Welcome back. If you've made it this far in the course, you have a solid background in all things circadian, basic properties of circadian systems, molecular mechanisms and genetics of the clock. Now we can start to understand not only how the clock is working, but the many things that it regulates and how it does this. By the end of the lecture you'll have a few new concepts under your belt. A more comprehensive list of what kinds of things are clock regulated, a concept of what this means with respect to the Eskin-o-gram -the cartoon that we use to suggest how the clock is put together- and several mechanisms that the clock uses for regulating outputs. In many cases, I'll show you examples of data so that you can get an idea of what sorts of experiments have been done to show clock control. We'll discuss some of the ways that the field of clocks research thinks about how to determine if something is clock regulated or simply driven by zeitgeber changes. We've already seen quite a long list of clock-regulated physiology and behaviour. In the first lecture, I showed these slides, listing physiologies and behaviours that oscillate in humans each day. This slide shows that blood pressure and lung function change with time of day. The next one says that many components in blood from immune cells to ions to gas concentration are changing with time of day. Hormones are going up and down, very often both with pulsatile patterns but also with longer daily expression patterns. The electrical activity in your brain shows different characteristics over the day and night -this is especially obvious during sleep, as you saw in the first lecture. But it also has indications on how well we perform over the course of the day. We are better at solving problems in the mid to late afternoon than at other times of day. Should you believe me, that so much physiology is regulated by the clock? I start to sound like some sort of fanatic, everything is run by the clock. Part of working in science is learning to be critical through knowledge and logical thinking. How should the experiment be done? And was it done in this way? I guess that you can appreciate how difficult it is to set up the simplest of experiments in for instance mice, to develop methods to evaluate them in constant darkness. So researchers use night vision goggles. We don't generally put humans into these kinds of conditions. I asked you in the first lecture to discuss how you would improve the protocols that Aschoff used in the bunker. Now I want to take a few minutes to discuss how the field of chronobiology research has approached measuring the human circadian clock. The protocols that I'll discuss are the constant routine, a modified constant routine (that includes sleep or a nap), temporal isolation, and forced desynchrony. In a constant routine, a subject is invited into the lab usually at least a day before the experiment starts. They go into a room that is dimly lit and the lighting remains exactly the same for the duration of the experiment. The subject is kept in bed, with a posture almost lying down. Semi recumbent. Meals are very small, similar in calorie content and generally boring (or rather they should not be too exciting) and they are given at short intervals. The goal is to keep the individual quiet and not allow anything eventful into the experiment that might be reflected in the behaviour or physiology that will be monitored. The first night, they're allowed to sleep a pseudo normal night, with wake time specified as part of the protocol. Then the next day they're required to stay awake for 40 hours, not sleeping for an entire day, night and following day. After the 40 hours awake, they then are allowed to sleep again for a night before being released to go home. So why no sleep for 40 hours? That's to prevent masking of certain measurements by sleep. (1) This graph shows how different core body temperature is when the subject is allowed to sleep in a constant routine, still with continuous bed rest, (2) compared to if they remain awake the entire time, marked here as sleep deprivation. The average temperature is higher with sleep deprivation and the amplitude of the daily oscillation is slightly larger (3) when the subject is allowed to sleep. The black line shows what happens when normal activity is allowed. The amplitude almost doubles. So sleep and activity both mask the circadian regulation of core body temperature by the clock. The constant routine is the experimental answer to the masking problem. Here are three more graphs that show results from constant routines, demonstrating that cognitive and physical performance are rhythmic in constant conditions. In the first, computation performance -how quickly and accurately the numbers are added or multiplied- varies by about 20% over the day. The speed of reaction to a visual stimulus changes by about 15% between day and night. How tightly something can be gripped with your hands changes by about 10% over the course of 24 hours. Constant routines, as were used here, are really difficult to do. They're expensive, requiring a lot of attention from scientists and technicians just to get a single data set. More important, they're pretty hard for the subjects, who have to stay awake in a dimly lit room while sort of lying down through an entire night and two days. The subjects are so often doing tests that somehow the time manages to pass. But constant routines also deliver only a single 40-hour time series of data for the entire protocol and although it's not masked for sleep and activity, it can show masking effects caused by accumulating sleep deprivation. So scientists have been looking for good alternatives. One is a simple modification of the constant routine, either allowing the subject to sleep for the intervening night or allowing a short nap once every 2-3 hours. By allowing sleep, sleep deprivation is reduced, thus eliminating that variable, despite introducing some effects due to sleeping. The data that I showed in the first lecture -for heart rate, core body temperature and melatonin levels- was from a modified constant routine where the patients were allowed to sleep overnight. All three of these parameters are also rhythmic in a constant routine with no sleep. The difficulties with constant routines led to yet another protocol for measuring circadian rhythms in humans, namely, the forced desynchrony protocols. This method is based on entrainment - or rather the lack of entrainment- of the human circadian clock. In the entrainment lecture, you learned that circadian systems are so robust that they can NOT entrain to zeitgeber cycles that are outside of a rather narrow limit, the so-called range of entrainment. Humans and many other mammals may have difficulties entraining to light dark cycles as short as 23 hours or as long as 25 hours. There is, of course, some inter-individual difference in this range of entrainment. But by going outside these limits, let's say 20 or 28 hours, one can be sure that subjects can't entrain. Under these conditions, one can watch their circadian rhythms in physiology and performance run through the short or long enforced sleep wake cycle, showing a circadian rhythm close to near 24 hours. This graph shows how the timing of melatonin production changes in a 28 hour constant routine. In some individuals it gets early relative to local time, and in others it gets later. This means, that in this protocol, some people have free running periods of less than 24 hours while others have periods longer than 24 hours. Interestingly, the average free running period in a forced desynchrony comes out very close to 24 hours. In the protocol I showed you in the first lecture, the experiment where subjects slept in apartments with no information about local or sun time, for around 30 days in a row, the free running periods were on average longer -about 25 hours. Why might this be? The protocols are fundamentally different, and it's up to us to figure out what it is telling us, what these differences mean. Perhaps you have some ideas about this, already. I suspect that it's related to the timing of self-selected light exposure, compared to the regimented one in the forced desynchrony. The reason that I think this is part of the explanation is that animals in constant darkness have a very different free running period than those in constant light. So for a starting point, we can already say that there's no single free running period, that it depends on the constant conditions and that the light in the constant conditions has a big effect. Hence, different free running periods in humans. In this first section of the fourth lecture I've told you about many clock controlled processes in humans and we discussed various methods that can be used to measure them. I went into detail using this example, but many of the same considerations are relevant for animals, plants, fungi, and probably anything with a circadian clock. Other animals generally show all the same sorts of clock-regulated processes but in the next section, I'll tell you about something really different.
