The third post in the series on entrainment, first written on April 10, 2005, starts slowly to get into the meat of things…As always, clicking on the spider-clock icon will take you to the site of the original post.
In the previous post, I introduced the concept of entrainment of circadian rhythms to environmental cycles. As I stated there, I will focus on non-parametric effects of light (i.e., the timing of onsets and offsets of light) on the phase and period of the clock.
Entrainment is a mechanism that forces the internal period (&tau – tau) of the biological clock to assume the same period (T) as the environmental cycle, i.e., &tau=T. In nature, at least on this planet, this means forcing the biological clock to oscillate with the period of 24h.
Non-parametric properties of light are binary: either the light is on, or it is off. For now, we will ignore parametric properties such as light intensity and light quality (spectral composition).
According to the theory, circadian rhythms are entrained by effects of transitions – lights-on or lights-off – on the phase of the rhythm. Thus, switching on the light in the morning, for instance, induces a phase-shift: either a phase-advance or a phase-delay. This is analogous to re-setting an imprecise wrist-watch every morning. If the watch is running slow, phase-advancing it every morning will reset it to the correct local time. If the watch is running fast, phase-delaying will reset it to the proper time.
One can conceptualize biological rhythms to be like slow watches if the endogenous freerunning period (&tau) is longer than 24 hours, or like fast watches if the &tau is shorter than 24 hours. Experimental evidence in a number of organisms points out that effects of lights-on in the morning are what is really important. The evidence for effects of lights-off in the evening is mixed.
If, for example, an animal has a clock with endogenous freerunning period (&tau) of 23 hours, it needs to be reset by phase-delaying it by exactly one hour (23 – 24 = -1h) each day. Another animal may have &tau=25 hours and needs to be phase-advanced by exactly one hour (25-24 = +1h) each day. How this requirement is met has been studied by systematic exploration of phase-shifting effects of light and construction of Phase-Response Curves (PRC).
There are eight different experimental protocols that can be used to construct a PRC. They have been shown in many cases to be equivalent to each other (I have published a study demonstrating that two of them are equivalent in my species), yet some questions still remain. Choice of the particular method will depend on the organism studied and the actual question that is asked. I will here describe the simplest method (Aschoff Type I), the protocol that is sure to make your peer-reviewers happy as it is the most straightforward and uncontroversial method and is considered to be “standard”.
Individual organisms are kept in isolation in prolonged constant conditions, including, most importantly, constant darkness (DD), and some type of output of the clock is continuously monitored (e.g., locomotor activity or body temperature in animals, leaf movements in plants, etc.) Every couple of weeks or so, each animal is exposed to a light pulse of predetermined duration, intensity and spectral composition. What is systematically varied is the phase of the rhythm at which the light-pulse is applied. How is the phase determined?
The animal kept in DD does not experience a real day and a real night. Yet, its behavior indicates that it continues to experience subjective day and subjective night. For instance, for a nocturnal animal, time when it is active is subjective night and time when it is asleep is subjective day. In a diurnal animal it is the reverse: animal is active during its subjective day and asleep during subjective night.
By convention, the time at which the animal initiates its day-specific activity (i.e., waking up for diurnal animals and falling asleep for nocturnal animals) is denoted as Circadian Time Zero (CT0), or sometimes refered to as Internal Time Zero (IT0). That is the time at which dawn would have arrived in the first cycle in DD, i.e., one cycle after the last experience of lights-on in LD12:12.
In diurnal animals, time of activity/wakefulness is subjective day and it spans CT0 – CT12. Its subjective night, when it is inactive/asleep, spans CT12-CT24. Likewise, by convention nocturnal animals start their activity (e.g,. wheel-running in rodents) at CT12 and their subjective day also spans CT0-CT12 and subjective night CT12-CT24.
When you look at the records of your experimental animals you will see that light pulses resulted in phase-shifts of the circadian rhythm. In most organisms, light-pulses applied during the subjective day have no effect on the phase, pulses that fell during the early subjective night resulted in phase delays, and pulses targeted at late subjective night produced phase-advances.
You will also notice that it often took several days of gradual phase-advancing before the rhythm assumes the new steady state after the pulse. For phase-delays, fewer days are needed for the assumption of steady-state. These apparent gradual phase-shifts are called transients.
However, remember that circadian systems are multioscillatory in nature. Like the pacemaker (e.g., the SCN in mammals), every other cell in the body also contains a clock that behaves like a slave oscillator. Experiments using two pulses given just hours apart determined that the pacemaker is phase-shifted by the light-pulse immediately. Activity, temperature, etc. are driven by brain centers other than the pacemaker and thus act as peripheral oscillators. What you see in the records of your animals is gradual resynchronization of these peripheral clocks by the pacemaker.