"Generalization" number 12 introduces another pioneer of circadian research, namely Jürgen Aschoff. Aschoff was a German physiologist who studied circadian rhythms in many different species, from house-flies, via starlings, to hamsters. But he was most famous for studying humans in temporal isolation by building a bunker into a hill in Bavaria. Although Pittendrigh and Aschoff often differed in their interpretations of circadian phenomena, they were close friends throughout their life. "Generalization" 12 refers to the observation that the period in constant conditions depends on light. Many free-running rhythms can be recorded both in constant darkness, called "DD" and in constant light, called "LL". Aschoff had observed that day-active animals produced shorter free-running rhythms when exposed to "LL" compared to "DD", while the opposite was true for nocturnal animals. Pittendrigh proposes to call this systematic observation "Aschoff's rule". But Aschoff himself later denied this being a "rule" because he found too many exceptions. Again, the whole argument shows how much circadian biologists were fascinated by the free-running period, rather than translating their observations into real-life and "phase of entrainment". We have already discussed the strong association between phase of entrainment in a light-dark cycle and free-running rhythms in constant conditions. The earlier the phase, the shorter the period. If we translate Aschoff's rule into entrained conditions, we come to the following scenario: In many nocturnal species, light slows down the clock compared to that in darkness. As indicated by the horizontal arrows in this figure, the two opposing influences on the circadian system emphasise the central role of dusk for nocturnal species. The effects of light and darkness are the opposite for many diurnal species... Light speeds up the clock compared to darkness. In this case, the two opposing influences on the entrained circadian clock emphasise the central role of dawn for diurnal species. The fact that there are many exceptions to the rule just tells us that the ecology and evolution of every species is unique and that many of the nocturnal and diurnal species have evolved without anchoring their daily programme to dusk or to dawn. In many ways, most of this lecture was already on entrainment of circadian clocks, despite focusing primarily on free-running clocks. Although an adaptable phase of entrainment is the most important function of circadian clocks, Pittendrigh officially comes to this feature of the system only in his 13th generalization. This "generalization" states that there are two dominant environmental signals that circadian clocks entrain to: light and temperature. And warm-blooded, homeothermic animals predominantly use light to entrain to their cyclic environment. Aschoff had coined the term "zeitgeber", (German for "time giver"), which has meanwhile been widely accepted as the term used for environmental signals that clocks can use for entrainment. We have experimented earlier in this lecture with zeitgebers that have a shorter period than 24 hours and presumed that the respective circadian clock manages to entrain to such a short zeitgeber cycle. Most Circadian clocks can indeed entrain to zeitgeber cycles longer and shorter than 24 hours, but do so only within certain limits. As the 13th generalization states, these limits appear to be wider in simple and more narrow in complex organisms. Aschoff and his collaborator Rüdger Wever exposed humans in their isolation units to a wide range of zeitgeber periods and found the following results: Subjects started out to be exposed to a normal LD 12:12. Every week, the length of the light dark cycle was shortened by 30 minutes. Up to 22.5 hours -namely 11 hours and 15 minutes of light and the same length of darkness- the subjects´ clocks entrained stably to these non-24 hour-cycles. Shorter cycles were, however, outside of the so-called "range of entrainment" for the human clock, so that subjects could not successfully organise their lives within the presented days and nights. As discussed earlier, the "phases of entrainment" delayed progressively in relationship to dawn with decreasing zeitgeber cycle length. While it was close to lights-on in LD 12:12 it lagged the light transition by several hours, when the LD cycle was only 22 and a half hours. The 14th generalization introduces the concept of phase-shifts which is at the heart of entrainment. These phase shifts are also the basis of the so-called "phase response curve", or PRC for short. The PRC and its role for entrainment where at the center of Pittendrigh´s research for most of his career. He was fascinated by the fact that a single light pulse could change the phase of the circadian clock and that in some organisms, this light-pulse could be as short as a millisecond. I would like to introduce the concept of phase response curve, the PRC, by assuming that a component of the circadian system is degradable by light. This assumption is very realistic because one or more molecular components of the circadian circuit are indeed light sensitive in all organisms investigated so far. Components of the clock oscillate with a circadian frequency as exemplified by the red curve shown in this graph. If one presumes that a light pulse is able to degrade this component, this intervention will shift the phase permanently in comparison to the control rhythm. Please compare the red curve representing the control rhythm with the blue curve representing the rhythm that received a light pulse. For a successful entrainment, the responsiveness of the system must be phase dependent, meaning that it responds differently to the same stimulus, depending on the internal time it encounters this stimulus. Phase-specific responses are a feature of every oscillator, as can be easily demonstrated by a swing. Depending on whether the swing moves away from you or towards you, your push will have a different effect. If you push in the direction that the swing is already moving, then you will speed up or advance its path. If you push the swing when it comes towards you, your push will decelerate or delay its path. This phase-specific responsiveness gives rise to the PRC. The phase response curve shown here is a PRC to light pulses. If light is switched on for a brief time, some time between internal midnight and internal noon, then the clock will be advanced, while it will be delayed during the other times of the internal day. Around internal mid-day, a light pulse will have little or no effect. This makes perfect sense for synchronizing a rhythm that has become out of phase with its environment: If a circadian clock encounters light in the second half of the night, this clock is too late or too slow and has to be advanced. If it encounters light in the first half of the night, it was too early or too fast and needs to be delayed. Since there are limits to how much a clock can be advanced or delayed, one can understand why clocks cannot entrain successfully to all zeitgeber cycles. Let's go back to the earlier graph about the limits of entrainment. To make things simple, we presume that the entire day of this human subject is approximately 24 hours long and that the maximum capacity of its circadian clock is an advance of 1.5 hours. In this case, the clock of this individual can only entrain to light-dark cycles as short as 22.5 hours, but is incapable of synchronizing to shorter days. Modern chronobiologists take advantage of these "limits of entrainment" and submit subjects to very short or very long days. These so-called forced-desynchrony experiments allow to separate between influences on our biology that are connected to the circadian clock and those that are connected simply to the fact that we haven't slept for a given number of hours. Although the concept of the PRC has taught us a lot about entrainment, the idea that entrainment is achieved by a single short light pulse is unrealistic for diurnal organisms, like us, for example, who are exposed to long stretches of light. To solve this dilemma, we have introduced a slightly modified version of the PRC that doesn't rely on single light pulses, but integrates light over long periods. I will come back to this integrating response curve in the lecture about human chronotypes. "Generalization" number 15 states that every change does not necessarily lead to an immediate response of the circadian clock. In a complex system, it is not surprising that all components need some time to adapt as a network to a new situation. Many of us have experienced such so-called "transients" after travelling across time zones. Usually, our circadian system takes one day for each hour of the time change to adjust to the new light-dark regime. The 16th and last of Pittendrigh's generalizations states that circadian rhythms are surprisingly immune against chemical perturbations. It is remarkable that at a time when researchers had just begun to understand the circadian clock, Pittendrigh's comes up with 15 correct and important generalizations and makes only one wrong prediction. Many biochemical and molecular experiments have meanwhile shown that given the right chemical, clocks can be extremely sensitive to these interventions.