We said that we wanted a model system for clocks research, because of the possibility to dissect the system. Rodents have been used to map out the organization of the mammalian circadian system. For many years, there was the debate, where the clock was located or how clock behavior develops. Is it an additive property of many individual cells, all with their own clocks? Or is it an emergent property of different parts of a complex system? This was nailed down in a variety of ways. First, a discrete region of the brain called the suprachiasmatic nucleus, the SCN, was identified as a circadian pacemaker. This means that is is largely responsible for dictating sleep - wake behavior. The SCN was indicated as the pacemaker in many ways, in many experimental protocols. One of the most dramatic examples were experiments where the SCN was excised. It was surgically removed showing that the animal became arrhythmic. In this series of experiments, an SCN, a suprachiasmatic nucleus was then transplanted into an animal that had an SCN removed. It was transplanted within a capsule, and it led to the restoration of rhythms. This demonstrated the role of soluble factors in circadian pacemaking, because the SCN was in that capsule. Interestingly, light entrainment of the clock was never restored in these transplant experiments. The intact SCN is directly innervated by cells from the retina. The graph shows here that the animal continued to free run even when they're held in light dark cycles. So we know that in mine there's a central pacemaker that organizes behavioral rhythms. But we still haven't answered the question of if this arises from individual cells, or if it's a property of the organ itself. The question was answered by dissociating SCN neurons and placing them on a microelectrode array. The spontaneous firing rate of the individual cells could be followed and it showed that each one possessed its own circadian rhythm. They had their own period and their own unique phases. They could fire at different times of day when they're separated from one another. Amazing experiment. Interestingly, if the cells are plated so densly that they form synapses, they can synchronize their period and phase. A follow-up question might be: Are neural cells and, indeed, SCN cells special or do other cells also show rhythms? This graph shows free running rhythms in a human osteosarcoma cell line in culture U2OS cells. Skin fibroblasts and immune cells from mice and humans are also rhythmic, as are cells from practically all tissues that have been tested. What have we learned about circadian organisation? The mammalian clock uses a central pacemaker, the SCN, the Suprachiasmatic Nucleus, and it's important for entrainment by light. Peripheral cells and organs have their own clock. They use many zeitgebers, signals from the SCN, endogenous temperature cycles, other sensory and metabolic inputs. There will be more on that in the coming lectures. The circadian clock is built from daily rhythms in almost every cell in our body. There's another extremely important animal model, namely <i>Drosophila melanogaster</i>, the fruit fly. Why the fly? If mice are cheap, flies so much cheaper. They're obviously smaller. There's a long tradition in genetics. They have a small genome. Many of their genes are homologous to human genes. And where a mice have billions of neurons, flies have about a hundred thousand, making mapping of neural networks somewhat less complicated. What does a circadian rhythm in a fly look like then? One of the pioneers in the field, Colin Pittendrigh, performed many fundamental circadian experiments on eclosion, the event in fly development where the animal emerges from the pupae. This graph shows how a population of pupae emerge at a discreet time of day. First in a light dark cycle, and then continuing at about the same time of day, or as a circadian rhythm, even when they're held in constant darkness. Like humans and mice, <i>Drosiphila</i> show rhythms in activity and rest that continue in constant darkness. These are measured in individual animals held in small tubes. When they fly around they break a beam of infrared light which they do not perceive. And this gives a graph of their activity. Much like was shown for mice. In addition to feeding, courtship, olfaction, the Malpighian tubules are kidneys of flies also shows circadian rhythms. These are the main animal models that are studied for circadian rhythms research. There are in addition, many more that are studied for specific questions. Next, I want to tell you about circadian rhythms in plants. Interestingly, it's often said, that modern chronobiology began in 1792, with an astronomer and the Mimosa plant. De Mairan noticed that the plant on his desk showed rhythms and leaf movement. He did an experiment namely to put the plant into a dark cabinet and check its leaf position through the night and day. He saw that although the plant was removed from the environmental light dark cycle, the leaves continued to go up and down during the day and night. Some of you might also be familiar with the flowering clock of Linnaeus showing which flowers open at different times of day. This likely reflects a co-evolution with the insects that pollinate them. For most experimental plant work, <i>Arabidopsis thaliana</i>, a small weed, is used for clocks research. What does this clock do? Similar to what de Mairan predicted, its leaves move in constant light, with a period of about 24 hours going up during the day, and coming down at night. This movie shows a sunflower changing its orientation over the course of a day. This is in a light dark cycle. The plant orients towards the sun and at night, it moves back around. Importantly, the plant is anticipating where the sun will come up even before sunrise. This anticipation is thought to be a key function of the circadian timing system. Anticipation has long been appreciated in plants since the photosynthesis machinery already increases before dawn. This graph shows that expression of chlorophyll ab binding protein is clock controlled continuing for many days in constant light. Here you see a time-lapse movie of gene expression in situ in a leaf of a small <i>Arabidopsis</i> plant. The chlorophyll ab binding protein promoter, is driving expression of firefly luciferase in constant darkness. A new model organism for green clocks is <i>Ostreococcus</i> ,a single celled marine algae. Like <i>Arabidopsis</i>, it also shows rhythms in chlorophyll ab binding protein expression. The last model eukaryote that I want to discuss is from the fungi. <i>Neurospora crassa</i> is a filamentous fungus, that spends most of its life in the asexual or vegetative state. It grows as mycelia or vegetative hyphae, growing along the surface of and into agar, that is when it's in the lab of course. About once a day in constant darkness, or exactly once a day in a 24 hour light dark cycle, a developmental switch is thrown and asexual spores called conidia form. These grow on aerial hyphae and thus make a distinctive appearance, a so-called band, a big fluffy mass. When the fungus is grown in a race tube like this, the linear growth is interspersed with the conidiation pattern and this can be highly quantitative. So with this very simple readout, a lot of clock biology can be determined. This system has been invaluable in working out the basic mechanistic principles that we'll be discussing in lecture three. The last model system that I want to describe today is cyanobacteria. <i>Synechococcus</i> is a freshwater photosynthetic prokaryote, hence it has no nucleus like the eukaryotic models that we work with. We might predict that this will make some differences in how they make a rhythm, and we can discuss this in a later session. This class of circadian rhythms, the prokaryotic class, the cyanobacteria class, was initially described due to the fascinating temporal segregation of incompatible metabolic processes, nitrogen fixation and photosynthesis. In species that feature organelles dedicated for these processes, the temporal segregation is lacking. Like higher plants, photosynthesis is clock regulated, as are many processes in this bacterium. That's a summary of circadian rhythms in the model systems, that we'll be discussing as we move through this course. I want to take a minute now to describe one of the other key clock properties. We talked extensively about entrainment and especially about free running rhythms in this lecture. But there's a third key clock component namely temperature compensation. This refers to the temperature sensitivity of most biological systems, whereby they increase rates at higher temperatures, and decrease them at lower ones. The rule of thumb is a twofold difference in rate over 10 degrees, Centigrade or a so called Q10 of 2. Circadian systems have a Q10 of close to 1, which is thought to protect their integrity as a clock, rather than serving as a thermometer. Here you can see the free running period in eclosion over 10 degrees. The eclosion occurs almost at the exact same time, while the rate of development slows down considerably at lower temperatures. These two exquisitely timed processes are clearly distinctly regulated with development, being temperature sensitive and the clock not so. The period of conidia formation in the fungus <i>Neurospra</i> also changes little over ten degrees. Here showing a Q10 of about 1.2. To summarize what I've told you in the second half of this lecture. I've shown you examples of rhythms in animals, plants, fungi, and cyanobacteria. You've learned about common properties of clocks, free running period, entrainment with zeitgebers, and temperature compensation. We'll discuss these more in coming lectures, especially entrainment, in the next session. We've also discussed how to interpret graphs showing circadian biology, single and double plots for instance, and about special considerations in performing chronobiology experiments. Up next, the wrap up session. [SOUND]
