And this is how fruit fly locomotor activity rhythms have been traditionally measured. Single flies are put in these tubes. The flies run back and forth. The tubes are snapped into a device
04:22
where there's infrared light source and a photo cell on the other side of the tube. And so this is a beam break device used for rodents and all kinds of activity measurements. And the fruit flies can't see in the infrared, so they're not affected by that wavelength of light. And one can measure their movement
04:42
and their rest period very accurately. And so this was one of the two assays that was used by this fantastic pioneering paper from Konopka and Benzer, now really a long time ago, in which they isolated the period mutants,
05:00
the mutants in this gene period. And this paper is sort of a lesson for the publication side of our business, something that a few of my colleagues here, our colleagues are gonna speak about in a couple of days. This paper received 10 citations in the first 10 years
05:22
after its publication, and it's considered one of the great papers. So cream rises to the top. The thermodynamics is inexorable, but the kinetics is uncertain, a major lesson of life. And so it was 10 to 15 years later
05:43
that Jeff Hall's lab and mine at Brandeis and independently Mike Young's lab at Rockefeller, both working independently, cloned this gene, showed that it was the gene by gene rescue, sequenced the gene. And contrary to our expectations,
06:02
that sequence turned out to be relatively uninformative because these were early days of DNA sequencing. The database was between slim and none. There were only 50 proteins or so, few families in the database. And so there were no relatives.
06:21
And it really wasn't until 20 years after Konopka and Benzer that Paul Hardin, who was a postdoc in my laboratory in collaboration with Jeff, did a really landmark experiment and discovered that the RNA from the period gene underwent circadian oscillations.
06:40
The RNA goes like this, transcription oscillates completely in synchrony with the behavioral rhythms that have been initially assayed. And the protein product, single amino acid changes in the protein, change the phase of the RNA oscillations
07:01
exactly in a manner one would predict if there were a negative feedback loop that governs the transcription. And we proposed that this was central to timekeeping. And over the course of the next decade or so, actually just about a decade,
07:20
many other players were identified in this transcriptional feedback loop. The positive transcription factor, another negative transcription factor, timeless that you'll hear about from Mike Young. And it also was shown that this feedback loop
07:40
is completely conserved between flies and mammals. And so these are orthologous proteins. There's only one substitution, if you will, in the two systems. That is Cryptochrome for timeless in this feedback loop. And so we really have a highly conserved system from flies to humans.
08:01
And this description ignores very important features of post-transcriptional regulation, which you'll hear about from Mike, I'm certain. And so because I made the title past, present and future, I want to highlight in the course of my 25 minutes some challenges for the present and also for the future.
08:26
Why should young people consider this field interesting? What remains to be discovered? And I think there's a very important and profound piece of information missing,
08:41
and that is what really does the timing? In biochemical terms, what is the rate-limiting step of the reaction, or it's almost certainly steps? What does account for the fact that this is almost always 24 hours, plus or minus a tiny bit?
09:00
And this turns out to be almost impossible to do without biochemical reconstruction and nothing along those lines has been done. So I think we really don't know what are the rate-limiting steps. And as a companion question, let me pose for you the question, how does temperature compensation work?
09:22
And of course that term is enigmatic, at least for some of you. And so I need to tell you that this is really an additional canonical property of circadian rhythms. That is the circadian period of an organism changes almost not at all with temperature. And this is not only true for insects,
09:42
cold-blooded animals like fruit flies, which have no change in endogenous period between 18 and 29 degrees, for example, their physiological range. In other words, the Q10 is almost exactly 1.0. But it's even true for mammalian tissues if you put them in tissue culture and then ask what's the circadian period
10:02
of the clock in culture where there's no homeothermic regulation, it's also the Q10 is almost exactly 1.0. So this must be, I pose to you that this must be somehow intimately related to timekeeping, the two are related.
10:21
Why doesn't the timekeeping change as a function of temperature? And I think when we solve one problem, that is the rate-limiting steps, we'll probably have some insight into temperature compensation or vice versa. So this problem is very important. That is the circadian clock is very important for physiology.
10:42
That is, not checking slides. So more than 70% of the genome is under circadian control somewhere. So this is a heat map from the liver and shows, indicates the fact that there are thousands of genes in the liver
11:00
which undergo circadian oscillation and the peak phase of transcription varies around the clock. In other words, some genes are morning genes, some are afternoon genes, some are evening genes. And if you add up from all the tissues that have been assayed, the oscillating genes, you come up with more than 70% of the genome.
11:21
And of course, this explains why so much of physiology is under circadian control because so much of the genome is regulated directly or indirectly by the clock. And of course, this is the second challenge for the present or future, leveraging circadian rhythm knowledge
11:40
to improve human health. And these are the things which we know or highly suspect are impacted by the clock. That is, aspects of human health and disease, diabetes, metabolic diseases, et cetera. And of course, everyone here knows about jet lag
12:02
as an important challenge. Nobody, people come up to me all the time, even here, what do you do? And I say, I suffer and I take drugs. Just like you.
12:21
So this effect of the clock on physiology on all of these various features includes sleep. And I want for a few minutes to talk about sleep in the context of the circadian clock and in particular, talk about fly sleep. And I think Mike will talk about human sleep
12:45
if I'm not mistaken. And so I'll simply point out to you that fly sleep really resembles mammalian sleep and I should have really had the title, it is almost identical to mammalian sleep.
13:01
So there's, for example, there's similar pharmacology if you treat flies with drugs which either increase or decrease sleep. Almost all of them which we know about and affect us in a very stereotype way affect the fruit fly in exactly the same way. There's an increased arousal threshold
13:21
when flies sleep, the simple concept. If you poke a person or a fly when they're sleeping, it takes more of a poke to get them to respond than if they're awake. And that arousal threshold undergoes characteristic changes during sleep. It's higher during the first three hours of sleep than during the last three hours of sleep.
13:43
That's true for fruit flies as well. The effects of aging are almost identical in fruit flies in humans. Old fruit flies wake up at three, four, five in the morning like many of us and sometimes they go back to sleep, sometimes they don't go back to sleep.
14:01
And so it is a really excellent substrate for studying the aging aspects of sleep. And then I think most interestingly from the scientific point of view is this fly sleep is under homeostatic regulation. So what do I mean by that? If you sleep deprive flies in any of a number of ways,
14:25
simplest is to shake them, then the amount of sleep that the flies experience shown here on that graph, the y-axis is sleep and the x-axis is time and the yellow strain is experiencing sleep deprivation.
14:43
And now if at the end of that 24 hour cycle, if the sleep deprivation is removed, then the flies will oversleep to make up for the missing sleep that they have missed during the deprivation period. So that's homeostatic regulation.
15:02
Something is keeping track of sleep need or sleep debt. And so of course it's not only under homeostatic regulation, but it's also under circadian regulation. That is the clock is making us tired at 10 30 at night, sort of in an inexorable metronome like fashion.
15:21
And then we're optimally alert at seven in the morning. And that circadian regulation is very regular and is contrasted with the homeostatic regulation, which is more plastic and more sensitive to past history and environmental circumstances.
15:41
So sleep is almost certainly principally controlled by the brain. And Drosophila has about a million fold fewer neurons than the human brain. So about a hundred million versus a hundred billion neurons. And of course that makes a brain problem
16:03
to the extent to which it's shared between fruit flies and humans much more accessible experimentally in fruit flies. And from the circadian neuron perspective, there are only 75 Drosophila clock neurons in the fruit fly brain.
16:20
They're stained in red on the left there. That's an immunohistochemistry picture with an anti period antibody. And one can visualize about 75 neurons. They're in small numbers of groups with names and functions and so forth. And of course this is an outstanding neural circuit problem
16:44
and something I've focused on for the past 15 years or so in my lab. That is how does this small group of neurons interact with each other and how do they control behavior? And so this is what I'm gonna talk about
17:01
for the next seven minutes or so, a couple of experimental directions that were led by a postdoc Fang Guo and a master's student Megana Hola who worked with principally with Fang. And so the backdrop for this goes back over these 15 years or so
17:20
and it is centered focused on this canonical activity pattern that is characteristic of Drosophila melanogaster for that matter is characteristic of mosquitoes. Lots of other insects. There is a peak of activity in the morning illustrated here by this AM peak. These are total activity events
17:43
in 30 minute windows in 30 minute sections across the day within an incubator with a light dark cycle. So what you see on the top is six hours of night, 12 hours of day and then six hours of night. And there's a morning peak and then a siesta in the middle of the day.
18:02
Flies are very smart and like people in much of the world they know about taking a nap in the middle of the day. They then have a peak of activity in the evening and then they sleep at night. And notice those activity peaks anticipate the discontinuous transitions in the incubator.
18:21
In other words, the lights snap on and snap off. There's no gradual accumulation of dawn and yet the animals ramp up their activity in advance of the discontinuous transition. They know that dawn and dusk are coming and that's the clock doing its thing. And so the different circadian neurons,
18:40
these 75 neurons can be divided into small little groups and one of them is responsible for the peak of morning activity. One of them is responsible for sleep, siesta and nighttime sleep and another group is responsible for evening locomotor activity. They work autonomously.
19:00
That is you can eliminate that group and that particular activity event will be eliminated. You can accelerate the clock in only one of those groups and then that particular event will shift in time whereas the other events stay temporally in their position. And so this is a quote from Sydney Brenner.
19:24
Progress in science depends on new techniques, new discoveries and new ideas probably in that order. Something that the students might wanna discuss with us is the importance of new ideas. How important really are they? One perspective is that ideas are easy and cheap
19:42
and most of them are wrong unless they're based in some new experimental discovery. Those brackets are mine actually because I think it's debatable whether discoveries, a chance piece of data in the lab or new techniques should be in first place there but I agree with Sydney
20:02
that those two are much more important than new ideas that are not based on an experiment. So here's a new piece of equipment that we built in order to do optogenetics and to record activity. So rather than using those tubes, we use 96 well plates of flies so that the flies can run around in the individual wells
20:23
and then we record the locomotor activity with a camera and most importantly because the insect cuticle is so thin, light can go in easily from LEDs and so we can optogenetically stimulate precisely even a single neuron carrying
20:41
a channelrhodopsin gene with a certain wavelength and then either activate or inhibit the neurons. And so here's an experiment in which those sleep promoting DN1 clock neurons are inhibited with green light when they carry a particular channelrhodopsin
21:03
which contains a chloride channel which inhibits those neurons and what you see in turquoise and yellow in the top and the bottom during the 24 hour period illustrated with the bracket is that the amount of sleep is dramatically suppressed
21:21
when just a handful of neurons, four neurons, carry that inhibitory chloride channel compared to the parental strains. And so these neurons really affect the circadian behavior and impact sleep in a very important fashion.
21:41
And so a second technical advance, if you will, is designed to address when do these neurons actually fire and how does this firing relate to locomotor activity? And of course the challenge, a profound challenge for neuroscience in general is how can you assay neural activity
22:02
in a freely moving behavioral animal in a neuron specific way? So how can you actually do this? The animal gets to run around and you want to know which neurons are firing when. And so the strategy that Fang took which is illustrated on the right hand side here,
22:21
the one we finally used on the right takes advantage of some tools that Li-Ching Lo's lab built and the key is that a transcription factor which is calcium sensitive is used to drive a reporter gene which expresses luciferase. And so when calcium is high,
22:41
that transcription factor is active and that generates a lot of luciferase and that's the key to the assay. And now the sort of version two of the fly box is these plates in which the flies are running around
23:01
and being video recorded gets slid into a top counter, a light monitoring machine which is used all over biotech for doing 96 or 384 well assays and individual flies are in there and of course the cuticle is light penetrant,
23:22
not only light on the way in but light on the way out. And so this is so sensitive that even a single neuron carrying this luciferase reporter will report the amount of light over circadian time over many days and that can be assayed.
23:41
And sure enough when one assays either wake promoting neurons or these DN1 sleep promoting neurons shown in red then one gets entirely distinct patterns of activity which correspond to the function of these particular neurons. For instance there's the blue peak on your left
24:01
corresponds to evening activity and there's a peak of neural activity or calcium I should say more properly from these cells which completely corresponds to that evening activity peak consistent with the idea that they're driving the activity. So we have both activation and inhibition
24:20
and we have tracing to indicate that these neurons are really important. And lastly I should say that these sleep promoting DN1 neurons in work which is very recent, they have been traced to a region of the central brain which has been identified as being really important for controlling sleep and arousal threshold.
24:43
So those circadian neurons track and trace directly to this region of the ellipsoid body and activating the circuit not surprisingly promotes sleep and actually raises the arousal threshold. And so to return to the question I asked at the beginning,
25:02
why are these neurons different? What makes them fire at different times of day? It's almost certain that differential gene expression probably explains the firing difference between neurons and we've just published a differential gene expression paper for these different neurons in eLife I should say.
25:22
So I've mentioned of course that fly sleep is regulated by homeostatic as well as circadian regulation and this is the third challenge I want to present to the young people here thinking what should I do with the rest of my career to address the question why do we sleep
25:40
and what's the conserved function of sleep? By conserved I mean identical in flies and humans and of course now I'm hoping that this field will recapitulate what's happened with circadian biology and of course another way of posing this question is where and what is keeping track of sleep need? What is the counter, the homeostatic counter
26:03
and where is the homeostatic counter? What's the biochemical principle? So with that I'm going to close on time I might add to the chairman who just went like this to me and I don't think this was a victory sign. And so I need to just say in two words
26:25
without the lengthy slides that I originally had that I owe a profound thank you for not only for being here for my wonderful career, my journey to the many people who've worked in my lab, my fantastic trainees and my awesome family,
26:43
some of whom are here as well and with that I look forward to chatting with all of you over the course of this week. Thank you very much.