Welcome to the lecture on circadian formalisms and entrainment. The circadian clock is quite unique, although it does not primarily produce a physiological, metabolic or behavioral outcome, there is practically no physiological, metabolic or behavioral outcome, that is not influenced or modulated by the circadian clock. The pioneers of circadian research were excellent in defining the properties and behaviors of this function in great detail. You have heard about many of these definitions in the wrap up of the introduction section. Although the independence of circadian rhythms from a cyclic environment was already discovered by de Mairan in the 18th century, and many botanists started to investigate daily rhythms experimentally in the first half of the 20th century, a broad and systematic research into circadian clocks was only established after the Second World War. One of the first international conferences dedicated to biological rhythms was organized in Cold Spring Harbor in 1960. The papers and discussions published in the proceedings of this conference are more or less our field's birth certificate. Colin Pittendrigh, one of the pioneers of circadian research, contributed a compendium of 16 definitions concerning circadian rhythms in these proceedings, which he called "Empirical generalizations". I will use these generalizations to introduce many important concepts that explain how circadian clocks function. The important impact of these commonly accepted definitions greatly contributed to the success of our field, ranging from characterizing many of its behaviors to eventually identifying the genes that work together in this temporal program. We already covered the first "generalization" in the introduction section. It introduces the concept that daily rhythms continue to oscillate in a constant environment with their own period, defined with the Greek letter "tau". Rhythms that oscillate with their own period in constant conditions are called free-running rhythms. Pittendrigh therefore adds FR to specify that "tau" is being measured in a free-run. Many of the generalizations concern the clock's behavior under constant conditions, but I will explain how all these relate to entrainment. Pittendrigh makes two points in his first "generalization". First, that true biological circa-rhythms continue to oscillate in a constant environment. And second, that circadian rhythms have a period around 24 hours. Most of the 16 "generalizations" have some accompanying statement. In this case, he clarifies that the capacity to free run is a pretty good proof for the oscillation to be endogenously generated, but that this is "by no means" the only evidence, for the clock's endogenous nature. Allow me to digress for a moment. After de Mairan discovered in 1729 that the leaves of the mimosa plant, continue to fold and unfold in constant darkness, researchers continued to be fascinated by the easily observable "free-run". The circadian clock is the product of an evolutionary process that involves mutations and their selection based on fitness. Since the presumed advantage of the circadian system, is to optimally adapt to a cyclic and predictable environment, the selection pressure must have also involved a cyclic environment. If this hypothesis is correct, then the evolution of this circadian system never involved selecting for clocks to free-run in constant conditions, but rather selected for an optimal timing of events WITHIN the cyclic environment. Consequently, the free-running period of circadian rhythms, observed in practically all organisms, must be rather a CONSEQUENCE than a prerequisite of circadian rhythms. One of the important characteristics of circadian clocks is that they are flexible in how they embed themselves into the light-dark cycle. This is exemplified in the next figure. The top blue rhythm peaks around dawn every day, the green rhythm around noon and the red one in the afternoon. The phase relationship that a circadian rhythm adopts to it's zeitgeber is called "phase of entrainment". "Entrainment" is used by chronobiologists to define the active mechanism of clocks to synchronize with their cyclic environment. The "phase of entrainment" can be measured arbitrarily by defining some characteristic phase of the zeitgeber, for example dawn. The phase of the zeitgeber is symbolized by the greek capital letter "Phi". We also have to define a characteristic phase of the biological rhythm, for example its maximum. The phase of the biological rhythms are symbolized by the Greek small letter "phi". The difference between these two phases defines the "phase of entrainment", which is symbolized by the Greek letter "Psi". I'm going into these phase relationships in such detail because they are a central function of the circadian clock in real life. The mechanisms that allow fine-tuning phase are based on an oscillator that is entrained to its environment. Pittendrigh stated in his first "generalization" that the free-running period is not the only evidence for endogenous generation of circadian rhythms. A stable but still adaptable "phase of entrainment" is an excellent alternative proof for an endogenous process. The second "generalization" emphasizes the ubiquitous nature of circadian rhythms in a lovely double meaning. The first states that circadian clocks are found in organisms of all phyla. This suggests that having a circadian clock is the default state of an organism -ranging from simple unicellular algae to complex human mammals. It also suggests that exceptions to this default state must have a good reason for NOT having a circadian clock. In the other meaning of this "generalization", Pittendrigh predicted that there will be practically no function in an organism that is not modulated by the circadian clock. The scope of this prediction exploded around the turn of the century when scientists found that every cell in the body potentially has the capacity to generate its autonomous circadian rhythms. The third "generalization" stresses the fact that circadian rhythms are NOT elicited purely by the environment, but they are generated by the organism itself. In view of the well known fact that circadian rhythms continue on constant conditions together with the fact that the free running period can deviate from 24 hours, this "generalization" is somewhat strange. The reason for this is that in the 1960s, there was a heated discussion between most circadian biologists and Frank Brown, a well-known endocrinologist from Northwestern University, Chicago. Brown insisted that it was impossible to ensure real constant conditions and therefore, some unknown signal (other than light or temperature) could be responsible for the daily rhythms to continue in constant darkness. In the published discussion part, Colin Pittendrigh called this unknown element a "ghost", and makes the following statement: "The question of the ghost is simple -either it is an aspect of living organization, or an unknown geophysical variable. My taste in ghosts suggests the latter, but as a scientist, I must agree that Dr. Brown may prove right; and as a scientist, HE will doubtlessly agree he may prove wrong. We will both have some fun in any case." "Generalization" number 4 addresses how rhythms change when released into constant conditions. The rhythm shown in this graph continues to be rhythmic in constant conditions, but its amplitude dampens until the rhythm only shows some residual random fluctuations. Even dampened rhythms are generated by an oscillator. They therefore can adopt a specific phase-relationship to the zeitgeber. Since most organisms hardly ever experience constant conditions, such a damped oscillator could adequately serve as a circadian clock. Although self-sustainment is obviously not a prerequisite for the circadian function, most circadian rhythms actually are self- sustained in constant conditions. Based on the hypothesis that the circadian system has been selected for under real-life conditions, I would argue the self-sustained free-run simply reflects how a circadian system was optimized for and under entrained conditions. We don't have to go into the fifth "generalization" too much here, since the genetics of circadian systems will be covered in other parts of the course. I will however address the comment attached to this "generalization". Pittendrigh and others had observed that circadian rhythms were sometimes absent when an organism was raised in constant conditions from the beginning of its development on. This graph exemplifies such an organism that was raised in constant conditions. Initially, it shows no evidence of a circadian daily rhythm. Yet, a single light pulse can jump start the clock so that it continues to oscillate even in an unstructured environment. At the time, researchers interpreted this as an actual jump start of the clock itself. Meanwhile, we attribute the appearance of circadian rhythmicity to the fact that the light-pulse simply synchronised the individual cellular clocks in an animal, each of which produced perfectly normal circadian rhythms all of which were out of phase with each other. Such a scenario could look like this and the average output of these oscillators would look like this. No rhythm is visible although the underlying oscillators are all perfectly rhythmic. If some external signal can synchronize the individual oscillators, then the rhythm would become apparent. In the case described in "generalization" number 5, the Drosophila oscillator could be synchronized with a flash of light as brief as half a millisecond. The scenario of how several oscillators interact to generate a rhythmic output leads us to the next "generalization" that addresses the levels of circadian organization within an organism. In 1960, circadian researchers did know that single cell organisms can produce perfect circadian rhythms with all the features known from the clocks and plants and animals. But in higher animals such as mammals, one believed that the dedicated center in the brain such as the SCN, was the heart of the circadian system which made everything else in the body go up and down at the appropriate time of day. What they didn't know at the time was that every cell in the body contained an autonomous circadian clock -similar to a single cell organism- and that the SCN was merely a conductor of all these independent circadian oscillators. Something like an internal zeitgeber. Pittendrigh speculated that a hierarchical circadian system may even exist within the cell. As you will hear later, he was completely right. "Generalization" 7 points out how precise circadian rhythms are. The period of a free-running rhythm may only vary by a couple of minutes per day. It is remarkable how much the field was focused on the phenomenon of a self sustained free run -and somehow still is. Any selection for precision in a free-running rhythm would have required the organism to live in constant conditions for a long time, which is highly unlikely. Yet the clock-like precision of free-running rhythms is indeed remarkable for a biological system. When we translate this precision back to normal real life, it means that -given the same daily conditions- the system can keep an extremely precise phase relationship, with its zeitgeber. For many of the circadian functions, this dependable precision in phase is crucial, for example, for bees, which use their circadian clock in combination with the position of the sun to orient precisely in space. We will come back to this later in the course. But how does the period of a rhythm that is free-running in constant conditions relate to a certain phase under entrainment? Let us presume three circadian systems that produce different periods in constant darkness. The top rhythm is slightly shorter than 24 hours, the middle one is more or less exactly 24 hours, and the bottom rhythm is slightly longer than 24 hours. As you will learn in more detail later, different mutations of clock genes can produce different periods in constant conditions. So let's presume these three rhythms represent different clock mutants of a given organism. If these three mutants are exposed to the same light-dark cycle, their predictable phase of entrainment will look something like this. The shortest rhythm will reach its peak earliest, let's say around dawn. The rhythm that is closest to 24 hours may peak around noon, and the slowest rhythm sometime in the afternoon. It is important to understand that if individuals of the same species have different periods in constant conditions they will produce systematically different phases under entrained conditions. The reverse is however not necessarily true. When we see individuals of the same species entrained later or earlier in a light-dark cycle, we cannot automatically presume that they will produce longer or shorter free-running rhythms. Different reasons may lead to different phase relationships, ranging from different light sensitivities of the respective circadian clock, to the robustness of the underlying oscillator. The next two "generalizations", number 8 and number 9, are again evidence for how much the field focused on constant conditions. Number 8 states that free-running rhythms may spontaneously change their period despite a constant environment. This can only mean that the rhythms are not only generated endogenously, but that their period may change due to events within the organism itself, such as aging and development, reproductive state, circannual time of year, etc. Number 9 states that free-running rhythms may be species specific. If one measures many individuals, the period of the free-running rhythms form a normal distribution. Mean and width of this distribution are specific for a given species. Again, I argue that the observed changes in free-running rhythms reflect changes in entrained phase. The mechanisms that produce the free-run in constant conditions are the same that fine-tune the phase of the rhythm within the structure of the zeitgeber. As much as an individual may spontaneously change its free run, it may change its phase of entrainment, for example, depending on age. Thus, phases of entrainment also form normal distributions. Their characteristics are specific for age, or time of year, or species. I will come back to this in much more detail when I talk about human chronotypes and how human clocks behave in real life. You may have noticed that the numbers on the x-axis go in the opposite direction as usual, namely from positive on the left to negative on the right. This is due to a convention in circadian biology. Processes that precede an event are given positive numbers and those that lag negative numbers. One can easily remember this convention because longitudes to the east of Greenwich where the sun rises earlier have positive and those to the west where the sun rises later have negative numbers.