Science requires controlled and well-planned experiments. Without correct set-up, results from experiments may not be reliable enough to be trusted. Circadian biology is no different in that regard, and especially when trying to find out if something has a working circadian clock, controlled experiments are crucial.

The basic experiment to properly measure whether or not a circadian clock works in the dark consists of three parts:

Firstly you need to chose an appropriate variable or output to observe and measure. This could be activity patterns, metabolic patterns, breathing rate, respiration rate patterns or the core gene transcription within the molecular clock itself. The choice of variable to measure is very important, as rhythms in these variables can be stronger or weaker depending on the organisms and Some measurements will tell you more about whether or not the circadian clock is ‘ticking’ than others. Almost exclusively, the few early studies that investigated the circadian clock in the dark measured activity or rhythmic behaviour – an output of the circadian clock – and struggled from highly inconsistent results, highly susceptible to experimental procedure. Even the once gold standard of measuring transcription, or the pattern of when genes turn on and off, might not be perfect – protein-only circadian rhythms have been detected in one species of cyanobacteria.

Secondly, you need to entrain the clock – give the organism a daily pattern in light, food or some other external stimulus, to allow the potential clock to follow a pattern or rhythm. For example, our body clocks follow patterns of light and dark very well – that is why jet lag is so obvious to us. Without this entrainment period, you may not be measuring the clock properly. In mammals especially, the circadian clock has a tendency to fall out of synchrony all over the body, sort of like the combined effect trying the tell the true time from many different watches that haven’t been wound up or reset to the same time. The master pacemaker that sets all these watches, the SCN in the brain, needs the input from external stimuli to strongly set these rhythms.

Finally, after giving the entraining stimulus for a few days or repeats, continue to measure your chosen output after taking the stimulus away. If it continues to repeat, and repeat with roughly the same rhythm as your entrainment programme, it is most likely being controlled by an internal timer, the clock. If not, it is just simply responding to your stimulus – when the stimulus is taken away, your chosen output stops repeating. Of course, without controlling the environment, other entraining stimuli could persist to keep generating rhythms – this argument lingered for many years during the early stages of the circadian clock field. In first description of rhythms persisting in the absence of external cycles in 1729, de Mairan did not directly attribute the rhythms to an internal clock as he could not exclude other potential factors that would trigger the behaviour: temperature cycles, or changes in other parameters like humidity. Acceptance that there is an internal circadian clock only came after further experiments in which other scientists such as du Monceau repeated de Mairan’s work while controlling for more and more factors each time. Even then, some scientists, like Frank Brown, continued to disagree, believing that circadian rhythms were generated by some unknown factor, yet to be controlled in experiments.

This experimental set up is key for exploring new animals; without it is very difficult to say for certain whether or not your animal has a working clock. In the next blog we’ll look at why this experimental set-up and choice of variable to measure has prevented solid conclusions from being made about whether or not animals in the dark have a working body clock.